SHROOM3-FYN Interaction Regulates Nephrin Phosphorylation and Affects Albuminuria in Allografts

BASIC RESEARCH www.jasn.org

Chengguo Wei,1 Khadija Banu,1 Felipe Garzon,1 John M. Basgen,2 Nimrod Philippe,1 Zhengzi Yi,1 Ruijie Liu,1 Jui Choudhuri,1 Miguel Fribourg,3 Tong Liu,4 Arun Cumpelik,1 Jenny Wong,1 Mubeen Khan,1 Bhaskar Das,1 Karen Keung,5 Fadi Salem,1 Kirk N. Campbell,1 Lewis Kaufman,1 Paolo Cravedi,1 Weijia Zhang,1 Philip J. O’Connell,5 John Cijiang He,1 Barbara Murphy,1 and Madhav C. Menon1

Due to the number of contributing authors, the afﬁliations are listed at the end of this article.

ABSTRACT Background We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin–binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development. Methods We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli. Results Expression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate.We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3–binding domain, distinct from its ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration. Conclusions We demonstrate a novel mechanism that may explain SHROOM3’s dichotomous associations in glomerular versus nonglomerular compartments in CKD

J Am Soc Nephrol 29: 2641–2657, 2018. doi: https://doi.org/10.1681/ASN.2018060573

CKD is defined by reduced eGFR or proteinuria, Received June 1, 2018. Accepted September 14, 2018. fi and characterized by interstitial brosis and C.W., K.B., and F.G. contributed equally to this work. glomerulosclerosis, reflecting damage to different Published online ahead of print. Publication date available at renal compartments. Genome-wide association www.jasn.org. studies (GWAS) have identified candidate susceptibility loci for CKD.1,2 Although mechanistic Correspondence: Dr. Madhav C. Menon or Dr. Barbara Murphy, Division of Nephrology, Department of Medicine, Icahn School studies have ascribed functional characteristics of Medicine at Mount Sinai, 1 Gustave L Levy Place, Annenberg to some of these loci,3 the detailed basis of these Building, 23-04A, New York, NY 10029. E-mail: Madhav.menon@ SNP-variant associations with renal function mssm.edu or [email protected] or histology remain largely undescribed. This Copyright © 2018 by the American Society of Nephrology

J Am Soc Nephrol 29: 2641–2657, 2018 ISSN : 1046-6673/2911-2641 2641 BASIC RESEARCH www.jasn.org hinders translation of GWAS information for the develop- Significance Statement ment of novel therapies. We previously identified the TCF7L2-dependent enhancer We previously identified that allograft SHROOM3 expression, and a function of the A-allele at rs17319721 in a cohort of renal CKD-associated intronic SHROOM3 locus with enhancer function, fi allograft recipients from the genomics of allograft rejection are associated with subsequent renal allograft brosis, whereas tubular Shroom3 knockdown reduced fibrosis. Although these data 4 fi study (GoCAR). Rs17319721, in the rst intron Shroom3, suggested potential to target SHROOM3 as therapy in kidneys is associated with CKD in GWAS.1,2,5 Shroom3 is an F-actin carrying the A-allele (risk locus), they contrast with evidence of a binding protein, important for epithelial morphogenesis via protective role for SHROOM3 in glomerular development. We rho-kinase (ROCK) binding.6,7 We observed enhanced show here the interesting divergent associations of glomerular and SHROOM3 SHROOM3 expression in A-allele donors at 3-month biopsy, nonglomerular expression with CKD, as well as the fi corresponding dichotomous associations of the enhancer A-allele associated with increased interstitial brosis score (CADI with eGFR and albuminuria in allografts. In adult glomeruli, score) in allografts by 1-year post transplant. Tubular-specific Shroom3 knockdown caused albuminuria without podocyte loss in Shroom3 knockdown in mice inhibited renal fibrosis in a ure- the short term. Shroom3 interacted with the Src-kinase FYN via teric obstruction model. Addition of a ROCK inhibitor abro- SH3-binding site in podocytes, and regulated FYN-activation and gated the enhanced collagen production when SHROOM3 downstream phosphorylation of Nephrin, and actin cytoskeletal organization. These current mechanistic data are essential before was overexpressed in tubular cells, suggesting that the the design of a therapeutic intervention in humans targeting ROCK binding function of the ASD2-domain of SHROOM3 SHROOM3 to inhibit renal fibrosis. contributed to profibrotic effects. Recent work has confirmed the TCF7L2-dependent transcription of a Shroom3 isoform in the presence of the A-allele and identified a 14–3-3 binding site, SHROOM3 SNP Analysis which could facilitate profibrotic Hippo signaling in renal cells.8 Targeted SNP genotyping for rs17319721 was done using These data suggested potential for testing the role of Shroom3 TaqMan SNP analysis assay (#4351379; Applied Biosystems, antagonism in renal tubules as a therapeutic strategy for renal Foster City, CA). DNA was extracted from preimplantation fibrosis in A/A or A/G genotype allografts. biopsies or blood for donor SNP and from peripheral blood However, other data suggest a protective role for Shroom3 for recipient SNP assay (approximately 20 ng per sample). 9,10 in glomerular development. In the fawn-hooded hyperten- In Vitro Studies fi sive rat model (FHH) of FSGS, mutations were identi ed within Cells the Shroom3 locus, and the FSGS phenotype was reversed with Human podocytes (Dr. Moin Saleem), and HEK-293 cells were wild-type Shroom3 gene.9 Homozygous Shroom3-knockout expanded using RPMI-1640 (1% ITS) and DMEM (Gibco) mice showed abnormal glomerulogenesis (e13.5–e18.5) and media. deficient myosin-II phosphorylation due to deficient ROCK- 10 binding function of Shroom3. When human CKD-GWAS Overexpression Studies SNPs were examined for associations with albuminuria, the A human SHROOM3 construct, and shRNA lentivirus has been A-allele was surprisingly associated with a mild beneficial described.4 Site-specific mutagenesis replacing -CCC/-CCA effect on albuminuria despite the increased risk of CKD.11 (Proline) with -GCC/-GCA (Alanine) by proprietary recombi- These data suggest a protective role for Shroom3 in pro- nase (GenScript Inc., Piscataway, NJ). Cell adhesion assay was teinuria and glomerular development, but do not explain the performed in b-Laminin–coated 96-well plates, as described.13 association of the A-allele with CKD, or allograft nephropathy. We hypothesized that SHROOM3 may have dichotomous roles shRNA Suppression Studies in glomerular and tubular compartments in order to explain the Human SHROOM3 short hairpin clones (Open Biosystems, associations of the enhancer SNP with lower albuminuria, Lafayette, CO) were tested for optimal suppression in 293-T and lower GFR. We used inducible, tissue-specific, shRNA- cells. The selected GFP-tagged hairpins were used to generate a mediated knockdown mice to examine the effect of Shroom3 mammalian VSV pseudotyped lentiviral expression construct. antagonism in postdevelopmental glomeruli, to describe the phe- Lentiviral medium was used to infect human podocytes at notype and ascribe mechanism. 33°C. Cells were passaged in puromycin (5 mg/ml)-RPMI 1640 for experiments after 7 days differentiation at 37°C.

METHODS Reverse Transcription For in vitro studies we used Superscript-III (Invitrogen, Life See Supplemental Material for details. Technologies, Grand Island, NY) with starting total RNA approximately 1000 ng. GoCAR Study Details of the observational GoCAR cohort, including eligibility Quantitative PCR and exclusion criteria are published elsewhere.4 eGFR was calcu- SHROOM3 expression was assayed in vitro/in vivo by real-time lated using Modiﬁed Diet in Renal Disease equation.12 protein-to-creatinine ratios (PCR) (7500; Applied Biosystems).

2642 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2641–2657, 2018 www.jasn.org BASIC RESEARCH

A non-Glomerular B non-Glomerular * * 2.0 3 Dataset-1 Dataset-2 1.5 2 1.0 1 0.5 (microarray) (microarray) 0 0.0 Log SHROOM3 Expression –0.5 Log SHROOM3 Expression –1 GFR<60 (n=17) GFR>60 (n=25) GFR<60 GFR 60-90 GFR>90 (n=14) (n=20) (n=15)

C non-Glomerular D Glomerular 200 Spearman r=-0.27 200 Dataset-2 Spearman r=0.34 P=0.05 Dataset-2 P=0.03 150 150

100 100 eGFR-baseline 50 50 eGFR ml/min (n=37)

0 0 0123 01234 Log SHROOM3 expression (microarray) Log SHROOM3 expression (microarray)

E F A/A A/G G/G A/A A/G G/G 300 2.5 n=9 n=27 n=34 n=13 n=39 n=23 p=0.08 2.0

200 p=0.05 1.5

1.0 100

0.5 Protein:Creatinine ratio (gm/gm)

Albumin/Creatinine ratio (mg/mmol) 0 0.0 3m 24m 3m 24m 3m 24m A/A 24m A/G 24m G/G 24m

Figure 1. Glomerular versus nonglomerular SHROOM3 expression and rs17319721 have dichotomous associations with eGFR and albuminuria in humans. Dot graphs plot SHROOM3 expression (log SHROOM3 expression by microarray) in nonglomerular fraction of (A) dataset 123 and (B) dataset 224 (glomerular and nonglomerular transcriptome) of renal biopsy samples plotted against eGFR (ml/min). Figure shows the correlation lines between eGFR (ml/min) and (C) nonglomerular and (D) glomerular SHROOM3 expression in dataset 2 (Spearman R=20.27 and 0.35; P=0.05 and 0.03, respectively). (E) Dot plots show serial mean ACR (mg/mmol) at 3 and 24 months in GoCAR Sydney cohort displayed by donor SNP type (paired t-test P=0.05 between 3- and 24-month A/A donors). (F) Dot plots show mean protein-to-creatinine ratios (mg/gm) at 24 month cross-sectionally by donor SNP type (unpaired t test A/A versus G/G =0.08). Dot plots/whiskers=mean/SEM; *P,0.05.

