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Loss of the Podocyte-Expressed Tcf21/Pod1 Results in Podocyte Differentiation Defects and FSGS

† ‡ Yoshiro Maezawa,* Tuncer Onay,* Rizaldy P. Scott,* Lindsay S. Keir,§ Henrik Dimke,* ‡ | Chengjin Li,* Vera Eremina,* Yuko Maezawa, Marie Jeansson,*¶ Jingdong Shan,** †† ‡‡ ‡ Matthew Binnie, Moshe Lewin, Asish Ghosh, Jeffrey H. Miner,§§ Seppo J. Vainio,** ‡ and Susan E. Quaggin*

*The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; †Department of Diabetes, Metabolism and Endocrinology, Chiba University Hospital, Chiba, Japan; ‡Feinberg Cardiovascular Research Institute and Division of Nephrology and Hypertension, Northwestern University, Chicago, Illinois; §Academic Renal Unit, University of Bristol, Bristol, United Kingdom; |Neuroscience and Mental Health Program, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; ¶Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; **Biocenter and Infotech Oulu, Laboratory of Developmental Biology, Faculty of Biochemisty and Molecular Medicine, Oulu Center for Cell Matrix Research, University of Oulu, Finland; ††Division of Respirology, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada; ‡‡Department of Nephrology, RAMBAM Health Care Campus, Haifa, Israel; and §§Renal Division, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri

ABSTRACT Podocytes are terminally differentiated cells with an elaborate cytoskeleton and are critical components of the glomerular barrier. We identified a bHLH transcription factor, Tcf21, that is highly expressed in developing and mature podocytes. Because conventional Tcf21 knockout mice die in the perinatal period with major cardiopulmonary defects, we generated a conditional Tcf21 knockout mouse to explore the role of this transcription factor in podocytes in vivo. Tcf21 was deleted from podocytes and podocyte progenitors using podocin-cre (podTcf21) and -cre (wnt4creTcf21) driver strains, respectively. Loss of Tcf21 from capillary-loop stage podocytes (podTcf21) results in simplified glomeruli with a decreased number of endothelial and mesangial cells. By 5 weeks of age, 40% of podTcf21 mice develop massive proteinuria and lesions similar to FSGS. Notably, the remaining 60% of mice do not develop proteinuria even when aged to 8 months. By contrast, earlier deletion of Tcf21 from podocyte precursors (wnt4creTcf21) results in a profound developmental arrest of podocyte differentiation and renal failure in 100% of mice during the perinatal period. Taken together, our results demonstrate a critical role for Tcf21 in the differentiation and maintenance of podocytes. Identification of direct targets of this tran- scription factor may provide new therapeutic avenues for proteinuric renal disease, including FSGS.

J Am Soc Nephrol 25: 2459–2470, 2014. doi: 10.1681/ASN.2013121307

Over the past decade, many landmark studies have of these can cause neonatal and adult human demonstrated a central role for podocytes in renal nephrotic syndromes.1 Furthermore, podocytes health and disease. Mature podocytes extend elab- orate foot processes that interdigitate with each Received December 16, 2013. Accepted February 8, 2014. other and are connected by a specialized intercel- Published online ahead of print. Publication date available at lular junction called the slit diaphragm. The slit www.jasn.org. diaphragm is composed of several , in- Correspondence: Dr. Susan E. Quaggin, Lurie Medical Research cluding nephrin, podocin, and Cd2-associated pro- Building, 303 E Superior, Chicago, IL 60611. Email: quaggin@ tein, that interact with actin-based cytoskeletal northwestern.edu proteins, such as a-actinin 4. Mutations in each Copyright © 2014 by the American Society of Nephrology

