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. Author manuscript; available in PMC: 2011 Apr 1.
Published in final edited form as: J Am Soc Nephrol. 2010 Aug 5;21(10):1691–1701. doi: 10.1681/ASN.2010030295

Glomerular Structure and Function Require Paracrine, Not Autocrine, VEGF–VEGFR-2 Signaling

Karen Sison *, Vera Eremina *, Hans Baelde †, Wang Min ‡, Masanori Hirashima §, I George Fantus *, Susan E Quaggin *,||
PMCID: PMC3013545  CAMSID: CAMS1632  PMID: 20688931

Abstract

VEGF is a potent vascular growth factor produced by podocytes in the developing and mature glomerulus. Specific deletion of VEGF from podocytes causes glomerular abnormalities including profound endothelial cell injury, suggesting that paracrine signaling is critical for maintaining the glomerular filtration barrier (GFB). However, it is not clear whether normal GFB function also requires autocrine VEGF signaling in podocytes. In this study, we sought to determine whether an autocrine VEGF–VEGFR-2 loop in podocytes contributes to the maintenance of the GFB in vivo. We found that induced, whole-body deletion of VEGFR-2 caused marked abnormalities in the kidney and also other tissues, including the heart and liver. By contrast, podocyte-specific deletion of the VEGFR-2 receptor had no effect on glomerular development or function even up to 6 months old. Unlike cell culture models, enhanced expression of VEGF by podocytes in vivo caused foot process fusion and alterations in slit diaphragm-associated proteins; however, inhibition of VEGFR-2 could not rescue this defect. Although VEGFR-2 was dispensable in the podocyte, glomerular endothelial cells depended on VEGFR-2 expression: postnatal deletion of the receptor resulted in global defects in the glomerular microvasculature. Taken together, our results provide strong evidence for dominant actions of a paracrine VEGF–VEGFR-2 signaling loop both in the developing and in the filtering glomerulus. VEGF produced by the podocyte regulates the structure and function of the adjacent endothelial cell.

Vascular endothelial growth factor (VEGF) was initially discovered as a tumor permeability factor in 1989.1,2 Subsequently, it has been shown that VEGF has major angiogenic stimulatory properties in development and pathologic angiogenesis of tumors, wound healing, diabetes, and arthritis. In the kidney, altered VEGF expression is associated with a variety of renal diseases that include thrombotic microangiopathy, diabetes, and primary glomerular disease.3–7 VEGF is a secreted dimeric glycoprotein that exists in a number of splice variants, each displaying different properties with regard to diffusibility and heparin-binding affinities.8 –11 The most abundant isoform expressed by the kidney is VEGF-165 (VEGF-164 in mouse).12,13 Two receptor tyrosine kinases have been identified for VEGF: VEGFR-1 (Flt1, fms-like tyrosyl kinase-1 where fms refers to feline McDonough sarcoma virus) and VEGFR-2 (human KDR, kinase domain receptor; mouse Flk1, fetal liver kinase-1).14,15 The majority of VEGF signaling described to date is mediated through VEGFR-2 whereas the signaling capacities of VEGFR-1 are still unclear. VEGF also binds to neuropilin-1 and neuropilin-2, both of which may function to enhance VEGFR-2 signaling.16,17

In the glomerulus, podocytes function as vasculature support cells and produce VEGF in vast amounts. Podocyte-specific deletion or overexpression of VEGF during development leads to dramatic and distinct glomerular phenotypes.18,19 Here, the major defects occur within the glomerular endothelial cell compartment, indicating that VEGF paracrine signaling is critical for the formation of a functional glomerular filtration barrier (GFB). More recently, Eremina et al. demonstrated that VEGF production by the podocyte is also necessary to maintain the integrity of the GFB once fully formed as pharmacologic and genetic inhibtion of VEGF in mature podocytes also results in glomerular endothelial cell damage and thrombotic microangiopathy.5 Although the role of VEGF production by podocytes is well established, the expression and/or function(s) of VEGF receptors within the podocyte is less clear and is an area of much discussion. Treating cultured mouse podocytes with VEGF-164 resulted in an eightfold increase in VEGFR-2 expression, enhanced cell survival, and upregulation of podocin and mediated the interaction between CD2-associated protein and podocin.20 Another group reported the expression of VEGFR-2 in podocyte foot processes in vivo using immunogold electron microscopic staining.21 In contrast, other groups have found VEGFR-1 but not VEGFR-2 in cultured mouse and human podocytes and have shown that VEGFR-1 mediates podocyte cell survival and production of extracellular matrix proteins.22,23

