A urea-activated nanocarrier for site-specific SGLT2 inhibition and metabolic rescue against cardiovascular-kidney-metabolic syndrome

Domain: biology
Published: 2026-04-03
Editorial outcome: major_revision

Abstract
Cardiovascular-kidney-metabolic (CKM) syndrome, driven by interlinked metabolic, renal
and cardiovascular dysfunction, remains therapeutically challenging due to its multi-organ
complexity. Although sodium-glucose cotransporter 2 (SGLT2) inhibitors such as empagliflozin
(EMPA) confer cardiorenal benefits, their efficacy is limited by poor renal specificity and systemic
off-target exposure. Here, we report a kidney-targeted and urea-responsive nanocarrier (T-PAAD
NPs) that enables renal tubule-selective release of EMPA in response to pathological urea
concentrations. This delivery strategy, rarely explored in nanomedicine, synergistically enhances
therapeutic precision while integrating reactive oxygen species (ROS) scavenging to mitigate
oxidative stress. In male mouse models of CKM, PT-PAAD NPs/EMPA effectively reprogram
cardiac and renal energy metabolism, restore filtration and contractile function, and achieve
superior glycemic, renal, and cardiovascular outcomes compared to free EMPA. By coupling
bioresponsive controlled release with metabolic modulation, this nanoplatform provides a
promising approach to treat CKM and other systemic metabolic disorders.

Introduction
Type 2 diabetes mellitus (T2DM), chronic kidney disease (CKD), cardiovascular disease
(CVD) have been defined as the most disruptive public health issues1, 2. Additionally, there exists
a strong interplay among T2DM, CKD and CVD, and they have the propensity to initiate and
perpetuate each other, leading to the increased morbidity and mortality3-5. In 2023, the American
Heart Association (AHA) coined a new term, cardiovascular-kidney-metabolic (CKM) syndrome,
to describe a systemic disease arising from the interconnectedness of obesity, diabetes, CVD and
CKD6. CKM health reflects the intricate interrelationships among metabolic risk factors, kidney
function, and the cardiovascular system. Therefore, it is essential to develop a comprehensive
strategy to manage the multidimensional risk factors from cardio‑renal‑metabolic connection for
CKM therapy7, 8.

Currently, in addition to healthy lifestyle, the out Global Burden of the Key Components of
Cardiovascular-Kidney-Metabolic Syndrome comes of several clinical trials have demonstrated
that sodium-glucose cotransporter-2 inhibitors (SGLT-2i) not only lower blood glucose level but
also have renal- and cardio-protective effects9-11. SGLT-2i acts on the SGLT-2 receptors expressed
in the renal proximal convoluted tubules to inhibit reabsorption of glucose for T2DM therapy12.
Furthermore, SGLT2i can reduce cardiac load by promoting the osmotic excretion of sodium and
urine through the kidneys, which in turn helps maintain cardiac ejection fraction and improve
cardiac energy metabolism13-16. Meanwhile, SGLT2i can also contribute to kidney protection by
improving renal hemodynamics, decreasing the proteinuria excretion, and promoting the renal
tubular energy metabolism17-19. These multifaceted actions underscore the therapeutic potential of
SGLT-2i in managing CKM syndrome. However, despite their promise, SGLT-2i also face several
limitations that may hinder their optimal clinical efficacy20. On one hand, poor aqueous solubility
of SGLT-2i, like empagliflozin (EMPA) makes them suffer from poor bioavailability21.
Additionally, the heterogeneity, complexity, and unique physiological structure of the kidney
present significant challenges for therapeutic interventions, especially with respect to safety,
specificity, and efficacy of small-molecule drugs22, 23. Both of these factors necessitate the high
doses of SGLT-2i for effective CKM management, causing the risk of adverse effects such as bone
fracture24, diabetic ketoacidosis25, 26, serious urinary tract infections27, etc.. On the other hand,
oxidative stress plays a pivotal role in the pathogenesis of CKM. Although SGLT-2i can indirectly
reduce oxidative stress through various mechanisms, they are unable to directly scavenge pre-
existing ROS in the damaged kidney28-30. Notably, several studies have demonstrated that the
combination of antioxidants with SGLT-2i can further enhance both renal- and cardio-protective
effects31, 32. Therefore, the strategies that improve the in vivo delivery efficiency of SGLT-2i and
simultaneously incorporate antioxidant therapies hold promise for enhancing their overall
therapeutic efficacy in CKM management.

Nano-drug delivery system is the results of merging nanotechnology with new drug
development, possessing the improved pharmacokinetics, the targeted delivery and the controlled
release of drugs to the specific tissues and cells33, 34. In addition, nanocarriers can be chemically
modified to possess multifunctional properties, such as ROS-scavenging capability, which can
suppress oxidative stress and provide additional therapeutic benefits for CKM management.
Despite these advantages, the nanomedicines for treating kidney diseases remain significantly
underdeveloped compared to nanomedicines in other fields, such as cancer nanomedicines, thereby
hindering the development of SGLT-2i-based nanomedicine35. Nevertheless, the kidney possesses
several unique features that can be exploited for the rational design of renal-targeted
nanomedicines36. Notably, the glomerular filtration barrier becomes leaky resulting from
impairment of the glomerular filtration barrier under diseased conditions, potentially facilitating
the nanomedicine across the physiological barriers to proximal tubule37. Additionally, the water
reabsorption via aquaporins further concentrates urea in the lumen, resulting in the urea
concentration in the renal tubule that are approximately 50% higher than in plasma38-40. Essentially,
urea levels within the S1 segment of the proximal tubule vary markedly across stages of kidney
injury, increasing from ~5 mM in healthy kidneys to ~10 mM in early injury and up to ~50 mM in
end-stage disease41, 42. This suggests that urea may serve as an endogenous stimulus to achieve the
efficient controlled drug release from nanomedicines within the kidney; however, this approach
has been rarely explored to date. Therefore, developing a urea-responsive nanocarrier with ROS-
scavenging ability to enable targeted delivery and controlled release of SGLT-2i in the proximal
tubule represents a promising strategy to improve therapeutic efficacy against CKM.

In our previous studies, a serial of smart polymers exhibiting an upper critical solution
temperature (UCST) have been designed and developed, with their phase inversion behavior
primarily driven by polymer-polymer hydrogen-bonds43, 44. Urea is known to disrupt
intramolecular hydrogen-bonded network; therefore, nanomedicine formulated from UCST
polymers are expected to disassemble in the presence of urea by breaking their intramolecular
hydrogen bonds, enabling rapid drugs release from UCST nanomedicines. Herein, we engineered
kidney-targeted and urea-responsive nanocarriers with intrinsic ROS-scavenging capacity by
employing multifunctional UCST polymer to achieve controlled release of the SGLT-2i
(empagliflozin, EMPA) specifically in the renal proximal tubules. This design synergistically
integrates oxidative stress mitigation with cardio-renal energy metabolism reprogramming,
offering enhanced therapeutic efficacy against CKM syndrome. Briefly, diallyl trisulfide (DATS),
a sulfur-rich compound derived from garlic, was introduced into polyethylene glycol-b-
poly(acrylonitrile-co-acrylamide) to synthesize the multifunctional UCST polymer
(poly(acrylamide-co-acrylonitrile-co-diallyl trisulfide),PAAD). This polymer self-assembles into
nanoparticles at physiological temperature via intramolecular hydrogen bonding, while the
trisulfide groups confer antioxidant and anti-inflammatory activities by scavenging existing ROS
in the damaged kidney. Meanwhile, the renal tubule targeting peptide, (Lys-Lys-Glu-Glu-Glu) -
3
Lys ((KKEEE) K), was conjugated to the surface of UCST nanoparticles to enhance proximal
3
tubule epithelial cell targeting. Upon administration, these EMPA-loaded nanomedicines
effectively accumulated in the kidney and targeted proximal tubules. Elevated urea levels in the
renal tubule disrupted the intramolecular hydrogen bonds of the UCST nanomedicine, triggering
nanoparticle disassembly and controlled EMPA release. Combined with the antioxidant properties
of the UCST polymer, EMPA effectively lowered blood glucose and blood pressure, while
improving cardiac and renal hemodynamics through reprogramming cardio-renal energy
metabolism. This novel and intelligent drug delivery system triggered by endogenous urea levels,
a mechanism rarely explored in nanomedicine, demonstrates promising potential for treating CKM.
This approach represents a significant advance toward nanomedicine-based co-management of
diabetes, cardiovascular disease, and kidney dysfunction, offering a compelling strategy to
improve clinical outcomes in patients with CKM.

Results
2.1. Synthesis and characterization of urea-responsive UCST copolymer with renal tubule
targeting capability
The multifunctional UCST copolymer, poly(acrylamide-co-acrylonitrile-co-diallyl trisulfide)
(PAAD), was synthesized via free radical polymerization. The (KKEEE) K peptide, a promising
3
renal tubule-targeting ligand, was subsequently conjugated to the PAAD copolymer through a
polyethylene glycol (PEG) linker (Supplementary Fig. 1). The chemical structures of the PAAD
and PEG-PAAD copolymers were confirmed by 1H-NMR spectroscopy (Supplementary Fig. 2a
and b). The molecular weight of these two copolymers were 21,908 Da and 24,804 Da, respectively
(Supplementary Fig. 2c). Furthermore, the bicinchoninic acid (BCA) assay confirmed successful
conjugation of the (KKEEE) K peptide to the PEG-PAADS copolymer, with a conjugation
3
efficiency of approximately 64.5% (Supplementary Fig. 2d).

As mentioned above, urea can disrupt the intramolecular hydrogen bonds among UCST
copolymers that govern their phase transition behavior. Based on this property, we hypothesized
that the UCST copolymer may exhibit urea -responsiveness. To demonstrate this hypothesis, the
phase transition temperatures of the PEG-PAAD copolymer were measured under varying urea
concentrations. As the urea concentration increased from 0 to 500 mM, the UCST of the copolymer
decreased markedly from 41.3 °C to 30.6 °C. Corresponding optical images revealed
concentration-dependeAnt changes in turbidity when the copolymer was incubated with urea at
different temperatures (Fig. 2a and Supplementary Fig. 3a). At 37 °C, the initially opaque PEG-
PAAD emulsion became transparent under high urea concentrations, and the Tyndall effect was
visibly diminished, indicating that the polymer aggregates disassembled in response to urea
(Supplementary Fig. 3b). Meanwhile, the Fourier Transform Infrared Spectroscopy (FTIR)
analysis of the PEG-PAAD copolymer was conducted in the presence of urea. As shown in Fig. 2b
and c, the characteristic FTIR peaks of PEG-PAAD copolymer were observed at 3350 cm-1 (N-H
stretching vibration), 1616 cm-1 (N-H bending vibration), and 1660 cm-1 (C=O stretching
vibration). However, due to the disruptive effect of urea on intramolecular hydrogen bonding, these
peaks shifted to higher wavenumbers, appearing at 3446 cm-1, 1624 cm-1, and 1667 cm-1,
respectively. To further validate this observation, molecular simulation analysis revealed that the
zipper-like intramolecular hydrogen bonds formed within PEG-PAAD copolymers were disrupted
in the presence of urea. Both the number and energy of hydrogen bonds were significantly reduced
(Fig. 2d and Supplementary Fig. 4), further confirming the urea-responsiveness of the PEG-PAAD
copolymer. To evaluate whether the urea-responsiveness of UCST copolymer could facilitate urea-
triggered drug release, the kidney-targeted UCST NPs (T-PAAD NPs) were fabricated using a
molar ratio of (KKEEE) K-PEG-PAAD to PEG-PAAD of 1:10. The resultant T-PAAD NPs were
3
employed to encapsulate EMPA, forming the spherical nanoparticles with an average diameter of
50.75 nm and a surface potential of -0.98 mV. Furthermore, T-PAAD NPs maintained excellent
colloidal stability in serum-containing medium at 37 °C for 72 hours without significant
aggregation. These NPs exhibited favorable drug loading capacity (233.3 g mg-1 NPs) and
encapsulation efficiency (47.6%) (Supplementary Fig. 5).

Importantly, the in vitro release profiles show that T-PAAD NPs enable stimulus-responsive
drug release across urea concentrations relevant to kidney injury. Under conditions mimicking
normal plasma or healthy proximal tubules (5 mM urea), T-PAAD NPs/EMPA showed a sustained
release pattern, reaching a cumulative release of 62.3% over 24 h. In contrast, as proximal tubular
injury progresses and urea levels rise, EMPA release was correspondingly accelerated. Notably,
exposure to a simulateAd severe-injury microenvironment (50 mM urea) triggered a rapid increase
in EMPA release, reaching ~93% within 24 h (Fig. 2e). This pronounced difference in release
kinetics supports the sensitivity of the T-PAAD NPs to the pathological microenvironment, driven
by elevated urea concentrations. Mechanistically, UCST-based nanomedicines are expected to
partially disassemble in the presence of urea, which disrupts intra- and interchain hydrogen
bonding; this urea-induced weakening lowers the UCST of T-PAAD and thereby facilitates faster
drug release. Taken together, because the urea concentration of the proximal tubule is markedly
elevated in injured kidneys, these UCST nanoparticles offer a promising platform for on-demand
drug release within the renal tubules.

2.2. Antioxidative capacity of T-PAAD NPs
In addition to their urea-responsiveness, T-PAAD NPs possess strong ROS-scavenging
activity due to the incorporation of the antioxidant DATS in PAAD copolymer. This feature is
expected to synergistically alleviate renal oxidative stress when combined with SGLT-2i therapy.
To assess the antioxidant capacity of T-PAAD NPs, three representative ROS species, namely
hydrogen peroxide (H O ), superoxide anion (O •−), and hydroxyl radical (·OH), were incubated
2 2 2
with T-PAAD NPs. As shown in Fig. 2f-h, the ROS clearance rate increased with nanoparticle
concentration, and effective scavenging activity was observed even at a low concentration of 0.125
mg mL-1. Additionally, at a concentration of 1 mg mL-1, T-PAAD NPs were able to decompose
approximately 90% of H O , 70% of the O •−, and 60% of the ·OH. Furthermore, the total
2 2 2
antioxidant capacity of T-PAAD NPs was evaluated using 2, 2’-azinobis (3-ethylbenzothiazoline
6-sulfonate) (ABTS) radical scavenging assay. There were more than 60% of ABTS •+ radicals
being scavenged in the group of T-PAAD NPs (1 mg mL-1), further confirming their potent
antioxidant activity (Fig. 2i). Given the favorable antioxidative properties of T-PAAD NPs, we
further evaluated their intracellular ROS-scavenging capacity. As shown in Fig. 2j, treatment of
proximal tubule epithelial cells (HK2 cells) with 150 M H O led to a marked increase in
2 2
intracellular ROS levels, indicated by a strong green fluorescent signal. In contrast, HK2 cells
treated with T-PAAD NPs exhibited a significant reduction in ROS levels. Quantitative analysis
by flow cytometry confirmed that even at a low concentration (12.5 g mL-1), T-PAAD NPs could
scavenge approximately 40.2% of intracellular H O (Fig. 2k and Supplementary Fig. 6a).
2 2
Consistently, T-PAAD NPs at this concentration improved HK2 cell viability from 78.9% to 93.5%
under oxidative stress induced by 150 μM H O , further confirming their effective intracellular
2 2
ROS-scavenging ability (Supplementary Fig. 6b). To further simulate the pathological
environment of CKD, HK2 cells were exposed to high levels of glucose (33 mmol L-1 D-glucose)
and palmitate (0.3 mmol L-1) to induce hyperglycemic and lipotoxic stress. This co-stimulation
significantly elevated the levels of malondialdehyde (MDA), a marker of lipid peroxidation, to
0.84 μmol g-1 protein, which were 1.67 and 1.80 folds higher than those observed in PBS and
mannitol control groups, respectively (Supplementary Fig. 6c, d). Notably, treatment with T-
PAAD NPs effectively normalized MDA levels, highlighting their potent antioxidative ability to
counteract cellular damage under metabolic stress. Together, these findings suggest that T-PAAD
NPs serve not only as a targeted nanocarrier for EMPA delivery but also as an effective antioxidant
agent. Their dual functionality offers strong potential to synergize with EMPA and enhance its
renal- and cardiovascular protective effects in CKM therapy.

2.3 Renal tubule targeting capability of T-PAAD NPs
In addition to controlled EMPA release and ROS-scavenging ability, the renal tubule-targeting
ability of T-PAAD NPs/EMPA is a critical determinant of their therapeutic efficacy. The
(KKEEE) K peptide targets megalin, a multiligand receptor highly expressed on renal tubular
3
epithelial cells45, 46, thereby facilitating the endocytosis of T-PAAD NPs. This targeting ability was
validated using coumarin 6 (C6)-loaded T-PAAD NPs, where flow cytometric analysis showed
significantly enhanced uptake by HK2 cells (Supplementary Fig.7). However, this uptake was
remarkedly suppressed by pretreatment with free (KKEEE) K peptide, confirming the receptor-
3
mediated and specific renal tubular cell-targeting ability of T-PAAD NPs (Fig. 3a-b).
To further assess renal targeting in vivo, we evaluate the biodistribution of T-PAAD NPs in a
CKM mouse model, established by feeding STZ-treated ApoE-/- mice with Western diet (WD) for
12 weeks (Fig. 3c). Following intraperitoneal (i.p) injection, IR780-loaded T-PAAD NPs exhibited
strong accumulation in the kidneys, with renal fluorescence intensities 1.60- and 1.80-fold higher
than those observed with free IR780 and PEG-PAAD NPs/IR780, respectively (Fig. 3d). In
contrast, this targeting effect was significantly attenuated in healthy mice fed a regular rodent chow
(RD) diet, further highlighting the disease-dependent targeting capability of T-PAAD NPs.
Moreover, time-dependent biodistribution analysis revealed rapid renal uptake and prolonged
kidney retention of T-PAAD NPs, with significantly higher fluorescence than non-targeted PAAD
NPs at 6, 12, and 24 h post-injection (Supplementary Fig. 8). To spatially evaluate the intrarenal
distribution of T-PAAD NPs, the T-PAAD NPs/C6 were i.p injected into CKM mice. At 6 h post-
injection, kidney tissues were collected, sectioned, and immune-stained for aquaporin-1 (AQP-1),
a biomarker specific to renal tubules. Confocal imaging revealed clear colocalization of the green
fluorescence from T-PAAD NPs/C6 with the red fluorescence from AQP-1, indicating that the NPs
successfully accumulated within renal tubular epithelial cells (Fig. 3e).
This enhanced-renal tubule accumulation can be attributed to two major factors. Firstly, the
CKM-induced kidney injury compromises the glomerular filtration barrier, facilitating the
enhanced translocation of nanoparticles into renal tubules. Meanwhile, due to the steep pressure
gradient along the nephron (50-10 mmHg) and the strong absorptive force of the peritubular
capillaries, NPs have increased chances to interact with capillary endothelial cell membranes and
can be effectively transported to renal tubule epithelial cells via the basolateral membrane23, 47.
Secondly, the (KKEEE) K peptide serves as a renal tubule-targeting ligand that is specifically
3
recognized and internalized by tubular epithelial cells, resulting in the high renal specificity and
rapid accumulation of T-PAAD NPs in the kidney. Collectively, this enhanced renal residency is
mediated by a composite mechanism of brush-border retention and cellular internalization, which
overcomes the rapid tubular transit flow and allows the pathological urea microenvironment to
trigger sustained and site-specific drug release.