J Am Soc Nephrol 25: 2459–2470, 2014 ISSN : 1046-6673/2511-2459 2459 BASIC RESEARCH www.jasn.org communicate with other cell types in the glomerulus, and are RESULTS able to monitor their extracellular environment and modify their shape and function accordingly.2 As a result, dysregula- Generation of a Floxed Tcf21 Founder Mouse Line tion in a number of signaling pathways and factors produced The Tcf21 conditional allele was designed with LoxP sites by podocytes, such as vascular endothelial growth factor inserted around the first , which contains the bHLH (VEGF), can also cause glomerular injury.3,4 domain (Figure 1A). After electroporation, embryonic stem During development, podocyte precursors appear at the (ES) cell clones were screened by Southern blot analysis with end of the S-shaped body farthest from the ureteric bud and probes outside the homology regions. One positive clone (5G differentiate through a well defined sequence of morphologic clone in Figure 1B) was used to generate chimeric mice. events from a columnar-shaped epithelium facing the vascular To confirm that the Cre-mediated deletion results in a cleft to mature podocytes with their elaborate foot processes. null Tcf21 allele, we generated inducible whole-body Tcf21 Several transcription factors have been identified that are knockout mice using the ROSA-rtTA/tetO-Cre system. Ad- expressed by podocyte precursors, including Wilms’ tumor ministration of doxycycline to the dam from the time of con- suppressor 1 (Wt1), Lmx1b, Foxc2, and MafB. Wt1 is a zinc ceptionresultedinmutantembryosthatshowpericardial finger–containing transcription factor, and genetic deletion bleeding and severely hypoplastic and kidneys at embry- of Wt1 leads to loss of nephrogenic induction and renal onic day 18.5, which are identical to the defects caused by agenesis.5 Glomeruli in knockout mice for Lmx1b or Foxc2 phenotype observed in conventional Tcf21 knockout mice are abnormal, with arrested podocyte differentiation, sim- (Figure 1C). This finding confirmed that the deleted condi- plified capillary loop structure, and defects in mesangial cell tional allele is indeed a null allele. Subsequent genotyping was ingrowth.6,7 Global deletion of MafB also results in abnor- performed by PCR (Figure 1D). mal foot process formation, with persistence of “flat feet” in podocytes.8 Normal Differentiation of Podocytes and Delay of Tcf21 (Pod1/capsulin/epicardin) is a basic helix-loop-helix Glomerular Maturation in Podocyte-Specific Tcf21 (bHLH) transcription factor whose expression is highest in Knockout Mice podocyte precursors and is maintained in mature podocytes. Previously we showed that conventional Tcf21 knockout mice However, at earlier stages of metanephric development, Tcf21 demonstrate an arrest of glomerular maturation (Figure 2A).9 is expressed in both Six2-expressing nephron progenitors and To examine the role of Tcf21 exclusively in podocytes, floxed Foxd1-expressing stromal .9–11 Global deletion Tcf21 mice were bred to transgenic podocin-cre mice to create studies of Tcf21 have shown that it is required for the devel- podocyte-specific knockout (podTcf21) mice. Using a Z/EG opment of , , , gonad, , and facial mus- reporter mouse,15 we confirmed that podocin-cre results in cles.9,12–14 The kidneys of Tcf21 knockout mice are severely gene excision from the capillary loop stage onward and not at hypoplastic because of a delay of nephrogenesis and abnormal the S-shape stage (Supplemental Figure 1A). At postnatal day branching morphogenesis of the ureteric bud.9 The few glo- 0, although Tcf21 expression was properly decreased in meruli found in kidneys of Tcf21 knockout mice appear to podocytes, podTcf21 glomeruli showed almost normal histo- arrest at the capillary loop stage of development. However, logic features and ultrastructure (Figure 2B). Mice were born because Tcf21 is also involved in the induction of nephrogen- in the expected Mendelian ratio, and podTcf21 pups appeared esis from the Six2-positive progenitor population, it has not healthy for 2 weeks (data not shown). These results indicate been possible to determine the roles of Tcf21 in developing that Tcf21 is not required for terminal differentiation of and mature podocytes. Indeed, the glomerular defects might podocytes after the capillary loop stage. reflect roles of Tcf21 in the progenitor or adjacent cell popu- However, immunostainings for podocin and endothelial lations, rather than in the podocyte itself. markers(Cd31andendomucin)revealedasurprisingly Here we report the phenotype in mice following podocyte- simplified glomerular structure at postnatal day 0 and 3 weeks selective knockout of Tcf21. Deletion of Tcf21 in relatively of age (Figure 2C). Immunostaining for a mesangial marker, mature podocytes (podocin-cre) allows podocyte differenti- desmin, showed a similar finding (Supplemental Figure 1B). ation to occur but results in a simplified glomerular structure. Interestingly, at P0, although the Wt1-positive podocyte num- However, 40% of podocyte-specific Tcf21 knockout mice ber did not differ, total cell number and nonpodocyte cell develop massive proteinuria at 3–5 weeks of age with prom- number in mutant glomeruli were reduced by 15% and inent glomerular lesions similar to human FSGS. By contrast, 25%, respectively (Figure 2D). Taken together, these findings earlier deletion of Tcf21 at the renal vesicle stage of glomerular suggest a delay in glomerular maturation with reduced influx development using a wnt4-cre driver line results in major de- and/or in situ proliferation of endothelial and mesangial cells. fects in podocyte differentiation. Finally, we show rapid development of kidney failure in a diabetic model in the “pro- A Subset of Podocyte-Specific Tcf21 Knockout Mice tected” podTcf21 knockout cohort. Together, our data dem- Develop Proteinuria and FSGS onstrate key roles of Tcf21 in podocytes in developing and By 5weeks of age, 40% of the podTcf21 mice developed massive mature animals, especially under disease conditions. proteinuria (Figure 3, A and C). Although there was large

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Figure 1. Generation of conditional allele for Tcf21. (A) Targeting construct for floxed Tcf21 allele. LoxP sites were inserted around exon1 (Ex1). FRT, FRT sequence; HA, homology arm; RI, EcoRI site; RV, EcoRV site; TG, targeted; WT, wild type. (B) Targeted clones were identified by Southern blot analysis using probes outside the 59 and 39 homology arms. Arrows indicate positively targeted clones. Clones 12F and 5G were used for ES cell aggregation; only the 5G clone resulted in germline transmission. (C) Validation of the floxed allele using the Rosa-rtTA/tetO-Cre system. Doxycycline induction to excise the floxed alleles in embryos from E0 (conception) resulted in a phenotype in newborn pups identical to that observed in conventional Tcf21 knockout pups. Note the severely hypoplastic kidneys (upper panels) and lungs (lower panels) in floxed mutants. Histology, hematoxylin and eosin; original magnification, 340. (D) PCR genotyping shows floxed Tcf21 allele (558 bp), Tcf21-LacZ allele (463 bp; conventional knockout), and floxed allele after Cre-mediated deletion (757 bp). Lane 3 shows the genotype from a mouse carrying one deleted floxed allele and a null LacZ allele. cont, control littermate; DOX, doxycycline; mut, mutant, Rosa-rtTA/tetO-Cre/Tcf21floxed/LacZ. variation between mutants, podTcf21 mice showed signifi- Glomeruli from the proteinuric podTcf21 show dramatic cant increase of the mean urinary -to-creatinine pathologic changes with focal and segmental sclerosis similar ratios (P,0.05 for 3 and 8 weeks; P,0.01 for 4 and 6 weeks- to human FSGS, with occasional crescent formation and of-age). Kidneys from the proteinuric podTcf21 mice were podocyte vacuolation (Figure 3E). Transmission electron mi- atrophic and pale, and the surface was irregular (Figure 3B). crographs (TEM) of kidneys from nonproteinuric podTcf21 Some mutants quickly reached ESRD and died at 3–6weeks mice at 3 weeks of age were similar to those of control kidneys. of age. Interestingly, 60% of podTcf21 mice never devel- However, scanning electron microscopy revealed subtle de- oped proteinuria even up until 8 months of age, suggesting fects with focal flattening and disorganization of foot pro- there are two populations of podTcf21 mutants: protected cesses (Figure 3F). In glomeruli from proteinuric podTcf21 and susceptible (Figure 3C). Susceptible PodTcf21 mice mice, podocyte foot processes were extensively effaced (Figure showed significant growth retardation at 6 and 8 weeks- 3F). At 6 weeks of age, glomeruli of proteinuric podTcf21 mice of-age (Figure 3D, Supplemental Figure 2A). Proteinuria showed foot process effacement with large bumpy subepithe- occurred in both male and female mutants to the same de- lial protrusion of the glomerular basement membrane (Sup- gree (41.7% and 38.5%, respectively), suggesting no sex plemental Figure 2B). Taken together, these results indicate differences. that Tcf21 is required for the maintenance of podocyte

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Figure 2. Delay of glomerular maturation in podTcf21 mice. (A) Periodic acid-Schiff (PAS)–staining (31000) of a glomerulus and a TEM (312,400) of the glomerular barrier in the kidney from conventional Tcf21 knockout at E18.5. (B) Upper panel: in situ hybridization for Tcf21 (3200) confirms loss of Tcf21 message in capillary loop stage podocytes in podTcf21 mice (arrowheads). Middle panel: periodic acid-Schiff–staining of glomeruli (31000). Lower panel: TEM that shows relatively normal glomerular filtration barrier of podTcf21 at postnatal day 0 (P0) (312,400). (C) Immunostainings for endothelial (CD31 or endomucin) markers and podocin demonstrate simplified structure of the glomerulus at P0 (upper) and 3 weeks (lower) in podTcf21 mice compared with control (3400). (D) Reduced non- podocyte cell number in podTcf21 glomeruli. Upper panel: immunostainings for a podocyte marker, Wt1 (31000). Lower panels: numbers of podocytes, non-podocytes, total cell number in each glomerulus, and non-podocyte/podocyte ratio. Total cell number and non-podocyte cell number in mutant glomeruli were reduced by 15% (51.8 cells in control versus 44.1 cells in podTcf21; P,0.05) and 25% (28.6 cells in control versus 21.4 cells in podTcf21; P,0.05), respectively. *P,0.05.

structure and the function of the glomerular filtration barrier, and F), presumably in podocytes. These observations sugges- but the extent of the effect varies among individual mice. ted that podocytes are injured. Laminin 111–positive area, normally seen in mesangium and immature glomerular base- PodTcf21 Mutants Show Podocyte Injury ment memrane,16 was expanded in podTcf21 glomeruli with To characterize the glomerular injury in kidneys of podTcf21 reduced capillary loops (Figure 4, G and H). Staining for Lam- mice, we performed in situ hybridization and immunostain- inin b2, a specific laminin in the glomerular basement mem- ings for glomerular markers. In injured glomeruli, Nephrin brane, shows an aberrantly tangled and clustered pattern mRNA expression and Podocin protein were partially lost (Figure 4, I and J). The mutant glomeruli showed prominent (Figure 4, A–D). Desmin expression was increased with a fibrinogen deposition, indicating the leakage of plasma com- punctate pattern at the edge of the glomeruli (Figure 4, E ponents (Figure 4, K and L). In addition, the mutant glomeruli

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Figure 3. Forty percent of podTcf21 mice develop proteinuria and FSGS. (A) Urinary protein-to-creatinine ratio in control and podTcf21 mice. Bars indicate mean6SEM. Forty percent of podTcf21 mutants developed massive proteinuria at 3 weeks onward. (B) Macroscopic picture of control and podTcf21 kidneys from mutant with massive proteinuria at 4 weeks of age. (C) Cumulative occurrence of pro- teinuria that is .3+ by dipstick or 20 mg/mg protein-to-creatinine ratio. Only 40% of the mutants develop proteinuria, identifying both a protected and a susceptible cohort. (D) Body weight of control versus podTcf21 mice. Bars indicate mean6SEM (24.5 g control versus 22.4 g podTcf21 at 6 weeks, 25.2 g control versus 22.5 g podTcf21 at 8 weeks; for both P,0.05). (E) Periodic acid-Schiff–staining of podTcf21 kidneys shows FSGS lesions. Various degrees of glomerulosclerosis and protein casts in tubules (arrowheads, middle panel), partial glomerulosclerosis (black arrowhead) with crescent formation (white arrowhead, right upper panel), and vacuolations in podocytes (white arrowheads, right lower panel) are shown. Middle panel, 3200; others, 31000. (F) Ultrastructural analysis of podTcf21 glomeruli. In non-proteinuric podTcf21 mice (middle panels), no abnormality was detectable by TEM, but scanning electron micro- graph (SEM) shows partial flattening and disorganization of podocyte foot processes (arrowhead). These findings are more prominent and global in proteinuric podTcf21 glomeruli (arrowheads, right panels). TEM, 39600; scanning electron micrograph, 312,400. demonstrated dynamic proliferation of cells in Bowman’scap- glomerular development or function. Some of these changes sule (Figure 4, M and N). were confirmed by quantitative realtime PCRs and in situ hy- bridization (Supplemental Figure 3; primer sequences are Microarray Analysis of Isolated Glomeruli shown in Supplemental Table 1). Complete lists of genes To characterize the molecular response in the glomeruli and to that show at least a 1.5-fold difference are provided (Supple- identify putative downstream targets for Tcf21, we performed a mental Tables 2 and 3). comprehensive analysis using Affymetrix microarray on total glomerular RNAs isolated from podTcf21 Podocyte Differentiation Is Abnormal in Wnt4creTcf21 and control mice. A total of 3065 genes whose transcript varied Mice significantly (P,0.05) were identified(8.6%ofthe35,556 We next hypothesized that Tcf21 may be most important when genes). Table 1 shows a partial list of genes associated with differentiation is actively occurring in podocyte precursors.

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Desmin staining also revealed a simplifica- tion of the glomerulus (Figure 5, I and J). Expression of synaptopodin, aPKCl,and Par3 (Figure 5, K–P) was decreased. Expres- sion of Nephrin, Wt1, and Zo1 (Figure 5, Q–V) remained unchanged. Finally, a pro- found defect of podocyte foot process for- mation was observed on TEM (Figure 5, W and X). Taken together, these data suggest a major defect in podocyte differentiation if Tcf21 is absent from the precursor stage on- ward. Notably, wnt4creTcf21 kidneys are slightly smaller but do not show any overt abnormality in tubular formation, suggest- ing that mesenchyme-to-epithelial trans- formation is not impaired (Supplementary Figure 4).

Tcf21 Is Required in Aging Podocytes and Protects against Diabetic Nephropathy Given the presence of two distinct popula- tions of adult podTcf21 knockout mice (proteinuric versus nonproteinuric), we Figure 4. Podocyte injury in glomeruli from proteinuric podTcf21 mice. (A and B) In were keen to examine the protected cohort situ hybridization shows decreased expression of Nephrin in proteinuric podTcf21 in more detail. To determine whether mice. (C–F) Immunostainings reveal decreased expression of Podocin (arrowheads, D) podTcf21 mice in the protected cohort and Desmin (arrowheads, F) in presumptive podocytes of proteinuric podTcf21 mice might develop proteinuria as they age, we compared with controls (C and E). (G and H) Expansion of Laminin111 expression followed nine podTcf21 mice (urinary domain was seen in proteinuric podTcf21 mutants. (I and J) Laminin b2(Lamb2)/ protein-to-creatinine,10 mg/mg at 8 weeks) Desmin staining shows a collapsed and tangled pattern (arrowhead, J) compared with until 1 year of age (Figure 6A, upper panel). a beautifully organized structure in controls (I). (K and L) Proteinuric podTcf21 glo- Outcomes for individual mice are shown in meruli show fibrinogen deposits (arrowheads). (M and N) Ki67 staining shows pro- Supplemental Table 4. One developed mas- liferation in the Bowman capsules and in crescents. A–FandK–N, 3400; G–J, 3600. siveproteinuriaat32weeksanddiedat38 weeks. At 1 year, among five surviving Therefore, we crossed the Tcf21 floxed mice to a wnt4-cre podTcf21, two showed mild proteinuria and focal glomerulo- driver mouse line (wnt4creTcf21).17 Wnt4 is an inducer of sclerosis (Figure 6B, upper panel). These results indicate that mesenchymal-to-epithelial transformation and is expressed podTcf21 mice develop proteinuria either before 5 weeks or after in pretubular aggregates, renal vesicles, and the proximal 32 weeks (Figure 6A, upper panel) but are unlikely to develop part of the S-shaped body of the developing nephron. Because proteinuria in the interim. Although incomplete or mosaic ex- Tcf21 is expressed in condensing mesenchyme but not in the cision may account for the milder phenotype, we found no dif- renal vesicle and reappears in podocyte precursors, wnt4-cre ference in the level of Tcf21 expression in glomeruli isolated mice should provide a selective knockout of Tcf21 from from kidneys of proteinuric (n=2) and nonproteinuric (n=5) podocyte precursors without affecting its expression in the mice, as determined by realtime quantitative PCR (not shown). condensing mesenchyme. To further examine the role of Tcf21 in podocytes in a In situ hybridization confirmed robust excision of Tcf21 in clinically relevant disease model, we chose the streptozotocin immature and mature podocytes from the S-shape stage model of diabetic nephropathy. We selected a cohort of onward, which is earlier than excision of Tcf21 using the nonproteinuric podTcf21 (urinary protein-to-creatinine ra- podocin-cre line (Figure 5, A and B [compare with Figure tio, 10 mg/mg) and control mice at 17 weeks of age. Diabetes 2B] Supplemental Figure 1A). Glomeruli of wnt4creTcf21 was induced according to the Diabetic Complications Consor- mice were primitive and reduced in size at embryonic stage tium streptozotocin protocol.18 Both groups were monitored E18.5. Podocytes remained columnar, and the complexity of for 18 weeks after injection. Sixty-six percent of the diabetic the glomerulus was reduced (Figure 5, C and D). The expression podTcf21 mice died before the end of the study compared with of podocin was largely reduced and showed granular and patchy 11.1% of nondiabetic podTcf21 mice (P,0.01) (Figure 6C). distribution at the lateral and apical sides (Figure 5, E–H). During the course of the disease, the diabetic podTcf21 mice

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Table 1. List of genes differentially expressed in glomeruli isolated from Pod1tcf21 versus wild-type mice podTcf21 Glomeruli Microarray Analysis (Categorized) Fold Change Ref Seq Gene Assignment Gene Symbol P Value (Mut/Con) NM_011545 Transcription factor 21 Tcf21 4.02E-11 0.116851 Podocyte-related genes NM_144783 Wt1 homolog Wt1 0.96 1.00341 AB513652 Nephrosis 1 homolog, nephrin (human) Nphs1 0.71 0.965275 NM_019459 Nephrosis 1 homolog, nephrin (human) Nphs1 0.82 0.967055 NM_130456 Nephrosis 2 homolog, podocin (human) Nphs2 0.69 0.952228 NM_015764 Gene regulated by estrogen in breast protein Greb1 0.04 1.09796 NM_009847 CD2-associated protein Cd2ap 0.04 0.913255 NM_001109975 Synaptopodin Synpo 0.01 0.834113 NM_001099331 R3H domain containing-like R3hdml ,0.001 0.195615 Mesangial genes NM_010043 Desmin Des 0.60 0.955636 NM_001146268 Pdgfrb Pdgfrb ,0.002 0.894954 Endothelial genes NM_01061 Kinase insert domain protein Kdr 0.09 0.953623 NM_001111059 CD34 antigen Cd34 0.41 1.02267 NM_010228 FMS-like tyrosine kinase 1 Flt1 ,0.002 0.883751 NM_013690 Endothelial-specific receptor tyrosine kinase Tek 0.01 0.890548 NM_008816 Platelet/endothelial cell adhesion molecule 1 Pecam1 0.22 0.953775 VEGF-related genes NM_001025250 VEGF-A Vegfa 0.01 0.858069 NM_011697 VEGF-B Vegfb ,0.001 1.20935 NM_009506 VEGF-C Vegfc ,0.006 1.46325 NM_008827 Placental growth factor Pgf 1.29E-05 0.432535 Notch pathway NM_008714 Notch gene homolog 1 (Drosophila) Notch1 0.25 0.959897 NM_010928 Notch gene homolog 2 (Drosophila) Notch2 0.54 0.975687 NM_019454 Delta-like 4 (Drosophila) Dll4 ,0.001 0.692084 NM_013904 Hairy/enhancer-of-split related with YRPW motif 2 Hey2 ,0.012 0.663724 NM_013905 Hairy/enhancer-of-split related with YRPW motif-like Heyl , 0.012 0.8704 Heparan sulfate proteoglycans NM_178870 Heparan sulfate (glucosamine) 3-O-sulfotransferase 3A1 Hs3st3a1 2.83E-10 0.130029 NM_016696 Glypican 1 Gpc1 4.04E-06 0.614059 NM_009929 Collagen, type XVIII, a1 Col18a1 6.17E-10 0.473699 NM_028072 Sulfatase 2 Sulf2 1.56E-07 1.84207 NM_001198565 Sulfatase 1 Sulf1 ,0.001 1.57147 NM_016771 Sulfotransferase family 1D, member 1 Sult1d1 ,0.001 2.43938 Collagens and laminins NM_007743 Collagen, type I, a2 Col1a2 6.51E-06 0.66233 NM_007739 Collagen, type VIII, a1 Col8a1 2.42E-05 2.39189 NM_007730 Collagen, type XII, a1 Col12a1 ,0.001 1.42416 NM_181277 Collagen, type XIV, a1 Col14a1 ,0.001 2.93957 NM_009929 Collagen, type XVIII, a1 Col18a1 6.17E-10 0.473699 NM_008480 Laminin, a1 Lama1 ,0.001 1.37389 NM_008485 Laminin, g2 Lamc2 0.02 1.3046 Wnt Signaling Cannonical Wnt pathway NM_007614 Catenin (cadherin associated protein), b1 Ctnnb1 0.04 0.957209 NM_010703 Lymphoid enhancer binding factor 1 Lef1 0.01 0.848828 NM_013685 Transcription factor 4 Tcf4 0.73 0.990435 NM_015732 Axin2 Axin2 0.61 0.951149 Wnt ligands NM_009523 Wingless-related MMTV integration site 4 Wnt4 0.03 0.894278

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Table 1. Continued

podTcf21 Glomeruli Microarray Analysis (Categorized) Fold Change Ref Seq Gene Assignment Gene Symbol P Value (Mut/Con) ENSMUST00000045747 Wingless-related MMTV integration site 4 Wnt4 0.16 0.913155 NM_009524 Wingless-related MMTV integration site 5A Wnt5a 1.30E-05 0.646918 NM_009528 Wingless-related MMTV integration site 7B Wnt7b 0.67 1.03785 NM_011720 Wingless related MMTV integration site 8b Wnt8b 2.26E-05 1.45817 NM_011719 Wingless-type MMTV integration site 9B Wnt9b 0.39 1.08941 NM_009519 Wingless-related MMTV integration site 11 Wnt11 0.05 0.882975 Wnt inhibitors and related NM_013834 Secreted -related protein 1 Sfrp1 .0.001 1.65044 NM_011915 Wnt inhibitory factor 1 Wif1 2.68E-11 3.57431 NM_011356 Frizzled-related protein Frzb ,0.001 2.78872 NM_021339 Cell adhesion molecule–related/downregulated by oncogenes Cdon 6.57E-08 2.6764 Wnt receptors NM_021458 Frizzled homolog 3 (Drosophila) Fzd3 ,0.001 1.29471 NM_008055 Frizzled homolog 4 (Drosophila) Fzd4 .0.007 1.15362 NM_008055 Frizzled homolog 7 (Drosophila) Fzd7 .0.001 1.34737 NM_001008231 associated activator of morphogenesis 2 Daam2 ,0.012 0.861158 Tgfs NM_031199 TGF-a Tgfa 5.28E-05 0.737485 NM_011577 TGF-b1 Tgfb1 0.78 1.01081 NM_009368 TGF-b3 Tgfb3 6.43E-05 1.52369 NM_009370 TGF-b receptor I Tgfbr1 0.72 1.01594 NM_009371 TGF-b receptor II Tgfbr2 0.02 0.875323 NM_011578 TGF-b receptor III Tgfbr3 ,0.003 0.853125 NM_008542 MAD homolog 6 (Drosophila) Smad6 ,0.001 1.20471 NM_001042660 MAD homolog 7 (Drosophila) Smad7 ,0.001 1.22369 Fgfs NM_010206 growth factor receptor 1 Fgfr1 4.12E-05 0.740399 NM_010207 Fibroblast growth factor receptor 2 Fgfr2 ,0.001 1.35853 NM_008010 Fibroblast growth factor receptor 3 Fgfr3 ,0.078 1.09647 NM_008011 Fibroblast growth factor receptor 4 Fgfr4 8.70E-08 0.559123 NM_054071 Fibroblast growth factor receptor-like 1 Fgfrl1 .0.004 1.13961 NM_011896 Sprouty homolog 1 (Drosophila) Spry1 5.09E-06 0.756577 Bmps NM_028472 Bone morphogenetic protein–binding endothelial regulator Bmper .0.002 1.24081 NM_007554 Bone morphogenetic protein 4 Bmp4 .0.001 1.35746 NM_007555 Bone morphogenetic protein 5 Bmp5 ,0.007 1.24253 NM_007557 Bone morphogenetic protein 7 Bmp7 0.01 0.867236 The affymetrix gene microarray platform was used to identify differences. Genes are categorized according to cell type expression or pathways. Complete lists of genes that show at least 1.5-fold difference are shown in Supplemental Tables 2 and 3. consistently developed massive proteinuria, with significant in- conditions, but diabetic podTcf21 mice showed a small decrease crease in protein-to-creatinine ratio to a mean6SEM of in body weight by study end (Supplemental Figure 5). Finally, we 18.6065.36 mg/mg in diabetic podTcf21 mice compared with examined how diabetes affects expression of Tcf21 in wild-type 5.2361.18 mg/mg in nondiabetic podTcf21 mice at 9 weeks of mice. Real-time PCR showed that glomerular Tcf21 expression is diabetes and 26 weeks of age, respectively (P,0.04) (Figure 6A). decreased to 36% in wild-type mice with streptozotocin-induced Diabetic control mice developed only minimal mesangial expan- diabetes (Figure 6E). Together, these data clearly demonstrate that sion; however, kidneys from proteinuric diabetic podTcf21 mice podTcf21 mice are susceptible to diabetic nephropathy. showed dramatic glomerulosclerosis with periodic acid-Schiff– positive nodular structures (Figure 6B). In diabetic podTcf21 mutants, cumulative incidence of proteinuria.10 mg/mg pro- DISCUSSION tein/creatinine was 87.8% at 18 weeks of diabetes (35 weeks of age) versus 22.2% in nondiabetic podTcf21 mice at the same age In recent years, several genetic discoveries have established the (P,0.001) (Figure 6D). Blood glucose did not differ between podocyte as a key player in glomerular health and disease. podTcf21 and control mice in diabetic and nondiabetic Although our understanding of podocyte-expressed cytoskel-

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factors and is highly expressed in podocyte precursors and mature podocytes. Previously we reported a severe defect in renal development in Tcf21 conventional knockout mice.9 Here, we show for the first time that loss of Tcf21 from podocyte pro- genitorsresultsinanarrestofpodocyte differentiation. However, somewhat sur- prisingly, despite persistent high levels of Tcf21 expression throughout podocyte devel- opment, deletion at a slightly later develop- mental time-point in capillary loop glomeruli does not appear to affect differentiation of the complex cytoskeletal architecture of the podocyte. Furthermore, glomerular barrier function remains intact until weaning, when the glomerulus must deal with a substantially increased filtration load. Indeed, in guinea pigs, the GFR increases 7-fold in the first month of life, which is likely similar in mice. In humans, it increases from 30 to 100 ml/min per 1.73 m2 during the first year of life.19–21 Although podTcf21 mice do not dem- onstrate any obvious defects in podocyte differentiation, they do show retardation in glomerular maturation, with fewer mesan- gial and endothelial cells per glomerulus. In the developing glomeruli, podocytes ex- press cytokines and growth factors, includ- ing VEGF-A, that are necessary to recruit endothelial and mesangial precursor cells into the vascular cleft, where they form the glomerular tuft and capillary loops.4,22 Ex- pression profiling and real-time PCR re- sults from podTcf21 glomeruli reveal the downregulation of several key angiogenic factors, including VEGF-A, Pgf, Dll4, Figure 5. Deletion of Tcf21 gene in podocyte progenitors results in arrest of podocyte Hey2, Flt1/Vegfr-1, and soluble Flt1. Inter- differentiation. (A and B) Tcf21 expression assessed by in situ hybridization. Ring-shaped or crescent-shaped podocyte pattern (arrowheads) is absent in wnt4creTcf21 mutants. estingly, all these factors are increased at 3 (C and D) Histology of wnt4creTcf21 glomeruli shows a severely simplified capillary loop weeks of age in glomeruli of control mice and a defect of mesangial ingrowth. (E–H) Podocin expression is severely decreased in compared with day 0, but this upregulation wnt4creTcf21 glomeruli. In controls, Podocin is detected alongside the capillary loops is attenuated in podTcf21 glomeruli, sug- (arrowheads, G) but is also seen at apical and lateral sides in wnt4creTcf21 podocytes gesting that Tcf21 is upstream of an angio- (arrowheads, H). (I and J) Desmin/Cd31 staining shows simplified capillary loops and genic network. mesangial migration defects in wnt4Tcf21 mutant glomeruli. (K–P) Synaptopodin, aPKCl, Another surprising finding in our study and Par3 expression are decreased, but the distribution doesn’t change. (Q and R) In situ was the bimodal penetrance pattern of ’ – hybridization for Nephrin doesn t show any change. (S V) Wt1 and Zo1 stainings are not glomerular injury in podTcf21 mice. De- changed. (W and X) TEM demonstrate a severe defect in podocyte foot process for- spite equivalent excision of the Tcf21 gene mation. A and B, 3200; C and D, G and H, 31000; E and F, K–V, 3400; I and J, 3600; (determined by glomerular mRNA expres- TEM, 39600. sion), approximately 40% of mice devel- oped massive proteinuria around 3–5 etal proteins and growth factors has grown extensively, less is weeks of age, while the remaining 60% appeared protected. known about the transcription factors that control podocyte Furthermore, most of the protected cohort never developed function. Tcf21 belongs to the bHLH family of transcription proteinuria even by 1 year of age. We suspect that this

