Pyrimidinergic calcium signaling links tubular metabolism to fibrosis in kidney disease

Domain: chemistry
Published: 2026-02-19
Editorial outcome: minor_revision

Abstract
Chronic kidney disease (CKD) is a major global health problem, with substantial associated morbidity and mortality. Fibrosis is the final common pathway of organ damage in CKD, so understanding how this arises during kidney injury is critical for building a holistic picture of the pathogenesis. Here, using gene expression data, intravital microscopy in mice and realistic cell models, we uncover evidence of a signaling pathway linking tubular pyrimidine metabolism and injury-evoked extracellular uridine diphosphate (UDP) release to activation of the P2Y6 receptor (P2Y6R) in surrounding fibroblasts. We show that P2Y6R activation triggers intracellular calcium rises, which stimulate fibroblast proliferation, migration, and conversion towards a myofibroblast phenotype. Conversely, genetic knockout or pharmacological blockade of the P2Y6R reduces fibrosis in mice with CKD. Thus, we reveal that pyrimidinergic calcium signaling couples fibroblast responses to changes in tubular metabolism in disease states, and represents a potential new target for therapeutic intervention.

Introduction
Chronic kidney disease (CKD) affects 1 in 10 people and can lead to kidney failure, which has major adverse effects on health systems, economies, and the environment1,2. CKD is a strong independent risk factor for cardiovascular disease and is predicted to become the 5th leading cause of death by 20403, so there is a pressing need to unravel the pathophysiology of CKD and develop new therapies.

The proximal tubule is the workhorse of the kidney, responsible for reabsorbing most of the glomerular filtrate. Metabolism in the proximal tubule is complex and closely entwined with solute transport4, and can change substantially in disease states5–7. Interstitial fibroblasts surround proximal tubules with long processes8; activation of these cells and transformation to a myofibroblast phenotype is a critical step in CKD progression, which leads to secretion of collagen and other extracellular matrix proteins, and ultimately replacement of healthy parenchyma7,9–12. Extracellular sensing of metabolite release could provide a possible mechanism to couple fibroblast responses with alterations in tubular activity.

Pyrimidines have multiple important roles in cell biology and can be generated by de novo synthesis or salvage pathways, with the latter generally predominating in differentiated cells13. Here, using gene expression analysis, we find evidence that pyrimidine metabolism is active in proximal tubules, and when injured, they upregulate a key enzyme in the pyrimidine salvage pathway. Moreover, induction of damage in cultured proximal tubular cells causes UDP release into the extracellular environment. Furthermore, we show that the target receptor for UDP (the P2Y6 receptor [P2Y6R]) is present in neighboring fibroblasts and that its expression increases in disease models. Thus, pyrimidinergic signaling represents a plausible pathway for tubulo-interstitial crosstalk in CKD.

Results
Pyrimidine metabolism is highly active in proximal tubules, which release UDP when injured

We began by interrogating a single-cell sequencing dataset derived from mice with renal ischemia-reperfusion injury14–16, and identified a high expression of genes involved in pyrimidine metabolism in proximal tubules (Fig. 1a, Supplementary Fig. 1). Moreover, both healthy tubules and emerging new and injured cell populations expressed cda (Fig. 1b), which encodes the key pyrimidine salvage pathway enzyme cytidine deaminase (CDA) that converts cytidine to uridine to maintain the cellular UDP pool17. Furthermore, antibody staining of fixed kidneys in two mouse models of CKD—unilateral ureteric obstruction (UUO) and folic acid nephropathy (FAN)—revealed a dramatic increase in abundance of CDA in aquaporin-1 positive proximal tubules, with evidence of activation of surrounding fibroblasts (increased expression of alpha smooth muscle actin [αSMA]) (Fig. 1c and Supplementary Fig. 2).

Expression of cda was also detected in human proximal tubule-derived cells (HK-2) in culture and increased significantly after induction of injury with cisplatin or folic acid (Fig. 1d). Crucially, this was accompanied by a rise in extracellular UDP concentration, denoting release from cells (Fig. 1e). However, due to technical constraints, we were not able to assess UDP release from tubules in vivo.

Expression of the P2Y6R increases in kidney fibroblasts in disease states

These data suggest that injured proximal tubular cells produce and release UDP, which is the endogenous ligand for the P2Y6R. We therefore analyzed the renal expression of this receptor in single-cell databases and found that it is not only expressed in pdgfrb positive stromal cells, but that its expression increases in models of CKD caused by ischemia-reperfusion injury14–16 or glomerulopathy (POD-ATTAC)18 (Fig. 2a, b and Supplementary Figs. 3 and 4). Although significant, changes in P2Y6R expression in these datasets were quite small; however, much larger increases were confirmed at the whole kidney level by RT-qPCR in mice with UUO or FAN, with global P2Y6R knockout (KO) mice used as a control19 (Fig. 2c, d).

Activation of the P2Y6R can induce calcium release within cells via phospholipase C20. To investigate the effects of P2Y6R stimulation on calcium signals in kidney tissue we used freshly cut slices from the kidneys of transgenic mice expressing the fluorescent calcium reporter GCaMP6s21, and observed that application of either UDP or a synthetic agonist (MRS2957)22 induced calcium rises in interstitial cells (Supplementary Fig. 5), which were inhibited by the non-specific purinergic blocker suramin or a specific P2Y6 antagonist (MRS2578) (Fig. 2e–g). In addition, both agonists stimulated increases in intracellular calcium in cultured rat renal fibroblasts transfected with GCaMP6s (NRK-49F), which appeared initially in cell processes before propagating towards the cell body (Fig. 2h–k).

In summary, these data indicate a high level of pyrimidinergic signaling machinery in the tubulo-interstitial compartment, which potentially ramps up in disease states.

Activation of the P2Y6 receptor stimulates proliferation, migration, and pro-fibrotic gene expression in renal fibroblasts

To investigate the functional consequences of activating the P2Y6R in renal fibroblasts, we incubated NRK-49F cells with a P2Y6R agonist (MRS2957) for 24 h and found that this significantly increased cell proliferation and migration compared to controls (Fig. 3a, b). Blockade of the P2Y6R with an antagonist (MRS2578) abolished these effects, as did chelation of intracellular calcium with BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (Fig. 3c, d), indicating a dependence on calcium release. In addition, P2Y6R activation increased expression of genes encoding the proliferation marker MKI67 and the pro-fibrotic markers PDGFRβ, αSMA, fibronectin, vimentin, and collagen 1 (Fig. 3e). Finally, increased abundance of fibronectin and vimentin was confirmed at protein level after 72 h with antibody staining (Fig. 3f, g). In contrast, knocking down the P2Y6R in NRK-49F cells with siRNA blunted agonist effects on cell proliferation and activation of fibrosis markers (Supplementary Fig. 6).

Fibroblasts in mouse kidneys display spontaneous calcium signals in processes surrounding tubules

Having identified that injured tubules release UDP and that this can induce calcium rises in renal fibroblasts in vitro or ex vivo, we proceeded to investigate the nature of fibroblast calcium signals in vivo by performing intravital multiphoton microscopy in GCaMP6s-expressing mice. To confirm expression of GCaMP6s in fibroblasts, we used antibody staining in fixed tissue for the well-established marker Platelet Derived Growth Factor Receptor beta (PDGFRβ), which labeled a population of interstitial cells distinct from MHC class II positive immune cells (Supplementary Fig. 7). During these experiments, we observed spontaneous slow calcium transients arising in proximal tubular cells, as previously reported21. In addition, we noted spontaneous calcium rises in interstitial cells surrounding tubules and exhibiting a fibroblast morphology (Fig. 4a–c). To confirm their identity, we marked the region of live imaging, fixed and sliced the tissue, and retrospectively stained it for PDGFRβ. Visualization of calcium traces from PDGFRβ-positive cells revealed a different dynamic from proximal tubules. In contrast to periodic, discrete transients, fibroblast calcium signals were more frequent and noisier (Fig. 4d, e).

