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Podocin Inactivation in Mature Kidneys Causes Focal Segmental Glomerulosclerosis and

Ge´raldine Mollet,*† Julien Ratelade,*† Olivia Boyer,*†‡ Andrea Onetti Muda,*§ ʈ Ludivine Morisset,*† Tiphaine Aguirre Lavin,*† David Kitzis,*† Margaret J. Dallman, ʈ Laurence Bugeon, Norbert Hubner,¶ Marie-Claire Gubler,*† Corinne Antignac,*†** and Ernie L. Esquivel*†

*INSERM, U574, Hoˆpital Necker-Enfants Malades, Paris, France; †Faculte´deMe´ decine Rene´ Descartes, Universite´ Paris Descartes, Paris, France; ‡Pediatric Nephrology Department and **Department of Genetics, Hoˆpital Necker- Enfants Malades, Assistance Publique-Hoˆpitaux de Paris, Paris, France; §Department of Pathology, Campus ʈ Biomedico University, Rome, Italy; Department of Biological Sciences, Imperial College London, London, England; and ¶Max-Delbruck Center for Molecular Medicine, Berlin, Germany

ABSTRACT Podocin is a critical component of the glomerular slit diaphragm, and genetic mutations lead to both familial and sporadic forms of steroid-resistant nephrotic syndrome. In mice, constitutive absence of podocin leads to rapidly progressive renal disease characterized by mesangiolysis and/or mesangial sclerosis and nephrotic syndrome. Using established Cre-loxP technology, we inactivated podocin in the adult mouse in a -specific manner. Progressive loss of podocin in the recapitu- lated albuminuria, hypercholesterolemia, hypertension, and renal failure seen in nephrotic syndrome in humans. Lesions of FSGS appeared after 4 wk, with subsequent development of diffuse glomerulosclerosis and tubulointerstitial damage. Interestingly, conditional inactivation of podocin at birth resulted in a gradient of glomerular lesions, including mesangial proliferation, demonstrating a developmental stage dependence of renal histologic patterns of injury. The development of significant albuminuria in this model occurred only after early and focal foot process effacement had progressed to diffuse involvement, with complete absence of podocin immunolabeling at the slit diaphragm. Finally, we identified novel potential mediators and perturbed molecular pathways, including cellular proliferation, in the course of progression of renal disease leading to glomerulosclerosis, using global gene expression profiling.

J Am Soc Nephrol 20: 2181–2189, 2009. doi: 10.1681/ASN.2009040379

FSGS is a clinicopathologic syndrome character- Recent discoveries have established the genetic ized by podocyte injury and progressive scarring bases of some familial forms of FSGS,4–8 as well as in the renal glomerulus. Epidemiologic studies the role of genes in enhancing susceptibility to glo- have shown an increasing incidence of FSGS in the United States,1,2 particularly among black in- Received April 8, 2009. Accepted June 10, 2009. dividuals, making it the most common cause of Published online ahead of print. Publication date available at ESRD as a result of primary glomerular diseases www.jasn.org. 2 in both white and black individuals. Given the J.R., O.B., and A.O.M. contributed equally to this work. rising prevalence of chronic kidney disease,3 ef- Correspondence: Dr. Corinne Antignac, INSERM U574, 6e`me forts to understand the pathophysiologic and ge- e´tage, Tour Lavoisier, Hoˆpital Necker-Enfants Malades, 149 rue netic mechanisms leading to FSGS are crucial for de Se`vres, 75015 Paris, France. Phone: ϩ33-1-4449-4552; Fax: developing strategies aimed at prevention and ϩ33-1-4449-0290; E-mail: [email protected] therapy. Copyright ᮊ 2009 by the American Society of Nephrology

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mice of constitutive absence of podocin or ex- pression of a podocin missense mutant and iden- tified genetic and environmental modifiers of the renal disease.17–19 Podocin mutant mice present with albuminuria at birth and develop lesions of mesangiolysis and mesangial sclerosis. Their early death (within the first few weeks of life, depend- ing on the genetic background17–19) precludes ex- tensive study into the mechanisms of glomerular disease. We therefore generated a novel murine model in which podocin was inactivated in the adult mouse kidney in a podocyte-specific man- ner, using established Cre-loxP technology, and here detail the physiologic, ultrastructural, and transcriptional changes leading to FSGS in this model.

