Serum Response Factor Is Essential for Maintenance of Podocyte Structure and Function

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Bing Guo,1,2 Qing Lyu,1 Orazio J. Slivano,1 Ronald Dirkx,1 Christine K. Christie,1 Jan Czyzyk,3 Aram F. Hezel,4 Ali G. Gharavi,5 Eric M. Small,1 and Joseph M. Miano1

1Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, New York; 2Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 3Department of Pathology and Laboratory Medicine and 4James P. Wilmot Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York; and 5Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York

ABSTRACT Podocytes contain an intricate actin cytoskeleton that is essential for the specialized SRF is expressed in podocytes of the function of this cell type in renal filtration. Serum response factor (SRF) is a master adult mouse and human kidney (Figure transcription factor for the actin cytoskeleton, but the in vivo expression and function 1A), with onset of expression in mice of SRF in podocytes are unknown. We found that SRF protein colocalizes with podo- occurring between embryonic day 18.5 cyte markers in human and mouse kidneys. Compared with littermate controls, mice in and postnatal day 0 (Supplemental Fig- which the Srf gene was conditionally inactivated with NPHS2-Cre exhibited early post- ure 1A). A genetic cross to inactivate Srf natal proteinuria, hypoalbuminemia, and azotemia. Histologic changes in the mutant in podocytes (Figure 1B) yielded an mice included glomerular capillary dilation and mild glomerulosclerosis, with reduced expected Mendelian ratio of pups (Sup- expression of multiple canonical podocyte markers. We also noted tubular dilation, cell plemental Figure 1B) with normal proliferation, and protein casts as well as reactive changes in mesangial cells and in- glomerular structure (Supplemental terstitial inflammation. Ultrastructure analysis disclosed foot process effacement with Figure 1C). Furthermore, quantitative loss of slit diaphragms. To ascertain the importance of SRF cofactors in podocyte func- immunofluorescence microscopy (IFM) tion, we disabled the myocardin-related transcription factor A and B genes. Although showed no change in the expression of loss of either SRF cofactor alone had no observable effect in the kidney, deficiency of SRF in podocytes of newborn, podocyte- both recapitulated the Srf-null phenotype. These results establish a vital role for SRF specific Srf KO mice (Supplemental Fig- and two SRF cofactors in the maintenance of podocyte structure and function. ure 1, D and E). Together, these results suggest that SRF plays little, if any, role in J Am Soc Nephrol 29: 416–422, 2018. doi: https://doi.org/10.1681/ASN.2017050473 glomerulogenesis during embryonic development. Asignificant reduction of SRF/WT1- positive cells was observed in Srf KO mice Podocytes are terminally differentiated Podocyte-specificknockout(KO)ofSRF- between 3 and 6 weeks after birth (Figure cells of the glomerulus with elaborate ex- dependent cytoskeletal-associated genes, 1, C and D, Supplemental Figure 2). Al- tensions of the cell body that terminate as such as Tln1,6 Cfl1,7 Itgb1,8 and Tjp1,9 re- though Srf KO mice were indistinguishable interdigitating foot processes separated sults in defective glomerular function. SRF by slit diaphragms essential for selective is regulated by PRKDC,10 a kinase that has filtration at the glomerular filtration been implicated as a susceptibility gene for Received May 1, 2017. Accepted October 11, 2017. barrier. adriamycin-induced nephropathy,11 and Maintenance of podocyte structure and INF2, which has mutations that contribute Published online ahead of print. Publication date function is dependent on a complex actin to FSGS.12 However, the specificroleof available at www.jasn.org. cytoskeleton.1 Serum response factor SRF in the kidney and particularly, within Correspondence: Dr. Joseph M. Miano, Aab (SRF) is a transcription factor that podocytes is ill defined. Here, evidence is Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, 601 binds a DNA cis-acting element known provided in support of an essential role of Elmwood Avenue, Rochester, NY 14642. Email: j.m. as a CArG box.2 Conserved CArG boxes SRF and two of its cofactors in maintaining [email protected] are adjacent to thousands of genes, includ- the structure and function of podocytes in Copyright © 2018 by the American Society of – ing numerous cytoskeletal genes.3 5 mice. Nephrology