2.4. Mitigating hyperglycemia and renal dysfunction by T-PAAD NPs/EMPA
CKM syndrome is a complex disorder comprising T2DM, kidney disease, cardiovascular
disease, and obesity. These interconnected conditions share common risk factors and often
exacerbate one another. To comprehensively investigate the therapeutic efficacy of T-PAAD
NPs/EMPA against CKM syndrome in vivo, we established a CKM mouse model by feeding ApoE-
/- mice with WD for 16 weeks in combination with STZ injection. This model successfully
mimicked the metabolic and pathophysiological features of CKM, including T2DM, renal
dysfunction, cardiovascular disease, and obesity. Mice were randomly divided into three treatment
groups: PBS (control), free EMPA, and T-PAAD NPs/EMPA (Fig. 4a). As a SGLT-2i, EMPA
reduces renal glucose reabsorption, thereby lowering blood glucose levels. Following treatment,
the blood glucose levels in CKM mice receiving T-PAAD NPs/EMPA dropped to 13.65 mmol L-1,
approaching levels observed in healthy controls, whereas free EMPA reduced blood glucose levels
only to 20.60 mmol L-1 (Fig. 4b). To further assess glucose homeostasis, intraperitoneal glucose
tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were conducted after three
weeks of treatment. The IPGTT results showed that the glycemic peak in CKM mice treated with
T-PAAD NPs/EMPA was 19.75 mmol L-1, which was 1.69- and 1.30- fold lower than that in the
PBS and free EMPA groups, respectively. Furthermore, the area under the curves (AUCs) for both
IPGTT and IPITT was significantly lower in the T-PAAD NPs/EMPA group, indicating improved
glucose tolerance and insulin sensitivity (Fig. 4c, d and Supplementary Fig. 9a, b). However, the
administration of blank T-PAAD NPs did not lead to any significant reduction in blood glucose
levels or improvement in glucose tolerance (IPGTT) compared to the untreated CKM group
(Supplementary Fig. 10). In addition, hemoglobin A1c (HbA1c), a key indicator of long-term
glycemic control and prognostic marker for diabetes, decreased by 0.95% in the T-PAAD
NPs/EMPA group, compared to only a 0.46% reduction in the free EMPA group (Supplementary
Fig. 9c). Beyond enhancing urinary glucose excretion, T-PAAD NPs/EMPA also promoted
natriuresis and osmotic diuresis without causing significant urinary tract infections
(Supplementary Fig. 11).
Collectively, these findings confirm that targeted and controlled EMPA release from T-PAAD
NPs to renal tubule cells plays a crucial role in glycemic regulation. This strategy not only achieves
superior glycemic manage but also improves glucose tolerance and insulin sensitivity in CKM
mice. These combined metabolic benefits contribute to reduced blood pressure, lower blood
volume, decreased cardiac pre- and afterload, and enhanced renal hemodynamics, ultimately
providing comprehensive cardio-renal protection.

Diabetic nephropathy (DN), a prevalent microvascular complication of diabetes, plays a
pivotal role in the progression of CKM syndrome. Diabetes-induced kidney injury disrupts the
cardiorenal axis, initiating bidirectional pathological crosstalk between the heart and kidneys48.
This mutual dysfunction further exacerbates systemic metabolic disturbances, ultimately
contributing to the hallmark triad of CKM syndrome. Given this central role of DN, we evaluated
the therapeutic efficacy of T-PAAD NPs/EMPA in mitigating diabetes-associated kidney damage.
To assess renal function, glomerular filtration rate (GFR) was measured by transcutaneous
monitoring of FITC-sinistrin clearance (Fig. 4e). The obvious reduction (24.6%) of GFR in
untreated CKM mice indicated that there existed the severe kidney dysfunction in CKM mice. In
contrast, EMPA treatments, especially T-PAAD NPs/EMPA, markedly preserved renal function.
Notably, GFR in the T-PAAD NPs/EMPA group decreased by only 0.18%, suggesting significant
renal protection (Fig. 4f and Supplementary Fig. 12). In addition to GFR, the urine albumin-
creatinine ratio (UACR) was measured as a key indicator of kidney damage. As shown in
Supplementary Fig. 13a, T-PAAD NPs/EMPA treatment effectively prevented albuminuria,
reducing the UACR to a level 2.16-fold lower than that in untreated CKM mice. Furthermore,
serum biomarkers of renal function, including creatinine (CRE), blood urea nitrogen (BUN), and
uric acid (UA), were significantly improved in the T-PAAD NPs/EMPA group. Specifically, CRE,
BUN, and UA levels were reduced to 11.9 mM, 39.2 μM, and 130.0 μM, respectively, closely
approximating values observed in healthy controls (Supplementary Fig. 13b-d). In contrast, free
EMPA treatment failed to achieve comparable improvements.
To further confirm the enhanced therapeutic effect of T-PAAD NPs/EMPA against DN,
histopathological evaluations of kidneys were conducted following various treatments (Fig. 4g).
Periodic acid-Schiff (PAS) staining revealed classic pathological features of DN in CKM mice,
including glomerular enlargement, basement membrane thickening, and mesangial cell
proliferation. Hematoxylin and eosin (H&E) staining further demonstrated serious tubular injury
characterized by vacuolization, tubular dilation and atrophy, brush border loss, and thickened
tubular basement membrane. However, these pathological alterations were significantly alleviated
in both free EMPA and T-PAAD NPs/EMPA treatment groups. In particular, there appeared no
distinguishable kidney pathological changes between the healthy mice and the T-PAAD
NPs/EMPA-treated CKM mice. Moreover, Masson staining corroborated these findings, showing
substantially reduced collagen deposition in the kidney after the T-PAAD NPs/EMPA treatment,
indicative of effective amelioration of renal fibrosis. Quantitative analysis confirms a significant
reduction in the fibrotic area, validating the enhanced anti-fibrotic efficacy of T-PAAD NPs/EMPA
(Supplementary Fig. 14a). Furthermore, terminal deoxynucleotidyl transferase dUTP nick-end
labeling (TUNEL) assays and immunostaining of kidney injury molecule-1 (KIM-1) were used to
evaluate renal tubular apoptosis and injury. Both parameters were markedly elevated in untreated
CKM mice but significantly reduced following EMPA-based therapies. Notably, T-PAAD
NPs/EMPA treatment resulted in the greatest reduction in TUNEL-positive cells and KIM-1
expression (Fig. 4h and Supplementary Fig. 14b, c), highlighting its superior protective effect
against renal tubular apoptosis and injury.
Taken together, these findings demonstrate that T-PAAD NPs/EMPA offer superior protection
against kidney dysfunction in CKM mice compared to free EMPA. This enhanced therapeutic
efficacy may be attributed to several key mechanisms. First and foremost, hyperglycemia is a
major risk factor for kidney damage, and tight glycemic control is essential for renal protection. T-
PAAD NPs/EMPA leverage a urea-triggered disassembly mechanism within the renal tubules to
achieve spatiotemporally controlled release of EMPA, thereby enhancing drug accumulation at the
site of SGLT-2 receptor expression in renal tubular epithelial cells. This precision targeted delivery
strategy amplifies the blockade of SGLT-2, leading to more pronounced natriuretic and glycosuric
effects and sustained urinary glucose excretion. Meanwhile, the improved glycemic control
achieved by T-PAAD NPs/EMPA is expected to positively influence renal hemodynamics, which
refers to the regulation of blood flow and pressure within the kidneys. As shown in Fig. 4i and
Supplementary Fig. 15, color Doppler ultrasound was used to assess renal hemodynamic
parameters, including the peak systolic velocity (PSV) and end-diastolic velocity (EDV) of the
right renal artery in mice. The resistance index (RI) and pulsatility index (PI) were calculated to
evaluate renal blood perfusion and the degree of kidney function impairment. Analysis of these
parameters revealed significant abnormalities in PSV and EDV in CKM mice, along with a marked
increase in PI and RI compared to the healthy control group, indicating elevated renal arterial blood
flow resistance and reduced renal blood perfusion. However, following treatment with T-PAAD
NPs/EMPA, renal hemodynamic parameters were largely restored to normal levels, suggesting that
T-PAAD NPs/EMPA effectively preserved glomerular filtration function, modulated renal blood
flow distribution, and alleviating renal dysfunction. Additionally, T-PAAD NPs could also
scavenge the existed ROS in kidney. Thus, the synergistic effect of EMPA and T-PAAD NPs further
normalized key oxidative stress markers (H₂O₂ and MDA), while enhancing renal antioxidant
enzyme activity (catalase (CAT) and superoxide dismutase (SOD)), and improving total
antioxidant capacity (T-AOC). These changes were accompanied by reduced renal inflammation
and fibrosis, thereby offering enhanced protection against kidney damage (Supplementary Fig. 16).

2.5. Improving cardiovascular function by T-PAAD NPs/EMPA
The cardiovascular system and kidneys are vital for maintaining systemic homeostasis and
are intricately linked through the bidirectional “cardio-renal axis”, which plays a central role in the
regulation of blood pressure, hemodynamics, and metabolic balance49, 50. The functional interplay
between these two organs is essential for sustaining energy homeostasis and overall physiological
equilibrium51. Based on this pathophysiological framework, we systematically evaluated the
therapeutic potential of T-PAAD NPs/EMPA in improving cardiovascular function in CKM mice.
As shown in the Fig. 5a-c and Supplementary Fig. 17, CKM mice exhibited pronounced
hemodynamic dysregulation compared with healthy controls, developing sustained hypertension
characterized by elevated systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean
arterial pressure (MAP), reaching 142.29 mmHg, 95.26 mmHg, and 111.31 mmHg, respectively.
Notably, after four weeks of treatment with T-PAAD NPs/EMPA, these values were significantly
reduced to 117.85 mmHg (SBP), 79.30 mmHg (DBP), and 92.47 mmHg (MAP). This
antihypertensive effect was superior to that of free EMPA, which reduced SBP, DBP, and MAP to
133.49, 84.69, and 99.84 mmHg, respectively. Similarly, multimodal cardiac imaging (M-mode
echocardiography and pulsed Doppler flowmetry) quantitatively demonstrated the profound
cardio-reparative effects of T-PAAD NPs/EMPA in CKM mice (Fig. 5d). The treatment of T-PAAD
NPs/EMPA significantly restored systolic performance, elevating the ejection fraction (EF) from
41.5% to 53.4% (Fig. 5e) and improving fractional shortening (FS) by 8.1% (Fig. 5f) in
comparison to the untreated CKM mice. In addition, the mitral ratio of peak early to late diastolic
filling velocity serves as a critical hemodynamic biomarker for evaluating left ventricular
compliance. Physiologically, the pressure gradient during early passive filling (E-wave)
predominates over atrial contraction-driven flow (A-wave) in healthy myocardium, maintaining
an E/A ratio >1.0. Pathological diastolic dysfunction fundamentally alters this dynamic, exhibiting
characteristic E/A inversion (<1.0) due to impaired ventricular relaxation and elevated left atrial
filling pressures. Thus, in CKM mice, echocardiographic quantification revealed severe diastolic
impairment with E/A ratio depression to 0.91 (Fig. 5g). However, T-PAAD NPs/EMPA restored
pseudo-normalized filling patterns, elevating E/A ratios to 1.49, demonstrating the capacity of T-
PAAD NPs/EMPA to concurrently reverse the systolic and diastolic dysfunction of CKM mice.
Collectively, these data demonstrate that T-PAAD NPs/EMPA effectively ameliorate
cardiovascular abnormalities, attenuating pathological cardiac remodeling while helping to re-
establish systemic circulatory homeostasis.
These benefits likely arise from two complementary mechanisms. Firstly, hemodynamic
unloading: improved renal filtration and enhanced natriuresis lower systemic blood pressure and
cardiac afterload, thereby reducing the hemodynamic stress that contributes to CKM-associated
cardiac dysfunction. Secondly, systemic metabolic correction: T-PAAD NPs/EMPA markedly
decreases circulating glucose and lipid levels, alleviating myocardial glucolipotoxicity and
improving cardiac substrate utilization. In summary, by unloading the heart via improved renal
function and blood pressure and correcting systemic metabolism via sustained glycosuria and lipid
reduction, T-PAAD NPs/EMPA would improve the cardiovascular function.

Given the established role of hypertension in accelerating atherosclerotic plaque development
through enhanced lipid deposition and endothelial injury, we further assessed the therapeutic
efficacy of T-PAAD NPs/EMPA against atherosclerosis, which is a primary pathological basis of
CVD and is closely related to the metabolic disorders underlying CKM syndrome. En face
quantitative analysis of Oil Red O (ORO)-stained aortas demonstrated that T-PAAD NPs/EMPA
significantly reduced atherosclerotic burden, decreasing the plaque area ratio from 38.29% in
untreated CKM mice to 20.50% (Fig. 5h). Histopathological evaluation of aortic root sections
supported these findings. H&E staining revealed a notable reduction in lesion area following T-
PAAD NPs/EMPA treatment (Fig. 5i), while ORO staining of aortic roots showed a 1.39-fold
greater reduction in plaque area compared to free EMPA, indicating the enhanced anti-
atherosclerotic effect of the nanomedicine (Fig. 5j). Additionally, Masson’s trichrome staining
indicated that collagen content within plaques increased to 49.21% in the T-PAAD NPs/EMPA
group, which was 1.35 and 1.14 folds higher than those in the CKM and free EMPA groups,
respectively (Fig. 5k). Since collagen deposition critically determined the integrity and stability of
plaques, these data collectively suggest that T-PAAD NPs/EMPA not only attenuated plaque
burden but also improved plaque stability, potentially reducing the risk of rupture and providing
enhanced therapeutic benefits.

2.6. Alleviating hyperlipidemia of hepatic steatosis by T-PAAD NPs/EMPA
Hyperlipidemia increases the risk of cardiovascular and chronic kidney diseases through
multiple mechanisms, including the promotion of atherosclerosis, endothelial injury, chronic
inflammation, and oxidative stress. These factors collectively contribute to the progression of
CKM syndrome52, 53. In addition to mitigating hyperglycemia, EMPA can also influence the lipid
metabolism to manage hyperlipidemia, which is also crucial for treating CKM syndrome54, 55.
Compared to free EMPA, T-PAAD NPs/EMPA exhibit superior blood lipid-lowering effects (Fig.
6a-d). In CKM mice, treatment with T-PAAD NPs/EMPA resulted in 1.68-, 1.60-, and 1.32-fold
reductions in serum triglyceride (TG), total cholesterol (TC), and low-density lipoprotein
cholesterol (LDL-C) levels, respectively. However, free EMPA failed to produce the comparable
lipid-lowering effect. These results highlight the enhanced lipid-modulating capacity of T-PAAD
NPs/EMPA and support their potential for effectively mitigating dyslipidemia in CKM mice.
Additionally, the liver plays a pivotal role as the metabolic command center for systemic lipid
homeostasis, precisely regulating lipid synthesis, storage, and secretion. In CKM mice, this
delicate balance is disrupted, leading to severe hepatic dysfunction characterized by ectopic lipid
accumulation and dyslipidemia. To assess the therapeutic potential of T-PAAD NPs/EMPA in this
context, its efficacy against hepatic steatosis was evaluated. As shown in Fig. 6e and f, the CKM
mice developed obvious hepatomegaly, with liver/body weight ratio increasing to 8.96%, however,
which was superiorly reversed by T-PAAD NPs/EMPA (6.26%) compared with free EMPA
(6.83%). This was accompanied by the substantial decreases of serum ALT and AST levels,
indicating the robust hepatoprotective effects of T-PAAD NPs/EMPA (Supplementary Fig. 18).
Meanwhile, histological examination demonstrated that T-PAAD NPs/EMPA significantly
resolved macrovesicular steatosis and achieved a 6.13% reduction in hepatic triglycerides (ORO-
positive area), significantly outperforming the free EMPA treatment (2.03% reduction) (Fig. 6g).
Meanwhile, adipose tissue functions not only as an energy reservoir but also as a dynamic
endocrine organ that plays a critical role in maintaining systemic metabolic homeostasis. In CKM
mice, we observed substantial remodeling of adipose tissue architecture. Specifically, white
adipose tissue (WAT) mass was reduced by 1.82% compared to the RD control group. Histological
analysis of epididymal WAT (eWAT) revealed significantly smaller adipocytes, along with
evidence of ectopic lipid deposition in visceral organs, reflecting severe metabolic dysregulation.
Concurrently, brown adipose tissue (BAT) in CKM mice also exhibited increased lipid droplet
accumulation compared to RD mice (Fig. 6h). Notably, T-PAAD NPs/EMPA exhibited the robust
therapeutic efficacy, resulting in a 2.24-fold increase in eWAT mass recovery compared to the
untreated CKM group and substantially outperforming the minimal 1.01-fold improvement
observed with free EMPA treatment (Fig. 6i). Moreover, BAT morphology was restored following
T-PAAD NPs/EMPA treatment, with lipid droplet size and distribution resembling those seen in
healthy controls. Since BAT contributes to enhanced insulin sensitivity, glucose utilization, and
overall metabolic regulation, these findings underscore the ability of T-PAAD NPs/EMPA to
restore both the mass and morphology of WAT and BAT. These results demonstrate the
therapeutic potential of T-PAAD NPs/EMPA for reversing systemic metabolic dysfunction
associated with CKM syndrome.
Mechanistically, these effects likely stem from kidney-targeted delivery that increases local
EMPA exposure in proximal tubules, thereby amplifying urinary glucose excretion. The resulting
sustained glycosuric energy loss generates a stronger systemic negative energy balance than free
EMPA, whose effective exposure can be attenuated by presystemic and hepatic metabolism. In
response, metabolism may shift away from hepatic lipogenesis toward lipid mobilization and
increased fatty acid oxidation. To rule out confounding by energy intake, we monitored food
consumption and body weight throughout the treatment period (Supplementary Fig. 19). Daily
food intake remained stable and comparable across groups, arguing against caloric restriction as a
driver of the hepatic changes. Moreover, whereas untreated CKM mice showed pathological
weight loss, mice treated with T-PAAD NPs/EMPA maintained physiologic weight stability.
Together, these findings support that the benefits arise from targeted metabolic reprogramming
rather than differences in energy intake or overall energy balance.

