In vivo electrochemical reduction-induced reconfiguration of Prussian blue for metal counterion-free potassium removal in hyperkalemia

Domain: chemistry
Published: 2025-12-23
Editorial outcome: major_revision

Abstract
Hyperkalemia, a life-threatening metabolic disorder, persists as an unmet medical need since current first-line pharmacotherapy—ion-exchange potassium binders—inevitably release metal counterions (e.g., sodium) that paradoxically aggravate metabolic comorbidities. Here, we present a paradigm-shifting in vivo reduction-induced ion-selective adsorption (IRISA) strategy, utilizing Prussian Blue and Vitamin C for metal counterion-free potassium removal. IRISA leverages VC to trigger structural remodeling of PB through Fe reduction, inducing spin-state transitions and lattice reconfiguration that create K+-specific channels with optimized charge distribution. Spectroscopic studies and DFT calculations reveal K+ adsorption through electron density redistribution at coordinatively unsaturated Fe sites. This ion adsorption mechanism enables selective adsorption of K+ without metal counterion release. In three male animal models of hyperkalemia, it rapidly normalizes serum K+ while preserving Na+ homeostasis, offering a critical advantage over current first-line therapies that perturb sodium balance. Our findings establish a new paradigm for electrolyte disorder treatment using in vivo reduction-induced material reconfiguration, merging electrochemical principles with precision medicine.

Introduction
Hyperkalemia is a life-threatening metabolic disorder characterized by elevated serum K+ levels1. It is particularly common in individuals with comorbidities such as impaired renal function, cancer, liver failure, severe diabetes, and polypharmacy2–6. Approximately 90% of K+ is excreted via the kidneys in physiological condition7,8. Decreased renal function leads to potassium retention, increasing the risk of hyperkalemia9. Current treatments, including potassium binders like sodium polystyrene sulfonate (SPS, Kayexalate®), calcium polystyrene sulfonate (CPS, Kalimate®), sodium zirconium cyclosilicate (SZC, Lokelma™) and patiromer sorbitex calcium (patiromer, Veltassa®), promote K+ removal via the gastrointestinal (GI) tract, compensating for reduced renal excretion10–14. However, SPS and CPS carry well-documented risks of severe GI complications, particularly colonic necrosis and intestinal perforation15–18. While patiromer is an alternative, it exhibits critical limitations relative to the inorganic crystalline matrix of SZC, including delayed therapeutic onset (7–48 h), significant drug interaction potential, nonspecific binding (e.g., Mg2+), and higher GI adverse event rates, making it unsuitable for emergent management19,20. Notably, all current therapeutic binders rely on ion-exchange mechanism, releasing metal counterions that can disrupt the fluid balance and cause metabolic disturbances, particularly in patients with underlying conditions21. Even SZC releases Na+, which increases the risk of edema, fluid retention and hypertension, especially in patients with chronic kidney disease or heart failure22–24. In previous work, we developed a nanoporous material based on K+-NH4+ ion exchange, where NH4+ replaces Na+. The release of NH4+ inhibits Na+ reabsorption while simultaneously promoting K+ reabsorption25. This mechanism exacerbates salt wasting, consequently resulting in suboptimal performance in managing conditions such as Pseudohypoaldosteronism type I (PHA-1) complicated by hyperkalemia25,26. Additionally, in patients with abnormal nitrogen metabolism complicated by hyperkalemia, releasing NH4+ increases the burden on the liver, posing additional risks4,27. To date, no potassium binder has been developed that completely avoids metal counterion release. Therefore, a novel potassium adsorption approach that minimizes metal counterion-related complications is urgently needed.

Prussian blue (PB), an organometallic-like framework with large interstitial sites that facilitates high K+ mobility, has shown promise as a host material for potassium-ion batteries28–30. This application leverages a fundamental property shared by PB and its analogue: their well-established ability to electrochemically intercalate and deintercalate various alkali metal ions, as extensively studied in systems like sodium-ion batteries31. In potassium-ion batteries, PB serves as the positive electrode, where significant amounts of K+ are inserted during the voltage-driven reduction process of electrochemical reaction32. The ability of PB to insert K+ is primarily due to the variable oxidation states of iron, which help maintain charge balance and enable efficient K+ selection and adsorption33,34. The insolubility of PB allows for gut-restricted use in humans, offering a unique safety profile for treating Cesium and Thallium poisoning, with zero systemic exposure35–37. Given these advantageous characteristics, including high K+ diffusion coefficient, remarkable storage capacity and structural stability, PB, as an FDA-approved drug, aligns well with therapeutic approaches of potassium binders for hyperkalemia38–40. However, K+ adsorption capacity of single PB is limited in both single-ion and multi-element systems, and it involves counterion release41. Therefore, PB cannot be directly used for hyperkalemia management.

This paradox motivates our reimagining of in vivo and electrochemical interfaces, we could use antioxidant to replicate voltage-driven ion insertion principles. Herein, by bridging electrochemical insertion concepts with gut-restricted therapeutic design, we present an innovative in vivo reduction-induced ion-selective adsorption (IRISA) strategy for hyperkalemia treatment. Our IRISA strategy combines PB with vitamin C (VC), a well-known safe antioxidant, to enhance K+ adsorption capacity and prevent metal counterion release of PB. VC can reduce Fe, facilitating K+ binding within diffusion channels of PB42,43. Material characterizations and theoretical calculations demonstrate that the presence of K+ is essential for VC to alter the valence state of Fe in PB. The reduction of PB promotes K+ removal from the GI tract, where it aids in maintaining K+ transfer from blood vessels until serum K+ levels normalize (Fig. 1a). Co-administration of PB and VC is safe and effective in both in vitro simulated solutions and three animal models. Unlike ion-exchanging binders, this strategy selectively adsorbs and removes K+ without releasing metal counterions, making it particularly beneficial for hyperkalemia patients with underlying medical conditions. In summary, our orally administered IRISA strategy, a potassium binder based on PB and VC (PB+VC), provides a promising therapeutic approach for hyperkalemia, mimicking the electrochemical reduction of an electrode. This strategy offers a novel framework with potential applications across medical therapy, electrochemistry, and beyond.

Results
The adsorption capacity of PB during reduction
The performance of potassium binders is assessed based on two critical parameters: K+ adsorption capacity and selectivity. Prussian blue (PB), a material widely used in potassium-ion batteries, demonstrates high K+ storage potential19. As an FDA-approved drug, PB is utilized for the removal of Cesium and Thallium, from the human body, with a recommended dose of 9 g day−1, highlighting its gut-restricted properties and non-toxic side effects35–37,44. In multi-element isoelectric (MEI) mixed solutions containing K+, Na+, Ca2+, and Mg2+, PB demonstrated an excellent selectivity for K+. However, it exhibited limited adsorption capacity (Fig. S1a). Inspired by electrochemical behavior of PB, where K+ insertion occurs during reduction in PB as a positive electrode, we hypothesized that altering the valence state of Fe in PB might enhance its selective adsorption of K+ by maintaining charge balance. A range of orally safe antioxidants was tested for their ability to improve K+ adsorption. Among these, VC significantly enhanced the K+ adsorption capacity when combined with PB in equal mass ratios (Fig. S1b). The limited efficacy of other reducing agents likely stems from their inadequate reducing power, or poor water solubility that restricts interaction with PB. Further analysis revealed that increasing VC concentration enhanced K+ adsorption capacity of PB up to a PB:VC mass ratio of 1:1, beyond which no significant improvement was observed (Fig. S1c). Considering the renal excretion of VC, excessive doses may impose additional strain on kidney function which is already impaired. Accordingly, a PB:VC mass ratio of 1:1 was selected as an optimal and relatively safe proportion, balancing maximal adsorption capacity.

To comprehensively evaluate the K+ adsorption efficacy of the PB and VC combination (PB+VC), we compared it with different potassium binders, in simulated GI fluids that mimic in vivo environments, including Simulated gastric fluid electrolyte solution (SGF), Simulated intestinal fluid electrolyte solution (SIF) and Simulated Colonic Fluids electrolyte solution (SCoF) (Fig. 1b). PB+VC demonstrated superior K+ adsorption capacity compared to PB alone, SPS, CPS and comparable capacity with SZC, with no significant difference between PB:VC mass ratios of 1:1 and 1:4. Additional adsorption tests in bio-relevant simulated GI solutions demonstrated that PB+VC retained exceptional K+ adsorption capacity and exhibited robust resistance to interference from complex components, underscoring its stability (Fig. S1d).

Given the competitive cations present in the human GI tract, we assessed the selectivity of PB+VC in the presence of interfering ions, such as Na+, Ca2+, and Mg2+. exhibited minimal adsorption of ions other than K+, demonstrating superior selectivity compared to other binder, while additionally avoiding the release of metal counterions (Fig. 1c). PB+VC demonstrates not only excellent potassium adsorption capacity but also exhibits remarkable potassium selectivity in vitro, showing negligible adsorption of Ca2+ and Mg2+ (with no significant difference compared to SZC). Importantly, unlike SZC, SPS (which releases Na+) or CPS (which releases Ca2+), PB+VC does not liberate any metal ions. Additionally, to address potential safety concerns, we measured Fe release (Fig. S1e). Results indicated PB+VC released significantly less Fe than FDA-approved PB, confirming that VC-induced reduction does not compromise its initial structural integrity or safety profile.

Structural properties of PB before and after VC reduction
To investigate the structural changes in PB contributing to its enhanced K+ adsorption performance upon VC addition, multiple characterizations were conducted. The XRD patterns (Fig. 2a) were consistent with Prussian blue (PDF#01-0239), indicating that the PB backbone remained stable after VC treatment during K+ adsorption. Analysis of ion contents in the solution before and after adsorption revealed significant K+ incorporation, accompanied by reduced lattice parameters. This reduction suggests that K+ was incorporated into the crystal structure of PB. Significantly, analysis of the recovered liquid samples following MEI adsorption by PB+VC revealed no detectable Fe release. These observations provide evidence that, in addition to potassium adsorption, the cyanide-bridged coordination network framework of PB+VC should maintain its original chemical composition, thereby largely excluding the possibility of dissolution-recrystallization occurring during the adsorption process. Fourier transform infrared (FTIR) and Raman spectroscopy (Fig. 2b, c) revealed no peaks corresponding to organic groups such as -CH2- or -CH3, confirming that VC and its reaction products did not enter the PB structure after ion adsorption. Observed peak shifts were attributed to changes in the valence state of Fe and the coordination environment of the ν(C≡N) bond, providing evidence of a reduction process in PB45,46. Room temperature 57Fe Mössbauer spectra (Fig. 2d, e and Table S1) clearly revealed the presence of high-spin Fe2+ in VC-treated PB. By comparing the spectral area ratios of Fe species in PB and PB+VC, it can be concluded that a significant amount of mol of Fe3+ in each mol of PB was reduced to Fe2+, resulting in a lower overall oxidation state of Fe in the PB structure.

Further analyses using X-ray photoelectron spectroscopy (XPS) and X-ray fluorescence spectroscopy (XRF) (Fig. 2f and Table S2) confirmed the successful incorporation of K+ into VC-treated PB without release of other ions. To determine the elemental content, quantitative analysis of the solids prior to and following adsorption was carried out via inductively coupled plasma optical emission spectroscopy (ICP-OES) (Table S3). The elemental composition analysis of PB+VC, with variations ranging from 0.11 wt% to 12.1 wt% of K, revealed that the molar ratio of K to Fe in the treated sample was approximately 1:1.8. This result aligns well with the observations from Mössbauer spectroscopy, which demonstrated the reduction of Fe3+ and achievement of maximum theoretical K+ adsorption capacity. Based on these consistent findings, we can reasonably infer that the maximum reduction of PB by VC during the preparation process results in the chemical formula of the treated product being K4Fe7C18N18 (Fig. 2g). Specific surface area measurements showed that both untreated and VC-treated PB retained permanent porosity (Fig. S2), although partial filling of pores with K+ in VC-treated PB reduced the available surface area. Both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images (Figs. 2h–j, and S3a-c, f-h) reveal uniformly-sized, well-dispersed nanocubes. Both PB and PB+VC exhibit particle sizes ranging from 30 to 70 nm, which facilitates rapid K+ diffusion through the structure. The highly consistent particle size distribution between PB and PB+VC further confirms that K+ adsorption was achieved not via dissolution-recrystallization, but rather through a reductive adsorption mechanism. High-angle dark-field scanning TEM (HAADF-STEM) and EDS elemental mapping (Figs. 2k, l and S3d, e, i, j) further confirmed uniform K+ incorporation within the PB structure, reinforcing the selective K+ adsorption capability.

Pseudo-in-situ XRD and Raman data (Fig. 3a, b) highlighted the role of VC concentration in enhancing the reduction capacity of PB. Increased VC concentration led to further decreases in PB lattice parameters and leftward shifts in the C≡N stretching vibration peak47. These results suggest that the volume of K+ incorporated into the PB crystal lattice and the cation valence state of Fe are positively correlated with the overall reduction capacity. Cyclic voltammetry (CV) measurements (Fig. 3c) provided additional insights into the redox behavior of PB. Significant redox peaks with higher current densities and enclosed areas were observed in MEI and KCl solutions, indicating efficient ion insertion and release. The incomplete alignment of oxidation and reduction peaks reflected limited reversibility, which may promote efficient ion adsorption while hindering ion desorption, thereby enabling more thorough K+ removal. Finally, XRD, Raman, and UV-vis characterizations of PB+VC treated with different electrolyte solutions (Fig. 3d, e, f) showed that only KCl and MEI solutions exhibited the same trend, further validating the reduction-induced adsorption mechanism.

Theoretical studies of PB selective adsorption
Density functional theory (DFT) calculations were conducted to understand the adsorption mechanism of PB for ions during the reduction process. A comparison of adsorption energies for different ions revealed that PB preferentially adsorbs K+ over Na+, Ca2+, and Mg2+ (Fig. 3g). Simulating the reduced state of PB by adding four electrons significantly enhanced ion adsorption, highlighting the role of reduction in improving K+ selectivity. The reduction mechanism was further validated by analyzing the electronic structures of cation-inserted PB. In K-PB, unoccupied spin-up states of Fe shifted below the Fermi level, suggesting that Fe more readily accept electrons and undergo redox reactions (Fig. S4).

To explore atomic-scale changes in PB during ion insertion, charge density differences were calculated to visualize the electronic state distribution (Fig. S5). In K-PB, increased charge density around Fe formed electron channels that facilitated efficient charge transfer, confirming that Fe gained electrons upon K+ insertion. Correspondingly, Bader charge analysis (Fig. 3h, i) showed an increase in the charge on Fe, establishing Fe acts as an active redox center during the charge transfer process triggered by K+ insertion. These charge transfer behaviors were consistent with the projected density of states (PDOS) and charge density differences. Collectively, the DFT calculations provided a detailed explanation of reduction mechanism and its enhanced K+ adsorption capacity during ion adsorption from electrolyte solutions.

Safety and therapeutic effects of PB+VC in animal models
To evaluate the feasibility of PB+VC as a potential treatment for hyperkalemia, in vitro and in vivo biosafety assays were conducted. Considering the gut-restriction of PB+VC, we tested three GI cells to assess the cytotoxicity of PB+VC, including GES-1, IEC-6 and Caco-2, based on the locations where the drug would interact48–50. The results of the cytotoxicity assay and calcein/PI assays revealed that PB+VC at 320 μg ml−1 (160 μg ml−1 PB+160 μg ml−1 VC) is non-toxic to GI cells (Figs. 4a and S6).

In vivo biocompatibility was further assessed in healthy rats (Fig. 4b). K+ excretion was monitored by collecting feces and urine on days 1, 8, and 15 (Fig. 4c, d). 14 days’ continuous PB+VC treatment resulted in a significant reduction in urinary K+ and an increase in fecal K+, demonstrating that PB+VC effectively adsorbs K+ and excretes it mostly via the feces. K+ was primarily excreted via urine in physiological condition, PB+VC reduces the renal excretion of K+ and potentially alleviates the burden on the kidneys8. Blood K+, Na+, Ca2+, and Mg2+ levels, alongside urinary Na+, Ca2+, and Mg2+ excretion and fecal Na+, Ca2+, and Mg2+ content, remained within physiologically normal ranges across all experimental groups (Figs. 4d and S7). Blood gas parameters (pH, HCO3-, CO2) and hematological parameters exhibited no clinically significant deviations (Figs. S8-10). Notably, Blood Fe2+, transferrin, and ferritin levels also fell within normal physiological ranges (Fig. 4f–h). These findings collectively demonstrate that our therapeutic strategy, even at higher PB+VC doses, induces neither hypokalemia nor other electrolyte disturbances, acid-base imbalances, or hematological abnormalities, thereby confirming its safety profile.

Since our strategy achieves K+ adsorption in the GI, we comprehensively assessed GI safety. The results indicated that both GI permeability and intestinal length remained within normal range (Figs. 4i and S11). Given that hyperkalemia is often accompanied with impaired renal function, we selected a therapeutic dose of 4 g kg−1 PB+VC (PV1: 2 g kg−1 PB+2 g kg−1 VC) and a high dose of 12 g kg−1 PB+VC (PV2: 6 g kg−1 PB+6 g kg−1 VC) to evaluate potential nephrotoxicity at different VC dosages. Urinalysis and blood biochemical parameters showed no significant differences compared to the control group, demonstrating that even at three times the therapeutic dose, VC did not cause additional renal burden or toxicity (Figs. 4j–m and S12-14). Histopathological analysis through hematoxylin and eosin (H&E) staining of stomach, small intestine, and colon tissues harvested on day 14 confirmed that PB+VC did not affect the physiological function of healthy rats (Figs. 5n–q and S15). In conclusion, PB+VC was found to be safe in rats even at a high dose. This is well above the therapeutic dose of SZC administered to animals in current studies (approximately 4 g kg-1 in rats), indicating a wide therapeutic window for PB+VC24,51–53.

The mechanism of action of amiloride involves blocking the epithelial sodium channel (ENaC) in the distal tubule of the kidney54. PHA-1 is commonly associated with hyperkalemia, which results from impaired ENaC function55. Given that both conditions involve a failure of ENaC function, we selected amiloride-induced hyperkalemia as a model to evaluate the efficacy of the drugs56. In contrast to the reduction in blood K+ observed with SZC, which may be attributed to Na+ release to alleviate amiloride-induced salt-wasting, as evidenced by the partial K+-lowering effect of NaCl administration alone, PB+VC demonstrated efficacy in this model through direct adsorption, reducing blood K+ to normal levels (Figs. 5a, b and S16)57,58. Additionally, amiloride caused only a mild, clinically insignificant decrease in blood Na+ through K+-sparing/Na+-excreting. We hypothesize that PB+VC attenuates this change by restoring K+ excretion and enhancing NHE3-mediated reabsorption of Na+ in response to increased VC-derived H+ concentration59. Concurrently, the Na+ release by SZC led to elevated blood Na+ levels, disrupting the electrolyte balance in mice (Fig. 5c).

Hyperkalemia is also commonly associated with impaired renal function and high-potassium diets. To mimic this disease and establish hyperkalemia models, we used male SD rats with chronic kidney disease (CKD) induced by 5/6 nephrectomy (Nx) (Fig. 5d, g)52,60,61. Both PB+VC and SZC were found effective in reversing the increase in blood K+ levels (Fig. 5e, h). As anticipated, the PB+VC treatment, as a gut-restricted strategy based on metal counterion-free release, effectively corrected abnormal blood K+ by promoting substantial fecal K+ excretion. KCl supplementation moderately reduced blood Na+ in rats, likely through tonic inactivation of NaCl cotransporter (a known antihypertensive mechanism) in the renal distal convoluted tubule62. This effect was exacerbated in the 5/6 Nx model due to impaired tubular Na+ reabsorption from nephron loss. The K+ excretion mechanism of PB+VC concurrently restored normal blood Na+ (Fig. 5f, I).

Unlike SZC, which exacerbates renal metabolic stress through excessive Na+ release, particularly detrimental given pre-existing nephron impairment where Na+ metabolism competitively disrupts other electrolytes (e.g., Mg2+), PB+VC not only corrects hyperkalemia-induced sodium imbalance but also prevents secondary electrolyte disturbances and metal counterion release, thereby avoiding additional renal metabolic burden (Figs. S17a, b, d, f and S18). Notably, PB+VC did not exacerbate acidosis, a frequent complication of hyperkalemia, and even demonstrated mild ameliorative effects during prolonged administration (Fig. S17c, f). This makes PB+VC a more suitable clinical treatment for hyperkalemia, particularly for patients with underlying health conditions such as CKD, PHA-1 and Gordon’s syndrome (hyperkalemia-hypertension syndrome), due to its distinctive adsorption mechanism that avoids ion exchange. To sum up, PB+VC treatment for hyperkalemia in animal models is both safe and effective, with the potential for broader clinical applications.