Ampliﬁcation curves were analyzed using automated as endogenous control. Similarly, primers were designed for 7500 software platform, via the DDCT method. Human glyc- mouse Gapdh, Nphs1, Podocin, and Synaptopodin (Supple- eraldehyde phosphate dehydrogenase (GAPDH)wasused mental Table 1).

J Am Soc Nephrol 29: 2641–2657, 2018 SHROOM3 Interacts with FYN in Podocytes 2643 BASIC RESEARCH www.jasn.org

Table 1. Demographics of GoCAR-Sinai and GoCAR-Sydney cohorts PBS with 1:10 AnV (BD-550474) and – – Demographics GoCAR-Sinai (n=70) GoCAR-Sydney (n=75) 1:1000 viability dye (65 0863 14; eBio- Donor age, mean6SD [range] 45.67614.9 yr [3–68] 40.77615.9 yr [14–76] science). As positive control, podocytes Donor sex (% females) 24 (34.3) 36 (48.0) were pretreated with 25% w/v H2O2 for Donor race 20 minutes at 4°C before staining. White (W) 33 (47.1) 68 (90.7) Black (AA) 15 (21.4) 0 (0.0) Mass Spectrometry Hispanic (H) 15 (21.4) 0 (0.0) PCDEST SHROOM3 and pcDNAcDNAM3 Other (O) 7 (10.0) 7 (9.3) lacZ (control vector) were overexpressed in Donor type 293 T cells. Protein lysates after 48 hours Deceased donors 57 (81.4) 38 (50.7) transfection were immunoprecipitated with Living donors 13 (18.6) 37 (49.3) anti–V5-tag, anti-SHROOM3, or control Recipient age, mean6SD [range] 56.57612.18 yr [26–83] 44.21612.6 yr [19–69] Recipient race IgG, and run on PAGE gels. Three resul- White (W) 19 (27.1) 61 (81.3) tant lanes were sent for mass spectrometry Black (AA) 33 (47.1) 0 (0.0) (protein mixture identification by LC/MS; Hispanic (H) 11 (15.7) 0 (0.0) LTQ Orbitrap Velos; ThermoFisher Scien- Other (O) 7 (10.0) 14 (18.7) tific). Proteins identified in both overexpres- Recipient sex (% females) 49 (70.0) 28 (37.3) sion and endogenous lanes were selected for Recipient ESRD further filtering analysis (See Supplemental Diabetes 29 (41.4) 29 (38.7) Material). Hypertension 24 (34.3) 21 (28.0) Glomerular disease 8 (11.4) 10 (13.3) In Vivo Studies Others 9 (12.9) 15 (20.0) Albuminuria, mean6SEMa NA Murine Shroom3 Knockdown Model Tetracycline-responsive, shRNAmir- 3-mo versus 24 mo 18.667.3 versus 5.762.2 A/A donors (n=13) 17.564.9 versus 17.267.1 mediated Shroom3 knockdown mouse A/G donors (n=39) 9.762.4 versus 30.1612.1 strain on the basis of tested shRNA guide G/G donors (n=23) sequences was developed with Mirimus Proteinuria, mean6SEMb NA Inc., NY. In double-transgenic CAGS- 2-yr visit 0.2560.21 rtTA/Podocin-RTTA/PAX8-RTTA;Shroom3 A/A donors (n=13) 0.4960.46 RNAi mice, shRNAmir-mediated knock- A/G donors (n=39) 0.6860.71 down was driven by the respective universal G/G donors (n=23) or tissue-specific promoters and inducible a Albumin: Creatinine ratio (mg/mol). by doxycycline (DOX) feeding.4 Male mice bProtein: Creatinine ratio (mg/g). (7–8 week old) were DOX-fed for 6 weeks (600 mg/g DOX chow; Envigo Inc.), and sub- Western Blotting $ Cells were lysed with a buffer containing 1% NP-40, a prote- jected to weekly urine collection (n 4micepergroupinalldata). ase inhibitor mixture, and tyrosine and serine/threonine After 6 or 8 weeks of DOX feeding, kidney tissues were collected fl phosphorylation and phosphatase inhibitors. Lysates were for histology, immuno uorescence, RNA isolation for quantita- subjected to immunoblot analysis using polyclonal rabbit tive PCR/RNA-sequencing, protein extraction for Western anti-SHROOM3 (#SAB3500818; Sigma), anti-V5 tag antibody blotting, and immunoperoxidase. Glomeruli and nonglomerular (A01724; GenScript Inc.), Phospho-SRC family Y416 rabbit fractions were extracted using DYNA-bead perfusion. mAb(#2101S;CellSignaling Technology),nonphosphorylated SRC family Y416 mouse mAb (#2102S; Cell Signaling Adriamycin Injection Study Technology), Fyn rabbit polyclonal antibody (#4023S; Cell Sig- Eight-week old male Podo-RTTA mice (C57B6/129SVJ/FVB naling Technology), mouse monoclonal Fyn (#610163; BD background) and littermates were fed DOX (6 weeks), and Biosciences), phospho-Nephrin (Y1176/1193; #Ab80299; were injected with retrobulbar injection (ADR 15 g/kg). Abcam), total Nephrin (gift from Dr. Lawrence Holzman to DOX was continued. Urine was collected 2-weekly till eutha- J.C.H.), and actin (mouse monoclonal, #A5441; Sigma). Anti- nasia at 8 weeks post injection. V5 magnetic beads (MBL International) were used for immunoprecipitation. Densitometry was performed on images of Aged Mice Western blots using ImageJ software. CAGS-TG mice were aged to 12 months on regular chow. DOX was initiated at 12 months for 12 weeks (n=3 each). Kidney Flow Cytometry tissue was collected for microscopy and IHC (P57). Mor- Human podocytes (Scramble or Si-2) were incubated for phometry on light microscopic images was performed after 20 minutes at 4°C in either annexin buffer (BD-556454) or thresholding using ImageJ or metamorph software.

2644 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2641–2657, 2018 www.jasn.org BASIC RESEARCH

Table 2. Multivariable regression analysis of donor SNP depletion was performed before Poly-A selection to improve genotype and proteinuria phenotype quality. Single-ended sequencing with 75 bp read length was GoCAR-Sydney (n=75) GoCAR-Sinai (n=70) carried out on NEXTSEQ (Illumina). The differential anal- Covariates Coefficient P Value Coefficient P Value ysis between transgenic and nontransgenic (NTG) samples by paired LIMMA was performed to identify significantly Donor , characteristics dysregulated genes at P value 0.05, which were then sub- Donor SNP 0.272 0.04 0.232 0.05 jected to Gene Ontology function and canonical pathway (ref-A/A) enrichment analysis by Fisher exact test. Genes uniquely Donor status 20.044 0.72 0.117 0.33 and exclusively downregulated in glomerular fraction, but (ref-DD) not in nonglomerular fraction were identified and com- Donor race 20.205 0.11 20.156 0.20 pared via Gene Ontology function and canonical pathway (ref-W) enrichment analysis. Podocyte-enriched genes were simi- Donor sex 0.111 0.38 0.093 0.44 larly compared on the basis of prior data.22 Complete data (ref-F) is available on the Gene Expression Omnibus database Recipient (GEO-GSE110092). characteristics Donor SNP 0.235 0.05 0.248 0.04 (ref-A/A) Statistical Analyses ESRD etiology 0.178 0.13 20.038 0.77 Descriptive statistics were used to summarize participants, (ref-DM) and compared by chi-squared/Fisher exact test. Spearman Recipient race 20.067 0.57 0.007 0.95 correlation was used to determine relationship between (ref-W) SHROOM3 expression, and eGFR (Nephroseq). Multivari- Recipient age 20.104 0.38 0.707 0.48 able linear regression was performed, with ACR and PCR as A/A, A/A genotype; DD, deceased donor recipients; W, white; F, female; outcomes. Predictors were assessed using baseline variables DM, diabetes mellitus. (SPSS version 23; SPSS Statistics).

