Overexpression of VEGF-A in podocytes of adult micecauses glomerular diseaseDelma Veron1, Kimberly J. Reidy2, Claudia Bertuccio1, Jason Teichman2, Guillermo Villegas2,Juan Jimenez3, Wa Shen2, Jeffrey B. Kopp4, David B. Thomas5 and Alda Tufro1

1Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut, USA; 2Department of Pediatrics, Albert EinsteinCollege of Medicine, Bronx, New York, USA; 3Analytical Imaging Facility, Albert Einstein College of Medicine, Bronx, New York, USA;4Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,Maryland, USA and 5Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA

We sought to examine the pathogenic role of excessive

VEGF-A expression in podocytes, since it has been reported

that diabetic nephropathy and other glomerular diseases are

associated with increased VEGF-A expression. The induction

of podocyte-specific VEGF164 overexpression in adult

transgenic mice led to proteinuria, glomerulomegaly,

glomerular basement membrane thickening, mesangial

expansion, loss of slit diaphragms, and podocyte effacement.

When doxycycline-mediated VEGF164 was stopped, these

abnormalities reversed. These findings were associated with

reversible downregulation of metalloproteinase 9 and

nephrin expression. Using transmission electron microscopy,

we established that VEGF-A receptor-2 (VEGFR2) was

expressed in podocytes and glomerular endothelial cells. We

also found that VEGF164 induced VEGFR2 phosphorylation in

podocytes. Further, we were able to co-immunoprecipitate

VEGFR2 and nephrin using whole kidney lysates, confirming

interaction in vivo. This implies that autocrine and paracrine

VEGF-A signaling through VEGFR2 occurs in podocytes and

may mediate the glomerular phenotype caused by VEGF164

overexpression. Thus, we suggest that podocyte VEGF164

overexpression in adult mice is sufficient to induce

glomerular filtration barrier structural and functional

abnormalities similar to those present in murine diabetic

nephropathy.

Kidney International advance online publication, 10 March 2010;

doi:10.1038/ki.2010.64

KEYWORDS: diabetic glomerulopathy; nephrin; podocyte; proteinuria; VEGF

Vascular endothelial growth factor-A (VEGF-A) is themajor determinant and regulator of angiogenesis.1 Inactiva-tion of a single VEGF-A allele causes embryonic lethality,showing that threshold levels of VEGF-A signaling arerequired for maintaining the differentiation, correct assem-bly, and maturation of endothelial cells.2,3 Similarly,podocyte-specific deletion of VEGF-A in mice showedthat over 50% allelic dose of VEGF-A is required for develop-ment and maintenance of a functional glomerular filtra-tion barrier.4 Conversely, massive VEGF-A overexpressionin podocytes caused glomerular collapse and perinatallethality.4 These developmental phenotypes precluded theanalysis of the role of VEGF-A expression in adult kidneydisorders.

Excessive renal VEGF-A expression has been documentedin diabetic nephropathy and glomerular diseases, whichare the leading causes of end-stage renal disease in humans.5

However, it is not clear whether changes in VEGF-Aexpression are a cause, a consequence, or a bystander inkidney diseases.5–7 Anti-VEGF antibody therapy reduced theseverity of diabetic nephropathy in rodents,8–10 whereas itinduced hypertension and glomerular disease in cancerpatients.11,12 VEGF-A was shown to accelerate the resolutionof experimental glomerulonephritis, ameliorate cyclosporinenephropathy, and protect remnant kidneys.13 Our previousin vitro studies showed that VEGF-A attracts endothelial cellsto developing glomeruli,14 promotes podocyte survivalthrough an autocrine pathway involving VEGF receptor-2(VEGFR2), induces podocin upregulation and its associationwith CD2AP.15 It is unclear whether autocrine podocyteVEGF-A signaling occurs in vivo. In this study we report thatinduction of moderate podocyte VEGF164 overexpressionin adult mice causes a glomerular disease characterized byglomerulomegaly, mesangial proliferation, glomerular base-ment membrane (GBM) thickening, loss of slit diaphragms,podocyte effacement, and proteinuria in the absence ofa diabetic milieu, which is reversible upon removal ofdoxycycline, and we provide insight into its molecularmechanism.

http://www.kidney-international.org o r i g i n a l a r t i c l e

& 2010 International Society of Nephrology

Received 6 October 2009; revised 2 January 2010; accepted 13 January

2010

Correspondence: Alda Tufro, Department of Pediatrics, Yale University

School of Medicine, 333 Cedar Street, P.O. Box 208064, New Haven,

Connecticut 06520–8064, USA. E-mail: alda.tufro@yale.edu

Kidney International 1

RESULTSInducible podocyte VEGF164 overexpression in vivo

To analyze the pathogenic role of VEGF-A in glomerulardisease and circumvent early lethality, we developed an indu-cible, podocyte-specific, tetracycline-regulated VEGF164 over-expression model (podocin-rtTA:tet-O-VEGF164) by breedingpodocin-rtTA mice16 with tet-O-VEGF164 mice.17 In podocin-rtTA:tet-O-VEGF164 mice, immunoreactive VEGF-A localizedto podocytes was increased upon doxycycline induction(Supplementary Figure S1a). VEGF-A protein in whole kidneylysate measured by enzyme-linked immunosorbent assayincreased twofold in doxycycline-treated podocin-rtTA:tet-O-VEGF164 mice (0.1±0.005 vs 0.2±0.01 pg/mg protein, �dox,n¼ 8 vs þ dox, n¼ 13, Po0.05, Supplementary Figure S1b),whereas urine VEGF-A increased 15-fold (13.1±1.2 vs206.7±110.2 pg/day, �dox, n¼ 9 vs þ dox, n¼ 6, Po0.05).