To elucidate the spatiotemporal pattern of intracellular calcium signals in renal fibroblasts in more detail, we imaged them in freshly cut slices of mouse kidney tissue, which allowed us to visualize cells with multiple processes contained within the same field of view (Fig. 4f–i). From this, we could ascertain that calcium signals in fibroblasts arose in processes, before propagating across cells (Fig. 4j and Supplementary Movie 1).

Taken together, these findings indicate that calcium signals in renal fibroblasts comprise (relatively) fast and irregular transients originating in processes wrapping around tubules, which raises the possibility that they could be triggered by signaling factors emanating from the latter.

Calcium activity in kidney fibroblasts increases in mouse models of tubular injury and kidney fibrosis

Next, we wanted to know if tubular injury stimulates calcium signaling in fibroblasts. To investigate this, we injected GCaMP6s-expressing mice with the tubular toxin cisplatin (20 mg/kg, intraperitoneal), according to an established protocol21. After 72 h, we observed that calcium transients were reduced in frequency in proximal tubules, denoting epithelial damage (Fig. 5b, c, e). Conversely, calcium activity in surrounding fibroblasts was significantly increased, indicating a signaling response (Fig. 5d and Supplementary Movie 2).

To explore the nature of calcium signaling in renal fibrosis during CKD, we used the UUO model. After 7 days, filtration and tubular uptake of a small fluorescently labeled dextran was severely impaired in UUO mice, signifying a considerable defect in kidney function, and calcium transients were almost completely abolished in proximal tubules (Fig. 5f, g, i). In contrast, calcium signaling was increased in fibroblasts (Fig. 5h and Supplementary Movie 3), and this was associated with proliferation of PDGFRβ-positive cells and elevated expression of αSMA, indicating activation and conversion to a myofibroblast phenotype (Fig. 5j–m).

Knockout or inhibition of the P2Y6 receptor decreases kidney fibrosis in ureteric obstruction

Having uncovered evidence of a signaling pathway involving UDP activation of the P2Y6R in renal fibroblasts, we wanted to know if disrupting this would impact on fibrosis. We therefore used global P2Y6R KO mice19 and subjected them to UUO for 7 days. In comparison to wild-type littermates, obstructed kidneys in KO mice displayed less fibroblast proliferation, lower expression of fibronectin, αSMA, and collagen 1, fewer infiltrating macrophages, and decreased total area of fibrosis (measured using Sirius red staining) (Fig. 6).

Next, to investigate the consequences of blocking the P2Y6R with a pharmacological approach, we performed 7-day UUO experiments in wild-type mice treated with daily injections of either a P2Y6R antagonist (MRS2578) or placebo, and found that the former produced similar beneficial effects on renal fibrosis to P2Y6 KO (Fig. 7). Moreover, P2Y6R blockade also decreased calcium signaling in fibroblasts in GCaMP6s-expressing mice subjected to UUO (Supplementary Fig. 8).

Knockout or inhibition of the P2Y6 receptor decreases kidney fibrosis in folic acid nephropathy

To validate findings with the UUO model, we repeated experiments in FAN and observed that kidneys in KO animals displayed lower fibroblast proliferation and expression of pro-fibrotic markers, decreased macrophage infiltration, and a smaller total area of fibrosis (Fig. 8a–c). Moreover, blood urea and creatinine levels were significantly lower in KO mice, denoting an improvement in kidney excretory function (Fig. 8d).

Finally, we performed folic acid experiments in wild-type mice treated either with a P2Y6R antagonist (MRS2578) or vehicle and observed that the former were protected against renal fibrosis (Fig. 9a–c). Mean urea and creatinine levels were lower in antagonist-treated mice, but differences between groups were not statistically significant, due to a high variability in values within treatment groups (Fig. 9d).

To conclude, these results demonstrate that blocking the UDP-P2Y6R signaling axis is protective against the development of renal fibrosis in mice with CKD.

Methods
Reagents
Unless stated otherwise, all reagents were purchased from Sigma Aldrich.

Dyes
Dextran Alexa 647 (D22914, Thermo Fisher Scientific) was purchased from the manufacturer.

Cell culture
Normal rat kidney fibroblasts (NRK-49F, CRL-1570, ATCC) were cultured at 37°C in a 5% CO2 humidified atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 5% fetal bovine serum and 1% penicillin/streptomycin. Human cortical proximal tubular cells (HK-2, CRL-2190, ATCC) were cultured in DMEM/F12 media supplemented with REGM Renal Epithelial Cell Growth Medium BulletKit (Lonza), 1% L-Glutamine, and 1% Pen Strep at 37°C in a 5% CO2 humidified atmosphere.

Cisplatin and folic acid cell models
HK-2 cells were seeded as previously described for both cisplatin and folic acid treatments31. Briefly, at 80% confluency, cells were washed with PBS and treated with 20 µM cisplatin and NaCl, as well as 1 mM folic acid and NaHCO3 in supplement-free DMEM/F12 media. For cisplatin experiments, the cells were incubated for 24–48 h. For folic acid experiments, the cells were incubated for 72 h. Extracellular UDP was quantified with AffiASSAY MicroMolar UDP assay kit (AFG-PRF-266, AffiGEN), by following the manufacturer’s protocol. Fluorescence was detected on a microplate reader (Cytation 5, BioTek), and UDP concentrations were quantified by using a standard UDP curve.

Fibroblast proliferation experiments
NRK-49F cells were grown in 96-well plates (655087, Greiner) adjusted for fluorescence image quality for 72 h in culturing medium. The effect of P2Y6R activation on fibroblast proliferation was examined by adding either 50 μM UDP or 5 μM P2Y6 specific agonist (MRS2957, Cat. No. 4260, Tocris) to serum-free medium for 24 h. Bonding and removal of intracellular calcium ions was achieved by incubation with 20 μM calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, LuBioScience GmbH) for 30 min at 37°C in a 5% CO2 humidified atmosphere. P2Y6R inhibition was achieved by adding 1 μM specific P2Y6R antagonist (MRS2578, Cat. No. 2146, Tocris) to medium containing a P2Y6 receptor agonist (MRS2957). After treatment, NRK-49F cells were washed with pre-warmed (37°C) PBS before being fixed with pre-warmed 4% PFA prepared in PBS for 10 min. Fixed cells were washed with PBS several times and stained with a blue, fluorescent nuclear stain (Hoechst 33342, Sigma). Thereafter, the cell number was counted using a wide field fluorescence microscope and a fully automated high-content imaging system (INCell Analyzer 2500HS, GE).

Fibroblast migration analysis (in vitro scratch assay)
The effect of P2Y6R activation on fibroblast migration was investigated by a well-established scratch assay32. NRK-49F cells were grown in a 12-well plate until confluency, followed by scraping the cell monolayer with a p200 pipette tip in a straight line. The debris was washed with PBS, and cells were further incubated in serum-free medium for 24 h containing either 50 μM UDP or 5 μM P2Y6R specific agonist (MRS2957). Images were captured at 0 h and 24 h with Personal AUtomated Lab Assistant PAULA Cell imager (Leica Microsystems).