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Figure 6. Tcf21 is required in aging podocytes and protects mice from diabetic nephropathy. (A) Upper panel: Long-term follow-up of urinary protein-to-creatinine ratios taken from control and protected podTcf21 mice (defined by protein-to-creatinine ratio,10 mg/mg at 8 weeks of age). PodTcf21 mutants show sporadic mild proteinuria after 32 weeks. Lower panel: Controls and podTcf21 mutants without proteinuria at 17 weeks were rendered diabetic by streptozotocin injection. Diabetic podTcf21 mutants develop massive proteinuria. (B) Representative histologic findings of glomeruli from each group at the end of the study. Aged podTcf21 mutants show mild sclerosis (arrowheads, upper right). Diabetic podTcf21 kidneys show severe glomerulosclerosis and nodular lesions (arrowheads, lower right), while control diabetic mice show mild mesangial expansion only (lower left). Diabetic podTcf21 mice show a significant decrease in survival (C) and increase in occurrence of proteinuria (D). (E) Wild-type diabetic mice show reduced expression of Tcf21 in glomeruli. Bars show mean6SEM. *P,0.05 compared with podTcf21; **P,0.01 compared with podTcf21. DM, diabetes mellitus. difference relates to major modifying loci that vary because of reported in biopsy specimens from diabetic patients.23 There- the mixed background strain (C57Bl6, 12931/SvJ, and CD1) fore, we wondered whether glomeruli in nonproteinuric of mice studied. podTcf21 mice were more susceptible to an injury because The different phenotypes observed in podTcf21 versus of diabetes and induced diabetes in a cohort of protected mu- wnt4creTcf21 mice supports an important role of Tcf21 in tants. PodTcf21 mice showed a dramatic increase in diabetic early stages of podocyte differentiation. Tcf21 mRNA was renal injury with a significantly increased incidence of pro- absent from S-shaped body glomeruli in wnt4creTcf21 mice teinuria, glomerular injury, and mortality compared with and from the later capillary loop stage in podTcf21 mice, control diabetic mice. Tcf21 levels are decreased in renal respectively. The excellent podocyte excision (65% at P0 and mRNA samples isolated from kidneys of patients with diabetic 97% by 3 weeks) in podTcf21 mice suggests that the earlier nephropathy.23 In db/db mice, Tcf21 levels are increased dur- timing of excision rather than mosaic excision explains the ing the early phase of diabetic nephropathy but decreased at more severe phenotype observed in wnt4creTcf21 mice. the late stage.24 We also confirmed its decrease in glomeruli Diabetic nephropathy is the leading cause of ESRD in North from diabetic control mice after 10 weeks of diabetes. These America, and alterations in Tcf21 expression have been results suggest that Tcf21 plays a protective role in diabetic

2468 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2459–2470, 2014 www.jasn.org BASIC RESEARCH nephropathy. It is interesting to speculate that Tcf21 protects Immunostaining the podocyte during times of increased glomerular filtration, For snap-frozen sections, dissected kidneys were immediately em- which occurs at the time of weaning and also in early phases of bedded in Tissue-Tek optimal cutting temperature (OCT) 4583 diabetic nephropathy and may occur in other renal diseases. compound (Sakura Finetek, Inc.) after dissection. Some kidney In summary, the current study demonstrates critical roles specimens were also fixed in 4% paraformaldehyde overnight at 4°C, for Tcf21 in developing and mature podocytes in both cryoprotected in 30% sucrose overnight, and then embedded in op- physiologic and disease conditions. Tcf21 works to modulate timal cutting temperature compound. For paraffin sections, kidneys podocyte function and is required when podocytes/glomeruli were fixed in 10% formalin in PBS and embedded in paraffin. Sections are undergoing dynamic differentiation, maturation, and 5 mm thick were cut, rehydrated, and boiled in TEG buffer (10 mM Tris, aging. In addition, our data reveal protective roles of Tcf21 0.5 mM ethylene glycol tetraacetic acid; pH, 9.0) for antigen retrieval. in diabetic nephropathy. Identification of the direct targets of After blocking (10% goat serum, 0.3% Triton X100, 3% albumin in Tcf21 may provide us new therapeutic approaches for PBS), primary antibodies were applied to sections overnight at 4°C. proteinuric renal diseases, including FSGS and diabetic After washing, sections were incubated with secondary antibodies for nephropathy. 1 hour at room temperature.