Glomerular Morphometry Study Approval Tissue Processing. Glutaraldehyde fixed samples were processed Institutional review board approval for human subjects in as described.14 GoCAR was obtained from all sites; informed written consent was taken from all participants (living donors/recipients). All Podocyte Number. The fractionator/dissector method was used animal studies were in accordance with protocols approved by to count podocytes. Podocyte nuclei were surrogates for podo- the Institutional Animal Care and Use Committee at Mount cytes assuming only one nucleus per podocyte. Using an ultra- Sinai. The study adheres to the STROBE checklist for cohort microtome, serial 1-mm-thick sections were cut from an epon studies (see Supplemental Material). tissue block. Using Adobe Photoshop, the images from each pair of sections were view together. Podocyte profiles from glomerulus 1 seen in the second section of a pair (the sample section) RESULTS but not present in the first section (the look-up section) were 2 counted as a Q s. This counting was repeated for all the pairs of Renal Biopsies Show Dichotomous Associations of sections from a glomerulus (Supplemental Figure 1E).15,16 Glomerular versus Nonglomerular Shroom3 Expression Glomerular and Glomerular Component Volumes. The Cavalieri with CKD Principle was used to measure glomerular volume (Supple- As the rs17319721 enhancer associated with lower GFR but – mental Figure 2).17 19 An average of 7.4 glomeruli was mea- lower albuminuria,11 we hypothesized that SHROOM3 ex- sured per kidney. An average of 841.5 points were counted per pression in renal compartments would show dichotomous kidney. The glomerulus was divided into four components: associations with CKD. We used public datasets that reported podocytes, mesangium, capillary lumens and endothelial CKD (by eGFR), and renal SHROOM3 expression (glomerular/ fi space, and other. The other component was de ned as nonglomerular fraction data).23,24 In the nonglomerular ’ Bowman s space, glomerular basement membrane, and non- transcriptome of dataset 1, CKD (eGFR,60 ml/min) was as- resolvable areas within the glomerulus, and were not used in sociated with significantly higher SHROOM3 compared with glomerular component analysis. biopsy samples with eGFR.60 ml/min (Figure 1A). Similarly, Foot Process Width. Average foot process width was determined in dataset 2, nonglomerular mean SHROOM3 in biopsy as described before (Supplemental Figure 1G).20,21 sampleswithCKDwassignificantly higher than those with eGFR.90 ml/min (Figure 1B). We observed an inverse RNA-Sequencing correlation between nonglomerular SHROOM3 and eGFR in RNA was isolated from glomerular/nonglomerular fractions dataset 2 (n=49; Figure 1C). These relationships between after homogenization (RNeasy kit; Qiagen). Ribosomal RNA nonglomerular SHROOM3 and CKD are consistent with

J Am Soc Nephrol 29: 2641–2657, 2018 SHROOM3 Interacts with FYN in Podocytes 2645 BASIC RESEARCH www.jasn.org

A PODO-NTG B PAX8-TG CAGS-TG PODO-TG PAX8-NTG CAGS-NTG 200 250 200 150 ** ** 150 100 100 50 ACR mcg/mg ACR mcg/mg 50 0 0

3-wks 4-wks 6-wks Baseline 3-wks 4-wks 6-wks 8-wks Baseline Refeed 3-wks WithdrawWithdraw 2-wks 3-wks

C CAGS-NTG CAGS-TG D 110 100 90 80

Glom 70 60 50 Podocyte number /

CAGS-TG CAGS-NTG

E SYNPO WT1 DAPI-MERGE

CAGS-NTG

CAGS-TG

FG 13 40 *** 12 30 11 20 10 9 10 Glomerulus Area SYNPO /

glomerular profile 8 0 WT-1 Positive cell per

CAGS-TG CAGS-TG CAGS-NTG CAGS-NTG

CAGS-NTG

CAGS-NTG CAGS-TG CAGS-TG

2646 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2641–2657, 2018 www.jasn.org BASIC RESEARCH

CKD-GWAS that identified rs173197211 and the enhancer male double-transgenic CAGS- (global), Podo-, and PAX8- function of this SNP.4 However, glomerular transcriptome RTTA/Shroom3-ShRNA mice (CAGS-TG, Podo-TG, PAX8- from dataset 2 showed a linear correlation between SHROOM3 TG), as well as NTG controls with DOX performing urine and eGFR, suggesting an opposite relationship between glo- ACR estimations. Glomerular extracts of CAGS-TG/Podo-TG, merular SHROOM3 and eGFR (n=37; Figure 1D). These but not PAX8-TG showed Shroom3 knockdown (Supplemental data suggest potential dichotomous roles of SHROOM3 in dis- Figure 1, A and B), whereas withdrawal of DOX (week 3) tinct renal compartments in CKD. restored Shroom3 protein in kidney lysates of CAGS-TG (Supplemental Figure 1C). CAGS- and Podo-TG mice developed Shroom3 SNP rs17319721 in the Donor Associates significant albuminuria by week 3 compared with respective with Reduced Albuminuria after First Transplant Year NTG- or PAX8-TG mice, suggesting a podocyte-specificeffect Because rs17319721 increased SHROOM3 expression in al- of Shroom3 knockdown (Figure 2, A and B; n=5 mice). Protein- lografts and associated with higher CADI score/lower uria reversed in Podo-TG upon DOX withdrawal (.2 weeks), eGFR,4 we examined the association of donor genotype and reappeared on reinitiation (Figure 2A). We examined and proteinuria using two cohorts within GoCAR: GoCAR- glomeruli of CAGS-TG at 6 and 8 weeks, compared with Sydney (n=75; 13 A/A, 39 A/G, 23 G/G), with serial albu- CAGS-NTG. We chose BALBc CAGS-TG mice because of min-to-creatinine ratios (ACRs); and GoCAR-Sinai (n=70; their stronger Shroom3 knockdown (Supplemental Figure 1, 9 A/A, 27 A/G, 34 G/G), with protein-to-creatinine ratios, C versus A) and to avoid strain-based heterogeneity. No loss of recorded at 2-year visit (Table 1, demographics). The demo- podocytes was identified in CAGS-TG mice by fractionator/ graphics of GoCAR enrollees have been previously pub- dissector technique (6 weeks; see Methods; Figure 2,C and D, lished.4,22 Because early proteinuria is confounded,25 we Supplemental Figure 1E), or by Wt-1/Synpo costaining assessed early (3-month) versus late (24-month) albumin- (8 weeks; Figure 2, E and F). Interestingly we identified a sig- uria in GoCAR-Sydney. By 24 months, A/A allografts had the nificant decrease in area of Synpo stain in glomeruli from lowest ACRs (paired t test P=0.05; A/A donors, 3-month versus CAGS-TG mice (Figure 2G). No glomerulosclerosis was ob- 24-month albuminuria, 18.267.3 versus 5.762.2 mg/mmol, served in the CAGS-TG group (Supplemental Figure 1F). Elec- respectively; NS for A/G or G/G allografts; Figure 1E). In mul- tron microscopy revealed extensive foot process effacement tivariable models (adjusted for recipient/donor characteris- (FPE), with higher mean foot process width in representative tics) A/A allografts had lower ACR at 24 months (Table 2). In CAGS-TG (Figure 2H, Supplemental Figure 1G; Methods). GoCAR-Sinai, protein-to-creatinine ratios were performed for clinical care at the 2-year visit, A/A allografts had signifi- Glomerular RNA-Sequencing Identifies cantly lower protein-to-creatinine ratios in multivariable Downregulated Key Podocyte Signal-Transduction analyses(Figure1F,Table2).Thesedatasuggestthatdonor Pathways with Shroom3 Knockdown A-allele has a mild but significant effect attenuating late al- Weperformed RNA-sequencing on glomerular/nonglomerular buminuria in allografts, despite increased CADI score. fractions (6 weeks DOX). Principal component analysis showed clustering of transgenic versus NTG glomeruli (Fig- Glomerular and Podocyte-Specific Shroom3 ure 3A; one failed QC). Of 1225 and 1102 genes significantly Knockdown Induce Reversible Albuminuria with up-anddownregulated(DEGs)inglomerulartranscrip- Podocyte Foot Process Effacement without tome between CAGS-TG and -NTG, 877 and 704 DEGs re- Podocyte Loss spectively, were unique to glomeruli (Figure 3B; LIMMA test We examined Shroom3 antagonism in adult glomeruli using P,0.05; GSE110092). Pathway analysis of unique DEGs global- and tissue-specific knockdown mice, as rs17319721 showed predominant downregulation of genes involved in translated into an approximately 1.5-fold increase in SHROOM3 tyrosine kinase/Small-GTPase/Integrin signaling, and Actin expression in allografts,4 and a milder effect on albuminuria,11 cytoskeletal regulation (Supplemental Figure 3A). Clustered than reported in Shroom3 knockout animals.9,10 We fed adult Gene Ontology analysis was done on 704 downregulated,

Figure 2. Glomerular Shroom3 knockdown causes albuminuria in adult mice with FPE without podocyte loss. (A) Line graphs compares trend lines of serial ACRs in Podo-RTTA mice (Podo-TG) and nontransgenic littermates (Podo-NTG) upon DOX feeding, withdrawal and reinitiation. (B) ACR trend lines in CAGS-TG, CAGS-NTG, PAX8-TG, and PAX8-NTG animals. (C) Representative images of CAGS-NTG and -TG glomeruli with identification of podocytes (black arrowhead) for quantification by the fractionator/dissector technique. (D) Quantification of podocytes/glomerulus (n=8 mice per group; mean6SD). (E) Right to left: representative 403 immunofluorescence images for SYNPO, WT1, and merge (with DAPI), respectively (top row: CAGS NTG-animals, second row: CAGS-TG). (F) Podocyte quantification (n=4 mice per group) by WT1/DAPI-merge nuclei/glomerular profile, and (G) shows quantification of area SYNPO stain/ glomerular profile (.30 glomeruli per kidney) (line/whiskers=mean/SD). (H) Right to left: representative electron microscopic images (30003) of CAGS-NTG and -TG animals; left panel shows sample inlay magnified; Mean foot process width (five glomeruli per mouse) in representative NTG and TG animals were 267 and 441 nm, respectively. *P,0.05; **P,0.01; ***P,0.001.

