AJP - Renal Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 291: F422-F428, 2006. First published April 4, 2006; doi:10.1152/ajprenal.00448.2005
0363-6127/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/F422    most recent
00448.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (17)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guan, F.
Right arrow Articles by Tufro, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guan, F.
Right arrow Articles by Tufro, A.

Autocrine VEGF-A system in podocytes regulates podocin and its interaction with CD2AP

Fangxia Guan,1 Guillermo Villegas,1 Jason Teichman,1 Peter Mundel,2 and Alda Tufro1

1Department of Pediatrics/Nephrology and Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx; and 2Department of Medicine, Mount Sinai School of Medicine, New York, New York

Submitted 9 November 2005 ; accepted in final form 29 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Vascular endothelial growth factor (VEGF-A) signaling is required for endothelial cell differentiation, vasculogenesis, angiogenesis, and vascular patterning. During kidney morphogenesis, podocyte VEGF-A guides endothelial cells toward developing glomeruli. Podocyte VEGF-A expression continues throughout life but its function after completion of development remains unclear. Here, we examined the expression of VEGF-A and its receptors VEGFR1, VEGFR2, NP1, and NP2 in conditionally immortalized mouse podocytes cultured in undifferentiated and differentiated conditions using RT-PCR and Western analysis. VEGF-A secretion was assessed by ELISA and Western analysis. Upon podocyte differentiation, VEGF-A protein expression and secretion increased threefold. Differentiated podocytes expressed eightfold higher VEGFR2 mRNA levels than undifferentiated podocytes, whereas VEGFR1, sVEGFR1, NP1, and NP2 mRNA levels were similar. We examined the regulation and function of the VEGF-A system by exposing differentiated podocytes to recombinant VEGF165 (20 ng/ml) or control media for 24 h. VEGF165 induced a twofold increase in VEGFR2 mRNA and protein levels, whereas VEGFR1, sVEGFR1, NP1, and NP2 mRNA levels remained unchanged. VEGF165 induced VEGFR2 phosphorylation. VEGF165 reduced podocyte apoptosis ~40%, whereas anti-VEGFR2 neutralizing antibody enhanced it twofold. We determined that VEGF-A signaling regulates slit diaphragm proteins by inducing a dose-response podocin upregulation and increasing its interaction with CD2AP. The data indicate that podocytes in culture have a functional autocrine VEGF-A system that is regulated by differentiation and ligand availability. VEGF-A functions in podocytes include promoting survival through VEGFR2, inducing podocin upregulation and increasing podocin/CD2AP interaction.

cell differentiation; apoptosis; VEGF-A secretion; slit diaphragm proteins; VEGF receptor 2

VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF)-A is critical for differentiation of endothelial cells and their correct assembly in early development (7, 25). Later on, VEGF-A is required for normal development of glomerular capillaries (6) and probably establishing their fenestrated phenotype (22). We showed that VEGF-A is a chemoattractant for endothelial cells and directs the migration of endothelial cell toward developing nephrons (32). Podocytes and glomerular endothelial cells are among the few cell types where VEGF-A is expressed throughout life (28, 33). The role of VEGF-A in podocytes upon completion of development remains poorly understood. It was recently shown that anti-VEGF therapy and soluble flt-1 cause proteinuria, foot processes fusion, and endothelial damage (30), suggesting that VEGF-A is also necessary for podocyte and glomerular endothelial cell integrity and function.

VEGF-A signals through VEGFR1 and VEGFR2 receptors, previously called Flt-1 and Flk1-KDR, respectively, located predominantly on the surface of endothelial cells (4, 31). VEGFR2 mediates proliferation, survival, and migration signals and is considered the main VEGF-A signaling receptor (7). The function of VEGFR1 is less clear, probably due to the fact that a soluble isoform of the receptor (sFlt-1) that is secreted and functions as a decoy preventing VEGF-A binding to signaling receptors is produced in several cell types (27). VEGF165 signals are amplified on binding to coreceptors neuropilin 1 (NP1) and neuropilin 2 (NP2) (29). In addition to endothelial cells, VEGF-A receptors are expressed by renal tubular epithelial cells, osteoblasts, mononuclear phagocytes, hematopoietic cells, and some cancer cells (11, 29, 34). In all these cell types, VEGFR2 is considered the main signaling receptor (7). Coexpression of VEGF-A and its receptors implies the presence of an autocrine VEGF system. In podocytes, only VEGFR1 and NP1 expression have been reported; it is presently unclear how VEGF antiapoptotic signals are transduced and whether VEGFR1 and NP1 soluble isoforms, which function as dominant negatives rather than signaling receptors, are produced by podocytes (8).

In the present studies, we sought to characterize the VEGF-A system in mouse podocytes in culture and to determine whether VEGF-A signaling regulates slit diaphragm proteins expression. We show that immortalized podocytes have a functional autocrine system including VEGFR2 that mediates survival and morphogenetic signals. This system is regulated by differentiation and VEGF-A. VEGF-A upregulates podocin and its association with CD2AP, consistent with a role for VEGF-A in the regulation of the slit diaphragm.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. A previously described conditionally immortalized mouse podocyte cell line (20) was maintained in RPMI 1640 (GIBCO) supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin (GIBCO) with or without recombinant mouse {gamma}-interferon (Sigma) in humidified incubators with air-5% CO2. Podocytes were propagated on collagen I-coated plates at 33°C in the presence of recombinant mouse {gamma}-interferon (10 U/ml). Removal of {gamma}-interferon and temperature switch to 37°C inactivated the SV40 T antigen and induced podocytes to differentiate. Differentiated podocytes were serum starved for 24 h and exposed to media with or without recombinant human VEGF165 (R&D Systems; 20 ng/ml) for 30 min to 24 h as indicated.