2.7. T-PAAD NPs/EMPA alleviating the CKM syndrome by reprogramming the cardio-renal
energy metabolism
In CKM mice, extensive transcriptional dysregulation was observed in both kidney and heart
(Fig. 7a-d). Volcano plots (Fig. 7 a and c) illustrated differentially expressed genes when
comparing CKM mice to healthy controls (RD group). In the kidneys of CKM mice, 1,875 genes
were significantly upregulated and 515 downregulated; while in the hearts, 511 genes were
upregulated and 220 downregulated. Pathway enrichment analyses using Gene Ontology (GO) and
Kyoto Encyclopedia of Genomes (KEGG) databases revealed strong activation of pro-diabetic and
inflammatory pathways in CKM mice (Fig. 7b, d and Supplementary Fig. 20). Conversely, key
metabolic pathways such as fatty acid degradation, pantothenate & CoA biosynthesis, and the
PPAR signaling pathway, which were critical for lipid oxidation and peroxisomal fatty acid
metabolism, were significantly downregulated in CKM mice. These pathways were closely related
to energy metabolism, indicating that severe metabolic dysfunction and inflammation in the heart
and kidney played an important role in CKM progression (Supplementary Fig. 21 a, b). Consistent
with these findings, Gene Set Enrichment Analysis (GSEA) showed downregulation of gene sets
associated with oxidative phosphorylation and fatty acid metabolism, along with upregulation of
gene sets associated with inflammatory response in CKM mice relative to the RD group (Fig. 7e).
However, following the T-PAAD NPs/EMPA treatment, the RNA transcriptomic profiles shifted
markedly toward a restorative pattern (Fig.7 f, h). T-PAAD NPs/EMPA treatment significantly
downregulated 634 genes and upregulated 415 genes in the kidney, while 368 genes were
downregulated and 121 upregulated in the heart. Notably, T-PAAD NPs/EMPA suppressed several
pathological pathways that were elevated in the heart and kidney tissues of CKM mice, including
those involved in pro-diabetic signaling, aberrant lipid metabolism, atherosclerosis, and IL-17-
mediated inflammation (Fig. 7g, i). In parallel, beneficial pathways such as glutathione metabolism
and drug metabolism were upregulated following treatment (Supplementary Fig. 21c, d and 22).
Further GSEA results confirmed that T-PAAD NPs/EMPA treatment led to upregulated expression
of genes involved in oxidative phosphorylation pathway and downregulated expression of genes
related to inflammatory response in both heart and kidneys when compared with untreated CKM
mice (Fig. 7j). Collectively, the observed transcriptomic reprogramming supported that T-PAAD
NPs/EMPA effectively attenuates inflammation while reactivating energy metabolism pathways at
the gene expression level.
To determine whether the observed transcriptional changes translated into downstream
metabolites alterations, the untargeted metabolomic analysis were performed. Partial least squares
Discriminant Analysis (PLS-DA) revealed that the untreated CKM mice formed a metabolically
distinct cluster, clearly separated from the healthy control group. This separation reflected the
profound metabolic disruptions caused by CKM syndrome. Importantly, the metabolic profiles of
the free-drug group closely resembled those of the CKM group, whereas T-PAAD NPs/EMPA-
treated group showed pronounced shifts, indicating that T-PAAD NPs/EMPA treatment
substantially remodeled renal and cardiac metabolic signatures (Supplementary Fig. 23).
Heatmaps and KEGG enrichment analysis further confirmed that T-PAAD NPs/EMPA
significantly reprogrammed key metabolic pathways. In the kidneys, T-PAAD NPs/EMPA
increased metabolites involved in glycerophospholipid metabolism and arginine metabolism,
while decreasing those associated with glycolysis (Fig. 7k). KEGG pathway enrichment analysis
further confirmed that arginine metabolism, glycolysis/gluconeogenesis, and lipid metabolism
were among the most significantly altered pathways (Fig. 7l). These findings suggest that the
energy metabolism of the kidney was substantially reprogrammed, consistent with transcriptomic
evidence of restored metabolic gene expression. Similarly, in the heart, T‑PAAD NPs/EMPA
treatment corrected abnormalities in lipid metabolism. The levels of free fatty acids and
triglyceride derivatives shifted toward those observed in healthy controls (Fig. 7m and
Supplementary Fig. 24). KEGG enrichment analysis (Fig. 7n) further identified fatty acid
biosynthesis and glycerolipid metabolism as key pathways affected by the T‑PAAD NPs/EMPA
treatment. Together, these coordinated metabolic improvements in both the kidney and heart
provide strong evidences that T‑PAAD NPs/EMPA effectively reprograms the cardio-renal energy
metabolism in CKM mice.
To further validate these findings, key metabolic readouts were quantitatively assessed to
reinforce the therapeutic reprogramming effects mediated by T‑PAAD NPs/EMPA. As shown in
Fig. 7o, q, CKM mice exhibited significantly reduced ATP levels in both heart and kidney,
reflecting impaired energy production. In contrast, T‑PAAD NPs/EMPA treatment markedly
increased ATP content in kidneys, nearly restoring it to the levels observed in healthy controls. A
similar improvement in ATP levels was observed in heart, suggesting a systemic enhancement of
energy metabolism. Fatty acid oxidation (FAO), a primary source of ATP in both renal and cardiac
tissues under physiological conditions, was also evaluated. As shown in Fig. 7p and r, FAO activity
was significantly suppressed in untreated CKM mice, consistent with the transcriptomic evidence
of downregulated PPAR signaling and fatty acid catabolism (Supplementary Fig. 21a). Notably,
T‑PAAD NPs/EMPA treatment restored the FAO capacity in both kidney and heart, indicating
improved mitochondrial oxidative function. Given that FAO serves as the dominant energy source
in normal cardiac and renal physiology, these results provide compelling functional evidence that
T‑PAAD NPs/EMPA enhances therapeutic outcomes in CKM by reprogramming cardio-renal
energy metabolism.
Overall, these findings demonstrate that T-PAAD NPs/EMPA therapy effectively
reprogramed both the transcriptional and metabolic landscapes in the heart and kidneys of CKM
mice. The treatment suppressed pro-inflammatory and diabetic signaling while restoring energy
metabolism. This integrated multi-omics evidence strongly supports the conclusion that T-PAAD
NPs/EMPA mitigates CKM syndrome by reprogramming the cardio-renal energy homeostasis.

2.8. Biocompatibility evaluation in vivo
Prior to in vivo toxicity assessment, the hemocompatibility of T-PAAD NPs was rigorously
evaluated. Hemolysis assays performed at 2, 4, and 6 h revealed that the hemolysis rates remained
consistently below the 5% safety threshold across all tested concentrations (up to 400 μg mL⁻¹),
with no visible erythrocyte rupture observed in the supernatants (Supplementary Fig. 25). These
results demonstrate the excellent hemocompatibility of T-PAAD NPs, supporting their suitability
for systemic administration. Finally, to further evaluate the biocompatibility of T-PAAD NPs, a
single high-dose injection (500 mg kg-1, ten-fold the therapeutic dose) was administered to healthy
C57BL/6J male mice (n = 6) to evaluate acute in vivo toxicity. Over a 14-day monitoring period,
body weight fluctuations were negligible (Supplementary Fig. 25). At the end of the study, H&E
staining of major organs revealed no signs of necrosis, hemorrhage, or tissue damage in the heart,
liver, spleen, lungs, or kidneys. Additionally, liver and kidney function indicators, as well as
routine blood parameters, remained within normal physiological ranges and showed no significant
differences between treated and control groups. These results demonstrate the excellent biosafety
and non-toxicity of T-PAAD NPs, supporting their potential for clinical translation
(Supplementary Fig. 26).

Methods
Materials
Acrylamide (AAm), Acrylonitrile (AN), DATS, 4,4'-Azobis(4-cyanovaleric acid) (ACVA),
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N – hydroxysuccinimide (NHS) ,
Hoechst 33342, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2′,7′-
dichlorofluorescein diacetate (DCFH-DA), streptozotocin (STZ), ORO, empagliflozin (EMPA),
coumarin 6 (C6), and IR780 were purchased from Sigma-Aldrich. NH -PEG2000-COOH were
2
purchased from Titan. (KKEEE)₃K peptide were purchased from ChinaPeptides (QYAOBIO).
Antibodies: AQP-1 (1:200, Catalog #PA5-78805), KIM-1 (1:600, Catalog #PA5-98302) were
obtained from Thermo Fisher Scientific.

Cells and animals
HK2 cells (derived from adult male human kidney) were acquired from the Shanghai Cell
Bank of the Chinese Academy of Sciences (China). Male apolipoprotein E-deficient (ApoE−/−)
mice (about 6 weeks old) and C57BL/6J mice (about 6 weeks old) were purchased from Beijing
Vital River Laboratory Animal Technology Co., Ltd (China). Mice were housed under a 12/12-h
light/dark cycle at a constant temperature (22 °C) and humidity (50–60%), with free access to
food and water ad libitum. All animal experimentation procedures were ethically approved by the
Animal Experimentation Ethics Committee of School of Life Science and Technology in Xi’an
Jiaotong University (approval no. XJTUSLST-2023-54).

Synthesis and characterization of PEG-PAAD copolymer
The PAAD copolymer was synthesized via free radical polymerization. Specifically, AAm
(502.9 mg), AN (300 mg), DATS (63.1 mg), and ACVA (12.1 mg) were dissolved in N, N-
dimethylformamide (DMF) (4 mL) at a monomer feed molar ratio of AAm: AN: DATS = 5:4:0.25.
The solution was deoxygenated through three cycles of “freeze–vacuum–thaw”, with each cycle
lasting 30 minutes and maintaining a constant vacuum pressure of 6×10⁻² Pa. The mixture was
subsequently stirred at a constant temperature of 70 °C for 24 hours. Upon completion of the
reaction, the resulting copolymer (isolated with an approximate yield of 51.8%) was precipitated
into an excess of cold diethyl ether to remove unreacted monomers and oligomers, followed by
collection via centrifugation.
Subsequently, PAAD (100 mg) and NH -PEG-COOH (Mw:2000, 12 mg) were dissolved in
2
dimethyl sulfoxide (DMSO) (3 mL), followed by the addition of NHS (0.7 mg), EDC (1.2 mg),
and triethylamine (TEA) (0.6 mg). The reactant molar ratio was PAAD: NH -PEG-COOH = 1:1.2.
2
The mixture was stirred at 50 °C for 24 hours. The product was then purified by dialysis against
ultrapure water for 48 hours, utilizing a dialysis membrane with a Molecular Weight Cut-Off
(MWCO) of 3500, which was sufficient to ensure the removal of organic solvents and unreacted
reagents. Finally, the solution was lyophilized to obtain the PEG-PAAD copolymer (isolated with
an approximate yield of 50%).
The synthesized polymers PAAD and PEG-PAAD were characterized using a Bruker Avance
nuclear magnetic resonance spectrometer (400 MHz) for 1H-NMR analysis. The average
molecular weight (Mw) of the polymer was determined through gel permeation chromatography
(Agilent PL-GPC 50) at a column temperature of 45 °C, employing DMSO as the mobile phase at
a flow rate of 1 mL min−1. Fourier transform infrared spectroscopy (FTIR) were recorded through
the KBr pellet method using a FTIR spectrometer (Thermo Scientific Nicolet iS50). The UCST of
polymer was measured using a variable-temperature ultraviolet spectrometer (Purkinje T6-1610F)
with the wavelength set at 650 nm. The analysis began at 4 °C with a heating rate of 0.5 °C min−1.
Transmittance values were recorded at various temperatures to calculate the UCST, which was
defined as the temperature when the transmittance reached 50%. The Tyndall effect of the polymer
PEG-PAAD aqueous solution was observed via laser irradiation with the wavelength set at 650
nm.

Molecular dynamics simulation
To investigate the potential disruption of zipper-like intramolecular hydrogen bonds in PEG–
PAAD copolymers by urea, density functional theory (DFT) calculations were performed using
the Vienna Ab initio Simulation Package (VASP). To mimic the physiological aqueous
environment, the implicit solvent model VASPsol was employed to account for dielectric screening
and solvent polarization effects. The exchange-correlation interactions were described using the
generalized gradient approximation of Perdew-Burke-Ernzerhof (GGA-PBE). The projector
augmented-wave (PAW) method was used to treat the interactions between ion cores and valence
electrons. A plane-wave cutoff energy of 500 eV was adopted. The structural models were fully
relaxed until the Hellmann–Feynman forces on each atom were below 0.02 eV/Å and the total
energy change between successive steps was less than 1×10⁻⁵ eV. During geometry optimization,
the Brillouin zone was sampled using a Γ-centered 3×3×3 k-point grid, which was determined
based on systematic convergence tests. Grimme’s DFT-D3 method was applied to account for
dispersion interactions in the urea–polymer systems. Subsequently, urea-induced changes in
hydrogen bond populations and binding energies within the polymeric system were systematically
quantified.

ROS scavenging assays of PEG-PAAD copolymer
Measurement of H O scavenging: H O scavenging capacity of PEG-PAAD was tested by
2 2 2 2
the hydrogen peroxide detection kit (Nanjing NJiancheng Bioengineering Institute). H O reacts
2 2
with ammonium molybdate to form a stable yellow complex, which displays an absorbance peak
at 405 nm. Various concentrations of PEG-PAAD (0.125-1 mg mL−1) were incubated with H
2
O
2
at 37 °C, respectively. After reaction, the concentration of remaining H O was determined
2 2
according to the manufacturer’s instructions, and the H O -eliminating capacity was calculated.
2 2
Measurement of AO
2
•− scavenging: The superoxide anion (O
2
•−) scavenging activity was
assessed using a superoxide anion assay kit (Nanjing Jiancheng Bioengineering Institute)
according to the manufacturer’s instructions. Different concentrations of PEG-PAAD (0.125-1 mg
mL−1) were added to the working solution. The absorbance at 550 nm was measured using a
microplate reader (Tecan M200) after standing for 10 min.
Measurement of ·OH scavenging: The scavenging activity of PEG-PAAD against ·OH was
determined based on the effective removal of ·OH originating from the Fenton reaction. In detail,
the working test solutions containing 500 μM 3,3′,5,5′-tetramethylbenzidine (TMB), 2 mM H O ,
2 2
1 mM FeSO and different concentrations of PEG-PAAD (0.125-1 mg mL−1) in sodium acetate
4
buffer (0.5 M, pH 4.5) were prepared in the dark and rest for 5 min. Afterwards, the absorbance at
630 nm of the solution was monitored using a microplate reader (Tecan M200).
Measurement of ABTS radical scavenging: The radical scavenging capacity of PEG-PAAD
was evaluated using an ABTS radical scavenging capacity assay kit (Beyotime Biotechnology).
Briefly, ABTS solution was incubated with potassium persulfate overnight to generate ABTS
radicals. PEG-PAAD at various concentrations (0.125-1 mg mL−1) were mixed with ABTS radical
solutions and incubated for 10 min. The absorption of ABTS radicals at 734 nm was measured.
The scavenging efficiency of ABTS radicals was calculated by the following equation:
ABTS scavenging efficiency (%) = ((A
ABTS
-A
Sample
)/A
ABTS
) ×100% (1)
where A represented the absorbance of ABTS without additional processing. A
ABTS Sample
represented the absorbance of ABTS with the addition of PEG-PAAD.

Synthesis and characterization of PEG-PAAD-(KKEEE)₃K copolymer
The PEG-PAAD (50 mg) was dissolved in DMSO (3 mL), and EDC (0.6 mg) and NHS (0.4
mg) were added for activation of the terminal carboxyl groups by stirring for 4 hours. Subsequently,
the (KKEEE)₃K peptide (5.7 mg) and TEA (0.3 mg) were added, and the reaction was carried out
at 35 °C under 300 rpm stirring for 20 hours. The final product was purified by dialysis against
ultrapure water for 48 hours, utilizing a dialysis membrane with a Molecular Weight Cut-Off
(MWCO) of 3500, and lyophilized to obtain the PEG-PAAD-(KKEEE)₃K copolymer. The
conjugation of (KKEEE)₃K peptides to PEG-PAAD copolymer was detected using the
bicinchoninic acid (BCA) assay.