Methods
Materials and chemicals. Prussian Blue (Fe7C18N18, PB, >99.0%) and L(+)-Ascorbic Acid (C6H8O6, Vitamin C, >99.0%) were purchased from Adamas-beta®. Sodium polystyrene sulfonate (SPS, Kayexalate®) was purchased from KALIMATE, calcium polystyrene sulfonate (CPS, Kalimate®) was purchased from SCOND. Sodium Zirconium Cyclosilicate Powder (Lokelma™) was purchased from AstraZeneca. Other reagents were purchased from Adamas-beta ® without any further purification.

Preparation of PB+VC. Briefly, PB (200 mg) and VC (200 mg) were mixed with MEI (40 mL), followed by incubation at 37°C with orbital shaking (180 rpm) for 2 h. Post-incubation, the mixture was centrifuged at 1600 g for 5 min to separate the supernatant. The resulting pellet underwent three washing cycles with deionized water (30 mL per cycle, vortex mixing for 30 s), each followed by identical centrifugation parameters. The purified precipitate was flash-frozen in liquid nitrogen and subsequently lyophilized for 48 h to obtain the final product for Structural and morphological characterization. In the processed PB+VC, the VC component and its oxidation products dissolve in aqueous solution, yielding a final product consisting primarily of K+-adsorbed PB.

Material characterizations. The textural properties of the samples were analyzed using the instruments listed below. The crystalline phase was characterized by X-ray diffraction (XRD, D8 Advance Da Vinci, Bruker, Germany) with 2θ ranging from 10° to 70°. The composition and structure of samples were recorded on Fourier transform infrared spectra (FT-IR, Nicolet 6700, Thermo Fisher Scientific, USA) and Raman spectra (inVia Qontor, Renishaw, UK). Quantification of the elements in the samples was performed using wavelength disperse X-ray fluorescence (XRF, S8 Tiger II, BRUKER AXS GmbH, China). Specific surface area and porosity in N2 were investigated with specific surface area and porosity analyzer (Autosorb-IQ3/EVO, Quantachrome, USA). Ultraviolet−visible (UV-vis) absorption spectra were collected using a CARY 5000 spectrophotometer (Lambda 950, PerkinElmer, USA). The 57Fe Mössbauer spectra of samples were recorded on Mössbauer spectrometer (SEECo W304, Wissel, Germany) using a 57Co/Rh source in transmission geometry. The data were fitted by using the MossWinn 4.0 software. The chemical composition was analyzed by X-ray photoelectron spectroscopy (XPS, NEXSA, Thermofisher Scientific, USA).

Microscopy images: A small amount of powdered sample was first adhered to conductive tape and sputter-coated with gold for 45 s at 10 mA using a Quorum SC7620 sputter coater. Subsequently, the sample morphology was examined using a ZEISS GeminiSEM 300 (Germany) scanning electron microscope (SEM) at an accelerating voltage of 3 kV with an SE2 secondary electron detector, while EDS mapping was performed at 15 kV. For further analysis, another portion of the powder was embedded and ultrathin-sectioned for characterization using a FEI Talos F200X field-emission transmission electron microscope (TEM, USA) equipped with a BM Ceta camera. Both TEM imaging and EDS mapping were conducted at 200 kV, with the EDS system achieving an energy resolution of 136 eV. All TEM data were processed and analyzed using Velox software.

Ex-situ X-ray diffraction (XRD) data were collected using a D8 ADVANCE Da Vinci diffractometer with Cu Kα radiation (λ = 0.154 nm). Ex-situ Raman spectra were recorded in the 2500–500 cm−1 Raman shift region using Renishaw inVia Qontor. Both were treated with different concentrations of reducing agents.

Electrochemical measurements. Preparation of PB electrodes: 10 mg PB catalyst was added to 950 μL water, 950 μL ethanol and 50 μL Nafion solution, then sonicated for 15 min. The resulting 100 μL sample was dropped onto a clean ITO electrode, dried at room temperature. The electrochemical adsorption performance of the electrode materials was studied by cyclic voltammetry (CV) (CHI 660E, CH Instruments Inc., USA). The platinum electrode was against electrode, reference electrode was Ag/AgCl electrode, electrolyte is different ionic solution and the voltage window is from −1. V to +1 V, and the scan rate is 50 mV s-1.

Quantum chemical calculation. All the calculations are performed in the framework of the density functional theory with the projector augmented plane-wave method, as implemented in the Vienna ab initio simulation package (VASP)63. The crystal structures input (CIF files) were uploaded as attachments. The exchange-correlation potential was chosen as the generalized gradient approximation (GGA) proposed by Perdew, Burke, and Ernzerhof (PBE)64. The long range van der Waals interaction is described by the DFT-D3 approach65. The cut-off energy for plane wave is set to 500 eV. Furthermore, the Brillouin zone was sampled using a Monkhorst–Pack k-point mesh (56) with 5×5×5 k-points for lattice optimizations and 9×9×9 k-points for static density of states (DOS) calculations, respectively. The energy criterion is set to 10−6 eV in iterative solution of the Kohn-Sham equatio. All the structures are relaxed until the residual forces on the atoms have declined to less than 0.03 eV/Å.

In these calculations, model anhydrous structures with a face-centered cubic F4̅3m unit cell were assumed. Four compensating charges are mimicked in the reduced state. The absorption energy was calculated on the basis of the PB crystal inserting different atoms as absorbate, given as Ead = (Ehost/adsorbate − Ehost − n Eadsorbate)/n, and Ehost/adsorbate, Ehost and Eadsorbate are the DFT total energies of the adsorbate on the host crystal, the host crystal and the adsorbate itself, respectively.

In vitro ions adsorption assays. The adsorption capacity of PB+VC was evaluated by weighing Prussian blue at a concentration of 5 mg/mL and adding it to a centrifuge tube containing the configured liquid, with different mass ratios ascorbic acid added as a reducing agent. Furthermore, the adsorption capacity of alternative adsorbents (PB, SZC, SPS, and CPS) was employed at a concentration of 5 mg/mL for comparative purposes. The treated tubes were subjected to shaking at 37°C for a period of two h at 180 rpm. Following this, the liquid phase underwent filtration and dilution before being characterized by inductively coupled plasma emission spectroscopy (ICP-OES). For this study, a Thermo iCAP7600 inductively coupled plasma emission spectrometer was utilized.

Adsorption capacity calculation formula as specified below:
Adsorption capacity = (C0 − Ce)V/m (mmol/g)
C0 = Initial concentration before adsorption (mmol/L)
Ce = Equilibrium concentration after adsorption (mmol/L)
V = Solution volume (L)
m = Mass of adsorbent (PB+VC, PB, SZC, SPS, and CPS) (g)

The components of the simulated fluids are listed below:
Multi-element isoelectric solution with K+, Na+, Ca2+, Mg2+ (MEI). 50 mM KCl, 50 mM NaCl, 25 mM CaCl2 and 25 mM MgCl2 were dissolved and made up to 1 L with purified water.

Simulated gastric fluid electrolyte solution (SGF). 8.625 mM KCl, 1.125 mM KH2PO4, 31.25 mM NaHCO3, 0.15 mM MgCl2, 0.625 mM (NH4)2CO3, 59 mM NaCl was mixed in 1000 mL of purified water. The pH was adjusted to 3 with 1 M HCl.66

Simulated intestinal fluid electrolyte solution (SIF). 8.5 mM KCl, 1 mM KH2PO4, 106.25 mM NaHCO3, 0.4125 mM MgCl2, and 48 mM NaCl was mixed in 1000 mL of purified water. The pH was adjusted to 7 with 1 M HCl66.

Fasted-state simulated gastric fluid electrolyte solution (FaSSGF). 80 mM Sodium taurocholate, 20 mM Lecithin, 8.625 mM KCl, 1.125 mM KH2PO4, 31.25 mM NaHCO3, 0.15 mM MgCl2, 0.625 mM (NH4)2CO3 and 34.2 mM NaCl was mixed in 1000 mL of purified water. The pH was adjusted to 1.6 with 1 M HCl or 1 M NaOH67.

Fed-state simulated gastric fluids electrolyte solution (FeSSGF). 17.12 mM Acetic acid, 29.75 mM Sodium acetate, 8.625 mM KCl, 1.125 mM KH2PO4, 31.25 mM NaHCO3, 0.15 mM MgCl2, 0.625 mM (NH4)2CO3 and 237.02 mM NaCl was mixed in 1000 mL of purified water. The pH was adjusted to 5.0 with 1 M HCl or 1 M NaOH67.

Fasted-state simulated intestinal fluids electrolyte solution (FaSSIF). 3 mM Sodium taurocholate, 0.75 mM Lecithin, 8.5 mM KCl, 1 mM KH2PO4, 106.25 mM NaHCO3, 8.7 mM NaOH, 28.7 mM NaH2PO4, 0.4125 mM MgCl2 and 105.9 mM NaCl was mixed in 1000 mL of purified water. The pH was adjusted to 6.50 with 1 M HCl or 1 M NaOH68.

Fed-state simulated intestinal fluid electrolyte solution (FeSSIF). 15 mM Sodium taurocholate, 3.75 mM Lecithin, 144 mM Glacial Acetic Acid, 8.5 mM KCl, 1 mM KH2PO4, 106.25 mM NaHCO3, 101 mM NaOH, 0.4125 mM MgCl2 and 173 mM NaCl was mixed in 1000 mL of purified water. The pH was adjusted to 5.0 with 1 M HCl or 1 M NaOH68.

Simulated Colonic Fluids electrolyte solution (SCoF). 5.5900 g K2HPO4, 0.4100 g KH2PO4 was mixed in 1000 mL of purified water. The pH was adjusted to 7.8 with 1 M HCl or 1 M NaOH67.

Fasted-state simulated colonic fluids (FaSSCoF). 0.0646 g sodium tauroglycocholate, 0.1932 g lecithin, 0.0305 g sodium oleate, 10.88 g NaOH, 5.4513 g tris(hydroxymethyl)aminomethane hydrochloride, 8.8210 g maleic acid, 5.5900 g K2HPO4, 0.4100 g KH2PO4 was mixed in 1000 mL of purified water. The pH was adjusted to 7.8 with 1 M HCl or 1 M NaOH.

Fed-state simulated colonic fluids (FeSSCoF). 0.2583 g sodium tauroglycocholate, 0.3219 g lecithin, 0.1609 g sodium oleate, 3.7600 g NaOH, 3.7553 g tris(hydroxymethyl)aminomethane hydrochloride, 3.4821 g maleic acid, 5.5900 g K2HPO4, 0.4100 g KH2PO4 was mixed in 1000 mL of purified water. The pH was adjusted to 6.0 with 1 M HCl or 1 M NaOH.

In vivo experiment part
Materials and chemicals. Rat Lipopolysaccharide (LPS) and Diamine Oxidase (DAO) ELISA kits were purchased from BYabscience (Nanjing, China). Rat Ferritin and transferrin ELISA kits were obtained from Jianglai Biotech (Shanghai, China). Purified, refined diet (AIN-93G) and refined diet (AIN-93G) supplemented with additional K (3% weight/weight) were purchased from Yihe Suyuan Bio-engineering (Nanjing, China).

Cell culture. Human gastric mucosal epithelial cells (GES-1), intestinal crypt epithelial cells (IEC-6) and human colon carcinoma cell line, Caco-2, was obtained from the National Collection of Authenticated Cell Cultures and cultivated in Dulbecco’s modified Eagle’s medium (DMEM) (Adamas life®) supplemented with 20% fetal bovine serum (FBS) (Adamas life®) and 1% antibiotic antimycotic solution (containing 10,000 units/ml penicillin G, 10 mg/ml streptomycin sulfate and 25 μg/ml amphotericin) (Adamas life®). All the cells were incubated at 37°C in 5% CO2 atmosphere. Before each experiment, the cultures were washed with phosphate-buffered saline (PBS, pH 7.4) (Thermo Fisher Scientific), detached with 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) solution (Beyotime Biotechnology), and then centrifuged at 1000 rpm for 5 min, followed by resuspension.

Cytotoxicity evaluation. The cytotoxicity of samples against GES-1, IEC-6 and Caco-2 cells (GI cells) was evaluated by live/dead staining and CCK-8 assay. First, 1×105 GI cells were seeded in six-well plates, after incubation at 37°C in a humidified atmosphere with 5% CO2 for 24 h, the culture medium was replaced by PB, VC and PB+VC (each concentration, 160, 160, and 320 μg ml−) for 24 h. Then, the cells were observed by a fluorescence microscope after being stained with calcein-AM (acetoxymethyl) and propidium iodide (PI) (Adamas life®). Meanwhile, an CCK-8 assay was conducted to assess the cytotoxicity. A total of 5×103 GI cells were seeded in 96-well plates and cultured for 24 h. Then, cells were treated with a series of concentrations of PB, VC and PB+VC and incubated for 24 h. After CCK-8 (Yeasen) addition for 4 h, the OD values of each well were measured at 450 nm on an enzyme standard. The cell viability calculation is as follows: (ODsamples − ODblank)/(ODcontrol − ODblank)×100%.

Animals. All procedures were conducted in agreement with the guideline of the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University. IACUC of Shanghai Jiao Tong University approved all protocols (No. 3 A2023215). Male SD rats (200~250 g body weight) were purchased from the Vital River Laboratory Animal Technology Co., Ltd (Nanjing, China). Male C57BL/6 mice (15–20 g body weight) were purchased from Jihui Laboratory Animal Care Co., Ltd (Shanghai, China). All animals were housed in an SPF facility at Shanghai Jiao Tong University and received human care. Prior to the experiments, the animals were maintained on a 12-hour light/dark cycle with constant temperature and humidity, and acclimated to the housing environment for at least seven days. Animals were randomly assigned to treatment groups using a random number generator. To control for potential covariates, such as body weight, animals were stratified before randomization to ensure equal distribution across groups.

Toxicity study in normal animals. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University. All SD rats were purchased from Shanghai Jihui Laboratory Animal Care Co., Ltd. 30 male rats were divided into 4 groups in the experiment. The control group received water intragastric injection twice a day during drug administration. As for 3 experimental groups, they respectively received 6 g kg−1 PB, 6 g kg−1 VC, 4 g kg−1 PB+VC (PV1: 2 g kg−1 PB+2 g kg−1 VC) or 12 g kg−1 PB+VC (PV2: 6 g kg−1 PB+6 g kg−1 VC) by intragastric injection twice a day, respectively.

1 h before the first intragastric administration on days 0, 7, and 14, whole blood samples (150 μL) were collected and analyzed using an automatic blood analyzer (XN-1000V, Sysmec Co., Japan). Approximately 100 μL whole blood samples were used for electrolyte concentration and arterial blood gas analysis. Indicators of respiratory function, acid-base balance, and plasma ions (K+, Na+, Ca2+) were obtained in rats using a portable blood gas analyzer (iSTAT-300, Abbott Laboratories, USA) equipped with EG7+ chips (EG7+, Abbott Laboratories, USA).

About 1 mL blood samples were put into a centrifuge tube, standing for 1 h, and then the samples were centrifuged for 10 min at a speed of 1600 g to collect serum. The serum (~200 μL) was used in the test of Mg2+, Fe2+, kidney function and liver function by automated analyzer including software (Cobas c 311, Roche Diagnostics, USA). ELISA Ferritin, transferrin, DAO, and LPS kits were used according to manufacturer’s instructions.

Stool and urine samples were collected on days 1, 8, and 15 of the experiment. All stool and urine samples were digested in nitric acid and hydrogen peroxide prior to analysis. K+, Na+, Ca2+, Mg2+ concentration of stool and urine were analyzed on the ICP-MS (NexION 2, PerkinElmer, USA) spectrometer after digestion.

At the end of the experiment, rats were treated with FITC-dextran (300 mg kg−1, p.o.) and whole blood was collected through orbits after 3 h for further testing. Then the rats were euthanatized by CO2. GI tracts were isolated and pathological sections were prepared to evaluate possible toxicity of the drugs.

Urinalysis of rats: Selected rats were placed in cages for 24 h to collect urine samples. Midstream urine samples were collected from rats during urination, mixed thoroughly, and 2 mL of the sample was used for urinalysis using an automated urine biochemical analyzer (URIT-500B, URIT, China). The rest data were semi-quantitative results. Semi-quantitative results in urine analysis were converted into scores as follows: urinary protein (0: not detected, 1: <0.15 g/L, 2: <0.3 g/L, 3: <1.0 g/L), urinary glucose (0: not detected, 1: <2.8 mmol/L, 2: <5.5 mmol/L, 3: 14 mmol/L).

Efficacy study in mice and rats with high blood K+. First, hyperkalemia was induced in 20 normal female C57BL/6 mice by intraperitoneal injection of 0.8 wt% KCl and 0.03 wt% amiloride (Adamas life®) and these mice were divided into 5 groups in the experiment56. The dose of intraperitoneal injection was 10 μl/g. 4 groups were followed by respectively intragastric injection of 4 g kg−1 PB, 4 g kg−1 VC, 4 g kg−1 PB+VC (2 g kg−1 PB+2 g kg−1 VC) or 4 g kg−1 SZC per day. 4 normal control mice received intragastric injection of deionized water.

Second, at the beginning of the experiment, Sprague-Dawley (SD) rats with chronic kidney disease were induced in male SD rats by 5/6 subtotal nephrectomy (Nx) 21 days before the experiment61. KCl solution was used by intragastric injection to evaluate the therapeutic effect of this drug on acute hyperkalemia under the condition of a high-potassium diet in chronic kidney disease. Then, 25 nephrectomized rats were divided into 1 control group, 1 blank group, and 4 experimental groups. Rats in 4 experimental groups respectively received 4 g kg−1 PB, 4 g kg−1 VC, 4 g kg−1 PB+VC (2 g kg−1 PB+2 g kg−1 VC) or 4 g kg−1 SZC per day. The control group received intragastric injection of water, and the blank group received KCl solution.

Third, refined diet (AIN-93G) supplemented with additional K (3% weight/weight) only and in combination with amiloride (0.05 mM) was used to evaluate the therapeutic effect of this drug on chronic hyperkalemia under the condition of a high-potassium diet in chronic kidney disease60. 16 nephrectomized rats were divided into 1 control group, 1 blank group, and 3 experimental groups. The control group received refined diet, and other group received high-potassium diet high-potassium diet for 14 days. Rats in experimental groups respectively received 4 g kg−1 PB, 4 g kg−1 PB+VC (2 g kg−1 PB+2 g kg−1 VC) or 4 g kg−1 SZC per day was started on day 5 for 8 days.

All the Blood samples were collected 1 h after the intragastric injection of drugs. K+, Na+, Ca2+ were analyzed on the Abbot iSTAT 1 analyzer with EG7+ chips. Mg2+ was analyzed on automated analyzer including software.

The effects of NH4+ and Na+ in the amiloride-induced hyperkalemia model. Hyperkalemia was induced in 9 normal male C57BL/6 mice by intraperitoneal injection of 0.8 wt% KCl and 0.03 wt% amiloride (Am) and these mice were divided into 3 groups in the experiment. The dose of intraperitoneal injection was 10 μl/g. As for 3 experimental groups, they respectively received deionized water, 1.79 g kg−1 NaCl and 1 g kg−1 NH4Cl per day by intragastric injection, divided into two daily doses administered after 1 h and 5 h apart. All the Blood samples were collected 1 h after the intragastric injection of drugs. K+, Na+ were analyzed on the Abbot iSTAT 1 analyzer with EG7+ chips.