Protein extraction and Western blotting. Control and VEGF-A-treated podocytes were harvested in RIPA lysis buffer (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and proteinase inhibitors) for Western blotting. Protein concentration was determined by BCA assay (Sigma) according to the manufacturer’s instructions. Fifty to one hundred micrograms protein/lane were resolved in 6–10% SDS PAGE. The following polyclonal primary antibodies were used for immunoblotting: anti-VEGF (sc-507, Santa Cruz Biotechnology), VEGFR2 (sc-315, Santa Cruz Biotechnology), VEGFR2-P (Sc-16629-R, Santa Cruz Biotechnology), anti-SV40 T large antigen (DP02, Oncogene), antisynaptopodin (20); antipodocin (26); anti-CD2AP was kindly provided by Dr. A. Shaw, Washington University School of Medicine; anti-Akt (610860, BD Biosciences), antiphosphorylated Akt pSer473 (9271, Cell Signaling), and antiactin (A2066, Sigma) was routinely used as internal control for protein loading. Secondary antibodies were antirabbit horseradish peroxidase (HRP) IgG or antimouse HRP-IgG (Amersham), as appropriate. Enhanced chemiluminescence (ECL, Amersham) was used for visualization following manufacturer’s instructions. To assess VEGF-A secretion, podocyte supernatants were collected at the end of the experiments and concentrated (x140) using Amicon Ultra filters (Millipore), and 40-µl samples were resolved by SDS-PAGE and immunoblotted with anti-VEGF-A (sc. 507, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies as described above.

Coimmunoprecipitation. Protein extracts were prepared as described above from control and VEGF-A-treated podocytes. For each immunoprecipitation, 1 mg protein extract was precleared with protein A-Sepharose (Roche) in 0.5 ml TNE buffer (250 mM NaCl, 5 mM EDTA, 10 mM Tris, pH 7.4, proteinase inhibitors) for 1 h at 4°C. Precleared extracts were immunoprecipitated with antipodocin at 4°C for 3 h and 50 µl of protein A-agarose were added and incubated at 4°C overnight. The beads were washed five times in TNE buffer before bound proteins were eluted by boiling the samples for 5 min in sample buffer at 95°C. Immunoprecipitated proteins were analyzed by Western blotting.

ELISA. VEGF-A concentration was measured in podocyte supernatant by ELISA using mVEGF Quantikine (R&D Systems) following the manufacturer’s instructions. VEGF-A was measured in duplicate 50-µl samples from undifferentiated and differentiated podocytes after 24 h culture in 95% air-5% CO2. Data were corrected for total protein concentration and reported as picograms of VEGF per microgram of protein.

Reverse transcription and semiquantitative PCR. Total RNA was isolated from control and VEGF-A-treated podocytes by standard methods using TRIzol reagent (Invitrogen). RNA concentration and quality were assessed by spectrophotometry at 260 and 280 nm. cDNA was generated using Superscript II RT first-strand cDNA synthesis kit (Invitrogen) according to manufacturer’s protocols. Briefly, 2–5 µg of total RNA were reverse-transcribed in the presence of random primers and reverse transcriptase according to the following sequence: 65°C for 5 min, followed by 42°C for 60 min, and 75°C for 15 min. One microliter of the resulting cDNA was used for each PCR amplification. Specific primers (Table 1) were designed and PCR amplifications were performed as follows: 94°C for 5 min once, 94°C for 45 s, annealing temperature as indicated in Table 1 for 30 s, and 72°C for 45 s, repeated 30 cycles, 72°C for 5 min on the last cycle. PCR products were resolved in 1% TAE agarose gels, imaged, and analyzed by image quantitative software (FC8000, Alpha Innotech). Glutaraldehyde phosphate dehydrogenase (GAPDH) was used as internal control. A negative control without cDNA was also performed to exclude contamination of buffers, primers, and enzymes.


View this table:
[in this window]
[in a new window]
  		Table 1. Primer sequences, product sizes, and annealing temperatures

 
Immunocytochemistry. Differentiated and undifferentiated mouse podocytes were grown on collagen type I-coated glass coverslips, washed with PBS, and fixed with 3.7% formaldehyde for 10 min at room temperature, permeabilized with 0.3% Triton X-100 in PBS for 5 min, rinsed with PBS, blocked with 2% FCS, 2% BSA, and 0.2% gelatin in PBS for 30 min, and incubated with primary antibody for 60 min at room temperature or overnight at 4°C. Anti-VEGFR2 (sc-315, Santa Cruz Biotechnology) and anti-phospho-Tyr397 FAK (05–537, Upstate) antibodies were used. Then, cells were washed with PBS, incubated with Cy2 donkey anti-rabbit IgG and/or Cy3 donkey anti-mouse IgG (Jackson Immunoresearch Laboratories). Cells were washed with PBS, rinsed with tap water, mounted on glass slides using Fluoromount-G (Southern Biotechnology Associates), and examined by confocal microscopy (FV300, Olympus).