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Signiﬁcance statement

The structure and function of podocytes are dependent on an intricate actin cytoskeleton. Serum response factor (SRF) is a master regulator of the actin cytoskeleton; however, there is little information about SRFinpodocytebiology.Podocyte-speciﬁc knockout of Srf in mice results in foot process effacement and renal failure, leading to early death. Combined genetic inactivation of the SRF cofactors Mkl1/Mkl2 phenocopies the Srf knockout. Cultured podocytes with reduced SRF exhibit de- fects in the actin cytoskeleton and dysregulated expression of several genes, including those necessary for a functional actin cytoskeleton. SRF and MKL1/MKL2 arecriticalforpodocytestructureand normal renal function. Future work should evaluate these factors in human renal disease and interrogate target genes essential for podocyte homeostasis.

Quantitative histopathology established progressive tubular dilation, protein cast formation, and glomerular capillary dilation in Srf KO mice (Figure 2, FandG).Lotus tetragonolobus lectin staining indicated dilation of both proximal and distal tubules (Supplemental Figure 3). Occasional instances of glomerulosclerosis were observed at 6 weeks of age with little to no interstitial fibrosis (Supplemental Figure 4), likely due to the rapid onset of kidney failure. IFM of Srf KO mice showed lower expression and irregular distribution of several podocyte markers, including a–Actinin-4, Synaptopodin, Podocin, and Nephrin (Figure 3, A–C). Furthermore, upregulation of smooth muscle a-actin and Figure 1. Generation of podocyte-specific Srf KO mice. (A) IFM of sections of human and vWF was seen in Srf KO glomeruli (Sup- mouse kidney shows SRF expression in both podocytes and nonpodocytes. Podocytes plemental Figure 5, A and B), suggestive were identified by immunolabeling of Synaptopodin or WT1. Note that efforts to reliably of reactive changes in mesangial cells show WT1 in human podocytes were unsuccessful. (B) General strategy for generating and glomerular capillary endothelial fi podocyte-speci c Srf KO mice. (C) Representative images of control and mutant glomeruli cells, respectively. Srf KO mice also ex- immunolabeled with anti-WT1 (podocyte marker) and anti-SRF antibodies (Supplemental fi Figure 2 shows single-channel images). (D) Quantitative IFM of control and mutant glo- hibited an in ltration of CD45-positive fl meruli (n=15) shows 44.5%, 68.8%, and 62.2% reductions of SRF+/WT1+ podocytes at 3, 4, in ammatory cells within the tubuloin- and 6 weeks, respectively. Scale bars, 50 mminA,upperpanel;20mm in A, lower panel and terstitium and Ki67 staining in tubular C. ***P,0.001. epithelial cells; no such findings were evident in control mice or within the from littermate controls at birth, they leukocyturia (Figure 2, C and D) as well as glomerulus of Srf KO mice (Supplemen- exhibited a notable decrease in body size elevated serum creatinine and BUN, with tal Figure 5, C–E). The presence of (Figure 2A) and body weight (Figure 2B) concomitant hypoalbuminemia (Figure inflammatory cells and proliferative tu- at 6 weeks of age. Srf KOs also displayed 2E). Death occurred by 7 weeks of age bular epithelial cells of Srf KO mice increased urinary protein, hematuria, and in both male and female Srf KO mice. likely represents a secondary response to