Podocyte VEGF164 overexpression causes proteinuria

Adult mice overexpressing podocyte VEGF164 for 1 monthdeveloped proteinuria (TOPO þ dox, Figure 1a–c). Incontrast, age-matched uninduced podocin-rtTA:tet-O-VEGF164 mice (TOPO �dox) did not develop proteinuria(Figure 1a and c), nor did single transgenic mice receivingdoxycycline (CON þ dox, Figure 1a), indicating that neitherthe transgenes nor the doxycycline per se caused proteinuria.Notably, VEGF164-induced proteinuria was reversible 2 weeksafter doxycycline removal from the diet (Figure 1b and c),suggesting a cause-and-effect relationship between VEGF164

and proteinuria. Moreover, proteinuria positively correlatedwith increased kidney VEGF-A content in mice overexpres-sing VEGF164 (Figure 1d), but did not correlate with VEGF-Aplasma levels, which remained unchanged (24±1.9 vs22±2.2 pg/ml, �dox, n¼ 9 vs þ dox, n¼ 12, P¼NS),suggesting that podocytes are not the main source ofcirculating VEGF-A, and that podocyte VEGF164 effects arelocal rather than systemic. VEGF164-induced albuminuriawas detected by immunoblotting (Figure 1c), and quantifiedby enzyme-linked immunosorbent assay (Figure 1e). Theurine albumin/creatinine ratio was 4- to 16-fold higher indoxycycline-induced than in uninduced podocin-rtTA:tet-O-VEGF164 mice (P¼ 0.0027, Figure 1e), consistent with thedata obtained by dipstick and western analysis.

Podocyte VEGF164 overexpression induces glomerulomegaly

We examined the morphologic changes associated withVEGF164-induced proteinuria. Podocyte VEGF164 overexpres-sion in adult mice induced glomerular enlargement,thickened GBM, and mild mesangial expansion (Figure 2a,Supplementary Figure S2a). Morphometric analysis confir-med VEGF164-induced glomerulomegaly, with 1.8- to 2-foldincrease in mean glomerular volume, which was partiallyreversible upon cessation of VEGF164 overexpression (Figure2a and b), and showed a right shift in the distributionof glomerular volumes indicating widespread enlargementand increased size heterogeneity (Figure 2b) and increasedmesangial matrix area (Supplementary Figure S2a).

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Figure 1 | Podocyte VEGF164 overexpression in adult mice causes proteinuria. (a) Podocin-rtTA:tet-O-VEGF164 adult mice induced withdoxycycline for 1 month (TOPO þdox) develop proteinuria, as compared with uninduced podocin-rtTA:tet-O-VEGF164 mice (TOPO �dox)and single transgenic controls receiving doxycycline or not (CON þdox and CON �dox). (b) Proteinuria is reversible upon removalof doxycycline induction (TOPO þdox vs TOPO ±dox). (c) Albumin immunoblot showing albuminuria in induced TOPO þdox, noalbuminuria in uninduced TOPO �dox, and minimal albuminuria in TOPO ±dox. (d) Proteinuria has a significant positive correlation withkidney vascular endothelial growth factor-A (VEGF-A) content in induced TOPO mice (þdox), whereas it does not in uninduced TOPO mice(�dox). (e) Urine albumin/creatinine ratio (mg/mg) in TOPO þdox (n¼ 8) is more than fourfold higher than in TOPO �dox (n¼ 6). Noticethat the scale on the right side of the figure is depicted to include the sample labeled (o), which showed 1562 mg/mg, and horizontal barsdepict the median for each experimental group. In all graphs *indicates Po0.05 TOPO þdox vs TOPO �dox mice, and TOPO þdox vsTOPO ±dox mice.

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Podocyte VEGF164 overexpression induces GBM thickeningand podocyte effacement in adult mice

To further evaluate these glomerular abnormalities, kidneyswere examined by transmission electron microscopy (TEM)1 month after VEGF164 transgene induction. TEM revealeda dramatic increase in GBM thickness and irregularity,and absence of lamina rara interna and externa in kidneysoverexpressing VEGF164 (Figure 2c, panels C and F) whencompared with controls: podocin-rtTA:tet-O-VEGF164 with-out doxycycline (Figure 2c, panels I and L) and singletransgenics on doxycycline (Figure 2c, panels A, B, D, and E)or off doxycycline (Figure 2c, panels G, H, J, and K),which were all normal. In addition to the GBM changes,doxycycline-induced mice showed partial foot processeffacement and fusion (Figure 2c, panels C and F), andincreased mesangial matrix deposition (SupplementaryFigure S2b). These ultrastructural changes were observedin all kidneys examined and involved all glomeruli (nX3/kidney). Surprisingly, no endothelial cell swelling or damagewas observed by TEM and fenestrations were intact inkidneys from doxycycline-treated mice.

Morphometric analysis showed a twofold increase inGBM thickness in induced podocin-rtTA:tet-O-VEGF164 mice(TOPO þ dox) when compared with uninduced controls(TOPO �dox, Figure 3a). Podocyte abnormalities wereapparent along most of the GBM length, and resulted inan average 66% increase in foot process width (268±5 vs445±28 nm, TOPO �dox vs TOPO þ dox, Po0.001),a 40% decrease in slit diaphragm density, and a threefoldincrease in occluding junction density (Figure 3b and c).VEGF164-induced GBM, podocyte, and mesangial abnor-malities were reversible at 2 weeks after doxycycline removal,underscoring the cause-and-effect relationship betweenexcess podocyte VEGF-A and development of glomerulardisease (Figure 3a–d, Supplementary Figure S1b).