RESULTS

Generation of Podocyte-Specific Nphs2 Knockout Mice To study the effects of podocin inactivation in the mature kidney, we generated triallelic Nphs2lox2/ ϩ Ϫ,Cre mice, bearing a floxed Nphs2 exon 2 allele (Figure 1A), a null Nphs2 allele,17 and a podocyte- expressed, tamoxifen-responsive Cre recombi- nase transgene20 (Supplemental Figures S1 and S2). Mice bearing a null allele in the heterozygous state do not demonstrate glomerular disease17 and neither do Nphs2lox2/lox2 mice, even after 8 wk of follow-up (data not shown). Administration of Figure 1. The Nphs2 gene encoding podocin shows conditional inactivation. tamoxifen to phenotypically normal 6-wk-old (A) The Nphs2 gene was targeted by homologous recombination using a Nphs2lox2/Ϫ, Creϩ mice resulted in nuclear trans- construct in which exon 2 was flanked by loxP sites. The Neo selection cassette location of Cre recombinase in approximately was removed by mating with an Flp deleter strain of mice, and excision of exon 70% of and in excision of exon 2. A 2 by a podocyte-expressed Cre recombinase was achieved on administration of progressive decrease in podocin expression was tamoxifen. (B) Real-time PCR revealed downregulation of podocin mRNA as early as 1 wk after Cre activation, using primers and probe that encompass seen as early as 7 d after Cre induction, both at the exons 2 to 3. Expression levels are shown relative to those in control mice, using mRNA level (shown using real-time PCR; Figure 18S rRNA as a reference gene. (C) Western blotting of total kidney extracts 1B) and at the protein level (demonstrated by revealed progressive decreases in podocin expression at the protein level over Western blotting [Figure 1C] and by quantifica- time. (D and E) Levels of podocin were quantified by measurement of pixel tion of fluorescence signal intensity of podocin in intensity of immunofluorescence labeling of podocin (in red) in glomeruli delin- glomeruli [Figure 1, D and E]). eated using an anti-nidogen antibody (in green) and normalized to glomerular surface area. *P Ͻ 0.0001. Podocin Loss Leads to Nephrotic Syndrome Inactivation of podocin resulted in death of Ϫ ϩ merular disease.9,10 The NPHS2 gene, which encodes the slit Nphs2lox2/ ,Cre mice at a median time of 11 wk after the start diaphragm protein podocin, not only accounts for 43% of fa- of tamoxifen administration (Figure 2A). Albuminuria was de- milial and 10% of sporadic forms of nephrotic syndrome, but tected on a Coomassie blue–stained protein gel (Figure 2B) also some genetic variants may increase the risk for glomerular after a mean of 10 d (range 8 to 13 d). This progressed to disease.11,12 Podocin acts as a structural scaffold in podocyte massive, nonselective proteinuria by 4 wk (Figure 2B). Tail- foot processes and interacts with slit diaphragm proteins to cuff plethysmography measurements, carried out 4 wk after facilitate cellular signaling events.13–16 podocin inactivation, showed a modest but significant increase Previously, we described the phenotypic consequences in in the BP of null mice (Figure 2C). Finally, plasma levels of

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consistently present only 6 wk after Cre recombi- nase induction. Mice that were killed after 9 wk demonstrated progression of tubular injury, with basement membrane thickening and interstitial fibrosis (Figure 3F).

Developmental Stage Effects on Renal Disease Given the discrepancy between the renal pheno- types resulting from constitutive and conditional inactivation of podocin, we investigated the effects of Nphs2 inactivation at birth, during which nephrogenesis is not completed in mice. After ad- ministration of tamoxifen to nursing mothers for 3 d after birth, resulting in tamoxifen delivery via Ϫ milk,21,22 albuminuria developed in Nphs2lox2/ , Creϩ pups after 2 wk (data not shown). When mas- sive proteinuria was present at 4 wk by Coomassie blue staining, mice were killed. The renal histology showed a gradient of lesions. Superficial outer cor- tical glomeruli, which were not present at the time Figure 2. Conditional Nphs2 knockout mice have various phenotypes. (A) of birth, were normal and podocin expression was Ϫ Kaplan-Meier survival curve of control and Nphs2lox2/ mice upon Cre activation intact (Figure 4, A and B). In midcortical and the by tamoxifen shows median survival of 11 wk upon podocin loss. (B) Mice majority of juxtamedullary glomeruli, in which Cre develop detectable albuminuria at 2 wk by Coomassie staining of 12% SDS- recombinase was active and podocin was dimin- PAGE. This progresses to massive nonspecific proteinuria by 4 wk. (C) Tail-cuff ished (Figure 4A), lesions of mesangial proliferation Ϫ plethysmography of Nphs2lox2/ mice 4 wk after Cre activation shows mild were observed (Figure 4B). In addition, in the most lox2/Ϫ hypertension. Experiments were performed on 12 control and 12 Nphs2 severely affected juxtamedullary glomeruli, podocin Ͻ mice. §P 0.05 after two-tailed t test. (D) Elevations in serum levels of choles- was absent (Figure 4A) and lesions of FSGS were terol 4 wk after podocin loss precede increases in serum urea and creatinine. present (Figure 4B). Control values at all time points were unchanged and were pooled. Data are means Ϯ SEM, with at least five mice in each group. #P Ͻ 0.001 by one-way Ultrastructural Studies ANOVA. We performed serial ultrastructural studies in cholesterol were significantly elevated by 4 wk, whereas plasma adult Nphs2lox2/Ϫ,Creϩ mice, after Cre induction, to under- urea and creatinine levels began to rise only at 6 wk (Figure stand the role of foot process effacement (FPE) in the develop- 2D). ment of proteinuria. Whereas foot processes in control mice were evenly spaced and separated by a slit diaphragm (Figure Ϫ ϩ Mice Develop FSGS and Tubular Damage 5A), foot processes in Nphs2lox2/ ,Cre mice were focally ef- Despite downregulation of podocin expression, the renal his- faced at 1 and 2 wk after Cre induction (Figure 5, B and C). tology of Nphs2lox2/Ϫ,Creϩ mice at the light microscopic level 1 Morphometric measurements revealed a near doubling of the wk (data not shown) after induction of Cre recombinase activ- mean foot process width as early as 1 wk (Figure 5E). The ity was similar to those of control (Nphs2lox2/Ϫ,CreϪ and degree of FPE progressed to diffuse effacement 4 wk (Figure 5D) Nphs2lox2/ϩ,Creϩ) mice (Figure 3A). After 2 wk, occasional after tamoxifen administration. Progression of FPE corresponded podocyte hypertrophy was noted, with minimal mesangial ma- to the worsening of albumin leak, eventually to massive, nonse- trix expansion in some glomeruli (Figure 3B). Four weeks after lective proteinuria. Contrary to the findings of others implicating Cre induction, FSGS was observed in many glomeruli, with podocyte detachment and subsequent glomerular basement varying degrees of severity (Figure 3C). Glomerular pseudo- membrane (GBM) denudation as a mechanism of glomerular crescents were observed in at least 30% of glomeruli beginning sclerosis,23 ultrastructural studies did not reveal areas of denuded at 4 wk (Figure 3D). GBM in Nphs2lox2/Ϫ,Creϩ mice at any of the time points investi- Glomerulosclerosis continued to worsen by 6 wk, with in- gated. creasing segmental involvement (Figure 3E). At or near the Using immunogold electron microscopic labeling, we local- time of death, global sclerosis was evident in the majority of ized podocin at the slit diaphragm in controls (Figure 6A) and glomeruli (Figure 3F). Tubulointerstitial injury characterized in areas where foot processes were maintained in Nphs2lox2/Ϫ, by diffuse tubular dilation, tubular atrophy and necrosis, and Creϩ mice at 1 and 2 wk (Figure 6B). No labeling was seen in the presence of proteinaceous casts (Figure 3, E and F) was parietal epithelial, endothelial, or mesangial cells. In some ar-

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S2) and canonical pathway perturbations (Supple- mental Table S3). Functional annotation, using the Ingenuity Pathway Analysis Program, showed early and significant perturbations of cell-cycle regulation and cellular proliferation pathways. Proliferation of resident cells in the glomerulus during the develop- ment of glomerulosclerosis has been inconsistently demonstrated in humans and animal models. In Nphs2lox2/Ϫ,Creϩ mice, immunostaining for Ki-67 identified a significant increase in the percentage of proliferating resident glomerular cells (Figure 7A), involving podocytes and endothelial and mesangial cells (Figure 7B). Immunostaining for CD133 and CD24, markers of progenitor cells, were negative, suggesting that proliferative cells were intrinsic to the glomerulus (data not shown).

DISCUSSION

Animal models of podocyte injury, using toxic agents, immunologic damage, renal ablation, gene targeting, and transgenesis or via expression of HIV component proteins, have offered tre- mendous insights into the pathophysiologic mechanisms leading to glomerulosclerosis.24 We chose to develop murine models of podocyte in- jury on the basis of the targeted inactivation of the podocin gene, mutations in which account for both familial and sporadic cases of steroid-resis- tant nephrotic syndrome.5,11,25 In addition, dys- Figure 3. Renal lesions progress upon Nphs2 inactivation in the mature kidney. regulation of podocin expression was described in (A) Control mice show normal glomerular and tubular morphologies. (B) At 2 wk, an experimental model of membranous nephrop- there were no significant glomerular changes besides mild glomerular enlarge- athy,26 in FSGS,27 and in other forms of acquired ment and occasional podocyte hypertrophy. (C) There is segmental accumula- 28,29 tion of collagen (arrow) in many glomeruli 4 wk after Cre induction. (D) In at least proteinuric diseases, therein highlighting its 30% of glomeruli starting at 4 wk, pseudocrescents were observed. (E) There is pivotal role in the pathogenesis of glomerular dis- progression of lesions of FSGS 6 wk later, accompanied by tubular dilation and ease development. protein casts. (F) At 9 wk, diffuse global sclerosis was present, along with marked The early demise of two previous mouse mod- tubular dilation, atrophy, and interstitial fibrosis. Bars ϭ 50 ␮m. Slides were els of podocin inactivation and the presence of stained with periodic acid-Schiff stain. Magnification: ϫ400 in A, B, C, and E; histologic lesions of mesangiolysis and mesangial ϫ1000 in D and F. sclerosis, which are not characteristic of human disease because of podocin mutations, impelled eas of focal effacement at 1 and 2 wk, gold particles were dis- us to generate a model of podocin inactivation in the mature placed within the cytoplasm of effaced podocytes, sometimes kidney. Using an established Cre deleter line,20 expression and clustering along the plasma membrane facing the urinary space nuclear translocation of Cre recombinase in approximately (Figure 6C). Only after 4 wk was podocin completely absent in 70% of podocytes resulted in a 50% downregulation of podo- diffusely effaced foot processes (Figure 6D). cin expression as early as 1 wk after induction. By 4 wk, podo- cin was nearly absent in all glomeruli, suggesting that podocin Global Gene Expression Profiling of Mutant Glomeruli loss was secondarily achieved through a vicious cycle of dam- To identify molecular pathways perturbed upon podocin loss, aged podocytes’ damaging neighboring podocytes.30,31 This we performed global gene expression profiling of glomeruli may explain immunogold labeling studies showing displaced isolated from mutant and control mice at weeks 1, 2, and 4. We podocin in some focally effaced foot processes, reflecting validated microarray results using quantitative real-time PCR podocytes in which Cre recombinase was not activated. Alter- (Supplemental Figure S3; Supplemental Table S1) and revealed a natively, these particles may represent residual podocin in the complex array of transcriptional changes (Supplemental Table process of cytosolic degradation. Indeed, failure of podocin