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Figure 2. Postnatal nephropathy and renal failure in podocyte-speciﬁc Srf KO mice. (A and B) Decreased body size and body weight in Srf KO mice compared with control mice at 6 weeks of age (n$5 age-matched male mice). (C) Coomassie Blue staining of urine samples shows massive proteinuria in 3-week-old mutant mice. (D) Dipstick analysis indicates increased urinary protein (left panel), red blood cells (RBCs; center panel), and white blood cells (WBCs; right panel) of mutant mice between 3 and 6 weeks of age (n$7). (E) Serum analysis reveals elevated creatinine (left panel) and BUN (center panel) and decreased albumin (right panel) in 6-week-old Srf KO mice (n$5). (F) He- matoxylin and Eosin staining of kidneys at 6 weeks of age. Note dilated tubules (arrowhead), glomerular capillary dilation (asterisk), and cast formation (arrow) in Srf KO kidneys (n$3). (G) Quantitative measures of dilated tubules, protein casts, and glomerular capillary dilation in control versus Srf KO kidneys at 3, 4, and 6 weeks of age (n$30 ﬁelds of view in three or more mice per condition). Scale bars, 100 mmin F, top panel; 40 mm in F, middle panel; 20 mminF,bottompanel.*P,0.05; **P,0.01; ***P,0.001.

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Figure 3. Podocyte marker expression and ultrastructural abnormalities in Srf KO mice. (A–C) IFM shows diminished and punctate staining of (A) a–Actinin-4, (B) Synaptopodin and Podocin, and (C) Nephrin in mutant mice (n=3) at 4 and 6 weeks of age. (D and E) Representative (D) scanning electron microscopy (SEM) and (E) transmission electron microscopy (TEM) of control and Srf KO kidneys (n=2 each for control and mutant). Note (D, lower panel) foot process effacement and (E, bottom panel) loss of slit diaphragms in Srf KO. Scale bars, 20 mmin A–C; 2 mm in D, upper panel; 5 mm in E, top panel; 1 mm in D, lower panel and E, middle panel; 0.2 mm, E bottom panel. the glomerulopathy-induced accumula- 3E). Collectively, these results suggest dysregulated expression of podocyte- tion of tubular urinary casts. Ultrastruc- that SRF plays an important role in main- enriched genes,14 including Acta2, Fgfr1, ture analysis by scanning and transmis- taining postnatal podocyte structure and and Actn4 (Supplemental Figure 6). The sion electron microscopy revealed function. upregulation of Fgfr1 is of particular rele- occasional foot process effacement in 3- To verify the regulatory role of SRF on vance in this context given its association week-old Srf KO mice (data not shown); podocyte actin cytoskeleton, we evaluated with experimental membranous nephrop- this phenotype was more pronounced at F-actinbyphalloidinstaininginacondi- athy15 and regulation by microRNA-16,16 a 6 weeks of age (Figure 3, D and E). Higher tional immortalized podocyte cell line.13 known SRF-dependent target gene.17 magniﬁcation showed loss of slit dia- siRNA-induced Srf knockdown attenuated Because the myocardin family of tran- phragms between adjacent foot processes, phalloidin staining in differentiated podo- scriptional cofactors controls expression with little evidence of alterations in glo- cytes, suggesting that low SRF in podocytes of contractile and actin cytoskeletal– merular basement membrane thickness results in dysregulation of the actin cyto- related genes,18–20 we hypothesized these or fenestrated endothelial cells (Figure skeleton. Quantitative RT-PCR revealed factors were key SRF cofactors mediating