We determined that random non-fasting plasma glucoselevels were normal in podocin-rtTA:tet-O-VEGF164 on doxy-cycline (157±24 mg/dl, n¼ 8), and not different fromglucose levels in uninduced mice (179±11 mg/dl n¼ 9,P¼NS). We also determined that systolic blood pressurewas normal in doxycycline-induced and uninduced podocin-rtTA:tet-O-VEGF164 adult mice (104±5 mm Hg, n¼ 5 vs99±5 mm Hg, n¼ 5, P¼NS).

Podocyte VEGF164 overexpression decreases renal expressionof matrix metalloproteinase 9 (MMP-9) and nephrin

GBM thickening and podocyte effacement are suggestive ofabnormal GBM composition and altered podocyte proteinexpression,18 respectively. Thus, we examined the expressionof major GBM components and podocyte-specific proteins.We determined that collagen IV and laminin had similarlocalization in the glomerular and tubular GBM of miceoverexpressing podocyte VEGF164 and uninduced controls(Figure 4a). Real-time PCR revealed that collagen IV a3,a4, a5, and laminin a1, a5, and b1 kidney mRNA levelswere unchanged after 1 month of VEGF164 overexpression

(Figure 4b). Similarly, Ndst1 and 2 and syndecan wereunchanged (Figure 4b). Integrin a3 and a5 kidney mRNAlevels did not change significantly, whereas integrin b1mRNA was significantly decreased after 1 month of VEGF164

overexpression (Figure 4b). However, b1-integrin proteinlevels were not significantly altered (data not shown). Incontrast, total kidney MMP-9 mRNA, protein level andactivity, as well as glomerular immunoreactivity weredecreased significantly in mice overexpressing podocyteVEGF164 (Figure 4b, c, f, and g). MMP-9 downregulationwas abrogated by removal of VEGF164 induction (Figure 4c).As doxycycline is a known inhibitor of metalloproteinases,19

we also examined the effect of doxycycline on renal MMP-9expression in single transgenic mice (CON þ dox). MMP-9was not altered in single transgenics receiving doxycycline(Figure 4c and g). Hence, VEGF164 overexpression specificallyinduced downregulation of MMP-9 expression and activity(Figure 5c and g). MMP-2 protein level remained unchanged inmice overexpressing podocyte VEGF164 (Figure 5d). Collectively,these data suggest that podocyte VEGF164 overexpression inadult mice reduces GBM turnover, as implied by the reversibledecrease in MMP-9 mRNA, protein, and activity levels.

Podocyte VEGF164 overexpression induced a significantdecrease in nephrin mRNA, protein levels, and immunor-eactivity, whereas nephrin phosphorylation increased them(Figure 4b, e, and f). Removal of doxycycline inductionabrogated nephrin downregulation and phosphorylation,underscoring the cause–effect relationship between VEGF164

overexpression and nephrin dysregulation (Figure 4e). Incontrast, podocin expression levels remained unchanged(data not shown), suggesting that VEGF164 overexpressiondoes not cause overall dysregulation of slit diaphragmproteins or podocyte loss. The concomitant reversibilityof nephrin downregulation and phosphorylation, podocyteeffacement, and proteinuria upon removal of VEGF164

induction suggests that nephrin dysregulation is the mole-cular basis for the VEGF164-induced podocyte effacement.

Podocytes express VEGFR2, and VEGFR2 interacts withnephrin in vivo

We next analyzed whether the ultrastructural changesobserved in podocytes from doxycycline-induced podocin-rtTA:tet-O-VEGF164 mice were caused by autocrine VEGF164

signaling. We identified the glomerular cell types expressingVEGFR2, the main VEGF-A signaling receptor, by TEM andhistochemistry in adult Flk-1þ /� mice, which carry one Flk-1allele with a LacZ gene in-frame insertion.20 As shown inFigure 5a–c, Flk-1þ /� mice express b-Gal in lieu of VEGFR2in both glomerular endothelial cells and podocytes. More-over, b-Gal electron-dense precipitates were observed in thepodocyte cell body (Figure 5b) and foot processes, closelyadjacent to slit diaphragms (Figure 5c). Neighboringmesangial cells were mostly devoid of b-Gal electron-denseprecipitates, and tubular cells, as well as unstained samplesfrom the same kidneys were negative (Figure 5d). Immuno-electron microscopy confirmed VEGFR2 protein expression

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D Veron et al.: VEGF overexpression causes glomerular disease o r i g i n a l a r t i c l e

in podocyte foot processes and cell bodies as well as inendothelial cells (Figure 5e and f). Tyrosine phosphorylatedVEGFR2 was detected in glomeruli from control mice butrarely colocalized with nephrin (Figure 5g, �dox), whereas

phosphorylated VEGFR2 was increased and colocalized withnephrin in podocytes from VEGF164-overexpressing mice(Figure 5g, þ dox), suggesting that increased VEGF164 signalsinduced VEGFR2 phosphorylation in podocytes.

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To examine a possible crosstalk between VEGF-A andnephrin signaling pathways we performed immunoprecipita-tion experiments. VEGFR2 co-immunoprecipitated withnephrin in kidney lysates (Figure 5h), indicating thatVEGFR2 and nephrin interact in vivo. Moreover, purifiedFLAG-tagged VEGFR2 protein associated with endogenousnephrin (Figure 5i), confirming the interaction in vitro.