Analysis of pro-fibrotic protein expression in fibroblasts
NRK-49F cells were grown in 8-well removable chamber slides for immunofluorescence (80841-IBI, Ibidi) for 72 h. Subsequently, cells were incubated for another 72 h in serum-free medium containing 10 μM P2Y6R specific agonist (MRS2957). After treatment, NRK-49F cells were washed with pre-warmed (37°C) PBS before being fixed with pre-warmed 4% PFA prepared in PBS for 10 min. Fixed cells were washed with PBS several times, and permeabilized for 5 min with 0.1% Triton X-100 in prepared in PBS at room temperature. After washing with PBS, cells were blocked at room temperature for 30 min with 10% donkey and/or goat serum prepared in 1% BSA in PBS. Cells were incubated with the following primary antibodies: mouse vimentin (a kind gift from Prof. Johannes Loffing, University of Zurich, 1:50) and rabbit fibronectin (Sigma, F3648, 1:150) overnight at 4°C. On the following day, cells were incubated at room temperature for 2 h with suitable secondary antibodies: donkey anti-rabbit-AF647 (711-606-152, Jackson ImmunoResearch Europe Ltd, 1:100) and goat anti-mouse-AF568 (A-11004, Thermo Fisher Scientific, 1:100). Imaging was performed on an inverted confocal laser scanning microscope ZEISS LSM 980 Airyscan by using an LD LCI Plan-Apochromat 25×/0.8 silicon-oil objective.

Live cell imaging of calcium signals in cultured fibroblasts
NRK-49F cells were grown in 8-well chamber slides for 72 h. Thereafter, transfection with the GCaMP6s plasmid (pGP-CMV-GCaMP6s, #40753, Addgene) followed. After 48 h, cells were perfused with 10 μM P2Y6R agonist (MRS2957) and changes in fluorescence intensity were detected by an inverted confocal laser scanning microscope ZEISS LSM 980 Airyscan by using an LD LCI Plan-Apochromat 25x/0.8 water objective. GCaMP6s signal was imaged at 488 nm, and images were recorded every 1.40 s.

P2Y6 receptor knockdown in fibroblasts
The silencing of the receptor was performed by transfecting wild-type NRK-49F cells with Dicer-substrate short interfering RNAs (DsiRNAs) targeting P2RY6 (TriFECTa RNAi Kit; IDT Technology) using the Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific). Briefly, for RT-qPCR experiments, 5×105 cells were transfected according to the manufacturer’s protocol using 10 nM of P2RY6 DsiRNA and a scramble guide as a control for 48 h. The DsiRNA effect was assessed using RT-qPCR. The sequence of DsiRNA is listed in Supplementary Table S1. The same protocols used for fibroblast proliferation and pro-fibrotic marker expression in wild-type NRK-49F cells were repeated in P2RY6-silenced NRK-49F fibroblasts (NRK-49FP2RY6KD). The effect of P2RY6 knockdown on fibroblast proliferation was assessed after incubation of NRK-49FP2RY6KD for 24 h and on pro-fibrotic markers expression for 72 h with 10 μM P2Y6 receptor agonist (during the project, MRS2957 was discontinued by the manufacturer, so in these experiments MRS2693 (Cat. No. 2502, Tocris) was used as a replacement). P2Y6R expression was detected by P2Y6-FITC antibody (Tresars, 1:100).

RNA extraction, cDNA reverse transcription, and real-time quantitative PCR (RT-qPCR) in cell lines
RNA was extracted, according to the manufacturer’s protocol, using the miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen). RNA concentration was determined with NanoDrop™ One (Thermo Fisher Scientific) and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s protocol. 500 nM of both the sense and anti-sense primers were added to the PowerUP SYBR Green Master Mix (Applied Biosystems). RT-qPCR was performed using a LightCycler 480 system (Roche). For quantification, the relative change of target genes was calculated over that of the housekeeping gene (GAPDH) using the 2-ΔΔCT formula33. Specific primers were designed for rat (P2RY6, FN1, VIM, COL1A1, ACTA2, PDGFRB, MKI67, and GAPDH) and human targets (NGAL, KIM1, CDA, and GAPDH) and are listed in Supplementary Table S2.

Animals
C57BL6 mice. Experiments were performed on 8- to 15-week-old male C57BL mice (supplied by Janvier), in accordance with the regulations of the Zurich cantonal veterinary office (License No. ZH021/2015 and License No. ZH140/2023). Mice were euthanized 7 days following UUO or 14 days following FAN.

GCaMP6s mice. GCaMP6s mice were created by breeding Ai96 (RCL-GCaMP6s)34 (JAX stock #024106) with CMV-Cre mice35 (JAX stock #006054). GCaMP6s+/CMV-Cre+ (GCaMP6s) mice are characterized and described previously21. Experiments were performed on 8- to 23-week-old male and female GCaMP6s mice, in accordance with the regulations of the Zurich cantonal veterinary office (License No. ZH021/2015 and License No. ZH194/16). Mice were euthanized 3 days after inducing cisplatin nephrotoxicity or 7 days following UUO.

P2Y6 receptor knockout mice. An established mouse line with a global, constitutive depletion of the P2y6r on a C57Bl6 background was generated in IRIBHM (University of Brussels, Belgium), characterized, and described previously36. Experiments in these mice were conducted according to the Ethical Committee of Animal Well-Being in Brussels, license number 25GOSIRIBHM. Experiments were performed on 8- to 15-week-old male KO mice and their wild-type littermates. Mice were euthanized 7 days following UUO or 14 days following FAN.

RNA extraction, cDNA reverse transcription, and real-time quantitative PCR (RT-qPCR) in the mouse kidney
Kidneys were homogenized in 1 ml of Tri Reagent® (Invitrogen). Total RNAs were isolated according to the manufacturer’s instructions. After a 30 min DNAse (Thermo Fisher Scientific) treatment, a reverse transcription of 1 µg total RNA was performed using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) using random hexamers. Quantitative RT-PCR was performed with iTaq™ Universal SYBR® Green Supermix (Bio-Rad Laboratories) according to the manufacturer’s instructions on the iCycler C1000 Real-Time PCR Detection System (Bio-Rad Laboratories). Primers sequences were listed in the Supplementary Table S2. All PCRs were performed in duplicate, and normalized mRNA expressions were calculated by the 2ΔCq method using HPRT and RPL32 as internal references: normalized expression = 2((CqHPRT+CqRPL32)/2)-CqP2RY6.

Imaging of freshly cut kidney slices
Mice were anesthetized with intraperitoneal injection of ketamine (170 mg/kg) and xylazine (10 mg/kg), and the kidneys were harvested. After the kidney capsule was removed, the kidney was mounted and cut with a vibratome (Microm HM650V, Thermo Scientific, Waltham, Massachusetts, USA) into 280 µm thick longitudinal sections. The slices were maintained until imaging at 4°C in a physiological buffer containing (in mM): 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.8 CaCl2, 1.44 MgSO4, 5 glucose, 10 NaHCO3, 10 HEPES, 5 sodium pyruvate, 2.5 sodium butyrate and 2.5 sodium lactate, adjusted to pH 7.4 and gassed with carbogen (95% O2/5% CO2). Thereafter, slices were placed into a heated chamber at 37°C (Warner Instruments, Hamden, Connecticut, USA) filled with gassed slice buffer for live imaging with continuous perfusion. For live imaging of kidney medullary slices, 50 μM UDP, 50 μM suramin, 10 μM MRS2975 (Tocris), and 10 μM MRS2578 (Tocris) were dissolved in physiological buffer. Video recordings were performed with a Multiphoton-Leica SP8 DIVE FALCON supplied with a Harmonic Compound Infrared Apochromatic L (HCIRAPOL) 25×/1.0 W mot CORR objective and an Insigth DS+ Dual (680–1300 nm and 1041 nm) ultrafast Near-Infrared (NIR) laser system. GCaMP6s was excited at 950 nm and visualized at 500–550 nm. GCaMP6s signal was captured every 0.43 s. After imaging, the area imaged was marked with a laser to enable later recognition, and slices were fixed in 4% PFA for immunofluorescence staining.