Apoptosis. Differentiated podocytes were starved for 36 h, exposed to rVEGF165 (20 ng/ml), media alone, media + anti-VEGFR2 neutralizing antibody (2 µg/ml; R&D Systems), or media + mouse IgG (2 µg/ml; Vector) for 24 h. The in situ apoptosis marker caspACE FITC-VAD- FMK (Promega) was added (10 µM), following the manufacturer’s protocol. Briefly, cells exposed to the apoptosis marker were placed at 37°C for 20 min in the dark, washed with PBS, and fixed in 10% formalin for 30 min at room temperature. Slides were washed three times in PBS, mounted, and examined with fluorescence microscopy.

Statistical analysis. All experiments were performed at least three times, and the number of each of them is indicated in the figure legends or in RESULTS. In ELISA experiments, results from duplicate samples were averaged and data are expressed as means ± SE. Densitometric analysis of RT-PCR amplifications and Western blot analyses of three independent experiments are expressed as means ± SE fold-changes. In apoptosis experiments, the total number of cells and the number of apoptotic cells were counted in five fields per slide per condition and expressed as means ± SE percentage of apoptotic cells in each condition. Control and experimental conditions were compared using unpaired t-test or ANOVA as appropriate. Statistical significance was deemed as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Conditionally immortalized mouse podocytes proliferated and maintained an epithelial phenotype under permissive conditions (33°C), whereas under nonpermissive conditions (37°C) proliferation ceased and differentiation ensued, resulting in sprouting of long processes from podocyte bodies and spindle-like secondary processes (Fig. 1A), as previously described (20). Accordingly, SV40-T antigen was abundantly expressed at 33°C in the presence of {gamma}-IFN, whereas it was barely detected at 37°C in the absence of {gamma}-IFN (Fig. 1B). Synaptopodin, a marker of podocyte differentiation, was not detected under permissive conditions and abundantly expressed in nonpermissive conditions (Fig. 1B).


Figure 1
View larger version (117K):
[in this window]
[in a new window]
  		Fig. 1. Morphology and expression of SV40 and synaptopodin by immortalized murine podocytes. A: different cell shape of podocytes in 33 and 37°C. B: SV40 large T antigen is expressed by undifferentiated podocytes (33°C), whereas the podocyte-specific marker synaptopodin is only expressed by differentiated podocytes (37°C). Right: molecular weights.

 
VEGF-A expression and secretion. To determine whether VEGF-A expression is regulated by cell differentiation, we examined the expression of VEGF-A in undifferentiated and differentiated podocytes by Western blotting. Three VEGF-A isoforms were detected in differentiated podocytes, whereas only the two larger isoforms were detected in undifferentiated podocytes (Fig. 2A). Upon podocyte differentiation, VEGF-A expression increased threefold (Fig. 2B). Podocyte VEGF-A secretion to the supernatant also increased threefold on cell differentiation, as indicated by ELISA (Fig. 2C). Supernatant VEGF-A was normalized for total protein content. Secreted VEGF-A isoforms were undetectable by immunoblotting in the supernatant of undifferentiated podocytes, whereas VEGF164 and VEGF120 were secreted into the supernatant by differentiated podocytes at ~2:1 ratio (Fig. 2D). Collectively, the qualitative and quantitative data obtained by Western analysis and ELISA demonstrate that VEGF-A expression and secretion by podocytes are regulated by differentiation.


Figure 2
View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2. Vascular endothelial growth factor (VEGF)-A expression in undifferentiated and differentiated podocytes. A: Western blot showing 3 VEGF-A isoforms detected in differentiated podocytes, whereas only the 2 larger isoforms were detected in undifferentiated podocytes. B: densitometric analysis showed that podocyte differentiation induced a 3-fold increase in VEGF expression, n = 3, *P < 0.05. VEGF-A secretion by undifferentiated and differentiated podocytes. C: mVEGF-A ELISA assay. Readings were corrected for total protein content; data are expressed as means ± SE of pg VEGF/µg protein, n = 7, *P < 0.05. Differentiated podocytes secrete 3-fold more VEGF-A than undifferentiated podocytes. D: Western blot analysis. Differentiated mouse podocytes secrete VEGF120 and VEGF164 to the supernatant, whereas these isoforms are not detected in the supernatant from undifferentiated podocytes.

 
VEGF-A receptor expression. VEGF-A receptors 1 and 2, as well as their coreceptors neuropilins 1 and 2 (NP1, NP2) mRNA levels, were detected by semiquantitative RT-PCR in undifferentiated and differentiated podocytes (Fig. 3A). VEGFR1, NP1, and NP2 as well as soluble VEGFR1 (sVEGFR1) were expressed at similar levels in both conditions, whereas on podocyte differentiation the signaling receptor VEGFR2 was upregulated eightfold at the mRNA level and twofold at the protein level (Fig. 3, B and C). Fluorescent immunocytochemistry detected VEGFR2 expression in the cell membrane and perinuclear regions, and colocalization with phosphorylated focal adhesion kinase (P-FAK) suggested VEGFR2 clustering at focal adhesions (Fig. 3D).