Together, our findings show that VEGFR2 is expressed inpodocytes and interacts with nephrin in vivo, suggesting thatthe phenotype induced by podocyte VEGF164 overexpressionresults from autocrine and paracrine signals, which involvenephrin and MMP-9 dysregulation, nephrin–VEGFR2 inter-action, and VEGF164-induced phosphorylation of bothproteins in vivo.

Figure 2 | Podocyte VEGF164 overexpression in adult mice causes glomerulomegaly, GBM thickening, podocyte effacement, andSD loss. (a) Gomorri’s silver methenamine stain in podocin-rtTA:tet-O-VEGF164 uninduced mice (TOPO �dox), mice induced for 1 month(TOPO þdox), and mice induced for 1 month, and then off induction for 2 weeks (TOPO ±dox), showing enlarged glomeruli with thickerGBM in TOPO þdox mice, and regression of these changes in TOPO ±dox mice. (b) Morphometric analysis of glomerular size: podocin-rtTA:tet-O-VEGF164 induced mice (TOPO þdox) had approximately twofold larger glomerular volume than uninduced mice (TOPO �dox)and controls (CON þdox); the glomerular enlargement partially subsided after 2 weeks off doxycycline (TOPO ±dox). The distribution ofglomerular volumes showed significantly increased size heterogeneity in induced mice (TOPO þdox), in addition to overall enlargement, asshown by the mean±s.e.m. difference (inset). *Po0.05, TOPO þdox vs TOPO �dox-treated mice. (c) TEM showing kidneys from podocin-rtTA mice (left panels), tet-O-VEGF164 mice (middle panels), and podocin-rtTA:tet-O-VEGF164 mice (right panels) induced with doxycycline for1 month (þdox), and uninduced (�dox). All left and middle panels (single transgenics) show normal kidney ultrastructure, whereas inducedpodocin-rtTA:tet-O-VEGF164 kidneys (top panels C and F) show diffuse GBM thickening (*), mesangial expansion, and podocyte effacement.Notice that uninduced podocin-rtTA:tet-O-VEGF164 kidneys are normal (bottom panels I and L). Scale bars¼ 1 mm in panels A–C and G–I, and¼ 200 nm in panels D–F and J–L. EC, endothelial cell; efp, effaced foot process, fp, foot process; GBM, glomerular basement membrane;P, podocyte; RBC, red blood cell; SD, slit diaphragm; TEM, transmission electron microscopy.

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Figure 3 | Ultrastructural abnormalities caused by podocyte VEGF164 overexpression are reversible. Morphometric comparisons weremade among podocin-rtTA:tet-O-VEGF164 mice uninduced, induced, and that in which induction was removed to test reversibility (TOPO�dox, TOPO þdox, and TOPO ±dox, respectively). (a) Glomerular basement membrane (GBM) thickness expressed in nm, showing atwofold increase in mice overexpressing VEGF164; (b) foot process width, a measure of podocyte effacement, increased 460% in miceoverexpressing VEGF164; (c) density of podocyte foot process interactions (‘pores’), measured as the number of slit diaphragms (SD) oroccluding junctions (OJ) per mm of GBM length, showing mirror image changes: increase in OJ and decrease in SD in mice overexpressingVEGF164. Changes in all these morphometric parameters were reversible upon removal of VEGF164 induction (TOPO ±dox). Total number ofmeasurements (n) for each parameter in TOPO �dox, TOPO þdox, and TOPO ±dox mice were as follows: GBM thickness: n¼ 241, 431, and272; foot process width: n¼ 422, 584, and 311; SD: n¼ 378, 409, and 254; and OJ: n¼ 32, 165, and 40. *Po0.05, TOPO þdox vs TOPO �doxor TOPO ±dox; **Po0.05, TOPO ±dox vs TOPO �dox. (d) Representative transmission electron microscopy (TEM) showing the reversibleultrastructural abnormalities quantified in (a–c), large * points out the thickened GBM. Notice the lack of endothelial cell abnormalities, scalebars¼ 200 nm.

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D Veron et al.: VEGF overexpression causes glomerular disease o r i g i n a l a r t i c l e

DISCUSSION

In this study we show that podocyte VEGF164 overexpressionin adult mice results in proteinuria and a distinct glomerularphenotype characterized by glomerulomegaly, mesangial

expansion, podocyte foot process effacement, and thickenedGBM. The cause-and-effect relationship between podocyteVEGF164 overexpression and glomerular phenotype wasclearly shown by the reversibility of the functional,

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Figure 4 | Podocyte VEGF164 induction in adult mice decreases matrix metalloproteinase 9 (MMP-9) and nephrin. (a) Fluorescentimmunohistochemistry showing that collagen IV and laminin are normally localized in mice overexpressing VEGF164. (b) Real-time PCRdata showing relative mRNA expression of glomerular basement membrane (GBM) components and nephrin in mice overexpressingVEGF164/uninduced mice of the same genotype (TOPO þ dox/ TOPO �dox), using pooled RNA from 9 and 8 mice, respectively. Data areexpressed as mean fold changes±s.e.m., n¼ 3, *Po0.05. (c) Western blot showing decreased MMP-9 protein in mice overexpressingVEGF164 (TOPO þdox) vs uninduced mice (TOPO �dox), controls are single transgenic on dox (CON þdox), MMP-9 downregulation isreversible (TOPO ±dox). (d) Western blot showing no change in MMP-2 levels in TOPO þdox, TOPO �dox, and TOPO ±dox. (e) Westernblots showing decreased nephrin expression and enhanced phosphorylation in VEGF164-overexpressing (TOPO þdox) vs uninducedkidneys (TOPO �dox), nephrin dysregulation is reversible (TOPO ±dox). In (c–e) whole kidney lysate was pooled from 13 (TOPO þdox) and8 (TOPO �dox) podocin-rtTA:tet-O-VEGF164 mice, 8 single transgenic mice (CON þdox), and 6 podocin-rtTA:tet-O-VEGF164 mice ondoxycycline for 1 month and off for 2 weeks (TOPO ±dox). Actin and tubulin blots are shown to document equal loading. Densitometricanalysis shows mean±s.e.m. fold change in arbritrary units when compared with controls, nX4. (f) MMP-9 and nephrinimmunohistochemistry. Podocin-rtTA:tet-O-VEGF164 induced (þdox) showed decreased MMP-9 and nephrin when compared withuninduced mice (�dox), with unchanged localization, scale bars¼ 20mm. (g) MMP-9 zymogram shows decreased MMP-9 activity in kidneysoverexpressing VEGF164 (TOPO þ dox), positive control¼ recombinant mouse MMP-9 (2.5 ng).

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morphologic, and molecular changes observed upon removalof doxycycline.

Podocyte VEGF164 overexpression-induced albuminuriawas 4- to 16-fold higher than control, the proteinuria had a

significant positive correlation with the kidney VEGF-Acontent, and most importantly, was fully reversible uponcessation of doxycycline. The association of glomerulome-galy, mildly increased mesangial matrix, GBM thickening,

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Figure 5 | Vascular endothelial growth factor receptor-2 (VEGFR2) is expressed in podocytes and endothelial cells. (a) Transmissionelectron microscopy (TEM) of LacZ-stained kidneys from adult Flk-1þ /� LacZ mice showing extensive b-Gal electron-dense deposits inglomerular capillaries and in podocytes, scale bar¼ 1mm; (b) b-Gal electron deposits in a podocyte body, scale bar¼ 500 nm; and (c) b-Galelectron deposits in foot processes, scale bar¼ 200 nm. (d) TEM of the same kidney, not stained for LacZ (negative control), scale bar¼ 200 nm.(e) Immunogold staining for VEGFR2 showing immunoreactive VEGFR2 in foot processes, scale bar¼ 200 nm, inset: higher magnification oftwo foot processes flanking a slit diaphragm showing immunogold VEGFR2 staining; (f) immunoreactive VEGFR2 in podocyte body andendothelial cell, scale bar¼ 500 nm. cap, capillary lumen; EC, endothelial cell; fp, foot process; P, podocyte. 4b Gal electron-dense deposits.(g) VEGF164 overexpression induces VEGFR2 phosphorylation in podocytes: immunoreactive phosphorylated (P)-VEGFR2 (green) is increased inglomeruli from VEGF164-overexpressing mice (þdox) as compared with uninduced glomeruli (�dox); nephrin (red) and P-VEGFR2 (green)colocalize (yellow) in podocytes from VEGF164-overexpressing mice (þdox). Scale bars¼ 20mm. (h) VEGFR2 and nephrin interact in vivo:immunoprecipitation (IP) with anti-VEGFR2 (VR2) antibody followed by western blotting (WB) with anti-nephrin antibody and VEGFR2 antibodyshowing co-immunoprecipitation; reverse co-IP experiment: IP with anti-nephrin antibody, followed by WB with anti-nephrin and anti-VEGFR2antibody; VR2, VEGFR2; NS, normal rabbit serum (IP negative control); input, whole kidney lysate. (i) VEGFR2 and nephrin interact in vitro: IPwith anti-nephrin, followed by WB with anti-Flag and anti-nephrin antibodies, showing that nephrin immunoprecipitate interacts with purifiedFlag-tagged VEGFR2; input 1, whole kidney lysate; input 2, Flag-VEGFR2 transfected COS cell lysate.

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D Veron et al.: VEGF overexpression causes glomerular disease o r i g i n a l a r t i c l e

and foot process widening with loss of slit diaphragmsinduced by podocyte VEGF164 overexpression in adult micewere similar to the classic glomerular abnormalities reportedin early diabetic nephropathy in humans and rodents.18,21

However, normal glucose levels in mice overexpressingpodocyte VEGF164 ruled out diabetes mellitus, and theabsence of hypertension ruled out VEGF164-induced hyper-tensive nephrosclerosis.

The dramatic GBM thickening and the lack of significantendothelial cell damage observed by TEM in adult miceoverexpressing podocyte VEGF164 clearly differ from theglomerular phenotypes described in VEGF-induced collap-sing glomerulopathy,4 and a rabbit model of liver and kidneyVEGF overexpression,22 probably because of timing of VEGFoverexpression, transgene dosage, and species differences.Our data show pathogenic effects of moderately increasedpodocyte VEGF164 in adult mice causing a distinct GBM andpodocyte phenotype that spares the glomerular endothelialcells. Furthermore, we show that the molecular mechanismresponsible for this phenotype involves decreased expressionof collagenase MMP-9 and nephrin.

We provide evidence of decreased MMP-9 expression andactivity in podocyte VEGF164-overexpressing mice. Asdoxycycline is a known inhibitor of metalloproteinases,19,23

we also examined the effect of doxycycline on renal MMP-2and MMP-9 expression in single transgenic mice. We deter-mined that downregulation of MMP-9 expression is clearlyinduced by VEGF164 overexpression, as it was abrogated byremoval of VEGF164 transgene induction, and remainedstable in all control mice receiving doxycycline. Surprisingly,MMP-2 protein expression levels were not altered in miceoverexpressing podocyte VEGF164. Taken together, thesefindings suggest that podocyte VEGF164 overexpressionin vivo reduces GBM turnover, rather than enhancing synthesisof GBM components such as collagen IV and perlecan, assuggested by in vitro data.24,25 Alternatively, the thickenedGBM could be due to deposition of another collagen typenot normally expressed in the GBM, such as collagentype III,18,26,27 but our quantitative reverse transcriptase-PCRdata do not support this possibility (data not shown).

Consistent with partial podocyte effacement and fusion,we detected downregulation of nephrin expression andincreased nephrin phosphorylation in podocyte VEGF164-overexpressing kidneys, which were reversible upon doxy-cycline removal, suggesting that increased VEGF-A signalingalters the nephrin signaling complex function, and therebypodocyte phenotype. Decreased nephrin expression has beendescribed in multiple human proteinuric diseases,28,29

including diabetic nephropathy.18,21,30 Increased nephrinphosphorylation has been observed during developmentand in a rodent model of foot process effacement,31 whereasdecreased nephrin phosphorylation was reported in ratpuromycin aminonucleoside nephrosis and in human mini-mal change disease.32 As deletion of Nck, an adaptor proteinthat selectively binds to phosphorylated nephrin, leads tocongenital nephrotic syndrome,33 it seems that signals

mediated by phosphorylated nephrin are necessary for theestablishment of mature podocyte foot processes and slitdiaphragms, whereas the role of dysregulated nephrinphosphorylation in postnatal glomerular diseases is less clear.

We sought to identify the glomerular cell types expressingVEGFR2, the main VEGF-A signaling receptor,34–36 tounderstand whether the ultrastructural abnormalities andthe associated nephrin dysregulation observed in podocyteVEGF164-overexpressing mice were caused by autocrine orparacrine signaling. Using TEM combined with either b-Galstaining37 or immunogold staining, we detected VEGFR2expression in glomerular endothelial cells and in podocytes,extensively distributed in the cell body, foot processes, andclose to slit diaphgrams. Furthermore, increased activatedVEGFR2 was detected by immunohistochemistry in podo-cytes from mice overexpressing VEGF164, suggesting thatincreased VEGF164 signals induced VEGFR2 phosphorylationin situ. Localization of VEGFR2, but not VEGFR1, topodocytes by immunogold staining was recently reported.38

We identified a novel interaction between endogenousVEGFR2 and nephrin by co-immunoprecipitation in vivo,and confirmed the association between purified Flag-taggedmouse VEGFR2 and endogenous nephrin from kidneylysates, suggesting a crosstalk between VEGF-A and nephrinpathways. As nephrin expression is limited to podocytes,this interaction independently confirms the expression ofVEGFR2 in podocytes. Moreover, our observation thatpodocyte VEGF164 overexpression induced nephrin phos-phorylation supports the notion that VEGFR2–nephrininteraction may have functional relevance, and represents anovel mechanism for crosstalk between VEGF-A and nephrinsignaling pathways. Collectively, our findings suggest thatpodocyte VEGF164 signaling is both autocrine and paracrine,and provide a plausible interpretation for the podocytephenotypic changes and the nephrin dysregulation observedin podocyte VEGF164-overexpressing kidneys. Further studiesare warranted to define the biochemistry of VEGFR2–nephrininteraction, and how it modulates nephrin signaling. AsFlk-1-null mice are embryonic lethal and heterozygous micedo not have a vascular or glomerular phenotype,20 only acomplete, inducible, and podocyte-specific VEGFR2 knock-out would definitively elucidate whether the mechanismproposed in this study is required for the integrity of theglomerular filtration barrier. Our studies do not excludepossible effects of VEGF164 overexpression through VEGFR1,which is considered to exert an effect as a negative modulatorof VEGFR2 signaling in most tissues by sequestering VEGF-Aligand, and thereby decreasing VEGFR2 activation, althoughthe mechanisms involved remain controversial.34–36

In summary, induction of podocyte VEGF164 overexpres-sion in the adult mouse causes proteinuric glomerular diseasethat mimics early diabetic nephropathy, in the absence of adiabetic milieu. Reversibility of these changes upon removalof doxycycline provides proof of principle of the causalrelationship. Our findings are consistent with an autocrinepathogenic role of increased podocyte VEGF-A in this

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process, involving nephrin and MMP-9 dysregulation,nephrin–VEGFR2 interaction, and VEGF164-induced phos-phorylation of both proteins in vivo. Our inducible modelshould prove useful to identify the downstream mechanismsof VEGF-induced glomerular disease in adult mice, and totest therapeutic interventions.

MATERIALS AND METHODSInducible, podocyte-specific VEGF164 overexpression in miceAll mouse protocols were approved by the Committee for AnimalUse and Experimentation of Albert Einstein College of Medicine. Wegenerated tetracycline-regulated transgenic mice (podocin-rtTA:tet-O-VEGF164) that overexpress VEGF-A in podocytes upon inductionwith doxycycline by breeding podocin-rtTA mice16 with tet-O-VEGF164 mice,17 which carry FVB background. Transgenic micewere genotyped by reverse transcriptase-PCR using reportedprimers.16,17 Adult podocin-rtTA:tet-O-VEGF164 mice (2–3 monthsold) received doxycycline (0.625 mg/g chow, Harlen-Teklar,Madison, WI, USA) for 1 month (TOPO þ dox, n¼ 14).Uninduced podocin-rtTA:tet-O-VEGF164 (TOPO �dox, n¼ 9),induced single transgenics (CON þ dox, n¼ 4), and uninducedsingle transgenics (CON �dox, n¼ 4) served as controls. Additionalpodocin-rtTA:tet-O-VEGF164 mice were induced with doxycycline for1 month, and then switched to regular diet for 2 weeks to examinethe reversibility of VEGF164-induced changes (TOPO ±dox, n¼ 6).Blood pressure was measured using the Coda Noninvasive-Blood-Pressure-System (Kent Scientific, Torrington, CT, USA).39

Proteinuria and VEGF-A analysisMice were anesthetized with ketamine, and blood and urine sampleswere obtained from venous and bladder puncture, kidneys wereharvested for phenotype analysis, and mice were then killed byexsanguination. Urinary creatinine was measured using a colori-metric assay (Oxford Biomedical Research, Oxford, MI, USA).Albuminuria was assessed by dipstick (Albustix; Bayer, Elkhart, IN,USA), enzyme-linked immunosorbent assay (Albuwell; Exocell,Philadelphia, PA, USA), and immunoblotting. Urine samples(normalized for creatinine) were resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotted withanti-bovine serum albumin antibody (Upstate Biotechnology, LakePlacid, NY, USA). Serum and whole kidney VEGF-A were measuredby enzyme-linked immunosorbent assay (mVEGF-A, R&D, Minnea-polis, MN, USA), following the manufacturer’s protocol. Serumglucose was measured by glucose oxidase biosensor (One-Touch-Ultra-2 Blood Glucose-Meter, LifeScan, Milpitas, CA, USA).

Histology and morphometryKidneys were processed for light and transmission electron micro-scopy or frozen in isopentane and mounted in optimum cuttingtemperature compound (Sakura, Torrance, CA, USA). Hematoxylin/eosin and Gomorri’s silver methenamine staining were performed.

Morphometric analysis. Glomerular diameters were measuredin 135±5 gloms/section at � 400 magnification. Glomerularvolume was determined as Gv¼ b/k.(p.r2)3/2, where b¼ 1.38 is theshape coefficient for spheres, k¼ 1.1 is the size distributioncoefficient, and (p.r2) is the glomerular area.26 Measurements werecarried out in four mice per group. Slit diaphragms, foot processwidth, and GBM thickness were measured on TEM scans usingImage J, Bethesda, MD, USA (NIH). Measurements were madeperpendicular to the axis of the GBM every 500 nm on 30–50 mm

GBM length/5K images. Occluding junctions and slit diaphragmswere counted and normalized to GBM length (mm), termed ‘OJ orSD density’. TEM measurements were carried out in five mice pergroup; counts for each parameter are depicted in figure legends.

ImmunohistochemistryKidneys were embedded in optimum cutting temperature com-pound, and snap-frozen in dry ice/isopentane or fixed in formalinand paraffin embedded. Paraffin sections were deparaffinized,incubated in 10 mM citrate, blocked, and incubated with anti-VEGFantibody (Dako, Carpinteria, CA, USA, M7273), anti-nephrin(Fitzgerald, North Acton, MA, USA, 20R-NP002), and anti-phospho-VEGFR2tyr1175 (Cell Signaling, Danvers, MA, USA,19A10). Bound primary antibodies were visualized by immunoper-oxidase (Dako EnVisionþ System), counterstained with hematox-ylin, or by fluorescent-labeled (Cy2 or Cy3, Jackson Immuno-research, West Grove, PA, USA) secondary antibodies. Cryosections(6mm) were fixed in cold acetone, permeabilized with 0.3% Triton-X, blocked, and incubated with primary antibodies: anti MMP-9(ab38898, Abcam, Cambridge, MA, USA), anti-podocin (gift from P.Mundel, University of Miami School of Medicine), anti-collagen IV(Southern Biotech, Birmingham, AL, USA) and anti-laminin(Sigma, St Louis, MD, USA), followed by appropriate Cy2- orCy3-labeled anti-rabbit, anti-mouse, or anti-goat secondary anti-bodies. All sections were examined by confocal microscopy (FV300,Olympus, Center Valley, PA, USA).

Transmission electron microscopy, b-Gal stain, andimmunoelectron microscopyKidney cortex was minced to 2 mm cubes, fixed with 2%paraformaldehyde, 2.5% glutaraldehyde in 0.1 M sodium-cacodylatebuffer, postfixed with 1% OsO4 followed by 2% uranyl acetate,dehydrated through a graded series of ethanol, and embeddedin LX112 (Ladd Research, Williston, VT, USA). Thin sections(80 nm) were cut and stained with 0.5% uranyl acetate and 0.5%lead citrate. For TEM b-Gal stain, kidneys were fixed and processedfor LacZ stain as described,20 postfixed as above, and processed forTEM using standard techniques, except that uranyl acetate and leadcitrate steps were omitted, as they obscure the detection of b-Galprecipitates.37 For immunogold labeling, kidneys were fixed with4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M sodiumcacodylate buffer and subsequently processed as for TEM andembedded in LR-white resin (London Resin Co, Basingstoke, UK).Sections were blocked with 0.05 M glycine, followed by goat blockingbuffer (Aurion, Nl), incubated with anti-VEGFR2 polyclonal anti-body (SC no. 315 or SC no. 504, Santa Cruz, CA, USA), incubatedwith 10 nm goat anti-rabbit gold (Aurion, Wageningen, The Nether-lands), counterstained with 0.5% uranyl acetate and 0.5% lead citrate,and examined using a JEOL 1200EX TEM (Tokyo, Japan).