J Am Soc Nephrol 29: 2641–2657, 2018 SHROOM3 Interacts with FYN in Podocytes 2647 BASIC RESEARCH www.jasn.org

A B Differential Expressed Genes 6

Cxcl13 Log2Rat Fgfbp1 1 C3 C1 Wif1 0 CB6R1G 5 −1 Kcng2 –0.4 −2 Astn1 G3 G1 G2 Lypd1 Olfml2b Fam78b –0.2 Clic3 4 Tlr7 Top1mt Ptgds 0 C2 H2−Q6 Tnfrsf19 Cdkn2d −log10(p) H2−Q8 G4 0.2 Nfil3 Lrtm2 3 Olfr1396 G5 Opcml Nfe2l3 Tnfrsf11b 0.4 Naa10 Fcmr Agbl3 Kcne4 0.2 CB6STG 0.4 0 3D PCA Prame Sla –0.2 C030034L19Rik –0.4 Luzp2 –0.6 –0.377 Spef2 Tmem265 1700112J16Rik Gfra3 –0.378 Rnf125 Mgat5b –0.8 –0.379 –0.38 2 Sh2d2a Naip5 Coro2a Socs3 Tmc5 C530008M17Rik

Klhdc1 Lcn6 Nat14 Tatdn3

6430706D22Rik Fosb 2810410L24Rik Gm3286

−2 −1 0 1 2 Log2Rat

Figure 3. Glomerular RNA-sequencing identifies downregulated tyrosine kinase and small GTPase intracellular signaling pathways affecting actin cytoskeleton in podocytes with Shroom3 knockdown. (A) Principal component analysis plot of glomerular transcriptome of CAGSNTG (black dots; n=4) and CAGS-TG (red dots; n=3). (B) Volcano plot of differentially expressed genes between NTG and TG glomeruli unique to the glomerular fraction (877 upregulated and 704 downregulated genes). (C) Bars show relevant clustered Gene Ontology analysis terms of downregulated genes unique to glomerular transcriptome (among top 50 Gene Ontology terms by P-value). (D) Top 12/15 metapathway terms ranked by P-value isolating podocyte-specific genes that were differentially expressed in CAGS-TG versus NTG comparisons, both plotted against negative log(10) P values. Both graphs demonstrate downregulation of key signaling pathways–tyrosine kinase and small GTPases involved in signal transduction in podocytes (G4, G5, and CB6ST=TG group; G1, G2, G3, CB6R1G=NTG group). glomerular-unique DEGS. Key downregulated Gene Ontol- Shroom3- (endogenous) antibodies. We identified 287 unique ogy terms in CAGS-TG overlapped with pathway analysis interactions common to endogenous and overexpressed (Figure 3C, Supplemental Table 2). Among podocyte-enriched SHROOM3. The top ranked 20 proteins are in Supplemental DEGs22 (Supplemental Figure 3B), Synpo downregulation was Table3.Weconfirmed ROCK2, ROCK1, and 14–3-3 as identified by RNA-sequencing and confirmed by quantitative binding partners for SHROOM3.7,8 Interestingly, we identi- PCR whereas NPHS1/NPHS2 mRNA-levels were not signifi- fied FYN among the top-ranking proteins (Supplemental cantly downregulated by RNA-sequencing or quantitative PCR Table 3). FYN is a nonreceptor tyrosine kinase and activator (Supplemental Figure 1D). Metapathway analysis of podocy- of several pathways,26 which were downregulated in Shroom3 te-specific genes revealed predominant downregulation of knockdown glomeruli (Figure 3, C and D). Kinase enrichment podocyte signal-transduction pathways (Figure 3D, 12/15 analysis (KEA; ENRICHR database),27 showed enrichment for ranked by P value). These data suggest glomerular Shroom3 multiple Src-kinases including FYN (Supplemental Figure 4). knockdown induces downregulation of key podocyte signal- In podocytes, FYN binds Nephrin, phosphorylating the transduction pathways implicated in the regulation of actin cytoplasmic tail of Nephrin with subsequent actin cytoskeletal cytoskeleton. organization by recruiting NCK adapter proteins.28 Hence we investigated whether FYN mediated effects of SHROOM3 Immunoprecipitation Identifies SHROOM3-FYN knockdown on podocytes. Interaction in Podocytes via SH3-Binding Domain We confirmed the interaction of endogenous FYN and To screen the protein interactome of SHROOM3, we per- SHROOM3 by immunoprecipitation in human podocytes formed mass spectrometry (LC/MS) on 293-Tcell lysates im- (Figure 4A). FYN has Src-homology-domain 3- (SH3) munoprecipitated with V5- (overexpressed SHROOM3), and and SH2 domains responsible for interactions with

2648 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2641–2657, 2018 www.jasn.org BASIC RESEARCH

C Biological Process GO:0007242~intracellular signaling cascade 60 (Top_Dn) Rasl11a, Cdc42ep5, Tgfa, Rasgrp4, Gem, Dcdc2a, Adcy1, Plce1, Calca, Cnksr1

GO:0007265~Ras protein signal transduction 10 (Top_Dn) Cdc42ep5, Cnksr1, Gna12, Srgap1, Rras2, Nras, Rasgrp3, Cdc42, Tax1bp3, Brap

GO:0007169~transmembrane receptor protein tyrosine kinase signaling pathway 17 (Top_Dn) Epha6, Tgfa, Gfra2, Vegfa, Ephb1, Pak1, Myo1e, Foxc2, Cacna1a, Epha2

GO:0007264~small GTPase mediated signal transduction 21 (Top_Dn) Rasl11a, Cdc42ep5, Rasgrp4, Gem, Plce1, Cnksr1, Gna12, Srgap1, Rasl11b, Rab13

GO:0007167~enzyme linked receptor protein signaling pathway 20 (Top_Dn) Epha6, Tgfa, Gfra2, Vegfa, Ephb1, Pdgfb, Pak1, Myo1e, Foxc2, Cacna1a

GO:0007155~cell adhesion 35 (Top_Dn) Astn1, Postn, Col15a1, Amtn, Ncam1, Itgb3, Klra3, Itgal, Cgref1, Tyro3

GO:0010647~positive regulation of cell communication 16 (Top_Dn) Fgfbp1, Tgfa, Rasgrp4, Ncam1, Itgal, Vegfa, Pdgfb, Csf1, Ntrk2, Hhex

GO:0009967~positive regulation of signal transduction 15 (Top_Dn) Fgfbp1, Tgfa, Rasgrp4, Ncam1, Itgal, Vegfa, Pdgfb, Csf1, Hhex, Nod1

GO:0033674~positive regulation of kinase activity 13 (Top_Dn) Tgfa, Plce1, Csf1, Map2k6, F2r, Dgkz, Dgkh, Irak2, Tlr4, Ptpn11

GO:0045860~positive regulation of protein kinase activity 12 (Top_Dn) Tgfa, Plce1, Csf1, Map2k6, F2r, Dgkz, Dgkh, Irak2, Tlr4, Ptpn11

GO:0007266~Rho protein signal transduction 6 (Top_Dn) Cdc42ep5, Cnksr1, Gna12, Srgap1, Cdc42, Tax1bp3 change GO:0007229~integrin−mediated signaling pathway 9 (Top_Dn) Itgb3, Itgal, Itgae, Itgb5, Itga3, Ctgf, Cib1, Itga4, Itgax Dn

GO:0045785~positive regulation of cell adhesion 6 (Top_Dn) Itgal, Edil3, Calca, Col8a1, Csf1, Thbs1

GO:0000165~MAPKKK cascade 10 (Top_Dn) Cdc42ep5, Tgfa, Plce1, Adra2b, Map2k6, F2r, Ptpn11, Mdfic, Myd88, Brap

02468 −log10(P)

D Cononical Pathway Analysis

0 (Top_Up) wiki_PodNet:_protein−protein_interactions_in_the_podocyte 16 (Top_Dn) Wif1, Sulf1, Magi2, Col4a3, Nck2, Fyn, Vegfa, Sh2d4a, Lmx1b, Plce1

2 (Top_Up) Carf, Invs wiki_XPodNet_−_protein−protein_interactions_in_the_podocyte_expanded_by_STRING 21 (Top_Dn) Wif1, Sulf1, Magi2, Gabra4, Col4a3, Nck2, Fyn, Vegfa, Sh2d4a, Lmx1b

0 (Top_Up) wiki_Primary_Focal_Segmental_Glomerulosclerosis_FSGS 5 (Top_Dn) Col4a3, Fyn, Lmx1b, Plce1, Synpo

0 (Top_Up) REACTOME_NEPHRIN_INTERACTIONS 3 (Top_Dn) Magi2, Nck2, Fyn

0 (Top_Up) PID_FAK_PATHWAY 4 (Top_Dn) Nck2, Fyn, Itgb5, Pak1

1 (Top_Up) Ptprj PID_VEGFR1_2_PATHWAY 3 (Top_Dn) Nck2, Fyn, Vegfa change 1 (Top_Up) Col4a6 wiki_Focal_Adhesion 5 (Top_Dn) Itgb8, Fyn, Vegfa, Itgb5, Pak1 Dn Up 1 (Top_Up) Col4a6 PID_INTEGRIN3_PATHWAY 2 (Top_Dn) Col4a3, Vegfa

0 (Top_Up) wiki_Integrin−mediated_Cell_Adhesion 4 (Top_Dn) Itgb8, Fyn, Itgb5, Pak1

1 (Top_Up) Col4a6 KEGG_mmu04510:Focal_adhesion 5 (Top_Dn) Itgb8, Fyn, Vegfa, Itgb5, Pak1

0 (Top_Up) REACTOME_CELL_CELL_COMMUNICATION 4 (Top_Dn) Magi2, Nck2, Fyn, Pak1

0.0 2.5 5.0 7.5 10.0 −log10(P)

Figure 3. Continued.