Preparation and characterization of T-PAAD NPs/EMPA
The amphiphilic polymers PEG-PAAD and PEG-PAAD-(KKEEE) K (10% w/w), along with
3
empagliflozin (EMPA), were dissolved in DMSO (250 μL). The mass ratio of the drug to the
polymer was set at 2:5 (w/w). The mixture was then added dropwise into an aqueous solution
under probe sonication at 40% amplitude for 1 minute with a pulse cycle of 3 s on and 3 s off to
ensure uniform dispersion. The resulting emulsion was dialyzed using a dialysis membrane
(MWCO 3,500 Da) against a large volume (1 L) of PBS to thoroughly remove the organic solvent.
The particle size, zeta potential, and polydispersity index (PDI) of the nanoparticles were
determined using dynamic light scattering (Malvern Zetasizer Nano ZS90), while their
morphology was characterized by transmission electron microscopy (Thermo Fisher Scientific FEI
Talos F200C).
The encapsulation efficiency and drug loading of EMPA were measured by high-
performance liquid chromatography (HPLC) using an Agilent 1100 system. A C18 column (250
mm × 4.6 mm, 5 μm) was maintained at 30 ℃ and eluted with a mobile phase consisting of
acetonitrile (32%) and 0.01% (V/V) trifluoroacetic acid (68%) in an isocratic elution manner at a
flow rate of 1 mL min-1. The content of EMPA was quantified by monitoring the UV signals at 225
nm.

In vitro drug release profiles
To investigate the in vitro drug release profiles, T-PAAD NPs/EMPA were incubated at 37 °C
in media simulating normal plasma/tissue (5 mM Urea) or pathological kidney injury conditions
(10 and 50 mM Urea). To accurately separate the released drug and minimize non-specific
adsorption, the samples were processed using Millipore Amicon Ultra centrifugal filters (100 kDa
MWCO) with low-binding regenerated cellulose (RC) membranes. At predetermined time
intervals, the samples were centrifuged to collect the filtrate, and the retentate was replenished
with fresh solution of the same volume. The amount of EMPA released was determined using
HPLC (Agilent 1100 system).

In vitro cellular uptake assessment
The targeting ability of T-PAAD NPs was investigated by incubating HK2 cells that were
pretreated with free (KKEEE) K peptide or left untreated with C6-loaded T-PAAD NPs to assess
3
their targeting capacity. After a 4 h incubation, the cells were collected and assessed using flow
cytometry (BD Biosciences) at an excitation wavelength of 488 nm. Moreover, to visualize the
intracellular distribution of T-PAAD NPs, Hoechst 33342 was used to stain the nuclei of the T-
PAAD NPs/C6-treated cells. Subsequently, imaging was performed using a Confocal Laser
Scanning Microscope (CLSM Leica TCS SP8 STED 3X).

In vitro ROS scavenging assays
HK2 cells were cultured under the standard condition. For the protection of cells from H O
2 2
induced oxidative stress, different concentrations of T-PAAD NPs (12.5, 25, 50 μg mL-1) were
cultured with HK2 cells in the absence or presence of H O (150 μM) for 24 hours, and the relative
2 2
cell viabilities were tested using the MTT assay. To qualitatively and quantitatively describe the
content of the intracellular ROS under H O stimulation, DCFHS-DA was used to stain intracellular
2 2
ROS. The HK2 cells incubated with H O (150 μM) and different concentrations of T-PAAD NPs
2 2
(12.5, 25, 50 μg mL-1) for 24 h were washed twice with serum-free medium and then DCFH-DA
dye working solution was added for another 30 min. Finally, the unbound DCFH-DA probe was
removed by washing twice with a serum-free medium. Then, the above treated cells were evaluated
using CLSM and flow cytometry, respectively.
To establish a pathophysiologically relevant in vitro model of CKM syndrome, we developed
a co-stimulation system using high glucose and palmitate to mimic systemic hyperglycemia and
hyperlipidemia in renal tubular epithelial cells. Specifically, serum-starved HK2 cells were
pretreated with glucose (33 mM) and palmitate (0.3 mM) in combination with varying
concentrations of T-PAAD NPs for 24 hours following 6-hour serum deprivation. Lipid
peroxidation was quantitatively assessed by measuring malondialdehyde (MDA) levels using a
colorimetric assay kit, thereby evaluating the antioxidative capacity of T-PAAD NPs against
oxidative stress in HK2 cells.

Establishment of the CKM model
To establish a CKM syndrome mouse model, 8-week-old APOE-/- mice were fed a Western
diet (WD, 21% fat and 0.15% cholesterol) for 8 weeks, followed by daily low-dose STZ injections
(50 mg kg−1) for 5 consecutive days, with a 12-hour fasting period prior to each injection. Four
weeks after STZ treatment, the mice exhibited significantly elevated fasting blood glucose levels
(>11 mM), along with pronounced polydipsia, polyuria, and polyphagia. Additionally, early signs
of kidney injury emerged, evidenced by the onset of microalbuminuria and a slight increase in the
urinary albumin-to-creatinine ratio (UACR), indicating the development of T2DM with early renal
impairment. By eight weeks post-STZ treatment, the glomerular filtration rate (GFR) was
markedly reduced, and urinary albumin levels were significantly elevated, reflecting progressive
renal dysfunction. Moreover, the mice displayed prominent atherosclerotic plaque formation,
elevated blood pressure, dyslipidemia, cardiac remodeling, and metabolic abnormalities.
Collectively, these findings demonstrate that this model integrates the pathophysiological
interactions among T2DM, CKD, and CVD, providing a simple and effective system for
investigating the interrelationship among metabolic disturbances, cardiac, vascular, and renal
dysfunction. This model offers a valuable platform for the comprehensive study of the onset and
progression of CKM syndrome.

Biodistribution of T-PAAD NPs in CKM mice
T-PAAD NPs/IR780 (0.35 mg kg−1 of IR780) were intraperitoneally injected to healthy or
CKM mice, while the control group received an equivalent dose of free IR780. At 6 h post-
injection, major organs including the heart, liver, spleen, lungs, and kidneys were harvested and
subjected to fluorescence imaging analysis using an in vivo imaging system (Lumazone, USA).
For fluorescence images of kidney tissue slices, the T-PAAD NPs/C6 were intraperitoneally
injected into CKM mice. At 6 h post-injection, the kidneys were collected, and the proximal
tubules and their cell nucleuses were stained with AQP1-PE (Thermo Fisher Scientific, PA5-78805)
and DAPI, respectively.

In vivo therapeutic efficacy of T-PAAD NPs/EMPA against CKM syndrome
Male ApoE-/- mice at 8 weeks of age were randomly assigned to 5 groups (n=5): regular diet
group (RD), Western diet group (WD), CKM model group (CKM), free EMPA treatment group
(Free EMPA), and T-PAAD NPs/EMPA treatment group (T-PAAD NPs/EMPA). The specific
treatment protocol is shown in Fig. 4a. The free EMPA and T-PAAD NPs/EMPA (the dose of
EPMA:10 mg kg-1 body weight) were intraperitoneally administered every other day for 4 weeks.
During the treatment period, the mice's body weight, blood glucose, and blood pressure were
regularly monitored. At the end of the treatment, the GFR, cardiac ultrasound, and renal
hemodynamics were assessed to evaluate heart and kidney function, respectively. Finally, the mice
were euthanized by cervical dislocation after anaesthetization with isoflurane, and the major organ
tissues and plasma were collected for subsequent biochemical assays, histological analysis, and
transcriptomic and metabolomic analysis.

Measurement of blood pressure
During the experimental period, systolic blood pressure (SBP), and diastolic blood pressure
(DBP) were measured weekly using a noninvasive blood pressure system for mice (Softron
Biotechnology BP-2010A). The mean arterial pressure (MAP) was calculated according to the
following formula:
MAP = 1/3 × (SBP+2 × DBP) (2)

Intraperitoneal glucose tolerance test and insulin tolerance test
In the final week of treatment, all mice underwent intraperitoneal glucose tolerance test
(IPGTT) and intraperitoneal insulin tolerance test (IPITT) following a 12-hour fasting period to
evaluate the long-term efficacy of T-PAAD NPs/EMPA in regulating glucose tolerance and insulin
sensitivity. For the IPGTT, mice were administered an intraperitoneal injection of glucose (2 mg
g-1 body weight). Blood glucose levels were measured at 0 (pre-injection), 15, 30, 60, 90 and 120
minutes post-injection. For the IPITT, insulin was intraperitoneally administered at a dose of 0.55
U kg-1 body weight, and blood glucose levels were measured at the same time points as in the
IPGTT. Blood glucose concentrations were determined using glucose test strips (Roche
Mannheim), and the area under the curve (AUC) was calculated using GraphPad Prism 9.0
software.

Echocardiographic measurement
Following isoflurane anesthesia, transthoracic echocardiography was performed using the
Vevo LAZR-X Imaging System (VisualSonics). M-mode echocardiography measurements in the
left ventricular long-axis view quantified: left ventricular internal diameter at end-diastole and end-
systole (LVIDd, LVIDs). Left ventricular ejection fraction (LVEF) was calculated as:
LVEF (%) = (LVEDV - LVESV) / LVEDV × 100% (3)
where LVEDV represent left ventricular end-diastolic volume, LVESV represent left
ventricular end-systolic volume. While fractional shortening (FS) was derived as:
FS (%) = (LVIDd - LVIDPs) / LVIDd × 100% (4)
the early (E) and late (A) diastolic mitral flow velocities were measured by pulsed Doppler
in the four-chamber view and the ratio of E /A was calculated.

Renal doppler ultrasonography
Renal blood flow was visualized using color Doppler mode, with red indicating flow toward
the transducer and blue representing flow away. Pulsed-wave Doppler measurements of renal
artery hemodynamics were performed. And Resistance Index (RI) reflecting distal vascular
resistance was calculated using following formulation:
RI = (PSV – EDV) / PSV (5)
where PSV represent peak systolic velocity, EDV represent end-diastolic velocity.
Pulsatility Index (PI) evaluating arterial compliance and impedance was measured as:
PI= (PSV - EDV) / MV (6)
where MV represent mean velocity.

Glomerular filtration rate (GFR) measurements
The percutaneous GFR measurement technique (MediBeacon Mannheim) was used to assess
the GFR in awake mice. 24 hours prior to the experiment, dorsal fur was removed to ensure optimal
signal acquisition. Before formal testing, the mice were briefly anesthetized with isoflurane, and
the MediBeacon transdermal Mini-GFR monitor was securely attached to the shaved area of the
skin using medical tape. Subsequently, FITC-sinistrin (70 mg kg-1 body weight) was injected via
the tail vein. The skin fluorescence signal was continuously monitored for approximately 1.5 hours
while the mice were allowed to move freely in their cages. Afterward, the device was removed
under isoflurane anesthesia, and data were analyzed using MB Studio v.22 software (MediBeacon).

Biochemical measurements
Serum creatinine (CRE), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C),
triglycerides (TG), malondialdehyde (MDA), glycated hemoglobin (HbA1c), urinary
microalbumin, catalase (CAT), hydrogen peroxide (H O ), uric acid (UA), alanine
2 2
aminotransferase (ALT), and aspartate aminotransferase (AST) levels were measured using
reagent kits (Nanjing Jiancheng Bioengineering Institute, catalogue number: c011-2-1, A111-1-1,
A113-1-1, A110-1-1, A003-1-2, A056-1-1, A028-2-1, A007-2-1, A064-1-1, C012-2-1, C009-3-1,
C010-3-1) according to the manufacturer's instructions, respectively. Serum urea nitrogen (BUN)
and renal tissue total antioxidant capacity (T-AOC) and fatty acid oxidation (FAO) levels were
determined using reagent kits (Elabscience, catalogue number: E-BC-K329-S, E-BC-K136-M, E-
BC-K784-M) according to the manufacturer's instructions, respectively. Renal ATP content was
quantified using reagent kits (Beyotime Biotechnology, catalogue number: S0026). All
biochemical analyses were performed following standardized operating procedure.

Histological and immunofluorescence analysis
Upon treatment completion, mice were euthanized and major organs (heart, kidneys, liver,
aortas, epididymal white adipose tissue (eWAT), and brown adipose tissue (BAT)) were harvested
for organ index calculation (organ-to-body weight ratio). Fresh tissues were fixed in 4%
paraformaldehyde and processed for either paraffin-embedded sections or frozen sections.
Comprehensive histological analyses included: Periodic acid-Schiff (PAS) staining for glycogen
detection, hematoxylin and eosin (H&E) for cellular morphology, Masson's trichrome for fibrosis
quantification, and ORO for lipid droplet visualization. Apoptosis was assessed by a one-step
terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Servicebio), while
kidney injury molecule-1 (KIM-1) expression was localized through immunofluorescence staining
using anti-KIM-1 antibody (Thermo Fisher Scientific, PA5-98302). Histological image analysis
was semi-quantitatively performed using Image-Pro Plus 6.0 software.

Transcriptomics analysis
Cardiac and renal tissues were collected from each treatment group in the CKM mice (RD
group, n=3; CKM group, n=3; T-PAAD NPs/EMPA treatment group, n=3) for total RNA extraction.
mRNA enrichment, fragmentation, reverse transcription, library construction, transcriptome
sequencing, and data analysis were performed by Genergy Biotechnology. Libraries were
subjected to high-throughput sequencing using the Illumina NovaSeq 6000 platform. Differentially
expressed genes (DEGs) between sample groups were identified using DESeq2 software, with the
threshold set at q-value <0.05 and fold-change >2 or <0.5. Functional annotation and pathway
enrichment analysis of DEGs were conducted using the GO and KEGG databases.

Metabolomics analysis
Samples of heart, kidney, and serum were collected from the CKM mouse model across
different treatment groups (RD group, n=3; CKM group, n=3; T-PAAD NPs/EMPA treatment
group, n=3). After grinding and extraction, these samples were analyzed using gas
chromatography-mass spectrometry (GC-MS) (Agilent 7890A and Agilent 5975C). Subsequent
data analysis was performed using the gas chromatography-specific SIMCA analysis software
(version 14.1). The criteria for differential metabolites selection were listed as following: a variable
importance projection (VIP) value > 1.0 obtained through orthogonal partial least squares
discriminant analysis (OPLS-DA), and a two-tailed Student's t-test p-value < 0.05.

Bio-safety analysis of T-PAAD NPs
The hemolysis tendency of blank T-PAAD NPs was also evaluated. Briefly, the erythrocytes
at the density of 1 × 107 cells mL−1 were incubated with blank T-PAAD NPs at concentrations
ranging from 1.5625 μg mL−1 to 400 μg mL−1 at 37 °C for 2, 4, and 6 h. PBS (pH 7.4, 0.01 M) and
Triton X-100 (1%, v/v) solution were also selected as negative and positive control, respectively.
The absorbance of hemoglobin at the wavelength of 540 nm in the supernatant was measured using
the microplate reader (Tecan M200). The collected blood samples were analyzed for routine blood
tests (Sysmex KX21 Sysmex).

Acute toxicity assessment of T-PAAD NPs
Twelve healthy C57BL/6J male mice were randomly allocated into two groups (n=6): the
control group received intravenous saline injection, while the mice in another group were
administered T-PAAD NPs at ten-fold higher than the therapeutic concentration (500 mg kg-1).
Body weight and general health status were recorded every other day with ad libitum access to
food and water throughout the 14-day observation period. Following euthanasia under anesthesia,
comprehensive analyses were performed including complete blood count, hepatic function
markers (ALT, AST), renal function parameters (BUN, CRE), precise organ weight measurements
(heart, liver, spleen, lungs, kidneys) for organ index calculation, and histopathological examination
via H&E staining of major organs.