Statistical analysis
All experiments were performed at least triplicated with repeated measures. The results are expressed either as means ± standard error of the mean (S.E.M). One-way ANOVA with Tukey’s multiple comparison test were used for testing differences among groups. For each statistical analysis, the sample size was greater or equal to three (n ≥ 3). Detailed statistics can also be found in figure legends. Data were approximately normally distributed, and the variance was similar among all the groups. No samples were excluded from the analysis, and no preprocessing of data was performed before statistical analysis. Statistical significance is indicated as n.s. p>=0.5 and precise p values were calculated and showed in figures.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper demonstrated the removal of K+ from blood using the reduction of PB. The combination of PB and VC can remove K+ to the same extent as SCZ. However, there are various problems with this manuscript, as listed below. Regarding the DFT calculations, there is a lack of information, so it is impossible to judge whether it is good or bad. . 1. There are no details of the DFT calculations, so it is unclear what structure was assumed for the calculations, or the conditions of the calculations. Since the details are unknown, it is unclear whether the description is correct or not. 2. It is unclear how to prepare PB+CV sample in Fig. 2 is. This should be stated in the caption. 3. Fig. 2 EDX should be made clear. Also Fig. S4. 4. Fig. 4g There is no description of the method for evaluating the adsorption energy. 5. Fig. 4 What is ρ? What is n.s.? 6. Fig. S1 There is no description of the evaluation conditions for capacity. Concentration, time, temperature, ***. How was mmol evaluated? 7. Fig. S1c: No explanation for the hatches. 8. Table S3: No units. 9. Fig. S5 S6: No details of DFT calculations. Crystal structure. Basis. Approximation. What about structure optimization?", "reviewer_2": "Wu and colleagues report a novel approach to treat hyperkalemia with counterion-free potassium removal. The current ion-exchange potassium binders indeed exchange potassium for sodium or calcium, which may cause unwanted side-effects, especially in patients with chronic kidney disease (CKD) – the main target patient population for potassium binders. Therefore, this is a clinically relevant topic. The study elegantly applies principles from material science and electrochemistry to in vivo disease models of hyperkalemia. That said, there are several issues that need to be addressed. Major points: 1. This may be a naïve question, but to maintain electroneutrality K+ cannot be removed in isolation (i.e., without an anion) – how does this work in your K+ adsorption approach? 2. Please provide a rationale why SZC is used as comparator in all studies. Why not sodium or calcium polystyrene sulfonate (SPS/CPS) or patiromer? SPS and CPS are also not mentioned in lines 30-31 whereas they are probably still the most commonly used K+ binders worldwide. Characterizing SZC as ‘best-in-class’ also lacks evidence (and this does not match with clinical perspective). Finally, the manuscript now focuses on avoiding Na+ overload, but this is equally true for Ca2+ (which may contribute to vascular calcification, esp. in CKD). 3. Line 106: Fig. 1c does not show superior selectivity to other binders. Only SZC is tested as comparator (so 1 other binder) and the effects on Mg2+ and Ca2+ appear similar (was there statistical analysis?) 4. High vitamin C dosing can have serious side-effects (acute kidney injury, kidney stone formation). How does the vitamin C dose given experimentally translate to humans? The 14 day safety study (Figure 4 f-i) would be strengthened by adding measurements of potential vitamin C toxicity. These considerations should also be added to the Discussion. 5. Along the same lines, blood Fe is not the best read-out to demonstrate a lack of iron toxicity (Figure 4i). Ferritin and transferrin should be measured and reported. 6. Figure 4 j-m is showing apparently normal kidney, stomach, small intestine and colon sections. However, did the investigators systematically investigate the entire GI tract for possible lesions? This is a relevant question as a rare complication of SPS is colonic necrosis, but this may be challenging to detect in animals. 7. Along the same lines, I suggest to add Figure S7 to the main body of the manuscript (i.e., add it to Figure 4), because these are important in vitro studies to complement the safety profile. 8. The major limitation of the study is that the in vivo studies were acute (up to 2 hours), whereas K+ binders are clinically used chronically (days, weeks and sometimes months, years). Therefore, more chronic experiments (e.g. 1-2 weeks) are needed to see the longer-term effects on blood K+ and Na+. I think this is a crucial aspect that is currently lacking. 9. The in vivo selectively for K+ of PB+VC is not demonstrated (only in vitro, Fig. 1c). All other K+ binders can also bind Mg2+ and therefore cause hypomagnesemia. In addition SZC increases plasma bicarbonate. Therefore, Mg2+ and bicarbonate should be added to the measurements of the in vivo studies. Furthermore, the statement “…did not cause hypokalemia or other electrolyte disorders” (line 211) is incorrect as no other electrolytes were tested. 10. The development of hypernatremia with SZC (Fig. 5c – not 6c, line 243) is unusual and has not been reported in clinical trials. This raises the possibility that very (or too) high doses of SZC were used – why is a dose of 4 g/kg in rats equivalent to 10 g in humans? The investigators should also clarify why hypernatremia developed with SZC. Although hypernatremia can be caused by sodium overload (which might occur secondary to the potassium-sodium exchange), it is usually caused by a deficiency of water. Therefore, data on changes in body weight and urinary excretions of electrolytes and osmolality are necessary to clarify the development of hypernatremia. 11. The statement that SZC treats hyperkalemia because Na+ release alleviates amiloride-induced salt wasting (lines 240-241) is not supported by data and also inconsistent with current understanding of the mechanism of action of SZC. Either delete this statement or support with data. 12. Similarly, how does PB+VC increase blood Na+ if there is only a selective effect on K+ (Fig. 5c)? Furthermore, the lower blood Na+ with Am+KCl was not observed in your previous study (ref. #18) - please clarify. 13. Fig. 5f: Why does KCl significantly decrease blood Na+? This seems unexpected and unphysiological. 14. The authors seem to mix up PHA1 (in which K+ binders are used) and Gordon’s syndrome (or PHA2) in which hyperkalemia can be successfully treated with thiazide diuretics (line 250). Minor comments -Figure 1b: The abbreviations SGF, SIF and SCoF are not explained (except in the supplement). -Figure 4a-b: For consistency, please report K+ in urine and feces in mmol/day instead of g/day -Figure 4f: You probably meant “Alanine Aminotransferase” instead of “Alanine Aomptransferase” -Lines 18-19, this should read: “This ion adsorption mechanism enables selective adsorption of K+ without counterion release” -Lines 27-28, references 3-6 appear to be selected rather randomly and are not appropriately supporting the sentence (e.g. 3 and 4 are case reports, 5 is not related, 6 deals with improving hyperkalemia risk with SGLT2i). -Lines 36-38: Please elaborate on your previous study to clarify why NH4+ exchange did not work well. -Line 46: remove “nature”. -Lines 46-48: I suggest removing the words “radioactive” and “contamination” as what you want to treat with PB is the intoxication rather than the contamination. You might also consider writing “Cesium” and “Thallium” for enhanced clarity instead of “Cs” and “Tl” -Lines 46-48: “zero systemic exposure”. I understand that insoluble Prussian blue will not be absorbed intestinally. However, I read reports from patients’ sweat turning blue after prolonged Prussian blue treatment. How can that be explained? -Line 49, references: The references are about sodium-ion batteries and do not tell us how “PB aligns well with therapeutic approaches.” -Line 51: remove “the” -Line 84: You refer to the “excellent safety profile” of PB, but relatively little is known about the safety profile in humans; no controlled trials have been performed and the FDA approval of the drug is primarily based on reviews and case reports. -Line 201: reference 37 does not explain why these 3 cell types should be used. -Line 203: what do you mean by “relatively non-toxic”? Looking at the graphs in S7a-c, there seems to be no difference in cell viability after adding PB+VC. -Lines 209-210: Did you mean to say hepatological parameters? Fig. 5 should read Fig. 4. -Line 235: chronic renal failure is now referred to as chronic kidney disease. -Lines 243 and 248: This should read Fig. 5. -Line 371: renumber the reference.", "reviewer_3": "The manuscript deals with the use of Prussian Blue (PB) for in vivo counterion-free potassium removal in the presence of vitamin C (VC). Inspired by the efficiency of PB and its analogues as battery electrode materials, VC is elegantly used as a reducing agent, to trap the potassium cations in the PB structure for further elimination by the organism. Given my skills, my report will concentrate on the experimental study of the PB sample in my report. In this regard, I would suggest that the authors make the demonstration of the reduction/adsorption process more convincing and, if possible, more quantitative. Some missing data and information should be added to the manuscript and some points should be further discussed or clarified. 1/ Missing information. The PB sample after treatment with VC and potassium cation solution was analyzed by several techniques. The procedure used to recover the sample after treatment should be specified (washing, drying…). This information would remove any doubt concerning the possible presence of surface-adsorbed K+ ions and the water content of the sample. It is not clear how the values in Table S2 were obtained (were all the elements titrated ?, by which method?, were some hypotheses made to propose the chemical formulae?, what about water molecules?…). This point should be clarified. These data, if reliable, could be used i) to show the unchanged chemical composition of the cyanide-bridged Fe network after treatment, and ii) to quantify the potassium content of the sample after treatment. In this case, I would suggest that these data be given in the main text. There is an error in the chemical formula of PB in Table S2. 2/ Clarification of the chemical formula used for PB. The chemical formula used for PB is not entirely clear, probably C18Fe7N18 (or Fe4[Fe(CN)6]3) as stated in the experimental part, but a different chemical formula is used in Table 2 (Fe5C12N12). The chemical formula used for PB should be clarified. 3/ Proof of the stability of the FeIII4[FeII(CN)6]3 cyanide-bridged coordination network. Page 5, line 122. The authors conclude from the XRD data and the presence of potassium in the sample, that the cyanide-bridged Fe coordination network remained stable after treatment with VC and K+ ion. However, a dissolution-recrystallisation phenomenon transforming the initial PB particles (FeIII4[FeII(CN)6]3) into potassium containing PB particles (KxFeIII(4-y)FeIIy[FeII(CN)6]3+z) cannot be excluded from the XRD data and the presence of potassium ions in the sample. The analyses of the chemical composition of the sample before and after treatment (in table S2) should be used to show the unchanged chemical composition of the Fe4[Fe(CN)6]3 coordination network before and after treatment. The comparison of microscopy images of the particles before and after treatment would further confirm that the morphology and size of the particles has remained unchanged after treatment. Without a change in the chemical composition of the cyanide-bridged Fe coordination network and the morphology of the particles a dissolution-recrystallisation process can be clearly ruled out. 4/ Quantification of the adsorption/reduction phenomena. Page 5 line 131. There is no experimental evidence that most of the Fe³⁺ coordinated to nitrogen in PB has been reduced to Fe²⁺, and such experimental evidence is difficult to provide here because the two transition metal sites are occupied by the same chemical element. Mössbauer spectroscopy shows the presence of both Fe3+ and Fe2+ HS ions, but this spectroscopy is not quantitative. The authors could again use the chemical formula of the PB sample after treatment, to support their statement. From the data given in Table 2, a chemical formula close to K4Fe4[Fe(CN)6]3 can be deduced for the sample after treatment, which indeed corresponds to a reduction of most of the Fe3+ ions and the adsorption of the maximum number of K+ ions. However, the VC/PB ratio used for the treatment raises questions, as the complete oxidation of VC provides two electrons, whereas the complete reduction of PB would require 4 electrons (using a chemical formula of Fe4[Fe(CN)6]3 for PB). A VC/PB molar ratio of 2 would be required to completely reduce PB. This point should be clarified. The conditions and apparatus used for XPS analysis should be described in the experimental part.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Respond to Reviewers\nTo better demonstrate the responds, point-by-point, we copy the origin comments from\nthe editor and reviewers in this respond letter. Our responses are written in blue next\nto the related comments. The parts of the manuscript that have been revised and\nsupplemented are marked in red. We really appreciate this opportunity and your efforts.\nAll your efforts make this manuscript better.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis paper demonstrated the removal of K+ from blood using the reduction of PB. The\ncombination of PB and VC can remove K+ to the same extent as SCZ. However, there\nare various problems with this manuscript, as listed below. Regarding the DFT\ncalculations, there is a lack of information, so it is impossible to judge whether it is\ngood or bad.\n1. There are no details of the DFT calculations, so it is unclear what structure was\nassumed for the calculations, or the conditions of the calculations. Since the details\nare unknown, it is unclear whether the description is correct or not.\nWe sincerely appreciate the reviewer’s attention to the DFT methodology details. We\nwould like to clarify that the quantum chemical calculation procedures were\ncomprehensively described in the original manuscript (line 386-395). However, we\napologize for the oversight in not providing the computational structural files and have\nnow uploaded these structure files during the calculation process to facilitate the\nconfirmation of the calculation details and determine whether our description is correct.\nMethod section, Line 386-395\n“All the calculations are performed in the framework of the density functional theory with\nthe projector augmented plane-wave method, as implemented in the Vienna ab initio\nsimulation package (VASP). The crystal structures input (CIF files) were uploaded as\nattachments. The exchange-correlation potential was chosen as the generalized gradient\napproximation (GGA) proposed by Perdew, Burke, and Ernzerhof (PBE). The long range\nvan der Waals interaction is described by the DFT-D3 approach. The cut-off energy for\nplane wave is set to 500 eV. Furthermore, the Brillouin zone was sampled using a\nMonkhorst–Pack k-point mesh (56) with 5 × 5 × 5 k-points for lattice optimizations and 9 ×\n9 × 9 k-points for static density of states (DOS) calculations, respectively. The energy\ncriterion is set to 10−6 eV in iterative solution of the Kohn-Sham equatio. All the structures\nare relaxed until the residual forces on the atoms have declined to less than 0.03 eV/Å.”\n2. It is unclear how to prepare PB+CV sample in Fig. 2 is. This should be stated in the\ncaption.\nWe thank the reviewer’s insightful comment regarding the PB+VC sample preparation\ndetails. We apologize for this oversight and have now included a concise description\nof the PB+VC preparation method in the Figure 2 caption (revised version line137-141).\nThe full experimental procedure has been added to the Methods section (line 346-354)\nfor completeness.\nFigure 2 caption, Line 137-141:\n“The PB+VC sample was prepared by mixing PB and VC (1:1 mass ratio) in MEI at 37°C\nwith shaking (180 rpm, 2 h), followed by centrifugation, water washing, lyophilization (48\nh), and finally structural characterization. In the processed PB+VC, the VC component and\nits oxidation products dissolve in aqueous solution, yielding a final product consisting\nprimarily of K+-adsorbed PB.”\nMethod section, Line 346-354:\n“Briefly, PB (200 mg) and VC (200 mg) were mixed with MEI (40 mL), followed by\nincubation at 37°C with orbital shaking (180 rpm) for 2 hours. Post-incubation, the\nmixture was centrifuged at 3500 rpm for 5 minutes to separate the supernatant. The\nresulting pellet underwent three washing cycles with deionized water (30 mL per cycle,\nvortex mixing for 30 seconds), each followed by identical centrifugation parameters. The\npurified precipitate was flash-frozen in liquid nitrogen and subsequently lyophilized for 48\nhours to obtain the final product for Structural and morphological characterization. In the\nprocessed PB+VC, the VC component and its oxidation products dissolve in aqueous\nsolution, yielding a final product consisting primarily of K+-adsorbed PB.”\n3. Fig. 2 EDX should be made clear. Also Fig. S4.\nWe appreciate the reviewer's suggestion regarding the elemental analysis. In our\nmanuscript, we used \"EDS\" (Energy Dispersive Spectrometer) rather than \"EDX\"\n(Energy Dispersive X-ray Spectroscopy) to align with the instrumentation\nnomenclature of our scanning/transmission electron microscopy system where the\nspectrometer is integrated as a detector module. To avoid potential confusion, we have\nnow explicitly defined the abbreviation in the revised caption of Figure 2(lines 136-137)\nand enhanced the resolution of all EDS elemental maps (Figure 2l and Figure S3),\nensuring clear visualization of elemental distributions.\nFigure 2 caption, Line 136-137:\n“k, l) HAADF-STEM image and corresponding energy dispersive spectrometer (EDS)\nelement.”\n4. Fig. 4g There is no description of the method for evaluating the adsorption energy.\nWe thank the reviewer for their attention to our adsorption energy evaluation method.\nWe would like to clarify that the calculation formula for adsorption energy was provided\nin the Methods section (line 396-400): E\nad\n= (E\nhost/adsorbate\n− E\nhost\n− n E\nadsorbate\n)/n, and\nE , E and E are the DFT total energies of the adsorbate on the host\nhost/adsorbate host adsorbate\ncrystal, the host crystal and the adsorbate itself, respectively. The absorption energy\nwas calculated on the basis of the PB crystal inserting different atoms as absorbate\nand the calculations were performed using density functional theory (DFT) by\noptimizing the adsorption configurations and computing the energy differences.\nInterpretation criteria:\nE < 0: Indicates energy release during adsorption (more negative values correspond\nad\nto stronger adsorption and higher system stability)\nE > 0: Indicates energy absorption during adsorption (higher energy state than\nad\nseparated components, suggesting unfavorable adsorption)\nMethod section, Line 396-400:\n“In these calculations, model anhydrous structures with a face-centered cubic F4̅3m unit\ncell were assumed. Four compensating charges are mimicked in the reduced state. The\nabsorption energy was calculated on the basis of the PB crystal inserting different atoms\nas absorbate, given as E = (E − E − n E )/n, and E , E\nad host/adsorbate host adsorbate host/adsorbate host\nand E are the DFT total energies of the adsorbate on the host crystal, the host\nadsorbate\ncrystal and the adsorbate itself, respectively.”\n5. Fig. 4 What is ρ? What is n.s.?\nWe sincerely apologize for not explaining the statistical symbols ρ and n.s. in the\nmanuscript and appreciate the reviewer's meticulous attention to methodological clarity.\nWe would like to clarify that the statistical symbols in the figures use p values rather\nthan ρ symbols. In the revised manuscript, we have added explanations for both p and\nn.s. in the Methods sections (line 490-497).\nStatistical Analysis, Line 490-497\n“All experiments were performed at least triplicated with repeated measures. The\nresults are expressed either as means ± standard error of the mean (S.E.M). One-way\nANOVA with Tukey's multiple comparison test were used for testing differences among\ngroups. For each statistical analysis, the sample size was greater or equal to three (n\n≥ 3). Detailed statistics can also be found in figure legends. Data were approximately\nnormally distributed, and the variance was similar among all the groups. No samples\nwere excluded from the analysis, and no preprocessing of data was performed before\nstatistical analysis. Statistical significance is indicated as n.s. p > = 0.5 and precise p\nvalues were calculated and showed in figures.”\n6. Fig. S1 There is no description of the evaluation conditions for capacity.\nConcentration, time, temperature, ***. How was mmol evaluated?\nWe sincerely apologize for the insufficient description of the Figure S1 and have\naddressed this in the revised manuscript. Detailed information on data acquisition\nmethods—Prussian blue at a concentration of 5 mg/mL and adding it to a centrifuge\ntube containing the configured liquid, with 5 mg/mL different reducing agents added.\nThe treated tubes were subjected to shaking at 37°C for a period of two hours at 180\nrpm—has been comprehensively added in the Methods section (line 403-416) and\nSupplementary Information. Additionally, we have included the adsorption capacity\ncalculation formula.\nMethod section, Line 403-416\n“The adsorption capacity of PB+VC was evaluated by weighing Prussian blue at a\nconcentration of 5 mg/mL and adding it to a centrifuge tube containing the configured\nliquid, with different mass ratios ascorbic acid added as a reducing agent. Furthermore,\nalternative adsorbents (PB, SZC, SPS, and CPS) were employed at a concentration\nof 5 mg/mL for comparative purposes. The treated tubes were subjected to shaking at\n37°C for a period of two hours at 180 rpm. Following this, the liquid phase underwent\nfiltration and dilution before being characterized by inductively coupled plasma\nemission spectroscopy (ICP-OES). For this study, a Thermo iCAP7600 inductively\ncoupled plasma emission spectrometer was utilized.\nAdsorption capacity calculation formula as specified below:\nAdsorption capacity = (C - C ) V / m (mmol/g)\n0 e\nC = Initial concentration before adsorption (mmol/L)\ne\nV = Solution volume (L)\nm = Mass of adsorbent (PB+VC, PB, SZC, SPS, and CPS) (g)”\n7. Fig. S1c: No explanation for the hatches.