J Am Soc Nephrol 29: 2641–2657, 2018 SHROOM3 Interacts with FYN in Podocytes 2649 BASIC RESEARCH www.jasn.org

AB IP: lgG SHROOM3 lgG FYN 210 KD 210 KD SHROOM3: NP_065910.3 SHROOM3 (196 KD) PDZ POSH-binding ASD1 ASD2 (31-106) (~295-444) (848-1065) (1667 - 1954)

--- P L P P xxxxxxxxxxxxxxPxxxxxxxxxxxxxPxxxxxxxxxxPxxxxxxxxxxxxxPxxxP P V K P ---

--- A L P A xxxxxxxxxxxxxxPxxxxxxxxxxxxxPxxxxxxxxxxPxxxxxxxxxxxxxPxxxP A V K A --- FYN 55 KD (59 KD) 55 KD M-1 mutant M-2 mutant

C D E FYN (total) V5-tag V5-tag IP: V5-tag WT M-1 M-2 M-1+2 FYN (total) FYN (total)

SHROOM3 Eluate: WT WT M-1 M-2 M-1+2 Eluate: WT WT M-1 M-2 (196 KD) P-FYN (Y418) non P-FYN (Y418)

Ms-FYN (total) Ms-FYN (total)

IP: Rb-lgG + - - - - IP: Rb-lgG + - - - IP: Rb-FYN - + + + + IP: Rb-FYN - + + + FYN (59 KD)

F Scramble Si-1 Si-2 G Scramble Si-2 H Scramble Si-2 SHROOM3 SHROOM3 FYN (total)

P-SRC (Y416) nonP-SRC (Y416) Eluate: Scramble Scramble Si-2 SHROOM3 FYN (total) P-NEPHRIN (Y1176) non P-FYN (Y418) P-NEPHRIN (Y1176) NEPHRIN (total) Ms-FYN (total) NEPHRIN (total) ACTIN IP: Rb-lgG + - - GAPDH IP: Rb-FYN - + +

Figure 4. SHROOM3 interacts with Src kinase FYN in podocytes in vitro, at SH3-binding domain, and regulates FYN activation by phosphorylation. (A) Immunoprecipitation of human podocyte cell lysates with anti-SHROOM3 and anti-FYN antibodies, run on PAGE, with probing for FYN and SHROOM3, respectively. Figure shows representative immunoblots. (B) Figure shows SHROOM3 protein with consensus domains, and putative POSH-binding conserved domain. This Proline rich sequence was mutated at two identiﬁed –PxxP- loci generating -AxxA- sequences, mutants M1, M2, and M1+2. (C–E) Lysates from 293T cells overexpressing SHROOM3, M1, M2, or M1+2 mutants were immunoprecipitated with anti-V5 tag, or Rb-FYN antibodies. Representative Western blots of cell lysates immunoblotted for (C–E) Rb-FYN, (C and D) V5 (as loading controls), and immunoblots of eluates with (D and E) Ms-FYN, (D) Phospho- SRC (Y416), or (E) nonphosphoY416 SRC antibodies, respectively, are shown. (F and G) Human podocyte cell lines were stably infected with control (Scramble) or Shroom3 knockdown (Si-1, Si-2) puromycin-selectable lentiviruses. Representative Western blots from differentiated Scramble, Si-1, Si-2 podocyte lysates probed for (F) SHROOM3, FYN, phospho SRC family (Y416), phospho-NPHS1 (Y1176/ Y1193), total NPHS1, GAPDH (G) SHROOM3, nonphospho SRC (Y416), phospho-NPHS1 (Y1176/Y1193), total NPHS1, ACTIN, respectively from independent experiments are shown. (H) Lysates from Scramble- and Si-2 podocyte lines were immunoprecipitated with Rb-IgG, or Rb-FYN. Immunoblots for Rb-Fyn (total lysate: top row), and SHROOM3, nonphospho FYN (Y418), and total FYN (eluates: bottom three rows) are shown. Ms, mouse; Rb, rabbit. complementary binding-domain containing proteins.28,29 for SH3-binding sites, and identiﬁed two -PxxP- loci, 333–37 Shroom3 binds Posh (Plenty of SH3-domains protein)30; and 445–8 (Figure 4B). SHROOM3-CDNA construct4 was where a conserved Shroom3 region (peptide approximately mutated at these sites individually (P➔A substitutions) gener- 295–444) was critical to Posh-binding. LC/MS-KEA data ating SHROOM3-M1, -M2, and M1+2 constructs (Figure 4B). also showed enrichment of multiple kinases with SH3 do- SHROOM3/mutants were overexpressed in 293-T cells mains (Supplemental Figure 4). Therefore, we examined this followed by immunoprecipitation with V5 beads. Immuno- proline-rich region in SHROOM3 (Refseq-NP_065910.3) blotting for FYN showed site 445–8(i.e., M2 mutation) is

2650 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2641–2657, 2018 www.jasn.org BASIC RESEARCH

A SCRAMBLE SCRAMBLE Si-2 Si-2

B 0.5 *** C 30000 *** D 110 0.4 * )

2 20000 0.3 100

0.2 90 Area (  m MFI in Edge 10000 0.1 Forward Scatter 80 0.0 0 (normalized MFI*1000) Scramble Si 2 Scramble Si-2 Scramble Si-2

E Capillary+ Podocyte Mesangial Endothelial F fraction fraction fraction 800 2.0 * ) * 3 600 1.5

1.0 400

0.5 200 Volume (mcm Single Podocyte (ratio to cags-ntg) 0.0 0 Normalized fraction Volume

CAGS-TG CAGS-TG CAGS-TG CAGS-TG CAGS-NTG CAGS-NTG CAGS-NTG CAGS-NTG

G 3000/ 2000/ 1000/ H well well well 0.8 * *** NS 0.6

0.4 0-hour Cells 0.2

Fraction of adherent 0.0 Si Si Si

Scramble Scramble Scramble 6-hour

I 40 **

20 24-hour 10

Podocytes migrating/ 0

hpf image at 6-hours (n) Scramble Si-2 Si-2

Scramble

J Am Soc Nephrol 29: 2641–2657, 2018 SHROOM3 Interacts with FYN in Podocytes 2651 BASIC RESEARCH www.jasn.org the critical FYN-binding site of SHROOM3 (Figure 4C). These cytometry of differentiated podocytes further identified re- data confirm the interaction of endogenous FYN and duced forward-scatter mean fluorescence intensity implying SHROOM3 in podocytes via SH3-SH3 binding domains, reduced cell size in Si-2 versus Scramble (Figure 5D, Supple- respectively. mental Figure 5, A and B). Correspondingly, in vivo,significant reductions in mean volume of the podocyte fraction/ SHROOM3-FYN Interaction Regulates FYN Activation glomerulus, and single podocyte volume were seen in and Phosphorylation of Cytoplasmic Tail of Nephrin CAGS-TG (n=7 glomeruli per mouse; n=8 mice; Figure 5, We interrogated FYN phosphorylation (Y418, activation) E and F), whereas nonpodocyte glomerular fractions were with/without SHROOM3-FYN interaction. Total-FYN im- not significant (see Methods). The differences in cell size munoprecipitation eluates from 293 cell lysates overexpress- were not because of diminished cell viability, increased cell ing SHROOM3 or mutants were probed for activation (using death, or increased apoptosis (Supplemental Figure 5, C–G). generic Y416 antibodies). In eluates, from M2 to M1+2 mutant To examine podocyte adhesion, differentiated scramble/ lysates, i.e., without SHROOM3-FYN interaction, phospho- Si-2 cells were plated in laminin-coated 96-well plates. We Y418-FYN was inhibited compared with SHROOM3- and observed significantly reduced adhesion in Si-2 at 1-hour M1-mutants (Figure 4D). M2 mutant FYN eluates had in- (Figure 5G). Using Scratch assay,33 Si-2 cells showed increased nonphospho-Y418-FYN (Figure 4E). creased podocyte migration versus Scramble (Figure 5, H and I), To evaluate FYN activation in podocytes without corresponding to FPE identified in vivo. SHROOM3, we created stable SHROOM3 knockdown human podocyte lines (Si–1 and 2). Si-2 demonstrated greater Shroom3 Knockdown Inhibits Fyn Activation and SHROOM3 knockdown of both isoforms [4F]. Here, immu- Nphs1-Phosphorylation In Vivo noblotting identified reduced phospho-Y416 and increased Glomerular lysates from CAGS-TG and -NTG mice were stud- nonphospho-Y416 with the respective total-SRC-kinase anti- ied after the development of stable proteinuria (6 weeks DOX). bodies in Si-2 versus Scramble lysates (Figure 4, F and G, Here, Shroom3-specific ShrRNA with DOX feeding is cotran- respectively). FYN phosphorylates tyrosine residues located scribed with GFP in cells with ShRNA expression.4 Human in conserved Nck-binding YDxV motifs within the cytoplas- FYN Y418 corresponds to Fyn Y423 in mice.34 Phospho- mic tail of NPHS1 (Y1176/Y1193).31 Phospho-NPHS1 Y416-Src (Figure 6A) and Nphs1-phosphorylation (Y1176/ was significantly inhibited in Si-1 and 2, downstream of Y1193) were inhibited, whereas total Nphs1 was unchanged FYN (Figure 4, F and G). Total FYN immunoprecipitation (Figure 6A, Supplemental Figure 6A) with Shroom3 knock- eluates from Scramble- and Si-2 lysates showed significantly down in glomerular extracts, with significantly reduced phos- increased nonphospho-Y418-FYN in Si-2 versus Scramble pho-Src (Y416):Fyn (Figure 6B) and p-Nphs1:Nphs1 ratios (Figure4H).Hence,SHROOM3-FYNinteractioninpodo- (Figure 6C). Glomerular cells of CAGS-TG mice also showed cytes regulates FYN activation, and phosphorylation of increased nonphospho-Y416 staining compared with CAGS- NPHS1. NTG by IHC (Figure 6D). Together, our data confirm that SHROOM3 interacts with FYN via SH3-binding domain SHROOM3 Knockdown Alters Podocyte Morphology (445–8), and regulating FYN activation, and downstream sig- and Actin Cytoskeleton In Vitro naling including NPHS1phosphorylation (Y1176/Y1193) and We examined podocyte cytoskeleton by phalloidin F-actin maintenance of podocyte actin cytoskeleton and phenotype. staining in Si-2 and Scramble cells,9,31,32 and identified altered In this context, the presence of the enhancer A-allele would be actin bundle distribution with greater marginal staining in Si- beneficial in podocytes (summary in Figure 7). 2 cells (Figure 5, A and B). Interestingly significantly reduced To examine whether Shroom3 knockdown increased sus- podocyte cellular area was seen in Si-2 cells (Figure 5C). Flow ceptibility to glomerular injury, we performed adriamycin

