Nephrin Preserves Podocyte Viability and Glomerular...

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Nephrin Preserves Podocyte Viability and GlomerularStructure and Function in Adult Kidneys

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,†† andJohn Cijiang He*‡‡

*Department of Medicine/Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York; †Department ofNephrology; 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, ShanghaiFirst Municipal Hospital, Shanghai Jiaotao University School of Medicine; Shanghai, China; |Research Institute ofNephrology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China; ¶State University of New York atUniversity at Binghamton, Binghamton, New York; **Mirimus, Inc. Cold Spring Harbor, New York; ††Renal Electrolyte andHypertension Division, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania; and ‡‡RenalSection, James J Peter Veterans Administration Medical Center, Bronx, New York

ABSTRACTNephrin is required during kidney development for the maturation of podocytes and formation of the slitdiaphragm junctional complex. Because nephrin expression is downregulated in acquired glomerulardiseases, nephrin deficiency is considered a pathologic feature of glomerular injury. However, whethernephrin deficiency exacerbates glomerular injury in glomerular diseases has not been experimentally con-firmed. Here, wegeneratedmicewith inducible RNA interference–mediated nephrin knockdown. Short-termnephrin knockdown (6weeks), starting after the completionof kidneydevelopment at 5weeks of age, did notaffect glomerular structure or function. In contrast, mice with long-term nephrin knockdown (20 weeks) de-veloped mild proteinuria, foot process effacement, filtration slit narrowing, mesangial hypercellularity andsclerosis, glomerular basement membrane thickening, subendothelial zone widening, and podocyte apopto-sis.When subjected to an acquired glomerular insult induced by unilateral nephrectomy or doxorubicin, micewith short-term nephrin knockdown developed more severe glomerular injury compared with mice withoutnephrin knockdown. Additionally, nephrin-knockdown mice developed more exaggerated glomerular en-largement when subjected to unilateral nephrectomy and more podocyte apoptosis and depletion afterdoxorubicin challenge. AKT phosphorylation, which is a slit diaphragm–mediated and nephrin-dependentpathway in the podocyte, was markedly reduced in mice with long-term or short-term nephrin knockdownchallenged with uninephrectomy or doxorubicin. Taken together, our data establish that under the basalcondition and in acquired glomerular diseases, nephrin is required tomaintain slit diaphragm integrity and slitdiaphragm–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 themolecular building blockof the slit diaphragm.1 Nephrin bridges the filtra-tion slit to form a 35-nm-long globular cross-strand that contributes to a porous slit diagramscaffold.2 It also serves as a signaling hub3,4 by in-teracting with other slit diagram proteins.5,6 How-ever, all ascribed functions of nephrin were derivedfrom studies of cultured podocytes or mice withinherited nephrin deficiency.7,8 The function of

Received April 24, 2014. Accepted November 19, 2014.

X.L. and P.Y.C. contributed equally to this work.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Peter Y. Chuang, Department of Medicine,Division of Nephrology, Icahn School of Medicine at Mount Sinai,One Gustave L Levy Place, Box 1243, New York, NY 10029.Email: [email protected]

Copyright © 2015 by the American Society of Nephrology

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nephrin and the effect of acquired nephrin loss in the adultmice are not known.

Nephrin is required for podocyte maturation during glomer-ular development.9 In humans and mice with inherited nephrindeficiency due to loss-of-function mutations of NPHS110 orNphs1 deletion (Nphs12/2),7,8 massive proteinuria develops inutero or soon after birth. A series of elegant studies from thelaboratories of Holzman,11,12 Quaggin,13 and Holthofer14,15

highlighted the function of nephrin in rodents. In humans,nephrin is downregulated inmany forms of acquired glomerulardiseases, such as diabetic nephropathy,16–19 minimal-changedisease,20–22 FSGS,23 and membranous nephropathy,21,23,24

andothers.24,25 It has been suggested that nephrin deficiency causespodocyte injury in vivo, but this has not been directly confirmed.Development of glomerular dysfunction in rodents injected withan antinephrin antibody,26,27 as well as recurrence of nephropathydue to alloimmunization against nephrin after kidney transplanta-tion in individuals with congenital nephropathy of the Finnishtype,28,29 suggests that antinephrin antibody is detrimental to glo-merular function.Themechanismof antinephrin antibodycausingglomerular dysfunction in these cases, however, is not known.26

Here we produced several lines of genetically engineeredmice with inducible nephrin knockdown mediated by shorthairpin RNA (shRNA). We sought to directly characterizenephrin functionbyexamining adultmicewithacquired ratherthan inherited nephrin loss.

RESULTS

Murine Model of Inducible Nephrin KnockdownWegenerated transgenicmicewith inducible shRNA-mediatedNphs1 knockdown using an in vivo RNA interference ap-proach.30,31 Three different lines of mice encoding anmiR30-based expression cassette under the transcriptionalcontrol of a doxycycline (DOX)-responsive promoter elementwere produced. Each line harbors a different 21-bp Nphs1guide sequence. These three lines are called sh1401, sh1416,and sh3703. The DOX-responsive transgene, TGM, was con-figured with a GFP "spacer" between the tetracycline-responsivepromoter element and the miR30-based expression cassette(Supplemental Figure 1A). Mice with a single copy of TGMwere generated and then bred withmice that possess widespreadexpression of reverse tetracycline transactivator (rtTA), hereafterreferred to as CAGs-rtTA mice,30 to produce double transgenicCAGs-rtTA/+; TGM/+ mice. These double transgenic mice wereborn at the expected Mendelian ratio and were indistinguish-able from wild-type mice and singly transgenic mice harbor-ing the CAGs-rtTA or TGM transgene.