J Am Soc Nephrol 29: 416–422, 2018 Podocyte-Specific Srf Knockout 419 BRIEF COMMUNICATION www.jasn.org the observed phenotype. We first deter- unremarkable (Supplemental Figure of available antibodies. However, the mined the relative mRNA expression of 8B), and there was no indication of pro- strong phenotype observed in the myocardin-related transcription factor A teinuria (Supplemental Figure 8C). In Mkl1/Mkl2 dKO but not observed in in- (Mkl1)andB(Mkl2) with another family contrast, the Mkl1/Mkl2 double knock- dividual Mkl KOs implies the presence of member, Myocardin. Both Mkl1 and Mkl2 out (dKO) exhibited dilated tubules both within podocytes. Of note, Mkl1/ mRNA were more abundant in the glo- with numerous urinary casts and en- Mkl2 dKO mice, but not three-allele merular-rich cortex than Myocardin larged glomerular capillary loops as KO mice, showed somewhat lower ex- (Supplemental Figure 7). Accordingly, well as proteinuria at 7 weeks (Supple- pression of SRF in podocytes (Supple- we inactivated Mkl1 as a conventional mental Figure 8, A and D), all of which mental Figure 9); a similar reduction in KO,21 floxed Mkl222 using the NPHS2- are consistent with the histopathologic Srf mRNA was seen in skeletal muscle Cre driver, or both Mkl1 and Mkl2 features of the podocyte-specific Srf KO lacking these cofactors.24 IFM disclosed (Figure 4A). Unlike Srf KO mice, loss (Figure 4B versus Figure 2F). Thus, a reduced expression of Synaptopodin and of either Mkl1 or Mkl2 showed no ev- single allele from either Mkl cofactor Podocin in glomeruli of Mkl1/Mkl2 dKO idence of glomerular or renal tubular seems to compensate for the loss of the mice but not in Mkl2 KO or three-allele abnormalities (Supplemental Figure other three alleles, a finding that con- KOs, which were comparable with wild- 8A). Furthermore, the histology of the trasts with a prior Mkl KO study.23 type mice (Figure 4C versus Figure 3B). kidney in three-allele Mkl KO mice (i.e., We were unsuccessful in reliably de- The lifespan of Mkl1/Mkl2 dKO mice mice with only one wild-type allele of tecting MKL cofactors in podocytes, was over 10 weeks, slightly longer than Mkl1 or Mkl2) up to 1 year of age was likely because of technical limitations Srf KO mice. Previous studies showed an injury-promoting role of SRF/MKL in kidney disease25–28; in contrast, data here support SRF and MKL1/MKL2 in the maintenance of normal podocyte structure and function. Continued expression of WT1 in podocytes of the Srf KO and Mkl1/Mkl2 dKO suggests that Wt1 transcription is SRF/MKL independent. The absence of conserved SRF binding CArG boxes within proximal regulatory regions of the WT1 locus further supports this concept (J.M. Miano, unpublished observations). We propose that WT1 and other com- plementary transcription factors29 spec- ify and direct the initial differentiation of podocytes, including the onset of cytoskeletal gene expression. Subse- quently, SRF and MKL1/MKL2 direct a later program of gene expression necessary to augment and maintain the complex actin cytoskeleton, which is essential for the increasing demand on postnatal kidney filtration. Transcriptomic data suggest that al- tered SRF expression may play a role in human kidney disease. For example, SRF mRNA is reduced in human diabetic glomeruli,30 and several SRF target genes Figure 4. Recapitulation of the Srf KO phenotype in Mkl1/Mkl2 dKO mice. (A) Schematic of are upregulated in human podocytes Mkl1 (conventional) and Mkl2 (conditional) KOs in podocytes (the NPHS2-Cre cross is not treated with glucocorticoids.31 More- shown). Based on references 21 and 22. (B) Hematoxylin and Eosin staining of kidneys from control (wild-type) and Mkl1/Mkl2 dKO mice at 7 weeks of age. The asterisk, arrow, and ar- over, human mutations exist in different rowhead indicate the dilated glomerular capillary, the protein cast, and the dilated renal tu- cytoskeletal genes, such as ACTN4,that bule, respectively. (C) IFM showing decreased and punctate expression of Synaptopodin and are probable SRF targets on the basis of Podocin in dKO mice but not in Mkl2 KO and three-allele KOs at 7–10 weeks of age. Scale the presence of conserved CArG boxes bars, 100 mm in B, top panel; 40 mm, B, middle panel; 20 mm in B, bottom panel and C. and ChIP-seq data (J.M. Miano,