Statistical analysis
All the statistical data were presented as the mean±standard deviation (s.d.) from a minimum
of three independent experiments. One-way analysis of variance (ANOVA) with Tukey’s post hoc
test or two-way ANOVA with Sidak multiple comparisons test was used for multiple comparisons.
Unpaired Student’s t-test was used to analyze the difference between two groups. *P < 0.05,
**P < 0.01, ***P < 0.001 and ****P < 0.0001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the synthesis part of the work , I have noticed that the reaction conditions are not described completely which is very\ncrucial to understand the method you have followed. The Cycle duration, correct vacuum pressure, monomer feed molar\nratio are missing from the data. Dialysis of the MWCO and solvent removal verification could be added in the method as the\nresidual organic solvents can affect biological results. Apart from this , a very important aspect of polymer yield data has not\nbeen mentioned.\nIn the nanoparticle synthesis and characterisation part, the drug-polymer ratio and sonication parameters are not mentioned.\nEncapsulation efficiency is very important data and I couldn’t find it mentioned or evaluated in the work. The nanoparticle\nsize distribution stability over time is not presented. Please confirm dialysis efficiency too.\nIn the in vitro drug release analysis, please verify the sink conditions which can account for adsorption losses. The drug\nintegrity should also be assessed.\nIn the computational modelling part, provide justification for k-point selection and vacuum gap size. The effects of solvent are\nnot described, as I feel that modelling in vacuum is not the right method to reproduce the biological or in vivo conditions.\nIn the biosafety analysis, the hemolysis assay duration is taken as 12 hours. But this cannot be physiologically relevant.\nShorten the hemolysis exposure .\nIt is interesting to note that by coupling bioresponsive controlled release with metabolic modulation, nanoplatform provides a\npromising approach to treat CKM and other\nsystemic metabolic disorders. Any side effects?\nThe manuscript contains promising results but requires significant methodological clarification, missing controls should be\nadded, and description of statistical and experimental precision to meet publication standards in a top-tier journal.\nMore Papers adn patents of 2025 should be cited", "reviewer_2": "The authors developed a urea-activated nanocarrier (T-PAAD NPs) that allows for the delivery and release of an SGLT2\ninhibitor (empagliflozin) to the proximal tubule (via targeting megalin). This NPs are coupled to an antioxidant component to\nenhance its protective effects in cardiovascular-kidney-metabolic- syndrome (CKM). The authors conclude that the delivery\nof Empa in the T-PAAD NPs was superior to free empagliflozin in a mouse model of CKM.\nThe idea of of T-PAAD NPs its interesting and would have an impact to increase SGLT2i therapies efficacy, however\nseveral concerns need to be addressed:\n1) The urea concentration in the S1 segment of the proximal tubule (the main site of SGLT2 expression) is approximately\nequal to that of plasma. It then increases progressively along the S2-S3 segments due to water reabsorption exceeding urea\nreabsorption. Given this, do the authors believe that the urea concentration in the early proximal tubule will be sufficient to\ntrigger Empa release? In the in vitro studies, Empa release was only accelerated at 100mM urea. Would this concentration\nbe physiological?\n2) In patients with reduced eGFR and elevated plasma urea levels (uremia), could this lead to premature release of Empa\ninto the circulation?\n3) The authors state that: “NPs can be effectively transported to renal tubule epithelial cells via the basolateral membrane”.\nCould the NPs enter the cell even if megalin expression is restricted to the apical membrane?\n4) One of the proposed strengths of the T-PAAD NPs is their specificity for the proximal tubule. However, in Figure 3C, the\nnanoparticles appear to localize to multiple organs, which may suggest off-target distribution.\nFurthermore, the evidence supporting selective localization to the proximal tubule could be strengthened. In Figure 3E, the\nco-localization with AQP1 is shown, but both AQP1 and the nanoparticle signals appear broadly distributed across various\nrenal tubules. Typically, AQP1 staining is more confined to the apical membrane rather than diffusely present within the\nentire cell.\nHigher magnification images would help clarify subcellular localization, and the inclusion of appropriate negative controls—\nsuch as autofluorescence controls—would enhance the specificity and interpretability of the findings.\n5) The authors frequently describe that T-PAAD NPs are superior to free empagliflozin. However, in several key parameters\n(renal function, albuminuria, kim-1+tubules, MDA) there is no statistical difference between NPs and free empa treated mice.\nThis suggests that both treatments have a similar protective effect for these parameters. The authors need to more precisely\ndescribe the comparative effects and clearly distinguish between statistically significant and non-significant differences.\n6) The IF of Kim-1 in Fig. 4H is not convincing enough. The signal is quite faint and difficult to distinguish in the images\nprovided.\n7) Please provide higher amplification for HandE and Masson staining, as well as quantification of the fibrotic area.\n8) For the renal function studies, it seems that only 3 mice per group were analyzed. Please clarify.", "reviewer_3": "This manuscript presents a highly innovative and thought-provoking strategy to improve the delivery of SGLT2 inhibitors\nusing a urea-responsive, kidney-targeted nanocarrier system. The authors demonstrate comprehensive evaluations in a\nCKM mouse model, including renal, cardiac, and hepatic outcomes, as well as multi-omics analyses, which together provide\nan impressive breadth of data. The concept of leveraging pathological urea concentrations for controlled release and\ncombining this with renal targeting and intrinsic antioxidant properties is novel and of high potential translational interest.\nNevertheless, while the study clearly demonstrates strengths and intriguing results, there remain fundamental concerns that\nlimit the reliability of the conclusions in its current form. Overall, this work is of considerable interest and holds potential\nimpact in the field of cardiorenal-metabolic therapeutics, but substantial revisions and additional data are required before the\nconclusions can be fully supported.\nMajor Concerns\nPhysiological validity of urea-responsive carrier degradation\nThe central hypothesis is that T-PAAD NPs disassemble in the proximal tubule lumen in response to elevated urea\nconcentrations. However, urea levels in the proximal tubule are nearly identical to plasma, and since the majority of urea is\nreabsorbed in the proximal tubule, a local increase in concentration is unlikely to occur. Significant accumulation of urea\nprimarily takes place in the collecting ducts and deep medulla. Thus, the proposed mechanism is physiologically\nquestionable. Moreover, plasma itself contains ~5 mmol/L urea, raising the possibility of premature systemic disassembly.\nDirect evidence for selective, proximal tubule luminal release is lacking, which undermines the core concept.\nConcerns regarding Fig. 3e immunostaining and mechanistic interpretation\nThe confocal immunostaining in Fig. 3e raises significant concerns. The signals for AQP1 (red) and NP/C6 (green) not only\noverlap but also display nearly identical intensity patterns, including areas of maximal fluorescence. Such a degree of\ncoincidence is highly unusual and suggests possible color reassignment or bleed-through rather than true colocalization.\nMoreover, the distribution of AQP1 appears diffuse and extensive rather than restricted to the apical brush border, which is\ninconsistent with established staining patterns. In addition, coumarin 6 is prone to leakage and nonspecific lipid staining,\npotentially mimicking membrane or cytoplasmic localization. Without appropriate controls (e.g., C6 alone, negative staining)\nor quantitative colocalization analysis, Fig. 3e does not convincingly demonstrate proximal tubule-specific targeting. Beyond\nthese imaging issues, there is a more fundamental inconsistency between the proposed mechanism and the interpretation of\nthe data. The central concept of this nanocarrier is that it disassembles within the proximal tubule lumen in response to\nelevated urea, thereby releasing EMPA to act luminally on SGLT2 located at the apical brush border. However, the authors\nrepeatedly emphasize endocytosis and intracellular accumulation of nanoparticles, which would\nnot provide luminal access to the SGLT2 target. This raises the question of why intracellular uptake is highlighted when the\ncarrier is not designed to function via an intracellular mechanism.\nClarification of this discrepancy is essential.\nLack of comparison with non-targeted NP group\nWhile Fig.3 compares PAAD/EMPA and T-PAAD/EMPA for renal accumulation, therapeutic outcome data (Fig.4 onward)\nomit the non-targeted PAAD/EMPA group. Without this comparator, it is unclear whether the enhanced effects result from\nkidney targeting or simply from nanoparticle formulation. Inclusion of non-targeted groups is essential.\nInsufficient explanation of cardiac functional improvement\nFig. 5 demonstrates cardiac functional recovery with T-PAAD/EMPA, but the underlying basis of this effect is not clearly\nexplained. It remains uncertain whether the observed improvement reflects secondary benefits from renal protection,\nsystemic metabolic changes, or direct antioxidant actions. The discussion does not adequately elaborate on these\npossibilities, and a clearer explanation of the likely mechanism(s) would strengthen the manuscript.\nInterpretation of hepatic lipid metabolism improvement\nFig.6 shows dramatic improvements in hepatic steatosis and serum lipid profile, far greater than typically reported in clinical\ntrials of SGLT2 inhibitors (which usually show modest LDL↑, HDL↑, TG↓, and mild NAFLD improvement). The mechanism\nlinking kidney-targeted EMPA delivery to liver outcomes is not explained. In addition, food intake data are absent, and body\nweight data—though reportedly monitored—are not shown. These omissions make it impossible to exclude confounding by\nenergy balance.\nLimitations in multi-omics analysis\nOmics data (Fig.7) are presented for CKM vs. T-PAAD/EMPA only, without free EMPA or non-targeted NP controls. Thus, it\nis impossible to determine whether the observed changes are specific to kidney-targeted delivery or general EMPA effects.\nThe polymer’s intrinsic antioxidant contribution cannot be ruled out. Proper control groups must be included.\nOff-target liver accumulation undermines kidney specificity\nBiodistribution (Fig.3c) shows not only kidney uptake but also strong liver accumulation, likely due to reticuloendothelial\nclearance. This undermines the claim of kidney specificity. The discussion does not address this limitation and instead\nframes hepatic accumulation as beneficial, which is not adequate.\nBiodistribution shown only at a single timepoint (Fig. 3c)\nOrgan accumulation is presented at only one timepoint. To more convincingly support kidney targeting, it would be\npreferable, if feasible, to include serial imaging at multiple timepoints (e.g., 1 h, 4 h, 8 h, 24 h). A single snapshot is\ninsufficient to substantiate claims of selective renal retention.\nUnclear contribution of SGLT2 inhibition versus intrinsic antioxidant effects\nAcross the study, it remains uncertain whether the observed benefits—particularly in extra-renal organs such as the heart\nand liver—arise from enhanced SGLT2 inhibition by kidney-targeted EMPA or from the intrinsic antioxidant properties of the\ncarrier itself. Since a vehicle-only T-PAAD control group is missing, the relative contributions of drug versus carrier cannot\nbe disentangled. Without this critical control, the conclusions attributing the therapeutic effects specifically to kidney-targeted\nSGLT2 inhibition remain inadequately supported."}, "author_response": "Response to reviewers’ comments:\nWe are sincerely thankful for the comments provided by the reviewers, as well as for\ntheir valuable suggestions that greatly aided in the revision of our manuscript. In this\nresponse, we address the reviewers’ comments and have highlighted all the revisions\nmade in red for clarity.\nReviewer #1:\n1. In the synthesis part of the work, I have noticed that the reaction conditions are not\ndescribed completely which is very crucial to understand the method you have followed.\nThe Cycle duration, correct vacuum pressure, monomer feed molar ratio are missing\nfrom the data. Dialysis of the MWCO and solvent removal verification could be added\nin the method as the residual organic solvents can affect biological results. Apart from\nthis, a very important aspect of polymer yield data has not been mentioned.\nResponse: Thank you very much for your detailed and insightful feedback on our work.\nIn the Methods section, we have now fully included the previously missing synthesis\nreaction parameters, and added the necessary specific details regarding the purification\nand verification steps, which is described as follows.\n“The PAAD copolymer was synthesized via free radical polymerization.\nSpecifically, AAm (502.9 mg), AN (300 mg), DATS (63.1 mg), and ACVA (12.1 mg) were\ndissolved in DMF (4 mL) at a monomer feed molar ratio of AAm: AN: DATS = 5:4:0.25.\nThe solution was deoxygenated through three cycles of “freeze-vacuum-thaw,” with\neach cycle lasting 30 minutes and maintaining a constant vacuum pressure of 6×10-2\nPa. The mixture was subsequently stirred at a constant temperature of 70 ℃ for 24\nhours. Upon completion, the resulting PAAD copolymer (isolated with an approximate\nyield of 51.8%) was precipitated into an excess of cold diethyl ether to remove\nunreacted monomers and oligomers, followed by collection via centrifugation.\nSubsequently, PAAD (100 mg) and NH -PEG-COOH (Mw:2000, 12 mg) were\n2\ndissolved in DMSO (3 mL), followed by the addition of NHS (0.7 mg), EDC (1.2 mg),\nand TEA (0.6 mg). The reactant molar ratio was PAAD: NH -PEG-COOH = 1:1.2. The\n2\nmixture was stirred at 50 ℃ for 24 hours. The PEG-PAAD product was then purified\n1\nby dialysis against ultrapure water for 48 hours, utilizing a dialysis membrane with a\nMolecular Weight Cut-Off (MWCO) of 3500, which was sufficient to ensure the removal\nof organic solvents and unreacted reagents. Finally, the solution was lyophilized to\nobtain the final PEG-PAAD copolymer (isolated with an approximate yield of 50%).”\n2. In the nanoparticle synthesis and characterisation part, the drug-polymer ratio and\nsonication parameters are not mentioned. Encapsulation efficiency is very important\ndata and I couldn’t find it mentioned or evaluated in the work. The nanoparticle size\ndistribution stability over time is not presented. Please confirm dialysis efficiency too.\nResponse: We sincerely appreciate the reviewer’s valuable comments and have revised\nthe manuscript to include the missing synthesis and characterization details.\nSpecifically, the mass ratio of the drug to the polymer was set at 2:5 (w/w), and the\nnanoparticles were prepared using probe sonication at 40% amplitude for 1 minute with\na pulse cycle of 3s on and 3s off to ensure uniform dispersion. To ensure the exhaustive\nremoval of unencapsulated drugs and residual organic solvents, the resultant drug-\nnanoparticle suspension was purified using a dialysis membrane (MWCO 3,500 Da)\nagainst a large volume (1 L) of PBS. Given the minimal initial volume of organic\nsolvent (250 μL) and the low mass of free drug relative to the vast dialysate volume,\nwe maintained a steep concentration gradient by frequently refreshing the external\nbuffer. This rigorous protocol, which is widely employed in previous literatures for\npreparing nano-drug delivery systems, ensures the efficient diffusion of small-molecule\nimpurities, thereby guaranteeing the high purity of the final nanoparticle formulation.\nTo evaluate the drug loading capacity of T-PAAD NPs, both drug loading content\nand encapsulation efficiency (EE) were measured, which were 233.3 μg/mL and 47.6%,\nrespectively (Supplementary Fig. 5e).\nSupplementary Fig. 5e | Drug loading and encapsulation efficiency of T-PAAD\nNPs/EMPA. Data are presented as mean ± s.d. (n = 3).\ncomplete medium containing fetal bovine serum (FBS) at 37 ℃ and monitored the size\ndistribution via dynamic light scattering (Malvern Zetasizer Nano ZS90) at 0, 12, 24,\n48, and 72 hours. As shown in Supplementary Fig. 5d, the overlapping particle size\ndistribution curves confirm that the nanoparticles remain stable without significant\naggregation in a physiological environment over the 72-hour period.\nSupplementary Fig. 5d | Size distribution of T-PAAD NPs in Dulbecco’s Modified\nEagle Medium (DMEM) containing fetal bovine serum (FBS) at different intervals at\n37 °C.\n3. In the in vitro drug release analysis, please verify the sink conditions which can\naccount for adsorption losses. The drug integrity should also be assessed.\nResponse: Previous studies have confirmed that tubular urea concentrations increase\nfrom approximately 5 mM to 10-50 mM during severe renal injury. As shown in Fig.\n2e, under conditions mimicking normal plasma or healthy proximal tubules (5 mM\nurea), T-PAAD NPs/EMPA exhibited a sustained release profile, with a cumulative\nrelease of 62.3% over 24 hours. In contrast, as proximal tubular injury progresses and\nurea levels rise, EMPA release was correspondingly accelerated. Notably, exposure to\na simulated severe renal injury microenvironment (50 mM urea) triggered a rapid\nincrease in EMPA release, reaching approximately 93% within 24 hours. This\n3\nsignificant difference in release kinetics underscores the sensitivity of T-PAAD NPs to\nthe pathological microenvironment, driven by elevated urea concentrations.\nFigure 2e. Cumulative in vitro release of EMPA from T-PAAD NPs over 24 h under\nsimulated physiological and pathological conditions. Three media were used to model\nnormal plasma and the Sl segment of the proximal tubule across healthy-to-diseased\nstates: Condition l (normal plasma/healthy S1, 5 mM urea), Condition 2 (early kidney\ninjury S1, 10 mM urea), and Condition 3 (end-stage kidney injury S1, 50 mM urea).\nData are presented as mean ± s.d. (n = 3)\nTo ensure accurate measurement of the released Empagliflozin (EMPA) while\nminimizing adsorption artifacts, we utilized Millipore Amicon Ultra centrifugal filters\n(100 kDa MWCO) with low-binding regenerated cellulose (RC) membranes, which\nallow free passage of the small-molecule drug (MW ≈ 408 Da) and substantially\nEMPA\nreduce hydrophobic adsorption relative to polypropylene or PES materials. Additionally,\nat the end of in vitro drug release experiment, we also measured the amount of un-\nreleased-EMPA in T-PAAD NPs to perform the mass-balance recovery analysis. And\nthe total drug recovery in all groups were more than 95%, demonstrating that adsorption\nlosses were effectively controlled in the final release quantification and met the sink\ncriterion.\nMore importantly, drug integrity was rigorously assessed using a stability-\nindicating HPLC method, confirming that Empagliflozin (EMPA) remained chemically\nstable with no detectable degradation products throughout the entire release duration,\n4\neven under the most severe conditions (50 mM Urea). As shown in Figure R1, the\nretention time of the drug released from the T-PAAD NPs (12.065 min) was highly\nconsistent with that of the standard Empagliflozin (12.089 min). The identical peak\npositions and the absence of any new impurity peaks further verified that the chemical\nstructure of the drug remained intact during the encapsulation and release processes.\nFigure R1 | Representative HPLC chromatograms of standard EMPA and EMPA\nreleased from T-PAAD NPs under the simulated severe kidney injury condition (50 mM\nUrea). The alignment of retention times (Standard: 12.089 min vs. Released: 12.065\nmin) confirms the chemical stability and structural integrity of the drug.\n4. In the computational modelling part, provide justification for k-point selection and\nvacuum gap size. The effects of solvent are not described, as I feel that modelling in\nvacuum is not the right method to reproduce the biological or in vivo conditions.