Murine kidney development is completed between post-natal days 7 and 8.32 Induction of nephrin knockdown startedat 5 weeks of age with DOX-supplemented chow (650 mg/kg).The nephrin protein and mRNA levels of 11-week-old miceinduced with DOX for 6 weeks were significantly lower in all

Figure 1. Amurine model of inducible nephrin knockdown. An inducible knockdown of nephrin is robust and sustainable up to 20 weeksof induction. Inducible nephrin knockdown mice (sh3703) and control Luci.1309 mice with inducible overexpression of an shRNAagainst the firefly luciferase gene received DOX in the chow (625 mg/kg) starting at 5 weeks of age and continued for 20 weeks until 25weeks of age. (A) Representative Western blots of nephrin in kidney protein lysates of sh3703 and Luci.1309 mice that received DOXfor 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 thegraph contains samples from at least three mice. ***P,0.001 compared with matched Luci.1309mice. (C) Nephrin immunostaining andGFP fluorescence on frozen kidney sections of mice after 0, 1, 3, 6, and 20 weeks of induction. Scale bars, 50 mm. Error bars, SEM.

2362 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2361–2377, 2015


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three lines of nephrin-knockdown mice than in controlLuci.1309 mice, which express an shRNA targeting the fireflyluciferase gene30 (Supplemental Figure 1, B and C). TwoWest-ern bands specific to nephrin with apparent molecular massesof 160 kD and 180 kD have been described.33 The 180-kDband corresponds to N-glycosylated nephrin.33 Green fluores-cent protein (GFP) expression was similar between Luci.1309and sh3703 mice. The sh3703 line demonstrated the most ro-bust knockdown. Nephrin expression in the renal cortex ofsh3703 mice was knocked down by approximately 80% com-pared with control Luci.1309mice after 6 weeks of induction.

Because sh3703mice have the most robustknockdown, they were used in all subse-quent experiments.

Robust knockdown was observed after3 weeks of DOX induction and was sus-tainable for up to 20 weeks of induction(Figure 1A). Nephrin expression of age-matched sh3703 mice without DOX feed-ing (sh3703 No DOX_20w) did not differfrom that of control Luci.1309 mice withand without DOX induction for 20 weeks(DOX_20w and No DOX_20w) and wasmarkedly higher than that of sh3703micewith DOX induction (Supplemental Figure1D). sh3703mice that were induced with ahigher dose of DOX (650 mg/kg DOX foodplus 2 mg/ml DOX in the drinking water[DOX food+H2O_20w]) had a degree ofnephrin knockdown similar to that in thoseinduced with only 650 mg/kg DOX food(DOX_20w) (Supplemental Figure 1D).The kinetics of nephrin knockdownfollowed a biphasic pattern of early, rapiddecay followed by a progressive decline(Figure 1A). This biphasic pattern is consis-tent with the thought that nephrin is an in-tegral transmembrane protein involved instable trans interaction with adjacent footprocesses and thus is not readily turnedover under the basal condition. Maximalknockdown appeared to plateau at approx-imately 80%–90% for both nephrin proteinand mRNA transcript, suggesting that theshRNA-mediated knockdown inour systemis robust but not complete. Immunostain-ing of nephrin confirmed that glomerularnephrin expression was markedly reducedin GFP-positive glomerular cells of sh3703mice but not Luci.1309 mice (Figure 1C).TheseGFP-positive glomerular cells are pre-dominantly podocytes and notmesangial orendothelial cells.31

Western blotting using an antibodyagainst the intracellular domain of neph-

rin revealed both of the nephrin-specific bands (180 kD and160 kD) in the kidney cortex lysate. Consistent with previousreports, we also identified the 160-kDa nephrin band inextrarenal organs: heart, spleen, pancreas, and brain (Supple-mental Figure 1E).7,34,35 The exclusive presence of the 180-kDband in the kidney lysate has been described previously.35 Be-cause DOX does not cross the blood-brain barrier, GFP andnephrin expressions did not different between mice with andwithout induction. Histologic features of the myocardium didnot appear to be different between sh3703 and Luci.1309 mice(Supplemental Figure 1F).

Figure 1. Continued.

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Long-term, but Not Short-term, Nephrin Knockdown inAdult Mice Promotes Proteinuriash3703mice fed with DOX starting at 5 weeks of age developedmore proteinuria than did control Luci.1309 mice after 24weeks of induction (Figure 2A). In contrast, age-matchedLuci.1309 mice with DOX induction did not develop markedproteinuria. The urinary albumin excretion in sh3703 micewas approximately 3-fold higher than that in Luci.1309 miceafter 24 weeks of induction (mean6SD, 0.17960.041 versus0.05260.014 mg of albumin per mg of creatinine; P,0.05).The serum creatinine of sh3703 and Luci.1309 mice did notdiffer before and after 20 weeks of induction (Figure 2B). Thekidney-to-body weight ratio and serum glucose level were alsonot different (Supplemental Figure 2, A and B). The poly-anionic charge of the glomerulus as assessed by Alcian bluestaining also appeared to be similar after 6 and 20 weeks ofknockdown (Figure 2C).

Long-term Nephrin Depletion Disrupts AllComponents of the Glomerular TuftAfter 20 weeks of induction, the glomerular cross-sectionalarea of sh3703mice was enlarged (Figure 3, A and B). In con-trast, the glomerular structure and glomerular cross-sectionalarea of Luci.1309 kidney at up to 20 weeks of induction andsh3703 kidney at up to 6 weeks of induction appeared normal(Figure 3, A and B). Glomeruli of age-matched sh3703 micewithout DOX induction appeared normal (Supplemental Fig-ure 1G), thus ruling out any off-target effects or leakiness ofshRNA as the cause of glomerular changes in DOX-inducedsh3703 mice. Inspection of the glomerular tuft by transmissionelectron microscopy revealed diffuse podocyte effacement andfoot process (FP) widening in more than half of the total glo-merular basement membrane (GBM) surface area for sh3703mice (Figure 3C). The GBM was irregularly thickened with ascalloped 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 at5 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 sh3703mice was significantlyhigher 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 ofa kidney section stained with Alcian blue stain to assess the polyanionic charge of the glomerulus. Scale bars, 50 mm. Error bars, SEM.






appeared disorganized with widening of the subendothelialzone, which was occupied by loose extracellular matrix (arrow-heads in Figure 3C). To our surprise, slit diagram complexeswere still present despite more than 80% nephrin knockdown(arrows in Figure 3C), albeit at a lower frequency because ofdiffuse FPeffacement. Someof the slit diagram appeared apicallydisplaced. The FP width in sh3703mice was 4-fold greater thanthat inLuci.1309mice after 20weeks of induction (15826139nm

versus 388646 nm; P,0.01) (Figure 3D) but did not differ be-tween sh3703 and Luci.1309mice after 6 weeks of induction.

The glomerular transcript level of genes encoding forprotein components of the slit diagram (e.g., Nphs2/podocin,Neph1, Fat1/proto-cadherin, Cdh3/P-cadherin, zona occludin1, F11r/JAM-A,Ocln/occludin, andCgn/cingulin) or involved inpodocyte cytoskeleton organization (e.g., CD2AP, Inf2/invertedformin 2, Actn4/a-actinin 4, synaptopodin/Synpo andWt1) was

Figure 3. Long-term nephrin depletion disrupts all components of the glomerular tuft. Glomerular histology and ultrastructure of micewith and without nephrin knockdown. (A) Representative images of period acid-Schiff–stained kidney sections of sh3703 and Luci.1309mice after 0, 3, 6, and 20 weeks of DOX induction. (B) Graph of glomerular cross-sectional area (mm2) for Luci.1309 and sh3703 miceafter 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 dia-phragms (arrows) are present in filtration slits of Luci.1309 and sh3703 mice after 6 and 20 weeks of induction. Widening of thesubendothelial zone of the glomerular basement membrane (arrowheads) occupied by amorphous extracellular matrix material isobserved. (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=3mice for Luci.1309 after 6 weeks of induction; n=4 mice for sh3703 after 20 weeks of induction. (E) Relative glomerular expression ofgenes that encode for slit diaphragm components and podocyte-specific genes in sh3703 mice compared with Luci.1309 mice after20 weeks of induction. Black squares, genes not statistically different between sh3703 and Luci.1309 mice. Red squares: genes sta-tistically different between sh3703 and Luci.1309 mice. GOI, genes of interest. n=3 Luci.1309 mice and n=5 sh3703 mice. None of thegenes examined were different by more than 2-fold. (F) Representative images of immunostaining for synaptopodin (Synpo) andpodocin on frozen kidneys sections of mice after 20 weeks of induction. Scale bar, 50 mm. Error bars, SEM.



not different by more than 2-fold between sh3703 and Luci.1309mice after 20 weeks of induction (Figure 3E). This lack of a dif-ference in the glomerular expression of podocyte-specific genes isconsistent with what has been described previously for Nphs1knockout mice.9 Of note, the glomerular expression of Neph1and Ocln in sh3703 mice was significantly less than in Luci.1309mice, but the difference in expression levels was less than 2-fold.As expected,Nphs1 expression of sh3703micewas reduced to lessthan 15% of that in Luci.1309 mice. Neph3 and Claudin3 areupregulated in nephrin-knockout mice.9,28 However, in sh3703mice with acquired nephrin knockdown, glomerular Neph3 ex-pression did not appear to be different and glomerular Claudin3expression was actually lower than that in Luci.1309 mice. Toconfirm that the transcript levels reflect the protein levels, weimmunostained for several slit diagram proteins: synaptopodin,podocin, and zona occludin 1. The intensity of immunostainingdid not appear to be different (Figure 3F).

Prenatal Nephrin Knockdown Causes MassiveProteinuriaTo determine the effect of congenital nephrin knockdown, malemice that are homozygous for the CAGs-rtTA transgene andcompound heterozygous for the Luci.1309 and sh3703 TGMtransgenes (CAGs-rtTA/CAGs-rtTA; Luci.1309/sh3703) werecrossed to wild-type C57BL6 female mice (+/+; +/+) (Figure4A). Inseminated wild-type female mice with vaginal plugswere separated from the male mice and then fed DOX startingonE7. In three litters resulting from this breeding scheme, a totalof 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 ofproteinuria 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 mg albumin/mg ofcreatinine; P,0. 001) (Figure 4B). Nephrin expression was re-duced in the CAGs-rtTA/+; sh3703/+ pups (Figure 4C). Tubularcasts, tubular resorption droplets, and glomerular collapse withexpanded Bowman capsule were observed in CAGs-rtTA/+;sh3703/+ kidneys but notCAGs-rtTA/+; Luci.1309/+ kidneys (Fig-ure 4D). Glomerular capillary loops appeared distended, and theoverlying glomerular basement membrane were denuded of vis-ceral epithelial cell coverage. Because CAGs-rtTA/+; Luci.1309/+

and CAGs-rtTA/+; sh3703/+ pups were born at the expectedMendelian ratio, we concluded that nephrin is not essential forintrauterine survival. The phenotype of sh3703 mice with con-genital nephrin knockdown is similar to what has been describedfor pups with Nphs1 inactivating mutations.

Nephrin Reduction Promotes Hyperfiltration-InducedGlomerular InjuryBecause glomerular hyperfiltration, which occurs in manypathologic conditions,29 transduces excessive hydraulic pressureon the filtration structure, we hypothesized that a nephrinknockdown could exacerbate hyperfiltration-induced glomeru-lar injury. To test this hypothesis, we performed unilateral ne-phrectomy (UNPX) to induce glomerular hyperfiltration in theremnant kidney of mice with nephrin knockdown. Induction ofnephrin knockdown started at 5 weeks of age and continueduntil 27 weeks of age (Figure 5A). UNPX was performed at11 weeks of age. UNPX sh3703 mice developed a progressiveincrease in albuminuria starting as early as 19 weeks of age(8w after UNPX). In contrast, albuminuria in Luci.1309 micewith UNPX did not change markedly (Figure 5B). The urinaryalbumin-to-creatinine ratio of sh3703 mice was 63-fold higherthan that ofLuci.1309mice at 27weeks of age (5.36962.804mg/mgversus 0.08560.034mg/mg;P,0.001). Albuminwas the predom-inant protein excreted in the urine as determined by Coomasieblue staining of urinary proteins resolved by gel electrophoresis(Figure 5C). The serum creatinine level of sh3703 mice was alsosignificantly higher than that of Luci.1309 mice (0.212860.034mg/dl versus 0.121660.011 mg/dl; P=0.05) (Figure 5D). Theremnant kidney of UNPX sh3703mice developedmarked diffuse




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 andwithout nephrin knockdown, sh3703 and Luci.1309, respectively. (C) Western blots of kidney lysates of sh3703 and Luci.1309 pups.



mesangial hypercellularity, glomerulomegaly, and tubular proteincasts, but not in the remnant kidney of UNPX Luci.1309 mice(Figure 5E, Table 1). Variable involvement of segmental andglobal sclerosis, tubular protein casts, and interstitial inflamma-tionwas observed in the sh3703 group (Table 1). In contrast, noneof the Luci.1309mice developed glomerulosclerosis, tubular pro-tein casts, or interstitial inflammation. The kidney-to-bodyweight ratio of the remnant kidney of sh3703 mice, however,was not significantly different from that of Luci.1309mice (Figure5F). The glomerular cross-sectional area of UNPX sh3703 micewas 72% larger than that of the UNPX Luci.1309 mice(89576261.1 versus 49756104 mm2; P,0.05; n=6 NPX sh3703mice; n=4NPX Luci.1309mice; 20 glomeruli per mouse) (Figure5G). The glomerular cross-sectional area of UNPX sh3703 micewas also significantly larger than that of matched sh3703with thesame duration of DOX feeding (sh3703_22w) (89576261.1 ver-sus 58146264.4 mm2; P,0.05; n=6 NPX sh3703 mice; n=2sh3703_22wmice; 20 glomeruli permouse) (Figure 5G). Electronmicroscopy of the glomeruli demonstrated FP effacement. Insome of the filtrations slits examined, the slit diagram appearedto be absent (Figure 5H).

Nephrin Knockdown Accelerates Doxorubicin-InducedGlomerulosclerosisNext, we evaluated the development of doxorubicin-inducedglomerulosclerosis in mice with nephrin knockdown. Mostinbredmouse strains, includingC57BL6 and FVBN, are resistantagainst doxorubicin-induced renal injury.36 Luci.1309 andsh3703mice are on a mixed genetic background with contribu-tion from C57BL6 and 129sv strains. We first evaluated thedose-dependent response of our mouse strain to doxorubicin-induced nephropathy. Mice that have been induced with DOXfor 6 weeks were injected with one of three different doses ofdoxorubicin: 25 mg/kg, 18.8 mg/kg, and 15 mg/mkg. At doxo-rubicin doses of 25 mg/kg and 18.8 mg/kg, all sh3703mice diedwithin 10 days after doxorubicin injection (n=2mice at 25 mg/kgand n=6 mice at 18.8 mg/kg). In contrast, all Luci.1309 miceinjected with doxorubicin 25 mg/kg or 18.8 mg/kg survived.When doxorubicin 15 mg/kg was given, 80% of the sh3703mice (n=5) and 100%of the Luci.1309micewere viable 4weeksafter doxorubicin injection. The renal findings of mice thatreceived doxorubicin 15 mg/kg were characterized.

Four weeks after doxorubicin injection, sh3703mice devel-oped significantly more weight loss, albuminuria, and serumcreatinine elevation compared with Luci.1309 mice (Figure 6,A–E). Semi-quantitative histologic evaluation of the Luci.1309kidneys revealed that tubular casts were present in ,10% ofthe tubular compartment and glomerular abnormality was

absent (Figure 6F, panel a and b, Table 2). In contrast, kidneysof sh3703mice that received doxorubicin had tubular proteinresorption droplets, tubular protein casts involving .20% ofthe tubular area (range, 20%–80%), and glomerulosclerosisinvolving 20%–60% of the glomeruli (Figure 6F, panels c–h,Table 2). Prominent interstitial inflammation was observed in5 of the 7mice in the sh3703 group (Figure 6F, panel f, Table 2).Mesangial expansion with increased mesangial cell numberand mesangial matrix deposition were present in all thesh3703 mice but in only 1 of 3 Luci.1309 mice. Marked glo-merular epithelial cell hyperplasia was present in all the sh3703mice (Figure 6F, panel g, arrows), suggesting a compensatoryresponse to podocyte depletion. Consistent with the possibil-ity of podocyte depletion in sh3703mice, segments of denudedglomerular basement membrane without podocyte coverage(Figure 6F, panel h, arrows) and dilated capillary loops withcapsular adhesion of the glomerular tuft (Figure 6F, panel h,arrowhead) were observed in the sh3703 kidneys, but not theLuci.1309 kidneys. Focal areas of denuded GBM without anyFP coverage were noted (Figure 6G). Diffuse FP effacement andirregularly thickened GBM were also present in doxorubicin-injected sh3703 kidneys (Figure 6G). Electron-dense junctionalcomplex bridging the FPs appeared to be absent in some filtra-tion slits (Figure 6G).

AKT Signaling and Podocyte Viability Are Impaired inNephrin Knockdown MiceNephrin is a major molecular component of the slit diagram,which is a key signaling hub of the podocyte. Nephrin stimulatesPI3K-dependent AKT signaling and prevents Bad-mediatedapoptosis of podocytes in culture.37 In our model, long-termnephrin knockdown (i.e., 20weeks)was associatedwith reducedglomerular AKTphosphorylation (Figure 7A). Glomerular AKTphosphorylation was significantly lower in Luci.1309 kidneyswith UNPX and doxorubicin nephropathy compared withLuci.1309 mice without kidney disease. AKT phosphorylationof sh3703 glomeruli was less than Luci.1309 glomeruli in micewithout glomerular injury (i.e., DOX 20w). In mice with glo-merular injury (UNPX or doxorubicin), AKT phosphorylationwas also less in sh3703 mice compared with Luci.1309 mice.Because AKT phosphorylation prevents podocyte apoptosis invitro,37 we assessed podocyte apoptosis by transferase deoxynu-cleotidyl transferase–mediated digoxigenin-deoxyuridine nickend labeling assay (TUNEL) staining and Wilms’ tumor-1(WT-1) coimmunolabeling. TUNEL andWT-1 double-positivecells were detected in glomeruli of sh3703mice after 20 weeks ofnephrin knockdown as well as sh3703mice injected with doxo-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 DOXfeeding. Yellow arrows in part c, dilatedBowman capulewith collapsedglomeruli. Green arrow in part d, protein resorption droplets withintubular cells. Asterisk marks a tubular cast within the tubular lumen. Green arrowheads in part e, visceral epithelial glomerular cells. Greenarrow in part e, segment of glomerular basement membrane without podocytes. Double-headed green arrow in part e, dilated capillaryloop. Error bars, SEM.



Figure 5. Nephrin reduction promotes hyperfiltration-induced glomerular injury in uninephrectomized mice. Glomerular function andstructure of nephrin knockdown mice with UNPX. (A) Schema of study design. Five-week-old sh3703 and Luci.1309 mice were inducedwith DOX, and UNPX was performed at 11 weeks of age. Mice were euthanized at age 27 weeks. DOX induction was continuous fromages 5 to 27 weeks. (B) Urinary albumin excretion as quantified by the urinary albumin-to-creatinine ratio (UACR). P,0.05 comparedwith 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 togetherwith 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 miceat 27 weeks of age as determined by an HPLC-based measurement. *P,0.05. (E) Graph of kidney-to-body weight ratios for theremnant kidney for the indicated mice at the end of the experiment. (F) Representative histologic sections of mice at age 27 weeks.



sh3703mice with UNPX, or sh3703mice after 6 weeks of DOXinduction (data not shown). Podocyte density as quantifiedby the number of WT-1–positive cells per glomerular cross-sectional was lower in the sh3703 group than the Luci.1309 groupafter 20 weeks of DOX induction, as well as in the doxorubicinmodel of acquired glomerular injury (Figure 7C). The glomer-ular density of WT-1–positive nuclei in the doxorubicin-treatedsh3703 kidney was 56.6% of the Luci.1309 kidney (2.00960.16versus 3.54960.21 nuclei per 1000 mm2 of glomerular cross-sectional area; P,0.001). In contrast, the density of WT-1–positive cells in glomeruli of sh3703 mice did not differ fromthat of Luci.1309 mice after 6 weeks of DOX induction or afterUNPX. These findings suggest that nephrin reduction in adultmice impairsAKTphosphorylation,which is anephrin-dependent,

slit diagram–mediated signaling event in the podocyte. Thisevent is associated with loss of podocyte viability and a reduc-tion in podocyte number, both under the basal condition aswell as in the doxorubicin model of acquired podocyte injury.

DISCUSSION

Here we observed that acquired, long-term nephrin knock-down 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 neph-rin deficiency. Prior studies have delineated cell-cell and cell-matrix cross-talks within the glomerular compartment.38 Inparticular, podocyte-specific injury causes mesangial cell prolif-eration,39 and disruptedVEGF-mediated podocyte-endothelialcrosstalk leads to endothelial injury40 and mesangial expan-sion.41 Because nephrin acts in concert with other slit diagramproteins 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 coulddirectly affect the podocytes or other glomerular cells that in-teract with podocytes.

It remains to be determined why proteinuria, FP efface-ment, and podocyte dropout occurred with long-term but notshort-term nephrin knockdown even though robust nephrindepletion 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 theobservation that birds lack an ortholog ofNPHS1 but still formslitdiagramwithin their kidneysandcanretain serumalbumin intheir blood.3,42,43 So perhaps, as previously suggested by oth-ers,3,42 it is not nephrin per se that is critical for preventing pro-teinuria but the slit diagram itself that is required tomaintain theglomerular ultrafilter. Consistent with this hypothesis, slit dia-grams were observed in mice with.80% nephrin depletion.

It appears that the degree of knockdown was greater for the160-kD than for the 180-kD nephrin band. Because shRNA-mediated nephrin knockdown depletes the total nephrin pool,one would not expect preferential targeting of the nephrin withand without N-glycosylation. We speculate the reason that 160-kD nephrin band is depleted to a greater extent is because theN-glycosylated (180-kD) nephrin participates in stable intercel-lular nephrin-to-nephrin interactions and has a slower turnovercomparedwithnonglycoslyated (160-kD)nephrin,whichmakesthe 180-kD nephrin more resistant to shRNA-mediated de-pletion. This supposition is consistent with our observation thatbothnephrinbandswere equally reduced innephrinknockdown

(G)Graphof the cross-sectional areaof theglomerular tuft formice at age27weeks. **P,0.01. Scalebar, 50mm. (H) Representative imagesfrom transmission electron microscopy for sh3703 and Luci.1309mice 16 weeks after UNPX. Glomerular basement membrane of sh3703mice exhibits irregular thickening. Black arrows, slit diaphragms. Red arrows, filtration slits without visible slit diaphragms. Scale bar, 1mm.Error bars, SEM.




mice with doxorubicin-induced podocyte injury, which is apathologic condition known to increase nephrin turnover.

Becausenephrin expression is reduced inglomerulardiseases,reduction of nephrin is considered to cause glomerular andpodocyte injury either directly or indirectly. Hauser et al. pro-posed that endothelin-1–mediated shedding of nephrin frompodocytes is a key determinant of urinary protein loss in thecontext of endothelial injury, such as pre-eclampsia, hyperten-sion, and diabetes.44 Others have postulated that nephrin is acritical determinant of insulin responsiveness in podocytes andnephrin loss could contribute to the development of diabeticnephropathy.1,4 In addition, pharmacologic blockade of angio-tensin II activity prevents the reduction of nephrin in experi-mental models of diabetic nephropathy45 and passive Heymannnephritis46 and clearly mitigates the progression of renal diseasein those models. However, the causal relationship betweennephrin reduction and glomerular injury has never been dem-onstrated. Here we provide the first direct experimental dem-onstration that nephrin loss causes podocyte and glomerularinjury under basal conditions as well as in experimental glomer-ular injury models.

Our findings suggest that nephrin is critical for glomerularfunction under basal conditions as well as in glomerular diseases.Thus,preservationofnephrinexpressionisapotential therapeuticapproach that should be explored in patients with glomerulardisease to mitigate podocyte loss and glomerular injury.

CONCISE METHODS

Transgenic Mice and Experimental Models ofGlomerular DiseaseGenotypingThegenotyping for thehairpinswasdoneusing theColA1-TGMprimers:

ColA1-For59-AATCATCCCAGGTGCACAGCATTGCGG-39,ColA1-

Rev 59- ACTTGAGGGCTCATGAACCTCCCAGG-39, SAdpa-Rev 59-

ATC AAGGAA ACC CTGGAC TAC TGCG-39 (amplicon size wild-ype

220-bp and mutant 295-bp). Thermo profile for the PCR reaction was as

follows: 95°C for 6 minutes followed by 35 cycles at 95°C for 40 seconds,

62°C for 45 seconds, and72°C for 1minute, then72°C for 10minutes. The

genotyping for theCAGs-rtTAmicewasdoneusing the following primers:

Ch8 For1 59- TGC CTATCATGT TGT CAA A-39. SApA For1 59- CTG

CTGTCCATTCCTTATTC-39, Ch8Rev259-CGAAACTCTGGTTGA

CATG-39 (amplicon size wild-type 363-bp andmutant 330-bp). Thermo

profile for thePCR reactionwas as follows: 95°C for 2minutes followedby

35 cycles at 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 45

seconds, then 72°C for 5 minutes. All animal studies were performed in

accordance with the guidelines of and approved by the Institutional An-

imal Care and Use Committee at the Icahn School of Medicine at Mount

Sinai.

Doxorubicin Murine ModelIn the doxorubicin murine model, the Luci.1309 mice and sh3703 mice

(both 5 weeks old) were fed with DOX (650 mg/kg) for 6 weeks and

received intravenous doxorubicin by tail-vein injection.36 Urine was col-

lected weekly to assess for proteinuria, and serum was collected to assess

for serum creatinine. Mice were euthanized 4 weeks after doxorubicin

injection.Unilateral NephrectomyMice were fed with DOX (650 mg/kg)

for 6 weeks and then the right kidney was removed through a flank sur-

gical approach, as previously described.47 Each mouse was anesthetized

and a skin incision was made parallel to the last rib beginning midway

between that rib and the iliac crest. The renal vessels and the ureter were

ligated and severed, and the kidney was withdrawn through the incision.

Mice BreedingFirst, 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/

sh3703) by breeding a female CAGs-rtTA/+; Luci1309/+ mouse

with a male CAGs-rtTA/+; sh3703/+ mouse. Male CAGs-rtTA/CAGs-

rtTA; Luci1309/sh3703 mice were then crossed with wild-type

C57BL6 female mice (+/+; +/+). Impregnated wild-type C57BL6 fe-

male mice (+/+; +/+) were then separated from the male mice and fed

with DOX (625 mg/kg chow) starting on E7. The day of conception

was determined by checking the vaginal plug.

AntibodiesA rabbit antibody against the intracellular domain of nephrin was pro-

duced by Lawrence Holzman (University of Pennsylvania, Philadelphia,

PA), and a mouse antibody to synaptopodin was a gift from Dr. Peter

Table 1. Histologic features of mice with glomerular hyperfiltration

Mouse Model

Glomerular Compartment Tubular Compartment

Glomeruli withSclerosis (%)

MesangialExpansion (1–3+)

Tubular Area withProtein Casts (%)

InterstitialInflammation (1–3+)

Luci.1309

1 0 0 0 02 0 1 0 03 0 0 0 0

sh3703

1 ,10 3 20 12 ,10 3 20 03 0 1 0 04 0 3 0 05 ,10 3 20 1



Mundel (Massachusetts General Hospital, Boston, MA). We also used

rabbit antibody to podocin (Sigma-Aldrich, St. Louis, MO), rabbit anti-

body toWT-1(SantaCruzTechnology, SantaCruz,CA),mouseantibody

toGFP(SantaCruzTechnology), and rabbit antibody to phospho-Serine

473-AKTand total-AKT (Cell Signaling Technology, Beverly, MA).

Glomeruli IsolationMouse glomeruli were isolated as described elsewhere.48 Briefly, ani-

mals were perfused with Hank’s buffered salt solution containing

2.5 mg/ml iron oxide and 1% BSA. At the end of perfusion, kidneys

were removed, decapsulated,minced into 1-mm3 pieces, and digested

Figure 6. Nephrin deficiency accelerates doxorubicin-induced glomerulosclerosis. Nephrin knockdown promotes doxorubicin-induceddisruption of glomerular function and structure. (A) Schema of study design. Five-week-old sh3703 and Luci.1309 mice were induced withDOX 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 veinand followed for 4 more weeks. (B) Graph of normalized body weights of sh3703mice at 11, 12, 13, 14, and 15 weeks of age relative to theirbody 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 sh3703mice at 13 and 15 weeks ofage 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 mlper 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 ofserum creatinine levels of the indicated mice before and after doxorubicin injection as determined by an HPLC-based measurement.P,0.001 for comparisons of sh3703mice at 13 and 15 weeks of age to matched sh3703mice at 11 weeks of age. *P,0.05 for a comparisonof 15-week-old Luci.1309 versus 15-week-old sh3703 mice. (F) Representative histologic sections of mice at 4 weeks after doxorubicin in-jection. #, tubular protein casts. Normal-appearing histologic sections of doxorubicin-injected Luci.1309 mice 4 weeks after doxorubicininjection (panels a and b). Doxorubicin-injected sh3703 mice developed tubular protein casts (c), glomerulosclerosis (d), tubular proteinresorption droplets (e), tubulointerstitial inflammation (f), glomerular epithelial cell proliferation (arrows in part g), dilated capillary loops withdenuded 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, magnifiedview 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 ofLuci.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.



in Hank’s buffered salt solution containing 1 mg/ml collagenase A

and 100 units/ml deoxyribonuclease I. Digested tissue was then passed

through a 100-mm cell strainer and collected by centrifugation. The

tissue was resuspended in 2 ml of Hank’s buffered salt solution, and

glomeruli were collected using a magnet. Total RNAwas isolated using

TRIzol (Invitrogen).


Table 2. Histology of mice with doxorubicin nephropathy

Mouse Model

Glomerular Compartment Tubular Compartment

Glomeruli withSclerosis (%)

MesangialExpansion (1–3+)

Severity ofGlomerular EpithelialCell Hyperplasia (1–3)

Tubular Area withProtein Casts (%)

InterstitialInflammation (1–3+)

Luci.1309

1 0 0 0 20 12 0 1 0 0 03 0 0 0 0 0

sh3703

1 40 2 2 60 32 60 3 2 20 33 60 3 2 80 34 20 1 1 20 15 60 3 2 80 36 60 3 2 20 37 20 3 1 40 2



Figure 7. AKT signaling and podocyte viability are impaired in nephrin knockdown mice. AKT phosphorylation, apoptosis, and glo-merular density of podocytes in mice with nephrin knockdown. (A) Representative Western blots of glomerular lysates from indicatedmice for phospho-AKT (Ser473), total AKT, nephrin, and b-actin. After 20 weeks of DOX induction (DOX 20w), AKT phosphorylation wasmarkedly reduced in sh3703 mice compared with Luci.1309, but not after 6 weeks of DOX induction (DOX 6w). AKT phosphorylationwas markedly reduced in the remnant kidney of Luci.1309 mice with UNPX and further reduced in the remnant kidney of sh3703 micewith UNPX compared with Luci.1309 mice without UNPX. AKT phosphorylation was also reduced in doxorubicin-treated Luci.1309mice and further reduced in doxorubicin-treated sh3703 mice. Bar graph shows the normalized ratio of phospho-to-total AKT banddensity plotted for each of the indicated group of mice. (B) Representative images of kidney sections from the indicated mice labeledwith an antibody against WT-1 to identify podocytes as well as TUNEL for apoptotic nuclei. Glomerular cells with colabeling of WT-1and TUNEL were detected in kidneys of sh3703 mice after 20 weeks of DOX induction (DOX 20w) and doxorubicin-injected sh3703mice induced with DOX. Arrows indicate cells with colocalization of the Hoescht nuclear stain, TUNEL, and WT-1. Scale bar, 50 mm.



Measurements of Serum CreatinineSerum creatinine was measured by an HPLC-based method as

described previously.49 Five microliters of serum per mouse was pro-

cessed as described and then assayed using a Shimadzu Prominence

HPLC unit (Columbia, MD) connected with a

cation exchange column (PRP-X200; Hamilton

Co., Reno, NV).

Histopathology andImmunohistochemistry Staining ofKidney TissueKidney 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 seg-

mental or global sclerosis, the severity of mesangial

expansion (graded on a semiquantitative scale from

0 to 3+), 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 com-

partment, we assessed the percentage of total renal

parenchyma occupied by tubular casts and/or

microcysts as well as the severity of interstitial

inflammation (graded on a semiquantitative

scale from 0 to 3+). For the latter parameter, in-

terstitial inflammation was graded as 0 (absent),

1+ (involving 1%–25% of the cortical paren-

chyma), 2+ (involving 25%–50%), and 3+ (involv-

ing. 50%).