Figure 5. SHROOM3 knockdown alters podocyte actin cytoskeletal organization, morphology, adhesion and migration. (A) Right to left: representative immunofluorescence images (203) of phalloidin F-actin cytoskeletal stain of Scramble versus Si-2 podocyte cell lines (labeled). Dot plots summarize (B) ACTIN bundle intensity at cellular margins and (C) cell area of phalloidin-stained podocytes (n=4 experiments; 150 cells per experiment). (D) Dot plots show mean flow cytometric forward scatter (size) of trypsinized cell sus- pensions of podocytes normalized to Scramble podocytes (n=3 experiments; see Supplemental Figure 5, A and B). (E) Graph compares glomerular component volumes (podocyte, mesangial, and capillary+endothelial fractions), and (F) average single podocyte volume, between CAGS-NTG and CAGS-TG mice (n=8 mice; 6 weeks DOX). (G) Scramble and Si-2 podocyte cell lines were differentiated and plated on laminin-coated 96-well plates at 1000, 2000, and 3000 cells/well densities (n=3 sets for each density). Bar graphs compare proportion of adherent podocytes: total podocytes in each plating density at 1 hour by turbidimetry (crystal violet method). (H) Dif- ferentiated Scramble and Si-2 podocytes were plated in 10-cm dishes. Scratches were made on fully confluent plates using sterile pipette tips. Panels show representative phase-contrast images (43) of Scramble and Si-2 conditions at 0, 6, and 24 hours. (I) Line dot graphs compare number of cells inside scratch margins of eight streaks in duplicate plates at 6 hours (line dot graphs/whiskers=mean/ SD; *unpaired t test P,0.05; **P,0.01; ***P,0.001.

2652 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2641–2657, 2018 www.jasn.org BASIC RESEARCH

A B C CAGS-RTTA: NTG NTG TG TG 8 ** 1.5 ** Shroom3 6 1.0 GFP 4

Nphs1 0.5 P-Src (Y416) 2 (Y416)/Total FYN

Densitometry P-Src 0 0.0 Fyn (total) Densitometry p-Nphs1:

Nphs1 (Y1176) CAGS-TG CAGS-TG CAGS-NTG CAGS-NTG Nphs1 (total)

Gapdh

D 10X 40X 40X CAGS-NTG Non-Phospho Src-family (Y416) CAGS-TG

E CAGS-NTG CAGS-TG F G *** * 25 n=140 n=146 10 20 9 15 8 10 (mean) 5 7 P-57 stain

Glomerular profile 0 6 Podocytes/glomerulus P57-positive nuclei per

CAGS-TG CAGS-TG CAGS-NTG CAGS-NTG

H CAGS-NTG CAGS-TG I J 50 *** 15

CAGS-NTG )

40 2 CAGS-TG 30 10 20 PAS 5 (3 /group) 10 (× 1000 mcm Glomerular area

Number of Glomeruli 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Glomerular area (× 1000 mcm2)

CAGS-TG CAGS-NTG

Figure 6. In vivo glomerular Shroom3 knockdown inhibits Fyn phosphorylation and downstream phosphorylation of cytoplasmic tail of Nephrin. CAGS-NTG and TG littermate male mice were fed DOX (6 weeks). (A) Representative Western blots (n=2 each group shown) demonstrate immunoblotting for Shroom3, TGFP, Phospho Src (Y416), total Fyn, Phospho Nphs1 (Y1176/Y1193), Nphs1, and Gapdh.

J Am Soc Nephrol 29: 2641–2657, 2018 SHROOM3 Interacts with FYN in Podocytes 2653 BASIC RESEARCH www.jasn.org

A/A genotype SHROOM3

SHROOM3 SH3 FYN

PODOCYTE P PODOCYTE

SH3 FYN

P NEPHRIN (Y1176/Y1193) NEPHRIN P CD2AP

NCKap

GBM

EC EC EC EC EC EC EC EC

Figure 7. Cartoon summarizes putative mechanism of protective effect of the enhancer locus in Shroom3 (rs17219731) and SHROOM3 protein on albuminuria in adults. Homozygosity for the CKD-associated allele (A/A) associates with higher SHROOM3 expression, preserved interaction with FYN in podocytes (via SH3-binding domain), activation of FYN kinase (Y418 phosphorylation), downstream Nephrin tail phosphorylation at NCK-adapter protein (NCKap) docking sites, and actin cytoskeletal stabilization. Depletion of SHROOM3 causes cytoskeletal disarray by inhibition of Fyn activation and nephrin phosphorylation. injections in Podo-TG mice (6 weeks DOX) versus littermates. Figure 6D). This was distinct from young mice (Supplemental To avoid confounding from tubular Shroom3 knockdown, we Figure 1F). After 12 weeks, aged CAGS-TG mice (n=3) showed used Podo-TG mice albeit in a hybrid background with litter- no weight loss (Supplemental Figure 6G) but signiﬁcant podo- mate controls (n=5 each). Proteinuria was similar in Podo-TG cyte loss by P57-positive nuclear staining (Figure 6, E–G), and NTG without FSGS lesions in either group (Supplemental and increased glomerular area with mesangial expansion Figure 6, B and C). Because Fyn knockout mice show FPE (Figure 6, H–J), supporting an age-dependent phenotype without FSGS up to 1 year of age,28 and heterozygous of Shroom3 knockdown. Interestingly, nonglomerular Shroom3 knockout mice showed FSGS at 1 year of age despite Shroom3 protein was similar in aged CAGS-TG and -NTG FPE,10 we aged CAGS-TG and -NTG mice (1 year), and DOX mice with glomerulosclerosis, whereas simultaneous glomer- fed these mice for 8 and 12 weeks. At 8 weeks, CAGS-TG ular Shroom3 was knocked down (Supplemental Figure 6, E mice showed mesangial expansion and protein reabsorption and F). This suggests upregulation of tubular Shroom3 dropletsintubulesversusagedcontrols(n=4; Supplemental with glomerulosclerosis. These data from the nonglomerular

(B) Bar graphs show densitometry of Phospho Src Y416: total Fyn and (C) Phospho Nphs1:total Nphs1 between CAGS TG and NTG mice (n$5 sets). (D) Representative 103 (left: two panels) and 403 (right panels) photomicrographs from two animals of each group, show immunoperoxidase stain for nonphospho SRC (Y416). CAGS NTG and TG mice were aged approximately 1 year (n=3 mice each), then DOX-fed (12 weeks). (E) Representative photomicrographs (203) of immunoperoxidase for P57-positive nuclei (podocyte nuclei) between CAGS NTG and TG mice, and dot graphs quantify podocyte number/glomerular profile, depicted as (F) number of profiles counted per experimental condition, and (G) per mouse. (H) Representative PAS stained sections (203) showing glomerular enlargement with mesangial sclerosis in CAGS-TG mice compared with NTG. (I) Histogram shows distribution of area of individual glomeruli (31000 mcm2)in CAGS NTG (dark grey) and TG (light grey) (mean of 47.67 glomerular profiles/mouse). (J) Dot graphs compare mean area/individual glomerular profile in CAGS NTG versus TG (bar graphs/whiskers=mean/SEM; *unpaired t test P,0.05); **P,0.01; ***P,0.001.