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Figure 4. Inactivation at birth reveals devel- opmental stage dependence of phenotypes. (A) At 4 wk of life, podocin expression (in red) demonstrated a gradient from the outer cor- tical (OC) to midcortical (MC) and to jux- tamedullary (JM) glomeruli, marked with ni- dogen (in green). Because OC glomeruli are not formed at birth, podocin expression is intact, whereas MC glomeruli, which are im- mature at birth, have decreased podocin ex- pression. At birth, JM glomeruli are mature and show near absence of podocin. (B) His- tologic analysis showed normal OC glomer- uli, glomerular hypertrophy and lesions of mesangial proliferation in MC glomeruli and lesions of FSGS in severely affected JM glo- meruli. Slides were stained with periodic acid- Schiff. Magnification, ϫ400. targeting to sites of action on the plasma membrane has been shown both in vitro and in vivo to result in loss of func- tion.18,32,33 Podocyte injury in our model leads to progressive renal disease, ultimately recapitulating features of nephrotic syn- drome in humans, such as hyperlipidemia, hypertension, and renal insufficiency. Heterogeneity between mutant mice in the evolution of renal disease may partly be due to individual differences resulting from their underlying mixed genetic background. Less phenotypic variability has been seen in ongoing studies on congenic mice (C.A., un- published observations). In contrast to our previously described podocin knockout model in which foot processes are effaced at birth,17 podocyte FPE evolved from focal to diffuse involvement with inactiva- tion in the mature kidney; however, despite the presence of focally effaced foot processes at 1 wk, we found no significant albuminuria until the second week. The absence of albumin- uria at 1 wk may reflect the limits of detection of our assay or, alternatively, may represent a time when the capacity of com- pensatory proximal tubular reabsorption of albumin34 has not yet been saturated. Studies to investigate the tubular mechanisms of albumin handling in these mice are ongoing. Our findings contrast with models in which proteinuria has been described in the absence of FPE35 but are in agreement with two previous studies conducted in human samples36 and in the puromycin aminoglycoside nephrosis model37 showing absence of proteinuria despite extensive FPE. Finally, the ap- Figure 5. FPE develops upon podocin loss. (A) Ultrastructural pearance of massive, nonselective albuminuria coincided in studies in a control mouse revealed elaboration of podocytes (P) our study with diffuse FPE, but we cannot exclude concomi- into regularly spaced foot processes (FP), on the opposite side of tant changes in GBM composition, although no gross abnor- the GBM as the fenestrated endothelial cells (En) lining the cap- illary lumen (L). (B and C) At 1 (B) and 2 wk (C) after Cre induction, malities in the GBM were noted by electron microscopy. FPE was focal. (D) After 4 wk, foot processes were globally ef- Our results demonstrate that the evolution of renal pheno- faced. (E) Morphometric studies revealed progressive increase in types of mice in which podocin has been inactivated in the foot process width over time. Calculations are based on 15 cap- mature kidney is different from those of the previous Nphs2 illary loops each in three mice at each time point and compared models in which functional podocin was absent during kidney with five control mice. Magnification, ϫ15,000. *P Ͻ 0.0001. development. Rather than displaying lesions of mesangiolysis