Frozen sections were used for immunofluo-

rescence staining for nephrin, podocin, synapto-

podin, and WT-1, and images were taken by

using a Zeiss Axioplan 2 IE microscope.

Alcian Blue StainingDeparaffinized tissue sections were stained in 1%

Alcian blue (Sigma-Aldrich) solution and coun-

terstainedwith0.1% fast red (Vector) asdescribed

previously.51

TUNEL StainingTUNEL was performed using TUNEL labeling

mix (Promega Corp., Fitchburg, WI), according

to the manufacturer’s instructions. At least 30 glomeruli from each

mouse were photographed at 4003 magnification. The number of

WT-1–positive nuclei were counted and the cross-sectional area of

the glomerular tuft encompassing the WT-1–positive nuclei was

(C) Representative images of kidney sections stainedwithWT-1. Bar graphs below represent quantification of the density ofWT-1–labeledcells per glomerular tuft area (cells/1000 mm2). The density of WT-1 cells was significantly reduced for sh3703mice after 20 weeks of DOXinduction (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.


Table 3. Primers for RT-PCR

Gene Forward Reverse

Gapdh 59-GCCATCAACGACCCCTTCAT 59-ATGATGACCCGTTTGGCTCCNphs1 59-GTGCCCTGAAGGACCCTACT 59-CCTGTGGATCCCTTTGACATNphs2 59-CTTGGCACATCGATCCCTCA 59-CGCACTTTGGCCTGTCTTTGCldn3 59-GTCCGTCAGTTTTCGAAGGG 59-AGGATGGACACCACGATGAGSynpo 59-CTTTGGGGAAGAGGCCGATTG 59-GTTTTCGGTGAAGCTTGTGCNeph1 59-TCACAAGCAGGGCTTTAGGA 59-CTGGCTGAAGCGAGTCTGAGNeph3 59-ACATTTCCGGAACCCAGGAG 59-CCACAATCCGGACAGTAGGCCD2AP 59-TGGTGGAGTGGAACCCTGAA 59-GTGAGGTAGGGCCAGTCAAAP-cadherin 59-GATTTTGAGGCTCAGGACCA 59-GACCTTGGAAGGTGGAACAAInf2 59-CACCATCTCTCGGCTGTACC 59-CGAGTAGTTGACCACCGAGGFat1 59-CCAGGGAAGTCCGTTCACAA 59-TTGACTCTGGGTGGATTGCCActn4 59-TTCGACAACAAGCACACCAA 59-CCAGTGCCCCGCCATWt1 59-GAGAGCCAGCCTACCATCC 59-GGGTCCTCGTGTTTGAAGGAAJAM-A 59-CGCGTCGGGATTGTAACTGT 59-CACCGAACCCTTGCCTTGTAOccludin 59-AGGTGAGCACCTTGGGATTC 59-TTCAAAAGGCCTCACGGACACingulin 59-TGTGAATCCGGGAGCACTGA 59-TAGCACCCTCTGGCTCTGTAZO-1 59-TAGCACGGACAGTAGACACA 59-ATGGAAGTTGGGGTTCATAG



measured using ImageJ software, version 1.47. The number ofWT-1–

positive nuclei per 1000 mm2 of glomerular tuft area assessed was

calculated.

Transmission Electron MicroscopyMice were perfused with phosphate-buffered saline and then imme-

diately fixed in glutaraldehyde. Sections were mounted on a copper

grid and photographed under a Hitachi H7650 microscope.

Urine Albumin and CreatinineUrine creatinine was quantified using commercial kits from BioAssay

Systems (Hayward, CA). Urine albumin was determined using a com-

mercial assay fromBethyl Laboratory Inc. (Houston, TX).Urine albumin

excretion was expressed as the ratio of urine albumin to creatinine.

Coomasie Blue Staining of Urinary ProteinsUrine was denatured with Laemmli sample buffer and then resolved

on 10% SDS-PAGE. Proteins on the gel were detected by Coomassie

blue staining. Each lane contains 7.5 ml of urine.

Quantitative RT-PCROne microgram of total RNA extracted from glomerular extracts was

reverse transcribed to complementary DNA, which was used for

quantitative RT-PCR. Primers for RT-PCR were designed by using

Primer-Blast (National Center for Biotechnology Information) (see

Table 3). Gene expression was normalized to Gapdh and fold change

in expression relative to the control group was calculated using the

2-ΔΔCt method. Two technical replicates per gene were used.

Western Blot AnalysisWestern blotting was performed using specific antibodies for proteins

of interest as described previously.52

Statistical AnalysesData are expressed asmean6SEM. For comparison ofmeans between

three or more groups, two-way ANOVA with Bonferroni post-test

was applied. For comparisons of means between two groups, two-

tailed, unpaired t tests were performed. Prism 5 (GraphPad Software,

La Jolla, CA) was used for statistical analyses.

ACKNOWLEDGMENTS

We thankDr. PeterMundel (Massachusetts GeneralHospital, Boston,

MA) for sharing the synaptopodin antibody.

The study was supported by National Institutes of Health grants

5K08-DK082760, 1R01-DK098126, and 1R01-DK078897; Chinese

973 fund grant 2012CB517601; and Veterans Affairs Merit Review

Award 1I01-BX000345.

DISCLOSURESP.K.P. is the chief executive officer of Mirimus, Inc., which specializes in

engineering customized mouse models with reversible gene silencing.

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This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2014040405/-/DCSupplemental.





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