Intravital imaging
Following induction of general anesthesia with inhaled isoflurane (1.5–5%) and oxygen (600 ml/min), the internal jugular vein was cannulated to permit intravenous injections of reagents and dyes. The left kidney was externalized for imaging as described previously37. Mice were positioned on a custom-built temperature-controlled stage with monitoring of body temperature during experiments. Imaging was conducted using a custom-built multiphoton microscope performing in an inverted configuration38, and equipped with a broadband tunable laser (InSight DeepSee Dual Ultrafast Ti:Sapphire, Spectraphysics). Intravital imaging was achieved with an XLPlan N ×25/1.05 water immersion objective (Olympus), and emitted light was collected through four gallium-arsenide-phosphide photomultiplier tubes (Hamamatsu) in a non-descanned epifluorescence detection mode. GCaMP6s and Dextran Alexa 647 (2 mg/kg) signals were imaged at 950 nm and 1120 nm, respectively. Signals were acquired every 1.35 s. After imaging, the area was marked with the laser to enable later recognition, and kidney sections were fixed in 4% PFA for immunofluorescence staining.

Analysis of calcium signals
To analyze calcium activity in renal fibroblasts, recorded videos were first processed using FIJI image analysis software, and regions of interest were manually drawn. An arbitrary threshold for detection of calcium transients was set as 2× the lowest fluorescence intensity recorded. Thereafter, calcium activity was evaluated as a percentage of fluorescence signal above the threshold.

Kidney disease models
Murine model of cisplatin-induced acute kidney injury. GCaMP6s mice were given Cis-diamminedichloroplatinum(II) (cisplatin, 20 mg/kg body weight) or vehicle (0.9% saline) by intraperitoneal injection, followed by imaging 72 h after treatment21.

Murine model of unilateral ureteric obstruction (UUO). The left ureter was ligated to produce blockage of one kidney (hydronephrosis) using previously established proocols39. The procedure took place under general anesthesia (isoflurane) and analgesia (buprenorphine 0.1 mg/kg via s.c. injection). Mice were transferred to a warming chamber for recovery and kept there for 1 day postoperatively. After 7 days, animals underwent intravital imaging under general anesthesia. To investigate the effect of P2Y6R inhibition, mice were injected with the antagonist MRS2578 (3 mg/kg, i.p.) 5 h after the ureteric ligation, and once daily until day 7.

Murine model of folic acid nephropathy (FAN). To induce CKD, mice received a single injection of folic acid (250 mg/kg, i.p.) as previously described40. To investigate the effect of P2Y6R inhibition, C57BL6 mice were injected with the antagonist MRS2578 (3 mg/kg, i.p.) 5 h after folic acid injection, and once daily until day 14. A kinetic UV test for the quantitative determination of urea and an enzymatic assay for the quantitative determination of creatinine in mouse serum were performed using an AU480 Clinical Chemistry System, Beckman Coulter.

Histological and immunofluorescence staining of kidney sections
Frozen kidneys were sectioned at 5 µm and 16 µm. PFA-fixed, paraffin-embedded blocks were sectioned at 5 µm, and antigen retrieval was performed at 110°C in citrate buffer at pH 6 for immunofluorescence staining. Thick cortical and medullary kidney sections after multi-photon imaging were fixed in 4% PFA. Kidney sections were permeabilized with 0.1% Triton X-100 (Surfact-Amps detergent solution, ThermoFisher Scientific) for 10 min for thin sections and 1 h for thick sections. Next, tissue sections were blocked with 1% BSA and 10% donkey or goat serum before incubation overnight at 4°C with primary antibody.

The following primary antibodies were used for immunofluorescence staining: rabbit anti-PDGFRβ (Abcam, ab32570, 1:300 and 1:500), chicken anti-GFP (Aves, GFP-1020, 1:300 and 1:400), mouse anti-αSMA-Cy3 (Sigma, C6198, 1:300 and 1:500), rabbit anti-Fibronectin (Sigma, F3648, 1:200); mouse anti-Col1a1 (DSHB, F1C3, 1:100), rat anti-F4/80 (Bio Rad, MCA497GA, 1:50), rabbit anti-CDA (ThermoFisher, PA5-76650, 1:100), mouse anti-Aquaporin 1 (Abcam, ab9566, 1:250). On the following day, sections were washed with PBS, and incubated at room temperature for 2 h with the following secondary antibodies: donkey anti-rabbit-AF647 (711-606-152, Jackson ImmunoResearch Europe Ltd, 1:300 and 1:500), donkey anti-chicken-Cy3 (703-165-15, Jackson, 1:300 and 1:500), goat anti-mouse-AF568 (A-11004, ThermoFisher Scientific, 1:500), goat anti-rat-Cy5 (112-607-003, Jackson ImmunoResearch Europe Ltd, 1:500). After incubation with secondary antibodies, sections were washed with PBS, and cellular DNA was stained with 10 µg/ml Hoechst 33342 (H1399, Molecular Probes) for 5 min at room temperature. Slices were mounted with Dako mounting medium (Agilent, Santa Clara). Visualization of the staining was performed with an Axio Scan.Z1 slide scanner using a Plan-Apochromat 40×/0.95 air immersion objective, a confocal Leica SP8 inverse STED 3X microscope using an HC PL APO CS2 20×/0.75 oil immersion objective, and an inverted confocal laser scanning microscope ZEISS LSM 980 Airyscan by using an LD LCI Plan-Apochromat 25×/0.8 silicon-oil objective.

Collagen fibers were detected by staining the kidney sections according to Sirius red/fast green protocol41. After deparaffinization, sections were washed with PBS and thereafter with distilled water for 5 min. Next, tissue was incubated in 0.04% fast green solution for 15 min and washed with distilled water. After incubating in 0.1% fast green/0.04% Sirius red in saturated picric acid solution for 30 min, sections were washed with acidified water (0.5% acetic acid in distilled water). Subsequently, sections were dehydrated in 70%, 80%, 96%, and 100% ethanol, cleared with three changes of Histoclear, and mounted. Visualization of the collagen staining was performed with an Axio Scan.Z1 slide scanner using a Plan-Apochromat 20x/0.8 air immersion objective.

Image analysis
All images for comparison were taken using the same settings. Image processing was done in FIJI42 and Zeiss ZEN. Regions of interest were drawn manually to obtain fluorescent signals. Brightness and contrast were optimized and applied to all parts of the figures equally for better visualization of the images. Nuclei counting in fibroblast proliferation experiments was performed in CellProfiler43. Figures were created with BioRender.com.

snRNA sequencing analysis
Datasets from the IRI models (GSE151167, GSE139107) and the POD-ATTAC model (GSE218376) were analyzed for P2ry6 and Cda gene expression. Visualization of gene expression was performed using the VlnPlot and FeaturePlot functions in Seurat. Inferred RNA expression was calculated using Magic RNA, while gene set enrichment scores were computed with the AddModuleScore function in Seurat. The analysis was conducted using R version 4.3.1 and Seurat object version 4.

Statistical analysis
Data are presented as mean values (±SEM). All data were analyzed and displayed using GraphPad Prism software (GraphPad Prism version 10.0.0 for Windows, GraphPad Software, Boston, Massachusetts, USA, www.graphpad.com). The following normality and log normality tests were used: D’Agostino & Pearson test, Anderson-Darling test, Shapiro–Wilk test, and Kolmogorov-Smirnov test. Based on normal or non-normal data distribution, a suitable test was used to compare two groups. P values of less than 0.05 were considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This article identifies a signaling pathways of uridine diphosphate release with kidney injury to activation its receptor (P2Y6) in fibroblasts to mediate proliferation and myelofibroblast transformation. In general, the studies appear to be well performed and the results are convincing. Identification of this pathway is a novel finding. However, there are some concerns with the data presentation.\n1) Figure 1c needs specific tubule markers. Although the authors assume this is from proximal tubule, it is not clear that these are the only source.\n2) Figure 2a and 2b:. Although there are statistically significant differences , with the variability it tis not clear that these small differences are biologically significant, especially compared to levels of the increased P2Y6 expression seen with UUO or folic acid\n3) Figure 7 and Figure 8 basically recapitulate in different injury models what is seen in Figure 6. These figures could be moved to supplemental material or 6, 7 and 8 could be somehow combined in to one figure.\n4) The results would be of greater impact if there were evidence that this pathway is also operative in human kidney fibrosis", "reviewer_2": "The manuscript entitled ‘Pyrimidinergic calcium signaling links tubular metabolism to fibrosis in kidney disease’ is well presented with insightful details. The authors presented and explained their findings in a clear and concise manner.\n1) Based on the results and limitations, What are the challenges in applying the findings to humans?\n2) Is there a possibility of using patient-derived kidney samples for the experiment in order to evaluate the translatability of the research to humans?\n3) (Page 9-10) Given that P2Y6R therapies are promising due to the lack of baseline phenotypes in KO mice and the dramatic increase in renal expression in CKD, as you mentioned, and noting a single cell sequencing study in humans that identified upregulation of calcium-dependent genes in activated fibroblasts. How do you reconcile your findings with the single-cell sequencing study showing the upregulation of calcium-dependent genes in human-activated fibroblasts? How might these findings guide the development of preclinical models to better predict human-specific outcomes and minimize risks?\n4) The authors mentioned in the discussion about the involvement of P2Y6R in other diseases. Are there any shared mechanisms across these conditions that could unify its role as a therapeutic target? Could this help prioritize drug development for certain diseases?\n5) The author mentioned, 'We began by interrogating a single cell sequencing dataset derived from mice with renal ischemia-reperfusion injury'. Ischemia-reperfusion injury is a cause of acute kidney injury. Is there any comparison between the AKI, FAN, and UUO models? Is there any reason the FAN and UUO models were chosen?\n6) The authors could present the expression levels of profibrotic markers and other key proteins.\n7) I would like to ask if the authors investigated the potential involvement of other P2Y isotypes as potential key players in the progression of renal fibrosis.\n8) The quality of Figure 3 appears to be low, making it difficult to interpret. Enhancing the contrast and overall clarity would greatly improve its readability and ensure that important details are more discernible to readers.\n9) (Line 287-289), the authors state that they have done RT-qPCR for KIM-1 and NGAL; however, there is no data for them.\n10) (Line 94), spelling error “pyridiminergic”", "reviewer_3": "This paper presents an intriguing study employing live imaging technology to investigate calcium (Ca) signaling in fibroblasts. The authors propose that uridine diphosphate (UDP) from proximal tubules induces Ca signaling via the P2Y6 receptor (P2Y6R) in fibroblasts, leading to fibrosis. However, the reviewer contends that the conclusions are inadequately supported by the experimental data, necessitating further experimentation to substantiate the authors' claims.\nFigure 1: The expression of cytidine deaminase (CDA) in the proximal tubule is insufficient to demonstrate UDP secretion from proximal tubule. Additionally, there is no evidence that the quantity of UDP or synthetic ligands applied to the kidney slices in Figure 2 corresponds to the amount secreted by the proximal tubule in vivo.\nFigure 2a, b: Although the authors assert that P2Y6R is strongly expressed in fibroblasts, the experimental data supporting this conclusion is absent. The authors should present P2Y6R expression as a violin plot, as depicted in Figure 1b. In the publicly available web-based database (Kidney Interactive Transcriptomics; https://humphreyslab.com/SingleCell/), P2ry6 expression is fairly restricted in the macrophages, not in the fibroblasts, throughout the time course of mouse kidney injury models, ischemia reperfusion injury and unilateral urethral obstruction.\nGiven the centrality of P2Y6R expression in fibroblasts to this study, more rigorous verification is required. (It is also known that macrophages express PDGFRb, necessitating caution.)\nFigure 2e: There is no evidence that the cells exhibiting elevated intracellular Ca are fibroblasts. The authors should employ fibroblast-specific GCaMP6s mice using Ai96 (https://www.jax.org/strain/028866) to conclude this. Furthermore, the administration of UDP or a synthetic agonist (MRS2957) to the kidney slices is highly artificial, and there is no evidence that the concentration of UDP secreted from the proximal tubules reaches the level claimed by the authors.\nFigure 2h, Figure 3: NRK49F cells were originally isolated from renal cell cultures and established as a subpopulation with a \"fibroblast-like\" appearance, raising questions about the extent to which they retain fibroblast characteristics in vivo. Several other cell types are known to maintain the characteristics of renal fibroblasts, and it would be preferable to utilize these lines in this experiment. It is also already established that intracellular Ca levels increase in response to P2Y6R stimulation.\nFigure 4: While the authors state, “Taken together, these findings indicate that calcium signals in renal fibroblasts comprise (relatively) fast and irregular transients originating in processes wrapping around tubules, consistent with the notion that they are triggered by signaling factors emanating from the latter”, the reviewer believes that the authors cannot draw this conclusion based solely on anatomical positioning.\nFigure 4: The preparation of kidney slices is inherently disruptive, necessitating the release of numerous factors by various cell types, not solely by proximal tubules. Consequently, the observed spontaneous calcium signaling may not accurately reflect that of healthy, intact kidneys. In addition, the authors' assertion that the signal is initiated by factors from \"proximal tubules\" is not substantiated.\nFigure 5: Similarly, the authors attribute interstitial calcium signaling to fibroblasts, yet the specific cell type exhibiting calcium signaling has not been rigorously identified. The interstitium comprises diverse cell types, including fibroblasts, macrophages, endothelial cells, and dendritic cells. Furthermore, although it is established that the number of αSMA-positive myofibroblasts increases in the (j) UUO model, there is no evidence linking this to fibroblast Ca signaling. Additionally, there is no evidence that this Ca signaling is induced by UDP originating from proximal tubules.\nFigures 6-9: Figures 6 and 8 demonstrate that P2Y6 knockout (KO) confers resistance to fibrosis. However, as this is a conventional KO, it does not substantiate the authors' claim that \"proximal-derived\" UDP stimulates fibrosis via P2Y6R expressed \"by fibroblasts.\" Figures 7 and 9 involve systemic administration of an inhibitor, which does not support the authors' conclusions, either. The authors should, at a minimum, conduct experiments utilizing fibroblast-specific conditional KO.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "Figurek et al. present a manuscript with state-of-the-art techniques including omics and imaging. The reported role of UDP signaling activating fibroblasts in kidney injury is novel and highly interesting. The calcium sensor GCaMP6 is expressed in all cells of the body, with a cell specific expression of this sensor the cells of interest could be identified during intravital imaging. But the autors elegantly circumvent this limitation by employing immunohistochemistry of the same kidney area after fixing this tissue. The manuscript is well written, concise in language and easy to follow. However, there are a few points which should be addressed to further improve the manuscript.