Figure 3
View larger version (50K):
[in this window]
[in a new window]
  		Fig. 3. VEGF-A receptor expression and regulation in podocytes. A: RT-PCR amplification of soluble VEGFR1, VEGFR1, VEGR2, NP1, and NP2 mRNAs from undifferentiated (33°C) and differentiated (37°C) podocytes. GAPDH was amplified for loading control of each reaction. Primers used and PCR product sizes are indicated in Table 1. B: quantification of PCR, VEGFR2 increased 7.9 ± 0.63-fold above control on podocyte differentiation, n = 3, *P < 0.05. C: Western blot showing VEGFR2 expression that increases 2-fold on exposure to VEGF165, the same blot probed for actin demonstrates equal protein loading. D: fluorescent immunocytochemistry showing VEGFR2 in undifferentiated and differentiated mouse podocytes. Colocalization of VEGFR2 (green) and phosphorylated FAK (red) in cell membrane and focal adhesions (yellow spots pointed by arrows) is shown. E: RT-PCR amplification of soluble VEGFR1, VEGFR1, VEGR2, NP1, and NP2 mRNAs from differentiated podocytes grown in control conditions or exposed to hVEGF165 (20 ng/ml) for 24 h. GAPDH was amplified for loading control of each reaction. F: RT-PCR quantification showing that VEGFR2 increased 2 ± 0.3-fold above control in podocytes exposed to VEGF-A, n = 4, *P < 0.05. G: Western blot showing VEGF165-induced total VEGFR2 upregulation (24 h), actin immunoblot of the same membrane is shown as loading control. H: Western blot showing VEGF165-induced VEGFR2 tyrosine phosphorylation (10 min), actin immunoblot of the same membrane is shown as loading control.

 
To determine whether VEGF-A receptors are regulated by their ligand, differentiated podocytes were exposed to recombinant VEGF165 (20 ng/ml) for 24 h. VEGF-A induced a twofold increase in VEGFR2 mRNA level, as assessed by semiquantitative RT-PCR, whereas VEGFR1, sVEGFR1, NP1, and NP2 mRNA levels remained unchanged (Fig. 3, E and F). VEGF165 induced a twofold VEGFR2 protein upregulation and significantly increased VEGFR2 tyrosine phosphorylation (2.1 ± 0.85-fold, n = 3; Fig. 3, G and H).

VEGF-A promotes podocyte survival by activating the Akt pathway via VEGFR2. We determined that VEGF-A decreases podocyte apoptosis rate following serum starvation (Fig. 4A). This antiapoptotic effect is abolished by anti-VEGFR2 neutralizing antibody and not affected by an irrelevant IgG, indicating that it is mediated by VEGFR2 (Fig. 4A). Interestingly, anti-VEGFR2 neutralizing antibody enhanced podocyte apoptosis twofold above control, suggesting that endogenous VEGF plays a significant role in podocyte survival in control conditions.


Figure 4
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 4. VEGF-A reduces podocyte apoptosis and induces Akt phosphorylation. A: quantitation of podocyte apoptosis in control conditions and on exposure VEGF-A, anti-VEGFR2 neutralizing antibody or IgG. Data are expressed as means ± SE, n = 6, *P < 0.05. B: Western blot showing phosphorylated Akt and total Akt podocyte levels in control conditions and after 15 min of VEGF-A exposure, indicating that VEGF-A increases Akt phosphorylation, whereas total Akt remains unchanged. C: densitometric analysis of Akt-P changes showing a 2.2 ± 0.36-fold increase on VEGF165 exposure (*P < 0.05 vs. control), and blunting of this effect by VEGFR2 blockade (1.3 ± 0.14-fold increase above control, *P < 0.05 vs. control and ***P < 0.05 vs. VEGF), n = 4.

 
Because VEGF-A is known to stimulate the PI3K-Akt pathway in other cell types (10, 14), we examined Akt expression and phosphorylation in podocytes exposed to VEGF-A. As shown in Fig. 4, B and C, VEGF-A did not alter total Akt expression but increased Akt serine phosphorylation twofold, indicating that VEGF-A stimulated Akt activity. Akt phosphorylation induced by VEGF-A was blunted by anti-VEGFR2 neutralizing antibody, indicating that this effect was mediated, at least in part, by VEGFR2.

VEGF regulates slit diaphragm proteins. We determined that VEGF-A signaling regulates the expression of slit diaphragm proteins. In cultured podocytes, VEGF165 induced a four- to sevenfold increase in podocin expression, as indicated by the dose response shown in Fig. 5, A and B. Upon exposure to VEGF165, CD2AP expression did not change (Fig. 5C), but its interaction with podocin increased significantly by 8 h, as shown by coimmunoprecipitation (Fig. 5D). Podocin immunoblotting shows equal loading and confirms the enhanced interaction between podocin and CD2AP (Fig. 5D).