Real-time PCRTotal RNA was isolated from whole kidney cortex from podocin-rtTA:tet-O-VEGF164 mice receiving doxycycline (TOPO þ dox,n¼ 9) or not (TOPO �dox, n¼ 8 age-matched) using TrizolReagent (Invitrogen, Carlsbad, CA, USA). After DNAse digestion,reverse transcription was performed using Quantitect ReverseTranscription Kit (Qiagen, Valencia, CA, USA). Reverse transcrip-tion products were combined into separate pools (TOPO þ dox andTOPO �dox). Real-time PCR amplifications were performed usingSYBR-Green PCR-Master-Mix (Applied Biosystems, Foster City, CA,USA), appropriate primers (Supplementary Table 1), and an

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Eppendorf Realplex2 Mastercycler (Hamburg, Germany). The cyclethreshold (Ct) of amplified products was detected using themanufacturer’s software. Samples were run in duplicate andexperiments were repeated a minimum of three times. Relativequantification was determined using the DCt method.40 Data werenormalized to glyceraldehyde-3-phosphate dehydrogenase andexpressed as fold change relative to uninduced podocin-rtTA:tet-O-VEGF164 mice (TOPO þ dox/TOPO �dox).

Immunoblotting, immunoprecipitation, and zymogramKidneys from age-matched podocin-rtTA:tet-O-VEGF164 mice recei-ving doxycycline (TOPO þ dox, n¼ 13) or not (TOPO �dox,n¼ 8) were lysed in RIPA buffer þ protease inhibitors (Roche,Indianapolis, IN, USA) þ 1 mM Na3VO4 þ 1 mM NaF. Proteinconcentration was determined by bicinchoninic acid assay (Sigma).Equal amount of protein lysate from individual kidneys werecombined into separate pools (þ dox and �dox), and 100–150 mgprotein/lane were resolved in 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Primary antibodies used forimmunoblotting were: anti-nephrin (20R-NP002, Fitzgerald); anti-phosphorylated nephrin;31 anti-MMP-2 (Chemicon, Billerica, MA,USA); and anti-MMP-9 (Chemicon). Anti-actin (A2066, Sigma) oranti-tubulin (sc-58667, Santa Cruz) was used as a control forprotein loading. For nephrin and VEGFR2 immunoprecipitationexperiments, 1 mg protein from whole kidney lysate was resus-pended in immunoprecipitation buffers as described,31,41 with slightmodifications (1% Triton X-100, 1% NP-40, 0.5% deoxycholate,10 mM Tris, pH 8, 0.15 M NaCl, 1 mM EDTA, 50 mM NaF and 1 mM

Na3VO4, protease inhibitor cocktail), pre-cleared, and incubatedwith polyclonal nephrin31 and VEGFR2 (sc-504, Santa Cruz)antibodies. Some nephrin immunoprecipitation experiments werefollowed by overnight incubation with 1 mg Flag-VEGFR2 proteinpurified from COS cells transfected with Flag-mouse VEGFR2,immunoblotted, and VEGFR2 was detected by anti-Flag monoclonalantibody (Sigma). Rabbit serum was used as negative control.Lysates from COS cells and whole kidney were used as positivecontrols. Secondary antibodies were anti-rabbit or anti-mouse-horseradish peroxidase-IgG (Amersham), detected by chemolumi-nescence (Amersham). Kidney lysate pools (100 mg) were denaturedin sodium dodecyl sulfate buffer, resolved in zymogram gels (Novex-Zymogram-Gel; Invitrogen), renatured, developed, and stainedfollowing the manufacturer’s instructions. Positive control wasrecombinant mouse MMP-9 (R&D). Gelatinolytic activity revealedclear bands on a blue background.

Statistical analysisAll values are expressed as mean±s.e.m. To determine statisticalsignificance, we used unpaired Student’s t-test and one-way analysisof variance for comparisons between two groups or multiple groups,respectively. The Bonferroni correction was used to adjust formultiple testing in quantitative PCR experiments. Mann–Whitney U-test was used for albumin:creatinine ratios, as these data did not havea Gaussian distribution. Po0.05 was deemed statistically significant.

DISCLOSUREAll the authors declared no competing interests.

ACKNOWLEDGMENTSWe thank A. Akeson and J. Whittsett (Cincinnati Children’s Hospital)for providing the tet-O-VEGF164 mice. We thank Lawrence Holzmanfor providing nephrin antibodies and thoughtful advice. We thank

Frank Macaluso and Leslie Cummings (AECOM Imaging Facility) fortechnical assistance with TEM. Portions of this work were presentedat the 2007 ASN meeting. This study was supported by the NIH RO1DK59333 and O’Brien Center Grant P50 DK64236 (to AT). KJR wassupported by the NIH training Grant T32 DK 007110. NIDDKintramural research program (to JBK).

SUPPLEMENTARY MATERIALFigure S1. Tetracycline-regulated inducible podocyte VEGF164

overexpression in adult mice.Figure S2. Podocyte VEGF164 overexpression in adult mice causesmild mesangial expansion.Table S1. PCR primers

Supplementary material is linked to the online version of the paper athttp://www.nature.com/ki

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