Although the existence of a paracrine interaction between podocytes and endothelial cells is well established, it remains unclear whether VEGF signals in an autocrine fashion in podocytes in vivo. In this study, we utilized an inducible gene-targeting system to eliminate VEGFR-2 from all cells postdevelopmentally. Interestingly, whole body deletion of VEGFR-2 resulted in multiorgan vascular defects that affected only specific vascular beds, such as the glomerulus, while sparing others completely. Importantly, deletion of VEGFR-2 recapitulated the glomerular phenotype observed in mice with a podocyte-specific deletion of the VEGF ligand. To determine whether autocrine and/or paracrine VEGF–VEGFR-2 signaling is important in the glomerulus, we examined the expression of VEGFR-2 in vivo in podocytes using available mouse reporter lines and in FACS-sorted primary podocytes and glomerular endothelial cells. To test for biologic function, we generated podocyte-specific VEGFR-2 knockout mice. We also sought to determine whether the genetic deletion of VEGFR-2 from podocytes could rescue the glomerular disease that develops in mice where VEGF levels are increased. In contrast to the whole body deletion studies, deletion of VEGFR-2 specifically from podocytes has no effect on glomerular function. Taken together, our data provide genetic evidence that VEGFR-2 autocrine signaling is not required for normal podocyte function in vivo. Instead, we propose that VEGF paracrine signaling through VEGFR-2 plays a critical and specific role in the maintenance of the integrity of certain highly specialized endothelial cells, including those in the glomerular microvasculature.

RESULTS

Whole Body Deletion of VEGFR-2 Results in Glomerular Endothelial Cell Abnormalities

To examine the involvement of VEGFR-2 signaling in adult tissues and specifically the kidney, we utilized the tetOn inducible system that allows us to delete the VEGFR-2 gene in a time-specific manner. Specifically, we bred VEGFR-2flx/+, tetO-Cretg/+, and rosa-rtTatg/+ mice to VEGFR-2flx/flx, tetO-Cretg/+, and rosa-rtTatg/+ mice (Figure 1A). Mice that carried all four transgenes (tetO-Cre and rosa-rtTA and two floxed VEGFR-2 alleles) were identified by PCR analysis. The quadruple transgenic system was activated by the addition of doxycyline (DOX) to drinking water at 9 days old and the Cre-mediated recombination event between the loxP sites was confirmed in tail genomic DNA by PCR at 3 weeks old (Figure 1B).

Figure 1.

Figure 1

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Whole body deletion of VEGFR-2 results in vascular defects, with glomerular thrombotic microangiopathy. (A) Conditional whole body VEGFR-2 knockout mice contain five transgenes. rtTA is expressed by all cells of the body and, with DOX, binds to the tetO-regulated Cre protein. Cre induces recombination between the loxP sites on the VEGFR-2 allele and results in VEGFR-2 deletion in all cells. (B) PCR analysis of tail genomic DNA shows Cre-mediated excision of the floxed VEGFR-2 region (floxed allele, 439 bp; wild-type allele, 322 bp; deleted allele, 218 bp). (C) Light micrographs show pristine glomeruli in control mice (i through iii). Glomeruli from mutant mice show global glomerular damage [(iv through ix); black arrows in (iv)]. Features of chronic TMA include fragmented red blood cells [black arrows in (vi)], interposition of mesangial cells [white arrowhead in (viii)] and marked thickening of the wall of a capillary loop [black arrowhead in (ix)]. Top row and (viii): periodic acid–Schiff stain; (ii and v): hematoxylin and eosin stain. Bottom row: Trichrome masson stain. Original magnifications: ×100 (i and iv); ×400 (ii and v); ×800 (iii, vi, and ix); ×1000 (viii).

Loss of VEGFR-2 from all cells of the body led to glomerular injury and ascites by 2.5 months old. Histologic examination revealed globally damaged glomerular microvascular beds with loss of viable endothelial cells, whereas the tubulointerstitial vasculature was remarkably spared (Figure 1C). The renal disease observed in mice lacking VEGF in podocytes resembles the renal disease observed in adult mice deficient in VEGFR-2, with prominent glomerular endothelial cell defects, suggesting that VEGFR-2 is essential for glomerular endothelial cell maintenance and survival. Furthermore, additional effects were observed in specific vascular beds of other organs including the liver and thyroid (S.E. Quaggin and V. Eremina, unpublished results). In contrast, whole body post-natal deletion of VEGFR-1 does not result in a phenotype resembling the loss of VEGF from podocytes (K. Sison and S.E. Quaggin, unpublished data), confirming that VEGF signaling through VEGFR-2 is essential in the glomerulus.

Podocytes Do Not Express Detectable Levels of VEGFR-2 Transcript or Protein In Vivo

To determine whether VEGF signals through VEGFR-2 within podocytes in an autocrine fashion, we first determined which glomerular cells express VEGFR-2 in vivo. To do this, we took advantage of the VEGFR-2–green fluorescence protein (GFP) transgenic reporter strain. Immunostaining for the GFP clearly demonstrates expression in endothelial cells at all stages of glomerular development, but never in podocytes (Figure 2A). To further analyze the expression levels of VEGF receptors in podocytes, we performed reverse transcriptase real-time PCR on RNA from FACS-sorted podocytes from isolated and dissociated glomeruli (Figure 2B). Podocytes were identified by expression of cyan fluorescence protein (CFP) fluorescence that is driven by the nephrin promoter in a transgenic mouse line.24 As a control, VEGF and nephrin mRNA levels were measured. Because VEGF and nephrin are known to be highly expressed by podocytes, values were expressed as fold change over the CFP+ podocyte fraction, whereas the values for the receptors were expressed as fold change over the CFP− fraction. VEGFR-2 mRNA expression was undetectable in podocytes (Figure 2C). Neuropilin-1 mRNA was expressed at relatively low levels; however, VEGFR-1 displayed the highest level of expression. Expression was also determined in primary FACS-sorted glomerular endothelial cells from VEGFR-2–GFP transgenic mice as a comparison and confirmed that, within the glomerulus, VEGFR-2 was primarily expressed by GFP+ endothelial cells (Figure 2D).