Figure 5
View larger version (22K):
[in this window]
[in a new window]
  		Fig. 5. VEGF-A regulates the expression of slit-diaphragm proteins in podocytes. A: Western blot (WB) showing a dose-response curve indicating that VEGF upregulates podocin expression; WB reprobed for actin is shown to document equal loading. B: quantitation of VEGF165-induced podocin upregulation (means ± SE, n = 4, *P < 0.05). C: WB showing that VEGF-A does not alter CD2AP expression; actin WB is shown to document equal loading. D: coimmunoprecipitation showing that VEGF-A increases the association of podocin and CD2AP; podocin WB shows even loading.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present studies, we show that VEGFR2 and VEGF-A expression in immortalized mouse podocytes is regulated by cell differentiation and availability of the ligand, indicating that podocytes possess a functional autocrine VEGF-A system. We show that VEGF-A protects podocytes from apoptosis via VEGFR2. We also demonstrate that VEGF-A signaling upregulates podocin and increases podocin/CD2AP interaction in vitro.

Podocytes are differentiated cells necessary for the development and maintenance of the glomerular basement membrane and the capillary tufts, as well as the function of the glomerular filtration barrier (21). Several proteins are known to be critical for podocyte phenotype and function, including the slit diaphragm proteins nephrin, podocin, CD2AP, actinin-4, {alpha}3-integrin, synaptopodin, and VEGF-A (1, 2, 15, 24). Ablation of VEGF-A in podocytes during kidney morphogenesis resulted in the absence of glomerular vascularization and abnormal podocyte morphology (6). However, it is not clear whether the abnormal podocyte phenotype observed was a direct result of the the absence of VEGF-A, or a consequence of missing endothelial-podocyte interactions. The availability of conditionally immortalized podocytes enabled us to address this issue in vitro and to study the role of VEGF-A in podocyte differentiation, phenotype, and function. The cell line used here was established by isolating podocytes from H-2Kb-tsA58 transgenic mice "immortomouse" (13, 20). As described before, podocytes proliferated in permissive conditions and differentiated in nonpermissive conditions (20). Upon differentiation, phenotypic changes were associated with expression of prototypical podocyte markers, such as synaptopodin, and inactivation of the SV40 T antigen.

VEGF-A is a chemoattractant for endothelial cells that directs the migration of endothelial cells toward developing nephrons and is necessary for the development of glomerular capillaries (6, 32). In the mature podocyte, VEGF-A is thought to have paracrine effects on endothelial cells, promoting survival and maintaining their fenestrated phenotype; however, its specific functions are not well defined (5). In human and rodent kidneys, VEGF-A mRNA and protein were detected predominantly in podocytes, distal tubules, and collecting ducts (28, 33). VEGF120, VEGF164, and VEGF188 are the most abundantly expressed isoforms (7). Inhibitory isoforms of VEGF-A have also been described, but their functional role remains unclear (3a). VEGF-A stimulates differentiation, proliferation, and survival of endothelial cells (7). Similarly, VEGF-A promotes renal tubular epithelial cell and podocyte proliferation and survival (8, 9, 14, 34). Recent in vitro data suggest that VEGF promote human podocyte survival via Ca+ decrease (8).

In this study, we show that mouse podocytes produce three VEGF-A isoforms. Undifferentiated podocytes express only the two larger isoforms in the cell lysate. It is unclear whether VEGF120 is expressed at a low level and constitutively secreted, because VEGF120+164 were clearly measurable by ELISA but neither isoforms was detectable by Western blot analysis. This discrepancy is likely due to the different sensitivity of these methods. In contrast, differentiated podocytes express the three major isoforms and secrete VEGF120 and VEGF164, suggesting that podocytes can generate a VEGF-A gradient. Upon podocyte differentiation, total VEGF-A expression and secretion increased threefold, as detected by Western blot analysis and ELISA, respectively.

It has been shown that SV40 T large antigen can induce VEGF expression in mesothelioma cells (3). The same mechanism could potentially be driving VEGF-A expression in undifferentiated podocytes that express SV40 T antigen. However, VEGF-A expression and secretion increased several-fold on differentiation, when the SV40 T antigen was not expressed, clearly demonstrating that cell differentiation is associated with podocyte VEGF-A upregulation. Our data on VEGF-A secretion are consistent with those reported by Kim et al. (16) indicating that rat podocyte VEGF protein expression is upregulated by hypoxia and glucose.

VEGF receptors are not only expressed in endothelial cells but also in other cells types, including renal tubular epithelial cells and podocytes (8, 34). Conditionally immortalized human podocytes express VEGFR-1, VEGFR-3, NP1, and NP2 but not VEGFR-2 (8). NP1 was also detected in primary cultures of human podocytes (11). Here, we present evidence indicating that VEGFR2 is expressed in mouse immortalized podocytes, and its expression is regulated by differentiation and ligand availability. Our data showed that all VEGF-A receptors, VEGFR1, VEGFR2, NP1, and NP2, are expressed by differentiated and undifferentiated murine podocytes. We showed that VEGFR2 mRNA and protein are upregulated by differentiation and by increased VEGF165 availability in mouse podocytes. Our results differ from previous reports suggesting that VEGFR2 is not present in podocytes (9, 11). The discrepancy may be species related, because prior studies were performed in human podocytes. It was recently shown that VEGF-A proliferative and antiapoptotic effects on podocytes are abolished by tyrosine kinase inhibitors, indirectly suggesting that they are mediated by a tyrosine kinase receptor (8). However, phosphorylation of VEGFR1 was not demonstrated (8). Here, we identified VEGFR2 protein and showed VEGF165-induced VEGFR2 tyrosine phosphorylation in murine podocytes. Podocyte VEGFR2 was the only VEGF-A receptor regulated by differentiation and ligand availability.

We confirmed and extended previous studies indicating that VEGF-A protects podocytes from apoptosis (9). VEGF-A protection from apoptosis was abolished by anti-VEGFR2- neutralizing antibody, demonstrating that VEGFR2 mediates the autocrine antiapoptotic role of VEGF-A in podocytes. Moreover, we showed that anti-VEGFR2-neutralizing antibody enhances apoptosis compared with baseline, suggesting that endogenous VEGF signaling plays a significant role in podocyte survival in control conditions. In addition, we showed that VEGF-A signaling activates the Akt pathway, as indicated by increased Akt phosphorylation that is blunted by anti-VEGFR2-neutralizing antibody. Taken together, these data demonstrate that VEGFR2 is the main VEGF-A signaling receptor in podocytes.

Soluble VEGFR1 is expressed by undifferentiated and differentiated podocytes, but its expression in vitro appears to be constitutive, as suggested by a lack of change on exposure to recombinant VEGF165. Because an antibody that recognizes solely the soluble isoforms of VEGFR1 is not available, it is presently impossible to determine whether this isoform is regulated at the translational or posttranslational level. In normal conditions, sVEGFR1 is thought to act as a decoy for excess VEGF-A, but due to VEGF’s role in podocyte survival, excessive circulating sVEGFR1 such as that occurring in preeclampsia may cause direct podocyte damage by interrupting the protective VEGF-A/VEGFR2 autocrine loop.

Our data indicate that VEGF-A is involved in the homeostasis of slit diaphragm proteins. We showed that VEGF-A induced a dose-dependent four- to sevenfold upregulation of podocin and increased its association with CD2AP, suggesting that VEGF-A promotes the structural integrity of the slit-diaphragm complex. Podocin upregulation has not been previously described. Podocin is a hairpin-like protein that localizes to the foot process cell membrane and associates via its COOH terminus with CD2AP and nephrin (26). Mutations in the podocin gene cause slit-diaphragm disruption and foot process fusion (2, 24), resulting in autosomal recessive familial focal glomerulosclerosis in humans and massive proteinuria associated with mesangial sclerosis in mice. Podocin downregulation has also been reported in puromycin aminonucleoside nephrosis (19) and in the transcription factor LMX1B mutant mice (23). VEGF-A did not alter CD2AP expression level but increased its interaction with podocin, suggesting that it modulates the dynamic interactions between these proteins. VEGF165 enhanced the interaction between podocin and CD2AP transiently (8 h), likely reflecting the half-life of the recombinant protein in the culture system. CD2AP is an adapter protein that functions as an integral member of the slit-diaphragm complex by interacting with podocin and nephrin and anchoring them to the actin cytoskeleton (18, 26). Disruption of the CD2AP gene in mice causes early massive proteinuria, foot process effacement, glomerulosclerosis, and eventually end-stage renal disease (17). VEGF-A induced mild changes in nephrin expression. Sugimoto et al. (30) reported a decrease in nephrin expression 5 h following exposure to anti-VEGF antibody, suggesting that podocyte VEGF supports nephrin expression in vivo. Recent reports linked nephrin phosphorylation or binding to PI3K to podocyte survival (9, 12). VEGF-A is known to activate the PI3K-Akt survival pathway in endothelial and tubular epithelial cells (10, 34). Our data indicate that VEGF-A acts similarly in podocytes, reducing apoptosis via VEGR2 and activation of Akt. Nephrin may regulate the process via protein-protein interactions. Thus previous data and those presented here suggest that VEGF-A supports and is probably necessary to maintain the expression, function, and interactions of the slit-diaphragm genes.

In summary, we showed that podocytes have a functional autocrine VEGF-A system that promotes survival through VEGFR2 and is regulated by differentiation and ligand availability. In addition, we demonstrated that VEGF-A signaling upregulates podocin and increases podocin/CD2AP interaction in vitro.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants RO1-DK-64187 (A. Tufro), DK-59333 (A. Tufro), and the O’Brien Center Grant P50-DK-64236 (A. Tufro and P. Mundel).


   ACKNOWLEDGMENTS
 
We thank A. Shaw, Washington University School of Medicine, for kindly providing the CD2AP antibody and V. Bautch, University of North Carolina, for sharing the sVEGR1 primers sequence.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Tufro, Dept. of Pediatrics/Nephrology and Dept. of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Forchheimer Bldg., Rm. 708, Bronx, NY 10461 (e-mail: atufro{at}aecom.yu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Benzing T. Signaling at the slit diaphragm. J Am Soc Nephrol 15: 1382–1391, 2004.[Free Full Text]
  2. Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, Dahan K, Gubler MC, Niaudet P, and Antignac C. NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet 24: 349–354, 2000.[CrossRef][Web of Science][Medline]
  3. Cacciotti P, Strizzi L, Vianale G, Iaccheri L, Libener R, Porta C, Tognon M, Gaudino G, and Mutti L. The presence of simian-virus 40 sequences in mesothelioma and mesothelial cells is associated with high levels of vascular endothelial growth factor. Am J Respir Cell Mol Biol 26: 189–193, 2002.[Abstract/Free Full Text]
  4. Cui TG, Foster RR, Saleem M, Mathieson PW, Gillatt DA, Bates DO, and Harper SJ. Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF165b) mRNA and protein. Am J Physiol Renal Physiol 286: F767–F773, 2004.[Abstract/Free Full Text]
  5. De Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, and Williams LT. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 255: 989–991, 1992.[Abstract/Free Full Text]
  6. Eremina V and Quaggin SE. The role of VEGF-A in glomerular development and function. Curr Opin Nephrol Hypertens 13: 9–15, 2004.[Web of Science][Medline]
  7. Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, Gerber HP, Kikkawa Y, Miner JH, and Quaggin SE. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest 111: 707–716, 2003.[CrossRef][Web of Science][Medline]
  8. Ferrara N, Gerber HP, and LeCouter J. The biology of VEGF and its receptors. Nat Med 9: 669–676, 2003.[CrossRef][Web of Science][Medline]
  9. Foster RR, Hole R, Anderson K, Satchell SC, Coward RJ, Mathieson PW, Gillatt DA, Saleem MA, Bates DO, and Harper SJ. Functional evidence that vascular endothelial growth factor may act as an autocrine factor on human podocytes. Am J Physiol Renal Physiol 284: F1263–F1273, 2003.[Abstract/Free Full Text]
  10. Foster R, Saleem M, Mathieson P, Bates D, and Harper S. Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes. Am J Physiol Renal Physiol 288: F48–F57, 2005.[Abstract/Free Full Text]
  11. Gerber HP, McMurtrey A, Kowalski J, Yan M, Keyt BA, Dixit V, and Ferrara N. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem 273: 30336–30343, 1998.[Abstract/Free Full Text]
  12. Harper SJ, Xing CY, Whittle C, Parry R, Gillatt DA, Peat D, and Mathieson PW. Expression of neuropilin by human glomerular epithelial cells in vitro and in vivo. Clin Sci (Colch) 101: 439–466, 2001.[Medline]
  13. Huber TB, Hartleben B, Kim J, Schmidts M, Schermer B, Keil A, Egger L, Lecha RL, Borner C, Pavenstadt H, Shaw AS, Walz G, and Benzing T. Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling. Mol Cell Biol 23: 4917–4928, 2003.[Abstract/Free Full Text]
  14. Jat PS, Noble MD, Ataliotis P, Tanaka Y, Yannoutsos N, Larsen L, and Kioussis D. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc Natl Acad Sci USA 88: 5096–5100, 1991.[Abstract/Free Full Text]
  15. Kanellis J, Fraser S, Katerelos M, and Power DA. Vascular endothelial growth factor is a survival factor for renal tubular epithelial cells. Am J Physiol Renal Physiol 278: F905–F915, 2000.[Abstract/Free Full Text]
  16. Kestila M, Lenkkeri U, Mannikko M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V, Morita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A, and Tryggvason K. Positionally cloned gene for a novel glomerular protein–nephrin–is mutated in congenital nephrotic syndrome. Mol Cell 1: 575–582, 1998.[CrossRef][Web of Science][Medline]
  17. Kim BS, Chen J, Weinstein T, Noiri E, and Goligorsky MS. VEGF expression in hypoxia and hyperglycemia: reciprocal effect on branching angiogenesis in epithelial-endothelial co-cultures. J Am Soc Nephrol 13: 2027–2036, 2002.[Abstract/Free Full Text]
  18. Kim JM, Wu H, Green G, Winkler CA, Kopp JB, Miner JH, Unanue ER, and Shaw AS. CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility. Science 300: 1298–1300, 2003.[Abstract/Free Full Text]
  19. Lehtonen S, Zhao F, and Lehtonen E. CD2-associated protein directly interacts with the actin cytoskeleton. Am J Physiol Renal Physiol 283: F734–F743, 2002.[Abstract/Free Full Text]
  20. Luimula P, Sandstrom N, Novikov D, and Holthofer H. Podocyte-associated molecules in puromycin aminonucleoside nephrosis of the rat. Lab Invest 82: 713–718, 2002.[Web of Science][Medline]
  21. Mundel P, Reiser J, Zuniga Mejia Borja A, Pavenstadt H, Davidson GR, Kriz W, and Zeller R. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236: 248–258, 1997.[CrossRef][Web of Science][Medline]
  22. Pavenstadt H, Kriz W, and Kretzler M. Cell biology of the glomerular podocyte. Physiol Rev 83: 253–307, 2003.[Abstract/Free Full Text]
  23. Roberts WG and Palade GE. Neovasculature induced by vascular endothelial growth factor is fenestrated. Cancer Res 57: 765–772, 1997.[Abstract/Free Full Text]
  24. Rohr C, Prestel J, Heidet L, Hosser H, Kriz W, Johnson RL, Antignac C, and Witzgall R. The LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes. J Clin Invest 109: 1073–1082, 2002.[CrossRef][Web of Science][Medline]
  25. Roselli S, Heidet L, Sich M, Henger A, Kretzler M, Gubler MC, and Antignac C. CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility. Science 300: 1298–1300, 2003.[Abstract/Free Full Text]
  26. Rossant J and Hirashima M. Vascular development and patterning: making the right choices. Curr Opin Genet Dev 13: 408–412, 2003.[CrossRef][Web of Science][Medline]
  27. Schwarz K, Simons M, Reiser J, Saleem MA, Faul C, Kriz W, Shaw AS, Holzman LB, and Mundel P. Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. J Clin Invest 108: 1621–1629, 2001.[CrossRef][Web of Science][Medline]
  28. Shibuya M. Structure and dual function of vascular endothelial growth factor receptor-1 (Flt-1). Int J Biochem Cell Biol 33: 409–420, 2001.[CrossRef][Web of Science][Medline]
  29. Simon M, Grone HJ, Johren O, Kullmer J, Plate KH, Risau W, and Fuchs E. Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney. Am J Physiol Renal Fluid Electrolyte Physiol 268: F240–F250, 1995.[Abstract/Free Full Text]
  30. Soker S, Takashima S, Miao HQ, Neufeld G, and Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92: 735–745, 1998.[CrossRef][Web of Science][Medline]
  31. Sugimoto H, Hamano Y, Charytan D, Cosgrove D, Kieran M, Sudhakar A, and Kalluri R. Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria. J Biol Chem 278: 12605–12608, 2003.[Abstract/Free Full Text]
  32. Terman BI, Dougher-Vermazen M, Carrion ME, Dimitrov D, Armellino DC, Gospodarowicz D, and Bohlen P. Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun 187: 1579–1586, 1992.[CrossRef][Web of Science][Medline]
  33. Tufro A. VEGF spatially directs angiogenesis during metanephric development in vitro. Dev Biol 227: 558–566, 2000.[CrossRef][Web of Science][Medline]
  34. Tufro A, Norwood VF, Carey RM, and Gomez RA. Vascular endothelial growth factor induces nephrogenesis, and vasculogenesis. J Am Soc Nephrol 10: 2125–2134. 1999.[Abstract/Free Full Text]
  35. Villegas Lange-Sperandio GB and Tufro A. Autocrine and paracrine functions of vascular endothelial growth factor (VEGF) in renal tubular epithelial cells. Kidney Int 67: 449–457, 2005.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Nephrol Dial TransplantHome page
A. B. Fogo
The multi-talented podocyte
Nephrol. Dial. Transplant., November 1, 2009; 24(11): 3269 - 3270.
[Full Text] [PDF]