2654 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2641–2657, 2018 www.jasn.org BASIC RESEARCH compartment in FSGS mice are consistent with dichotomous small molecule antagonists will require complete crystallographic expression patterns seen in glomerular/nonglomerular tran- Shroom3 structure including binding pockets to FYN and scriptomes in human CKD biopsy samples. ROCK. Such inhibitors using homology modeling without com- peting domain effects have been designed before.40,41 Addition- ally, Shroom3-Rock interaction is essential for Rock activation in DISCUSSION other epithelia,6,7 and off-target effects will need to be calibrated against potential antifibrotic benefits. Hence tissue-specificde- We previously identified that allograft SHROOM3 expression livery systems such as biodegradable nanoparticle attachment associated with subsequent renal allograft fibrosis, whereas will need to be considered.42,43 tubular Shroom3 knockdown reduced fibrosis. We show Among proteinuric kidney diseases, inhibited Fyn- here the interesting divergent associations of SHROOM3 ex- phosphorylation has been associated with human minimal pression within glomerular/nonglomerular compartments change disease (MCD),44,45 where irreversible renal dysfunction with CKD, as well as the corresponding dichotomous associ- is uncommon.46 Fyn knockout mice also showed mild albumin- ations of the enhancer A-allele with eGFR and albuminuria uria with FPE without FSGS,28 distinct from phosphorylation- phenotypes in allografts. In adult glomeruli, Shroom3 knock- deficient nephrin mice.31 These data may suggest that aside from down caused FPE without podocyte loss in the short term. We inhibition of Nephrin-phosphorylation, impaired Fyn-activation identified the interaction of the Src-kinase FYN with may affect podocytes by other mechanisms, some of which may SHROOM3 via SH3-SH3 binding sites in podocytes, which indeed be protective. An interesting observation in CAGS-TG regulates FYN activation, Nephrin phosphorylation, and actin was the reduced podocyte volume. Fyn responds to cellular vol- cytoskeletal organization. In vitro, these corresponded to al- ume changes by activation or inhibition.47 Whether changes in tered podocyte morphology, adhesion, and migration. Aside podocyte volume observed with Shroom3 knockdown are fyn from revealing new biology regarding Shroom3 and FYN, dependent or independent and/or contribute to the milder phe- these data are an essential first step before the design of ther- notype observed when Fyn activation is disrupted, and whether apeutics in humans for renal fibrosis by inhibiting this interaction plays a role in human proteinuric kidney disease, SHROOM3. need to be examined further. It must be simultaneously noted Prior data reported severe glomerular phenotypes in ho- that Fyn hypo-phosphorylation may be an epiphenomenon in mozygous Shroom3 knockout mice from inhibited Shroom3- human MCD, and small scale GWAS studies in pediatric MCD Rock binding via the ASD2-domain, and in FHH rats via the have not identified susceptibility loci within the Fyn locus.48 ASD1-domain.9,10 Notably, heterozygous mice with early FPE The GWAS associations of rs1731921 with CKD1 and lower developed FSGS only by 1 year. The mechanistic basis for albuminuria,11 Nephroseq, and our data together indicate the the different phenotypes between homo- and heterozygotes association of SHROOM3 expression with the A-allele, and in was unexplained. In our study, downregulation of rho- turn, its opposing associations with albuminuria and eGFR. signaling pathways (Gene Ontology analysis) and Synpo Cumulatively, these provide mechanistic basis for the associ- (not Nphs1), and increased podocyte motility (implying ations of the A-allele with both phenotypes in humans. Our rho-Rac1 imbalance)35,36 are all consistent with Rock data also confirms the reported interaction of Shroom3 with inhibition in podocytes of CAGS-TG mice. However, Rock 14–3-3 (Supplemental Table 3).8 Cathepsin-L–dependent inhibitors are reported to reduce albuminuria, which is cleavage of Synpo depends on 14–3-3 binding,49 and de- inconsistent with the increased albuminuria in CAGS-TG creased Synpo protein in CAGS-TG mice may be related to mice, implying a Rock-independent effect of Shroom3 on impaired Shroom3 and 14–3-3 interaction. However de- podocytes.37–39 Thus, the extent and timing of Shroom3 creased Synpo mRNA levels likely result from inhibited deficiency could decide the glomerular phenotype, with ROCK action and not 14–3-3 binding.35 developmental complete knockout (knockout mice or Our human data are limited by small sample sizes and dif- FHHrats)showingearlyFSGSvia ASD2- or ASD1-domain fering urine protein measurements, although reduced protein- functions, respectively, whereas heterozygotes or CAGS-TG uria was consistent in both cohorts and in a larger GWAS mice with incomplete Shroom3 deficiency show an age- study.11 We used late albuminuria (24 months) to identify dependent milder phenotype related to deficient Fyn-binding association of rs17319721 with proteinuria because early function. post-transplant albuminuria (,6 months) is confounded by We reported that in tubular cells, facilitation of TGF-b early events.25 Interestingly, the odds ratio of CKD in GWAS signaling with excess SHROOM3 was abrogated by Rock (1.07 [95% confidence intervals (95% CI), 1.00 to 1.15])1 inhibitors, suggesting that the profibrotic effect may be ASD2- associated with the risk allele was lower than the odds ratio domain–dependent, and distinct from Fyn-binding site. Hence, for high CADI score (1.9 [95% confidence intervals (95% CI), the specific roles of the distinct protein binding domains of 1.10 to 3.59])4. The accelerated phenotype in allografts may SHROOM3 in proteinuria, as well as in the nonglomerular com- be from alloimmunity, immunosuppression, or two-kidney partment, should be examined in future work by designing spe- to one-kidney transition, which should be investigated in cific small molecule antagonists. Designing domain-specific uninephrectomy or transplant models. The mechanism for

J Am Soc Nephrol 29: 2641–2657, 2018 SHROOM3 Interacts with FYN in Podocytes 2655 BASIC RESEARCH www.jasn.org age-dependent effect of glomerular Shroom3 knockdown is salt-sensitive hypertension and kidney damage by increasing ur- not explained in our data and needs further study. Fyn was omodulin expression. Nat Med 19: 1655–1660, 2013 downregulated (approximately 1.4-fold) by RNA-sequencing 4. Menon MC, Chuang PY, Li Z, Wei C, Zhang W, Luan Y, et al.: Intronic fi locus determines SHROOM3 expression and potentiates renal allograft (Figure 3B), although no signi cant differences in Fyn fibrosis. J Clin Invest 125: 208–221, 2015 protein could be identified in glomeruli in vivo (Figure 5. Deshmukh HA, Palmer CN, Morris AD, Colhoun HM: Investigation of 6A), a finding we cannot fully explain. FYN and SHROOM3 known estimated glomerular filtration rate loci in patients with type 2 also correlated in glomerular transcriptome from Nephroseq diabetes. Diabet Med 30: 1230–1235, 2013 dataset 2 (Spearman r=0.44; P,0.01). In Nephroseq, these 6. Hildebrand JD, Soriano P: Shroom, a PDZ domain-containing actin- fi binding protein, is required for neural tube morphogenesis in mice. Cell ndings could relay the association of both FYN and 99: 485–497, 1999 SHROOM3 with podocyte number, although CAGS-TG 7. Nishimura T, Takeichi M: Shroom3-mediated recruitment of Rho ki- mice showed no podocytopenia. nases to the apical cell junctions regulates epithelial and neuro- In summary, we describe the proteinuria phenotype from epithelial planar remodeling. Development 135: 1493–1502, 2008 Shroom3 knockdown in adult glomeruli, and describe a novel 8. Prokop JW, Yeo NC, Ottmann C, Chhetri SB, Florus KL, Ross EJ, et al.: Characterization of coding/noncoding variants for SHROOM3 in pa- protein-protein interaction with FYN, which explains this tients with CKD. J Am Soc Nephrol 29: 1525–1535, 2018 phenotype. Our work ascribes mechanism to the intronic 9. Yeo NC, O’Meara CC, Bonomo JA, Veth KN, Tomar R, Flister MJ, et al.: CKD-associated enhancer locus explaining its dichotomous Shroom3 contributes to the maintenance of the glomerular filtration association with albuminuria and renal function in humans. barrier integrity. Genome Res 25: 57–65, 2015 10. Khalili H, Sull A, Sarin S, Boivin FJ, Halabi R, Svajger B, et al.: De- velopmental origins for kidney disease due to Shroom3 deficiency. JAmSocNephrol27: 2965–2973, 2016 ACKNOWLEDGMENTS 11. Ellis JW, Chen MH, Foster MC, Liu CT, Larson MG, de Boer I, et al.: CKDGen Consortium; CARe Renal Consortium: Validated SNPs for Research/study design: B.M., J.C.H., and M.C.M.; experimentation: eGFR and their associations with albuminuria. Hum Mol Genet 21: – C.W., N.P., R.L., A.C., K.B., F.G., P.C., J.W., and M.C.M.; clinical data: 3293 3298, 2012 12. Levey AS, Coresh J, Greene T, Stevens LA, Zhang YL, Hendriksen S, M.K., K.K., P.J.O., B.M., and M.C.M.; analyzing data: Z.Y., W.Z., M.F., et al.: Using standardized serum creatinine values in the modifica- T.L., F.S., A.C., J.M.B., J.C., K.B., and M.C.M.; providing reagents: tion of diet in renal disease study equation for estimating glomerular L.K., J.W., K.N.C., and B.D.; manuscript: B.M., J.C.H., and M.C.M. All filtration rate. Ann Intern Med 145: 247–254, 2006 authors contributed to editing of manuscript. 13. Tian X, Kim JJ, Monkley SM, Gotoh N, Nandez R, Soda K, et al.: Po- We thank the shared microscopy research facility and Dr. Richard docyte-associated talin1 is critical for glomerular filtration barrier – Gordon of the electron microscopy core at Icahn School of medicine maintenance. JClinInvest124: 1098 1113, 2014 14. Schwartzman M, Reginensi A, Wong JS, Basgen JM, Meliambro K, Nicholas at Mount Sinai, as well as Electron Microscopy Facility at the Uni- SB,etal.:Podocyte-specific deletion of yes-associated protein causes FSGS versity of Minnesota Imaging Center. Dr. Jenny Xiang at The Ge- and progressive renal failure. JAmSocNephrol27: 216–226, 2016 nomics Core Research Facility at Weill-Cornell School of Medicine, 15. Bai XY, Basgen JM: Podocyte number in the maturing rat kidney. Am New York. We acknowledge critical feedback from Prof Detlef JNephrol33: 91–96, 2011 Schondorff and Prof. Peter Heeger during different stages of this work 16. Nyengaard JR: Stereologic methods and their application in kidney research. J Am Soc Nephrol 10: 1100–1123, 1999 and manuscript. We acknowledge Dr. Lawrence Holzman for the gift 17. Gundersen HJ, Jensen EB: The efficiency of systematic sampling in of Nphs1 antibody. M.C.M. would like to acknowledge philanthropy stereology and its prediction. JMicrosc147: 229–263, 1987 from Nina and Homer Eaton, and Pablo Legorreta. 18. Basgen JM, Sobin C: Early chronic low-level lead exposure produces This work was supported by American Heart Association glomerular hypertrophy in young C57BL/6J mice. Toxicol Lett 225: 48– grant 15SDG25870018 (to M.C.M.) and National Institutes of 56, 2014 Health grant RO1 DK102420 (to B.M.). 19. Steffes MW, Brown DM, Basgen JM, Mauer SM: Amelioration of mesangial volume and surface alterations following islet transplantation in diabetic rats. Diabetes 29: 509–515, 1980 20. Toyoda M, Najafian B, Kim Y, Caramori ML, Mauer M: Podocyte de- DISCLOSURES tachment and reduced glomerular capillary endothelial fenestration in None human type 1 diabetic nephropathy. Diabetes 56: 2155–2160, 2007 21. Gundersen HJ: Estimators of the number of objects per area unbiased by edge effects. Microsc Acta 81: 107–117, 1978 22. Fu J, Wei C, Lee K, Zhang W, He W, Chuang P, et al.: Comparison of REFERENCES glomerular and podocyte mRNA profiles in streptozotocin-induced diabetes. J Am Soc Nephrol 27: 1006–1014, 2016 1. Köttgen A, Glazer NL, Dehghan A, Hwang SJ, Katz R, Li M, et al.: 23. Ju W, Nair V, Smith S, Zhu L, Shedden K, Song PXK, et al.: ERCB, Multiple loci associated with indices of renal function and chronic kid- C-PROBE, NEPTUNE, and PKU-IgAN Consortium: Tissue transcriptome- ney disease. Nat Genet 41: 712–717, 2009 driven identification of epidermal growth factor as a chronic kidney dis- 2. Böger CA, Gorski M, Li M, Hoffmann MM, Huang C, Yang Q, et al.: ease biomarker. Sci Transl Med 7: 316ra193, 2015 CKDGen Consortium: Association of eGFR-related loci identified by 24. Sampson MG, Robertson CC, Martini S, Mariani LH, Lemley KV, Gillies GWAS with incident CKD and ESRD. PLoS Genet 7: e1002292, 2011 CE, et al.: Nephrotic Syndrome Study Network: Integrative genomics 3. Trudu M, Janas S, Lanzani C, Debaix H, Schaeffer C, Ikehata M, et al.: identifies novel associations with APOL1 risk genotypes in black NEP- SKIPOGH team: Common noncoding UMOD gene variants induce TUNE subjects. JAmSocNephrol27: 814–823, 2016