Figure 2.

Figure 2

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Podocytes do not express VEGFR-2 in vivo. (A) GFP is knocked into the VEGFR-2 locus. GFP staining shows that VEGFR-2 is expressed only in endothelial cells in the glomerulus (arrows). (B) FACS sorting of primary podocytes. Glomeruli were isolated from Nephrin-CFP transgenic mice which contain a CFP reporter gene that is driven by the podocyte-specific nephrin promoter. Glomerular cells were dissociated enzymatically and were sorted into two fractions on the basis of fluorescence, CFP-expressing (CFP+) primary podocytes and CFP-negative (CFP−) endothelial and mesangial cells. RNA were extracted from each fraction and converted into cDNA. (C and D) VEGF receptor mRNA expression in FACS-sorted cells was assessed by reverse transcriptase real-time PCR (CFP+ = podocytes, CFP− = endothelial cells and mesangial cells, GFP+ = endothelial cells, GFP− = podocytes, mesangial cells). Values are expressed as fold increase over CFP−, CFP+, or GFP+ cell fraction and represent mean ± SEM (n = 3 to 4 in all groups). t test was used for statistical analyses (*P < 0.05, **P < 0.01, ***P < 0.001).

Deletion of VEGFR-2 from Developing Podocytes Does Not Affect Glomerulogenesis

Although we were not able to identify expression of VEGFR-2 in podocytes in vivo, we considered the possibility that very low levels of expression at the mRNA level might represent meaningful protein expression below the sensitivity of our assays. To definitively determine whether VEGFR-2 is required in the podocyte for glomerular and podocyte development, we bred mice with a podocyte-specific deletion of one VEGFR-2 allele (VEGFR-2flx/+, Nephrin-Cretg/+) with mice homozygous for the floxed VEGFR-2 allele (VEGFR-2flx/flx) (Figure 3A). In addition, another breeding strategy was established to generate VEGFR-2flx/GFP, Nephrin-Cretg/+ mice. In the latter model, GFP is knocked into the endogenous VEGFR-2 locus and is equivalent to a VEGFR-2 null allele; the goal is to enhance degree of excision of VEGFR-2 from podocytes. To confirm the genetic deletion of VEGFR-2, a PCR-based method was used to analyze the recombination event in glomerular genomic DNA. PCR primers that detect the recombined deleted allele have been previously designed,25 and with these primers, a PCR product was detected in VEGFR-2flx/flx, Nephrin-Cretg/+ mice (Figure 3C), confirming appropriate excision of the VEGFR-2 allele in genomic DNA. Podocyte-specific VEGFR-2 knockout mice were born and were followed for 6 months postnatal; these animals never developed proteinuria (Figure 3D), renal insufficiency, or glomerular injury. Light micrographs show normal glomeruli and transmission electron microscopy (TEM) studies reveal an intact GFB (Figure 3E). These data confirm that autocrine signaling through VEGFR-2 is not required for differentiated podocytes in vivo. As our Cre line excises at the capillary loop stage of glomerular development, we cannot exclude the possibility of an early role in S-shape stage podocyte precursors.

Figure 3.

Figure 3

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Genetic deletion of VEGFR-2 from podocytes does not affect glomerular development or function. (A) Generation of podocyte-selective VEGFR-2 knockout mice (Pod–VEGFR-2 KO). Heterozygous VEGFR-2flx/+, Neph-Cretg/+ mice were bred with homozygous VEGFR-2flx/flx or VEGFR-2flx/GFP mice and podocyte-specific Cre expression leads to the deletion of the VEGFR-2 gene. (B) Genotyping analysis. The floxed VEGFR-2 allele is identified by a 439-bp band, the wild-type VEGFR-2 allele is 322 bp, and the Cre transgene is 300 bp. (C) PCR analysis using primers designed to detect the deleted VEGFR-2 allele shows the Cre-mediated recombination event in glomerular genomic DNA from Pod–VEGFR-2 KO (deleted allele, 218 bp) [Lanes: (MW) molecular weight marker; (i) VEGFR-2flx/flx; (ii) VEGFR-2flx/flx, Nephrin-Cretg/+; (iii) VEGFR-2flx/+, Nephrin-Cretg/+; (iv and v) VEGFR-2flx/flx, tetO-Cretg/+, rosa-rtTAtg/+; (vi) wild type]. (D) Urine protein quantification. Values are expressed as urinary protein/creatinine (U-P/C) ratios and represent mean ± SEM (control, n = 4 to 5; Pod–VEGFR-2 KO, n = 5 to 6). (E) Light micrographs of glomeruli of Pod–VEGFR-2 KO mice are normal and are indistinguishable from controls (periodic acid–Schiff stain). TEM shows normal ultrastructure of the GFB.