\nResponse: We sincerely thank the reviewer for this insightful comment regarding the\ncomputational methodology and model validity. Regarding the parameter selection, the\nk-point grid of 3×3×3 was determined based on systematic convergence tests for the\nchosen supercell. In these tests, further refinement of the k-point mesh led to total\nenergy changes below ~1 meV per atom, indicating that 3 × 3 × 3 is sufficient to achieve\nconverged electronic energies for our system. With respect to the environmental\nconditions, we apologize for any confusion caused by the initial description. The\ncalculations were not performed in a pure vacuum; instead, they were carried out in an\naqueous environment using the implicit solvent model VASPsol. This framework\nallows us to incorporate dielectric screening and solvent polarization effects that are\nessential for approximating in vivo conditions. Importantly, the primary objective of\nour simulations is to elucidate the intrinsic quantum-mechanical energetic trends\n5\nassociated with urea-induced disruption of intramolecular hydrogen bonds in the\npolymer. The combined use of the implicit solvent model and Grimme’s DFT-D3\ndispersion correction provides a balanced and computationally feasible approach that\ncaptures both solvent screening and long-range intermolecular interactions, thereby\nenabling a reliable description of the urea-driven hydrogen-bond destabilization\nmechanism.\n5. In the biosafety analysis, the hemolysis assay duration is taken as 12 hours. But\nthis cannot be physiologically relevant. Shorten the hemolysis exposure.\nResponse: Thanks for your suggestion. We reassessed the hemolytic tendency of blank\nT-PAAD NPs at different incubation times. Briefly, the erythrocytes at a density of 1 ×\n107 cells mL-1 were incubated with blank T-PAAD NPs at concentrations ranging from\n1.5625 μg mL-1 to 400 μg mL-1 at 37 °C for 2, 4, and 6 h, respectively. PBS (pH 7.4,\n0.01 M) and 1% (v/v) Triton X-100 were used as the negative and positive controls.\nThe absorbance of hemoglobin at the wavelength of 540 nm in the supernatant was\nmeasured using the microplate reader (Tecan M200). As illustrated in Supplementary\nFig. 25, the supernatants of all T-PAAD NP-treated groups remained transparent,\nsimilar to the PBS negative control, indicating no visible rupture of red blood cells.\nQuantitatively, the hemolysis rates across all tested concentrations remained\nconsistently below the safety threshold of 5%, even after extended incubation (6 h) at\nthe highest concentration (400 μg mL⁻¹). These results robustly demonstrate that T-\nPAAD NPs possess excellent hemocompatibility suitable for systemic administration.\nSupplementary Fig. 25 | Hemolytic activity of T-PAAD NPs at different\n6\nconcentrations and incubation times. a–c, Quantitative analysis of hemolysis rates\nof red blood cells incubated with T-PAAD NPs at various concentrations for 2 h (a),\n4\nh (b), and 6 h (c). Insets show representative photographs of erythrocyte suspensions\nafter centrifugation. Data are presented as mean ± s.d. (n = 3).\n6. It is interesting to note that by coupling bioresponsive controlled release with\nmetabolic modulation, nanoplatform provides a promising approach to treat CKM and\nother systemic metabolic disorders. Any side effects?\nResponse: Thank you for highlighting the crucial aspect of biosafety. We specifically\nevaluated the potential side effects and biocompatibility of the T-PAAD NPs. Even at a\nhigh dose of 500 mg kg-1 (ten-fold the therapeutic dose), the nanocarrier exhibited\nexcellent biosafety with no signs of tissue damage in major organs or hematological\nabnormalities over a 14-day period (Supplementary Fig. 26). Crucially, our strategy\naddresses key limitations of administering the drug in its free form. Conventional\nSGLT2 inhibitors (e.g., empagliflozin, EMPA) can be constrained by suboptimal\nbioavailability and systemic exposure that contributes to off-target effects. In contrast,\nour kidney-targeted delivery system achieves the desired pharmacodynamic outcomes,\nwhile avoiding a marked increase in urinary tract infections (UTIs), a well-recognized\nrisk associated with free EMPA (Supplementary Fig. 11 a and b). Collectively, this\nnanoplatform reduces systemic exposure and thereby limits both systemic toxicities\nassociated with nanomaterials and EMPA-specific adverse effects linked to non-target\ndistribution.\n7. The manuscript contains promising results but requires significant methodological\nclarification, missing controls should be added, and description of statistical and\nexperimental precision to meet publication standards in a top-tier journal.\nResponse: We sincerely thank the reviewer for recognizing the promise of our work\nand for raising important concerns regarding methodological clarity, control\ncompleteness, and statistical rigor. In response, we have substantially revised the\nmanuscript to meet top-tier publication standards. Specifically, we have (i) greatly\nexpanded the Methods section to provide full experimental transparency and\n7\nreproducibility; (ii) performed additional experiments to include the previously missing\ncontrol groups, which further validate the mechanistic conclusions; and (iii)\nstandardized all statistical reporting by clearly specifying sample sizes (n), defining\nerror bars (s.d.), and providing precision and variability descriptions in every figure\nlegend. These revisions collectively strengthen the robustness and interpretability of\nour study in accordance with the reviewer’s recommendations.\n8. More Papers and patents of 2025 should be cited.\nResponse: We sincerely appreciate the reviewer’s valuable suggestion. We agree that\nkeeping the bibliography up-to-date is crucial to reflect the fast-moving nature of the\nnanomedicine and CKM therapy fields. In the revised manuscript, we have conducted\na comprehensive literature search and incorporated 3 additional references from 2025,\nincluding key academic papers (e.g., Ref. [4], [5], [20]), primarily in the Introduction\nsections, covering both high-impact academic papers to highlight the latest industrial\nand scientific progress. These additions have been primarily integrated into the\nIntroduction sections. The newly added references further strengthen the discussion of\nthe current state-of-the-art.\nReviewer #2:\nThe authors developed a urea-activated nanocarrier (T-PAAD NPs) that allows for the\ndelivery and release of an SGLT2 inhibitor (empagliflozin) to the proximal tubule (via\ntargeting megalin). This NPs are coupled to an antioxidant component to enhance its\nprotective effects in cardiovascular-kidney-metabolic- syndrome (CKM). The authors\nconclude that the delivery of Empa in the T-PAAD NPs was superior to free\nempagliflozin in a mouse model of CKM.\nThe idea of T-PAAD NPs its interesting and would have an impact to increase SGLT2i\ntherapies efficacy, however several concerns need to be addressed:\n1. The urea concentration in the S1 segment of the proximal tubule (the main site of\nSGLT2 expression) is approximately equal to that of plasma. It then increases\n8\nprogressively along the S2-S3 segments due to water reabsorption exceeding urea\nreabsorption. Given this, do the authors believe that the urea concentration in the early\nproximal tubule will be sufficient to trigger Empa release? In the in vitro studies, Empa\nrelease was only accelerated at 100mM urea. Would this concentration be physiological?\nResponse: We appreciate the reviewer’s insightful comments. Indeed, the proximal\ntubular microenvironment in kidney injury deviates significantly from the\nphysiological state. Previous literature has confirmed that tubular urea concentrations\nescalate from approximately 5 mM to 10-50 mM during severe renal injury (Refs. a, b).\nUnder these conditions, urea plays a critical role in facilitating drug release. We\nmeasured the in vitro release profiles of T-PAAD NPs/EMPA under varying\nphysiological and pathological conditions mimicking the proximal tubule. Under\nconditions resembling normal plasma or healthy proximal tubules (Conditions 1), the\nnanoparticles displayed a sustained release profile, with 62.3% cumulative release over\n24 hours. However, as injury progresses in the proximal tubule, both urea levels and\nthe release rate of EMPA increase, reflecting the increased responsiveness of T-PAAD\nNPs to the elevated urea concentrations. Notably, exposure to a simulated severe renal\ninjury microenvironment (Condition 3, 50 mM urea) triggered a rapid acceleration of\ndrug release, reaching approximately 93% within 24 hours (Fig. 2e). This significant\ndifference in release kinetics confirms the sensitivity of the nanocarrier to the stimuli\nof elevated urea concentrations.\nMechanistically, UCST-based nanomedicines are expected to partially\ndisassemble in the presence of urea, which disrupts intra- and inter-chain hydrogen\nbonding. This urea-induced weakening lowers the UCST of T-PAAD and facilitates\nfaster drug release. Taken together, because urea concentrations in the proximal tubule\nare markedly elevated in injured kidneys, these UCST nanoparticles offer a promising\nplatform for on-demand drug release within the renal tubules.\nsimulated physiological and pathological conditions. Three media were used to model\nnormal plasma and the Sl segment of the proximal tubule across healthy-to-diseased\nstates: Condition l (normal plasma/healthy S1, 5 mM urea), Condition 2 (early kidney\ninjury S1, 10 mM urea), and Condition 3 (end-stage kidney injury S1, 50 mM urea).\n[a] Vanholder, R. et al. Review on uremic toxins: classification, concentration, and\ninterindividual variability. Kidney Int. 63, 1934-1943 (2003).\n[b] Duranton, F. et al. Normal and pathologic concentrations of uremic toxins. J. Am.\nSoc. Nephrol. 23, 1258-1270 (2012).\n2. In patients with reduced eGFR and elevated plasma urea levels (uremia), could this\nlead to premature release of Empa into the circulation?\nResponse: We appreciate the reviewer’s important question regarding the possibility\nof premature EMPA release in uremic patients, which is indeed a critical consideration\nfor systemic safety. The likelihood of significant premature release into the circulation\nremains low for two main reasons. First, while elevated plasma urea levels in uremic\npatients may slightly increase the basal release rate, the systemic environment does not\nreach the conditions necessary to trigger the accelerated release mechanism observed\nin our in vitro studies. In contrast, rapid and extensive release (~93%) is observed when\nurea concentrations are elevated to 50 mM, a condition representative of severe renal\ninjury rather than systemic circulation (Figure 2e). This confirms that EMPA release is\n10\nprimarily driven by the concentrations of urea found in the pathological\nmicroenvironment of the kidney, rather than being prematurely triggered by systemic\nuremic conditions.\nSecond, our biodistribution studies confirm that T-PAAD NPs exhibit preferential\nrenal accumulation, indicating that the nanocarriers remain largely intact during\nsystemic transit and preferentially localize to the kidney before encountering the injury-\nassociated stimulus (Supplementary Fig. 8). This targeting behavior, together with the\nkidney injury-responsive design, minimizes systemic drug leakage and ensures that\nefficient release occurs only within the pathological microenvironment of the proximal\ntubule, thereby maintaining systemic safety while maximizing therapeutic specificity.\n3. The authors state that: “NPs can be effectively transported to renal tubule epithelial\ncells via the basolateral membrane”. Could the NPs enter the cell even if megalin\nexpression is restricted to the apical membrane?\nResponse: We appreciate the reviewer’s insightful comment regarding the cellular\nuptake pathway and agree that megalin is predominantly expressed on the apical\nmembrane of proximal tubular epithelial cells. However, the ability of T-PAAD NPs to\nenter renal tubular cells is not attributed to basolateral megalin, but rather to two\nsynergistic pathological processes characteristic of CKM-associated kidney injury. First,\nCKM-induced renal damage compromises both the glomerular filtration barrier and\ntubular epithelial integrity, enabling a greater fraction of circulating nanoparticles to\npass into the tubular lumen. Once in the lumen, the nanoparticles directly encounter the\napical membrane, where they undergo specific megalin-mediated endocytosis,\nconsistent with established apical trafficking biology. Second, as described in our\nmanuscript, the steep pressure gradient along the nephron (≈50 → 10 mmHg) and the\nstrong absorptive forces of the peritubular capillaries promote nanoparticle movement\ntoward the basolateral interface. Although megalin is apical, injured proximal tubular\ncells are known to exhibit upregulated non-specific basolateral uptake pathways,\nincluding non-specific absorptive endocytosis or transcytosis. These processes increase\nintracellular access from the basolateral side independently of megalin. Taken together,\n11\nthe enhanced renal retention of T-PAAD NPs results from a combination of (i) apical\nmegalin-mediated uptake following increased luminal entry due to filtration barrier\nimpairment, and (ii) megalin-independent basolateral internalization facilitated by\naltered hemodynamics and injury-induced endocytic activity.\n4. One of the proposed strengths of the T-PAAD NPs is their specificity for the\nproximal tubule. However, in Figure 3C, the nanoparticles appear to localize to multiple\norgans, which may suggest off-target distribution.\nFurthermore, the evidence supporting selective localization to the proximal tubule\ncould be strengthened. In Figure 3E, the co-localization with AQP1 is shown, but both\nAQP1 and the nanoparticle signals appear broadly distributed across various renal\ntubules. Typically, AQP1 staining is more confined to the apical membrane rather than\ndiffusely present within the entire cell.\nHigher magnification images would help clarify subcellular localization, and the\ninclusion of appropriate negative controls—such as autofluorescence controls—would\nenhance the specificity and interpretability of the findings.\nResponse: We sincerely thank the reviewer for these critical observations regarding\nbiodistribution specificity and interpretation of renal co-localization. We agree that\nnanoparticles may exhibit detectable signals in multiple organs, particularly the liver,\ndue to reticuloendothelial system (RES) uptake. This phenomenon is widely observed\nfor nanoscale formulations and does not contradict renal targeting. Importantly, our\nnewly added time-resolved biodistribution analysis (Supplementary Fig. 8)\ndemonstrates that T-PAAD NPs accumulate in the kidney more rapidly and are retained\nfor significantly longer durations than non-targeted PAAD NPs, confirming their\nrelative renal specificity despite baseline RES clearance.\ndependent biodistribution profiles of targeted versus non-targeted nanoparticles. Ex\nvivo fluorescence images of major organs (heart, liver, spleen, lung, and kidney)\nharvested from CKM mice at 6, 12, and 24 h following the administration of IR780-\nloaded T-PAAD NPs (targeted) or PAAD NPs (non-targeted). b, Quantitative analysis\nof the mean fluorescence intensity of the major organs in the ex vivo images over time.\nc, Quantitative analysis of the average fluorescence intensity specifically in the kidneys\nat different time point. Data points are means ± s.d. (n = 3). Two-tailed Student’s t-test\nwas used for statistical analysis (c). **P < 0.01 and ***P < 0.001.\nTo strengthen the evidence for proximal-tubule targeting, we have included\nhigher-magnification confocal images. These images show robust co-localization of T-\nPAAD NPs (green) with both AQP1 (Figure R2a, red) and SGLT2 (Figure R2b, red),\ntwo well-established proximal tubule markers. The observed intracellular distribution\nof the nanoparticles-distinct from the strictly apical staining of the membrane markers-\nis consistent with the proposed mechanism of megalin-mediated endocytosis, indicating\nthat the nanoparticles are successfully internalized by the proximal tubular cells.\nFurthermore, to ensure signal specificity, we have incorporated the reviewer’s\nrecommendation by including autofluorescence controls. These control groups exhibit\nnegligible background staining, confirming that the observed fluorescence in Figures\nR2a and b arises from true nanoparticle uptake and marker expression rather than\nimaging artifacts.\n(representing proximal tubule marker, red) in the kidney after T-PAAD NPs/C6 i.p.\nadministration. Nuclei were stained with DAPI (blue). b, Fluorescence images of C6\n(green) and SGLT-2 (representing the therapeutic target, red) in the kidney. For both (a)\nand (b), negative controls (bottom rows) were performed on kidney sections containing\nT-PAAD NPs by omitting the primary antibodies and incubating sections only with the\ncorresponding fluorescent secondary antibodies to verify signal specificity. The\ncomplete absence of red signal, despite the persistent intrinsic fluorescence of the\nnanoparticles (green), confirms the specificity of the immunolabeling and definitively\nrules out spectral bleed-through or non-specific secondary antibody binding.\n5. The authors frequently describe that T-PAAD NPs are superior to free empagliflozin.\nHowever, in several key parameters (renal function, albuminuria, kim-1+tubules, MDA)\nthere is no statistical difference between NPs and free empa treated mice. This suggests\nthat both treatments have a similar protective effect for these parameters. The authors\nneed to more precisely describe the comparative effects and clearly distinguish between\nstatistically significant and non-significant differences.\nResponse: We sincerely appreciate the reviewer’s thoughtful and rigorous assessment\n14\nof our comparative therapeutic analysis. After re-examining the statistical outcomes,\nwe fully agree that the distinctions between T-PAAD NPs/EMPA and free EMPA must\nbe described with precision and in strict accordance with statistical significance. Our\nupdated analysis confirms that for several downstream renal injury markers, including\nalbuminuria, KIM-1-positive tubules, and MDA levels, both treatments conferred\ncomparable protective effects. Although these differences did not consistently reach\nstatistical significance, the T-PAAD NPs group also exhibited numerically greater\nimprovement (Supplementary Fig. 14).\nSupplementary Fig. 14 | Quantitative analysis of renal fibrosis, cellular apoptosis,\nand KIM-1 expression in kidney tissue sections. a, Quantitative analysis of renal\nfibrosis in different groups. b, c, The positive areas in the kidneys were analyzed.\nQuantitative analysis of TUNEL- (b) and KIM-1-positive (c) areas in kidney tissue\nsections by immunofluorescence staining. The data were analyzed using one-way\nANOVA with Tukey’s post hoc test, and shown as mean ± s.d. (n =5). **P < 0.01,\n***P < 0.001, ****P < 0.0001.\nAdditionally, the T-PAAD NPs platform demonstrated statistically significant\nsuperiority over free EMPA in several critical systemic drivers of CKM progression,\nincluding glycemic control (blood glucose: 13.64 vs. 22.80 mmol/L), blood pressure\nregulation (MAP reduction), and attenuation of metabolic dysfunction (notably reduced\nhepatic steatosis). These parameters represent fundamental pathological drivers that\nultimately shape the renal injury trajectory, and the significant improvements observed\nwith T-PAAD NPs provide mechanistic justification for the enhanced therapeutic\npotential of the nanoplatform.\nIn response to the reviewer’s request, we have now precisely distinguished\nbetween statistically significant and non-significant effects throughout the revised text\nand figure legends, ensuring that our claims are rigorously aligned with the quantitative\n15\ndata. Statements of “superior therapeutic efficacy” are now reserved exclusively for\nendpoints where statistical significance was achieved, while all other parameters are\ndescribed using neutral, statistically accurate terminology.\n6. The IF of Kim-1 in Fig. 4H is not convincing enough. The signal is quite faint and\ndifficult to distinguish in the images provided.\nResponse: We sincerely thank the reviewer for this careful observation and agree that\nthe KIM-1 immunofluorescence images in Fig. 4h were not sufficiently clear in the\noriginal submission. In the revised manuscript, we refined the image processing\nuniformly across all groups by applying standardized contrast and intensity\nnormalization, allowing clearer discrimination of true KIM-1 signals without altering\nthe underlying data (Fig.R3).\nFigure R3 | Fluorescence images of KIM-1(red) and 4’,6-diamidino-2-phenylindole\n(DAPI, blue) of kidney tissues from different groups. Scale bars, 50 µm.\n7. Please provide higher amplification for H and E and Masson staining, as well as\nquantification of the fibrotic area.