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作者：Fangxia Guan, Guillermo Villegas, Jason Teichman, Peter Mundel, and Alda Tufro作者单位：1 Department of Pediatrics/Nephrology and Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx; and 2 Department of Medicine, Mount Sinai School of Medicine, New York, New York 6 b( R( l) u9 e& |9 ^
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【摘要】% w O5 S* P0 Z4 Z! t- H0 D T
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 VEGF 165 (20 ng/ml) or control media for 24 h. VEGF 165 induced a twofold increase in VEGFR2 mRNA and protein levels, whereas VEGFR1, sVEGFR1, NP1, and NP2 mRNA levels remained unchanged. VEGF 165 induced VEGFR2 phosphorylation. VEGF 165 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. : i# o" ` K' h. F: @
【关键词】 cell differentiation apoptosis VEGFA secretion slit diaphragm proteins VEGF receptor
9 W: V! S: K2 \5 l/ g/ }: b% Y 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.
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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 ). VEGF 165 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 ).
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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.& ^/ g) u& L0 w# O5 L8 {- j: g
7 Q5 v. a6 k. t, NMATERIALS AND METHODS
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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 -interferon (Sigma) in humidified incubators with air-5% CO 2. Podocytes were propagated on collagen I-coated plates at 33°C in the presence of recombinant mouse -interferon (10 U/ml). Removal of -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 VEGF 165 (R&D Systems; 20 ng/ml) for 30 min to 24 h as indicated.2 \* u6 Q, @7 Y1 J1 o% C2 {
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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 pSer 473 (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 ( x 140) 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.% T" U; t' I6 L7 Z* L9 r
' U. ]7 ~+ u% A) uCoimmunoprecipitation. 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.5 q2 q7 |! ?. }, ]: }2 a
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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% CO 2. