View Because It May Allow Manipulation of Podocyte Signaling in a Cell-Speciﬁc Fashion

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Depletion of Gprc5a Promotes Development of Diabetic Nephropathy

Xiaojie Ma ,1 Angelina Schwarz,1 Sonia Zambrano Sevilla,1 Anna Levin,2 Kjell Hultenby,3 Annika Wernerson,2 Mark Lal,4 and Jaakko Patrakka1

1Karolinska Institutet/AstraZeneca Integrated Cardio Metabolic Center, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden; 2Division of Renal Medicine, Department of Clinical Sciences, Intervention and Technology, and 3Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; and 4Bioscience, Cardiovascular, Renal and Metabolic Diseases, Innovative Medicines Biotech Unit, AstraZeneca, Gothenburg, Sweden

ABSTRACT Background Renal glomeruli are the primary target of injury in diabetic nephropathy (DN), and the glomerular podocyte has a key role in disease progression. Methods To identify potential novel therapeutic targets for DN, we performed high-throughput molecular profiling of G protein–coupled receptors (GPCRs) using human glomeruli. Results We identified an orphan GPCR, Gprc5a, as a highly podocyte-specific gene, the expression of which was significantly downregulated in glomeruli of patients with DN compared with those without DN. Inactivation of Gprc5a in mice resulted in thickening of the glomerular basement membrane and activation of mesangial cells, which are two hallmark features of DN in humans. Compared with wild-type mice, Gprc5a-deficient animals demonstrated increased albuminuria and more severe histologic changes after induction of diabetes with streptozotocin. Mechanistically, Gprc5a modulated TGF-b signaling and activation of the EGF receptor in cultured podocytes. Conclusions Gprc5a has an important role in the pathogenesis of DN, and further study of the podocyte- specific signaling activity of this protein is warranted.

J Am Soc Nephrol 29: 1679–1689, 2018. doi: https://doi.org/10.1681/ASN.2017101135

Diabetic nephropathy (DN) develops in 20%–40% of develop more kidney-speciﬁc treatment modalities. patients with diabetes mellitus.1 The disease is respon- However, we still understand rather poorly patho- sible for .40% of new ESRD cases.2 Today the treat- genic mechanisms of diabetic renal damage and ment options of DN are based largely on repurposing therefore lack distinct molecular targets. of existing therapeutics and are neither kidney- DN is a microvascular complication of diabetes target–based nor designed to address the underlying that affects primarily renal glomeruli.2 The hallmark molecular mechanisms. Obviously, it is important to

Signiﬁcance Statement Received October 27, 2017. Accepted March 15, 2018. Podocytes play a key role in the progression of diabetic Published online ahead of print. Publication date available at nephropathy (DN). This manuscript describes a new po- www.jasn.org. docyte-associated G protein–coupled receptor (GPCR), Correspondence: Dr. Jaakko Patrakka, Karolinska Institutet/As- Gprc5a, which modulates the progression of DN by traZeneca Integrated Cardio Metabolic Center, Karolinska In- regulating EGF receptor (EGFR) and TGF-b signaling. stitutet at Karolinska University Hospital Huddinge, 14186 Gprc5a can be an interesting molecule from the phar- Stockholm, Sweden. Email: [email protected] maceutic point of view because it may allow manipulation of podocyte signaling in a cell-speciﬁc fashion. Copyright © 2018 by the American Society of Nephrology

J Am Soc Nephrol 29: 1679–1689, 2018 ISSN : 1046-6673/2906-1679 1679 BASIC RESEARCH www.jasn.org sign of the disease is albuminuria secondary to glomerular dam- western blotting and RT-PCR. Diabetes was induced in 8-week-old age. Glomerular pathology includes thickening of the glomeru- littermate and mutant mice by intraperitoneal injections of strep- lar basement membrane (GBM), mesangial cell activation with tozotocin (50 mg/kg) for 5 days. Induction of hyperglycemia was expansion of mesangial matrix, and dedifferentiation and loss of confirmed by measuring nonfasting blood glucose 2 weeks after the podocyte cells.2 Glomerular damage leads eventually to sclerosis last injection and by analyzing HbA1c at the end of the experiment. and decreased renal function. Signaling pathway mediated by Urine was collected every second week and mice were euthanized 24 TGF-b has been pinpointed to play a major role in the patho- weeks after injections. genesis of glomerular disease.1 However, mechanisms modulat- ing the pathway in DN are not well understood. Cell Culture Gprotein–coupled receptors (GPCRs) constitute a protein Immortalized human podocytes were grown as described.8 family of receptors that sense molecules outside the cell and Stable overexpressing cells were established by transfecting activate a great diversity of intracellular signal transduction path- pcDNA3.1-hGprc5a construct using Lipofectamine. Silencing ways.3 Because of their central involvement in biologic processes, of Gprc5a expression was done using siRNA (Invitrogen). Ef- GPCRs have been a very successful protein class for drug discovery fects of Gprc5a on EGF-mediated signaling were studied by because the market share of drugs targeting GPCRs is 20%–30%.3 adding 25 ng/ml of HB-EGF on culture media. Because the glomerulus is the main target of injury in DN, we analyzed glomerular GPCRs by large-scale expressional profiling. Luciferase Reporter Assay We identified Gprc5a as a novel highly podocyte-specificGPCR Effects of Gprc5a on Smad2/3-mediated target gene transcrip- whose expression was significantly downregulated in patients with tion were assessed further using a dual-luciferase reporter sys- DN. Studies in Gprc5a-deficient mouse line and cell culture in- tem (Promega). dicate that the receptor modulates EGFreceptor (EGFR) and TGF- b signaling in podocytes. Gprc5a knockout mice phenocopy key Statistical Methods features of glomerular pathology in human DN, including the Real-time PCR experiments were analyzed using t test or one- thickening of the GBM and mesangial cell activation. Moreover, way ANOVAwith Tukey’s post hoc test. Immunohistochemical Gprc5a deficiency in mice promotes the progression of nephrop- and morphologic data were analyzed using t test. athy in diabetes, highlighting its central role in the pathogenesis of the disease. Gprc5a enables manipulation of pathogenic signaling pathways in a podocyte-specificfashionandcanthereforebea RESULTS new pharmaceutic target to treat DN. High-Throughput Expressional Profiling of Isolated METHODS Human Glomeruli Reveals a Number of Novel Glomerulus-Enriched GPCRs Because GPCRs have been an outstanding protein family to Human Kidney Tissues target pharmacologically, we performed a large-scale qPCR Control tissue was collected from kidneys removed due to renal analysis of the glomerular GPCR transcriptome using a pre- carcinoma or from living kidney donors (Karolinska University designed 384-well panel. The analysis of isolated human glo- Hospital,Stockholm,Sweden).Renalbiopsysamplesfrompa- meruli revealed a number of GPCRs that were putatively tients with diabetes showing DN were collected from Karolinska enriched in the glomerulus as compared with the kidney frac- University Hospital. tion devoid of glomeruli (Supplemental Table 1). We identi- fi PCR Experiments ed several GPCRs known to be associated with glomeruli, 9 Glomeruli were isolated using standard sieving and perfusion such as PTGER4 (PG E receptor 4) and EDNRB (Endothelin 10 protocols. The expression of human GPCRs was analyzed receptor type B), but also several targets that have previously using a predesigned 384-well plate (GPCR Tier 1–4 H384; notbeendescribedtobepresentintheglomerulus.TheHu- Bio-Rad) and Gapdh was used for normalization. Primer se- man Protein Atlas (www.proteinatlas.com) was used to con- fi fi quences are listed in Supplemental Table 6. Relative mRNA rm the glomerular protein expression of some the identi ed gene transcripts with highest expression (ELTD1, GPR160, levels of genes were calculated using the DDCt method. F2R, S1PR1, CD97; Supplemental Figure 1) but still many Western Blotting, Immunostaining, and genes remain to be confirmed by alternative methodologies. Immunoelectron Microscopy Among the identified genes, we focused on an orphan GPCR, Western blotting and immunohistochemical experiments were Gprc5a, because it has not previously been ascribed a role in performed as previously described.4–7 kidney function.

Mouse Models Gprc5a Is Highly Speciﬁc to Glomerular Podocytes Gprc5a mutant mouse in FVB background was generated Expression of Gprc5a in different human tissues was studied by using TALENs (Cyagen). Inactivation of Gprc5a was veriﬁed using analyzing two publicly available RNA sequencing databases

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(www.medicalgenomics.org, www.proteinatlas.org). Both da- experiment in RNA isolated from different mouse organs tabases showed that Gprc5a was highly enriched in lung tissue showed highly restricted and lung-enriched expression pat- and only sparsely expressed in other organs (Figure 1A, Sup- tern (Supplemental Figure 2B). However, when we performed plementalFigure2A).Inlinewiththis,theRT-PCR PCR using isolated kidney fractions, we could amplify a band for

Figure 1. Gprc5a is highly enriched in renal podocyte cells in humans. (A) Gprc5a transcript is highly expressed by lung tissue and only sparsely by other human tissues as detected by RNAseq analysis (data extracted from www.medicalgenomics.org). (B and C) In isolated human kidney fractions, RT-PCR and qPCR show Gprc5a expression in the glomerulus (Glom) and not in the fraction lacking glomeruli (Rok, rest of kidney). Nephrin was used to control the purity of glomerular fractions and 28S as a loading control. (D) Immunofluo- rescence for Gprc5a in adult human kidney tissue demonstrates strong immunoreactivity in glomeruli and no significant signal is detected in extraglomerular areas. (E–H) Double labeling with podocyte marker nephrin (red) shows almost complete overlapping (yellow) with Gprc5a (green) indicating localization to podocyte foot processes. (I) In western blotting, anti-Gprc5a antibody detects a 40-kD protein almost solely in the glomerulus fraction. Nephrin antibody validates the purity of isolated glomeruli and calnexin was used as a loading control (CL, capillary lumen). (J) Immunoelectron microscopy for Gprc5a shows gold labeling mostly in podocytes where it often localizes (arrowheads) close to the plasma membrane (fp, foot process; ec, endothelial cell). Original magnifications, 3150 (D); 3400 (F–H). (I) Scale bar=200 nm.

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Gprc5a that was restricted to the human and mouse glomerulus analyzed data previously generated by Woroniecka et al.12 in (Figure 1B, Supplemental Figure 2B). This was confirmed by which they performed microarray on glomerular tufts isolated qPCR that showed 16-fold upregulation of the transcript in the from healthy patients and patients with DN. In the study, glomerulus in comparison to the rest of the kidney (Figure 1C). Gprc5a was one of the most highly downregulated genes in Next, we validated the expression of Gprc5a at the protein DN glomeruli (Figure 2G). Taken together, Gprc5a expression level. In immunofluorescence staining, a strong signal for in glomeruli is significantly downregulated in patients with Gprc5a was detected in human glomeruli whereas no reactivity DN, suggesting a role in the disease process. was detected outside glomerular tufts (Figure 1D). In double labeling experiments Gprc5a showed almost complete over- Generation and Characterization of Gprc5a Knockout lapping reactivity with podocyte foot process marker nephrin Mouse Line (Figure 1, E–H). In western blotting, we detected a strong To analyze the role of Gprc5a in the glomerulus in vivo,we band sized approximately 40 kD in human glomeruli that generated a mouse line in which Gprc5a gene was mutated. We was not readily detected in the tubulo-interstitial fraction de- targeted the first coding exon of Gprc5a gene using TALENs void of glomeruli (Figure 1I). No expression was detected in and mutagenesis generated a 2-bp deletion as validated by glomerular endothelial or mesangial cells as shown by double Sanger sequencing (Figure 3A). The coding sequence is pre- labeling experiments with endothelial marker CD31 and me- dicted to go out of frame due to the deletion and produce a sangial marker PDGF receptor b (PDGFRB) (Supplemental short 22-residue protein sequence. Figure 3A). The genotyping of Gprc5a mouse line was performed by Immunoelectron microscopy was performed to localize amplifying a 340-bp product from the genomic region Gprc5a in more detail in podocytes. Immuno-gold label was around the mutation followed by digestion with Kpn1 re- detected mostly at the plasma membrane of podocytes (Figure striction enzyme. The wild-type allele generates two 170-bp 1J). No significant labeling was observed outside podocytes fragments, whereas mutated animals lack the restriction site (Figure 1J, Supplemental Figure 3B). This was validated with a and generate an intact 340-bp fragment (Figure 3B). As ex- semiquantification of gold labeling (Supplemental Figure 3C). pected, the mutation generates a null allele because no Taken together, our detailed molecular profiling unraveled Gprc5a protein was detected using western blotting (Figure Gprc5a as a novel highly podocyte-specific GPCR. 3C). Moreover, the mutation seems to result in an instable The specificity of anti-Gprc5a antibody was validated by mRNA because no Gprc5a transcript could be amplified us- transfecting HEK293 cells with a Gprc5a construct. Western ing RT-PCR (Figure 3D). Taken together, we generated blotting showed a clear band of about 40 kD in transfected cells successfully a Gprc5a-deficient mouse line (2/2). whereas control cells showed no reactivity (Supplemental Fig- Knockout mice were born in an expected Mendelian her- ure 3D). Moreover, we used an additional Gprc5a antibody itance, developed normally, and did not exhibit any obvious that was directed against a different part of Gprc5a protein.11 macroscopic or histologic abnormalities in major organs (data This antibody gave nearly an identical staining pattern in kid- not shown). Renal histology was normal at birth and at 2 ney tissue as the first antibody (Supplemental Figure 3E). months of age as observed in routine light and electron microscopic examination (Figure 3, E–H). The glomerular filtra- Gprc5a Expression Is Downregulated in DN tion barrier seemed to be also functionally intact because no To study whether Gprc5a plays a role in human glomerular albuminuria was detected at 2 months of age (data not diseases,weanalyzeditsexpressioninbiopsysamplescollected shown). from patients with DN. Clinical parameters of patients included are found in Supplemental Tables 2 and 3. We Gprc5a Deficiency Results in Thickening of the GBM detected a clearly reduced Gprc5a expression in DN glomeruli and Activation of Profibrotic Signaling Pathways by immunohistochemistry (Figure 2, A and B). This was val- Toevaluate theeffects of agingon Gprc5a-deficient glomeruli, idated by a semiquantitative scoring of glomerular staining we analyzed albuminuria, blood chemistry, and kidney his- intensity (Figure 2C). Because podocytes can be lost in ad- tology inmiceat12monthsofage.Nosignificantalbuminuria vanced stages of DN, we performed a semiquantitative im- or increase in BUN was detected in 12-month-old 2/2 mice munoelectron microscopy study in which we analyzed (Supplemental Table 4). In histologic examination, PAS Gprc5a expression in remaining podocytes. This analysis staining revealed a significant expansion of mesangial matrix showed significantly decreased labeling for Gprc5a protein in 2/2 mice (Figure 4, A and B). This was validated by mea- in DN (Figure 2, D–F). suring mesangial index and by a semiquantitative scoring of To analyze Gprc5a mRNA levels in DN glomeruli, we first mesangial expansion (Figure 4C, Supplemental Figure 4E). To analyzed our unpublished RNA sequencing dataset generated analyze mesangial cell phenotype in more detail, we performed from microdissected glomeruli. The expression of Gprc5a immunostaining for a–smooth muscle actin (aSMA), a mRNA was significantly reduced in isolated glomeruli of 20 marker for the activation of mesangial cells.13 The expression patients with DN when compared with 20 control patients of aSMA was clearly upregulated in knockout glomeruli (Fig- (A. Levin et al., manuscript under preparation). Finally, we ure 4, D and E). The increased aSMA expression was validated

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Figure 2. Gprc5a expression in the glomerulus is downregulated in patients with DN. (A and B) Immunohistochemistry in paraffin- embedded kidney biopsy samples shows clearly reduced glomerular staining in patients with DN in comparison to controls. (C) Visual scoring of staining intensity shows significant downregulation of Gprc5a expression. (D and E) Immunoelectron microscopy shows less gold labeling for Gprc5a in podocytes of patients with DN. (F) Semiquantification validates the reduced labeling intensity. (G) RNA levels of Gprc5a are significantly reduced in microdissected DN glomeruli in comparison to controls as detected by microarray using two different probes (left and right). No significant expression is detected in tubular fractions. Red, DN; blue, control. (A and B) Original magnifications, 3200. (D and E) Scale bars=150 nm. (C and F) Data shown as the mean6SEM; *P,0.05 was considered statistically significant. Ctrl, control; FP, foot process; Glo; glomerulus. by semiquantitative scoring of immunofluorescence staining DN,14 we analyzed EGFR expression in 2/2 glomeruli. The (Figure 4F). No tubulointerstitial changes were detected in expression of EGFR was five-fold upregulated as detected by 12-month-old 2/2 kidneys (Supplemental Figure 4, A and B). qPCR (Figure 4J). Taken together, lack of Gprc5a in podocytes In electron microscopic examination, we detected a striking results in the activation of profibrotic signaling in glomeruli and thickening of the GBM in 12-month-old 2/2 mice (Figure 4, glomerular phenotype that closely phenocopies histopathology G–I). Slit diaphragms appeared intact and endothelial mor- of human DN. phology was unaffected (Supplemental Figure 4, C and D). Moreover, podocytes showed occasional foot process efface- Gprc5a Deficiency Promotes Glomerular Injury in ment but this did not reach statistical significance when slits/GBM Diabetes length were analyzed (Supplemental Figure 3F). Because Gprc5a expression was downregulated in human DN, we Because the GBM and mesangial matrix were expanded in wanted to analyze its pathogenic role in a mouse model of diabetes. 12-month-old 2/2 kidneys, we analyzed profibrotic signaling Diabetes was induced in 2/2 and littermate control mice at the pathways in isolated glomeruli. Using qPCR, we detected a age of 8 weeks with intraperitoneal streptozotocin injections and 35-fold upregulation of TGF-b, a cardinal driver of fibrosis, the mice were followed for 24 weeks. All mice developed diabetes in 2/2 glomeruli when compared with age-matched littermates as indicated by blood glucose values of .200 mg/dl measured (Figure 4J). In line with this, Col1a1, a downstream effector of 2 weeks after the induction (Supplemental Table 5 with other key TGF-b, was significantly upregulated (20-fold). Because EGFR parameters). Gprc5a-deficient mice were significantly more albu- has been shown to modulate TGF-b signaling in podocytes in minuric than control STZ-treated mice as indicated by Alb/Crea

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Figure 3. Mutant mice do not express Gprc5a and do not show any obvious renal phenotype at young age. (A) TALENs were used to generate a 2-bp deletion in exon 2 of Gprc5a gene (marked with green in DNA sequence). The gene goes out of frame and is predicted to generate a short 22-residue truncated protein. (B) Genotyping of Gprc5a mice. Wild-type mice show 170 bp, homozygous mutant 340 bp, and heterozygotes both fragments. (C and D) Mutation generates a null allele because no Gprc5a protein or mRNA is detected in mutant animals. (E and F) Kidney histology is normal in 2-month-old 2/2 animals as demonstrated by PAS staining. (G and H) In electron microscopy, no abnormalities are detected in glomerular endothelial cells, the GBM, or podocytes. (E and F) Original magnifications, 3200. (G and H) Scale bars=250 nm. KO, knockout; PAS, periodic acid-Schiff staining; TALEN, transcription activator-like effector nuclease. ratio measurements and by analyzing urine samples on SDS- Electron microscopic examination showed segmental foot process PAGE gel (Figure 5, A and B). Albuminuria was higher in 2/2 effacement in 2/2 kidneys, whereas control STZ-treated animals mice already 5 weeks after the induction of diabetes and remained exhibited intact foot process architecture (Figure 5, I–J). elevated until the end of the experiment. Mice were euthanized 24 weeks after the injection and kidneys Overexpression of Gprc5a Inhibits EGFR and TGF-b were analyzed. Macroscopically, 2/2kidneys were indistinguishable Signaling in Cultured Podocytes from littermate controls (data not shown). Histologic examination Next, we analyzed Gprc5a in a well established podocyte cell revealed, however, more significant changes in Gprc5a-deficient culture line.8 Because endogenous expression of Gprc5a was glomeruli. FSGS as well as few totally sclerotic glomeruli were ob- rather low in cultured cells, we started by generating a stable served in 2/2 kidneys of STZ-treated mice, whereas control STZ- overexpressing cell line. The overexpression was validated by treated mice exhibited only discrete mesangial expansion (Figure 5, western blotting (Figure 6A) and localization of Gprc5a to the C and D). The semiquantification of sclerotic changes in glomeruli plasma membrane was shown by immunofluorescence staining validated the significant difference between 2/2 and control kid- (Supplemental Figure 4A). We analyzed signaling mediated by neys (Figure 5E). Moreover, mesangial index and semiquantification EGFR because Gprc5a has been linked to EGFR in the lung.11 of mesangial changes revealed significantly more prominent The stable overexpression of Gprc5a in podocytes inhibited expansion of mesangial matrix in KO animals (Figure 5, F–H). EGFR activation after EGF stimulation as detected by decreased

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Figure 4. Aging Gprc5a mice develop DN-like changes in the glomerulus. (A and B) Significant mesangial expansion was detected in Gprc5a knockout animals at 12 months of age as detected by PAS staining. (C) Mesangial index shows significant expansion in 2/2 animals in comparison to littermate controls. (D and E) Staining for aSMA (green) is increased in 2/2 glomeruli as shown by immunofluorescence staining. Double labeling was performed with anti-nephrin antibody (red). (F) Semi- quantitative scoring of glomerular staining validates the induction of aSMA expression in 2/2 mice in comparison to controls. (G and H) In electron microscopy, the thickening of the GBM (arrow) is observed and podocytes show focal foot process effacement. (I) The quantification validates the significant thickening of the GBM. (J) Expression of TGF-b,Col1a1,and EGFR genes in isolated glomeruli is upregulated in 12-month-old 2/2 mice as shown by qPCR. Original magnifications, 3200 (A and B); 3100 (D and E). (G and H) Scale bars=250 nm. (C, F, I, and J) Data shown as the mean6SEM; *P,0.05 was considered statistically significant. CTRL, control; KO, knockout; PAS, periodic acid-Schiff staining; qPCR, quantitative-PCR. receptor phosphorylation (Figure 6A). Overexpression also blun- Gprc5a showed decreased luciferase activity after EGF stimulation ted downstream signaling to TGF-b pathway as detected by de- (Figure 6B). To validate that Gprc5a modulated also down- creased TGF-b levels and Smad2/3 phosphorylation (Figure 6A). stream targets of TGF-b signaling, we performed qPCR experi- Expressional changes were validated by calculating normalized ments for collagen 1 a 1(COL1A),fibronectin, and aSMA. As densities in the blot (Supplemental Figure 4B). Inhibition of expected, overexpression of Gprc5a blunted the activation of TGF-b signaling was further confirmed in a luciferase assay using COL1A1, fibronectin, and aSMA expression after EGF exposure Smad2/3 responsive element because podocytes overexpressing (Figure 6, C–E).

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week week

Figure 5. Gprc5a deficiency promotes diabetic damage in the glomerulus. (A and B) After induction of diabetes with streptozotocin (STZ) (week 0), 2/2 mice develop higher albuminuria than STZ-treated littermate controls as shown by albumin-to-creatinine ratios and analysis of urine samples on SDS-PAGE gel. BSA was used as a positive control. (C–E) Kidney histology at 24 weeks after the induction of diabetes shows more sclerotic changes in 2/2 glomeruli in comparison to controls. (F and G) Mesangial index and semiquantification of mesangial changes shows significant expansion of mesangial matrix in 2/2 animals. (H–J) Electron microscopic examination exhibits segmental podocyte foot process effacement in 2/2 glomeruli, whereas foot process structure is intact in control animals. (C and D) Original magnifications, 3400. (F and G) Scale bars=250 nm. (E–H) Data shown as the mean6SEM; *P,0.05 was considered statistically significant. Alb, albumin; Cre, creatinine; Ctrl, control; KO, knockout.

Silencing Gprc5a Expression Promotes EGFR and TGF-b had remaining effects on cellular signaling. The silencing Signaling in Cultured Podocytes of Gprc5a in podocytes promoted EGFR activation after EGF Although endogenous expression of Gprc5a in cultured podo- stimulation as detected by increased receptor phosphorylation cytes was clearly weaker than in vivo (data not shown), we (Figure 7A). Silencing promoted also downstream signaling to silenced the gene using siRNA to see whether this expression TGF-b pathway as detected by increased TGF-b levels and

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Figure 6. Overexpression of Gprc5a inhibits EGFR activation and TGF-b signaling. (A) Western blotting shows blunted activation of EGFR and Smad2/3, as well as decreased expression of TGF-b1 after exposure to HB-EGF in Gprc5a-overexpressing cells. Actin was used as a loading control. (B) In the luciferase assay, the activation of Smad2/3 is diminished after exposure to HB-EGF in cells overexpressing Gprc5a. (C–E) Overexpression of Gprc5a suppresses expression of Col1A1, fibronectin, and aSMA genes after exposure to HB-EGF as detected by qPCR. (B–E) Data shown as the mean6SEM; *P,0.05 was considered statistically significant. 5a, Gprc5a; FN, fibronectin; HB-EGF, heparin-binding EGF-like growth factor; UT, untreated cells; Vec, empty vector.

Smad2/3 phosphorylation (Figure 7A). Expressional changes diabetes, and (4) Gprc5a modulates EGFR and TGF-b signaling were validated by calculating normalized densities in the blot pathways. Thus, Gprc5a plays a pathogenic role in the nephrop- (Supplemental Figure 5C). Activation of TGF-b signaling was athy and we propose that Gprc5a can be an interesting novel further confirmed in a luciferase assay using Smad2/3 responsive molecular target providing the possibility to develop a cell-specific element because podocytes overexpressing Gprc5a showed in- therapy option for DN. creased luciferase activity after EGF stimulation (Figure 7B). To Renal fibrosis is a hallmark of CKD and predicts poor prog- analyze whether the silencing of Gprc5a also had effects on down- nosis for renal insufficiency.16 TGF-b plays a critical role in the stream targets of TGF-b, we analyzed the expression of COL1A1, progression of renal fibrosis.1 Therefore, the pharmaceutic fibronectin, and aSMA using qPCR. In line with our results in industry has tried to develop molecular compounds that overexpression experiments, silencing of Gprc5a with siRNA pro- would inhibit the signaling pathway.17 However, targeting moted the expression of COL1A1, fibronectin, and aSMA in TGF-b in a systemic fashion may induce unexpected effects be- comparison to control cells (Figures 7, C–E, and 8). cause TGF-b can have both profibrotic and anti-inflammatory roles.18 This dual role can be one of the reasons for a recently failed clinical trial with anti–TGF-b therapy in DN.17 Sim- DISCUSSION ilarly, EGFR has gained a lot of attention in kidney diseases because its activation is involved in the pathogenesis of acute Podocyte damage, mediated for instance by TGF-b and EGF sig- and chronic renal injuries.19,20 Activation of EGFR can result naling, is a central event in the pathogenesis of DN.15 In this study, in beneficial or detrimental consequences in the kidney, de- we have identified a novel highly podocyte-specificGPCR, pending on the particular setting.20 In AKI, EGFR activation Gprc5a, which can allow us to manipulate these pathogenic sig- promotes recovery of renal function and structure. In naling pathways in a cell-specific fashion. We show that: (1) chronic disease models, however, EGFR activation contrib- Gprc5a is expressed highly by podocytes and not by other cells utes to development and progression of renal diseases.19,20 in the kidney, (2) Gprc5a expression is significantly downregula- Opposing effects of EGFR in kidney diseases have com- ted in patients with DN, (3) inactivation of Gprc5a in mice results promised possibilities to use it as a target of pharmaceutic in DN-like phenotype and promotes glomerular injury in intervention.

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Figure 7. Silencing of Gprc5a expression promotes EGFR activation and TGF-b signaling. (A) Western blotting shows increased activation of EGFR and Smad2/3, as well as increased expression of TGF-b1 after exposure to HB-EGF in Gprc5a silenced cells. Actin was used as a loading control. (B) In the luciferase assay, the activation of Smad2/3 is promoted after exposure to HB-EGF in cells with silenced Gprc5a expression. (C–E) Silencing of Gprc5a expression promotes the expression of Col1A1, fibronectin, and aSMA genes after exposure to HB-EGF as detected by qPCR. (B–E) Data shown as the mean6SEM; *P,0.05 was considered statistically significant. FN, fibronectin; HB-EGF, heparin-binding EGF-like growth factor; UT, untreated cells.

In this study, we show that a highly podocyte-speciﬁc to modulate these two critical signaling pathways in a cell- GPCR, Gprc5a, regulates both EGFR and TGF-b signaling speciﬁc fashion and in that way escape potential effects result- pathways in podocytes. Thus, Gprc5a offers us an opportunity ing from systemic modulation of cellular signaling. Previous

Figure 8. Hypothetical model showing how Gprc5a-depletion activates EGFR-mediated matrix production in DN. In normal conditions (left), Gprc5a inhibits the activation of EGFR. In DN (right), the expression of Gprc5a is downregulated leading to loss of inhibitory signals that results in phosphorylation of EGFR. EGFR activates TGF-b signaling that causes the activation of mesangial cells and increased matrix deposition in both the GBM and the mesangium.

1688 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1679–1689, 2018 www.jasn.org BASIC RESEARCH studies have shown that inactivation of EGFR exclusively in 3. Wacker D, Stevens RC, Roth BL: How ligands illuminate GPCR molec- podocytes protects kidneys from DN,13 which underlines the ular pharmacology. Cell 170: 414–427, 2017 key role of this pathway in podocytes. The activation of EGFR 4. Patrakka J, Xiao Z, Nukui M, Takemoto M, He L, Oddsson A, et al.: Expression and subcellular distribution of novel glomerulus-associated plays a critical role also in the development of rapidly pro- proteins dendrin, ehd3, sh2d4a, plekhh2, and 2310066E14Rik. JAm gressive GN.21 It will be interesting to evaluate how Gprc5a is Soc Nephrol 18: 689–697, 2007 modulated in other glomerular disease processes. 5. Patrakka J, Ruotsalainen V, Ketola I, Holmberg C, Heikinheimo M, On the basis of our findings in human DN and in Gprc5a- Tryggvason K, Jalanko H. Expression of nephrin in pediatric kidney J Am Soc Nephrol – deficient mice, we believe that chronic reduction in Gprc5a ex- diseases. 12(2): 289 296, 2001 6. Sistani L, Dunér F, Udumala S, Hultenby K, Uhlen M, Betsholtz C, et al.: pression plays a pathogenic role in the progression of DN. One Pdlim2 is a novel actin-regulating protein of podocyte foot processes. possibility is that the reduced Gprc5a expression drives the dedif- Kidney Int 80: 1045–1054, 2011 ferentation of podocytes and in that way promotes disease pro- 7. Sistani L, Rodriguez PQ, Hultenby K, Uhlen M, Betsholtz C, Jalanko H, gression. Our cell culture studies have, on the other hand, only et al.: Neuronal proteins are novel components of podocyte major limited value in this respect because the short-term nature of these processes and their expression in glomerular crescents supports their role in crescent formation. Kidney Int 83: 63–71, 2013 experiments cannot copy the chronic nature of DN. However, the 8. Saleem MA, O’Hare MJ, Reiser J, Coward RJ, Inward CD, Farren T, et al.: A results in cell culture support our other findings in mice showing conditionally immortalized human podocyte cell line demonstrating that Gprc5a modulates EGFR and TGF-b signaling in podocytes. nephrin and podocin expression. J Am Soc Nephrol 13: 630–638, 2002 Gprc5a is a novel molecular player restricted to the glomerular 9. Stitt-Cavanagh EM, Faour WH, Takami K, Carter A, Vanderhyden B, JAm podocyte that plays an important role under normal physiologic Guan Y, et al.: A maladaptive role for EP4 receptors in podocytes. Soc Nephrol 21: 1678–1690, 2010 conditions and that additionally possesses disease-modifying prop- 10. Lenoir O, Milon M, Virsolvy A, Hénique C, Schmitt A, Massé JM, et al.: erties. Although these observations highlight the involvement of Direct action of endothelin-1 on podocytes promotes diabetic glo- Gprc5a in determining podocyte and renal health, a druggability merulosclerosis. JAmSocNephrol25: 1050–1062, 2014 assessment of this receptor reveals a number of challenges. Although 11. Zhong S, Yin H, Liao Y, Yao F, Li Q, Zhang J, et al.: Lung tumor sup- fi pressor GPRC5A binds EGFR and restrains its effector signaling. Cancer classi ed as a GPCR of receptor class C (metabotropic glutamate Res – – 75: 1801 1814, 2015 receptor like), Gprc5a does not possess the long amino-terminal 12. Woroniecka KI, Park AS, Mohtat D, Thomas DB, Pullman JM, Susztak K: domain that characterizes the metabotropic glutamate receptors of Transcriptome analysis of human diabetic kidney disease. Diabetes 60: this family and that is requisite for ligand binding, receptor activa- 2354–2369, 2011 tion, and G-protein coupling.22 Gprc5a is an orphan receptor and 13. Nagai K, Tominaga T, Ueda S, Shibata E, Tamaki M, Matsuura M, et al.: there is currently no evidence indicating that this GPCR couples to Mesangial cell mammalian target of rapamycin complex 1 activation results in mesangial expansion. J Am Soc Nephrol 28: 2879–2885, 2017 23 G-proteins. Needless to say, it is challenging to develop a screening 14. Chen J, Chen JK, Harris RC: EGF receptor deletion in podocytes attenuates cascade without identification of ligands and substantial pathway diabetic nephropathy. JAmSocNephrol26: 1115–1125, 2015 investigation. Whether Gprc5a can be ligand activated or whether 15. Herman-Edelstein M, Weinstein T, Gafter U: TGFb1-dependent po- alternative strategies to manipulate its signaling function are feasible docyte dysfunction. Curr Opin Nephrol Hypertens 22: 93–99, 2013 fi fi J Clin will require dedicated investigation. 16. Duf eld JS: Cellular and molecular mechanisms in kidney brosis. Invest 124: 2299–2306, 2014 17. Voelker J, Berg PH, Sheetz M, Duffin K, Shen T, Moser B, et al.: Anti- TGF-b1 antibody therapy in patients with diabetic nephropathy. JAm ACKNOWLEDGMENTS Soc Nephrol 28: 953–962, 2017 18. Sureshbabu A, Muhsin SA, Choi ME: TGF-b signaling in the kidney: Profibrotic and protective effects. Am J Physiol Renal Physiol 310: This study was supported by KI/AZ Integrated Cardio Metabolic Center, F596–F606, 2016 Marianne and Marcus Wallenberg Foundation, Swedish Diabetes Founda- 19. Harskamp LR, Gansevoort RT, van Goor H, Meijer E: The epidermal tion, Westmans Foundation, Karolinska Institute Foundation and through growth factor receptor pathway in chronic kidney diseases. Nat Rev the regional agreement on medical training and clinical research (ALF) Nephrol 12: 496–506, 2016 between the Stockholm County Council and the Karolinska Institute. 20. Tang J, Liu N, Zhuang S: Role of epidermal growth factor receptor in acute and chronic kidney injury. Kidney Int 83: 804–810, 2013 21. Bollée G, Flamant M, Schordan S, Fligny C, Rumpel E, Milon M, et al.: Epidermal growth factor receptor promotes glomerular injury and renal DISCLOSURES failure in rapidly progressive crescentic glomerulonephritis. Nat Med M.L. is employed by AstraZeneca. J.P.’s research is supported by AstraZeneca. 17: 1242–1250, 2011 22. Bräuner-Osborne H, Krogsgaard-Larsen P: Sequence and expression pattern of a novel human orphan G-protein-coupled receptor, GPRC5B, a family C Genomics – REFERENCES receptor with a short amino-terminal domain. 65: 121 128, 2000 23. Zhou H, Rigoutsos I: The emerging roles of GPRC5A in diseases. On- coscience 1: 765–776, 2014 1. Chang AS, Hathaway CK, Smithies O, Kakoki M: Transforming growth factor-b1 and diabetic nephropathy. Am J Physiol Renal Physiol 310: F689–F696, 2016 2. Reidy K, Kang HM, Hostetter T, Susztak K: Molecular mechanisms of This article contains supplemental material online at http://jasn.asnjournals. diabetic kidney disease. JClinInvest124: 2333–2340, 2014 org/lookup/suppl/doi:10.1681/ASN.2017101135/-/DCSupplemental.

J Am Soc Nephrol 29: 1679–1689, 2018 Gprc5a in Diabetic Nephropathy 1689 Supplemental Figure 1.

F2R S1PR1

GPR160 ELTD1

CD97 Supplemental Figure 2.

A Human Protein Atlas RNAseq database

B Expression in mouse ssues

lung glom brain heart liver kidney spleen testis skm rok

Gprc5a Gapdh Supplemental Figure 3.

A Gprc5a Pdgfrb Merged

Gprc5a CD31 Merged CL

B C 1.4 * 1.2 mc 1 matrix 0.8

0.6

0.4 matrix 0.2

0 pod end 1 2 mes3

D Vector Gprc5a E

40 kD - - Gprc5a

- acn

Supplemental Figure 4.

Control 12 month-old KO 12 month-old A B

C D

E F 1 2 0.8 * 1.5 score 0.6 m µ 1 0.4

0.2 Slits/ 0.5

Mesangial 0 0 Ctrl KO Ctrl KO Supplemental Figure 5. A

B 1.2 Vector Gprc5a 1 * 0.8 0.6 * 0.4 * NormalizedDensity 0.2 0 pEGFR/tEGR pSmad/tSmad TGF-β1

C 1.8 siCON 1.6 siGprc5a * 1.4 * * 1.2 1 0.8 0.6

NormalizedDensity 0.4 0.2 0 pEGFR/tEGR pSmad/tSmad TGF-β1 Supplemental table 1. List of glomerulus-expressed GPCRs as detected by qPCR. Data shown as mean ± standard deviation (Glom=glomerulus, Rok=rest of kidney).

GPCR Glom Rok Glom/Rok LPAR6 41892,11 ± 38478,89 1040,06 ± 1370,12 39,28 ELTD1 30275,64 ± 14085,26 33,23 ± 46,99 910,13 GPR116 24020,06 ± 7789,84 55,1 ± 47,75 434,96 PTH1R 15402,81 ± 17644,32 7521,01 ± 3264,57 1,05 CALCRL 14096,09 ± 3854,84 199,06 ± 222,52 69,81 HPRT1 13342,04 ± 10677,69 1824,77 ± 1767,23 6,31 S1PR5 9474,7 ± 10124,3 110,84 ± 29,54 84,48 LPHN2 8645,89 ± 914,74 256,04 ± 293,87 32,77 FZD1 8176,2 ± 4947,45 1321,97 ± 1311,91 5,18 CXCR4 7097,31 ± 4388,91 535,98 ± 640,28 12,24 GPR160 6446,59 ± 1550,07 4816,3 ± 5918,99 0,34 NPY1R 6177,74 ± 6282,76 209,64 ± 296,48 28,47 PTGER4 5323,66 ± 3789,51 179,13 ± 210 28,72 RXFP1 4785,68 ± 1531,16 1,13 ± 1,6 4234,74 ADRB1 4404,61 ± 6229,06 307,91 ± 348,03 13,3 F2R 4140,07 ± 1977,93 167,62 ± 174,95 23,7 GPR4 3969,84 ± 833,92 91,04 ± 112,2 42,61 NPR1 3934,21 ± 3350,55 95,16 ± 45,01 40,34 S1PR1 3456,24 ± 275,58 18,02 ± 20,35 190,78 P2RY13 2889 ± 1976,44 300,27 ± 334,26 8,62 GPR18 2638,88 ± 55,61 274,22 ± 387,81 8,62 CD97 2473,76 ± 1908,21 81,07 ± 22,28 29,51 FZD4 2276,04 ± 1152,38 133,56 ± 118,89 16,04 LGR4 2066,89 ± 917,68 636,44 ± 782,06 2,25 EDNRB 1968,09 ± 1987,51 175,56 ± 203,42 10,21 FZD8 1883,8 ± 1522,78 286,02 ± 164,32 5,59 CMKLR1 1843,35 ± 1319,13 24,54 ± 20,92 74,12 FZD7 1812,88 ± 909,53 334,15 ± 417,1 4,43 CRCP 1618,04 ± 927,95 886,3 ± 190,45 0,83 GPRC5B 1585,22 ± 371,1 43,6 ± 3,61 35,36 APLNR 1437,43 ± 1565,72 17,14 ± 12,4 82,88 P2RY8 1396,83 ± 492,35 116,37 ± 116,17 11 CCRL2 1334,77 ± 648,96 27,91 ± 35,84 46,83 TBP 1306,6 ± 143 201,97 ± 132,33 5,47 LTB4R2 1255,64 ± 913,91 31,48 ± 35,77 38,88 GPR56 1216,47 ± 42,82 707,7 ± 478,7 0,72 NPR2 1208,39 ± 764,5 82,62 ± 83,16 13,63 CYSLTR1 1173,21 ± 16,82 26,73 ± 21,52 42,89 SSTR1 1098,23 ± 906,65 600,49 ± 680,75 0,83 XPR1 1071,68 ± 136,26 861,57 ± 892,77 0,24 GPBAR1 1053,72 ± 873,55 87,5 ± 27,04 11,04 GPR34 1037,58 ± 4,29 456,5 ± 606,21 1,27 TAS2R13 1027,12 ± 214,18 340,33 ± 481,29 2,02 FPR1 1001,44 ± 1104,68 48,7 ± 54,58 19,57 VIPR1 957,99 ± 516,73 1,36 ± 0,22 702,22 GPR65 948,64 ± 345,01 37,8 ± 43,95 24,1 CHRM3 929,86 ± 118,76 3,65 ± 2,57 253,9 DRD5 921,67 ± 1168,33 441,64 ± 624,57 1,09 AGTR1 920,76 ± 1302,15 151,15 ± 205,5 5,09 CX3CR1 873,69 ± 145,36 49,2 ± 46,77 16,76 F2RL2 869,62 ± 159,18 99,39 ± 132,67 7,75 TAS2R14 832,8 ± 731,27 165,07 ± 217,96 4,05 GABBR1 828,79 ± 283 9,51 ± 13,45 86,17 CXCR2 825,56 ± 752,67 61,89 ± 54,6 12,34 TBXA2R 821,6 ± 93,03 0 ± 0 - TAS2R10 773,93 ± 567,76 280,27 ± 396,37 1,76 P2RY1 747,01 ± 345,04 352,9 ± 480,6 1,12 NPY5R 743,98 ± 35,34 372,79 ± 527,21 1 LANCL1 743,76 ± 113,51 320,23 ± 256,55 1,32 S1PR3 720 ± 15,8 36,34 ± 24,18 18,81 TAAR1 709,53 ± 873,23 291,99 ± 393,48 1,43 CCR2 679,16 ± 594,11 83,9 ± 97,54 7,1 EDNRA 678,55 ± 384,23 156,17 ± 169,42 3,35 TAS2R4 632,56 ± 386,52 78,5 ± 95,15 7,06 TAS2R50 620,65 ± 656 254,29 ± 359,62 1,44 DRD4 614,74 ± 725,98 26,47 ± 9,55 22,23 GPR173 597,31 ± 337,05 22,35 ± 15,48 25,73 FPR2 585,44 ± 649,47 29,28 ± 41,41 18,99 PTGER2 564,8 ± 15,77 20,19 ± 14,01 26,97 SORT1 555,94 ± 510,36 202,98 ± 178,16 1,74 HCAR3 548,79 ± 387,63 139,57 ± 128,38 2,93 HTR2B 525,1 ± 250,96 2,72 ± 1,27 191,86 GPR125 517,17 ± 38,73 176,27 ± 185,54 1,93 GPRC5A 504,97 ± 100,44 30,38 ± 42,97 15,62 OPN3 502,64 ± 266,96 497,29 ± 540,34 0,01 GPR180 498,51 ± 99,04 139,09 ± 156,46 2,58 CYSLTR2 497,64 ± 408,53 84,87 ± 112,11 4,86 CXCR7 461,02 ± 70,52 142,67 ± 141,63 2,23 C3AR1 457,53 ± 380,95 56,79 ± 63,58 7,06 P2RY10 456,65 ± 374,09 73,95 ± 104,58 5,18 C5AR1 454,69 ± 13,83 62,87 ± 19,31 6,23 GNRHR 432,27 ± 347,56 125,42 ± 177,37 2,45 CHRM2 414,87 ± 488,42 71,04 ± 100,46 4,84 ADORA1 403,89 ± 418,78 18,65 ± 4,59 20,66 VN1R1 397,47 ± 37,81 52,85 ± 74,74 6,52 TACR1 387,12 ± 315,38 0 ± 0 - GPR182 383,91 ± 53,67 25,6 ± 32,14 14 FZD3 382,1 ± 342,42 198,69 ± 160,84 0,92 MRGPRX1 377,21 ± 533,45 43,62 ± 54,99 7,65 TAS2R1 368,95 ± 454,33 84,16 ± 119,01 3,38 GPRC5D 368,66 ± 6,33 38,84 ± 45,44 8,49 GPR17 363,04 ± 146,32 39,34 ± 24,72 8,23 CCR5 362,98 ± 416,77 63,96 ± 90,45 4,68 GPR22 357,86 ± 387,31 272,84 ± 379,27 0,31 GRM3 356,14 ± 426,03 135,92 ± 192,23 1,62 TAS2R3 354,53 ± 319,34 38,11 ± 53,89 8,3 MC4R 352,89 ± 455,32 28,4 ± 40,17 11,43 CHRM5 351,54 ± 289,38 64,06 ± 79,25 4,49 TAS2R38 345,33 ± 411,82 34,93 ± 49,4 8,89 TAS2R16 341,94 ± 408,13 102,01 ± 143,66 2,35 NPY2R 333,89 ± 401,95 34,59 ± 44,44 8,65 GPR174 331,94 ± 304,56 50,64 ± 62,28 5,55 GPR21 330,87 ± 201,16 22,17 ± 31,35 13,93 HCAR1 328,57 ± 38,97 32,52 ± 7,04 9,11 MRGPRF 324,32 ± 8,37 34 ± 31,08 8,54 GRM7 322,15 ± 414,84 100,95 ± 132,24 2,19 TAAR6 318,12 ± 439,81 143,85 ± 190,89 1,21 TRHR 310,23 ± 364,33 109,31 ± 154,59 1,84 LTB4R 307,81 ± 103,47 53,55 ± 26,61 4,75 PTGFR 304,11 ± 361,65 204 ± 288,5 0,49 CHRM1 304,01 ± 40,17 25,64 ± 23,98 10,86 GPR63 301,93 ± 163,86 77,84 ± 96,33 2,88 GPR77 301,08 ± 10,09 114,49 ± 137,33 1,63 CCR1 296,15 ± 259,11 44,6 ± 60,08 5,64 HRH1 294,74 ± 132,58 14,06 ± 19,89 19,96 TAAR8 294,33 ± 294,18 131,53 ± 186,01 1,24 CCBP2 288,98 ± 79,88 43,81 ± 29,52 5,6 MRGPRD 287,27 ± 110,03 16,43 ± 4,18 16,48 HTR1F 286,18 ± 313,64 74,35 ± 105,14 2,85 CNR1 274,6 ± 289,21 28,29 ± 32,82 8,71 LPAR4 271,22 ± 379,55 53,53 ± 75,7 4,07 SIGMAR1 264,55 ± 83,92 89,27 ± 92,51 1,96 CXCR6 263,58 ± 184,94 21,16 ± 18,62 11,45 HTR2A 262,4 ± 331,83 75,59 ± 79,94 2,47 CRHR1 262,06 ± 203,66 46,65 ± 14,84 4,62 GPR133 261,06 ± 150,82 13,61 ± 5,87 18,18 OXTR 259,08 ± 125,38 174,66 ± 247 0,48 TAAR5 253,51 ± 288,62 13,28 ± 18,78 18,09 LPHN1 253,39 ± 141,85 8,64 ± 1,89 28,32 FFAR3 248,12 ± 43,77 19,57 ± 11,02 11,68 CCR3 246,09 ± 231,83 19,26 ± 18,36 11,78 GPR148 243,33 ± 182,91 43,23 ± 47,31 4,63 SMO 242,8 ± 54,27 120,53 ± 109,58 1,01 GPR135 242,24 ± 63,81 139,66 ± 29,34 0,73 CCR6 240,99 ± 259,32 103,99 ± 125,83 1,32 CCR9 239,91 ± 202,09 27,06 ± 38,27 7,87 HRH4 239,04 ± 271,63 65,48 ± 86,85 2,65 XCR1 236,17 ± 296,39 17,81 ± 11,59 12,26 HCAR2 234,9 ± 240,19 19,82 ± 11,32 10,85 GPR101 234,28 ± 283,61 27,14 ± 31,22 7,63 TAS2R5 233,81 ± 62,51 66,22 ± 93,64 2,53 P2RY4 232,72 ± 155,12 11,93 ± 16,87 18,51 FFAR2 231,93 ± 1,68 24,31 ± 28,17 8,54 GPER 230,11 ± 124,41 81,1 ± 21,48 1,84 GP1BA 226,76 ± 36,22 60,93 ± 70,9 2,72 PROKR1 220,45 ± 201,84 21,55 ± 30,47 9,23 MC2R 216,25 ± 257,15 62,26 ± 88,05 2,47 GPR111 213,32 ± 52,92 0 ± 0 - OPN4 204,93 ± 166,61 11,79 ± 4,99 16,38 P2RY2 201,19 ± 1,34 18,27 ± 14,68 10,01 GPR55 199,48 ± 83,54 10,77 ± 15,24 17,51 TAAR2 197,67 ± 250,16 75,54 ± 96,42 1,62 LGR5 196,84 ± 266,28 33,68 ± 47,63 4,84 HRH2 194,7 ± 64,78 18,79 ± 14,31 9,36 PTAFR 194,34 ± 2,19 28,85 ± 13,65 5,74 MLNR 191,37 ± 200,51 30,62 ± 43,3 5,25 GPR15 190,84 ± 247,23 29,86 ± 31,5 5,39 HTR1E 190,33 ± 239,58 12,52 ± 17,71 14,2 MAS1 189,34 ± 213,7 42,13 ± 59,58 3,49 BAI3 187,98 ± 205,11 1,73 ± 0,69 107,35 FZD6 182,78 ± 99,26 98,54 ± 107,33 0,85 GPR152 178,37 ± 113,66 70,7 ± 80,38 1,52 CCKBR 178,04 ± 243,5 17,89 ± 25,29 8,95 GPR61 176,47 ± 38,93 62,61 ± 88,54 1,82 GPR84 176,27 ± 123,87 14,71 ± 20,81 10,98 GPR119 175,94 ± 190,27 7,25 ± 10,25 23,28 MTNR1B 175,73 ± 207,88 9,6 ± 13,57 17,31 GRM8 173,63 ± 226,38 39,47 ± 45,15 3,4 RXFP4 171,85 ± 74,77 14,47 ± 9,67 10,88 OGFR 169,46 ± 109,84 80,55 ± 27,87 1,1 GRM2 169,07 ± 59,58 13,78 ± 13,66 11,27 GHSR 166,68 ± 169,44 18,77 ± 3,36 7,88 LPAR5 162,57 ± 124,99 40,58 ± 36,86 3,01 FZD10 161,37 ± 51,15 71,26 ± 96,86 1,26 HCRTR1 159,08 ± 33,45 16,84 ± 8,37 8,45 AVPR2 156,8 ± 178,87 48,82 ± 42,82 2,21 MC3R 155,41 ± 175,64 9,33 ± 13,2 15,65 TPRA1 155,37 ± 59,24 48,26 ± 65,01 2,22 BDKRB1 154,82 ± 18,21 24,03 ± 17,34 5,44 DARC 153,22 ± 160,04 9,89 ± 13,99 14,49 MC5R 152,1 ± 201,46 34,39 ± 36,86 3,42 GPR45 148,4 ± 132,96 20,85 ± 22,94 6,12 CXCR1 148,02 ± 39,73 3,04 ± 4,3 47,71 EMR3 146,19 ± 22,84 3,52 ± 4,98 40,49 GPR151 144,81 ± 64,8 44,32 ± 51,35 2,27 RGR 143,86 ± 98,89 26,49 ± 37,46 4,43 GPR161 142,66 ± 30,59 0 ± 0 - BDKRB2 137,96 ± 71,68 78,04 ± 58,34 0,77 AVPR1A 134,18 ± 158,56 14,83 ± 4,43 8,05 CNR2 133,25 ± 80,12 16,99 ± 16,51 6,84 GPR6 132,89 ± 178,58 21,92 ± 22,53 5,06 HTR1B 131,64 ± 135,78 9,49 ± 13,42 12,87 GPR113 127,85 ± 81,53 27,5 ± 13,61 3,65 LPAR2 124,31 ± 81,06 21,88 ± 9,6 4,68 CALCR 123,07 ± 44,31 6,26 ± 8,85 18,66 GPR32 122,37 ± 57,27 10,47 ± 3,69 10,69 MCHR1 120,55 ± 48,19 17,68 ± 14,64 5,82 GPR171 120,54 ± 31,36 26,51 ± 19,32 3,55 MC1R 120,43 ± 27,51 41,37 ± 29,77 1,91 HTR1D 118,9 ± 80,43 7,36 ± 2,86 15,15 CELSR3 118,22 ± 17,4 27,33 ± 15,17 3,33 DRD1 117,96 ± 138,52 9,93 ± 14,04 10,88 HTR1A 113,9 ± 96,34 14,64 ± 12,65 6,78 GNRHR2 113,13 ± 81,34 21,82 ± 2,88 4,19 GPR3 112,36 ± 83,66 32,91 ± 38,6 2,41 PTGDR 112,09 ± 70,35 6,72 ± 0,36 15,67 GPR68 111,25 ± 50,67 28,17 ± 2,93 2,95 GPR39 108,77 ± 101,5 29,51 ± 14,39 2,69 PROKR2 103,62 ± 110,51 8,63 ± 12,2 11,01 OXER1 102,84 ± 12,81 51,84 ± 0,62 0,98 TACR3 101,86 ± 22,89 1,86 ± 2,63 53,87 GPR12 101,46 ± 119,46 10,22 ± 14,45 8,93 CCR10 100,19 ± 7,59 21,44 ± 3,68 3,67 ADRA2B 100,08 ± 141,54 6,9 ± 9,75 13,51 CXCR3 98,01 ± 24,72 15,41 ± 4,54 5,36 GALR2 95,6 ± 10,66 46,08 ± 56,43 1,07 EMR2 95,51 ± 29,83 0,87 ± 1,24 108,16 SCARF2 95,22 ± 68,04 3,69 ± 5,22 24,81 FZD2 92,77 ± 42,17 19,99 ± 18,6 3,64 GPR31 92,41 ± 45,2 10,31 ± 3,16 7,96 GPR78 91,78 ± 37,18 25,59 ± 8,29 2,59 GPR124 89,58 ± 17,92 2,83 ± 4,01 30,61 GPR153 89,18 ± 7 43,2 ± 12,48 1,06 CHRM4 88,15 ± 7,66 26,72 ± 25,43 2,3 GALR3 87,01 ± 11,41 13,34 ± 3,2 5,52 ADORA2A 84,88 ± 120,04 32,79 ± 15,47 1,59 PRLHR 83,9 ± 19,77 58,36 ± 82,54 0,44 S1PR4 82,74 ± 10,19 51,78 ± 66,95 0,6 MRGPRE 81,71 ± 8,33 13,1 ± 5,29 5,24 S1PR2 81,14 ± 46,36 16,18 ± 12,06 4,02 RXFP3 77,7 ± 66,69 13,15 ± 5,11 4,91 P2RY6 76,78 ± 48,3 18,23 ± 7,73 3,21 FPR3 74,98 ± 30,43 8,9 ± 12,59 7,42 GPR1 74,41 ± 29,23 0 ± 0 - SSTR4 72,57 ± 62,15 11,37 ± 0,93 5,38 SSTR3 71,97 ± 27,75 3,76 ± 5,32 18,15 GPR27 70,08 ± 64,84 13,75 ± 15,03 4,1 BAI2 65,84 ± 30,39 23,84 ± 16,95 1,76 FFAR1 65,61 ± 15,82 18,58 ± 14,7 2,53 GPR143 59,51 ± 27,72 19,19 ± 3,21 2,1 P2RY14 55,48 ± 24,68 9,73 ± 13,76 4,7 GRM6 54,95 ± 58,21 13,16 ± 8,16 3,18 TAS1R3 49,87 ± 0,26 15,74 ± 16,46 2,17 BAI1 47,64 ± 52,15 24,81 ± 26,43 0,92 GRPR 47,48 ± 38,44 0 ± 0 - TMEM11 47,07 ± 6,85 25,03 ± 0,74 0,88 NPBWR2 46,93 ± 22,73 13,04 ± 12,9 2,6 EMR1 43,74 ± 40,75 7,92 ± 4,36 4,52 HTR6 43,73 ± 55,25 23,54 ± 6,11 0,86 GPR162 42,66 ± 0,59 10,93 ± 1,72 2,9 CCR4 37,09 ± 29,8 2,55 ± 3,61 13,54 NPBWR1 36,43 ± 48 25,89 ± 24,71 0,41 FZD9 34,76 ± 13,04 12,62 ± 4,37 1,75 OPRL1 30,49 ± 27,68 2,65 ± 0,01 10,51 NMUR1 29,43 ± 4,97 0 ± 0 - ADORA3 29,17 ± 41,26 11,17 ± 12,82 1,61 GPR35 28,44 ± 28,79 16,63 ± 6,3 0,71 UTS2R 26,92 ± 13,87 8,45 ± 6,99 2,19 GPR98 26,1 ± 33,49 0,87 ± 1,22 29,17 PDGFRL 24,68 ± 8,92 2,36 ± 3,33 9,48 P2RY12 23,86 ± 9,39 2,33 ± 3,3 9,22 TACR2 18,61 ± 16,35 2,66 ± 1,89 5,98 CCR7 17,61 ± 23,07 3,71 ± 5,25 3,74 GPR114 16,54 ± 7,29 1,79 ± 2,53 8,24 ADORA2B 16,3 ± 23,05 0,53 ± 0,75 29,59 CCRL1 16,06 ± 10,79 0 ± 0 - OPCML 15,79 ± 17,97 6,22 ± 8,79 1,54 CELSR2 15,79 ± 5,38 2,88 ± 4,08 4,48 GPR85 15,46 ± 0,72 1,77 ± 2,5 7,75 GPR123 15,28 ± 13,6 5,19 ± 0,81 1,95 GPR19 12,73 ± 6,65 0 ± 0 - GPR75 11,85 ± 10,02 0,42 ± 0,6 27,1 PTGIR 11,32 ± 7,81 3,8 ± 2,29 1,98 RRH 10,06 ± 3,16 7,76 ± 10,97 0,3 GPR97 10,02 ± 14,17 2,1 ± 2,97 3,77 PTH2R 6,64 ± 0,07 3,42 ± 4,84 0,94 GRM5 6,59 ± 6,22 4,04 ± 0,48 0,63 LHCGR 5,73 ± 7,41 3,58 ± 2,93 0,6 GIPR 5,32 ± 1,11 0,65 ± 0,92 7,17 HTR7 4,7 ± 6,65 0 ± 0 - TSHR 4,65 ± 0,81 0 ± 0 - GPR83 3,9 ± 5,51 0 ± 0 - ADRA1B 3,69 ± 5,22 1,38 ± 1,38 1,67 CCR8 3,46 ± 3,34 0 ± 0 - KISS1R 2,84 ± 3,89 0,6 ± 0,86 3,7 OPN5 2,61 ± 3,69 0 ± 0 - CASR 2,41 ± 2,37 2,16 ± 3,05 0,12 LGR6 2,16 ± 3,06 0,92 ± 1,3 1,36 P2RY11 2,12 ± 0,66 0,61 ± 0,39 2,45 OPRK1 1,77 ± 2,01 0,88 ± 1,24 1,01 GPR37 1,2 ± 0,58 0,6 ± 0,85 1,01 NMUR2 1,2 ± 1,7 0 ± 0 - VIPR2 0,7 ± 0,99 0 ± 0 - GRM4 0,62 ± 0,88 0,52 ± 0,74 0,19 GPR44 0,62 ± 0,88 0 ± 0 - RXFP2 0,53 ± 0,75 0,29 ± 0,41 0,81 NTSR1 0,48 ± 0,69 0 ± 0 - GHRHR 0,48 ± 0,68 0 ± 0 - HCRTR2 0,42 ± 0,59 0 ± 0 - NPFFR2 0,3 ± 0,42 0 ± 0 - HTR4 0,26 ± 0,37 0 ± 0 - LPAR3 0,21 ± 0,29 0 ± 0 - PCR 0,18 ± 0,25 0 ± 0 - GALR1 0,15 ± 0,21 0 ± 0 - F2RL3 0,14 ± 0,2 0 ± 0 - GLP2R 0,03 ± 0,04 0 ± 0 -

Supplemental Table 2. Parameters at the time of biopsy for DN patients and for control patients included in immunohistochemical studies. NA = not available. As controls we used tissue from kidneys nephrectomized due to renal cancer. ______Histological Age Gender P-Crea U-Alb Ischemia time Other relevant clinical diagnoses diagnosis (µmol/mol) (mg/L) (min) and histological findings ______DN 50 Male 626 3436 <5 retinopathy DN 68 Male 367 16 <5 hypertension, congestive heart failure DN 55 Male 131 NA1 <5 hypertension, congestive heart failure DN 63 Male 189 3576 <5 hypertension, retinopathy DN 54 Female 161 NA2 <5 MGUS3 (no amyloidosis in biopsy) Control 48 Male 96 NA 40 none Control 58 Male 84 NA <5 none Control 59 Male 83 NA 45 MALT lymphoma Control 80 Male 77 NA 30 hypertension Control 52 Male 52 NA <5 hypertension ______1U-Albumin/creatinine 328 mg/mmol 2U-Albumin/creatinine 470 mg/mmol 3Monoclonal gammopathy of undetermined significance Supplemental Table 3. Parameters at the time of biopsy for DN patients and for controls included in immunoelectron microscopic studies. DN=diabetic nephropathy; NA = not available. As controls we used biopsies from living related donors. ______Histological Age Gender P-Crea U-Alb/Cre Ischemia time Other relevant clinical diagnoses diagnosis (µmol/mol) (µg/mg) (min) and histological findings ______DN 64 Female 118 NA* <5 hypertension DN 64 Male 183 479 <5 hypertension, ischemic heart disease DN 70 Male 142 163 <5 hypertension DN 68 Female 93 9 <5 none DN 68 Male 75 224 <5 hypertension Control 52 Female 72 0.3 <5 none Control 43 Female 58 0.7 <5 none Control 42 Female 61 <5.0 <5 none Control 43 Female 73 <5.0 <5 none Control 49 Female 79 <5.0 <5 none ______*Albuminuria 8.1g/24h Supplemental Table 4. Parameters for Gprc5a KO mice at 12 months of age. No significant difference was observed between KO animals and litter-mate controls. ______Mouse Weight Kidney Blood urea nitrogen U-Alb/Crea (n) (g) (g) (mg/dl) ______KO (6) 32.72±1.32 0.51±0.09 22.08±1.31 32.04±15.41

Control (5) 32.73±0.82 0.49±0.11 22.61±1.41 30.69±27.05 ______

Supplemental Table 5. Parameters for mice treated with STZ to induce diabetes. ______Mouse Weight Kidney weight B-Gluc 7 days HbA1c 24 weeks BUN 24w Mortality (n) (g) (g) after STZ (%) (mg/dl) (n) (mg/dl) ______KO (7) 27.79±2.75 0.595±0.208 >2001 7.91±0.552 22.19±1.48 0

Control (5) 26.75± 2.54 0.546±0.201 >2001 8.34±0.872 23.15± 1.43 0 ______1Animals that did not show diabetic levels of blood glucose were sacrificed and not included in the study. 2Non-diabetic values in mouse (n=5) using this method were <5.00 Supplemental table 6. Primers used in this study.

Gene Species Primer sequences (Forward; Reverse) ACTA2 Human 5’-AGACCCTGTTCCAGCCATC-3’; 5’-GCTAGGGCCGTGATCTCC-3’ ACTA2 Mouse 5’-TGCTGTCCCTCTATGCCTCT-3’; 5’-GAAGGAATAGCCACGCTCAG-3’ Col1a1 Human 5’-GTGCTAAAGGTGCCAATGGT-3’; 5’-GGGTCCTTGAACACCAACAG-3’ Col1a1 Mouse 5’-TGACTGGAAGAGCGGAGAGT-3’; 5’-GAATCCATCGGTCATGCTCT-3’ EGFR Mouse 5’-CTTGCATGTTTGCACTCGTT-3’; 5’-GAGGCAAGAGAAGCAGATGG-3’ Fibronectin Human 5’- AAACCTCGGCTTCCTCCAT-3’; 5’-CGGTGGCTGTCAGTCAAAG-3’ Gapdh Mouse 5’-TGTTCCTACCCCCAATGTGT R-TGTGAGGAGATGCTCAGTG-3’ Gprc5a Human 5’-GCTGCTCACAAAGCAACGAA R-ATAGAGCGTGTCCCCTGTCT-3’ Gprc5a Mouse 5’-CTTCTTCGCATCCTTCTTGG R-AGTGTGTCCCCCATCTCAAG-3’ Gprc5a (genotyping) Mouse 5’-TTGCGGGGTATAGGTGTGT R-GCATATGTGGAACCCGTGTC-3’ Nephrin Human 5’-GAGAGCCCCATTCAAAGGCT R-AGAAGGAGCTCACGGTTTCG-3’ TGF-β Mouse 5’-GGCTACCATGCCAACTTCTG-3’; 5’-GTTGGACAACTGCTCCACCT-3’ 28S Human 5’-TTGAAATCCGGGGGAGAG R-AGATTGTTCCAACATGCCAG-3’ ______

SUPPLEMENTAL MATERIAL AND METHODS

Human kidney tissues and study approval

Control human tissue for stainings and Western blotting was collected from normal parts of kidneys removed due to renal carcinoma at Karolinska

University Hospital (Stockholm, Sweden). For immunoelectron microscopy studies, biopsy material from living kidney donors was used. Renal biopsies from diabetic patients showing DN were collected from archives of Karolinska

University Hospital (Stockholm, Sweden). The biopsies were evaluated using light microscopy, electron microscopy and immunofluorescence following standard procedures. Clinical data of these patients can be found in supplemental Tables 2 and 3. The collection of human tissue was accepted by the local ethical committee.

GPCR expression analysis using qPCR

Glomeruli isolated through sieving were used in the analysis. We isolated

RNA using standard methods and analysed the expression of human GPCRs using a pre-designed 384-well plate (GPCR Tier 1-4 H384, Bio-Rad).

Experiment was repeated twice and Gapdh was used for normalization.

RT-PCR and real-time PCR

Total RNA from mouse/human glomeruli, kidney tissue devoid of glomeruli and cultured podocytes was isolated using Trizol reagent (Invitrogen) and

RNA was reverse-transcribed using iScriptTM Reverse Transcription kit (Bio- rad). PCR experiments were performed using standard procedures. Relative mRNA levels of genes were calculated using the ΔΔCt method. Gapdh

(mouse) and 28S (human) were used for normalization. Primer sequences used for amplication are listed in supplemental table 4. Experiments were repeated at least three times, and data are expressed as mean ± S.E.

Statistical analysis was performed using Student's t test or one-way analysis of variance with Tukey's post hoc test. Glomeruli were isolated from human and mouse kidney tissue using standard procedures.

Western blotting

Proteins from kidney fractions and cultured cells were extracted by RIPA buffer and Western blotting was performed as described previously (23).

Antibodies used were directed against Gprc5a (Atlas Antibodies,

HPA007928), nephrin (22), p-EGFR (Tyr1068) (Cell Signaling, #2234), total

EGFR (Cell Signaling, #4627), p-Smad2/3, (Cell signaling, #8828), total

Smad2/3 (Cell signaling, #8685), TGF beta1 (Abcam, ab155264), actin

(Abnova, #DH1003) and calnexin (Abcam, #ab10286).

Immunostaining

Immunofluorescence and immunoperoxidase stainings were performed as previously described (4). Primary antibodies used were the same as used for

Western blotting. Additionally, anti-CD31 (BDbioscience, #560984), anti-

ASMA (Sigma, #H5228), PDGFRB (RandD system, #MAB126) and Gprc5a

(12) antibodies were used. Staining intensity in renal biopsies for Gprc5a and for ASMA in mouse kidneys was scored in each glomeruli as: 3 = strong; 2 = moderate; 1 = weak; 0 = absent. For Gprc5a, 87 glomeruli in 5 DN and 100 glomeruli in 5 control cases were analyzed. For ASMA, 30 glomeruli/mouse were analyzed in 5 -/- and 5 control kidneys.

Immunoelectron microscopy

Immunolabeling was performed as described previously (Wernerson et al,

2003). To semi-quantify labeling in immunoelectron microscopy, 30 images covering podocyte cytoplasm were chosen randomly in each biopsy. The area of the podocyte cytoplasm was calculated by point counting using a 1,5 x1,5 cm square lattice, and expressed as µm2. The number of gold particles was counted in the images and the mean concentration was calculated in each biopsy.

Electron microscopy

For transmission electron microscopy kidney samples were fixed with 2% paraformaldehyde and 2,5% glutaraldehyde. To quantify the thickness of the

GBM and number of slits, we selected 5 random capillaries from each sample.

In each capillary the distance between the podocyte and endothelia in 5 randomly chosen areas were measured and the mean value from the 5 capillaries was calculated and expressed as nm. In each capillary, the length of the GBM was measured and the number of podocyte slits was counted.

Thus from 5 capillaries, the mean value was calculated and expressed as slits/µm.

Mouse models Gprc5a mutant mouse strain in pure FVB background was generated using

TALEN technology (Cyagen). We targeted exon 2 of Gprc5a mouse gene and the mutation was verified using Sanger sequencing. Genotyping was performed by PCR-amplifying a 340 bp fragment around the deletion followed by digestion with Kpn1 enzyme. The amplified product is digested to two

170bp fragments in wild-type alleles, whereas mutant allele lacks the digestion site and thus only 340bp is detected. Inactivation of Gprc5a gene was verified using Western blotting and RT-PCR.

Diabetes was induced in 8-week old littermate control and Gprc5a mutant mice by giving intraperitoneal injections of streptozotocin (50 mg/kg, Sigma-

Aldrich) for 5 consecutive days. Induction of hyperglycemia was confirmed by measuring non-fasting blood glucose 2 weeks after the last injection and by measuring HbA1c at the end of the experiment. Urine was collected every second week starting from 5 weeks after the last injection. Mice were euthanized 24 weeks after injections. Key parameters of these mice are summarized in Table 5. The animal work was accepted by the local ethical committee.

Histological analyses

Kidneys for histological analysis were prepared according to standard procedures. Mesangial matrix expansion was evaluated in 12 month-old and

STZ-treated animals using PAS staining. The measurements were done in 5 -

/- and 5 control mice using two methods: 1) By calculating mesangial index

(using standard procedures with ImageJ software) in 5 random glomeruli/mouse; 2) By semi-quantitative scoring of 30 glomeruli/mouse as follows: 0 = no expansion, 1 = mild expansion, 2 = moderate expansion; 3 = severe expansion.

The presence of sclerotic changes in STZ-treated mice was quantified as follows: 30 glomeruli from each mouse was analyzed, score was 0 if no sclerotic lesions was present; 1 if segmental or global sclerosis was detected.

A total of 5 -/- and 5 control mice were analyzed.

Cell culture and generation of stable cell line

Immortalized human podocytes were grown as described previously (5).

Stable Gprc5a overexpressing cells were established by Lipofectamine 2000 transfection of pcDNA3.1-hGprc5a construct using standard procedures.

Silencing of Gprc5a expression was done using siRNA (Invitrogen) according to standard procedures. To study effects of Gprc5a on EGF mediated signaling, we added 25ng/ml of HB-EGF on culture media and collected cell lysates at 0 and 5 minutes. Experiments were repeated at least three times.

Luciferase reporter Assay

Effect of Gprc5a on Smad2/3 mediated target gene transcription was assessed further by using the dual-luciferase reporter system (Promega). The podocityes were transfected with pGL3-CAGA-luc plasmid and with renilla luciferase construct pRL-TK-Rluc. The pGL3-CAGA-luc is a reporter of the

Smad2/3 activation as the CAGA-box in the vector is transcribed by activated

Smad2/3 resulting in luciferase activity. Luciferase assays were performed according to standard procedures in normal culture media and with addition of 25ng/ml HB-EGF. Data is presented as relative luciferase units. Experiments were repeated at least three times.

FIGURE LEGENDS FOR SUPPLEMENTAL FIGURES

Supplemental Figure 1. Immunoperoxidase staining for F2R, S1PR1,

GPR160, ELTD1 and CD97 in human kidney tissue shows clear signal in glomeruli. Data extracted from Human Protein Atlas database

(proteinatlas.org). Magnifications: x400.

Supplemental Figure 2. (A) Expression of Gprc5a mRNA in different human tissues as detected by RNA sequencing (data extracted from www.humanproteinatlas.org). Gprc5a mRNA is highly expressed in lung tissues and only sparsely in other human organs. (B) Expression of Gprc5a mRNA in different mouse tissues as detected by RT-PCR analysis. Gprc5a can be detected only in lung tissue and in isolated glomerular capillary tufts.

Gapdh was used as a loading control.

Supplemental Figure 3. (A) Double labeling experiment with mesangial marker PDGFRB (red) does not show co-localization with Gprc5a (green).

Double staining with endothelial marker CD31 (red) does not show overlapping reactivity with Gprc5a (green). CL=capillary loop. (B)

Immunoelectron microscopy for Gprc5a does not show significant labeling in mesangial cells (mc). (C) Quantification of immune-gold experiment shows significant enrichment of Gprc5a signal in podocytes (pod) in comparison to endothelial (end) and mesangial cells (mes). (D) HEK293 cells transfected with Gprc5a construct show a strong band of about 40kDa whereas cells transfected with a control vector show no reactivity. Actin was used as a loading control. (E) Immunostaining with a previously characterized anti-

Gprc5a antibody (12) shows strong podocyte staining around glomerular capillary loops in both immunofluorescence and immunoperoxidase stainings.

Magnifications: x1000 in A; x200 in E. Data shown as the mean ± s.e in C;

*P<0.05 was considered statistically significant.

Supplemental Figure 4. (Analysis of 12-month old mice. (A-B) Knockout kidneys show normal normal histology in tubulointerstitial space despite obvious glomerular abnormalities. (C-D) In electron microscopy, podocyte slit diaphragms (black arrows) are intact and glomerular endothelial cells show normal morphology (red arrows). (E) Significant mesangial expansion is observed in 12-month old animals by semi-quantitative scoring. (F) No significant foot process effacement is detected as measured by counting podocyte filtration slits / GBM length. Data shown as the mean ± s.e in E.

*P<0.05 was considered statistically significant.

Supplemental Figure 5. Gprc5a in cultured human podocytes. (A)

Immunofluorescence staining for Gprc5a shows immunoreactivity at the plasma membrane (arrows) in the podocyte cell line transfected with Gprc5a.

(B) Normalized densities for pEGFR and pSmad2, as well as density for TGF-

β1 in control and Gprc5a overexpressing cells. The activation of EGFR and

Smad2, as well as expression of TGF-β1, is significantly reduced in overexpressing cells. (C) Normalized densities for pEGFR and pSmad2, as well as density for TGF-β1 in control and Gprc5a silenced cells. The activation of EGFR and Smad2, as well as expression of TGF-β1, is significantly promoted in cells with silenced Gprc5a expression. Data shown as the mean

± s.e in B and C; *P<0.05 was considered statistically significant. SIGNIFICANCE STATEMENT

Podocytesplaya key role intheprogression ofdiabetic nephropathy (DN). This manuscript describes a new podocyte-associated G protein–coupled receptor (GPCR), Gprc5a, which modulates the progression of DN by regulating EGF receptor (EGFR) and TGF- b signaling. Gprc5a can be an interesting molecule from the pharmaceutic point of view because it may allow manipulation of podocyte signaling in a cell- speciﬁcfashion.