Podocyte-Specific Loss of VEGFR-2 Does Not Rescue the Proteinuric Phenotype in Mice with Upregulated VEGF Expression in the Glomerulus

It has been reported that exposure of cultured podocytes to VEGF increases levels of VEGFR-2 expression and that these VEGF-treated cells exhibit alterations in slit diaphragm (SD)–associated proteins and enhanced survival.20 Inhibition of VEGFR-2 blocked the effects of VEGF. On the basis of these results, it was concluded that VEGF signals through VEGFR-2 via an autocrine loop in podocytes. To investigate the possibility that enhanced levels of VEGF might enhance VEGFR-2 expression and/or function in podocytes, we generated additional transgenic mouse models: (1) a gain-of-function VEGF-164 transgene expressing high levels of VEGF specifically in podocytes and (2) double-mutant mice with deletion of the VEGFR-2 gene in podocytes on the VEGF overexpressor background.

Transgenic mice carrying three transgenes (Nephrin-Cre, rosa-rtTA, and tetO–VEGF-164) were induced with DOX (Figure 4A) and are referred to as VEGF overexpressors. DOX was administered in the drinking water of VEGF overexpressors and control (includes wild-type mice and mice that did not carry all transgenes) littermates at postnatal day 0 (P0) and 3 weeks old. Furthermore, as additional controls, uninduced triple transgenics were included and confirmed the nonleaky tetOn system. To verify increased VEGF levels, glomeruli were isolated from 3-week old mice that were induced at P0. Reverse transcriptase real-time PCR revealed a significant fivefold increase in VEGF mRNA in the VEGF overexpressors compared with controls (Figure 4B). The upregulated VEGF transcript levels were accompanied by an increase in VEGFR-2 tyrosine phosphorylation in glomerular lysates as shown by Western blot analysis (Figure 4C).

Figure 4.

Figure 4

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Upregulation of VEGF from developing podocytes leads to glomerular defects by 3 weeks old. (A) Generation of podocyte-specific VEGF overexpressors (PodVEGF+++). Podocyte-specific Cre expression leads to the excision of the “floxed” STOP codon, resulting in the expression of rtTA within the podocyte. In the presence of DOX, rtTA binds to the tetO sequence located upstream of the VEGF-164 gene, activating its transcription only in podocytes. (B) VEGF mRNA is increased in glomeruli isolated from VEGF overexpressors compared with controls as assessed by reverse transcriptase real-time PCR analysis (control, n = 4; PodVEGF+++, n = 10). t test was used for statistical analysis (**P < 0.01, significantly different from control mice). (C) Western blotting of lysates of isolated glomeruli show increased phosphorylation of VEGFR-2 in VEGF overexpressors compared with control mice (V+ = VEGF overexpressors, C = control). (D) Mice induced to overexpress VEGF at P0 leads to proteinuria after 3 weeks of DOX induction with protein deposition in the tubules [* in (i)] and glomerular alterations in rare glomeruli (ii) whereas other glomeruli are normal on light microscopy (iii) (hematoxylin and eosin stain). TEM shows focal foot process fusion [arrows in (iv)].

Upregulation of the VEGF ligand in podocytes leads to significant proteinuria at all stages examined. Induction at P0 results in proteinuria at 3 weeks old by dipstick analysis and urine protein:creatinine measurements (Figure 6B). TEM show focal effacement of podocytes and no defects were observed ultrastructurally in the glomerular endothelial cells [Figure 4D(iv)]. At 3 weeks of induction, the majority of glomeruli appear morphologically normal on light microscopic examination, although protein casts are easily found in tubules [Figure 4D(i and iii)]. By 6 weeks of induction, rare glomeruli exhibit obvious structural defects on light microscopy including sclerosis and periglomular infiltrates [Figure 4D(ii)]. Up-regulation of VEGF in the adult (3-weeks induction time point) also led to proteinuria, although only 50% of mice developed the glomerular phenotype (Table 1). This incomplete penetrance may be due to modifying effects of the mixed strain background or to differences in the efficiency of Cre-mediated excision. In the adult overexpressors that developed a glomerular phenotype, 4 days of induction led to massive proteinuria as assessed by dipstick and SDS-PAGE [Figure 5A(ii)] and Western blot analysis of urine [Figure 5A(iii)]. At the 4-day time point, no gross ultrastructural defects were observed on TEM in podocytes, endothelial compartment, or mesangium [Figure 5A(i)]. Proteinuria is still reversible at this time, as discontinuation of DOX resulted in resolution of proteinuria and re-induction resulted in recurrence of proteinuria (data not shown, n = 3). After 4 weeks of continuous induction, light microscopy revealed a higher degree of glomerular damage with mesangial expansion and periglomerular infiltrates that stained positive for the macrophage marker F4/80 (Figure 5B). TEM studies also showed podocyte foot process effacement, endothelial defects, and a thickened glomerular basement membrane (Figure 5B).