420 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 416–422, 2018 www.jasn.org BRIEF COMMUNICATION unpublished observations). Of note, we dipstick assay (Fisher Scientific, Hampton, DNAase I (Invitrogen), and reverse tran- previously reported the existence of NH) and Coomassie Blue staining. For quan- scribed into cDNA with iScript Reverse Tran- CArG SNPs in the human genome, tification of dipstick data, different indices scription Supermix (Bio-Rad, Hercules, CA). with such variants exhibiting defective were assigned to corresponding ranges of SYBR Green Supermix (Bio-Rad) was used SRF binding and target promoter activ- protein, white blood cell, and red blood cell for quantitative PCR; relative mRNA levels ity.3 Given the abundant expression of readings. Urine was resolved by SDS-PAGE to Gapdh or Rhoa were normalized to the 2ΔΔ SRF in human podocytes as reported gel and stained by SimplyBlue SafeStain (In- control sample using the 2 Ct method. Se- here, it will be important to determine vitrogen, Waltham, MA) according to the quences of real-time primers are provided in if levels of SRF protein change in podo- manufacture’s protocol. Blood samples were Supplemental Table 3. cytopathies and whether CArG SNPs harvested via heart puncture and submitted Cell culture, Srf knockdown, and phallo- exist that portend kidney disease. In to the Cobas 6000 Analyzer to measure serum din staining are described in Supplemental contrast to SRF,severalmutationsin levels of BUN, creatinine, and albumin. Body Material. MKL1 and MKL2 are associated with hu- weight data were obtained from age-matched man diseases, although none have been male control and Srf KO mice. linked to kidney disease (https://www. ACKNOWLEDGMENTS ebi.ac.uk/gwas/). Histopathology and IFM In conclusion, SRF and MKL1/MKL2 Kidney tissues were formalin fixed, paraffin are essential for postnatal podocyte embedded, and sectioned at 5-mmthickness. We thank Karen L. De Mesy Bentley and the structure and function, likely through Slides were deparaffinized and rehydrated in University of Rochester Medical Center Elec- the maintenance of a normal actin cyto- PBS. Hematoxylin and Eosin staining, Masson tron Microscopy Core for generating the elec- skeleton. Future work should interrogate trichrome staining (American MasterTech, tron microscopic images. SRF and MKL1/MKL2 target genes Lodi, CA), and immunofluorescence staining B.G. is supported by a grant from Union within podocytes through integrative were conducted according to standard proce- Hospital, Tongji Medical College, Huazhong computational, RNA-seq, and ChIP-seq dures. Immunofluorescence images were taken University of Science and Technology. A.G.G. studies. Such information could shed on a confocal microscope (Olympus FV 1000). is supported by Department of Defense Peer new light on the pathogenesis of some The primary antibodies and detailed proce- Reviewed Medical Investigator-Initiated Re- podocytopathies and open up new op- dures for immunostaining are provided in search award PR151419, and E.M.S. is supported portunities for therapeutic interventions Supplemental Material and Supplemental Ta- by National Institutes of Health (NIH) grants in CKD. ble 2. Quantitation of kidney histology and HL120919 and HL133761. J.M.M. is supported WT1+/SRF+ cells was conducted by two inde- by NIH grants HL112793 and HL117907. pendent observers blind to genotype. CONCISE METHODS DISCLOSURES Electron Microscopy Animals and Human Subjects Forscanningelectronmicroscopy,the kidneys None. fl fl 2 2 fl fl Srf / , Mkl1 / , Mkl2 / ,andNPHS2-Cre were fixed in 0.1 M sodium cacodylate–buffered mice21,22,32,33 were used to produce podocyte- 2.5% glutaraldehyde/4.0% paraformalde- fl fl REFERENCES specific Srf KO mice (Srf / /NPHS2-Cre)and hyde fixative, postfixedinbuffered1.0%

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