\n- Figure 2f: The authors show a strong calcium increase after UDP or a P2Y6 agonist, but suramin or the antagonist are only started after the strong peak declined. There is no set of experiment where the calcium transient is shown without an antagonist. The panel 2g shows time above an arbitrary threshold, but this could be a strong signal or weak artifacts as the threshold is not reported\n- The manuscript contains beautiful imaging studies of calcium signaling in wildtype animals and injured animals in vivo. The effect of a P2Y6R antagonist on kidney slices is only shown in acute kidney slices. While the first half of the manuscript is centered around UDP and calcium, in the second half when P2Y6R mice and P2Y6 antagonists are used together with disease models no in vivo calcium imaging is provided. This would be necessary to claim the importance of calcium signaling in the development of kidney fibrosis (which is nicely demonstrated in Figures 6-9). This could be either done by crossing P2Y6R knockout mice to GCaMP6 mice and employing one disease model or adding a P2Y6R antagonist in the existing GCaMP6 mouse line in health and disease to demonstrate the central role of the P2Y6R in calcium signaling.\n- The group has a reputation for tubular physiology and in the omics data the proximal tubule is subdivided into S1/S2/S3 segments. However, for the imaging data no detailed explanation is given if the shown results are true for all tubular segments or if there are differences between the segments are provided.\n- In the provided movies it is hard to discriminate proximal tubule cells from fibroblasts due to the speed of changing calcium signals. I would recommend to provide annotations or a IF staining picture next to the movies to be able to know where fibroblasts and PT cells are supposed to be (similar to the panels in the figures)\n- Figure 4: the authors demonstrate different patterns in calcium signals of fibroblasts and PT cells, what is shown seems to be a single measurement. It remains unclear on how many measurements this important message was based.\nMinor points:\n- Half of the introduction is a summary of the paper (line 55-63), this is a repetition of the abstract\n- Line 67: it initially was clear to me which was the dataset used (3 papers from independent groups). It should be mentioned early on, that more information about this dataset can be found in Suppl. Figure 1 (which I found in the end)"}, "author_response": "We thank the reviewers for their positive feedback on our study and helpful comments. We have performed new experiments and analyses to address the issues raised, and feel that the manuscript is much improved. We therefore hope that it is now suitable for publication. Some of the additional experiments were performed by a new co-author (Dr. Imene Sakhi) who has been added to the manuscript.\nReviewer #1 (Remarks to the Author):\nThis article identifies a signaling pathways of uridine diphosphate release with kidney injury to activation its receptor (P2Y6) in fibroblasts to mediate proliferation and myelofibroblast transformation. In general, the studies appear to be well performed and the results are convincing. Identification of this pathway is a novel finding. However, there are some concerns with the data presentation.\n1) Figure 1c needs specific tubule markers. Although the authors assume this is from proximal tubular, it is not clear that these are the only source.\nWe apologize that this information was missing. We have co-stained specimens for a proximal tubular marker (aquaporin 1) and these new images have been added to figure 1 (zoomed in) and supplementary figure 2 (zoomed out).\n2) Figure 2a and 2b:. Although there are statistically significant differences , with the variability it is not clear that these small differences are biologically significant, especially compared to levels of the increased P2Y6 expression seen with UUO or folic acid\nThis is a good point, which we have acknowledged in the revised manuscript. The differences with UUO and FA experiments may reflect increased sensitivity with PCR.\n3) Figure 7 and Figure 8 basically recapitulate in different injury models what is seen in Figure 6. These figures could be moved to supplemental material or 6, 7 and 8 could be somehow combined in to one figure.\nWe understand the point that the reviewer is making, but because the two CKD models are quite different and each have their particular strengths we prefer to keep these data in the main figures. We thank the reviewer for their understanding.\n4) The results would be of greater impact if there were evidence that this pathway is also operative in human kidney fibrosis\nWe agree that this is an important question, which we also considered. However, upon interrogating publicly available human gene expression databases, we observed some variability in findings concerning pyrimidinergic genes, which might be explained by differences in methodology, sensitivity etc. Therefore, at the moment, we prefer to focus our manuscript on cell and mouse models, where we can conduct detailed functional studies in a manner that is not possible in humans. This limitation of the study was acknowledged in the original manuscript discussion. Moreover, we referenced a previous study that highlighted activation of calcium-dependent genes in fibroblasts in human CKD (PMID: 33176333), which provides some indication that calcium signaling might play a role.\nReviewer #2 (Remarks to the Author):\nThe manuscript entitled ‘Pyrimidinergic calcium signaling links tubular metabolism to fibrosis in kidney disease’ is well presented with insightful details. The authors presented and explained their findings in a clear and concise manner.\n1) Based on the results and limitations, What are the challenges in applying the findings to humans?\nPlease see our response to reviewer 1 above.\n2) Is there a possibility of using patient-derived kidney samples for the experiment in order to evaluate the translatability of the research to humans?\nUnfortunately, we are not able to perform detailed functional tests in human kidney in the same manner that we can in mouse tissue.\n3) (Page 9-10) Given that P2Y6R therapies are promising due to the lack of baseline phenotypes in KO mice and the dramatic increase in renal expression in CKD, as you mentioned, and noting a single cell sequencing study in humans that identified upregulation of calcium-dependent genes in activated fibroblasts. How do you reconcile your findings with the single-cell sequencing study showing the upregulation of calcium-dependent genes in human-activated fibroblasts? How might these findings guide the development of preclinical models to better predict human-specific outcomes and minimize risks?\nThe increase in expression of calcium-dependent genes in human fibroblasts in CKD is consistent with the possibility that calcium signaling is activated in these cells. We do not know at present to what extent this is stimulated by P2Y6R activation or by other signaling pathways (e.g. angiotensin II). It would therefore be interesting in the future to screen human-derived fibroblasts to establish which signaling molecules can induce calcium responses.\n4) The authors mentioned in the discussion about the involvement of P2Y6R in other diseases. Are there any shared mechanisms across these conditions that could unify its role as a therapeutic target? Could this help prioritize drug development for certain diseases?\nThis is an interesting question. Activation of intracellular calcium release could be one shared mechanism, but there might be others. Interestingly, since we submitted our article, another group has shown that blocking the P2Y6R is protective against atherosclerosis in mouse models of CKD (PMID: 40049228) – we have added this citation to the revised discussion to further highlight the therapeutic potential of the pathway in cardio-renal disease.\n5) The author mentioned, 'We began by interrogating a single cell sequencing dataset derived from mice with renal ischemia-reperfusion injury'. Ischemia-reperfusion injury is a cause of acute kidney injury. Is there any comparison between the AKI, FAN, and UUO models? Is there any reason the FAN and UUO models were chosen?\nThe ischemia-reperfusion data were used to explore expression profiles of pyrimidinergic genes in healthy mouse kidneys and in injured tubules, which we hypothesized would emit signaling factors that activate surrounding interstitial cells during the transition to chronic damage. We chose UUO and FAN because they are well-established models of renal fibrosis and CKD, respectively.\n6) The authors could present the expression levels of profibrotic markers and other key proteins.\nOur apologies, but we are not sure what the reviewer is referring to here. Activation of profibrotic genes markers was shown in both cells (figure 3) and mouse kidney tissue (figures 6-9).\n7) I would like to ask if the authors investigated the potential involvement of other P2Y isotypes as potential key players in the progression of renal fibrosis.\nThis is an interesting question, but to date we have focused only on the P2Y6R, because this is the endogenous receptor for UDP.\n8) The quality of Figure 3 appears to be low, making it difficult to interpret. Enhancing the contrast and overall clarity would greatly improve its readability and ensure that important details are more discernible to readers.\nWe apologize for this – the figure quality was affected by integration into a pdf file. For the final version we will upload a separate figure file.\n9) (Line 287-289), the authors state that they have done RT-qPCR for KIM-1 and NGAL; however, there is no data for them.\nThese data are in figure 1D.\n10) (Line 94), spelling error “pyridiminergic”\nOur apologies, thanks for pointing this out – it has now been corrected.\nReviewer #3 (Remarks to the Author):\nThis paper presents an intriguing study employing live imaging technology to investigate calcium (Ca) signaling in fibroblasts. The authors propose that uridine diphosphate (UDP) from proximal tubules induces Ca signaling via the P2Y6 receptor (P2Y6R) in fibroblasts, leading to fibrosis. However, the reviewer contends that the conclusions are inadequately supported by the experimental data, necessitating further experimentation to substantiate the authors' claims.\nFigure 1: The expression of cytidine deaminase (CDA) in the proximal tubule is insufficient to demonstrate UDP secretion from proximal tubule. Additionally, there is no evidence that the quantity of UDP or synthetic ligands applied to the kidney slices in Figure 2 corresponds to the amount secreted by the proximal tubule in vivo.\nSecretion of UDP was detected from proximal tubular-derived cells in figure 1. Unfortunately, we are unaware of a suitable technique that could demonstrate this from proximal tubules in vivo. For the slices experiments, it is important to explain that UDP could be degraded in the perfusion system and by extracellular enzymes in the kidney tissue. Therefore, we do not know how much of the initial concentration actually reaches the fibroblasts. This is a limitation of the approach, but we are not aware of any other methodology to directly deliver UDP (or agonists) to cells within mouse kidney tissue and record calcium responses in real time.\nFigure 2a, b: Although the authors assert that P2Y6R is strongly expressed in fibroblasts, the experimental data supporting this conclusion is absent. The authors should present P2Y6R expression as a violin plot, as depicted in Figure 1b. In the publicly available web-based database (Kidney Interactive Transcriptomics; https://humphreyslab.com/SingleCell/), P2ry6 expression is fairly restricted in the macrophages, not in the fibroblasts, throughout the time course of mouse kidney injury models, ischemia reperfusion injury and unilateral urethral obstruction.\nGiven the centrality of P2Y6R expression in fibroblasts to this study, more rigorous verification is required. (It is also known that macrophages express PDGFRb, necessitating caution.)\nWe stated that the P2Y6R is expressed in fibroblasts and that its expression increases in CKD (line 79). However, RNA and protein abundance do not always correlate, which is why we performed experiments to show functional activity of the P2Y6R in renal fibroblasts. We agree that the P2Y6R is also expressed in other cells – including immune cells - and have provided this data as requested by the reviewer in supplementary figure 3. Indeed, expression in immune cells might contribute to the anti-fibrotic effects of the P2Y6R antagonist and we have added this possibility to the revised discussion. However, it is important to note that expression of the P2Y6R at RNA level in immune cells does not necessarily mean that the protein is abundant and active – this needs to be established in future studies. Moreover, it can be seen in supplementary figure 3 that the expression in immune cells decreases post-injury (in contrast to the increase in stromal cells). We did not observe PDGFRb expression in immune cells in our dataset.\nFigure 2e: There is no evidence that the cells exhibiting elevated intracellular Ca are fibroblasts. The authors should employ fibroblast-specific GCaMP6s mice using Ai96 (https://www.jax.org/strain/028866) to conclude this. Furthermore, the administration of UDP or a synthetic agonist (MRS2957) to the kidney slices is highly artificial, and there is no evidence that the concentration of UDP secreted from the proximal tubules reaches the level claimed by the authors.\nWe used mice expressing GCaMP6s in both tubules and interstitial cells to simultaneously record changes in calcium signals in each in disease states. PDGFRb expression was then used to retrospectively identify fibroblasts - this marker has been widely used in previous studies. Regarding UDP secretion from proximal tubular cells, please see our earlier comment. We do not know how much is secreted in vivo and are not aware of a method to measure this. However, due to the close apposition of tubular cells and fibroblast processes in the intact kidney, it is possible that even small amounts of UDP release might trigger responses.\nFigure 2h, Figure 3: NRK49F cells were originally isolated from renal cell cultures and established as a subpopulation with a \"fibroblast-like\" appearance, raising questions about the extent to which they retain fibroblast characteristics in vivo. Several other cell types are known to maintain the characteristics of renal fibroblasts, and it would be preferable to utilize these lines in this experiment. It is also already established that intracellular Ca levels increase in response to P2Y6R stimulation.\nNRK49F cells have been widely used in previous studies of kidney fibrosis (e.g. see PMID: 32139089, PMID: 37626891, PMID: 39259608, PMID: 30154246). Like all cell lines, they are not a perfect model of fibroblasts in vivo. Nevertheless, findings from these cells were in line with in vivo results in our hands. Moreover, the cells expressed the P2Y6R and displayed extensive processes – two important criteria for our specific study. We agree that P2Y6R activation is known to trigger calcium responses in various different cell types, but the potential relevance of this to renal fibroblast activation/fibrosis was not previously investigated.\nFigure 4: While the authors state, “Taken together, these findings indicate that calcium signals in renal fibroblasts comprise (relatively) fast and irregular transients originating in processes wrapping around tubules, consistent with the notion that they are triggered by signaling factors emanating from the latter”, the reviewer believes that the authors cannot draw this conclusion based solely on anatomical positioning.\nWe believe that our observations are consistent with this notion, but agree that this is somewhat speculative and have reworded the section accordingly.\nFigure 4: The preparation of kidney slices is inherently disruptive, necessitating the release of numerous factors by various cell types, not solely by proximal tubules. Consequently, the observed spontaneous calcium signaling may not accurately reflect that of healthy, intact kidneys. In addition, the authors' assertion that the signal is initiated by factors from \"proximal tubules\" is not substantiated.\nWe agree that calcium signals may be altered in the slice model, which is why we also performed experiments in vivo. However, the slice approach has distinct advantages, e.g. a higher level of resolution to delineate intracellular signal propagation and the opportunity to deliver substances directly to the tissue. Our gene expression data suggest that pyrimidine metabolism is most active in the proximal tubule. However, we cannot exclude the possibility that signals might emanate from other sources and have made this clear in the revised discussion.\nFigure 5: Similarly, the authors attribute interstitial calcium signaling to fibroblasts, yet the specific cell type exhibiting calcium signaling has not been rigorously identified. The interstitium comprises diverse cell types, including fibroblasts, macrophages, endothelial cells, and dendritic cells. Furthermore, although it is established that the number of αSMA-positive myofibroblasts increases in the (j) UUO model, there is no evidence linking this to fibroblast Ca signaling. Additionally, there is no evidence that this Ca signaling is induced by UDP originating from proximal tubules.\nWe used a fibroblast specific marker (PDGFRb) to identify fibroblasts. We did not observe co-staining with an immune cell marker (MHCII – please see new supplementary figure 7). Endothelial cells have a distinct morphology and location. Therefore, we believe that the calcium traces we extracted were specific to fibroblasts. Increased calcium signaling was associated with both fibroblast proliferation and activation in cell and in vivo models. Moreover, P2Y6R KO or inhibition reduced fibrosis in vivo. These findings are suggestive that calcium signaling is playing a role. Additionally, in response to a request from reviewer 5 below, we have added new experiments showing a reduction in fibroblast calcium signaling in UUO mice treated with a P2Y6R antagonist (Supplementary figure 8).\nFigures 6-9: Figures 6 and 8 demonstrate that P2Y6 knockout (KO) confers resistance to fibrosis. However, as this is a conventional KO, it does not substantiate the authors' claim that \"proximal-derived\" UDP stimulates fibrosis via P2Y6R expressed \"by fibroblasts.\" Figures 7 and 9 involve systemic administration of an inhibitor, which does not support the authors' conclusions, either. The authors should, at a minimum, conduct experiments utilizing fibroblast-specific conditional KO.