Home page
 Nephrol Dial TransplantHome page
J. Nystrom, K. Hultenby, S. Ek, J. Sjolund, H. Axelson, K. Jirstrom, M. A. Saleem, K. Nilsson, and M. E. Johansson
CRIM1 is localized to the podocyte filtration slit diaphragm of the adult human kidney
Nephrol. Dial. Transplant., July 1, 2009; 24(7): 2038 - 2044.
[Abstract] [Full Text] [PDF]

Home page
 Nephrol Dial TransplantHome page
E. Letavernier, P. Bruneval, S. Vandermeersch, J. Perez, C. Mandet, M.-F. Belair, J.-p. Haymann, C. Legendre, and L. Baud
Sirolimus interacts with pathways essential for podocyte integrity
Nephrol. Dial. Transplant., February 1, 2009; 24(2): 630 - 638.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
M. Saint-Geniez, T. Kurihara, and P. A. D'Amore
Role of Cell and Matrix-Bound VEGF Isoforms in Lens Development
Invest. Ophthalmol. Vis. Sci., January 1, 2009; 50(1): 311 - 321.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
C.-H. Ku, K. E. White, A. Dei Cas, A. Hayward, Z. Webster, R. Bilous, S. Marshall, G. Viberti, and L. Gnudi
Inducible Overexpression of sFlt-1 in Podocytes Ameliorates Glomerulopathy in Diabetic Mice
Diabetes, October 1, 2008; 57(10): 2824 - 2833.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
S. N. Korgaonkar, X. Feng, M. D. Ross, T.-c. Lu, V. D'Agati, R. Iyengar, P. E. Klotman, and J. C. He
HIV-1 Upregulates VEGF in Podocytes
J. Am. Soc. Nephrol., May 1, 2008; 19(5): 877 - 883.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
B. Haraldsson, J. Nystrom, and W. M. Deen
Properties of the Glomerular Barrier and Mechanisms of Proteinuria
Physiol Rev, April 1, 2008; 88(2): 451 - 487.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
B. A. Bryan, T. E. Walshe, D. C. Mitchell, J. S. Havumaki, M. Saint-Geniez, A. S. Maharaj, A. E. Maldonado, and P. A. D'Amore
Coordinated Vascular Endothelial Growth Factor Expression and Signaling During Skeletal Myogenic Differentiation
Mol. Biol. Cell, March 1, 2008; 19(3): 994 - 1006.
[Abstract] [Full Text] [PDF]

Home page
 CJASNHome page
E. Letavernier, P. Bruneval, C. Mandet, J.-P. D. Van Huyen, M.-N. Peraldi, I. Helal, L.-H. Noel, and C. Legendre
High Sirolimus Levels May Induce Focal Segmental Glomerulosclerosis De Novo
Clin. J. Am. Soc. Nephrol., March 1, 2007; 2(2): 326 - 333.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/F422    most recent
00448.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (17)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guan, F.
Right arrow Articles by Tufro, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guan, F.
Right arrow Articles by Tufro, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.