Nephrin Preserves Podocyte Viability and Glomerular Structure and Function in Adult Kidneys

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† ‡ | Xuezhu Li,* Peter Y. Chuang,* Vivette D. D’Agati, Yan Dai,*§ Rabi Yacoub,* Jia Fu, †† Jin Xu,* Oltjon Taku,¶ Prem K. Premsrirut,** Lawrence B. Holzman, and ‡‡ John Cijiang He*

*Department of Medicine/Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York; †Department of Nephrology; Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China; ‡Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York; §Department of Nephrology, Shanghai First Municipal Hospital, Shanghai Jiaotao University School of Medicine; Shanghai, China; |Research Institute of Nephrology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China; ¶State University of New York at University at Binghamton, Binghamton, New York; **Mirimus, Inc. Cold Spring Harbor, New York; ††Renal Electrolyte and Hypertension Division, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania; and ‡‡Renal Section, James J Peter Veterans Administration Medical Center, Bronx, New York

ABSTRACT Nephrin is required during kidney development for the maturation of podocytes and formation of the slit diaphragm junctional complex. Because nephrin expression is downregulated in acquired glomerular diseases, nephrin deficiency is considered a pathologic feature of glomerular injury. However, whether nephrin deficiency exacerbates glomerular injury in glomerular diseases has not been experimentally confirmed. Here, we generated mice with inducible RNA interference–mediated nephrin knockdown. Short-term nephrin knockdown (6 weeks), starting after the completion of kidney development at 5 weeks of age, did not affect glomerular structure or function. In contrast, mice with long-term nephrin knockdown (20 weeks) developed mild proteinuria, foot process effacement, filtration slit narrowing, mesangial hypercellularity and sclerosis, glomerular basement membrane thickening, subendothelial zone widening, and podocyte apoptosis. When subjected to an acquired glomerular insult induced by unilateral nephrectomy or doxorubicin, mice with short-term nephrin knockdown developed more severe glomerular injury compared with mice without nephrin knockdown. Additionally, nephrin-knockdown mice developed more exaggerated glomerular en- largement when subjected to unilateral nephrectomy and more podocyte apoptosis and depletion after doxorubicin challenge. AKT phosphorylation, which is a slit diaphragm–mediated and nephrin-dependent pathway in the podocyte, was markedly reduced in mice with long-term or short-term nephrin knockdown challenged with uninephrectomy or doxorubicin. Taken together, our data establish that under the basal condition and in acquired glomerular diseases, nephrin is required to maintain slit diaphragm integrity and slit diaphragm–mediated signaling to preserve glomerular function and podocyte viability in adult mice.

J Am Soc Nephrol 26: 2361–2377, 2015. doi: 10.1681/ASN.2014040405

Nephrin is considered the molecular building block Received April 24, 2014. Accepted November 19, 2014. of the slit diaphragm.1 Nephrin bridges the ﬁltra- X.L. and P.Y.C. contributed equally to this work. tion slit to form a 35-nm-long globular cross- Published online ahead of print. Publication date available at strand that contributes to a porous slit diagram www.jasn.org. scaffold.2 It also serves as a signaling hub3,4 by in- Correspondence: Dr. Peter Y. Chuang, Department of Medicine, 5,6 teracting with other slit diagram proteins. How- Division of Nephrology, Icahn School of Medicine at Mount Sinai, ever, all ascribed functions of nephrin were derived One Gustave L Levy Place, Box 1243, New York, NY 10029. from studies of cultured podocytes or mice with Email: [email protected] inherited nephrin deﬁciency.7,8 The function of Copyright © 2015 by the American Society of Nephrology

J Am Soc Nephrol 26: 2361–2377, 2015 ISSN : 1046-6673/2610-2361 2361 BASIC RESEARCH www.jasn.org nephrin and the effect of acquired nephrin loss in the adult RESULTS mice are not known. Nephrin is required for podocyte maturation during glomer- Murine Model of Inducible Nephrin Knockdown ular development.9 In humans and mice with inherited nephrin Wegenerated transgenic mice with inducible shRNA-mediated deficiency due to loss-of-function mutations of NPHS110 or Nphs1 knockdown using an in vivo RNA interference ap- 2 2 Nphs1 deletion (Nphs1 / ),7,8 massive proteinuria develops in proach.30,31 Three different lines of mice encoding an utero or soon after birth. A series of elegant studies from the miR30-based expression cassette under the transcriptional laboratories of Holzman,11,12 Quaggin,13 and Holthofer14,15 control of a doxycycline (DOX)-responsive promoter element highlighted the function of nephrin in rodents. In humans, were produced. Each line harbors a different 21-bp Nphs1 nephrin is downregulated in many forms of acquired glomerular guide sequence. These three lines are called sh1401, sh1416, diseases, such as diabetic nephropathy,16–19 minimal-change and sh3703. The DOX-responsive transgene, TGM,wascon- disease,20–22 FSGS,23 and membranous nephropathy,21,23,24 figured with a GFP "spacer" between the tetracycline-responsive and others.24,25 It has been suggested that nephrin deficiency causes promoter element and the miR30-based expression cassette podocyte injury in vivo, but this has not been directly confirmed. (Supplemental Figure 1A). Mice with a single copy of TGM Development of glomerular dysfunction in rodents injected with were generated and then bred with mice that possess widespread an antinephrin antibody,26,27 as well as recurrence of nephropathy expression of reverse tetracycline transactivator (rtTA), hereafter due to alloimmunization against nephrin after kidney transplanta- referred to as CAGs-rtTA mice,30 to produce double transgenic tion in individuals with congenital nephropathy of the Finnish CAGs-rtTA/+; TGM/+ mice. These double transgenic mice were type,28,29 suggests that antinephrin antibody is detrimental to glo- born at the expected Mendelian ratio and were indistinguish- merular function. The mechanism of antinephrin antibody causing able from wild-type mice and singly transgenic mice harbor- glomerular dysfunction in these cases, however, is not known.26 ing the CAGs-rtTA or TGM transgene. Here we produced several lines of genetically engineered Murine kidney development is completed between post- mice with inducible nephrin knockdown mediated by short natal days 7 and 8.32 Induction of nephrin knockdown started hairpin RNA (shRNA). We sought to directly characterize at 5 weeks of age with DOX-supplemented chow (650 mg/kg). nephrin function byexamining adult micewithacquired rather The nephrin protein and mRNA levels of 11-week-old mice than inherited nephrin loss. induced with DOX for 6 weeks were significantly lower in all

Figure 1. A murine model of inducible nephrin knockdown. An inducible knockdown of nephrin is robust and sustainable up to 20 weeks of induction. Inducible nephrin knockdown mice (sh3703) and control Luci.1309 mice with inducible overexpression of an shRNA against the firefly luciferase gene received DOX in the chow (625 mg/kg) starting at 5 weeks of age and continued for 20 weeks until 25 weeks of age. (A) Representative Western blots of nephrin in kidney protein lysates of sh3703 and Luci.1309 mice that received DOX for 0, 1, 3, 6, and 20 weeks. A bar graph of normalized ratios of Western band densities of nephrin/b-actin for the Western blots shown. (B) Nephrin mRNA transcript levels of sh3703 mice relative to age-matched, DOX-fed Luci.1309 mice in the kidney. Each point on the graph contains samples from at least three mice. ***P,0.001 compared with matched Luci.1309 mice. (C) Nephrin immunostaining and GFP fluorescence on frozen kidney sections of mice after 0, 1, 3, 6, and 20 weeks of induction. Scale bars, 50 mm. Error bars, SEM.

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Because sh3703 mice have the most robust knockdown, they were used in all subse- quent experiments. Robust knockdown was observed after 3 weeks of DOX induction and was sustainable for up to 20 weeks of induction (Figure 1A). Nephrin expression of age- matched sh3703 mice without DOX feeding (sh3703 No DOX_20w) did not differ from that of control Luci.1309 mice with and without DOX induction for 20 weeks (DOX_20w and No DOX_20w) and was markedly higher than that of sh3703 mice with DOX induction (Supplemental Figure 1D). sh3703 mice that were induced with a higher dose of DOX (650 mg/kg DOX food plus 2 mg/ml DOX in the drinking water [DOX food+H2O_20w]) had a degree of nephrin knockdown similar to that in those induced with only 650 mg/kg DOX food (DOX_20w) (Supplemental Figure 1D). The kinetics of nephrin knockdown followed a biphasic pattern of early, rapid decay followed by a progressive decline (Figure 1A). This biphasic pattern is consistent with the thought that nephrin is an in- tegral transmembrane protein involved in stable trans interaction with adjacent foot processes and thus is not readily turned over under the basal condition. Maximal knockdown appeared to plateau at approximately 80%–90% for both nephrin protein and mRNA transcript, suggesting that the shRNA-mediated knockdown in our system is robust but not complete. Immunostain- ing of nephrin confirmed that glomerular nephrin expression was markedly reduced in GFP-positive glomerular cells of sh3703 mice but not Luci.1309 mice (Figure 1C). These GFP-positive glomerular cells are pre- dominantly podocytes and not mesangial or 31 Figure 1. Continued. endothelial cells. Western blotting using an antibody against the intracellular domain of neph- three lines of nephrin-knockdown mice than in control rin revealed both of the nephrin-specific bands (180 kD and Luci.1309 mice, which express an shRNA targeting the firefly 160 kD) in the kidney cortex lysate. Consistent with previous luciferase gene30 (Supplemental Figure 1, B and C). Two West- reports, we also identified the 160-kDa nephrin band in ern bands specific to nephrin with apparent molecular masses extrarenal organs: heart, spleen, pancreas, and brain (Supple- of 160 kD and 180 kD have been described.33 The 180-kD mental Figure 1E).7,34,35 The exclusive presence of the 180-kD band corresponds to N-glycosylated nephrin.33 Green fluores- band in the kidney lysate has been described previously.35 Be- cent protein (GFP) expression was similar between Luci.1309 cause DOX does not cross the blood-brain barrier, GFP and and sh3703 mice. The sh3703 line demonstrated the most ro- nephrin expressions did not different between mice with and bust knockdown. Nephrin expression in the renal cortex of without induction. Histologic features of the myocardium did sh3703 mice was knocked down by approximately 80% com- not appear to be different between sh3703 and Luci.1309 mice pared with control Luci.1309 mice after 6 weeks of induction. (Supplemental Figure 1F).

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Long-term, but Not Short-term, Nephrin Knockdown in Long-term Nephrin Depletion Disrupts All Adult Mice Promotes Proteinuria Components of the Glomerular Tuft sh3703 mice fed with DOX starting at 5 weeks of age developed After 20 weeks of induction, the glomerular cross-sectional more proteinuria than did control Luci.1309 mice after 24 area of sh3703 mice was enlarged (Figure 3, A and B). In con- weeks of induction (Figure 2A). In contrast, age-matched trast, the glomerular structure and glomerular cross-sectional Luci.1309 mice with DOX induction did not develop marked area of Luci.1309 kidney at up to 20 weeks of induction and proteinuria. The urinary albumin excretion in sh3703 mice sh3703 kidney at up to 6 weeks of induction appeared normal was approximately 3-fold higher than that in Luci.1309 mice (Figure 3, A and B). Glomeruli of age-matched sh3703 mice after 24 weeks of induction (mean6SD, 0.17960.041 versus without DOX induction appeared normal (Supplemental Fig- 0.05260.014 mg of albumin per mg of creatinine; P,0.05). ure 1G), thus ruling out any off-target effects or leakiness of The serum creatinine of sh3703 and Luci.1309 mice did not shRNA as the cause of glomerular changes in DOX-induced differ before and after 20 weeks of induction (Figure 2B). The sh3703 mice. Inspection of the glomerular tuft by transmission kidney-to-body weight ratio and serum glucose level were also electron microscopy revealed diffuse podocyte effacement and not different (Supplemental Figure 2, A and B). The poly- foot process (FP) widening in more than half of the total glo- anionic charge of the glomerulus as assessed by Alcian blue merular basement membrane (GBM) surface area for sh3703 staining also appeared to be similar after 6 and 20 weeks of mice (Figure 3C). The GBM was irregularly thickened with a knockdown (Figure 2C). scalloped appearance (Figure 3C). The endothelial layer

Figure 2. Long-term nephrin knockdown promotes mild proteinuria without effecting serum creatinine or glomerular polyanionic charge. Glomerular function and polyanionic charge of mice with and without nephrin knockdown. DOX-inducible nephrin knockdown started at 5 weeks of age and continued for 20 weeks. (A) Urinary albumin excretion as quantiﬁed by the ratio of urinary albumin to creatinine ratio (UACR, mg of albumin/mg of creatinine) after 0, 1, 2, 4, 6, 8, 12, 16, and 20 weeks of DOX induction. UACR of sh3703 mice was signiﬁcantly higher than that of Luci.1309 mice (P=0.02) after 20 weeks of induction. *P,0.05 compared with sh3703 mice at 0 week of induction. (B) Serum creatinine of sh3703 and Luci.1309 mice was not statistically different at 20 weeks of induction. (C) Representative images of a kidney section stained with Alcian blue stain to assess the polyanionic charge of the glomerulus. Scale bars, 50 mm. Error bars, SEM.

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Figure 3. Long-term nephrin depletion disrupts all components of the glomerular tuft. Glomerular histology and ultrastructure of mice with and without nephrin knockdown. (A) Representative images of period acid-Schiff–stained kidney sections of sh3703 and Luci.1309 mice after 0, 3, 6, and 20 weeks of DOX induction. (B) Graph of glomerular cross-sectional area (mm2)forLuci.1309 and sh3703 mice after 0, 3, 6, and 20 weeks of DOX feeding (50556150 mm2 for Luci.1309_20w versus 72146250 mm2 for sh3703_20w; **P,0.001). (C) Representative images of transmission electron microscopy for Luci.1309 and sh3703 after 6 and 20 weeks of induction. Slit diaphragms (arrows) are present in ﬁltration slits of Luci.1309 and sh3703 mice after 6 and 20 weeks of induction. Widening of the subendothelial zone of the glomerular basement membrane (arrowheads) occupied by amorphous extracellular matrix material is observed. (D) Graph of the FP widths of indicated mice. **P,0.01; n=3 mice for Luci.1309 and sh3703 after 6 weeks of induction; n=3 mice for Luci.1309 after 6 weeks of induction; n=4 mice for sh3703 after 20 weeks of induction. (E) Relative glomerular expression of genes that encode for slit diaphragm components and podocyte-speciﬁcgenesinsh3703 mice compared with Luci.1309 mice after 20 weeks of induction. Black squares, genes not statistically different between sh3703 and Luci.1309 mice. Red squares: genes statistically different between sh3703 and Luci.1309 mice. GOI, genes of interest. n=3 Luci.1309 mice and n=5 sh3703 mice. None of the genes examined were different by more than 2-fold. (F) Representative images of immunostaining for synaptopodin (Synpo) and podocin on frozen kidneys sections of mice after 20 weeks of induction. Scale bar, 50 mm. Error bars, SEM. appeared disorganized with widening of the subendothelial versus 388646 nm; P,0.01) (Figure 3D) but did not differ be- zone, which was occupied by loose extracellular matrix (arrow- tween sh3703 and Luci.1309 mice after 6 weeks of induction. heads in Figure 3C). To our surprise, slit diagram complexes The glomerular transcript level of genes encoding for were still present despite more than 80% nephrin knockdown protein components of the slit diagram (e.g., Nphs2/podocin, (arrows in Figure 3C), albeit at a lower frequency because of Neph1, Fat1/proto-cadherin, Cdh3/P-cadherin, zona occludin diffuse FPeffacement. Some of the slit diagram appeared apically 1, F11r/JAM-A, Ocln/occludin, and Cgn/cingulin) or involved in displaced. The FP width in sh3703 mice was 4-fold greater than podocyte cytoskeleton organization (e.g., CD2AP, Inf2/inverted that in Luci.1309 mice after 20 weeks of induction (15826139 nm formin 2, Actn4/a-actinin 4, synaptopodin/Synpo and Wt1) was

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Prenatal Nephrin Knockdown Causes Massive Proteinuria To determine the effect of congenital nephrin knockdown, male mice that are homozygous for the CAGs-rtTA transgene and compound heterozygous for the Luci.1309 and sh3703 TGM transgenes (CAGs-rtTA/CAGs-rtTA; Luci.1309/sh3703)were crossed to wild-type C57BL6 female mice (+/+; +/+) (Figure 4A). Inseminated wild-type female mice with vaginal plugs were separated from the male mice and then fed DOX starting on E7. In three litters resulting from this breeding scheme, a total of 12 pups were born: Six (50%) were CAGs-rtTA/+; Luci.1309/+ and 6 (50%) were CAGs-rtTA/+; sh3703/+. All CAGs-rtTA/+; Luci.1309/+ pups survived to .6 weeks of age; all CAGs-rtTA/+; sh3703/+ pups died within 72 hours after birth. The amount of proteinuria in the 3 CAGs-rtTA/+; sh3703/+ pups that survived .24 hours was .90-fold higher than that of the 6 CAGs-rtTA/+; Luci.1309/+ pups (85.465.1 versus 0.9960.3 mgalbumin/mgof creatinine; P,0. 001) (Figure 4B). Nephrin expression was reduced in the CAGs-rtTA/+; sh3703/+ pups (Figure 4C). Tubular casts, tubular resorption droplets, and glomerular collapse with expanded Bowman capsule were observed in CAGs-rtTA/+; sh3703/+ kidneys but not CAGs-rtTA/+; Luci.1309/+ kidneys (Fig- ure 4D). Glomerular capillary loops appeared distended, and the overlying glomerular basement membrane were denuded of visceral epithelial cell coverage. Because CAGs-rtTA/+; Luci.1309/+ and CAGs-rtTA/+; sh3703/+ pups were born at the expected Mendelian ratio, we concluded that nephrin is not essential for intrauterine survival. The phenotype of sh3703 mice with congenital nephrin knockdown is similar to what has been described for pups with Nphs1 inactivating mutations.

Nephrin Reduction Promotes Hyperfiltration-Induced Glomerular Injury Because glomerular hyperfiltration, which occurs in many pathologic conditions,29 transduces excessive hydraulic pressure on the filtration structure, we hypothesized that a nephrin Figure 3. Continued. knockdown could exacerbate hyperfiltration-induced glomerular injury. To test this hypothesis, we performed unilateral ne- not different by more than 2-fold between sh3703 and Luci.1309 phrectomy (UNPX) to induce glomerular hyperfiltration in the mice after 20 weeks of induction (Figure 3E). This lack of a dif- remnant kidney of mice with nephrin knockdown. Induction of ference in the glomerular expression of podocyte-specific genes is nephrin knockdown started at 5 weeks of age and continued consistent with what has been described previously for Nphs1 until 27 weeks of age (Figure 5A). UNPX was performed at knockout mice.9 Of note, the glomerular expression of Neph1 11 weeks of age. UNPX sh3703 mice developed a progressive and Ocln in sh3703 mice was significantly less than in Luci.1309 increase in albuminuria starting as early as 19 weeks of age mice, but the difference in expression levels was less than 2-fold. (8w after UNPX). In contrast, albuminuria in Luci.1309 mice As expected, Nphs1 expression of sh3703 mice was reduced to less with UNPX did not change markedly (Figure 5B). The urinary than 15% of that in Luci.1309 mice. Neph3 and Claudin3 are albumin-to-creatinine ratio of sh3703 mice was 63-fold higher upregulated in nephrin-knockout mice.9,28 However, in sh3703 than that of Luci.1309 mice at 27 weeks of age (5.36962.804 mg/mg mice with acquired nephrin knockdown, glomerular Neph3 ex- versus 0.08560.034 mg/mg; P,0.001). Albumin was the predom- pression did not appear to be different and glomerular Claudin3 inant protein excreted in the urine as determined by Coomasie expression was actually lower than that in Luci.1309 mice. To blue staining of urinary proteins resolved by gel electrophoresis confirm that the transcript levels reflecttheproteinlevels,we (Figure 5C). The serum creatinine level of sh3703 mice was also immunostained for several slit diagram proteins: synaptopodin, significantly higher than that of Luci.1309 mice (0.212860.034 podocin, and zona occludin 1. The intensity of immunostaining mg/dl versus 0.121660.011 mg/dl; P=0.05) (Figure 5D). The did not appear to be different (Figure 3F). remnant kidney of UNPX sh3703 mice developed marked diffuse

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Figure 4. Prenatal nephrin knockdown causes massive proteinuria. Prenatal DOX feeding for nephrin knockdown during development. (A) A schematic of the breeding scheme with the genotype of parents and pups speciﬁed. Inseminated female was fed DOX on day E7. The observed percentage of pups with a given genotype indicated within parentheses. (B) Urinary albumin excretion of pups with and without nephrin knockdown, sh3703 and Luci.1309, respectively. (C) Western blots of kidney lysates of sh3703 and Luci.1309 pups.

J Am Soc Nephrol 26: 2361–2377, 2015 Nephrin Function in Adult Mice 2367 BASIC RESEARCH www.jasn.org mesangial hypercellularity, glomerulomegaly, and tubular protein absent (Figure 6F, panel a and b, Table 2). In contrast, kidneys casts, but not in the remnant kidney of UNPX Luci.1309 mice of sh3703 mice that received doxorubicin had tubular protein (Figure 5E, Table 1). Variable involvement of segmental and resorption droplets, tubular protein casts involving .20% of global sclerosis, tubular protein casts, and interstitial inflamma- the tubular area (range, 20%–80%), and glomerulosclerosis tion was observed in the sh3703 group (Table 1). In contrast, none involving 20%–60% of the glomeruli (Figure 6F, panels c–h, of the Luci.1309 mice developed glomerulosclerosis, tubular pro- Table 2). Prominent interstitial inflammation was observed in tein casts, or interstitial inflammation. The kidney-to-body 5ofthe7miceinthesh3703 group (Figure 6F, panel f, Table 2). weight ratio of the remnant kidney of sh3703 mice, however, Mesangial expansion with increased mesangial cell number was not significantly different from that of Luci.1309 mice (Figure and mesangial matrix deposition were present in all the 5F). The glomerular cross-sectional area of UNPX sh3703 mice sh3703 mice but in only 1 of 3 Luci.1309 mice. Marked glo- was 72% larger than that of the UNPX Luci.1309 mice merular epithelial cell hyperplasia was present in all the sh3703 (89576261.1 versus 49756104 mm2; P,0.05; n=6 NPX sh3703 mice (Figure 6F, panel g, arrows), suggesting a compensatory mice; n=4 NPX Luci.1309 mice; 20 glomeruli per mouse) (Figure response to podocyte depletion. Consistent with the possibil- 5G). The glomerular cross-sectional area of UNPX sh3703 mice ity of podocyte depletion in sh3703 mice, segments of denuded was also significantly larger than that of matched sh3703 with the glomerular basement membrane without podocyte coverage same duration of DOX feeding (sh3703_22w) (89576261.1 ver- (Figure 6F, panel h, arrows) and dilated capillary loops with sus 58146264.4 mm2; P,0.05; n=6 NPX sh3703 mice; n=2 capsular adhesion of the glomerular tuft (Figure 6F, panel h, sh3703_22w mice; 20 glomeruli per mouse) (Figure 5G). Electron arrowhead) were observed in the sh3703 kidneys, but not the microscopy of the glomeruli demonstrated FP effacement. In Luci.1309 kidneys. Focal areas of denuded GBM without any some of the filtrations slits examined, the slit diagram appeared FP coverage were noted (Figure 6G). Diffuse FP effacement and to be absent (Figure 5H). irregularly thickened GBM were also present in doxorubicin- injected sh3703 kidneys (Figure 6G). Electron-dense junctional Nephrin Knockdown Accelerates Doxorubicin-Induced complex bridging the FPs appeared to be absent in some filtra- Glomerulosclerosis tion slits (Figure 6G). Next, we evaluated the development of doxorubicin-induced glomerulosclerosis in mice with nephrin knockdown. Most AKT Signaling and Podocyte Viability Are Impaired in inbred mouse strains, including C57BL6 and FVBN, are resistant Nephrin Knockdown Mice against doxorubicin-induced renal injury.36 Luci.1309 and Nephrin is a major molecular component of the slit diagram, sh3703 mice are on a mixed genetic background with contribu- which is a key signaling hub of the podocyte. Nephrin stimulates tion from C57BL6 and 129sv strains. We first evaluated the PI3K-dependent AKT signaling and prevents Bad-mediated dose-dependent response of our mouse strain to doxorubicin- apoptosis of podocytes in culture.37 In our model, long-term induced nephropathy. Mice that have been induced with DOX nephrin knockdown (i.e., 20 weeks) was associated with reduced for 6 weeks were injected with one of three different doses of glomerular AKT phosphorylation (Figure 7A). Glomerular AKT doxorubicin: 25 mg/kg, 18.8 mg/kg, and 15 mg/mkg. At doxo- phosphorylation was significantly lower in Luci.1309 kidneys rubicin doses of 25 mg/kg and 18.8 mg/kg, all sh3703 mice died with UNPX and doxorubicin nephropathy compared with within 10 days after doxorubicin injection (n=2 mice at 25 mg/kg Luci.1309 mice without kidney disease. AKT phosphorylation and n=6 mice at 18.8 mg/kg). In contrast, all Luci.1309 mice of sh3703 glomeruli was less than Luci.1309 glomeruli in mice injected with doxorubicin 25 mg/kg or 18.8 mg/kg survived. without glomerular injury (i.e., DOX 20w). In mice with glo- When doxorubicin 15 mg/kg was given, 80% of the sh3703 merular injury (UNPX or doxorubicin), AKT phosphorylation mice (n=5) and 100% of the Luci.1309 mice were viable 4 weeks was also less in sh3703 mice compared with Luci.1309 mice. after doxorubicin injection. The renal findings of mice that Because AKT phosphorylation prevents podocyte apoptosis in received doxorubicin 15 mg/kg were characterized. vitro,37 we assessed podocyte apoptosis by transferase deoxynu- Four weeks after doxorubicin injection, sh3703 mice devel- cleotidyl transferase–mediated digoxigenin-deoxyuridine nick oped significantly more weight loss, albuminuria, and serum end labeling assay (TUNEL) staining and Wilms’ tumor-1 creatinine elevation compared with Luci.1309 mice (Figure 6, (WT-1) coimmunolabeling. TUNEL and WT-1 double-positive A–E). Semi-quantitative histologic evaluation of the Luci.1309 cells were detected in glomeruli of sh3703 mice after 20 weeks of kidneys revealed that tubular casts were present in ,10% of nephrin knockdown as well as sh3703 mice injected with doxo- the tubular compartment and glomerular abnormality was rubicin (Figure 7B), but not in glomeruli of Luci.1309 mice,

Each lane is a lysate from a different mice. (D) Period acid-Schiff–stained kidney sections of Luci.1309 and sh3703 pups with prenatal DOX feeding. Yellow arrows in part c, dilated Bowman capule with collapsed glomeruli. Green arrow in part d, protein resorption droplets within tubular cells. Asterisk marks a tubular cast within the tubular lumen. Green arrowheads in part e, visceral epithelial glomerular cells. Green arrow in part e, segment of glomerular basement membrane without podocytes. Double-headed green arrow in part e, dilated capillary loop. Error bars, SEM.

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Figure 5. Nephrin reduction promotes hyperﬁltration-induced glomerular injury in uninephrectomized mice. Glomerular function and structure of nephrin knockdown mice with UNPX. (A) Schema of study design. Five-week-old sh3703 and Luci.1309 mice were induced with DOX, and UNPX was performed at 11 weeks of age. Mice were euthanized at age 27 weeks. DOX induction was continuous from ages 5 to 27 weeks. (B) Urinary albumin excretion as quantiﬁed by the urinary albumin-to-creatinine ratio (UACR). P,0.05 compared with the sh3703 group at 15 weeks of age. P,0.001 compared with the Luci.1309 group at 27 weeks of age. (C) Urine (7.5 ml per lane) from each of the indicated mice obtained at 15 and 27 weeks of age was analyzed on a 10% denaturing polyacrylamide gel together with 15, 7.5, 1.5, and 0.75 mg BSA and then stained with Coomassie brilliant blue R250. (D) Bar graph of serum creatinine levels of mice at 27 weeks of age as determined by an HPLC-based measurement. *P,0.05. (E) Graph of kidney-to-body weight ratios for the remnant kidney for the indicated mice at the end of the experiment. (F) Representative histologic sections of mice at age 27 weeks.

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slit diagram–mediated signaling event in the podocyte. This event is associated with loss of podocyte viability and a reduction in podocyte number, both under the basal condition as well as in the doxorubicin model of acquired podocyte injury.

DISCUSSION

Here we observed that acquired, long-term nephrin knockdown affects all components of the glomerulus—podocytes, mesangial cells and matrix, GBM, and endothelial cells— which had not been seen previously in other models of nephrin deficiency. Prior studies have delineated cell-cell and cell- matrix cross-talks within the glomerular compartment.38 In particular, podocyte-specific injury causes mesangial cell proliferation,39 and disrupted VEGF-mediated podocyte-endothelial crosstalk leads to endothelial injury40 and mesangial expansion.41 Because nephrin acts in concert with other slit diagram proteins to regulate the organization of membrane lipid rafts, we reasoned that acquired nephrin deficiency disrupts cluster- ing of membrane raft microdomains to prevent nephrin- mediated signaling (i.e., AKT phosphorylation), which could directly affect the podocytes or other glomerular cells that in- teract with podocytes. It remains to be determined why proteinuria, FP effacement, and podocyte dropout occurred with long-term but not short-term nephrin knockdown even though robust nephrin depletion was achieved after the third week of knockdown. Others have questioned the central role of nephrin in guiding, forming, and maintaining the slit diagram on the basis of the observation that birds lack an ortholog of NPHS1 but still form slitdiagramwithin their kidneysand canretainserumalbumin in their blood.3,42,43 So perhaps, as previously suggested by oth- Figure 5. Continued. ers,3,42 it is not nephrin per se that is critical for preventing proteinuria but the slit diagram itself that is required to maintain the sh3703 mice with UNPX, or sh3703 mice after 6 weeks of DOX glomerular ultrafilter. Consistent with this hypothesis, slit dia- induction (data not shown). Podocyte density as quantified grams were observed in mice with .80% nephrin depletion. by the number of WT-1–positive cells per glomerular cross- It appears that the degree of knockdown was greater for the sectional was lower in the sh3703 group than the Luci.1309 group 160-kD than for the 180-kD nephrin band. Because shRNA- after 20 weeks of DOX induction, as well as in the doxorubicin mediated nephrin knockdown depletes the total nephrin pool, model of acquired glomerular injury (Figure 7C). The glomer- one would not expect preferential targeting of the nephrin with ular density of WT-1–positive nuclei in the doxorubicin-treated and without N-glycosylation. We speculate the reason that 160- sh3703 kidney was 56.6% of the Luci.1309 kidney (2.00960.16 kD nephrin band is depleted to a greater extent is because the versus 3.54960.21 nuclei per 1000 mm2 of glomerular cross- N-glycosylated (180-kD) nephrin participates in stable intercel- sectional area; P,0.001). In contrast, the density of WT-1– lular nephrin-to-nephrin interactions and has a slower turnover positive cells in glomeruli of sh3703 mice did not differ from comparedwith nonglycoslyated (160-kD) nephrin, which makes that of Luci.1309 mice after 6 weeks of DOX induction or after the 180-kD nephrin more resistant to shRNA-mediated de- UNPX. These findings suggest that nephrin reduction in adult pletion. This supposition is consistent with our observation that mice impairs AKT phosphorylation, which is a nephrin-dependent, bothnephrinbandswere equally reducedinnephrin knockdown

(G) Graph of the cross-sectional area of the glomerular tuft for mice at age 27 weeks. **P,0.01. Scale bar, 50 mm. (H) Representative images from transmission electron microscopy for sh3703 and Luci.1309 mice 16 weeks after UNPX. Glomerular basement membrane of sh3703 mice exhibits irregular thickening. Black arrows, slit diaphragms. Red arrows, ﬁltration slits without visible slit diaphragms. Scale bar, 1mm. Error bars, SEM.

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Table 1. Histologic features of mice with glomerular hyperfiltration Glomerular Compartment Tubular Compartment Mouse Model Glomeruli with Mesangial Tubular Area with Interstitial Sclerosis (%) Expansion (1–3+) Protein Casts (%) Inflammation (1–3+) Luci.1309 10000 20100 30000 sh3703 1 ,10 3 20 1 2 ,10 3 20 0 30100 40300 5 ,10 3 20 1 mice with doxorubicin-induced podocyte injury, which is a genotyping for the CAGs-rtTA mice was done using the following primers: pathologic condition known to increase nephrin turnover. Ch8 For1 59- TGC CTA TCA TGT TGT CAA A-39. SApA For1 59-CTG Because nephrin expression is reduced in glomerular diseases, CTG TCC ATT CCT TAT TC-39,Ch8Rev259- CGA AAC TCT GGT TGA reduction of nephrin is considered to cause glomerular and CAT G-39 (amplicon size wild-type 363-bp and mutant 330-bp). Thermo podocyte injury either directly or indirectly. Hauser et al. pro- profile for the PCR reaction was as follows: 95°C for 2 minutes followed by posed that endothelin-1–mediated shedding of nephrin from 35 cycles at 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 45 podocytes is a key determinant of urinary protein loss in the seconds, then 72°C for 5 minutes. All animal studies were performed in context of endothelial injury, such as pre-eclampsia, hyperten- accordance with the guidelines of and approved by the Institutional An- sion, and diabetes.44 Others have postulated that nephrin is a imal Care and Use Committee at the Icahn School of Medicine at Mount critical determinant of insulin responsiveness in podocytes and Sinai. nephrin loss could contribute to the development of diabetic nephropathy.1,4 In addition, pharmacologic blockade of angio- Doxorubicin Murine Model tensin II activity prevents the reduction of nephrin in experi- In the doxorubicin murine model, the Luci.1309 mice and sh3703 mice mental models of diabetic nephropathy45 and passive Heymann (both 5 weeks old) were fed with DOX (650 mg/kg) for 6 weeks and nephritis46 and clearly mitigates the progression of renal disease received intravenous doxorubicin by tail-vein injection.36 Urine was col- in those models. However, the causal relationship between lected weekly to assess for proteinuria, and serum was collected to assess nephrin reduction and glomerular injury has never been dem- for serum creatinine. Mice were euthanized 4 weeks after doxorubicin onstrated. Here we provide the first direct experimental dem- injection.Unilateral NephrectomyMice were fed with DOX (650 mg/kg) onstration that nephrin loss causes podocyte and glomerular for 6 weeks and then the right kidney was removed through a flank sur- injury under basal conditions as well as in experimental glomer- gical approach, as previously described.47 Each mouse was anesthetized ular injury models. and a skin incision was made parallel to the last rib beginning midway Our findings suggest that nephrin is critical for glomerular between that rib and the iliac crest. The renal vessels and the ureter were function under basal conditions as well as in glomerular diseases. ligatedandsevered,andthekidneywas withdrawn through the incision. Thus,preservationofnephrinexpressionisapotentialtherapeutic approach that should be explored in patients with glomerular Mice Breeding disease to mitigate podocyte loss and glomerular injury. First, we generated male mice that were homozygous for the CAGs- rtTA transgene and compound heterozygous for the Luci.1309 and sh3703 ColTGM transgene (CAGs-rtTA/CAGs-rtTA; Luci1309/ CONCISE METHODS sh3703)bybreedingafemaleCAGs-rtTA/+; Luci1309/+ mouse with a male CAGs-rtTA/+; sh3703/+ mouse. Male CAGs-rtTA/CAGs- Transgenic Mice and Experimental Models of rtTA; Luci1309/sh3703 mice were then crossed with wild-type +/+ +/+ Glomerular Disease C57BL6 female mice ( ; ). Impregnated wild-type C57BL6 fe- Genotyping male mice (+/+; +/+) were then separated from the male mice and fed ThegenotypingforthehairpinswasdoneusingtheColA1-TGMprimers: with DOX (625 mg/kg chow) starting on E7. The day of conception ColA1-For 59-AATCATCCC AGG TGC ACAGCATTG CGG-39,ColA1- was determined by checking the vaginal plug. Rev 59- ACT TGA GGG CTC ATG AAC CTC CCA GG-39, SAdpa-Rev 59- ATC AAG GAA ACC CTG GAC TAC TGC G-39 (amplicon size wild-ype Antibodies 220-bp and mutant 295-bp). Thermo profile for the PCR reaction was as A rabbit antibody against the intracellular domain of nephrin was pro- follows: 95°C for 6 minutes followed by 35 cycles at 95°C for 40 seconds, duced by Lawrence Holzman (University of Pennsylvania, Philadelphia, 62°C for 45 seconds, and 72°C for 1 minute, then 72°C for 10 minutes. The PA), and a mouse antibody to synaptopodin was a gift from Dr. Peter

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Figure 6. Nephrin deficiency accelerates doxorubicin-induced glomerulosclerosis. Nephrin knockdown promotes doxorubicin-induced disruption of glomerular function and structure. (A) Schema of study design. Five-week-old sh3703 and Luci.1309 mice were induced with DOX until 15 weeks of age. At 11 weeks of age, both groups of mice were injected with doxorubicin (15 mg/kg body weight) via the tail vein and followed for 4 more weeks. (B) Graph of normalized body weights of sh3703 mice at 11, 12, 13, 14, and 15 weeks of age relative to their body weights at 11 weeks of age. *P,0.05 for a comparison of 15-week-old Luci.1309 versus sh3703 mice. (C) Graph of urinary albumin-to- creatinine ratios (UACR) of mice at 11, 13, and 15 weeks of age. P,0.05 and P,0.001 for comparisons of sh3703 mice at 13 and 15 weeks of age to matched sh3703 mice at 11 weeks of age. *P,0.05 for a comparison of 15-week-old Luci.1309 versus sh3703 mice. (D) Urine (7.5 ml per lane) from each of the indicated mice before (11 weeks of age) and after (15 weeks of age) doxorubicin injection was analyzed on a 10% denaturing polyacrylamide gel together with 15, 7.5, 1.5, and 0.75 mg BSA and then stained with Coomassie brilliant blue R250. (E) Graph of serum creatinine levels of the indicated mice before and after doxorubicin injection as determined by an HPLC-based measurement. P,0.001 for comparisons of sh3703 mice at 13 and 15 weeks of age to matched sh3703 mice at 11 weeks of age. *P,0.05 for a comparison of 15-week-old Luci.1309 versus 15-week-old sh3703 mice. (F) Representative histologic sections of mice at 4 weeks after doxorubicin injection. #, tubular protein casts. Normal-appearing histologic sections of doxorubicin-injected Luci.1309 mice 4 weeks after doxorubicin injection (panels a and b). Doxorubicin-injected sh3703 mice developed tubular protein casts (c), glomerulosclerosis (d), tubular protein resorption droplets (e), tubulointerstitial inflammation (f), glomerular epithelial cell proliferation (arrows in part g), dilated capillary loops with denuded basement membrane (arrows in part h), and adhesion of the capillary tuft to the Bowman capsule (arrowhead in part h). Scale bar, 50 mm. (G) Representative electron microscopy images. Doxorubicin-injected Luci.1309 mice display focal FP effacement (part a, magnified view in part b, and another magnified view in part c), but GBM is always covered by FP. Slit diaphragms are present in the filtration slits of Luci.1309 mice (arrows in part d). In contrast, denuded GBM without any FP coverage is observed in doxorubicin-injected sh3703 mice (arrowheads in part e and magnified view in f). Diffuse effacement of FP (part g) and occasional absence of slit diaphragm from filtration slits (arrowheads in part h) are present in doxorubicin-injected sh3703 mice. E, endothelial cell; P, podocytes. Error bars, SEM.

Mundel (Massachusetts General Hospital, Boston, MA). We also used Glomeruli Isolation rabbit antibody to podocin (Sigma-Aldrich, St. Louis, MO), rabbit anti- Mouse glomeruli were isolated as described elsewhere.48 Brieﬂy, ani- body to WT-1(Santa Cruz Technology, Santa Cruz, CA), mouse antibody mals were perfused with Hank’s buffered salt solution containing to GFP (Santa Cruz Technology), and rabbit antibody to phospho-Serine 2.5 mg/ml iron oxide and 1% BSA. At the end of perfusion, kidneys 473-AKT and total-AKT (Cell Signaling Technology, Beverly, MA). were removed, decapsulated, minced into 1-mm3 pieces, and digested

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Figure 6. Continued. in Hank’s buffered salt solution containing 1 mg/ml collagenase A tissue was resuspended in 2 ml of Hank’s buffered salt solution, and and 100 units/ml deoxyribonuclease I. Digested tissue was then passed glomeruli were collected using a magnet. Total RNA was isolated using through a 100-mm cell strainer and collected by centrifugation. The TRIzol (Invitrogen).

Table 2. Histology of mice with doxorubicin nephropathy Glomerular Compartment Tubular Compartment Severity of Mouse Model Glomeruli with Mesangial Tubular Area with Interstitial Glomerular Epithelial Sclerosis (%) Expansion (1–3+) Protein Casts (%) Inﬂammation (1–3+) Cell Hyperplasia (1–3) Luci.1309 1 0 0 0 20 1 2010 00 3000 00 sh3703 14022 603 26032 203 36032 803 42011 20 1 56032 80 3 66032 20 3 72031 40 2

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Figure 7. AKT signaling and podocyte viability are impaired in nephrin knockdown mice. AKT phosphorylation, apoptosis, and glomerular density of podocytes in mice with nephrin knockdown. (A) Representative Western blots of glomerular lysates from indicated mice for phospho-AKT (Ser473), total AKT, nephrin, and b-actin. After 20 weeks of DOX induction (DOX 20w), AKT phosphorylation was markedly reduced in sh3703 mice compared with Luci.1309, but not after 6 weeks of DOX induction (DOX 6w). AKT phosphorylation was markedly reduced in the remnant kidney of Luci.1309 mice with UNPX and further reduced in the remnant kidney of sh3703 mice with UNPX compared with Luci.1309 mice without UNPX. AKT phosphorylation was also reduced in doxorubicin-treated Luci.1309 mice and further reduced in doxorubicin-treated sh3703 mice. Bar graph shows the normalized ratio of phospho-to-total AKT band density plotted for each of the indicated group of mice. (B) Representative images of kidney sections from the indicated mice labeled with an antibody against WT-1 to identify podocytes as well as TUNEL for apoptotic nuclei. Glomerular cells with colabeling of WT-1 and TUNEL were detected in kidneys of sh3703 mice after 20 weeks of DOX induction (DOX 20w) and doxorubicin-injected sh3703 mice induced with DOX. Arrows indicate cells with colocalization of the Hoescht nuclear stain, TUNEL, and WT-1. Scale bar, 50 mm.

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HPLC unit (Columbia, MD) connected with a cation exchange column (PRP-X200; Hamilton Co., Reno, NV).

Histopathology and Immunohistochemistry Staining of Kidney Tissue Kidney samples were fixed in 10% formalin, embedded in paraffin, and sectioned to 4-mm thickness. Periodic acid-Schiff–stained sections were used to assess kidney histologic features. Renal histologic abnormalities were scored as previously described.50 The glomerular parameters scored in- cluded the percentage of glomeruli exhibiting segmental or global sclerosis, the severity of mesangial expansion (graded on a semiquantitative scale from 0to3+), and the severity of podocyte hyperplasia (graded on a semiquantitative scale from 0 to 3+). For the latter parameter, podocyte hyperplasia was graded as 0 (absent), 1+ (involving 1%–25% of all glomeruli sampled), 2+ (involving 26%–50% of glomeruli), and 3+ (involving .50% of glomeruli). For the evaluation of the tubulointerstitial compartment, we assessed the percentage of total renal Figure 7. Continued. parenchyma occupied by tubular casts and/or microcysts as well as the severity of interstitial inflammation (graded on a semiquantitative Table 3. Primers for RT-PCR scale from 0 to 3+). For the latter parameter, in- fl Gene Forward Reverse terstitial in ammationwasgradedas0(absent), 1+ (involving 1%–25% of the cortical paren- Gapdh 59-GCCATCAACGACCCCTTCAT 59-ATGATGACCCGTTTGGCTCC + – + Nphs1 59-GTGCCCTGAAGGACCCTACT 59-CCTGTGGATCCCTTTGACAT chyma), 2 (involving 25% 50%), and 3 (involv- . Nphs2 59-CTTGGCACATCGATCCCTCA 59-CGCACTTTGGCCTGTCTTTG ing 50%). Cldn3 59-GTCCGTCAGTTTTCGAAGGG 59-AGGATGGACACCACGATGAG Frozen sections were used for immunofluo- Synpo 59-CTTTGGGGAAGAGGCCGATTG 59-GTTTTCGGTGAAGCTTGTGC rescence staining for nephrin, podocin, synapto- Neph1 59-TCACAAGCAGGGCTTTAGGA 59-CTGGCTGAAGCGAGTCTGAG podin, and WT-1, and images were taken by Neph3 59-ACATTTCCGGAACCCAGGAG 59-CCACAATCCGGACAGTAGGC using a Zeiss Axioplan 2 IE microscope. CD2AP 59-TGGTGGAGTGGAACCCTGAA 59-GTGAGGTAGGGCCAGTCAAA P-cadherin 59-GATTTTGAGGCTCAGGACCA 59-GACCTTGGAAGGTGGAACAA 9 9 Alcian Blue Staining Inf2 5 -CACCATCTCTCGGCTGTACC 5 -CGAGTAGTTGACCACCGAGG Deparaffinized tissue sections were stained in 1% Fat1 59-CCAGGGAAGTCCGTTCACAA 59-TTGACTCTGGGTGGATTGCC Alcian blue (Sigma-Aldrich) solution and coun- Actn4 59-TTCGACAACAAGCACACCAA 59-CCAGTGCCCCGCCAT terstained with 0.1% fast red (Vector) as described Wt1 59-GAGAGCCAGCCTACCATCC 59-GGGTCCTCGTGTTTGAAGGAA 51 JAM-A 59-CGCGTCGGGATTGTAACTGT 59-CACCGAACCCTTGCCTTGTA previously. Occludin 59-AGGTGAGCACCTTGGGATTC 59-TTCAAAAGGCCTCACGGACA Cingulin 59-TGTGAATCCGGGAGCACTGA 59-TAGCACCCTCTGGCTCTGTA TUNEL Staining ZO-1 59-TAGCACGGACAGTAGACACA 5 9-ATGGAAGTTGGGGTTCATAG TUNEL was performed using TUNEL labeling mix (Promega Corp., Fitchburg, WI), according Measurements of Serum Creatinine to the manufacturer’s instructions. At least 30 glomeruli from each Serum creatinine was measured by an HPLC-based method as mouse were photographed at 4003 magnification. The number of described previously.49 Five microliters of serum per mouse was pro- WT-1–positive nuclei were counted and the cross-sectional area of cessed as described and then assayed using a Shimadzu Prominence the glomerular tuft encompassing the WT-1–positive nuclei was

(C) Representative images of kidney sections stained with WT-1. Bar graphs below represent quantification of the density of WT-1–labeled cells per glomerular tuft area (cells/1000 mm2). The density of WT-1 cells was significantly reduced for sh3703 mice after 20 weeks of DOX induction (DOX 20w) as well as doxorubicin-injected sh3703 compared with their corresponding matched control Luci.1309 mice. ***P,0.001. Error bars, SEM. NS, not significant.

J Am Soc Nephrol 26: 2361–2377, 2015 Nephrin Function in Adult Mice 2375 BASIC RESEARCH www.jasn.org measured using ImageJ software, version 1.47. The number of WT-1– REFERENCES positive nuclei per 1000 mm2 of glomerular tuft area assessed was calculated. 1. Welsh GI, Saleem MA: Nephrin-signature molecule of the glomerular podocyte? J Pathol 220: 328–337, 2010 2. Wartiovaara J, Ofverstedt LG, Khoshnoodi J, Zhang J, Mäkelä E, Sandin Transmission Electron Microscopy S, Ruotsalainen V, Cheng RH, Jalanko H, Skoglund U, Tryggvason K: Mice were perfused with phosphate-buffered saline and then imme- Nephrin strands contribute to a porous slit diaphragm scaffold as re- diately fixed in glutaraldehyde. Sections were mounted on a copper vealed by electron tomography. J Clin Invest 114: 1475–1483, 2004 grid and photographed under a Hitachi H7650 microscope. 3. Grahammer F, Schell C, Huber TB: The podocyte slit diaphragm— from a thin grey line to a complex signalling hub. 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