In Situ Hybridization CONCISE METHODS For in situ hybridization, the samples were fixed in diethylpyrocarbonate- treated 4% paraformaldehyde overnight at 4°C, cryoprotected in Mouse Lines 30% sucrose, embedded in OCT compound, and frozen. Digoxigenin- The floxed Tcf21 targeting vector was created using bacterial artificial conjugated RNA probes were made using a DIG RNA labeling kit (BAC) recombineering, detailed procedures were de- according to the manufacturer’s instruction (Roche). The detailed scribed previously.25 Briefly, a first loxP site was inserted at a non- procedure of in situ hybridization is described elsewhere.27 conserved region before the first exon of the Tcf21 gene. A second loxP site and PGK-neo selection cassette were inserted in a noncon- Isolation of Glomeruli served region of intron 1. ES cell clones were screened using Southern The detailed procedure of glomerular isolation was described pre- blot analysis with probes located outside the homology arms. One viously.28 Briefly, mice were anesthetized and perfused with magnetic correctly targeted ES cell clone was used for ES cell aggregation that microbeads (Dynabeads M-450 tosylactivated; Dynal) from the left produced chimeras and gave germline transmission. ventricle. Kidneys were minced, digested with collagenase A (Roche), The Tcf21-LacZ mouse is a conventional knockout mouse for and filtered through a cell strainer. Glomeruli were then isolated by Tcf21 and was described elsewhere.9 Tcf21-lacZ and Tcf21-floxed using magnetic apparatus (Dynal). mice were bred to the podocin-cre driver strain to generate podocyte fl specific Tcf21 knockout mice (podocin-cre/Tcf21 oxed/LacZ, referred to as podTcf21). The Wnt4-cre driver mouse line is described else- RNA Extraction, Microarray Analysis, and Quantitative where, and the gene excision occurs in renal vesicles, pretubular ag- Real-Time PCR gregates, and stromal cells.17 The Tcf21 floxed and Tcf21-LacZ mice Total RNA was extracted with an RNeasy mini kit (Qiagen), and the fl were bred to wnt4-cre mice, and wnt4-cre/Tcf21 oxed/LacZ mice were quality was evaluated by a 2100 Bioanalyzer (Agilent Technologies). generated (referred to as wnt4creTcf21). Littermates that lack at least Affymetrix Mouse GeneChip 1.0 ST was used for RNA microarray one of the transgenes were used as controls. analysis, which was done in the Center for Applied Genomics (The Additional information on methods is available in the Supple- Hospital for Sick Children, Toronto, ON, Canada). Microarray data mental Material. were analyzed using Genomics Suite software (Partek). Reverse transcription was performed using an iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’sinstruction.cDNAsam- Phenotypic Analysis fi Urine was collected at designated time-points and tested using urine ples were ampli ed by iTaq SYBR Green Supermix (Bio-Rad). Com- dipsticks (Chemstrip 5L; Roche). Additionally, urinary protein parative gene expression was calculated using the delta-delta CT concentration was quantified by Bradford assay. Briefly, the protein method (Applied Biosystems), and described relative to the house- Hprt fi assay dye (500–0006; Bio-Rad) was added to urine samples; after 10 keeping gene, . Speci c primers for the genes are provided in minutes’ incubation, absorbance was measured at 595 nm. Urinary Supplemental Table 1. creatinine concentration was measured by the Jaffe method.26 The results are reported as urinary protein-to-creatinine ratio (mg/mg). Diabetes Mice were euthanized at designated time-points as described, and Seventeen-week-old control mice (n=12) and nonproteinuric micrographs were obtained using a dissection microscope (Leica podTcf21 mice (n=12) were made diabetic by 50 mg/kg intraperito- MZ6). Organs were fixed in 10% formalin in PBS and subjected to neal injection of streptozotocin (Sigma-Aldrich) for 5 days according histologic analysis. For ultrastructural analysis using an electron to the protocol for animal models from the Diabetic Complications microscope, kidneys were fixed in 0.1 M sodium cacodylate buffer Consortium (www.diacomp.org). Mice with blood glucose.20 mM with 2% glutaraldehyde. were used in the experiment.

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Statistical Analyses pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Statistical analyses were performed using a two-tailed t test using Am J Pathol 176: 85–97, 2010 Graphpad Prism. For survival and cumulative occurrence of protein- 11. Kobayashi A, Valerius MT, Mugford JW, Carroll TJ, Self M, Oliver G, McMahon AP: Six2 defines and regulates a multipotent self-renewing uria, log-rank analysis was used. P,0.05 was considered to fi nephron progenitor population throughout mammalian kidney de- represent a statistically signi cant difference. velopment. Cell Stem Cell 3: 169–181, 2008 12. Cui S, Ross A, Stallings N, Parker KL, Capel B, Quaggin SE: Disrupted gonadogenesis and male-to-female sex reversal in Pod1 knockout ACKNOWLEDGMENTS mice. Development 131: 4095–4105, 2004 13. Lu J, Chang P, Richardson JA, Gan L, Weiler H, Olson EN: The basic We are grateful to Ken Harpal and Douglas Holmyard for histology helix-loop-helix transcription factor capsulin controls spleen organo- genesis. Proc Natl Acad Sci USA 97: 9525–9530, 2000 and EM analysis. We thank Chao Lu and staff for help with microarray 14. Lu JR, Bassel-Duby R, Hawkins A, Chang P, Valdez R, Wu H, Gan L, analysis and Dr. Paul Thorner, Mazdak Bagherie, and Antoine Re- Shelton JM, Richardson JA, Olson EN: Control of facial muscle de- ginensi for helpful discussions. This work is supported by Canadian velopment by MyoR and capsulin. Science 298: 2378–2381, 2002 Institutes of Health Research grant 62931 and a Terry Fox grant. 15.NovakA,GuoC,YangW,NagyA,LobeCG:Z/EG,adoublereporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28: 147–155, 2000 16. Miner JH: Renal basement membrane components. Kidney Int 56: DISCLOSURES 2016–2024, 1999 None. 17. 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