Figure 6.

Figure 6

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Genetic loss of VEGFR-2 from podocytes does not rescue the glomerular phenotype of mice with upregulated VEGF levels. (A) Generation of quintiple transgenic mice that lack VEGFR-2 and overexpress the 164 isoform of VEGF from the podocyte. Podocyte-specific Cre expression simultaneously deletes the floxed VEGFR-2 allele and results in the expression of rtTA within the podocyte, activating VEGF transcription. (B) Urine protein quantification. Values are expressed as urinary protein/creatinine (U-P/C) ratios and represent mean ± SEM (control, n = 5; Pod-VEGF+, n = 5; Pod-VEGF+/VEGFR-2 DM (double mutant), n = 5). One-way ANOVA, followed by Bonferroni’s multiple comparison test was used for statistical analysis (***P < 0.001, significantly different from control mice). (C) Semiquantification of glomerular damage. Values are scores of glomerular changes based on an assigned index and represent mean ± SEM (control, n = 2; Pod-VEGF+, n = 3; Pod-VEGF+/VEGFR-2 DM, n = 4). (D) Hematoxylin and eosin staining shows a range of glomerular lesions in double mutant mice at 6 weeks old that progressively worsen by 9 weeks old.

Table 1.

Genotype and phenotype of transgenic mice

Genotype Phenotype No. Affected Mice No. Unaffected Mice Total No. Mice % Affected
VEGFR-2flx/flx, Neph-Cretg/+ (6 to 12 weeks old) Normal 0 15 15 0
VEGFR-2flx/GFP, Neph-Cretg/+ (6 to 24 weeks old) Normal 0 30 30 0
Neph-Cretg/+, rosa-rtTAtg/+, tetO–VEGF-164tg/+ (induced at P0) Proteinuria and glomerular disease at 3 weeks old 10 1 11 91
Podocin-Cretg/+, rosa-rtTAtg/+, tetO–VEGF-164tg/+ (induced in the adult) Proteinuria within 1 day of induction, progressive glomerular disease with continuous induction 5 5 10 50
VEGFR-2 flx/GFP, Neph-Cretg/+, rosa-rtTAtg/+, tetO–VEGF-164tg/+ (induced at P0) Proteinuria and glomerular disease at 3 weeks old 6 0 6 100
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Affected mice develop proteinuria and glomerular disease.

Figure 5.

Figure 5

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Increased VEGF expression by podocytes in adult glomeruli leads to ultrastructural defects in the GFB depending on length of induction. (A) Short 4-day induction of podocyte VEGF overexpression leads to reversible proteinuria with no changes in the GFB architecture. (i) Periodic acid–Schiff staining from kidney of mice induced to overexpress VEGF for 4 days show protein casts in tubules but normal glomeruli. No ultrastructural defects where observed in podocytes or the endothelial compartment by TEM. (ii) SDS-PAGE analysis of urine shows albuminuria in podocyte-specific VEGF overexpressors (lanes 1 to 5) and uninduced triple transgenic control littermate (lane 6). (iii) Western blotting reveals mouse heavy-chain IgG in urine of VEGF overexpressors (lanes 1 to 4) which are absent in triple transgenic control littermate (lane 5). (B) By contrast, 4 weeks of continuous VEGF overexpression results in marked protein deposition in the renal tubules [* in (i)], glomerular structural changes including mesangial expansion and sclerosis (ii) (periodic acid–Schiff stain), and the accumulation of F4/80 positive periglomerular macrophages (iv and v). TEM studies show podocyte foot process fusion [arrow in (iii)] and increased and irregular GBM thickness [● in (vi)].

To study whether VEGFR-2 in the podocyte plays a role in the development of proteinuria and glomerular pathology in the VEGF overexpressors, we sought to determine whether the loss of VEGFR-2 can rescue their glomerular disease. Double mutant mice that contained all five transgenes (tetO–VEGF-164, Nephrin-Cre, rosa-rtTA, VEGFR-2 floxed allele, and VEGFR-2GFP allele) in addition to control and VEGF overexpressors were induced with DOX at P0 (Figure 6). This early time point for induction of VEGF overexpression was chosen to avoid the variability of phenotype observed in the older mice. As before, podocyte-specific VEGF overexpressors developed proteinuria at 3 weeks old when induced with DOX at P0. Double mutants with deletion of VEGFR-2 in podocytes also developed proteinuria (Figure 6) and glomerular disease at 3 weeks old (Figure 6) after induction at P0, with a tempo and severity that were indistinguishable from VEGF overexpression alone. Thus, genetic deletion of VEGFR-2 from the podocyte does not rescue the proteinuria and glomerular pathology caused by upregulation of the VEGF within the glomerulus. These data demonstrate that the defects observed in the VEGF overexpressors do not result from enhanced autocrine signaling through VEGFR-2 in podocytes.