\nResponse: Thanks for your suggestion. We have provided higher-magnification images\nof the H&E- and Masson-stained kidney sections, along with quantitative analysis of\nthe fibrotic areas (Fig. R4).\nFigure R4 | Histological assessment of renal pathological changes and fibrosis. a,\nbars, 50 µm) of kidney slices from mice after different treatments. b, Quantitative\nanalysis of renal fibrosis in different groups. Data were analyzed using one­way\nANOVA with Tukey’s post hoc tests (b) and are shown as mean ± s.d. (n=5). **P < 0.01,\n***P < 0.001.\n8. For the renal function studies, it seems that only 3 mice per group were analyzed.\nPlease clarify.\nResponse: We appreciate the reviewer’s attention to the sample size used in the renal\nfunction studies. We would like to clarify that although each treatment group originally\nincluded n = 5 mice to ensure adequate biological variability, the Glomerular Filtration\nRate (GFR) measurements were performed on a subset of n = 3 mice per group. This\nwas necessitated by the high cost of the FITC-sinistrin tracer and the specialized real-\ntime transcutaneous detection required by the MediBeacon system-both of which are\nwidely acknowledged in the field to limit sample size for GFR assays.\nImportantly, the GFR results obtained from these biological replicates were highly\nconsistent across animals and reached statistical significance. Furthermore, the same\nrenal-protective trend was independently confirmed using multiple complementary\nrenal function markers, including BUN, serum creatinine, and UACR, all of which were\nassessed in the full n = 5 cohort. The convergence of these independent functional\nreadouts supports the robustness of our conclusions despite the smaller sample size used\nspecifically for GFR analysis.\nReviewer #3:\nThis manuscript presents a highly innovative and thought-provoking strategy to\nimprove the delivery of SGLT2 inhibitors using a urea-responsive, kidney-targeted\nnanocarrier system. The authors demonstrate comprehensive evaluations in a CKM\nmouse model, including renal, cardiac, and hepatic outcomes, as well as multi-omics\nanalyses, which together provide an impressive breadth of data. The concept of\nleveraging pathological urea concentrations for controlled release and combining this\n17\nwith renal targeting and intrinsic antioxidant properties is novel and of high potential\ntranslational interest. Nevertheless, while the study clearly demonstrates strengths and\nintriguing results, there remain fundamental concerns that limit the reliability of the\nconclusions in its current form. Overall, this work is of considerable interest and holds\npotential impact in the field of cardiorenal-metabolic therapeutics, but substantial\nrevisions and additional data are required before the conclusions can be fully supported.\n1. Physiological validity of urea-responsive carrier degradation\nThe central hypothesis is that T-PAAD NPs disassemble in the proximal tubule lumen\nin response to elevated urea concentrations. However, urea levels in the proximal tubule\nare nearly identical to plasma, and since the majority of urea is reabsorbed in the\nproximal tubule, a local increase in concentration is unlikely to occur. Significant\naccumulation of urea primarily takes place in the collecting ducts and deep medulla.\nThus, the proposed mechanism is physiologically questionable. Moreover, plasma itself\ncontains ~5 mmol/L urea, raising the possibility of premature systemic disassembly.\nDirect evidence for selective, proximal tubule luminal release is lacking, which\nundermines the core concept.\nResponse: We appreciate the reviewer’s thoughtful comments regarding the\nphysiological urea gradient along the nephron and its implications for our proposed\nrelease mechanism. We fully agree that urea concentrations in the S1 segment (≈ 5\nmM) are comparable to plasma and, on their own, are insufficient to significantly\naccelerate EMPA release. As addressed in response to Reviewer 2, urea in the S1\nsegment rises from ~5 mM in healthy kidneys to ~10 mM in early injury, and can reach\n~50 mM in end-stage renal disease. These markedly elevated luminal urea levels\nprovide a robust endogenous trigger for our nanocarrier. Thus, in CKM mice the\nproximal tubule microenvironment offers sufficient urea to activate the T-PAAD NPs,\nenabling selective EMPA release in diseased kidneys. Consequently, our in vitro release\ndata show minimal acceleration under physiological conditions (5 mM urea), while\npathologically relevant urea levels (50 mM) trigger an obvious release response,\nreaching approximately 93% at 24 hours (Figure 2e). This release behavior aligns with\n18\nthe intended design principle: the nanosystem remains stable under healthy plasma and\nearly tubular fluid conditions but becomes rapidly activated only under pathological\nstimuli.\nFig.2e Cumulative in vitro release of EMPA from T-PAAD NPs over 24 h under\nsimulated physiological and pathological conditions. Three media were used to model\nnormal plasma and the Sl segment of the proximal tubule across healthy-to-diseased\nstates: Condition l (normal plasma/healthy S1, 5 mM urea), Condition 2 (early kidney\ninjury S1, 10 mM urea), and Condition 3 (end-stage kidney injury S1, 50 mM urea).\nFurthermore, our biodistribution analysis (Supplementary Fig. 8) demonstrates\nrobust and preferential renal accumulation of T-PAAD NPs, ensuring that the majority\nof the injected payload is rapidly delivered to the kidney. This efficient targeting\nsubstantially increases the local nanoparticle concentration within the diseased tubular\nenvironment, thereby enhancing the effectiveness of the urea-responsive release\nmechanism while minimizing the likelihood of premature systemic disassembly.\n2. Concerns regarding Fig. 3e immunostaining and mechanistic interpretation\nThe confocal immunostaining in Fig. 3e raises significant concerns. The signals for\nAQP1 (red) and NP/C6 (green) not only overlap but also display nearly identical\nintensity patterns, including areas of maximal fluorescence. Such a degree of\ncoincidence is highly unusual and suggests possible color reassignment or bleed-\nthrough rather than true colocalization. Moreover, the distribution of AQP1 appears\n19\ndiffuse and extensive rather than restricted to the apical brush border, which is\ninconsistent with established staining patterns. In addition, coumarin 6 is prone to\nleakage and nonspecific lipid staining, potentially mimicking membrane or cytoplasmic\nlocalization. Without appropriate controls (e.g., C6 alone, negative staining) or\nquantitative colocalization analysis, Fig. 3e does not convincingly demonstrate\nproximal tubule-specific targeting. Beyond these imaging issues, there is a more\nfundamental inconsistency between the proposed mechanism and the interpretation of\nthe data. The central concept of this nanocarrier is that it disassembles within the\nproximal tubule lumen in response to elevated urea, thereby releasing EMPA to act\nluminally on SGLT2 located at the apical brush border. However, the authors\nrepeatedly emphasize endocytosis and intracellular accumulation of nanoparticles, which\nwould not provide luminal access to the SGLT2 target. This raises the question of why\nintracellular uptake is highlighted when the carrier is not designed to function via an\nintracellular mechanism. Clarification of this discrepancy is essential.\nResponse: We sincerely appreciate the reviewer’s detailed and rigorous assessment.\nWe have carefully addressed both the imaging-related concerns and the mechanistic\ninterpretation in the revised manuscript.\nTo resolve the concern that the AQP1 (red) and NP/C6 (green) signals in Fig. R2a\ndisplayed unusually similar intensity patterns, we have re-acquired the\nimmunofluorescence images using a higher-resolution confocal system to eliminate\nspectral bleed-through. The high-resolution immunofluorescence imaging clarifies the\nintrarenal localization. AQP-1 (red) specifically delineates the surface membranes of\nproximal tubule epithelial cells. Notably, the T-PAAD NP signals (green) are\npredominantly distributed surrounding the AQP-1 fluorescence, providing robust\nevidence that the nanoparticles are effectively internalized and enriched within the\nproximal tubular epithelium. It is important to note that C6 is a highly hydrophobic\nfluorescent probe commonly used for nanoparticle tracking, and its inclusion here\nallows precise visualization of nanoparticle distribution.\nThis correction directly addresses the reviewer’s concern regarding identical\nintensity profiles. In addition, we conducted stringent negative controls, including C6-\n20\nloaded T-PAAD NPs and secondary-antibody only controls, which confirmed the\nabsence of autofluorescence or nonspecific staining. These controls verify that the\nobserved fluorescence represents true biological localization rather than imaging\nartifacts.\nRegarding the mechanistic interpretation, we appreciate the reviewer’s concern\nand apologize for any confusion caused by insufficient clarity in the initial submission.\nOur system involves two complementary and non-conflicting processes. First, the\nprimary pharmacological action occurs at the apical brush border, where EMPA inhibits\nSGLT2. The nanocarrier was intentionally designed to undergo urea-responsive\ndisassembly within the tubular lumen, ensuring direct luminal access of EMPA to its\nmembrane target and remaining fully consistent with our conceptual framework.\nSecond, although luminal release is the principal route of action, intracellular\nnanoparticle uptake provides an important supportive benefit. In the high-flow\nproximal tubule, internalization creates a transient intracellular reservoir that reduces\nwashout and sustains local EMPA availability, an effect widely documented for tubular\nnanocarriers. Moreover, the T-PAAD polymer confers intrinsic antioxidant activity,\nallowing it to scavenge intracellular ROS, protect mitochondria, and stabilize tubular\nenergy metabolism. Thus, intracellular localization does not replace luminal SGLT2\ninhibition but instead offers a synergistic auxiliary pathway that enhances therapeutic\nefficacy in CKM.\nFig. R2a | Fluorescence images of C6 (representing T-PAAD NPs, green) and AQP1\n(representing proximal tubule marker, red) in the kidney after T-PAAD NPs/C6 i.p.\n21\nadministration. Nuclei were stained with DAPI (blue). Negative controls (bottom rows)\nwere performed on kidney sections containing T-PAAD NPs by omitting the primary\nantibodies and incubating sections only with the corresponding fluorescent secondary\nantibodies to verify signal specificity. The complete absence of red signal, despite the\npersistent intrinsic fluorescence of the nanoparticles (green), confirms the specificity of\nthe immunolabeling and definitively rules out spectral bleed-through or non-specific\nsecondary antibody binding.\n3. Lack of comparison with non-targeted NP group\nWhile Fig.3 compares PAAD/EMPA and T-PAAD/EMPA for renal accumulation,\ntherapeutic outcome data (Fig.4 onward) omit the non-targeted PAAD/EMPA group.\nWithout this comparator, it is unclear whether the enhanced effects result from\nkidney targeting or simply from nanoparticle formulation. Inclusion of non-targeted groups is\nessential.\nResponse: We sincerely appreciate the reviewer’s insightful comment regarding the\ninclusion of a non-targeted PAAD/EMPA group in the therapeutic studies. We would\nlike to clarify that the decision to exclude this group from the long-term efficacy\nexperiments was guided by the definitive biodistribution results presented in\nSupplementary Fig. 8. These data clearly demonstrate that non-targeted PAAD NPs\nexhibit markedly lower renal accumulation and substantially faster systemic clearance\ncompared with T-PAAD NPs. Without the megalin-mediated uptake mechanism, the\nnon-targeted nanoparticles reach the kidney at much lower levels, leading to an\nintratubular EMPA concentration that is insufficient to achieve a therapeutically\nmeaningful local exposure. Because the non-targeted formulation cannot deliver the\nrequired intrarenal drug levels, it would not serve as a meaningful therapeutic\ncomparator in this CKM model. For this reason, we focused our therapeutic evaluation\non comparing the optimized targeted nanoplatform (T-PAAD NPs/EMPA) with the\nclinical standard of care (free EMPA), which more clearly illustrates the translational\nrelevance and therapeutic advantages conferred by active renal targeting. Nonetheless,\nin response to the reviewer’s suggestion, we have added explicit clarification in the\nrevised manuscript to articulate this rationale and to explain how the biodistribution\nfindings directly informed our efficacy study design.\ndependent biodistribution profiles of targeted versus non-targeted nanoparticles. Ex\nvivo fluorescence images of major organs (heart, liver, spleen, lung, and kidney)\nharvested from CKM mice at 6, 12, and 24 h following the administration of IR780-\nloaded T-PAAD NPs (targeted) or PAAD NPs (non-targeted). b, Quantitative analysis\nof the mean fluorescence intensity of the major organs in the ex vivo images over time.\nc, Quantitative analysis of the average fluorescence intensity specifically in the kidneys\nat different time point. Data points are means ± s.d. (n = 3). Two-tailed Student’s t-test\nwas used for statistical analysis (c). **P < 0.01 and ***P < 0.001.\n4. Insufficient explanation of cardiac functional improvement\nFig. 5 demonstrates cardiac functional recovery with T-PAAD/EMPA, but the\nunderlying basis of this effect is not clearly explained. It remains uncertain whether the\nobserved improvement reflects secondary benefits from renal protection, systemic\nmetabolic changes, or direct antioxidant actions. The discussion does not adequately\nelaborate on these possibilities, and a clearer explanation of the likely mechanism(s)\nwould strengthen the manuscript.\nResponse: We appreciate the reviewer’s thoughtful comment regarding the\nmechanisms underlying the observed cardiac functional improvement and agree that a\nclearer mechanistic explanation is warranted. In the revised manuscript, we have\nexpanded the discussion to explicitly clarify that the cardiac benefits are multifactorial\nand largely reflect the restoration of the cardio-renal metabolic axis, rather than a direct\ncardiotropic effect of the nanoparticles.\nSpecifically, the cardiac recovery is primarily attributable to two interconnected\nsecondary mechanisms:\nnatriuresis reduce systemic blood pressure and cardiac afterload, thereby alleviating the\nhemodynamic stress that drives CKM-associated cardiac dysfunction. For example,\nenhanced natriuresis and diuresis with T-PAAD NPs/EMPA led to significantly lower\nblood pressure in CKM mice. After 4 weeks, treated mice’s systolic/diastolic pressures\ndropped to ~118/79 mmHg, versus ~142/95 mmHg in CKM controls (and ~133/85\nmmHg with free EMPA). This reduction in afterload and volume overload (a\nhemodynamic unloading effect) relieves cardiac stress, allowing the heart to pump\nmore efficiently.\nSecond, systemic metabolic correction: T-PAAD NPs/EMPA significantly lowers\ncirculating glucose and lipid levels, reducing myocardial glucolipotoxicity and\nimproving substrate utilization. Importantly, our multi-omics analysis (Fig. 7) provides\nmolecular evidence supporting these systemic contributions, demonstrating that the\ncombined hemodynamic and metabolic improvements synergistically promote cardiac\nenergy reprogramming, including the restoration of fatty acid oxidation (FAO)\npathways and ATP production. This mechanistic profile aligns with the known energy-\nstarvation phenotype of CKM-induced heart failure and explains the observed\nimprovement in cardiac function.\nIn summary, by unloading the heart (via improved renal function and blood\npressure) and correcting systemic metabolism (via sustained glycosuria and lipid\nreduction), our therapy rescued both systolic and diastolic function. Additionally, we\nhave expanded the discussion to explicitly link the renal and metabolic improvements\nto the observed cardiac benefits.\n5. Interpretation of hepatic lipid metabolism improvement\nFig.6 shows dramatic improvements in hepatic steatosis and serum lipid profile, far\ngreater than typically reported in clinical trials of SGLT2 inhibitors (which usually\nshow modest LDL↑, HDL↑, TG↓, and mild NAFLD improvement). The mechanism\nlinking kidney-targeted EMPA delivery to liver outcomes is not explained. In addition,\nfood intake data are absent, and body weight data—though reportedly monitored—are\n24\nnot shown. These omissions make it impossible to exclude confounding by energy\nbalance.\nResponse: We appreciate the reviewer’s rigorous assessment and agree that the hepatic\nmetabolic improvements observed in Fig. 6 are remarkably pronounced compared with\nthe modest NAFLD benefits typically reported for orally administered SGLT2\ninhibitors. We have now clarified in the revised manuscript that this enhanced effect\narises from the unique pharmacokinetic advantages of kidney-targeted EMPA delivery\nafforded by the T-PAAD nanoplatform, rather than from systemic confounding factors.\nBy achieving substantially higher local EMPA concentrations at the proximal\ntubules, the nanoplatform markedly amplifies urinary glucose excretion (glycosuria).\nThis produces a stronger and more sustained systemic negative-energy-balance signal\nthan is achievable with free EMPA, which undergoes substantial presystemic and\nhepatic metabolism. The intensified glycosuric drive forces a metabolic shift from\nhepatic lipogenesis toward lipid mobilization and enhanced fatty acid oxidation (FAO)\nto meet systemic energy demands. This mechanistic link is directly supported by our\nmulti-omics analysis (Fig. 7), which demonstrates robust upregulation of FAO-related\npathways and restoration of hepatic ATP production, consistent with effective reversal\nof lipid overload.\nTo address the reviewer’s concern regarding potential confounding by energy\nintake, we have now included the full monitoring dataset (body weight and food intake).\nAs shown in Supplementary Fig. 19, food intake remained stable and comparable\namong groups, indicating that reduced caloric consumption did not contribute to the\nobserved hepatic improvements. Moreover, body weight trajectories (Supplementary\nFig. 19) confirm that the T-PAAD/EMPA group maintained physiological weight\nstability, in contrast to the pathological weight loss seen in untreated CKM mice. These\nfindings rule out dietary restriction or cachexia as drivers of the improved hepatic\nphenotype and support the conclusion that targeted metabolic reprogramming, rather\nthan altered energy balance, underlies the dramatic improvement in hepatic lipid\nmetabolism.\nintake monitoring of mice in different groups during the 4-week therapeutic\nintervention period. b, Dynamic changes in body weight of mice in different groups\nthroughout the experimental period (from -1 to 8 weeks). Data are presented as mean ±\ns.d. (n=5).\n6.Limitations in multi-omics analysis\nOmics data (Fig.7) are presented for CKM vs. T-PAAD/EMPA only, without free EMPA\nor non-targeted NP controls. Thus, it is impossible to determine whether the observed\nchanges are specific to kidney-targeted delivery or general EMPA effects. The\npolymer’s intrinsic antioxidant contribution cannot be ruled out. Proper control groups\nmust be included.\nResponse: We appreciate the reviewer’s rigorous critique regarding the interpretation\nof the omics data. In our untargeted metabolomics, we included a free EMPA group,\nand the results showed that the metabolic profile of free EMPA-treated mice clustered\nalmost identically with that of untreated CKM mice. In other words, free EMPA\nproduced minimal shifts in the global metabolite patterns, underscoring that significant\nmetabolic remodeling only occurred with the T-PAAD NPs/EMPA therapy. This\njustifies why our figures highlight the T-PAAD NPs/EMPA vs. CKM contrast\n(Supplementary Fig. 23). We have clarified this point in the revision, mentioning that\nfree EMPA did not substantially alter key pathways, which is why those data were not\nshown in the main multi-omics figures.\nnew in vivo data (Supplementary Fig. 10) demonstrating that the vehicle-only control\n(Blank T-PAAD NPs) failed to improve glucose tolerance or lower blood glucose levels\ncompared to the CKM group. This indicates that the polymer itself does not confound\nthe omics results with off-target effects. The T-PAAD polymer was designed to be\nbiologically inert apart from its ROS-scavenging capability. Indeed, we observed that\nBlank T-PAAD NPs can mitigate oxidative stress due to the antioxidant DATS groups,\nbut they do not meaningfully influence glucose on their own. Collectively, combined\nwith the fact that T-PAAD NPs exhibited statistically superior therapeutic efficacy in\nall phenotypic “end-points” (e.g., blood glucose, blood pressure, and lipid profiles)\ncompared to free EMPA, we conclude that the omics changes in Fig. 7 reflect the\nenhanced efficacy specific to the pharmacological action of EMPA combined with\ntargeted delivery.