\nWe sincerely apologize for omitting the description of hatches in Figure S1c and thank\nthe reviewer for the careful scrutiny. In the revised manuscript, we have added the\nfollowing clarification to the Figure S1c caption: Curve fitting was performed via simple\nlinear regression using least-squares minimization, with the hatched areas\nrepresenting the 95% confidence interval of the fit.\n8. Table S3: No units.\nWe apologize for the clerical error in our previous manuscript, and we express our\nsincere gratitude to the reviewer for the meticulous attention. The missing units in our\ndata pertain to the bond lengths of PB and PB+VC. All calculated bond lengths are\nreported in angstroms (Å). In our revised version, we have corrected the error (Table\nS3).\n9. Fig. S5 S6: No details of DFT calculations. Crystal structure. Basis. Approximation.\nWhat about structure optimization?\nWe sincerely appreciate the reviewer's attention to the DFT methodology and structural\ndetails in Figures S4 and S5 (Updated figures). We would like to clarify that the\ncalculation process for the projected density of states (PDOS) spectra in Figure S4\nwas fully described in the original manuscript (line 386-395). However, we\nacknowledge and apologize for the oversight in not initially providing the computational\nstructural files for Figure S5. In the revised manuscript, we have now uploaded these\ncritical structural files as supplementary files, enabling verification of the DFT\ncalculations in Figure S5.\nMethod section, Line 386-395\n“All the calculations are performed in the framework of the density functional theory with\nthe projector augmented plane-wave method, as implemented in the Vienna ab initio\nsimulation package (VASP). The crystal structures input (CIF files) were uploaded as\nattachments. The exchange-correlation potential was chosen as the generalized gradient\napproximation (GGA) proposed by Perdew, Burke, and Ernzerhof (PBE). The long range\nvan der Waals interaction is described by the DFT-D3 approach. The cut-off energy for\nplane wave is set to 500 eV. Furthermore, the Brillouin zone was sampled using a\nMonkhorst–Pack k-point mesh (56) with 5 × 5 × 5 k-points for lattice optimizations and 9 ×\n9 × 9 k-points for static density of states (DOS) calculations, respectively. The energy\ncriterion is set to 10−6 eV in iterative solution of the Kohn-Sham equatio. All the structures\nare relaxed until the residual forces on the atoms have declined to less than 0.03 eV/Å.”\nReviewer #2 (Remarks to the Author):\nWu and colleagues report a novel approach to treat hyperkalemia with counterion-free\npotassium removal. The current ion-exchange potassium binders indeed exchange\npotassium for sodium or calcium, which may cause unwanted side-effects, especially\nin patients with chronic kidney disease (CKD) – the main target patient population for\npotassium binders. Therefore, this is a clinically relevant topic. The study elegantly\napplies principles from material science and electrochemistry to in vivo disease models\nof hyperkalemia.\nThat said, there are several issues that need to be addressed.\nMajor points:\n1. This may be a naïve question, but to maintain electroneutrality K+ cannot be\nremoved in isolation (i.e., without an anion) – how does this work in your K+ adsorption\napproach?\nWe sincerely appreciate the reviewer’s valuable feedback regarding the raised\nconcerns. In our electrochemical mechanism, vitamin C acts as a reducing agent that\ninitiates redox reactions with Prussian blue. During the reduction process of Prussian\nblue, potassium ions (K+) are adsorbed onto the coordinatively unsaturated iron sites\nthrough electron density redistribution. It should be clarified that this redox process is\nnot unidirectional—while vitamin C exerts its reducing effect, it is simultaneously\noxidized, releasing hydrogen ions (H+) through its inherent metabolic pathway. In the\nrevised manuscript, we have further elucidated the mechanism of PB+VC in K+\nadsorption, clearly demonstrating that while PB binds K+, VC releases H+ upon\noxidation during the process (Figure 2g).\nFigure 2g. Schematic diagram of the reaction mechanism of adsorption/reduction mechanism.\nAt safe dosage levels (as validated by our animal experiments and supplementary\ndata), the generated H⁺ is effectively neutralized by the bicarbonate buffer system\nthrough the physiological reaction: H+ + HCO - CO + H O. This biochemical\n3 2 2\nconversion transforms the electrically neutral substrate (vitamin C) into nonionic\nproducts (CO ) which are eliminated via respiration without disrupting normal\n2\nmetabolic processes (Kidney Int 1983, 24, 709-713.)\nTo clarify, our original use of \"counterion-free\" in the manuscript was specifically\nintended to emphasize the absence of metal ions, given that most metal ions are\nrenally metabolized. Since hyperkalemia frequently occurs in patients with renal\ndysfunction, the release of metal ions would not alleviate renal burden. In contrast, H+\nparticipates in various biochemical pathways with multiple metabolic routes, making it\nrelatively safer than metal ions. For greater precision, we have now changed the term\nas \"metal counterion-free\" in the revised manuscript.\nIn our cell experiments, we observed that when PB+VC was administered in vitro, the\noxidized VC was safely released without causing toxicity to GI cells (Figure 4a and S6).\nTo further validate whether the H⁺ released during the redox process of our\nadministered VC dose could be effectively neutralized by the bicarbonate buffer\nsystem, we have supplemented safety experiments in the revised manuscript. These\ninclude monitoring blood pH and HCO - levels in rats following long-term administration\n3\nof both therapeutic and high doses (3 times therapeutic dose) of PB+VC (Figure S8).\nOur results demonstrate that even at triple the therapeutic dose, the acid-base balance\nin rats remained within normal physiological ranges. This confirms that the H⁺ released\nfrom VC at these dosage levels can be effectively buffered by the endogenous\nbuffering system.\nFigure S8, Line 248-249\n“Blood gas parameters (pH, HCO -, CO ) and hematological parameters exhibited no\n3 2\nclinically significant deviations (Figure S8).”\n2. Please provide a rationale why SZC is used as comparator in all studies. Why not\nsodium or calcium polystyrene sulfonate (SPS/CPS) or patiromer? SPS and CPS are\nalso not mentioned in lines 30-31 whereas they are probably still the most commonly\nused K+ binders worldwide. Characterizing SZC as ‘best-in-class’ also lacks evidence\n(and this does not match with clinical perspective). Finally, the manuscript now focuses\non avoiding Na+ overload, but this is equally true for Ca2+ (which may contribute to\nvascular calcification, esp. in CKD).\nWe appreciate the reviewer’s constructive feedback regarding the experimental design.\nAs correctly noted, sodium or calcium polystyrene sulfonate (SPS/CPS) remain the\nmost commonly used clinical treatments for hyperkalemia. However, accumulating\nclinical evidence has revealed significant limitations of SPS/CPS: SPS induces sodium\nretention while CPS exhibits limited potassium-binding capacity; high-dose\nadministration correlates with constipation, colonic necrosis, and intestinal perforation;\nlower doses demonstrate reduced adverse effects but only manage mild hyperkalemia\n(Drugs R D 2018, 18, 231-235., J Korean Med Sci 2009, 24, 1207-1211. J Am Soc\nNephrol 2010, 21, 733-735., PLoS One 2017, 12, e0173542.). Crucially, clinical trials\nhave established that SZC outperforms CPS/SPS in K+ adsorption capacity, composite\nreduction of mortality and hyperkalemia-related hospitalization rates, and potential\nimprovement of hyperkalemia in CKD patients (Front Med (Lausanne) 2023, 10,\n1137981., Clin Kidney J 2024, 17, sfae021.).\nPatiromer exhibits a delayed onset of action (7-48 hours) and carries drug-drug\ninteraction risks, precluding its use in emergent management of severe hyperkalemia,\nwhile demonstrating higher gastrointestinal adverse event rates compared to the\ninorganic lattice-structured SZC (Pharmacotherapy 2017, 37, 401-411.). Importantly,\nSZC shows superior potassium ion specificity—patiromer's nonspecific binding affinity\nextends to Mg2+, potentially inducing hypomagnesemia and altering calcium\nhomeostasis (Pharmacotherapy 2017, 37, 401-411., Eur Heart J Suppl 2019, 21, A28-\nA33.).\nThe well-documented suboptimal efficacy and frequent adverse events of CPS/SPS\nas well as the delayed therapeutic onset and non-selective cation adsorption of\npatiromer, contrasted with the superior clinical performance of SZC This underlies our\nrationale for not selecting SPS/CPS and patiromer as comparators. Nevertheless, the\nreviewer’s comments have highlighted the necessity for enhanced contextual\nclarification. Accordingly, we have incorporated an expanded discussion of SPS/CPS\nand patiromer in the revised manuscript to better orient readers (line 33-38).\nTo better compare the efficacy of these agents, we also conducted additional in vitro\nexperiments with potassium binders (Figure 1b, c and line 111-113, 117-120). The in\nvitro results clearly demonstrate that SZC shows superior performance compared to\nboth SPS and CPS.\nTo comprehensively address potential concerns regarding Ca2+ metabolism\ndisturbances, we have supplemented detailed analyses of Ca2+ homeostasis in both\nsafety assessments and chronic hyperkalemia models, including measurements of\nblood Ca2+ levels as well as fecal and urinary calcium excretion (line 246-248, line 307-\n311 and Figure S7, S17, S18). Our results demonstrate that PB+VC administration at\nboth therapeutic doses and high doses (3 times therapeutic dose) did not significantly\nalter systemic Ca2+ balance or its physiological distribution patterns. These findings\nprovide robust evidence that our electrolyte-sparing K+ adsorption strategy effectively\nmaintains normal Ca2+ homeostasis without inducing calcium-related complications,\nthereby fundamentally addressing any potential concerns about Ca2+ metabolism\ninterference.\nIntroduction, Line 33-38\n“However, SPS and CPS carry well-documented risks of severe GI complications,\nparticularly colonic necrosis and intestinal perforation. While patiromer is an alternative,\nit exhibits critical limitations relative to the inorganic crystalline matrix of SZC, including\ndelayed therapeutic onset (7–48 hours), significant drug interaction potential,\nnonspecific binding (e.g., Mg2+), and higher GI adverse event rates, making it\nunsuitable for emergent management.”\nFigure1b, Line 111-113:\n“PB+VC demonstrated superior K⁺ adsorption capacity compared to PB alone, SPS,\nCPS and comparable capacity with SZC, with no significant difference between PB:VC\nmass ratios of 1:1 and 1:4.”\nFigure1c, Line 117-120:\n“Given the competitive cations present in the human GI tract, we assessed the selectivity\nof PB+VC in the presence of interfering ions, such as Na+, Ca2+, and Mg2+. exhibited\nminimal adsorption of ions other than K+, demonstrating superior selectivity compared to\nother binder, while additionally avoiding the release of metal counterions (Figure 1c).”\nFigure 4d, Figure S7, Line 246-248:\n“Blood K+, Na+, Ca2+, and Mg2+ levels, alongside urinary Na+, Ca2+, and Mg2+ excretion\nand fecal Na+, Ca2+, and Mg2+ content, remained within physiologically normal ranges\nacross all experimental groups (Figure 4d, and Figure S7).”\nFigure S17a, b, d, f, Figure S18, Line 307-311:\n“Unlike SZC, which exacerbates renal metabolic stress through excessive Na+ release,\nparticularly detrimental given pre-existing nephron impairment where Na+ metabolism\ncompetitively disrupts other electrolytes (e.g., Mg2+), PB+VC not only corrects\nhyperkalemia-induced sodium imbalance but also prevents secondary electrolyte\ndisturbances and metal counterion release, thereby avoiding additional renal metabolic\nburden (Figure S17a, b, d, f and Figure S18).”\n3. Line 106: Fig. 1c does not show superior selectivity to other binders. Only SZC is\ntested as comparator (so 1 other binder) and the effects on Mg2+ and Ca2+ appear\nsimilar (was there statistical analysis?)\nWe thank the reviewer for the valuable recommendation to conduct comparative in\nvitro studies with other potassium binders to further validate our strategy's superiority.\nIn response, we have supplemented CPS and SPS evaluations using ion adsorption\nprotocols in SGF, SIF, SCoF, and MEI, consistent with our previous methodology\n(Figures 1b, c and line 111-113, 117-120). To quantitatively assess cation selectivity,\nwe performed one-way ANOVA with Tukey's multiple comparison test on Figure 1c\ndata, with statistical significance explicitly indicated. The updated in vitro results\ndemonstrate that our strategy outperforms conventional binders in K+ adsorption\ncapacity, particularly showing superior K⁺ selectivity compared to CPS/SPS. While our\nselectivity profile shows no significant difference from SZC (p = 0.6402 for Mg2+, p =\n0.8456 for Ca2+), it markedly differs from the CPS/SPS for Ca2+ and Na+ (line 120-123).\nThese in vitro findings align with clinical evidence establishing the therapeutic\nsuperiority of SZC over CPS/SPS, justifying SZC is used as the comparator in\nsubsequent animal studies.\nThe assessment effectively complements the text and contributes to a comprehensive\npresentation of our findings. Again, we greatly appreciate the reviewer for the useful\nsuggestions and comments.\nFigure1b, Line 111-113:\n“PB+VC demonstrated superior K⁺ adsorption capacity compared to PB alone, SPS,\nCPS and comparable capacity with SZC, with no significant difference between PB:VC\nmass ratios of 1:1 and 1:4.”\nFigure1c, Line 117-120:\n“Given the competitive cations present in the human GI tract, we assessed the selectivity\nof PB+VC in the presence of interfering ions, such as Na+, Ca2+, and Mg2+. exhibited\nminimal adsorption of ions other than K+, demonstrating superior selectivity compared to\nother binder, while additionally avoiding the release of metal counterions (Figure 1c).”\nFigure1c, Line120-123:\n“PB+VC demonstrates not only excellent potassium adsorption capacity but also\nexhibits remarkable potassium selectivity in vitro, showing negligible adsorption of Ca2+\nand Mg2+ (with no significant difference compared to SZC). Importantly, unlike SZC,\nSPS (which releases Na+) or CPS (which releases Ca2+), PB+VC does not liberate any\nmetal ions.”\n4. High vitamin C dosing can have serious side-effects (acute kidney injury, kidney\nstone formation). How does the vitamin C dose given experimentally translate to\nhumans? The 14-day safety study (Figure 4 f-i) would be strengthened by adding\nmeasurements of potential vitamin C toxicity. These considerations should also be\nadded to the Discussion.\nWe appreciate the reviewer’s comment on the dosing. In the updated manuscript, we\nconducted a comprehensive 14-day safety evaluation using two dosage regimens:\nthe therapeutic dose (2 g kg⁻¹ PB + 2 g kg⁻¹ VC) and a high dose (3 times therapeutic\ndose: 6 g kg⁻¹ PB + 6 g kg⁻¹ VC). Renal safety was systematically assessed through\nblood biochemistry (liver and kidney function indicators), urinary biochemical analysis,\nand renal histopathology (H&E staining) (Figure 4j-q and Figure S12-14, line 259-264).\nOur results demonstrate that even at 3 times therapeutic VC dose, no renal impairment\nwas observed, with all parameters remaining within normal physiological ranges.\nFurthermore, evaluation of blood pH and HCO - levels revealed no significant\n3\ndifferences compared to controls (Figure S8 and line 245-246), confirming\nmaintenance of normal acid-base balance throughout treatment.\nTo better understand the clinical relevance, we have converted the experimental VC\ndosage to human equivalent dose. It is crucial to emphasize that when making dose\nadjustments between different species, a conversion relationship that takes into\naccount the species' surface area should be used.\nFigure B. Human equivalent dose calculation based on body surface area (J Basic Clin Pharm 2016, 7,\n27-31.). Data was obtained from FDA draft guidelines.\nAccording to FDA draft guidelines, the rat dose range of 2000 mpk is considered\nequivalent to 324 mpk in humans. Translating this to a 60 kg human, it would\ncorrespond to a daily medication dosage of 1.9 g. Current clinical practice defines the\ntolerable upper intake level of vitamin C as 2 grams, with excess doses primarily\ncausing diarrhea as the main adverse effect (J Oncol Sci 2016, 1, 10-12.).Oral vitamin\nC administration generally poses minimal health risks, as evidenced by studies\nshowing no significant differences in survival rates or quality of life between groups\nreceiving high-dose supplementation (10 g daily) and controls (N Engl J Med 1979,\n301, 687-690.). Furthermore, epidemiological data suggest only weak associations\nbetween increased vitamin C intake and nephrolithiasis risk (J Am Soc Nephrol 2004,\n15, 3225-3232.). Notably, serious renal complications such as acute kidney injury and\ncalculi formation are predominantly associated with high-dose intravenous vitamin C\nadministration (>50 g) rather than oral supplementation (Kidney Int Rep 2020, 5, 1815-\n1822.).\nOur 14-day safety study demonstrated that even at a high PB+VC dose, renal function\nremained within normal physiological ranges confirming that there should be no renal\ntoxicity. We need to explain the vitamin C component solely functions as a reductant\nin the therapeutic strategy, as evidenced by our in vitro data showing maximal\npotassium-binding efficiency at PB:VC mass ratio = 1:1 (Figure S1c). Increasing VC\ndosage beyond this stoichiometric equivalence yielded no significant enhancement in\ntherapeutic efficacy. This mechanistic saturation phenomenon, coupled with the\nestablished clinical safety profile of oral VC (2 g/day UL), scientifically justifies our\nexperimental design. Moreover, all administered doses in our therapeutic regimen\nwere strictly maintained within the documented safety ranges established in the\nliterature.\nFigure S8, Line 248-249\n“Blood gas parameters (pH, HCO -, CO ) and hematological parameters exhibited no\n3 2\nclinically significant deviations (Figure S8-10).”\nFigure 4j-m and Figure S12-14, Line 259-261\n“Urinalysis and blood biochemical parameters showed no significant differences\ncompared to the control group, demonstrating that even at three times the therapeutic\ndose, VC did not cause additional renal burden or toxicity (Figure 4j-m and Figure S12-\n14).”\n5. Along the same lines, blood Fe is not the best read-out to demonstrate a lack of iron\ntoxicity (Figure 4i). Ferritin and transferrin should be measured and reported.\nWe appreciate the reviewer suggested us to add more information on the\nmeasurement of ferritin and transferrin to evaluate the toxicity caused by possible Fe\nrelease. We concurrently evaluated blood Fe2+, along with transferrin and ferritin\nconcentrations. Compared to the control group, no significant differences were\nobserved in these three parameters, indicating that PB+VC is unlikely to release Fe\ninto the circulatory system or disrupt systemic iron homeostasis under physiological\nconditions. The suggestions were already accepted and changed in the revised version.\n(line 249-250 and Figure 4f-h)\nFigure 4f-h, Line 249-250:\n“Notably, Blood Fe2+, transferrin, and ferritin levels also fell within normal physiological\nranges (Figure 4f-h).”\n6. Figure 4 j-m is showing apparently normal kidney, stomach, small intestine and colon\nsections. However, did the investigators systematically investigate the entire GI tract\nfor possible lesions? This is a relevant question as a rare complication of SPS is colonic\nnecrosis, but this may be challenging to detect in animals.\nWe sincerely appreciate the reviewer's concern regarding the GI safety profile of our\ntherapeutic agent. To comprehensively evaluate potential GI pathology, we conducted\nmulti-faceted safety assessments:\n1. Intestinal Barrier Function Biomarkers:\nLipopolysaccharide (LPS): As a primary component of Gram-negative bacterial outer\nmembranes derived from gut microbiota, systemic LPS levels reflect intestinal barrier\nintegrity. Healthy barriers confine LPS to the intestinal lumen, preventing translocation\ninto circulation (Am J Physiol Gastrointest Liver Physiol 2016, 311, G1-G15.).\nDiamine Oxidase (DAO): This intracellular enzyme in small intestinal mucosa protects\nenterocytes. Elevated serum DAO indicates mucosal barrier failure or epithelial\nnecrosis (Dig Dis Sci 1984, 29, 872-874.).\nOur data demonstrate no significant differences in serum LPS or DAO concentrations\nbetween treated and healthy control groups (FigureS9a, b), indicating preserved\nbarrier function.\n2. Quantitative GI Permeability Assessment:\nUsing FITC-dextran assays, we quantified paracellular transport. Lower serum\nfluorescence intensity correlates with reduced GI permeability. Results confirm no\ntreatment-induced increase in permeability or leakage (Figure 4i).\n3. Macroscopic GI Integrity Evaluation:\nSince GI tract shortening is a hallmark of necrotizing colitis (Inflamm Bowel Dis 2014,\n20, 2198-2206.), we measured intact GI lengths. Treated animals showed no\nsignificant reduction compared to healthy controls (Figure S9c, d).\nThese complementary findings—consistent with the absence of inflammation, necrosis,\nor mucosal erosion in histopathological analyses—collectively demonstrate the\nfavorable safety profile of our approach at both therapeutic and high-dose (Figure 4n,\no, p, q and Figure S15).\nIn the revised manuscript, we have supplemented both the aforementioned\ngastrointestinal safety assessments and the discussion regarding complete\ngastrointestinal safety to better highlight the safety profile of our strategy (line 254-256).\nFigure 4i and Figure S11, Line 254-256:\n“Since our strategy achieves K+ adsorption in the GI, we comprehensively assessed\nGI safety. The results indicated that both GI permeability and intestinal length remained\nwithin normal range (Figure 4i and Figure S11).”\n7. Along the same lines, I suggest to add Figure S7 to the main body of the manuscript\n(i.e., add it to Figure 4), because these are important in vitro studies to complement\nthe safety profile.\nWe sincerely appreciate the reviewers' constructive suggestion. In response, we have\nintegrated a part of Figure S7 from the original manuscript into Figure 4, which\nstrengthens the safety profile validation through in vitro studies (Figure 4a and Figure\nS6).\n8. The major limitation of the study is that the in vivo studies were acute (up to 2 hours),\nwhereas K+ binders are clinically used chronically (days, weeks and sometimes\nmonths, years). Therefore, more chronic experiments (e.g. 1-2 weeks) are needed to\nsee the longer-term effects on blood K+ and Na+. I think this is a crucial aspect that is\ncurrently lacking.\nWe sincerely thank the reviewer for their interest in the chronic hyperkalemia treatment\napplication of our material. We fully acknowledge the need for extended-duration\nstudies, given that potassium binders are often used long-term clinically. In the revised\nmanuscript, we have supplemented the data by conducting an additional experiment,\na 14-day chronic hyperkalemia study featuring an 8-day treatment period. Throughout\nthis experiment, we collected and analyzed blood from the animals. Detailed results\nare presented (Figure 5 g-i) and the main text (lines 300-306). The results demonstrate\nthat PB+VC effectively restored serum potassium to normal levels throughout the\nchronic phase, exhibiting efficacy comparable to SZC; while SZC treatment induced a\nmoderate elevation in serum sodium, no statistically significant differences were\nobserved among the Control, PB+VC, and SZC groups. These collective findings\nconfirm the sustained therapeutic efficacy of our PB+VC strategy for long-term\nmanagement of chronic hyperkalemia.\nFigure 5 g-h, Line 300-306\n“As anticipated, the PB+VC treatment, as a gut-restricted strategy based on metal\ncounterion-free release, effectively corrected abnormal blood K+ by promoting\nsubstantial fecal K+ excretion. KCl supplementation moderately reduced blood Na+ in\nrats, likely through tonic inactivation of NaCl cotransporter (a known antihypertensive\nmechanism) in the renal distal convoluted tubule61. This effect was exacerbated in the\n5/6 N model due to impaired tubular Na+ reabsorption from nephron loss. The K+\nx\nexcretion mechanism of PB+VC concurrently restored normal blood Na+ (Figure 5f, I).”\n9. The in vivo selectively for K+ of PB+VC is not demonstrated (only in vitro, Fig. 1c).\nAll other K+ binders can also bind Mg2+ and therefore cause hypomagnesemia. In\naddition SZC increases plasma bicarbonate. Therefore, Mg2+ and bicarbonate should\nbe added to the measurements of the in vivo studies. Furthermore, the statement\n“…did not cause hypokalemia or other electrolyte disorders” (line 211) is incorrect as\nno other electrolytes were tested.\nWe appreciate the reviewer’s concern regarding the K+ selectivity of our strategy. We\nacknowledge that in the original manuscript, we only provided in vitro experimental\nvalidation, which could not fully demonstrate its in vivo efficacy. Therefore, in the\nrevised manuscript, we have supplemented the study with:\nMeasurements of Blood K+, Na+, Ca2+ and Mg2+, and bicarbonate levels and 24-hour\nurinary and fecal excretion of K+, Na+, Ca2+ and Mg2+ of safety assessments and chronic\nhyperkalemia animal experiments.\nThe experimental results clearly demonstrate that PB+VC restores Blood K+ to normal\nlevels primarily by enhancing fecal K+ excretion, while not affecting Blood Ca2+ and Mg2+\nconcentrations or their baseline metabolic pathways (Figure 4d, Figure S7, Figure\nS17a, b, d, f, Figure S18, and line 246-248, line 307-311). This finding further supports\nour original statement that the strategy “did not cause hypokalemia or other electrolyte\ndisorders” (Line 251-252).\nRegarding SZC, its binding efficiency for Mg2+ is notably low (reference), and it does\nnot induce hypomagnesemia. Interestingly, our data revealed that SZC slightly\nelevated Blood Mg2+ levels in chronic kidney disease (CKD) rats (Figure S17). This\nphenomenon may be attributed to Na+-Mg2+ excretion competition: Both Na+ and Mg2+\nare excreted via active or passive transport in the renal proximal tubule and thick\nascending limb of the loop of Henle. Elevated Blood Na+ (as evidenced by SZC-\ninduced significant urinary Na+ excretion, Figure S18, ) likely triggers preferential Na+\nexcretion to maintain electrolyte balance, thereby inhibiting Na+-Mg2+ exchange and\npromoting tubular magnesium reabsorption (J Clin Invest 1959, 38, 1733-1745.). Given\nthe limited metabolic capacity of impaired nephrons, this mechanism could lead to\ntransient hypermagnesemia (J Lab Clin Med 1969, 73, 763-771., Pol Med Sci Hist Bull\n1976, 15-16, 359-362).\nAdditionally, while SZC moderately restored plasma bicarbonate levels, our PB+VC\nstrategy showed no statistically significant difference in bicarbonate concentration\ncompared to SZC. Moreover, prolonged PB+VC administration even exhibited an\nupward trend in bicarbonate levels, indicating that it does not exacerbate acidosis (i.e.,\nplasma bicarbonate decline) in CKD rats and may even exert a mild corrective effect\n(line 311-313).\nFigure 4d, Figure S7, Line 246-248:\n“Blood K+, Na+, Ca2+, and Mg2+ levels, alongside urinary Na+, Ca2+, and Mg2+ excretion\nand fecal Na+, Ca2+, and Mg2+ content, remained within physiologically normal ranges\nacross all experimental groups (Figure 4d, and Figure S7).”\nLine 251-253:\n“These findings collectively demonstrate that our therapeutic strategy, even at higher\nPB+VC doses, induces neither hypokalemia nor other electrolyte disturbances, acid-base\nimbalances, or hematological abnormalities, thereby confirming its safety profile.”\nFigure S17a, b, d, f, Figure S18, Line 307-311:\n“Unlike SZC, which exacerbates renal metabolic stress through excessive Na+ release,\nparticularly detrimental given pre-existing nephron impairment where Na+ metabolism\ncompetitively disrupts other electrolytes (e.g., Mg2+), PB+VC not only corrects\nhyperkalemia-induced sodium imbalance but also prevents secondary electrolyte\ndisturbances and metal counterion release, thereby avoiding additional renal metabolic\nburden (Figure S17a, b, d, f and Figure S18).”\nFigure S17c, Line 311-313:\n“Notably, PB+VC did not exacerbate acidosis, a frequent complication of hyperkalemia,\nand even demonstrated mild ameliorative effects during prolonged administration\n(Figure S17c, f).”\n.\n10. The development of hypernatremia with SZC (Fig. 5c – not 6c, line 243) is unusual\nand has not been reported in clinical trials. This raises the possibility that very (or too)\nhigh doses of SZC were used – why is a dose of 4 g/kg in rats equivalent to 10 g in\nhumans? The investigators should also clarify why hypernatremia developed with SZC.\nAlthough hypernatremia can be caused by sodium overload (which might occur\nsecondary to the potassium-sodium exchange), it is usually caused by a deficiency of\nwater. Therefore, data on changes in body weight and urinary excretions of electrolytes\nand osmolality are necessary to clarify the development of hypernatremia.\nWe sincerely appreciate the reviewer’s critical inquiry regarding the dosing strategy.\nWe acknowledge the error in equating the 4 g/kg rat dose to a 10 g human equivalent\nin the original manuscript, which has been corrected in the revised version (line 264-\n266). The selected dose (4 g/kg) (human dose equivalent of 42 g/day) was\nmeticulously justified by referencing FDA-approved maximum safety thresholds for\nsodium zirconium cyclosilicate (SZC) in rats (6 g/kg), aligning with AstraZeneca's\nestablished experimental range (2-6 g/kg) and corresponds to the conventional\ntherapeutic dose used in rats with renal failure-induced hyperkalemia. (Kidney Int 2015,\n88, 404-411., Expert Opin Pharmacother 2015, 16, 1727-1734., Am J Physiol Renal\nPhysiol 2023, 324, F464-F471., J Cardiovasc Pharmacol Ther 2024, 29,\n10742484241227580.).\nAccording to the US Prescribing Information, each gram of SZC contains 80 mg of Na\nand has a K exchange capacity of 3.5 ± 10% mEq/g, meaning nearly all the Na in SZC\ncan be released to exchange for K (PLoS One 2014, 9, e114686.). During the\ntreatment, the 4g/kg dose of SZC administered to the rats adds a sodium burden of\n320 mg/kg. A healthy rat's unilateral kidney contains approximately 30,000-40,000\nnephrons. When metabolizing 320 mg of sodium (approximately 13.9 mmol), sodium\nexcretion is primarily regulated through glomerular filtration (accounting for 99%) and\ntubular reabsorption. The glomerular filtration rate (GFR) in healthy rats is\napproximately 10-15 ml/min/kg (about 2.5-3.75 ml/min for a 250g rat). With a Na\nexcretion rate of about 1-2 μmol/min/kg, metabolizing 320 mg Na takes approximately\n4 hours. In contrast, 5/6 nephrectomized rats (a chronic kidney disease model we\nselected) retain only 16.7% of their original nephrons. Although single nephron GFR\n(SNGFR) increases by 50% due to compensatory hypertrophy, the overall GFR\ndecreases to 40-50% of normal, significantly reducing sodium excretion capacity. We\nmeasured serum electrolyte levels 1 hour after administration. Within this short period,\nthe sodium released by SZC caused elevated Na levels that the remaining kidney\ntissue could not fully compensate. Concurrently, due to damage in the renal collecting\nducts, the baseline ability to reabsorb water to lower serum sodium levels was also\nimpaired. This combination led to a transient hypernatremia. Over time, renal\ncompensatory excretion and water reabsorption by the collecting ducts would restore\nserum sodium levels, but there is a high likelihood of fluid retention (in clinical practice,\nedema is a frequently reported adverse reaction to SZC).\nIn our supplementary chronic hyperkalemia experiments, SZC treatment similarly\ninduced a mild elevation in serum sodium levels. However, unlike the acute phase, this\nchronic elevation remained within normal physiological ranges for blood Na+.\nThis observation confirms that while renal metabolic capacity is compromised during acute\ntreatment, it gradually recovers to normal physiological levels during prolonged\nmetabolic adaptation (Figure 5g).\nTo further validate these findings, we additionally measured various ion concentrations\nin urine and blood, along with body weight changes during chronic SZC administration.\nThe results demonstrated no significant difference in weight recovery between SZC-\ntreated and PB+VC-treated rats, indicating no apparent edema symptoms (Figure\nS18i, ). The elevated blood Mg2+, as addressed in comment9, may be attributed to Na+-\nMg2+ excretion competition (Figure S17d). This further suggests that the potassium-\ncapturing process of SZC, accompanied by sodium release, may impose additional\nmetabolic burden on already compromised kidneys.\nThe 24-hour urine analysis revealed substantial Na+ excretion, confirming renal\nelimination of Na+. However, impaired nephrons may temporarily retain Na+ due to\ninsufficient metabolic capacity, leading to transient blood Na+ elevation. Collectively,\nthese data provide a clear explanation: the compromised renal function may result in\nmild hypernatremia during acute administration due to insufficient metabolic capacity,\nwhile Na+ homeostasis is gradually restored over time, as evidenced in the chronic\ntreatment phase.\nLine 264-266\nThis is well above the therapeutic dose of SZC administered to animals in current\nstudies (approximately 4 g kg-1 in rats), indicating a wide therapeutic window for\nPB+VC.\n11. The statement that SZC treats hyperkalemia because Na+ release alleviates\namiloride-induced salt wasting (lines 240-241) is not supported by data and also\ninconsistent with current understanding of the mechanism of action of SZC. Either\ndelete this statement or support with data.\nWe thank the reviewer for the attention to the mechanism of “Na+ release alleviates\namiloride-induced salt wasting.” We wish to clarify that our animal model employs\namiloride-induced ENaC blockade to mimic PHA-1, a salt-wasting syndrome. ENaC\ndysfunction impairs renal Na+ reabsorption, inhibiting the Na+-K+ exchange mechanism,\ntriggering Na+ excretion and K+ retention mechanisms that can manifest clinically as\nhyperkalemia. As referenced in the updated manuscript (Refs. 56 and 57), βENaC-\nknockout mice develop classic PHA-1 symptoms (weight loss, hyperkalemia) under\nlow-salt conditions, which resolve with NaCl supplementation. Simultaneously, sodium\nchloride supplements are clinically used to treat hyperkalemia in PHA-1. To better\nelucidate this phenomenon, we supplemented NaCl and NH Cl administration to\n4\ninvestigate whether Na+ or NH + (as in our original protocol) would alter electrolyte\n4\nlevels in this model (Figure S16). The results demonstrated that administering a\nclinically equivalent dose of Na+ (matching the Na+ release of SZC during Na+/K+\nexchange) partially alleviated hyperkalemia (Pediatr Nephrol 1996, 10, 501-503.).\nNotably, NH + release slightly elevated Blood K+, further confirming its K+-sparing/ Na+-\n4\nexcreting effect (Pediatr Nephrol 2017, 32, 933-942.). This demonstrates that Na+\nreplenishment compensates for ENaC-mediated salt wasting and counteracts\npathological K⁺ retention. In our amiloride-induced hyperkalemia model, SZC corrects\nhyperkalemia through Na+/K+ exchange while simultaneously releasing Na+ that\ncompensates for ENaC dysfunction, whereas PB+VC achieves potassium removal\nwithout introducing supplemental electrolytes; despite lacking Na+ provision, PB+VC's\nexceptional potassium-binding capacity enables complete serum K+ normalization\nsolely through enteric potassium sequestration.\nIn the updated manuscript, we have supplemented data-supported evidence that SZC\nalleviates amiloride-induced salt depletion through Na+ release during hyperkalemia\ntreatment, along with additional discussion to enhance comprehension (line 286-290).\nFigure 5a, b, Figure S16, Line 286-290\nIn contrast to the reduction in blood K+ observed with SZC, which may be attributed to\nNa+ release to alleviate amiloride-induced salt-wasting, as evidenced by the partial K+-\nlowering effect of NaCl administration alone, PB+VC demonstrated efficacy in this\nmodel through direct adsorption, reducing blood K+ to normal level.\n12. Similarly, how does PB+VC increase blood Na+ if there is only a selective effect\non K+ (Fig. 5c)? Furthermore, the lower blood Na+ with Am+KCl was not observed in\nyour previous study (ref. #18) - please clarify.\nWe sincerely appreciate the reviewers' insightful comments regarding the changes in\nBlood Na+ induced by amiloride administration. Here we would like to clarify that Na+\nand K+ homeostasis in vivo is maintained through dynamic mutual regulation.\nAmiloride treatment promotes K+ retention and Na+ excretion. Concurrently, the\nelevated peritubular K+ concentration directly downregulates luminal NaCl\ncotransporter (NCC) activity in the distal convoluted tubule (DCT). This results in\nimpaired sodium reabsorption in the DCT segment, representing an additional adaptive\nresponse to hyperkalemia (Am J Physiol Renal Physiol 2014, 306, F1059-F1068.).\nConsequently, the Blood Na⁺ levels in the model group become abnormally low\ncompared to the Control group.\nOur PB+VC therapy effectively corrects hyperkalemia and compensates for the\namiloride-inhibited K+ excretion pathway, thereby restoring sodium reabsorption.\nImportantly, during the reduction process of VC, H⁺ are released. The proximal tubules,\nthrough Na⁺-H⁺ exchanger 3 (NHE3), respond to the increased H⁺ concentration by\nenhancing Na⁺ reabsorption, which contributes to the restoration of normal Blood Na⁺\nlevels (Compr Physiol 2011, 1, 2083-2100).\nIn contrast, our previous strategy primarily relied on NH + - K+ exchange. The release\n4\nof NH + promoted K+ reabsorption while simultaneously inhibiting Na⁺ reabsorption\n4\n(Figure S16), which exacerbated salt wasting in PHA-1 and failed to restore normal\nsodium Na⁺ (line 44-46) (Pediatr Nephrol 2017, 32, 933-942., Adv Mater 2024, 36,\n2404688.).\nIn the revised manuscript, we have supplemented the explanation that the combination\nof PB and VC (vitamin C) moderately restores blood sodium levels primarily due to\ninternal homeostasis, and clarified why previous studies did not observe such effects.\nIn the revised manuscript, we have supplemented the explanation that the combination\nof PB+VC restores blood Na⁺ primarily due to internal homeostasis (line 290-293).\nMain, Line 44-46:\n“This mechanism exacerbates salt wasting, consequently resulting in suboptimal\nperformance in managing conditions such as Pseudohypoaldosteronism type I (PHA-1)\ncomplicated by hyperkalemia.”\nFigure 5c，Line 290-293:\n“Additionally, PB+VC can partially mitigate the hyponatremia induced by the K+-\nsparing/Na+-excreting effect of amiloride, primarily by restoring K+ excretion and\nenhancing NHE -mediated reabsorption of Na+ to respond the increased VC-derived\n13. Fig. 5f: Why does KCl significantly decrease blood Na+? This seems unexpected\nand unphysiological.\nWe sincerely appreciate the reviewers' insightful questions regarding the observed\nchanges in blood Na+. As addressed in our response to comment 12, Na+ and K+\nhomeostasis in vivo is maintained through dynamic mutual regulation.\nUnder conditions of elevated Blood K+ (hyperkalemia), the following key physiological\nalterations occur:\nA high dietary K+ intake causes a rapid dephosphorylation, and hence inactivation,\nof the thiazide‐sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule\n(DCT). Inactivation of NCC significantly inhibits the reabsorption of Na⁺ by DCT,\nresulting in more Na⁺ remaining in the lumen and being excreted with urine.28 Based\non the above mechanism, potassium supplementation is often used as a dietary\ntreatment to suppress the increase in blood pressure in the body caused by high\nsodium (Am J Cardiol 1961, 8, 527-532., Hypertension 1984, 6, 184-192.). This\nmechanism explains why the model group (5/6 nephrectomy) exhibited abnormally low\nserum sodium levels compared to the Control group. Importantly, the impaired renal\nmetabolic capacity in this model further exacerbates sodium imbalance due to:\nReduced tubular reabsorption efficiency (primarily in distal convoluted tubules and\ncollecting ducts) and structural loss of functional nephrons, diminishing overall sodium\nrecovery capacity.\nIn the revised manuscript, we have briefly supplemented the discussion on how KCl\nmay affect blood Na+, which will facilitate a better interpretation of the data presented\nin the paper (line 302-306).\nFigure 5f, I, Line 302-306:\n“KCl supplementation moderately reduced blood Na+ in rats, likely through tonic\ninactivation of NaCl cotransporter (a known antihypertensive mechanism) in the renal\ndistal convoluted tubule. This effect was exacerbated in the 5/6 N model due to\nx\nimpaired tubular Na+ reabsorption from nephron loss. The K+ excretion mechanism of\nPB+VC concurrently restored normal blood Na+ (Figure 5f, I).”\n14. The authors seem to mix up PHA1 (in which K+ binders are used) and Gordon’s\nsyndrome (or PHA2) in which hyperkalemia can be successfully treated with thiazide\ndiuretics (line 250).\nWe sincerely appreciate the reviewers' inquiries regarding our discussion of disease\ntreatment strategies. We would like to clarify that our reference to\nPseudohypoaldosteronism type 2 (PHA2), also known as Gordon syndrome or\nhyperkalemia-hypertension syndrome, was intentional due to its characteristic triad of\nhyperkalemia, hypertension, and metabolic acidosis.\nOur original intention was to highlight that, in contrast to SZC's Na+-K+ exchange\nstrategy (which cannot simultaneously address both hyperkalemia and hypertension\nin this condition), our electrolyte-free ion release approach shows potential for\nmanaging the hyperkalemic component of Gordon syndrome. However, as rightly\nnoted by the reviewers, while current diuretics can treat PHA2 (primarily targeting\nhypertension), their efficacy becomes limited when managing severe hyperkalemia\nepisodes.\nIn direct response to these valuable insights, we have substantially enhanced our\nmanuscript by: (1) After the disease name, a brief addition was made regarding the\ncomplications of Gordon's syndrome, providing stronger justification for our treatment\napproach focused on electrolytic counterion-free (2) Incorporating PHA1 into our\nstrategy expands its potential future therapeutic applications. This extension is\nparticularly critical because PHA1 carries a risk of life-threatening hyperkalemia, which,\nif improperly managed, may lead to cardiac arrest. Furthermore, we have\nexperimentally validated and demonstrated the efficacy of our strategy using PHA1-\nmimicking animal models of hyperkalemia (line 314-315).\nLine 313-316:\n“This makes PB+VC a more suitable clinical treatment for hyperkalemia, particularly\nfor patients with underlying health conditions such as CKD, PHA-1 and Gordon's\nsyndrome (hyperkalemia-hypertension syndrome), due to its distinctive adsorption\nmechanism that avoids ion exchange.”\nMinor comments\n-Figure 1b: The abbreviations SGF, SIF and SCoF are not explained (except in the\nsupplement).\nWe thank the reviewer for the comments. In the revised manuscript, the abbreviations\nSGF (Simulated gastric fluid electrolyte solution), SIF (Simulated intestinal fluid\nelectrolyte solution), and SCoF (Simulated Colonic Fluids electrolyte solution) have\nbeen spelled out and explicitly defined (line 108-111).\nLine 108-111:\n“To comprehensively evaluate the K⁺ adsorption efficacy of the PB and VC combination\n(PB+VC), we compared it with different potassium binders, in simulated GI fluids that\nmimic in vivo environments, including Simulated gastric fluid electrolyte solution (SGF),\nSimulated intestinal fluid electrolyte solution (SIF) and Simulated Colonic Fluids\nelectrolyte solution (SCoF) (Figure 1b).”\n-Figure 4a-b: For consistency, please report K+ in urine and feces in mmol/day instead\nof g/day\nWe thank the reviewer for their valuable feedback. In the revised manuscript, we have\nupdated the units for ion concentrations in urinary and fecal contents to mmol/day\n(Figure 4).\n-Figure 4f: You probably meant “Alanine Aminotransferase” instead of “Alanine\nAomptransferase”\nWe apologize for the writing errors and thank the reviewer for their careful review. The\ncorrections have been made in the updated manuscript (Figure 4m).\n-Lines 18-19, this should read: “This ion adsorption mechanism enables selective\nadsorption of K+ without counterion release”\nWe sincerely appreciate the reviewer's constructive feedback regarding the wording in\nour manuscript. In the revised version, the problematic statements have been\nappropriately revised to ensure scientific precision (line 18-19).\nAbstract, Line 18-19:\n“This ion adsorption mechanism enables selective adsorption of K+ without metal\ncounterion release.”\n-Lines 27-28, references 3-6 appear to be selected rather randomly and are not\nappropriately supporting the sentence (e.g. 3 and 4 are case reports, 5 is not related,\n6 deals with improving hyperkalemia risk with SGLT2i).‘\nWe sincerely appreciate the reviewers' suggestions regarding the references.\nReferences 3, 4, and 6 were originally cited to demonstrate the disease-induced\nhyperkalemia mentioned in the text, while reference 5 was included due to our\noversight. Upon re-evaluation, we agree that these references did not adequately\nsupport the statement. We have therefore updated references 3-6 in the revised\nmanuscript (line 27-28).\n-Lines 36-38: Please elaborate on your previous study to clarify why NH4+ exchange\ndid not work well.\nWe sincerely appreciate the reviewer's interest in our previous work. In previous\nstudies, the nanoporous materials we developed primarily achieved adsorption\nthrough K+-NH + ion exchange. During treatment, the release of NH + inhibits Na+\n4 4\nreabsorption while simultaneously promoting K+ reabsorption27. This mechanism\nexacerbates salt loss, consequently leading to suboptimal performance in managing\nconditions such as PHA-1 complicated by hyperkalemia. Moreover, for patients with\nhyperkalemia accompanied by abnormal nitrogen metabolism, the release of NH4⁺\nincreases the burden on the liver, posing additional risks. To better clarify the\nimportance of our IRISA strategy, we have added detailed explanations regarding the\ntherapeutic limitations of our previous approach in PHA1 management in the revised\nmanuscript (line 42-47).\nLine 42-47:\n“In previous work, we developed a nanoporous material based on K+-NH + ion\n4\nexchange, where NH + replaces Na+. The release of NH + inhibits Na+ reabsorption\n4 4\nwhile simultaneously promoting K+ reabsorption. This mechanism exacerbates salt\nwasting, consequently resulting in suboptimal performance in managing conditions\nsuch as Pseudohypoaldosteronism type I (PHA-1) complicated by hyperkalemia.\nAdditionally, in patients with abnormal nitrogen metabolism complicated by\nhyperkalemia, releasing NH + increases the burden on the liver, posing additional risks.”\n4\n-Line 46: remove “nature”.\nWe apologize for the mistake in our manuscript. In our revised version, we have\ncorrected the error (line 55).\nLine 55-56:\n“The insolubility of PB allows for gut-restricted use in humans, offering a unique safety\nprofile for treating Cesium and Thallium poisoning, with zero systemic exposure.”\n-Lines 46-48: I suggest removing the words “radioactive” and “contamination” as what\nyou want to treat with PB is the intoxication rather than the contamination. You might\nalso consider writing “Cesium” and “Thallium” for enhanced clarity instead of “Cs” and\n“Tl”\nWe thank the reviewer for the constructive suggestions. In the revised manuscript, the\nidentified sections have been methodically refined to enhance clarity, thereby\nsignificantly improving the manuscript's readability regarding the research objectives\n(line 55-56).\nLine 55-56:\n“The insolubility of PB allows for gut-restricted use in humans, offering a unique safety\nprofile for treating Cesium and Thallium poisoning, with zero systemic exposure.”\n-Lines 46-48: “zero systemic exposure”. I understand that insoluble Prussian blue will\nnot be absorbed intestinally. However, I read reports from patients’ sweat turning blue\nafter prolonged Prussian blue treatment. How can that be explained?\nWe appreciate the reviewers' concerns regarding the safety of Prussian blue (PB). We\nhave carefully reviewed reports from official sources such as the U.S. Food and Drug\nAdministration (FDA) and the U.S. Centers for Disease Control and Prevention (CDC),\nwhich indicate that PB does not result in systemic exposure and is excreted through\nthe intestines. However, it is important to note that PB is not recommended for patients\nwith gastrointestinal issues. We suspect that in very rare cases, patients with\ncompromised intestinal barriers may experience minimal absorption, allowing trace\namounts to enter the circulatory system and potentially be excreted through sweat.\n-Line 49, references: The references are about sodium-ion batteries and do not tell us\nhow “PB aligns well with therapeutic approaches.”\nWe sincerely appreciate the reviewer's comments regarding the reference citations.\nWe would like to clarify that these cited references primarily serve to demonstrate PB's\nintrinsic advantages for K+ adsorption. The battery-related references specifically\nsubstantiate PB's exceptional properties, including: High K+ diffusion coefficient,\nremarkable storage capacity and structural stability. These characteristics highlight the\noutstanding potential of PB for selective potassium adsorption, which inspired our\ninnovative approach of adapting battery ion adsorption principles for treating in vivo\nions overload conditions. To facilitate understanding of this sentence, we have\nspecifically listed the corresponding references for the intrinsic advantages of PB in\nthe updated manuscript (line 56-59).\nLine 56-59:\n“Given these advantageous characteristics, including high K+ diffusion coefficient,\nremarkable storage capacity and structural stability, PB, as an FDA-approved drug,\naligns well with therapeutic approaches of potassium binders for hyperkalemia.”\n-Line 51: remove “the”\nWe apologize for the mistake in our manuscript. In our revised version, we have\ncorrected the error (line 60).\n-Line 84: You refer to the “excellent safety profile” of PB, but relatively little is known\nabout the safety profile in humans; no controlled trials have been performed and the\nFDA approval of the drug is primarily based on reviews and case reports.\nWe sincerely appreciate the reviewer's concerns regarding the safety profile of PB. We\nacknowledge the reviewer's valid point that FDA approval alone does not constitute\ncomprehensive proof of the safety of PB in humans. In response, we have\nsupplemented Reference 43 in the revised manuscript, which explicitly states that: (1)\nPB causes no adverse effects on mammalian health and reproduction, and (2) no toxic\neffects have been observed in humans when PB is used for experimental or\ntherapeutic purposes. To more accurately reflect these findings, we have replaced the\noriginal phrase \"excellent safety profile\" with the more precise description \"non-toxic\nside effects\" throughout the text (line 95).\n-Line 201: reference 37 does not explain why these 3 cell types should be used.\nWe sincerely appreciate the reviewer's inquiry regarding our cell selection rationale. In\nour initial submission, we cited only Reference 37 (now updated to Reference 47 in\nthe revised manuscript) to explain our choice of Caco-2 tumor cell line for safety\nevaluation. This selection was based on the well-established fact that while Caco-2 is\na colon carcinoma cell line, it exhibits structural and functional characteristics of\nabsorptive intestinal epithelial cells, making it a standard model for studying small\nintestinal epithelial function.\nOur strategy, being an intestine-restricted compound (as evidenced by in vitro\ndissolution data, Figure S1e, and Figure 4e), must undergo GI excretion. Since its\nprimary contact would be with epithelial cells throughout the GI, we selected three\nrepresentative cell models: human gastric mucosal epithelial cells (GES-1),\nintestinal\ncrypt epithelial cells (IEC-6), and human colon carcinoma cells (Caco-2) for\ncomprehensive safety assessment.\nTo better substantiate our selection of GES-1 and IEC-6 for safety evaluation, we have\nsupplemented References 48 and 49 in the revised manuscript. These references\nconfirm that both cell types are widely accepted as normal GI cell models for\npharmacological safety assessment.\n-Line 203: what do you mean by “relatively non-toxic”? Looking at the graphs in S7a-\nc, there seems to be no difference in cell viability after adding PB+VC.\nWe sincerely apologize for our inaccurate terminology. The phrase \"relatively non-\ntoxic\" used in our previous manuscript was intended to convey that the data showed\nno statistically significant differences. In the revised manuscript, we have modified the\ndescription from \"relatively non-toxic\" to \"non-toxic\" to better align with the conclusions\nsupported by our data and to more precisely convey the excellent in vitro safety profile\nof our material.\n-Lines 209-210: Did you mean to say hepatological parameters? Fig. 5 should read\nFig. 4.\nWe sincerely apologize for the incorrect labeling in our figures and greatly appreciate\nthe reviewer's careful scrutiny. In the revised manuscript, we have now corrected this\nerror accordingly.\n-Line 235: chronic renal failure is now referred to as chronic kidney disease.\nWe sincerely appreciate the reviewer's valuable suggestions regarding our terminology\nusage. In the updated manuscript, all instances of \"chronic renal failure\" have been\nsystematically replaced with the more clinically appropriate term \"chronic kidney\ndisease\" throughout the text.\n-Lines 243 and 248: This should read Fig. 5.\nWe sincerely apologize for the incorrect labeling in our figures and greatly appreciate\nthe reviewer's careful scrutiny. In the revised manuscript, we have now corrected this\nerror accordingly.\n-Line 371: renumber the reference.\nWe apologize for the incorrect citation in the references and have now updated the\nreference numbering in the revised manuscript (line 480).\nReviewer #3 (Remarks to the Author):\nThe manuscript deals with the use of Prussian Blue (PB) for in vivo counterion-free\npotassium removal in the presence of vitamin C (VC). Inspired by the efficiency of PB\nand its analogues as battery electrode materials, VC is elegantly used as a reducing\nagent, to trap the potassium cations in the PB structure for further elimination by the\norganism. Given my skills, my report will concentrate on the experimental study of the\nPB sample in my report. In this regard, I would suggest that the authors make the\ndemonstration of the reduction/adsorption process more convincing and, if possible,\nmore quantitative. Some missing data and information should be added to the\nmanuscript and some points should be further discussed or clarified.\n1/ Missing information.\nThe PB sample after treatment with VC and potassium cation solution was analyzed\nby several techniques. The procedure used to recover the sample after treatment\nshould be specified (washing, drying…). This information would remove any doubt\nconcerning the possible presence of surface-adsorbed K+ ions and the water content\nof the sample.\nIt is not clear how the values in Table S2 were obtained (were all the elements titrated ?,\nby which method?, were some hypotheses made to propose the chemical formulae?,\nwhat about water molecules?…). This point should be clarified. These data, if reliable,\ncould be used i) to show the unchanged chemical composition of the cyanide-bridged\nFe network after treatment, and ii) to quantify the potassium content of the sample after\ntreatment. In this case, I would suggest that these data be given in the main text.\nThere is an error in the chemical formula of PB in Table S2.\nWe sincerely appreciate the reviewer's valuable suggestions regarding our sample\nprocessing protocol and the identified issue with the XRF matrix selection in Table S2.\nIn response, we have thoroughly revised our methodology and analyses as follows:\nFirst, we have added detailed sample processing procedures in the revised manuscript\n(line 347-354), specifying that recovered PB+VC samples were washed three times\nwith deionized water to remove surface-adsorbed K⁺ ions, followed by flash-freezing\nin liquid nitrogen and lyophilization for 48 hours to eliminate both free and adsorbed\nwater.\nRegarding Table S2, we sincerely apologize for the initial matrix selection error\n(Fe5C12N12) and have corrected it to Fe7C18N18, with all elemental ratios\nrecalculated accordingly. The XRF data were obtained through standardless analysis\nusing wavelength-dispersive X-ray fluorescence spectroscopy, which involves X-ray\nexcitation of characteristic fluorescence, intensity measurement via spectroscopic\ndispersion, and elemental content calculation through fundamental parameter\nmethods and Sherman equation for matrix effect correction (Table S2 caption). While\nthis approach is inherently semi-quantitative, our data clearly demonstrate the\nselective K+ adsorption capability of PB+VC (variations ranging from 0.12 wt% to 14.3\nwt% of K), as further corroborated by ICP analyses of treated solutions (Figure 1c and\nFigure S1e) showing >90% K+ removal. Furthermore, ICP analysis confirmed minimal\nvariations in other ionic species (Na, Ca, and Mg) and negligible Fe leaching\n(Quantitative analysis reveals that 1 mol of PB containing 7 mol of Fe releases no more\nthan 0.06 mol Fe when exposed to simulated fluid conditions). leaching, demonstrating\nthat our material selectively binds potassium ions without inducing significant changes\nto other chemical components in the solution system These comprehensive results\nconfirm the chemical stability of PB+VC's cyanide-bridged Fe network and validate our\ncorrected matrix for accurate quantification of K⁺ uptake, while demonstrating the\nmaterial's exceptional ion selectivity without significant elemental composition changes.\nWe fully concur with the reviewer's perspective that the elemental variation data\npresented in Table S2 significantly strengthens our demonstration of the successful\nimplementation of the intended reductive adsorption strategy. In response to this\nvaluable suggestion, we have now incorporated a discussion of these elemental\nchanges in the main text. This enhanced analysis enables us to make well-supported\ninferences regarding the chemical formula of the PB+VC composite, drawing upon\nmultiple lines of experimental evidence including: The systematic potassium content\nincrease (0.12 wt% to 14.3 wt%), corresponding Fe valence state changes (Mössbauer\nspectroscopy), minimal interference from other ions (ICP data) and structural\ncharacterization (XRD, FTIR)\nThe convergence of these independent measurements provides robust confirmation\nof our proposed chemical formulation (K4Fe4[Fe(CN)6]3) and its reductive adsorption\nmechanism. We believe this additional discussion substantially improves the\nmanuscript's scientific rigor by explicitly connecting the elemental composition data\nwith the material's functional performance.\nLine 148-152:\n“Significantly, analysis of the recovered liquid samples following MEI adsorption by\nPB+VC revealed no detectable Fe release. These observations provide evidence that,\nin addition to potassium adsorption, the cyanide-bridged coordination network\nframework of PB+VC should maintain its original chemical composition, thereby largely\nexcluding the possibility of dissolution-recrystallization occurring during the adsorption\nprocess.”\nLine 162-168:\n“The elemental composition analysis of PB+VC, with variations ranging from 0.12 wt% to\n14.3 wt% of K, revealed that the chemical composition of the treated samples closely\napproximates K Fe C N . This formulation aligns well with the observations from\n4 7 18 18\nMössbauer spectroscopy, which demonstrated the reduction of Fe3+ and achievement of\nmaximum theoretical K+ adsorption capacity. Based on these consistent findings, we can\nreasonably infer that the maximum reduction of PB by VC during the preparation process\nresults in the chemical formula of the treated product being K Fe [Fe(CN) ] (Figure 2g).”\n4 4 63\nMethod section, Line 347-354:\n“Briefly, PB (200 mg) and VC (200 mg) were mixed with MEI (40 mL), followed by\nincubation at 37°C with orbital shaking (180 rpm) for 2 hours. Post-incubation, the\nmixture was centrifuged at 3500 rpm for 5 minutes to separate the supernatant. The\nresulting pellet underwent three washing cycles with deionized water (30 mL per cycle,\nvortex mixing for 30 seconds), each followed by identical centrifugation parameters. The\npurified precipitate was flash-frozen in liquid nitrogen and subsequently lyophilized for 48\nhours to obtain the final product for Structural and morphological characterization. In the\nprocessed PB+VC, the VC component and its oxidation products dissolve in aqueous\nsolution, yielding a final product consisting primarily of K+-adsorbed PB.”\n2/ Clarification of the chemical formula used for PB.\nThe chemical formula used for PB is not entirely clear, probably C18Fe7N18 (or\nFe4[Fe(CN)6]3) as stated in the experimental part, but a different chemical formula is\nused in Table 2 (Fe5C12N12). The chemical formula used for PB should be clarified.\nWe apologize for the erroneous matrix selection (Fe5C12N12) in the initial XRF\nanalysis presented in Table S2 and deeply appreciate the reviewer's meticulous\nattention. The elemental quantification data in XRF are matrix-dependent, and our\nprevious incorrect matrix choice led to deviations in reported ratios. In the revised\nmanuscript, we have corrected the matrix to Fe7C18N18 (noting that XRF cannot\ndetermine oxidation states) and updated all elemental ratios in Table S2 based on\nrecalibrated calculations.\n3/ Proof of the stability of the FeIII4[FeII(CN)6]3 cyanide-bridged coordination network.\nPage 5, line 122. The authors conclude from the XRD data and the presence of\npotassium in the sample, that the cyanide-bridged Fe coordination network remained\nstable after treatment with VC and K+ ion. However, a dissolution-recrystallisation\nphenomenon transforming the initial PB particles (FeIII4[FeII(CN)6]3) into potassium\ncontaining PB particles (KxFeIII(4-y)FeIIy[FeII(CN)6]3+z) cannot be excluded from the\nXRD data and the presence of potassium ions in the sample. The analyses of the\nchemical composition of the sample before and after treatment (in table S2) should be\nused to show the unchanged chemical composition of the Fe4[Fe(CN)6]3 coordination\nnetwork before and after treatment. The comparison of microscopy images of the\nparticles before and after treatment would further confirm that the morphology and size\nof the particles has remained unchanged after treatment. Without a change in the\nchemical composition of the cyanide-bridged Fe coordination network and the\nmorphology of the particles a dissolution-recrystallisation process can be clearly ruled\nout.\nWe appreciate the reviewer's attention to the stability of the FeII4[FeII(CN)6]3 cyanide-\nbridged coordination network. While the reviewer's concern regarding the XRD data\nand potassium ion presence not completely ruling out dissolution-recrystallization is\nreasonable, our current data provide conclusive evidence that recrystallization does\nnot occur in our strategy. First, the Prussian blue we selected is a well-documented\nwater-insoluble material. Our entire experimental process was conducted at 37°C,\nwhich inherently limits dissolution conditions. Furthermore, to evaluate the stability of\nour strategy, we tested for iron leaching: no iron was detected in MEI solution, and only\nnegligible iron release was observed in complex simulated body fluid environments\n(Figure S1e and line148-152). This confirms that neither PB nor PB+VC undergoes\ndissolution at 37°C, effectively precluding any possibility of dissolution-recrystallization.\nThe iron valence changes observed in Mössbauer spectroscopy of post-processed\nsamples and the potassium ion ratio in chemical composition analysis (Table S2)\nfurther validate that our strategy effectively achieves potassium ion adsorption through\na reductive mechanism. Additionally, during PB+VC preparation, MEI exhibited\nselective adsorption of potassium ions with only minor disturbance of other ions and\nno detectable iron leaching, proving the chemical composition remains unchanged\nexcept for potassium adsorption. To more definitively demonstrate the absence of\nrecrystallization in our strategy, we have supplemented the revised manuscript with\nparticle size distribution data for PB and PB+VC samples (Figure 2i and Figure S3b).\nThe results show minimal differences in particle size between the two samples and no\nmorphological alterations. Collectively, this evidence conclusively demonstrates that\nboth the chemical composition of the cyanide-bridged iron coordination network and\nthe particle morphology remain unaltered, definitively excluding any dissolution-\nrecrystallization process. To enhance comprehension of our adsorption mechanism\nand substantiate the aforementioned analytical results, we have expanded the\ndiscussion in the revised manuscript.\nLine 148-152:\n“Significantly, analysis of the recovered liquid samples following MEI adsorption by\nPB+VC revealed no detectable Fe release. These observations provide evidence that,\nin addition to potassium adsorption, the cyanide-bridged coordination network\nframework of PB+VC should maintain its original chemical composition, thereby largely\nexcluding the possibility of dissolution-recrystallization occurring during the adsorption\nprocess.”\nFigure 2 caption, Line 134-137:\n“i) TEM image delineating the article size distribution of PB+VC at low magnification. j)\nTEM image delineating the structural details of PB+VC at high magnification. k, l) HAADF-\nSTEM image and corresponding energy dispersive spectrometer (EDS) element.”\nLine 170-175：\n“Both scanning electron microscopy (SEM) and transmission electron microscopy (TEM)\nimages (Figure 2h, i, j, and Figure S4a, b) reveal uniformly-sized, well-dispersed\nnanocubes. Both PB and PB+VC exhibit particle sizes ranging from 30 to 70 nm, which\nfacilitates rapid K+ diffusion through the structure. The highly consistent particle size\ndistribution between PB and PB+VC further confirms that K+ adsorption was achieved not\nvia dissolution-recrystallization, but rather through a reductive adsorption mechanism.”\n4/ Quantification of the adsorption/reduction phenomena.\nPage 5 line 131. There is no experimental evidence that most of the Fe³⁺ coordinated\nto nitrogen in PB has been reduced to Fe²⁺, and such experimental evidence is difficult\nto provide here because the two transition metal sites are occupied by the same\nchemical element. Mössbauer spectroscopy shows the presence of both Fe3+ and\nFe2+ HS ions, but this spectroscopy is not quantitative.\nThe authors could again use the chemical formula of the PB sample after treatment,\nto support their statement. From the data given in Table 2, a chemical formula close to\nK4Fe4[Fe(CN)6]3 can be deduced for the sample after treatment, which indeed\ncorresponds to a reduction of most of the Fe3+ ions and the adsorption of the\nmaximum number of K+ ions.\nHowever, the VC/PB ratio used for the treatment raises questions, as the complete\noxidation of VC provides two electrons, whereas the complete reduction of PB would\nrequire 4 electrons (using a chemical formula of Fe4[Fe(CN)6]3 for PB). A VC/PB molar\nratio of 2 would be required to completely reduce PB. This point should be clarified.\nThe conditions and apparatus used for XPS analysis should be described in the\nexperimental part.\nWe sincerely appreciate the reviewer's attention to the quantification of our\nadsorption/reduction phenomena. First, we would like to clarify that Mössbauer\nspectroscopy allows comprehensive analysis of iron's valence state, spin state, and\ncoordination environment. Under normal conditions, PB typically exhibits an alternating\nFe3+-N≡C-Fe2+ stable structure. This configuration stems from electronic structure and\ncoordination preferences: Fe3+ (high-spin, d5), being electron-deficient, preferentially\ncoordinates with the more electronegative N (providing lone pair electrons), while Fe2+\n(d6), with sufficient electrons, favors coordination with the less electronegative C.