2656 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2641–2657, 2018 www.jasn.org BASIC RESEARCH

25. Naesens M, Lerut E, Emonds MP, Herelixka A, Evenepoel P, Claes K, et al.: 39. Kolavennu V, Zeng L, Peng H, Wang Y, Danesh FR: Targeting of Proteinuria as a noninvasive marker for renal allograft histology and failure: RhoA/ROCK signaling ameliorates progression of diabetic ne- An observational cohort study. J Am Soc Nephrol 27: 281–292, 2016 phropathy independent of glucose control. Diabetes 57: 714–723, 26. Thomas SM, Brugge JS: Cellular functions regulated by Src family ki- 2008 nases. Annu Rev Cell Dev Biol 13: 513–609, 1997 40. Cichonska A, Ravikumar B, Parri E, Timonen S, Pahikkala T, Airola A, 27. Lachmann A, Ma’ayan A: KEA: Kinase enrichment analysis. Bio- et al.: Computational-experimental approach to drug-target in- informatics 25: 684–686, 2009 teraction mapping: A case study on kinase inhibitors. PLOS Comput 28. Verma R, Wharram B, Kovari I, Kunkel R, Nihalani D, Wary KK, et al.: Fyn Biol 13: e1005678, 2017 binds to and phosphorylates the kidney slit diaphragm component 41. Jelic D, Mildner B, Kostrun S, Nujic K, Verbanac D, CulicO,etal.: Nephrin. JBiolChem278: 20716–20723, 2003 Homology modeling of human Fyn kinase structure: Discovery of ros- 29. Verma R, Kovari I, Soofi A, Nihalani D, Patrie K, Holzman LB: Nephrin marinic acid as a new Fyn kinase inhibitor and in silico study of its ectodomain engagement results in Src kinase activation, nephrin possible binding modes. JMedChem50: 1090–1100, 2007 phosphorylation, Nck recruitment, and actin polymerization. JClinIn- 42. Alsaggar M, Liu D: Organ-based drug delivery. JDrugTarget26: 385– vest 116: 1346–1359, 2006 397, 2018 30. Taylor J, Chung KH, Figueroa C, Zurawski J, Dickson HM, Brace EJ, 43. Tuffin G, Waelti E, Huwyler J, Hammer C, Marti HP: Immunoliposome et al.: The scaffold protein POSH regulates axon outgrowth. Mol Biol targeting to mesangial cells: A promising strategy for specificdrug Cell 19: 5181–5192, 2008 delivery to the kidney. JAmSocNephrol16: 3295–3305, 2005 31. New LA, Martin CE, Scott RP, Platt MJ, Keyvani Chahi A, Stringer CD, 44. Zhang SY, Kamal M, Dahan K, Pawlak A, Ory V, Desvaux D, et al.: c-mip et al.: Nephrin tyrosine phosphorylation is required to stabilize and impairs podocyte proximal signaling and induces heavy proteinuria. Sci restore podocyte foot process architecture. JAmSocNephrol27: Signal 3: ra39, 2010 2422–2435, 2016 45. Audard V, Zhang SY, Copie-Bergman C, Rucker-Martin C, Ory V, 32. Verma R, Venkatareddy M, Kalinowski A, Patel SR, Salant DJ, Garg P: Candelier M, et al.: Occurrence of minimal change nephrotic syndrome Shp2 associates with and enhances nephrin tyrosine phosphorylation in classical Hodgkin lymphoma is closely related to the induction of c- and is necessary for foot process spreading in mouse models of po- mip in Hodgkin-Reed Sternberg cells and podocytes. Blood 115: 3756– docyte injury. Mol Cell Biol 36: 596–614, 2015 3762, 2010 33. Lee HW, Khan SQ, Faridi MH, Wei C, Tardi NJ, Altintas MM, et al.: A 46. Vivarelli M, Massella L, Ruggiero B, Emma F: Minimal change disease. podocyte-based automated screening assay identifies protective small Clin J Am Soc Nephrol 12: 332–345, 2017 molecules. JAmSocNephrol26: 2741–2752, 2015 47. Kapus A, Szászi K, Sun J, Rizoli S, Rotstein OD: Cell shrinkage regulates 34. Senis YA, Mazharian A, Mori J: Src family kinases: At the forefront of Src kinases and induces tyrosine phosphorylation of cortactin, in- platelet activation. Blood 124: 2013–2024, 2014 dependent of the osmotic regulation of Na+/H+ exchangers. JBiol 35. Wang L, Ellis MJ, Gomez JA, Eisner W, Fennell W, Howell DN, et al.: Chem 274: 8093–8102, 1999 Mechanisms of the proteinuria induced by Rho GTPases. Kidney Int 81: 48. Debiec H, Dossier C, Letouzé E, Gillies CE, Vivarelli M, Putler RK, et al.: 1075–1085, 2012 Transethnic, genome-wide analysis reveals immune-related risk alleles 36. Blattner SM, Hodgin JB, Nishio M, Wylie SA, Saha J, Soofi AA, et al.: and phenotypic correlates in pediatric steroid-sensitive nephrotic Divergent functions of the Rho GTPases Rac1 and Cdc42 in podocyte syndrome. JAmSocNephrol29: 2000–2013, 2018 injury. Kidney Int 84: 920–930, 2013 49. Buvall L, Wallentin H, Sieber J, Andreeva S, Choi HY, Mundel P, et al.: 37.GojoA,UtsunomiyaK,TaniguchiK,YokotaT,IshizawaS,KanazawaY, Synaptopodin is a coincidence detector of tyrosine versus serine/ et al.: The Rho-kinase inhibitor, fasudil, attenuates diabetic nephropathy in threonine phosphorylation for the modulation of rho protein crosstalk streptozotocin-induced diabetic rats. Eur J Pharmacol 568: 242–247, 2007 in podocytes. J Am Soc Nephrol 28: 837–851, 2017 38. Matoba K, Kawanami D, Okada R, Tsukamoto M, Kinoshita J, Ito T, et al.: Rho-kinase inhibition prevents the progression of diabetic nephropathy by downregulating hypoxia-inducible factor 1a. Kidney Int This article contains supplemental material online at http://jasn.asnjournals.org/ 84: 545–554, 2013 lookup/suppl/doi:10.1681/ASN.2018060573/-/DCSupplemental.

AFFILIATIONS

1Division of Nephrology, Department of Medicine and 3Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, New York; 2Morphometry and Stereology Laboratory, Charles R. Drew University of Medicine and Science, Los Angeles, California; 4Center for Advanced Proteomics, Department of Biochemistry and Molecular Biology, New Jersey Medical School, Newark, New Jersey; and 5Renal Unit, University of Sydney at Westmead Hospital, Sydney, Australia

J Am Soc Nephrol 29: 2641–2657, 2018 SHROOM3 Interacts with FYN in Podocytes 2657