DISCUSSION

Local production of VEGF by podocytes is essential for development and maintenance of the GFB. When VEGF production is reduced to zero, endothelial cells fail to migrate into the glomerular tuft at all, whereas slightly higher levels (approximately 50% of normal) result in endothelial swelling, loss of fenestrations, decreased survival, and thrombotic microangiopathy.5,18,19 Greater reductions in the level of VEGF result in more dramatic injury in the adjacent endothelial compartment. These results demonstrate the role of cross talk between podocytes and endothelial cells during development and in the adult glomerulus (Figure 7A).

Figure 7.

Figure 7

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VEGF signals via a paracrine loop to VEGFR-2 in the glomerulus. (A) Summary of genetic experiments: Deletion of VEGFR-2 from all cells results in specific injury to the glomerular microvasculature that resembles the glomerular endothelial injury observed in mice with specific deletion of the VEGF ligand from podocytes. Deletion of VEGFR-2 from podocytes does not cause significant glomerular disease. Together, these results do not support a major role for autocrine VEGF signaling through VEGFR-2 in the glomerulus. (B) VEGF is produced by the podocytes and functions through a paracrine signaling loop to act on VEGFR-2 expressed by glomerular endothelial cells.

Here we show that a similar glomerular injury occurs when the tyrosine kinase receptor, VEGFR-2, is deleted postnatally. VEGFR-2 is expressed by all endothelial cells; accordingly, all vasculature is severely and equally affected in conventional VEGFR-2 knockout mice that die at embryonic day 9.5.26 In stark contrast, only specific vascular beds are affected when VEGFR-2 is knocked out after completion of developmental angiogenesis. In the kidney, glomerular microvascular beds are globally affected and exhibit thrombotic microangiopathy, whereas other renal vascular beds are spared, underscoring the exquisite sensitivity/dependence of the GFB on the VEGF–VEGFR-2 axis (Figure 7B). Together these results support a model whereby podocyte-produced VEGF signals via a paracrine loop to the VEGFR-2 receptor expressed by the glomerular endothelial cells in the filtering glomerulus.5

Although analysis of podocyte-specific VEGF knockout mice strongly support this model, some questions remain. Specifically, does autocrine signaling of VEGF–VEGFR-2 in podocytes also contribute to maintenance and function of the GFB? In this regard, autocrine VEGF signaling loops have been described in other cell types such as hematopoietic stem cells, mammary tumor cell lines, and endothelial cells themselves.27,28 In cultured podocytes, it has been reported that podocytes respond to VEGF through VEGFR-2.20 In the reported series of transgenic VEGF mice described above,5,18,19 there is clear evidence for podocyte injury. Although this may be a by-product of the severe endothelial injury, a contribution of interruption of autocrine signaling cannot be excluded in these studies. Finally, up-regulation of VEGF developmentally in podocytes results in glomerular collapse and nephrotic range proteinuria. As these VEGF gain-of-function mice died before founder lines could be established, detailed analysis of the timing and severity of endothelial versus podocyte defects could not be studied.

In this paper, we took a genetic approach to further define the VEGF–VEGFR-2 axis in the glomerulus in vivo. Studies were undertaken in mice expressing endogenous levels of VEGF and also in transgenic mice expressing higher levels of VEGF in their podocytes. These latter mice develop podocyte foot process fusion and proteinuria suggestive of a cell autonomous or “autocrine” effect of VEGF.

As a first step, we looked for expression of the VEGFR-2 receptor in vivo and in primary podocytes. The VEGFR-2 reporter mouse line is highly sensitive, allowing the identification of single cells expressing VEGFR-2 with immunostaining. Nonetheless, we failed to detect any expression of VEGFR-2 in podocytes using this system. We also FACS-sorted primary podocytes and endothelial cells from glomeruli of transgenic mice carrying a CFP or GFP lineage tag, respectively, and performed real-time PCR. Again, with this exquisitely sensitive system, we easily detected VEGFR-2 mRNA expression in glomerular endothelial cells but not in podocytes. This finding contrasts reports from other groups who have detected VEGFR-2 on podocytes in vivo by immunogold techniques.21 A naturally occurring soluble version of VEGFR-2 has been identified and serves a function in the regulation of lymphatic vessels.25 Whether the reported podocyte VEGFR-2 is the soluble or full-length transmembrane form is unknown but may explain the differences.

Although we were unable to detect VEGFR-2 expression in podocytes in vivo, we speculated that a functionally significant level of expression might exist even if it was below the sensitivity of detection in our assays, or that expression of functionally relevant levels of the receptor may be induced in the presence of a ligand or in pathologic circumstances. Accordingly, as a definitive test, we performed genetic studies to determine whether VEGFR-2 has a functional role in the podocyte.

Deletion of the VEGFR-2 receptor from podocytes did not result in a glomerular developmental defect or in functional alteration of the GFB. To ensure excision of the receptor, we identified the excised allele in genomic DNA purified from glomerular lysates of transgenic mice and also analyzed the phenotype in mice carrying one null VEGFR-2GFP allele (VEGFR-2 deleted from the entire animal) over a floxed allele, so the degree of excision would be optimized (only one floxed allele needs to be excised). In both cases, mice developed normally and never developed proteinuria even until 6 months old.

In vitro data suggest that VEGF itself might upregulate VEGFR-2 expression in podocytes. To determine whetherVEGFR-2 plays a functional role in the setting of increased VEGF production, we generated an inducible gain-of-function VEGF mouse.

Upon treatment with doxycycline, the VEGF mRNA is upregulated fivefold in Nephrin-Cre, rosa-rtTA, and tetO–VEGF-164 mice. Induction at postnatal day 0 resulted in enhanced phosphorylation of the VEGFR-2 receptor in glomeruli and a consistent glomerular phenotype. Mice developed proteinuria by 3 weeks old accompanied by foot process fusion and GBM thickening on electron micrographs. In contrast, induction in older mice (4 weeks old) resulted in a variable phenotype with 50% of mice developing profound proteinuria. The heterogeneity is likely the result of inefficient induction and/or modifying effects of the mixed strain background. For this reason, we focused our studies on the younger time point.

Immortalized mouse podocytes treated with VEGF demonstrate changes in SD-associated proteins and increased survival and this can be inhibited by VEGFR-2 blockade.20 To determine whether the defects observed in podocytes in our transgenic overexpressors are due to autocrine signaling through VEGFR-2, we generated double mutants overexpressing VEGF but lacking VEGFR-2 in podocytes. Loss of the VEGFR-2 receptor from podocytes failed to rescue the phenotype or alter the course of disease in VEGF overexpressors.

In conclusion, our results demonstrate that VEGFR-2, if expressed by podocytes, is expressed at very low levels and is not required for podocyte self-support. We propose that the main target of VEGF in the glomerulus is the endothelial cell compartment even in a filtering glomerulus. In addition, we provide further evidence for the role of VEGFR-2 as the main effector of VEGF-mediated glomerular endothelial cell maintenance and survival, which provides a plausible explanation for the observed incidence of glomerular injury in patients receiving anti-angiogenic agents that target the VEGF pathway.

CONCISE METHODS

Transgenic Lines

Transgenic lines used include the following: tetO–VEGF-164, tetO-Cre, Podocin-Cre, Nephrin-Cre, rosa-rtTA, Nephrin-CFP, VEGFR-2–GFP, and floxed VEGFR-2 mice. tetO–VEGF-164 mice carry the cDNA for the murine VEGF-164 isoform that is driven by the tetO-CMV minimal promoter (kindly provided by J. Whitsett, Cincinnati, Ohio). Generation and characterization were previously described.29,30 In rosa-rtTA transgenic mice, the coding sequence for reverse tetracycline transactivator (rtTA) was targeted into the ubiquitously expressed ROSA26 locus. Because a floxed STOP codon precedes the coding sequence for rtTA, expression is dependent on a Cre excision.31 Presence of DOX results in the formation of an active transcriptional activator and the activation of the tet-responder transgene, allowing for temporal regulation.31 In Nephrin-Cre and Nephrin-CFP transgenic mice, Cre and CFP expression is under the control of the podocyte-specific Nephrin promoter, allowing for spatial regulation.24 Floxed VEGFR-2 and VEGFR-2–GFP mice were kindly provided by J. Rossant’s group (Toronto, Ontario, Canada). Floxed VEGFR-2 mice contain loxP sites around exon 1.25 VEGFR-2–GFP mice were generated by replacing exon 1 of the endogenous VEGFR-2 gene with EGFP cDNA and has been validated to be equivalent to a null allele.32 All the studies were performed on mixed strain backgrounds because of the large number of transgenes required and breeding strategies. Littermates were used as controls for all experiments.

Genotyping

Genomic DNA was isolated from mouse tails as described previously.33 PCR was used to identify transgenic mice using the following primers: Nephrin-Cre (sense 5′-ATG TCC AAT TTA CTG ACC G-3′, antisense 5′-CGC CGC ATA ACC AGT GAA-3′), tetO–VEGF-164 (sense 5′-TGG ATC CAT GAA CTT TCT GCT-3′, anti-sense 5′-GAA TTC ACC GCC TCG GCT TGT C-3′), and rosa-rtTA (sense 5′-GAG TTC TCT GCT GCC TCC GC-3′, antisense 5′-AAG ACC GCG AAG AGT TTG TG-3′). VEGFR-2 mutant mice were identified using the following primers: VEGFR-2–S1 (5′-TGGAGAGCAAGGCGCTGC-TAGC-3′), VEGFR-2–A (5′-CTTTCCACTCCTG-CCTACCTAG-3′), and VEGFR-2–S2 (5′-AATTT-GGGTGCCATAGCCAATC-3′). VEGFR-2–S1 and VEGFR-2–A generate a 439-bp fragment of the VEGFR-2 allele with 1 loxP site and a 322-bp DNA fragment for the wild-type allele. VEGFR-2–A and VEGFR-2–S2 detect the recombined allele and generate a 218-bp fragment.