\nAccording to ligand field theory, the strong ligand field causes Fe coordinated with the\nC of CN groups to adopt a low-spin state, whereas Fe coordinated with N remains\nhigh-spin.\nOur Mössbauer spectra clearly demonstrate the original structure of PB. The isomer\nshift (IS), reflecting electron density around the nucleus and characterizing\nelectrostatic interactions, shows that increased electron density in 57Fe yields negative\nIS values. In the cyanide-bridged structure, the more electronegative N atoms exhibit\nhigher electron density than C atoms. When Fe coordinates with the C atom of cyanide,\nelectron clouds shift toward C, increasing electron density on 57Fe (negative IS).\nConversely, coordination with the high electronegativity of N reduces the electron\ndensity of Fe (positive IS). As clearly shown in Table S1, LS-Fe2+ exhibits IS < 0 (C-\ncoordinated), while HS-Fe3+ shows IS > 0 (N-coordinated), consistent with these\nprinciples.\nThe appearance of HS-Fe2+ doublets in post-treatment PB+VC samples, displaying\nhigh-spin states and IS > 0, indicates N-coordination characteristics. This confirms that\nin VC-containing reducing solutions, Fe3+ (primarily N-coordinated) is reduced to Fe2+,\nas reflected in the spectra. We have refined our Mössbauer spectroscopy discussion\nto precisely convey two key objectives: (1) demonstrating the iron valence state\nchanges, and (2) validating the reductive adsorption mechanism. By comparing\nMössbauer peak areas, we calculate that approximately four Fe3+ are reduced to Fe2+,\ncorresponding to the adsorption of four K+. To eliminate any potential ambiguity, we\nremoved “Fe3+ coordinated with nitrogen” and modified it to “approximately 4 mol of\nFe3+ in each mol of PB was reduced to Fe2+” (line 158). This adjustment facilitates a\nmore intuitive representation of the content depicted in the figures and clearly\ndemonstrates that our strategy successfully achieved the reductive adsorption process.\nThe data of Table S2, supported by ICP analysis of treated solutions (Figure S1c) and\nparticle size distribution curves (Figure 2 and Figure S1c), verify that the Fe7C18N18\nmatrix remains chemically intact without recrystallization, preserving the cyanide-\nbridged iron network of PB. As the reviewer noted, the data presented in Table 2,\ncombined with the Mössbauer spectroscopy results, collectively demonstrate that the\nchemical composition of the treated samples approximates K Fe [Fe(CN) ] .\n4 4 6 3\nThis formulation quantitatively confirms: (1) the near-complete reduction of Fe3+, and (2) the\nmaximal adsorption of K+, thereby providing definitive evidence for both the Fe redox\nprocess and the stoichiometric relationship of potassium ion adsorption. The analytical\nresults quantitatively indicate the Fe redox process and K⁺ adsorption stoichiometry.\nWe have substantially enhanced the discussion of this aspect in the revised manuscript\nto provide a more comprehensive understanding of our strategy's adsorption process\nand underlying mechanism.\nRegarding the VC/PB ratio, we clarify that the 1:1 mass ratio (PB:VC) treanslates to a\n1:5 molar ratio, allowing VC to provide up to 10 electrons for complete PB reduction.\nThis ratio was optimized through systematic studies of K⁺ capacity versus PB-VC mass\nratios, representing the optimal balance between maximum adsorption capacity and\nprocess safety.\nWe apologize for omitting XPS analysis conditions and instrumentation details, which\nhave now been added to the Methods section (Line 370-371) of the revised manuscript.\nMethod section, Line 370-371:\n“The chemical composition was analyzed by X-ray photoelectron spectroscopy (XPS,\nNEXSA, Thermofisher Scientific, USA).”\nRespond to Reviewers\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nI have reviewed the author's responses to the two reviewers' comments and their\nrevisions. I believe that the issues regarding the description of experimental and\ncalculation conditions have been at least partially corrected. Since\nIn terms of condensed matter physics and energy storage, it is well known that PBA\ncan electrochemically intercalate and deintercalated alkali metal ions. In fact, PBA has\nbeen actively studied as an active material for sodium-ion secondary batteries. For\nexample, see Chem. Commun.2013, 49, 2750.I believe that providing this broader\nbackground would improve this paper.\nWe sincerely thank the reviewer for the constructive feedback and for acknowledging\nthe revisions made regarding the experimental and calculation conditions. We fully\nagree with the reviewer’s suggestion to provide broader context on Prussian blue\nanalogues (PBAs) within condensed matter physics and energy storage.\nTo address this point, we have integrated the following addition into the Main section\n(Line 51-54): “This application leverages a fundamental property shared by PB and its\nanalogue: their well-established ability to electrochemically intercalate and\ndeintercalate various alkali metal ions, as extensively studied in systems like sodium-\nion batteries31.”\nReviewer #2 (Remarks to the Author):\nThank you for addressing my comments and for providing the additional data.\nI have two remaining points:\n1. “Additionally, PB+VC can partially mitigate the hyponatremia induced by the K+-\nsparing/Na+-excreting effect of amiloride, primarily by restoring K+ excretion and\nenhancing NHE3-mediated reabsorption of Na+ to respond the increased VC-derived\nH+ concentration, thereby counteracting these effects”. This explanation remains\nspeculative and should be classified as such. More generally speaking, in their rebuttal,\nthe authors frequently explained the changes in plasma sodium by discussing changes\nin kidney sodium handling - however, plasma sodium is determined by changes in\nwater balance regulation, not sodium balance regulation (osmoregulation vs. volume\nregulation). This should be clarified.\nWe sincerely thank the reviewer for raising these valuable and insightful points\nregarding our manuscript. In response to the first point, we acknowledge that the\nmechanism proposed in our previous revision —whereby PB+VC alleviates amiloride-\ninduced the slight reduction in blood sodium — was indeed a speculative interpretation\nbased on established physiological principles. To enhance the rigor of our work, we\nhave explicitly labeled this explanation as \"speculative\" in the revised Discussion\nsection (Line 292-295: “Additionally, amiloride caused only a mild, clinically\ninsignificant decrease in blood Na+ through K+-sparing/Na+-excreting. We hypothesize\nthat PB+VC attenuates this change by restoring K⁺ excretion and enhancing NHE -\n3\nmediated reabsorption of Na+ in response to increased VC-derived H⁺ concentration\n59.). We have revised the statement that amiloride causes hyponatremia, as the data\nshow only a slight decrease in serum sodium levels that remained within the normal\nphysiological range. \nRegarding plasma sodium regulation, we deeply appreciate the reviewer’s crucial\nclarification on the conceptual distinction between osmoregulation and volume\nregulation. Amiloride increases the risk of hyponatremia due to enhanced urinary\nsodium loss in the distal convoluted tubule (Postgraduate Medical Journal. 1993, 69\n(816): 797–799). As observed in Figure 5f of our study, amiloride administration caused\na minor decrease in serum sodium levels (though remaining within the normal\nphysiological range). Additionally, it should be noted that hyponatremia secondary to\ndistal diuretic intake primarily results from sodium depletion rather than water retention\n(Nephron. 2018, 140 (1): 31–38, J. Clin. Med. 2022, 11(19), 5810).\nConsistent with our hypothesis, PB+VC facilitates sodium reabsorption in renal tubules\nthrough physiologically-based mechanisms, thereby preventing sodium depletion and\nconsequently avoiding reductions in serum sodium at its source. It should also be\nclarified that, given the acute nature of our single-dose experimental model, sodium\nmeasurements were taken shortly after treatment. The role of water balance in sodium\nconcentration regulation may require longer observation periods to manifest.\nImportantly, the serum sodium fluctuations in Figure 5f were minor and consistently\nwithin normal physiological limits, with no safety concerns observed post-\nadministration.\nWe acknowledge that the serum sodium changes usually reflect alterations in water\nbalance rather than sodium metabolism itself. However, sodium metabolism remains\na critical factor influencing serum sodium levels. Within the context of this acute diuretic\nadministration model, our analysis focused on acute renal sodium handling as the\nprimary driver. This emphasis is justified because although amiloride increases sodium\nexcretion, causing a minor reduction in serum sodium, levels remained within the\nnormal physiological range. Such minor osmotic changes are unlikely to significantly\ntrigger compensatory water balance mechanisms in the acute phase. Consequently,\nrenal sodium excretion likely constitutes the dominant factor affecting plasma sodium\nconcentrations over this short timeframe.\nWe sincerely appreciate the reviewer's critical guidance on sodium homeostasis, which\nprovides essential direction for our future research. Our group will prioritize\ninvestigating water balance dynamics on plasma sodium concentration in subsequent\nstudies.\n2. The Legend to Figure S16 should be clarified: “After 1h and 5h, the administrations\nof NaCl in dose of 0.895 g kg−1 and 1 g kg−1 NH4Cl per day.” It is unclear if the 0.895\ng/kg and 1 g/kg refer to the doses that were given after 1h and 5h or to the total daily\ndose - please clarify.\nWe sincerely thank the reviewer for the valuable feedback regarding the legend of\nFigure S16. We apologize for the ambiguity in the original description and hereby\nclarify as follows:\nThe “0.895 g kg−1 \" refers to the per-administration dose of NaCl (each dose: 0.895 g\nkg−1), with a total daily dose of 1.79 g kg−1. The “1 g kg−1” indicates the total daily dose\nof NH Cl (administered in two doses of 0.5 g kg−1 each).\n“Rats were respectively administered with 1.79 g kg−1 NaCl (divided into two doses of\n0.895 g kg−1) and 1 g kg−1 NH Cl (divided into two doses of 0.5 g kg−1) per day, after 1\n4\nh and 5 h apart.”\nWe greatly appreciate the careful review of reviewer, which has been essential for\nimproving the clarity of our manuscript.\nReviewer #3 (Remarks to the Author):\nConcerning the experimental study of the PB sample, the manuscript has clearly been\nimproved by experimental details addition. However, two points remain to be clarified\nabout the reduction/adsorption process before I can recommend it for publication.\nA question remains about the provided data and the mechanism for K+ uptake\nproposed by the authors.\nThe authors propose a mechanism based on the reduction of 4 Fe3+(HS) ions per mol\nof PB (line 158 and Figure 2g) accompanied by the uptake of 4 mol of K+ ions\ncorresponding to the chemical formula change of the powder from\nFe3+(HS)4[Fe2+(LS)(CN)6]3 to K4Fe4[Fe(CN)6]3 (line 167-168). If dissolution-\nrecrystallization phenomena do not occur during the treatment, the chemical formula\nof the powder after treatment is K4Fe2+(HS)4[Fe2+(LS)(CN)6]3. This means that all\nthe Fe3+(HS) ions have been transformed into Fe2+(HS) ions.\nThe presence of Fe2+(HS) ions in the powder after treatment is clearly demonstrated\nby Mössbauer spectroscopy. Nevertheless, Mössbauer spectroscopy also indicates\nthe presence of a significant amount of Fe3+(HS) ions in the powder after treatment,\nas the areas under the bands corresponding to the Fe2+(HS) and Fe3+(HS) ions are\ncomparable (Table S1). On the contrary, this data suggests that not all Fe3+(HS) ions\nwere reduced to Fe2+(HS) ions. This is inconsistent with the uptake of 4 K+ ion and\nreduction of 4 Fe3+(HS) ions per mol of PB during the treatment. Either the K+ ion\ncontent of the treated sample has been overestimated, or dissolution-recrystallization\nphenomena have occurred, modifying the Fe ions ratio in the two Fe sites of PB.\nWe thank the reviewer for raising questions regarding the valence change and the\nmechanism of K⁺ uptake. Concerning the possibility of dissolution–recrystallization\nphenomena, we have supplemented the particle size distribution of PB before and after\nMEI treatment. The results clearly show no change in particle size, indicating that\ndissolution–recrystallization is highly unlikely.\nTo further investigate the redox behavior of PB, we also conducted cyclic voltammetry\n(CV) measurements (Fig. 3c). The results clearly demonstrate that the structure of PB\nremains stable even under an applied electric field. Thus, we speculate that short-term\ntreatment with a reducing agent would not significantly affect its crystal structure.\nMoreover, if structural reconstruction had occurred, it would likely have led to the\nrelease of ferrocyanide ions. However, no abnormal iron concentrations were detected\neither in in vitro dissolution tests or in blood samples from animal dosing studies (Fig.\n4f and Fig. S1e).\nThe reviewer pointed out an inconsistency between the K⁺ content determined by XRF\nand the Mössbauer spectroscopy data. We would like to clarify that analysis of the\nMössbauer data for the as-purchased PB sample indicates an initial Fe³⁺:Fe²⁺ ratio of\napproximately 6:1. Although the ideal chemical formula of Prussian blue is often\nrepresented as Fe [Fe(CN) ] , the [Fe(CN) ] octahedron serves as the fundamental\n4 6 3 6\nbuilding block of the structure, with Fe3+ primarily acting as connectors to form the\nthree-dimensional network. In practice, the as-synthesized material is not a perfect\ncrystal and may contain excess Fe3+.\nThis is supported by the certificate of analysis (COA) of the commercially purchased\nPB reagent, which provides a molecular formula of C Fe N without specifying the\n18 7 18\niron valence states. Moreover, it is generally accepted that the face-centered cubic\nstructure used to represent PB is oversimplified and cannot fully explain the actual\nstructure of real PB samples, since synthesized crystals are never perfectly ideal.\nPrevious studies have also consistently observed that PB often contains an excess of\nFe3+ compared to ferrocyanide ions (Ionics. 2020, 26: 531–5547,). We believe that PB\ninherently possesses a complex crystal structure, and suspect that the presence of\ncertain defects in the material is an entirely normal phenomenon.\nFurther analysis of the Mössbauer data for the treated PB sample reveals a Fe3+ : Fe2+\nratio of about 1.6:5.4. This suggests that approximately 4.4 Fe3+ ions were reduced to\nFe2+, which is consistent with the XRF data indicating the uptake of about 4 mol of K⁺\nper mol of PB (Line 159-161: By comparing the spectral area ratios of Fe species in\nPB and PB+VC, it can be concluded that approximately 4.4 mol of Fe3+ in each mol of\nPB was reduced to Fe2+, resulting in a lower overall oxidation state of Fe in the PB\nstructure.).\nIn the previous manuscript, we employed the pressed pellet method for XRF analysis,\nwhich is a semi-quantitative analytical technique. This testing method primarily\ninvolves X-ray fluorescence penetrating into the material to a depth of several tens of\nmicrometers. Due to the relatively high surface adsorption of K+ and potential material\ninhomogeneity, this approach might overestimate the K+ content. To achieve more\naccurate and rigorous quantification of elemental content, we conducted two additional\nquantitative elemental analyses. The first involved digesting the PB in a reaction vessel\nfollowed by measurement using Inductively Coupled Plasma (ICP) analysis (Table S3).\nThe second method involves mixing the powder sample with a flux mixture of\nanhydrous lithium tetraborate and lithium metaborate, followed by fusion at high\ntemperature to form a homogeneous glass-like disk for XRF analysis via the fusion\nmethod (Table 1). The results show that the quantitative ICP data are highly consistent\nwith the fusion method XRF data and do not show significant differences from the\npressed pellet XRF data. As the results from both newly supplemented methods\nconfirm the solid elemental analysis, we have selected the ICP results to include in the\nSupporting Information (SI) to clarify the logic of the article. These results further\nconfirm that approximately 4 mol of K+ are absorbed and 4 Fe3+(HS) ions are reduced\nper mol of PB (Line 164-167).\nTable 1. Mass ratio of each element in PB an PB+VC obtained by Fusion method\nXRF and elemental analysis\nSamples Fe(wt%) K (wt%) Na (wt%)\nPB 35.0 0.11 1.22\nPB+VC 32.1 12.8 0.31\nThe data from Mössbauer spectroscopy, XRF, and ICP collectively confirm that 4\nmoles of Fe3+ are reduced per mole of PB. However, due to uncertainties in the initial\nvalence states, we have revised the chemical formula to Fe C N . The formulas\n7 18 18\nrepresenting the material before and after adsorption in Figure 2g have been updated\naccordingly.\nTable S3, Line 164-167: To determine the elemental content, quantitative analysis of\nthe solids prior to and following adsorption was carried out via inductively coupled\nplasma optical emission spectroscopy (ICP-OES) (Table S3). The elemental\ncomposition analysis of PB+VC, with variations ranging from 0.11 wt% to 12.1 wt% of\nK, revealed that the molar ratio of K to Fe in the treated sample was approximately\n1:1.8.\nThere is an inconsistency between the data provided on the Mössbauer spectrum in\nFigure 2e and the data provided in Table S1. The pink contribution in Figure 2e is most\nlikely due to HS-Fe2+ ions rather than the LS-Fe2+ contribution indicated on the\nspectrum.\nThese points need to be clarified.\nWe sincerely appreciate the reviewer's careful observation and apologize for the\nlabeling error in our figure. The data analysis in Table S1 is correct. Regarding Figure\n2e, the pink contribution corresponds to HS- Fe2+. We have corrected this figure in the\nrevised manuscript.\nIn their response to the reviewers, the authors state that they have added electron\nmicroscopy images of the PB powder before treatment to the manuscript (Figure 2i).\nThey use the microscopy images of the particles before and after PB+VC treatment to\nsupport the absence of dissolution-recrystallization phenomena during the treatment.\nNevertheless, the caption for Figure 2i states that the image shown corresponds to the\nparticles after treatment (PB+VC). Figure S3 also shows microscopy images after\ntreatment (after MEI treatment). Either the images of the PB before the treatment are\nmissing, or one of the figure captions is incorrect. Images of the PB powder before and\nafter treatment should be provided and clearly labelled.\nWe sincerely thank the reviewer for the meticulous attention to detail in identifying the\ninconsistencies in the figure captions related to the microscopy images. We\nacknowledge the confusion in the original submission regarding the labeling of PB\npowder images before versus after treatment. We deeply apologize for this error and\nany confusion it may have caused.\nWe have carefully addressed this issue in the revised manuscript. Figure 2i shows the\nimage corresponding to particles after treatment (post PB+VC treatment). In the\npreviously revised manuscript, Figure S3 displayed microscopy images of untreated\nPB powder (before treatment) - this was an error in our explanatory text labeling. To\nmore accurately utilize comparative microscopy images of particles before and after\ntreatment to support the absence of dissolution-recrystallization phenomena during the\ntreatment process, we have supplemented microscopy images of pure PB after\ntreatment (post-MEI treatment) for clearer demonstration.\nThe revised figures and captions now unambiguously provide the required\ncomparative microscopy evidence: this clear comparison of particle size distribution\nstrongly supports our conclusion regarding the absence of significant dissolution-\nrecrystallization phenomena during VC and MEI treatments, as the particle\nmorphology remains largely intact with unchanged dimensions.\nWe greatly appreciate the reviewer’s vigilance in ensuring the accuracy and clarity of\nour data presentation. These corrections ensure the microscopy observations are\npresented accurately and effectively, thereby strongly supporting our research\nconclusions.\nConcerning this point, there is an inversion with the citations of figures in the main text\n(line 170-173). Figure S4 does not correspond to the microscopy images, and Figure\nS3 does not correspond to the physisorption measurements. This must be revised.\nWe apologize for the erroneous citations of supplementary figures and thank the\nreviewer for detecting these issues. Corrections have now been implemented in the\nrevised manuscript.\nRespond to Reviewers\nREVIEWER COMMENTS\nReviewer #2 (Remarks to the Author):\nThank you for addressing my final comments. I have nothing further. This is an\ninteresting study.\nWe sincerely thank the reviewer for the constructive feedback and are grateful for their\nrecognition of our research and the revisions we have made.\nReviewer #3 (Remarks to the Author):\nThe manuscript has clearly been improved, and I can now recommend it for\npublication. However, given the complexity of the compounds and the experimental\ndata provided, the amount of reduced Fe3+ ions should be given with less accuracy\n(‘approximately 4.4’, line 160). I would suggest ‘A significant amount of reduced Fe3+\nions’ for instance, would be more appropriate.\nWe sincerely appreciate the reviewer’s constructive feedback and their recognition of\nthe improvements made to our manuscript. Regarding the comments on the\ncomplexity of the compound and the revised description of the experimental data, we\nfully agree with the suggestions and have modified the relevant statement to “ a\nsignificant amount of mol of Fe3+” (Line 137).\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nWe are deeply grateful to the reviewer for the time and effort in evaluating our\nmanuscript and for providing such valuable constructive feedback."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have reviewed the author's responses to the two reviewers' comments and their revisions. I believe that the issues regarding the description of experimental and calculation conditions have been at least partially corrected. Since In terms of condensed matter physics and energy storage, it is well known that PBA can electrochemically intercalate and deintercalated alkali metal ions. In fact, PBA has been actively studied as an active material for sodium-ion secondary batteries. For example, see Chem. Commun.2013, 49, 2750.I believe that providing this broader background would improve this paper.", "reviewer_2": "Thank you for addressing my comments and for providing the additional data. I have two remaining points: 1. “Additionally, PB+VC can partially mitigate the hyponatremia induced by the K+-sparing/Na+-excreting effect of amiloride, primarily by restoring K+ excretion and enhancing NHE3-mediated reabsorption of Na+ to respond the increased VC-derived H+ concentration, thereby counteracting these effects”. This explanation remains speculative and should be classified as such. More generally speaking, in their rebuttal, the authors frequently explained the changes in plasma sodium by discussing changes in kidney sodium handling - however, plasma sodium is determined by changes in water balance regulation, not sodium balance regulation (osmoregulation vs. volume regulation). This should be clarified. 