\nSupplementary Fig. 10 | Evaluation of the hypoglycemic effect of blank T-PAAD\nNPs. a, Dynamic changes in fasting blood glucose levels monitored over 3 weeks. b,\nIntraperitoneal glucose tolerance test (IPGTT) performed after 3 weeks of treatment. c,\nCorresponding area under the curve (AUC) quantitative analysis calculated from the\nIPGTT data. Statistical analysis was performed with one-way ANOVA (c), and two-\nway ANOVA with Sidak multiple comparisons test (a, b). Data are presented as mean\n± s.d. (n=5). **P < 0.01; ns, not significant (compared to the CKM group).\n7.Off-target liver accumulation undermines kidney specificity\nBiodistribution (Fig.3c) shows not only kidney uptake but also strong liver\naccumulation, likely due to reticuloendothelial clearance. This undermines the claim of\nkidney specificity. The discussion does not address this limitation and instead\nframes hepatic accumulation as beneficial, which is not adequate.\nResponse: We appreciate the reviewer’s rigorous assessment and agree that hepatic\n27\naccumulation, primarily resulting from reticuloendothelial system (RES) clearance, is\na well-recognized limitation in nanoparticle delivery and does not reflect absolute\nkidney specificity. In the revised manuscript, we have adjusted our terminology\naccordingly, shifting from “kidney-specific delivery” to the more accurate “enhanced\nrenal targeting efficiency.” We explicitly clarify that, while complete renal exclusivity\nis inherently difficult to achieve for nanoscale systems, our biodistribution data\ndemonstrate that T-PAAD NPs achieve substantially higher renal accumulation and\nprolonged retention than non-targeted controls, which is the relevant metric for\nevaluating targeting performance.\nRegarding the concern that hepatic uptake was previously framed as beneficial,\nwe have revised the discussion to more appropriately contextualize this finding.\nAlthough liver accumulation is technically off-target with respect to kidney delivery,\nCKM syndrome is a systemic, multi-organ metabolic disorder in which hepatic steatosis\nis a central pathological feature. Therefore, EMPA delivered to the liver may exert\nancillary metabolic benefits, but this is presented as a secondary and opportunistic\neffect, not as justification for hepatic uptake or as evidence of targeting. Importantly,\nour toxicity analyses (ALT/AST) confirm that this hepatic exposure does not induce\nhepatotoxicity (Supplementary Fig. 26).\nTogether, these clarifications acknowledge the limitations of nanoparticle\nbiodistribution while accurately conveying that the enhanced renal selectivity of T-\nPAAD NPs-rather than hepatic accumulation-underpins the therapeutic rationale of our\ndelivery strategy.\n8. Biodistribution shown only at a single timepoint (Fig. 3c)\nOrgan accumulation is presented at only one timepoint. To more convincingly support\nkidney targeting, it would be preferable, if feasible, to include serial imaging at multiple\ntimepoints (e.g., 1 h, 4 h, 8 h, 24 h). A single snapshot is insufficient to substantiate\nclaims of selective renal retention.\nResponse: We sincerely thank the reviewer for this constructive suggestion and agree\nthat a longitudinal assessment provides stronger evidence of renal targeting. In response,\n28\nwe have performed additional ex vivo biodistribution experiments comparing the\naccumulation of targeted T-PAAD NPs vs. non-targeted PAAD NPs at multiple\ntimepoints (6 h, 12 h, and 24 h) post-injection (Supplementary Fig. 8). As evident\nfrom the serial imaging, T-PAAD NPs exhibited rapid accumulation and sustained\nretention specifically in the kidney tissue across the entire observation window, whereas\nthe non-targeted PAAD NPs displayed significantly lower renal fluorescence intensity.\nThese temporal dynamics robustly validate the superior kidney-targeting capability and\nextended retention of our peptide-modified nanoplatform compared to the non-targeted\ncontrol.\nSupplementary Fig. 8 | Time-dependent biodistribution assessment. a, Time-\ndependent biodistribution profiles of targeted versus non-targeted nanoparticles. Ex\nvivo fluorescence images of major organs (heart, liver, spleen, lung, and kidney)\nharvested from CKM mice at 6, 12, and 24 h following the administration of IR780-\nloaded T-PAAD NPs (targeted) or PAAD NPs (non-targeted). b, Quantitative analysis\nof the mean fluorescence intensity of the major organs in the ex vivo images over time.\nc, Quantitative analysis of the average fluorescence intensity specifically in the kidneys\nat different time point. Data points are means ± s.d. (n = 3). Two-tailed Student’s t-test\nwas used for statistical analysis (c). **P < 0.01 and ***P < 0.001.\n9. Unclear contribution of SGLT2 inhibition versus intrinsic antioxidant effects\nAcross the study, it remains uncertain whether the observed benefits-particularly in\nextra-renal organs such as the heart and liver-arise from enhanced SGLT2 inhibition by\nkidney-targeted EMPA or from the intrinsic antioxidant properties of the carrier itself.\nSince a vehicle-only T-PAAD control group is missing, the relative contributions of\ndrug versus carrier cannot be disentangled. Without this critical control, the conclusions\nattributing the therapeutic effects specifically to kidney-targeted SGLT2 inhibition\nremain inadequately supported.\nResponse: We sincerely thank the reviewer for identifying this critical control gap. We\nagree that distinguishing the contribution of the drug (EMPA) from the intrinsic\nantioxidant effects of the carrier is essential for supporting our conclusions. To address\nthis, we have performed additional in vivo experiments including a vehicle-only control\ngroup (T-PAAD NPs without EMPA). As shown in the newly added Supplementary Fig.\n10, the administration of blank T-PAAD NPs did not lead to any significant reduction\nin blood glucose levels or improvement in glucose tolerance (IPGTT) compared to the\nuntreated CKM group (indicated by “ns” in the figures). These results confirm that the\nnanocarrier alone, despite its local ROS-scavenging capabilities, is insufficient to\nreverse the systemic metabolic dysregulation (i.e., hyperglycemia and insulin resistance)\ncharacteristic of CKM. Consequently, the profound therapeutic efficacy observed in the\nT-PAAD NPs/EMPA group-including the beneficial remodeling of extra-renal organs\nlike the heart and liver-can be confidently attributed to the kidney-targeted SGLT2\ninhibition and the subsequent reprogramming of systemic energy metabolism. The\nintrinsic antioxidant property of the carrier serves as a synergistic, rather than the\nprimary, driver of therapeutic outcome.\nSupplementary Fig. 10 | Evaluation of the hypoglycemic effect of blank T-PAAD\nNPs. a, Dynamic changes in fasting blood glucose levels monitored over 3 weeks. b,\nIntraperitoneal glucose tolerance test (IPGTT) performed after 3 weeks of treatment. c,\nCorresponding area under the curve (AUC) quantitative analysis calculated from the\nIPGTT data. Statistical analysis was performed with one-way ANOVA (c), and two-\nway ANOVA with Sidak multiple comparisons test (a, b). Data are presented as mean\n± s.d. (n=5). **P < 0.01; ns, not significant (compared to the CKM group).\nWe are sincerely thankful for the comments provided by the editor and the reviewers,\nas well as for their valuable suggestions that greatly aided in the revision of our\nmanuscript. In this response, we address the reviewers’ comments and have highlighted\nall the revisions made in red for clarity.\nReviewer #3 (Remarks to the Author):\nI still have important concerns regarding the proposed mechanism that the nanocarrier\nreleases EMPA within the proximal tubule lumen, particularly in the S1 segment, in\nresponse to urea stimulation, thereby exerting site-specific SGLT2 inhibition. While the\nin vitro release assays are evaluated on an hourly timescale (hours to 24 h), tubular\ntransit of filtrate through the S1 segment in vivo occurs on the order of seconds. This\napparent mismatch between release kinetics and physiological transit time makes a\npurely luminal, urea-triggered release mechanism in S1 difficult to reconcile.\nMoreover, the immunofluorescence images suggest substantial cell-associated and/or\nintracellular localization of the nanoparticles in proximal tubular epithelial cells. Rather\nthan confinement to the tubular lumen. This raises the possibility that the observed\ntherapeutic effects may be mediated by alternative mechanisms, such as brush-border\nretention with slow release or intracellular release following cellular uptake, rather than\nrapid urea-triggered luminal release.\nIn addition, the urea nitrogen (UN) concentration within the S1 microenvironment of\nthe CKM mouse model remains unclear. It is not demonstrated whether urea levels\napproaching those used to induce accelerated release in vitro (approximately 50 mM)\nare physiologically attainable within the S1 segment in this model. Therefore. it remains\nto be clarified whether the observed in vivo effects are primarily driven by urea-\nresponsive luminal release, or whether non-urea-dependent processes play a dominant\nrole.\nIf direct experimental clarification of these points is not feasible, the authors should\nexplicitly acknowledge these limitations and clearly define the range of mechanisms\nthat are currently supported by the data. At a minimum, the limitations should address:\n(i) the mismatch between in vitro release kinetics and in vivo S1 transit time;\n(ii) the lack of direct evidence for luminal release within the S1 segment;\n(iii) the potential contribution of cellular uptake and intracellular or membrane-\nproximal release; and\n(iv) the absence of direct measurements of urea concentration within the S1\nmicroenvironment of the CKM model.\nResponse: We sincerely thank the reviewer for this insightful and important comment.\nWe fully agree that the tubular transit time through the S1 segment occurs on the order\nof seconds in vivo, whereas our in vitro release assays were conducted on an hourly\ntimescale. Accordingly, we acknowledge that the in vitro release kinetics should not be\ninterpreted as a direct temporal surrogate for instantaneous luminal release within the\nS1 segment. Importantly, our intention was not to suggest that EMPA is released\nexclusively or instantaneously within the tubular lumen during a single pass through\nS1. Rather, we propose a composite mechanism involving brush-border retention,\ncellular internalization, and pathology-enhanced release mechanism. We have revised\nthe manuscript to clarify that the urea-responsive behavior of the nanocarrier is intended\nto reflect stimulus sensitivity and release acceleration under pathological conditions,\nrather than a purely rapid, luminal-triggered release confined to S1.\nFirstly, regarding the kinetic mismatch and localization (Points i, ii, & iii):\nConsistent with the reviewer’s observation, the immunofluorescence data indicate\nsubstantial cell-associated and intracellular localization of the nanoparticles within\nproximal tubular epithelial cells. This supports a mechanism in which T-PAAD\nnanoparticles undergo apical brush-border retention and megalin-mediated endocytosis,\nfollowed by intracellular or pericellular drug release, rather than remaining confined to\nthe tubular lumen.\nSecondly, the urea-responsive property of the nanocarrier is best interpreted as a\npathology-amplified release modulator rather than a strictly luminal, S1-restricted\ntrigger. While tubular fluid residence time in S1 is short, nanoparticles that are retained\nat the brush border or internalized by proximal tubular cells are exposed to the local\nintracellular and pericellular microenvironment for substantially longer durations,\nallowing urea-induced weakening of hydrogen bonding within the UCST polymer to\nprogressively accelerate EMPA release.\nThirdly, regarding the physiological attainable urea concentration (Point iv): We\nagree that the precise urea concentration within the S1 microenvironment of the CKM\nmodel is difficult to measure directly. Regarding the urea concentration, our in vivo data\nprovides a physiological rationale for using elevated urea levels (up to ~50 mM) in our\nin vitro release assays. As shown in Figure R1, the CKM mouse model exhibits severe\nrenal impairment, evidenced by a significant decline in Glomerular Filtration Rate\n(GFR) and marked albuminuria (high Urinary Alb/Cre ratio). Furthermore, histological\nanalysis (PAS and H&E staining) confirms substantial tubular injury, including the loss\nof brush border integrity and glomerular damage. In the context of such severe filtration\ncompromise and tubular dysfunction, the clearance of nitrogenous waste is significantly\nimpaired, leading to local retention and concentration of urea within the damaged\nnephrons. Previous studies on uremic toxins define the physiological range of urea in\nsevere renal failure. According to Vanholder et al. (Ref. a), systemic urea concentrations\nin uremic conditions can fluctuate significantly and reach levels exceeding 40-50 mM.\nWhile the average systemic BUN in our CKM mice at the measured timepoint was\napprox. 20-25 mM (Figure R1f), local concentrations within obstructed or severely\ndamaged nephrons-where fluid stasis occurs-are likely higher. Therefore, the 50 mM\nconcentration was selected not to represent healthy physiological levels, but to simulate\nthe pathological microenvironment characteristic of severe kidney injury, where urea\naccumulation is expected to trigger the accelerated release of the drug. In vivo, we\nanticipate that urea-dependent and urea-independent processes (including cellular\nuptake, prolonged tubular retention, and intracellular release) jointly contribute to the\nobserved therapeutic efficacy. To reflect these points more accurately, we have revised\nthe manuscript to (i) avoid overstatement of a purely luminal, rapid S1 release\nmechanism, and (ii) explicitly describe a composite mechanism involving brush-border\nretention, cellular internalization, and pathology-enhanced release kinetics. We believe\nthis revised framework better reconciles the in vitro release data with renal physiology\nand the in vivo imaging observations.\nFinally, we acknowledge that the kidney is an organ of immense structural and\nfunctional complexity. While our current study provides robust evidence for the\ntherapeutic efficacy and proposed mechanism, we recognize that static in vitro assays\ncannot fully replicate the dynamic fluidic gradients and metabolic heterogeneity of the\nrenal tubule in vivo. In future investigations, we aim to employ more sophisticated\nmodels, such as kidney-on-a-chip microfluidic systems, to simulate the drug release\nprocess within the complex renal microenvironment with greater precision and\ngranularity. Meanwhile, we also addressed these limitations in the “Conclusion section”\nof the revised manuscript, which were highlighted in red for clarity.\n[a] Vanholder, R. et al. Review on uremic toxins: classification, concentration, and\ninterindividual variability. Kidney Int. 63, 1934-1943 (2003).\nFigure R1 | Evaluation of renal function and pathological changes in the CKM mouse\nmodel. a, Schematic illustration of non-invasive GFR measurement using FITC-\nsinistrin clearance. b, c, Longitudinal monitoring of GFR from week 0 to 4 (b) and\nendpoint comparison (c) between RD and CKM groups. d-g, Quantification of kidney\ninjury biomarkers: Urinary albumin-urinary creatinine ratio (urinary Alb/CRE) (a) and\nserum levels of CRE (b), BUN (c) and UA (d). h, Representative histological images\nof kidney tissue stained with PAS (scale bars, 50 µm), H&E (scale bars, 50 µm), and\nMasson’s trichrome (scale bars, 50 µm) showing glomerular morphology and fibrosis.\nData were shown as mean ± s.d. (n=3-5), and analyzed using two-tailed Student's t-test\n(b-g). *P < 0.05, ***P < 0.001."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Accept the comments", "reviewer_2": "The authors addressed all my comments.", "reviewer_3": "I still have important concerns regarding the proposed mechanism that the nanocarrier releases EMPA within the proximal\ntubule lumen, particularly in the S1 segment, in response to urea stimulation, thereby exerting site-specific SGLT2 inhibition. While the\nin vitro release assays are evaluated on an hourly timescale (hours to 24 h), tubular transit of filtrate through the S1 segment in vivo occurs on the order of seconds. This apparent mismatch between release kinetics and physiological transit time makes a\npurely luminal, urea-triggered release mechanism in S1 difficult to reconcile.\nMoreover, the immunofluorescence images suggest substantial cell-associated and/or intracellular localization of the\nnanoparticles in proximal tubular epithelial cells, rather than confinement to the tubular lumen. This raises the possibility that the observed\ntherapeutic effects may be mediated by alternative mechanisms, such as brush-border retention with slow release or intracellular release following cellular uptake, rather than rapid urea-triggered luminal release.\nIn addition, the urea nitrogen (UN) concentration within the S1 microenvironment of the CKM mouse model remains unclear.\nIt is not demonstrated whether urea levels approaching those used to induce accelerated release in vitro (approximately 50\nmM) are physiologically attainable within the S1 segment in this model. Therefore, it remains to be clarified whether the\nobserved in vivo effects are primarily driven by urea-responsive luminal release, or whether non–urea-dependent processes\nplay a dominant role.\nIf direct experimental clarification of these points is not feasible, the authors should explicitly acknowledge these limitations\nand clearly define the range of mechanisms that are currently supported by the data. At a minimum, the limitations should\naddress:\n(i) the mismatch between in vitro release kinetics and in vivo S1 transit time;\n(ii) the lack of direct evidence for luminal release within the S1 segment;\n(iii) the potential contribution of cellular uptake and intracellular or membrane-proximal release; and\n(iv) the absence of direct measurements of urea concentration within the S1 microenvironment of the CKM model."}, "author_response": "Response to reviewers’ and editor’s comments:\nWe are sincerely thankful for the comments provided by the editor and the reviewers,\nas well as for their valuable suggestions that greatly aided in the revision of our\nmanuscript. In this response, we address the reviewers’ comments and have highlighted\nall the revisions made in red for clarity."}, {"round": 3, "reviewer_comments": {"reviewer_3": "I appreciate the authors’ thorough revisions, which have largely resolved my major concerns. I do not believe that additional\nexperimental validation is strictly required for the current manuscript. Nevertheless, given the high level of interest and\nconceptual novelty of this work, a more definitive clarification of the EMPA release mechanism would have further enhanced the\nmechanistic depth of the study and will be of great interest in future investigations."}, "author_response": "Response: We sincerely thank the reviewer for the positive assessment of our work and\nfor recognizing the conceptual novelty of the urea-responsive nanoplatform. We are\nparticularly grateful that our previous revisions have addressed the reviewer’s major\nconcerns.\nRegarding the EMPA release mechanism, we fully agree that a more definitive\nclarification of the in vivo release process would be of substantial scientific interest.\nWhile our current results support urea-triggered release mediated by intramolecular\nhydrogen-bond disruption, we acknowledge that the in vivo behavior is likely\nmodulated by additional factors, including tubular fluid dynamics, epithelial\ninteractions, and cellular uptake processes.\nFurthermore, our future research directions will aim to employ more\nphysiologically relevant experimental platforms-such as advanced microfluidic models\nand high-resolution in vivo tracking methods-to further dissect the spatiotemporal\ndynamics of nanocarrier disassembly and drug release within the renal\nmicroenvironment. We would like to reiterate our gratitude to the reviewer for this\ninsightful and constructive suggestion, which provides a clearer framework for our\nfuture investigations."}]

Decision letter