Urinalysis

Urine was collected and examined for the presence or absence of protein using a urine dipstick (Chemstrip 5L; Roche Diagnostics Corporation) and the standard colorimetric assay was performed according to the manufacturer’s instructions. In addition, total protein levels were assessed by using the Bradford colorimetric-based Bio-Rad assay. Briefly, the protein assay dye concentrate containing the Comassie blue dye (Bio-Rad, Catalog No. 500-0006) was added to the urine sample and was incubated at room temperature for 10 minutes. Absorbance was measured at 595 nm. Protein levels were normalized to the urine creatinine levels as measured by the Jaffe method.34 Results are expressed as total protein/creatinine ratios (mg/mg).

Histologic and Ultrastructural Analysis

Freshly dissected kidneys were fixed in 10% formalin/PBS, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin, periodic acid–Schiff or trichrome masson. Sections were examined and photographed with a DC200 Leica camera and Leica DMLB microscope (Leica Microsystems Inc.). For ultrastructural analysis using electron microscopy, kidney tissue was fixed in 1.5% glutaraldehyde, embedded in Spurr (Canemco Inc.), and sectioned.

Glomerulosclerosis Scoring Index

Periodic acid–Schiff stained paraffin kidney sections were used to determine the severity of glomerular damage. With use of light microscopy, a glomerular score for each mouse was derived as the mean of 40 glomeruli. The extent of glomerulosclerosis was assigned on the basis of a scale from 0 to 4: 0, normal glomerulus; 1, mesangial matrix expansion; 2, severe mesangial matrix expansion; 3, severe mesangial matrix expansion/segmental glomerulosclerosis; 4, global glomerulosclerosis (>50% of the glomerulus). The resulting index for each group of animals was expressed as a mean of all scores.

FACS-Sorting Primary Podocytes

Glomeruli were isolated from Nephrin-CFP and VEGFR-2–GFP mice using the sieving method. Nephrin-CFP mice contain a CFP reporter gene that is driven by the nephrin promoter. VEGFR-2–GFP mice carry a GFP cassette that is knocked into the VEGFR-2 locus and is under the control of an endogenous promoter. In these mice, CFP is expressed in Nephrin-expressing podocytes and GFP is expressed in VEGFR-2–positive endothelial cells, respectively.32,24 After enzymatic digestion with collagenase, trypsin, and EDTA in PBS for 1 hour, 37°C, cells were washed three times in PBS. Dissociated glomerular cells were sorted using a BD FACSAria flow cytometer (BD Biosciences). CFP-expressing (CFP+) primary podocytes and GFP-expressing (GFP+) endothelial cells were separated from glomeruli on the basis of CFP and GFP expression, respectively.

Real-Time PCR

Total RNA was extracted from FACS-sorted primary podocytes and endothelial cells with Trizol reagent (Invitrogen) and reverse-transcribed into cDNA using M-MLV reverse transcriptase (Fermentas) and random hexamer primers (Fermentas) according to the manufacturer’s protocol. cDNA samples and standards were amplified in qPCR MasterMix Plus for SYBR Green I (BioRad). Samples were analyzed in triplicate. Relative quantification of target gene expression was evaluated using the comparative CT method.35 The ΔCT value was determined by subtracting the HPRT CT value of each sample from its respective target CT. Fold changes in gene expression of the target gene were equivalent to 2−ΔΔCT. The values obtained were then entered into a t test.

Western Blot Analysis

Glomeruli were isolated from control and mutant mice using the sieving method and were lysed in RIPA lysis buffer (0.1% SDS, 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 1 mM DTT and supplemented with fresh protease and phosphatase inhibitors). Protein concentration was determined by the BioRad Assay according to the manufacturer’s instructions. Samples were resolved on an 8% SDS-PAGE gel and were transferred to a polyvinylidene difluoride membrane. Membranes were blocked in 5% nonfat dried milk for 1 hour, incubated with the appropriate primary antibodies diluted in blocking solution at 4°C overnight, and washed three times in 0.05% Tween 20 in PBS rinse buffer. Membranes were incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies for 1 hour at room temperature, washed, and developed with enhanced chemiluminescence (ECL, Amersham). The following primary antibodies were used for immunoblotting: rabbit anti–VEGFR-2, rabbit anti–PY1054/1059-VEGFR-2 (all used at 1:1000; provided by Dr. Wang Min, Yale University, New Haven, Connecticut) and mouse anti–β-actin (1:5000, Santa Cruz). Secondary antibodies were goat–anti-rabbit IgG-HRP and goat–anti-mouse IgG-HRP (1:10000; Santa Cruz).

Statistical Analysis

All values are expressed as means ± SEM. Statistical analysis was performed with unpaired, two-tailed t tests for single comparisons or one-way ANOVA with post hoc test using Bonferonni’s method for multiple comparisons. A level of significance for comparison was set at P < 0.05.

Acknowledgments

We thank D. Holymard for electron microscopy and K. Harpal for histologic stainings and P. Thorner for helpful discussions. We also thank K. Vintersten for the CFP fluorescence image provided. This work was funded by CIHR grants MOP 77756 and 62931, TF grant 016002, and KFOC grant to S.E.Q. S.E.Q. holds the Gabor-Zellerman Chair in Renal Research, UHN, University of Toronto, and is the recipient of a CRC Canada Research Chair Tier II.

Footnotes

DISCLOSURES

None.

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