2. The Legend to Figure S16 should be clarified: “After 1h and 5h, the administrations of NaCl in dose of 0.895 g kg−1 and 1 g kg−1 NH4Cl per day.” It is unclear if the 0.895 g/kg and 1 g/kg refer to the doses that were given after 1h and 5h or to the total daily dose - please clarify.", "reviewer_3": "Concerning the experimental study of the PB sample, the manuscript has clearly been improved by experimental details addition. However, two points remain to be clarified about the reduction/adsorption process before I can recommend it for publication. A question remains about the provided data and the mechanism for K+ uptake proposed by the authors. The authors propose a mechanism based on the reduction of 4 Fe3+(HS) ions per mol of PB (line 158 and Figure 2g) accompanied by the uptake of 4 mol of K+ ions corresponding to the chemical formula change of the powder from Fe3+(HS)4[Fe2+(LS)(CN)6]3 to K4Fe4[Fe(CN)6]3 (line 167-168). If dissolution-recrystallization phenomena do not occur during the treatment, the chemical formula of the powder after treatment is K4Fe2+(HS)4[Fe2+(LS)(CN)6]3. This means that all the Fe3+(HS) ions have been transformed into Fe2+(HS) ions. The presence of Fe2+(HS) ions in the powder after treatment is clearly demonstrated by Mössbauer spectroscopy. Nevertheless, Mössbauer spectroscopy also indicates the presence of a significant amount of Fe3+(HS) ions in the powder after treatment, as the areas under the bands corresponding to the Fe2+(HS) and Fe3+(HS) ions are comparable (Table S1). On the contrary, this data suggests that not all Fe3+(HS) ions were reduced to Fe2+(HS) ions. This is inconsistent with the uptake of 4 K+ ion and reduction of 4 Fe3+(HS) ions per mol of PB during the treatment. Either the K+ ion content of the treated sample has been overestimated, or dissolution-recrystallization phenomena have occurred, modifying the Fe ions ratio in the two Fe sites of PB. There is an inconsistency between the data provided on the Mössbauer spectrum in Figure 2e and the data provided in Table S1. The pink contribution in Figure 2e is most likely due to HS-Fe2+ ions rather than the LS-Fe2+ contribution indicated on the spectrum. These points need to be clarified. In their response to the reviewers, the authors state that they have added electron microscopy images of the PB powder before treatment to the manuscript (Figure 2i). They use the microscopy images of the particles before and after PB+VC treatment to support the absence of dissolution-recrystallization phenomena during the treatment. Nevertheless, the caption for Figure 2i states that the image shown corresponds to the particles after treatment (PB+VC). Figure S3 also shows microscopy images after treatment (after MEI treatment). Either the images of the PB before the treatment are missing, or one of the figure captions is incorrect. Images of the PB powder before and after treatment should be provided and clearly labelled. Concerning this point, there is an inversion with the citations of figures in the main text (line 170-173). Figure S4 does not correspond to the microscopy images, and Figure S3 does not correspond to the physisorption measurements. This must be revised.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Respond to Reviewers\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nI have reviewed the author's responses to the two reviewers' comments and their\nrevisions. I believe that the issues regarding the description of experimental and\ncalculation conditions have been at least partially corrected. Since\nIn terms of condensed matter physics and energy storage, it is well known that PBA\ncan electrochemically intercalate and deintercalated alkali metal ions. In fact, PBA has\nbeen actively studied as an active material for sodium-ion secondary batteries. For\nexample, see Chem. Commun.2013, 49, 2750.I believe that providing this broader\nbackground would improve this paper.\nWe sincerely thank the reviewer for the constructive feedback and for acknowledging\nthe revisions made regarding the experimental and calculation conditions. We fully\nagree with the reviewer’s suggestion to provide broader context on Prussian blue\nanalogues (PBAs) within condensed matter physics and energy storage.\nTo address this point, we have integrated the following addition into the Main section\n(Line 51-54): “This application leverages a fundamental property shared by PB and its\nanalogue: their well-established ability to electrochemically intercalate and\ndeintercalate various alkali metal ions, as extensively studied in systems like sodium-\nion batteries31.”\nReviewer #2 (Remarks to the Author):\nThank you for addressing my comments and for providing the additional data.\nI have two remaining points:\n1. “Additionally, PB+VC can partially mitigate the hyponatremia induced by the K+-\nsparing/Na+-excreting effect of amiloride, primarily by restoring K+ excretion and\nenhancing NHE3-mediated reabsorption of Na+ to respond the increased VC-derived\nH+ concentration, thereby counteracting these effects”. This explanation remains\nspeculative and should be classified as such. More generally speaking, in their rebuttal,\nthe authors frequently explained the changes in plasma sodium by discussing changes\nin kidney sodium handling - however, plasma sodium is determined by changes in\nwater balance regulation, not sodium balance regulation (osmoregulation vs. volume\nregulation). This should be clarified.\nWe sincerely thank the reviewer for raising these valuable and insightful points\nregarding our manuscript. In response to the first point, we acknowledge that the\nmechanism proposed in our previous revision —whereby PB+VC alleviates amiloride-\ninduced the slight reduction in blood sodium — was indeed a speculative interpretation\nbased on established physiological principles. To enhance the rigor of our work, we\nhave explicitly labeled this explanation as \"speculative\" in the revised Discussion\nsection (Line 292-295: “Additionally, amiloride caused only a mild, clinically\ninsignificant decrease in blood Na+ through K+-sparing/Na+-excreting. We hypothesize\nthat PB+VC attenuates this change by restoring K⁺ excretion and enhancing NHE -\n3\nmediated reabsorption of Na+ in response to increased VC-derived H⁺ concentration\n59.). We have revised the statement that amiloride causes hyponatremia, as the data\nshow only a slight decrease in serum sodium levels that remained within the normal\nphysiological range. \nRegarding plasma sodium regulation, we deeply appreciate the reviewer’s crucial\nclarification on the conceptual distinction between osmoregulation and volume\nregulation. Amiloride increases the risk of hyponatremia due to enhanced urinary\nsodium loss in the distal convoluted tubule (Postgraduate Medical Journal. 1993, 69\n(816): 797–799). As observed in Figure 5f of our study, amiloride administration caused\na minor decrease in serum sodium levels (though remaining within the normal\nphysiological range). Additionally, it should be noted that hyponatremia secondary to\ndistal diuretic intake primarily results from sodium depletion rather than water retention\n(Nephron. 2018, 140 (1): 31–38, J. Clin. Med. 2022, 11(19), 5810).\nConsistent with our hypothesis, PB+VC facilitates sodium reabsorption in renal tubules\nthrough physiologically-based mechanisms, thereby preventing sodium depletion and\nconsequently avoiding reductions in serum sodium at its source. It should also be\nclarified that, given the acute nature of our single-dose experimental model, sodium\nmeasurements were taken shortly after treatment. The role of water balance in sodium\nconcentration regulation may require longer observation periods to manifest.\nImportantly, the serum sodium fluctuations in Figure 5f were minor and consistently\nwithin normal physiological limits, with no safety concerns observed post-\nadministration.\nWe acknowledge that the serum sodium changes usually reflect alterations in water\nbalance rather than sodium metabolism itself. However, sodium metabolism remains\na critical factor influencing serum sodium levels. Within the context of this acute diuretic\nadministration model, our analysis focused on acute renal sodium handling as the\nprimary driver. This emphasis is justified because although amiloride increases sodium\nexcretion, causing a minor reduction in serum sodium, levels remained within the\nnormal physiological range. Such minor osmotic changes are unlikely to significantly\ntrigger compensatory water balance mechanisms in the acute phase. Consequently,\nrenal sodium excretion likely constitutes the dominant factor affecting plasma sodium\nconcentrations over this short timeframe.\nWe sincerely appreciate the reviewer's critical guidance on sodium homeostasis, which\nprovides essential direction for our future research. Our group will prioritize\ninvestigating water balance dynamics on plasma sodium concentration in subsequent\nstudies.\n2. The Legend to Figure S16 should be clarified: “After 1h and 5h, the administrations\nof NaCl in dose of 0.895 g kg−1 and 1 g kg−1 NH4Cl per day.” It is unclear if the 0.895\ng/kg and 1 g/kg refer to the doses that were given after 1h and 5h or to the total daily\ndose - please clarify.\nWe sincerely thank the reviewer for the valuable feedback regarding the legend of\nFigure S16. We apologize for the ambiguity in the original description and hereby\nclarify as follows:\nThe “0.895 g kg−1 \" refers to the per-administration dose of NaCl (each dose: 0.895 g\nkg−1), with a total daily dose of 1.79 g kg−1. The “1 g kg−1” indicates the total daily dose\nof NH Cl (administered in two doses of 0.5 g kg−1 each).\n“Rats were respectively administered with 1.79 g kg−1 NaCl (divided into two doses of\n0.895 g kg−1) and 1 g kg−1 NH Cl (divided into two doses of 0.5 g kg−1) per day, after 1\n4\nh and 5 h apart.”\nWe greatly appreciate the careful review of reviewer, which has been essential for\nimproving the clarity of our manuscript.\nReviewer #3 (Remarks to the Author):\nConcerning the experimental study of the PB sample, the manuscript has clearly been\nimproved by experimental details addition. However, two points remain to be clarified\nabout the reduction/adsorption process before I can recommend it for publication.\nA question remains about the provided data and the mechanism for K+ uptake\nproposed by the authors.\nThe authors propose a mechanism based on the reduction of 4 Fe3+(HS) ions per mol\nof PB (line 158 and Figure 2g) accompanied by the uptake of 4 mol of K+ ions\ncorresponding to the chemical formula change of the powder from\nFe3+(HS)4[Fe2+(LS)(CN)6]3 to K4Fe4[Fe(CN)6]3 (line 167-168). If dissolution-\nrecrystallization phenomena do not occur during the treatment, the chemical formula\nof the powder after treatment is K4Fe2+(HS)4[Fe2+(LS)(CN)6]3. This means that all\nthe Fe3+(HS) ions have been transformed into Fe2+(HS) ions.\nThe presence of Fe2+(HS) ions in the powder after treatment is clearly demonstrated\nby Mössbauer spectroscopy. Nevertheless, Mössbauer spectroscopy also indicates\nthe presence of a significant amount of Fe3+(HS) ions in the powder after treatment,\nas the areas under the bands corresponding to the Fe2+(HS) and Fe3+(HS) ions are\ncomparable (Table S1). On the contrary, this data suggests that not all Fe3+(HS) ions\nwere reduced to Fe2+(HS) ions. This is inconsistent with the uptake of 4 K+ ion and\nreduction of 4 Fe3+(HS) ions per mol of PB during the treatment. Either the K+ ion\ncontent of the treated sample has been overestimated, or dissolution-recrystallization\nphenomena have occurred, modifying the Fe ions ratio in the two Fe sites of PB.\nWe thank the reviewer for raising questions regarding the valence change and the\nmechanism of K⁺ uptake. Concerning the possibility of dissolution–recrystallization\nphenomena, we have supplemented the particle size distribution of PB before and after\nMEI treatment. The results clearly show no change in particle size, indicating that\ndissolution–recrystallization is highly unlikely.\nTo further investigate the redox behavior of PB, we also conducted cyclic voltammetry\n(CV) measurements (Fig. 3c). The results clearly demonstrate that the structure of PB\nremains stable even under an applied electric field. Thus, we speculate that short-term\ntreatment with a reducing agent would not significantly affect its crystal structure.\nMoreover, if structural reconstruction had occurred, it would likely have led to the\nrelease of ferrocyanide ions. However, no abnormal iron concentrations were detected\neither in in vitro dissolution tests or in blood samples from animal dosing studies (Fig.\n4f and Fig. S1e).\nThe reviewer pointed out an inconsistency between the K⁺ content determined by XRF\nand the Mössbauer spectroscopy data. We would like to clarify that analysis of the\nMössbauer data for the as-purchased PB sample indicates an initial Fe³⁺:Fe²⁺ ratio of\napproximately 6:1. Although the ideal chemical formula of Prussian blue is often\nrepresented as Fe [Fe(CN) ] , the [Fe(CN) ] octahedron serves as the fundamental\n4 6 3 6\nbuilding block of the structure, with Fe3+ primarily acting as connectors to form the\nthree-dimensional network. In practice, the as-synthesized material is not a perfect\ncrystal and may contain excess Fe3+.\nThis is supported by the certificate of analysis (COA) of the commercially purchased\nPB reagent, which provides a molecular formula of C Fe N without specifying the\n18 7 18\niron valence states. Moreover, it is generally accepted that the face-centered cubic\nstructure used to represent PB is oversimplified and cannot fully explain the actual\nstructure of real PB samples, since synthesized crystals are never perfectly ideal.\nPrevious studies have also consistently observed that PB often contains an excess of\nFe3+ compared to ferrocyanide ions (Ionics. 2020, 26: 531–5547,). We believe that PB\ninherently possesses a complex crystal structure, and suspect that the presence of\ncertain defects in the material is an entirely normal phenomenon.\nFurther analysis of the Mössbauer data for the treated PB sample reveals a Fe3+ : Fe2+\nratio of about 1.6:5.4. This suggests that approximately 4.4 Fe3+ ions were reduced to\nFe2+, which is consistent with the XRF data indicating the uptake of about 4 mol of K⁺\nper mol of PB (Line 159-161: By comparing the spectral area ratios of Fe species in\nPB and PB+VC, it can be concluded that approximately 4.4 mol of Fe3+ in each mol of\nPB was reduced to Fe2+, resulting in a lower overall oxidation state of Fe in the PB\nstructure.).\nIn the previous manuscript, we employed the pressed pellet method for XRF analysis,\nwhich is a semi-quantitative analytical technique. This testing method primarily\ninvolves X-ray fluorescence penetrating into the material to a depth of several tens of\nmicrometers. Due to the relatively high surface adsorption of K+ and potential material\ninhomogeneity, this approach might overestimate the K+ content. To achieve more\naccurate and rigorous quantification of elemental content, we conducted two additional\nquantitative elemental analyses. The first involved digesting the PB in a reaction vessel\nfollowed by measurement using Inductively Coupled Plasma (ICP) analysis (Table S3).\nThe second method involves mixing the powder sample with a flux mixture of\nanhydrous lithium tetraborate and lithium metaborate, followed by fusion at high\ntemperature to form a homogeneous glass-like disk for XRF analysis via the fusion\nmethod (Table 1). The results show that the quantitative ICP data are highly consistent\nwith the fusion method XRF data and do not show significant differences from the\npressed pellet XRF data. As the results from both newly supplemented methods\nconfirm the solid elemental analysis, we have selected the ICP results to include in the\nSupporting Information (SI) to clarify the logic of the article. These results further\nconfirm that approximately 4 mol of K+ are absorbed and 4 Fe3+(HS) ions are reduced\nper mol of PB (Line 164-167).\nTable 1. Mass ratio of each element in PB an PB+VC obtained by Fusion method\nXRF and elemental analysis\nSamples Fe(wt%) K (wt%) Na (wt%)\nPB 35.0 0.11 1.22\nPB+VC 32.1 12.8 0.31\nThe data from Mössbauer spectroscopy, XRF, and ICP collectively confirm that 4\nmoles of Fe3+ are reduced per mole of PB. However, due to uncertainties in the initial\nvalence states, we have revised the chemical formula to Fe C N . The formulas\n7 18 18\nrepresenting the material before and after adsorption in Figure 2g have been updated\naccordingly.\nTable S3, Line 164-167: To determine the elemental content, quantitative analysis of\nthe solids prior to and following adsorption was carried out via inductively coupled\nplasma optical emission spectroscopy (ICP-OES) (Table S3). The elemental\ncomposition analysis of PB+VC, with variations ranging from 0.11 wt% to 12.1 wt% of\nK, revealed that the molar ratio of K to Fe in the treated sample was approximately\n1:1.8.\nThere is an inconsistency between the data provided on the Mössbauer spectrum in\nFigure 2e and the data provided in Table S1. The pink contribution in Figure 2e is most\nlikely due to HS-Fe2+ ions rather than the LS-Fe2+ contribution indicated on the\nspectrum.\nThese points need to be clarified.\nWe sincerely appreciate the reviewer's careful observation and apologize for the\nlabeling error in our figure. The data analysis in Table S1 is correct. Regarding Figure\n2e, the pink contribution corresponds to HS- Fe2+. We have corrected this figure in the\nrevised manuscript.\nIn their response to the reviewers, the authors state that they have added electron\nmicroscopy images of the PB powder before treatment to the manuscript (Figure 2i).\nThey use the microscopy images of the particles before and after PB+VC treatment to\nsupport the absence of dissolution-recrystallization phenomena during the treatment.\nNevertheless, the caption for Figure 2i states that the image shown corresponds to the\nparticles after treatment (PB+VC). Figure S3 also shows microscopy images after\ntreatment (after MEI treatment). Either the images of the PB before the treatment are\nmissing, or one of the figure captions is incorrect. Images of the PB powder before and\nafter treatment should be provided and clearly labelled.\nWe sincerely thank the reviewer for the meticulous attention to detail in identifying the\ninconsistencies in the figure captions related to the microscopy images. We\nacknowledge the confusion in the original submission regarding the labeling of PB\npowder images before versus after treatment. We deeply apologize for this error and\nany confusion it may have caused.\nWe have carefully addressed this issue in the revised manuscript. Figure 2i shows the\nimage corresponding to particles after treatment (post PB+VC treatment). In the\npreviously revised manuscript, Figure S3 displayed microscopy images of untreated\nPB powder (before treatment) - this was an error in our explanatory text labeling. To\nmore accurately utilize comparative microscopy images of particles before and after\ntreatment to support the absence of dissolution-recrystallization phenomena during the\ntreatment process, we have supplemented microscopy images of pure PB after\ntreatment (post-MEI treatment) for clearer demonstration.\nThe revised figures and captions now unambiguously provide the required\ncomparative microscopy evidence: this clear comparison of particle size distribution\nstrongly supports our conclusion regarding the absence of significant dissolution-\nrecrystallization phenomena during VC and MEI treatments, as the particle\nmorphology remains largely intact with unchanged dimensions.\nWe greatly appreciate the reviewer’s vigilance in ensuring the accuracy and clarity of\nour data presentation. These corrections ensure the microscopy observations are\npresented accurately and effectively, thereby strongly supporting our research\nconclusions.\nConcerning this point, there is an inversion with the citations of figures in the main text\n(line 170-173). Figure S4 does not correspond to the microscopy images, and Figure\nS3 does not correspond to the physisorption measurements. This must be revised.\nWe apologize for the erroneous citations of supplementary figures and thank the\nreviewer for detecting these issues. Corrections have now been implemented in the\nrevised manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_2": "Thank you for addressing my final comments. I have nothing further. This is an interesting study.", "reviewer_3": "The manuscript has clearly been improved, and I can now recommend it for publication. However, given the complexity of the compounds and the experimental data provided, the amount of reduced Fe3+ ions should be given with less accuracy (‘approximately 4.4’, line 160). I would suggest ‘A significant amount of reduced Fe3+ ions’ for instance, would be more appropriate.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Respond to Reviewers\nREVIEWER COMMENTS\nReviewer #2 (Remarks to the Author):\nThank you for addressing my final comments. I have nothing further. This is an\ninteresting study.\nWe sincerely thank the reviewer for the constructive feedback and are grateful for their\nrecognition of our research and the revisions we have made.\nReviewer #3 (Remarks to the Author):\nThe manuscript has clearly been improved, and I can now recommend it for\npublication. However, given the complexity of the compounds and the experimental\ndata provided, the amount of reduced Fe3+ ions should be given with less accuracy\n(‘approximately 4.4’, line 160). I would suggest ‘A significant amount of reduced Fe3+\nions’ for instance, would be more appropriate.\nWe sincerely appreciate the reviewer’s constructive feedback and their recognition of\nthe improvements made to our manuscript. Regarding the comments on the\ncomplexity of the compound and the revised description of the experimental data, we\nfully agree with the suggestions and have modified the relevant statement to “ a\nsignificant amount of mol of Fe3+” (Line 137).\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nWe are deeply grateful to the reviewer for the time and effort in evaluating our\nmanuscript and for providing such valuable constructive feedback."}]

Decision letter