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ing from congenital to late-onset disease has been described in NPHS2 cases, but renal bi- opsy data have been limited. Contrary to mice, in which nephrogenesis continues until the sec- ond week of life, nephrogenesis in humans is complete at birth. One could speculate that cer- tain podocin mutations, however, may exert deleterious effects that affect developmental pathways in the renal glomerulus. A deeper un- derstanding of the role of podocin in intraglo- merular cross-talk is necessary to clarify whether a developmental stage dependence of renal disease evolution in nephrotic syndrome is of clinical relevance in humans. The transcriptional changes occurring during the course of glomerular disease development are Figure 6. Podocin is immunolocalization with progressive renal disease. (A) poorly understood. We used microarrays to re- Immunogold labeling of podocin shows discrete localization to the slit dia- veal the perturbations in gene expression in iso- phragms bridging foot processes (FP) in control mice. (B) At 1 wk, podocin lated glomeruli at various time points after podo- remained localized mostly to the slit diaphragms. (C) At 2 wk, podocin was cin inactivation and found a molecular signature localized mostly to cytosolic areas of podocytes (P), especially in areas of focal unique from other, previously published models FP effacement. (D) Podocin expression was essentially absent in areas of dif- of FSGS,43–45 thus, confirming the heterogeneity fusely effaced FP. No podocin expression was observed in glomerular endothe- of FSGS depending on the mechanism by which ϫ lial cells (En) lining the capillary lumen (L). Magnifications: 25,000 in A through podocyte injury is incurred. ϫ C; 15,000 in D. Intriguingly, we found that genes involved and mesangial sclerosis seen in Nphs2 knockout or the in cell-cycle regulation were the earliest genes to be tran- Ϫ ϩ Nphs2R140Q/R140Q mutant mice,17,18 Nphs2lox2/ ,Cre mice de- scriptionally activated in our model. Several other mouse veloped lesions of FSGS in the setting of nephrotic-range pro- models of glomerular disease failed to reveal a prominent teinuria, recapitulating the most common lesion found in re- role of proliferation of resident cells of the glomerulus, be- nal biopsies of patients bearing NPHS2 mutations.5,38 When sides parietal epithelial cells.46–48 Conversely, we found inactivation was carried out at birth, during which nephrons proliferation not only of podocytes, as seen in models of are continuing to form and mature, the spectrum of lesions HIV-associated nephropathy,49 but also of mesangial and included mesangial proliferation in the intermediate zone of endothelial cells. This may represent dedifferentiation of the renal cortex. These data suggest that podocin loss disrupts terminally differentiated cells in the glomerulus in response podocyte–mesangial cell–endothelial cell cross-talk in the glo- to podocyte injury in our model. It will be interesting to merulus differently in mature and in developing glomeruli. determine whether cellular proliferation is a compensatory The importance of cross-talk in glomerular assembly and mat- or a deleterious event in glomerulosclerosis. uration, mediated by the vascular endothelial growth factor Conditional inactivation of podocin in the adult murine pathway, for instance, has been highlighted by recent stud- kidney represents a novel model system of nephrotic syndrome ies.39,40 The developmental stage dependence of cystic renal as a result of FSGS, thereby recapitulating human disease re- disease evolution has similarly been elucidated in a Pkd1 sulting from mutations in the podocin gene and providing a knockout mouse model.21 vehicle for better understanding key pathophysiologic mecha- In humans, the age of onset of renal disease as a result of nisms of glomerular disease development. Furthermore, it re- podocin mutations is broad, and although minimal change vealed important phenotypic differences from previous consti- and FSGS are the predominant histologic lesions, mesangial tutive knockout models of podocin, therein highlighting the lesions have likewise been reported.38 It is noteworthy that importance of conditional targeting of other genes implicated the predominant renal biopsy findings in patients who bear in nephrotic syndrome. mutations in the NPHS1 gene, which encodes , and largely present with nephrotic syndrome at birth are lesions of mesangial hypercellularity with varying degrees of mes- CONCISE METHODS angial sclerosis.41 Recently, we identified a cohort of pa- tients who bear compound heterozygous NPHS1 mutations Generation of Mice and present with later onset nephrotic syndrome (mean 3 The Nphs2 mutant mouse line was established at the Mouse Clinical yr), in whom renal biopsy, by contrast, revealed minimal- Institute–Institut Clinique de la Souris (http://www.mci.u-strasbg.fr; change nephropathy and FSGS.42 A similar spectrum rang- Illkirch, France). The mouse genome was modified to allow for exci-