\nWe elected to use an existing global KO model, because it is well-established and displays a complete rather than partial KO. The KO is not fibroblast specific and this limitation was clearly acknowledged in the original manuscript (line 199-200). We found that KO and inhibitor treated mice were protected against fibrosis, which we believe is an interesting and potentially important translational observation that is consistent with a role for pyrimidinergic signaling in tubulo-interstitial cross-talk in CKD. Please see our earlier comment, we have acknowledged in the revised discussion that expression in immune cells could also contribute to these effects (if the receptor is active in these cells). We agree it would be interesting to investigate cell specific KOs in the future to dissect out their relative contributions, but this is beyond the scope of the current study.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #5 (Remarks to the Author):\nFigurek et al. present a manuscript with state-of-the-art techniques including omics and imaging. The reported role of UDP signaling activating fibroblasts in kidney injury is novel and highly interesting. The calcium sensor GCaMP6 is expressed in all cells of the body, with a cell specific expression of this sensor the cells of interest could be identified during intravital imaging. But the authors elegantly circumvent this limitation by employing immunohistochemistry of the same kidney area after fixing this tissue. The manuscript is well written, concise in language and easy to follow. However, there are a few points which should be addressed to further improve the manuscript.\n- Figure 2f: The authors show a strong calcium increase after UDP or a P2Y6 agonist, but suramin or the antagonist are only started after the strong peak declined. There is no set of experiment where the calcium transient is shown without an antagonist. The panel 2g shows time above an arbitrary threshold, but this could be a strong signal or weak artifacts as the threshold is not reported\nWe apologize for this missing information and have added new data showing the calcium signal in the absence of an antagonist (supplementary figure 5). The threshold to detect calcium rises was set at 2 x the lowest fluorescence intensity (methods line 350), to account for the fact that different cells have different baseline fluorescence values.\n- The manuscript contains beautiful imaging studies of calcium signaling in wildtype animals and injured animals in vivo. The effect of a P2Y6R antagonist on kidney slices is only shown in acute kidney slices. While the first half of the manuscript is centered around UDP and calcium, in the second half when P2Y6R mice and P2Y6 antagonists are used together with disease models no in vivo calcium imaging is provided. This would be necessary to claim the importance of calcium signaling in the development of kidney fibrosis (which is nicely demonstrated in Figures 6-9). This could be either done by crossing P2Y6R knockout mice to GCaMP6 mice and employing one disease model or adding a P2Y6R antagonist in the existing GCaMP6 mouse line in health and disease to demonstrate the central role of the P2Y6R in calcium signaling.\nWe thank the reviewer for this important suggestion and have performed new experiments showing that treatment with a P2Y6R antagonist reduces fibroblast calcium signals in UUO mice (Supplementary figure 8).\n- The group has a reputation for tubular physiology and in the omics data the proximal tubule is subdivided into S1/S2/S3 segments. However, for the imaging data no detailed explanation is given if the shown results are true for all tubular segments or if there are differences between the segments are provided.\nThis is an interesting question and we indeed considered to explore differences in tubular segments. However, due to the 3D structure of the kidney, it is possible that individual fibroblasts may have contact to multiple different segments, which could confound this analysis. Therefore, we do not feel able to comment further at this stage – we thank the reviewer for their understanding.\n- In the provided movies it is hard to discriminate proximal tubule cells from fibroblasts due to the speed of changing calcium signals. I would recommend to provide annotations or a IF staining picture next to the movies to be able to know where fibroblasts and PT cells are supposed to be (similar to the panels in the figures)\nWe apologize that this was not clear and have annotated the movies accordingly.\n- Figure 4: the authors demonstrate different patterns in calcium signals of fibroblasts and PT cells, what is shown seems to be a single measurement. It remains unclear on how many measurements this important message was based.\nThese traces are representative of observations from multiple cells in 3 healthy animals. Quantifications of calcium signals in tubular cells and fibroblasts from control mice were provided in figure 5.\nMinor points:\n- Half of the introduction is a summary of the paper (line 55-63), this is a repetition of the abstract\nThis section has been condensed to reduce repetition.\n- Line 67: it initially was clear to me which was the dataset used (3 papers from independent groups). It should be mentioned early on, that more information about this dataset can be found in Suppl. Figure 1 (which I found in the end)\nWe apologize for this, the link to supplementary figure 1 has now been placed earlier in the manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have partially responded to the concerns raised in the previous review. However, there is still concern that what is seen in the mouse studies is not translatable to humans. The manuscript would be stronger with some indication of human translatability, even if it is immunofluorescent staining of human kidney biopsies.", "reviewer_2": "The authors answered the questions properly.", "reviewer_3": "The authors have provided responses to each of the reviewers' questions. While experimental results are presented for some questions, for several points the reviewers considered important, the authors have responded solely by modifying the text. The reviewers understand that these experiments are technically challenging, but it is regrettable that they were not performed, as they would have been crucial for supporting the mechanism proposed by the authors.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "The authors added the requested in vivo calcium imaging experiments, which also show a reduction of calcium signaling after P2Y6 knockout. They adressed all major points raised from my side. The manuscript was further improved by this revision. Therefore, I have no further requests before publication."}, "author_response": "We thank the reviewers and editors for their further feedback on our study and are delighted that it is suitable for publication. Our responses to the comments are below and changes to the manuscript are in red.\nEditorial comments:\nFirst, we ask you to revise your paper to address our editorial requests (in the attached Author Checklist) and any remaining comments from reviewers. Please note that we agree with Reviewer 3 that the absence of evidence showing UDP release in vivo is an important limitation, and we expect you to clearly discuss it as a limitation of your study and tone down relevant statements regarding the mechanism. We also think that the limitation of not having human samples should be stated (as indicated by Reviewer 1). Additionally, we also ask you to clearly state that the use of the global KO is another important limitation of your work.\nThe author checklist has been completed and the figure legends updated as requested. We have added new text to the manuscript explaining the limitations of the study.\nReviewer #1 (Remarks to the Author):\nThe authors have partially responded to the concerns raised in the previous review. However, there is still concern that what is seen in the mouse studies is not translatable to humans. The manuscript would be stronger with some indication of human translatability, even if it is immunofluorescent staining of human kidney biopsies.\nPlease see our response to the editors above, we have acknowledged this limitation in the revised manuscript discussion.\nReviewer #2 (Remarks to the Author):\nThe authors answered the questions properly.\nWe thank the reviewer for taking the time to review the manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have provided responses to each of the reviewers' questions.\nWhile experimental results are presented for some questions, for several points the reviewers considered important, the authors have responded solely by modifying the text.\nThe reviewers understand that these experiments are technically challenging, but it is regrettable that they were not performed, as they would have been crucial for supporting the mechanism proposed by the authors.\nWe are sorry that we were not able to perform all of the experiments requested by the reviewer and thank them for their understanding. Please see our response to the editors above, we have ensured that the limitations of the study are clear in the revised manuscript.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank the reviewer for taking the time to review the manuscript.\nReviewer #5 (Remarks to the Author):\nThe authors added the requested in vivo calcium imaging experiments, which also show a reduction of calcium signaling after P2Y6 knockout. They addressed all major points raised from my side. The manuscript was further improved by this revision. Therefore, I have no further requests before publication.\nWe thank the reviewer for taking the time to review the manuscript."}]

Decision letter