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Phenotypic Characterization Survival was assessed by following a cohort of mice until death. There- after, five control and five null mice were killed at weeks 1, 2, 4, and 6 after tamoxifen administration. Blood was obtained via cardiac punc- ture, centrifuged at 5000 rpm for 5 min to obtain plasma, and blood urea nitrogen, creatinine, and cholesterol were measured using an Olympus AU-400 multiparametric analyzer. Time of onset of albu- minuria was determined by daily collection of spontaneously voided urine after initiation of tamoxifen induction, and 2 ␮l of urine was examined on a 12% SDS-PAGE gel stained with Coomassie blue. One kidney was extracted and snap-frozen in liquid nitrogen for protein and RNA analysis. When podocin was inactivated at birth, pups were genotyped at day 5 and albuminuria was tested thereafter. Litters were killed at 4 wk and characterized. Four weeks after initiation of tamoxifen administration, mice weighing 20 to 25 g were habituated to tail-cuff plethysmography over a 2-d period. Thereafter, repeated BP measurements (at least 10 per session per mouse) were obtained over 3 d, during which the arterial pulse was detected by a piezoelectric detector. Signal acquisition and processing were performed by PowerLab 4sp instruments using Chart 4.1.1 (ADInstruments, Spechbach, Germany). Kidneys fixed in alco- holic Bouin solution were paraffin-embedded, and 3-␮-thick sections were stained with periodic acid-Schiff and Masson-Trichrome.

Protein and RNA Analysis See supplemental data for detailed protocols on Western blotting, immunofluorescence, real-time PCR, and microarray analysis.

Figure 7. Cell proliferation in resident glomerular cells with Transmission and Immunogold Electron Microscopy progressive glomerulosclerosis is shown. (A) Quantification of Methods for tissue preparation for electron microscopy may be found Ki-67–positive cells in 10 glomeruli each in two control and three in supplemental data. Electron micrographs were obtained using a null mice at each time point showed an increase in proliferating Jeol JEM-100CX II transmission electron microscope. Eight to 10 cells as early as week 1 and progressing until week 4, with a photographs, covering at least 15 open random capillary loops in subsequent decrease at 6 wk. $P Ͻ 0.01; *P Ͻ 0.0001. (B) Immu- several glomeruli per mouse were taken; negatives were digitized, and lox2/Ϫ ϩ nofluorescence labeling in a representative Nphs2 ,Cre images with a final magnification of ϫ15,000 were obtained. With the mouse after 4 wk demonstrated proliferation of all resident cells in use of ImageJ software (National Institutes of Health; http://rsbweb.nih. the glomerulus, including podocytes (labeled with anti-nephrin gov/ij/features.html), the length of the peripheral GBM was measured; Ab, red), mesangial cells (labeled with anti-desmin Ab, red), and the number of slit pores overlying this GBM length was then manually endothelial cells (labeled with anti-CD31 Ab, red). Proliferating counted. The average foot process width, expressed in microns, was cal- cells were labeled with anti–Ki-67 Ab (green) and nuclei with Topro 3 (blue). Magnification, ϫ400. culated by dividing the total GBM length measured in the single glomer- ulus by the total number of slits counted. The value obtained was multi- ␲ sion of exon 2 of the Nphs2 gene upon tamoxifen administration. See plied by /4, a correction factor for the random orientation in which the supplemental data for further details regarding construct generation foot processes were sectioned.50 and mating scheme. Except for studies on neonatal mice, experiments were performed on 6-wk-old male and female triallellic Nphs2lox2/Ϫ, Statistical Analysis ϩ ϩ ϩ ϩ Ϯ Cre mice and their littermate controls: Nphs2lox2/ ,Cre , Nphs2lox2/ , All data are presented as means SEM and compared using two- CreϪ, or Nphs2lox2/Ϫ,CreϪ. No gender effects were seen. Mice were of tailed t test or Mann-Whitney test for independent samples, using Ͻ mixed genetic background. Cre recombinase was induced by intra- GraphPad Prism. P 0.05 was considered statistically significant. peritoneal administration of tamoxifen (33 mg/kg per d for 5 d; Sigma, Steinheim, Germany). In nursing mothers, 5 mg of tamoxifen was administered intraperitoneally during the first 3 d after delivery. ACKNOWLEDGMENTS Mice were maintained in a pathogen-free environment, and experi- ments were conducted in accordance with French government poli- This work was supported by grants from the EuReGene Network, an cies (Services Ve´te´rinaires de la Sante´et de la Production Animale, integrated project (5085) of the 6th Framework of the European Com- Ministe`re de l’Agriculture). mission (C.A.), GIS-Institut des Maladies Rares (C.A.), Agence Nationale

J Am Soc Nephrol 20: 2181–2189, 2009 Conditional Podocin Loss and FSGS 2187 BASIC RESEARCH www.jasn.org de la Recherche (C.A. and E.L.E.), and Genzyme Renal Innovations Pro- Daskalakis N, Kwan SY, Ebersviller S, Burchette JL, Pericak-Vance MA, gram (C.A. and E.L.E.). E.L.E. received a National Institutes of Health/ Howell DN, Vance JM, Rosenberg PB: A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science National Institute of Diabetes and Digestive and Kidney Diseases Ruth 308: 1801–1804, 2005 Kirchstein National Research Service Award (DK065409). In addition, 9. Kim JM, Wu H, Green G, Winkler CA, Kopp JB, Miner JH, Unanue ER, grants from the Ministe`re de l’Enseignement Supe´rieur et de la Re- Shaw AS: CD2-associated protein haploinsufficiency is linked to glo- cherche and the Fondation de la Recherche Me´dicale provided PhD merular disease susceptibility. Science 300: 1298–1300, 2003 thesis and master’s thesis support to J.R. and O.B., respectively. The 10. Kopp JB, Smith MW, Nelson GW, Johnson RC, Freedman BI, Bowden DW, Oleksyk T, McKenzie LM, Kajiyama H, Ahuja TS, Berns JS, Briggs mutant mouse line was established at the Mouse Clinical Institute W, Cho ME, Dart RA, Kimmel PL, Korbet SM, Michel DM, Mokrzycki (Institut Clinique de la Souris, Illkirch, France) in the Targeted MH, Schelling JR, Simon E, Trachtman H, Vlahov D, Winkler CA: MYH9 Mutagenesis and Transgenesis Department. is a major-effect risk gene for focal segmental glomerulosclerosis. Nat We thank Viviane Beau at INSERM U574 and Nicholas Sorhaindo Genet 40: 1175–1184, 2008 and Martine Muffat-Joly at Hoˆpital Bichat (IFR2-Laboratoire de Bio- 11. Machuca E, Hummel A, Nevo F, Dantal J, Martinez F, Al-Sabban E, Baudouin V, Abel L, Grunfeld JP, Antignac C: Clinical and epidemio- chimie, Paris, France) for mouse genotyping and biochemical, and BP logical assessment of steroid-resistant nephrotic syndrome associated measurements; Me´riem Garfa (Plateau d’imagerie cellulaire, IRNEM, with the NPHS2 R229Q variant. Kidney Int 75: 727–735, 2009 Paris, France) for confocal microscopy and Metamorph analysis; and 12. McKenzie LM, Hendrickson SL, Briggs WA, Dart RA, Korbet SM, Mokr- members of the Animal Facility at Hoˆpital Necker/IRNEM, Paris, for zycki MH, Kimmel PL, Ahuja TS, Berns JS, Simon EE, Smith MC, Tracht- the care and maintenance of the mice used for this study. man H, Michel DM, Schelling JR, Cho M, Zhou YC, Binns-Roemer E, Kirk GD, Kopp JB, Winkler CA: NPHS2 variation in sporadic focal segmental glomerulosclerosis. J Am Soc Nephrol 18: 2987–2995, 2007 13. 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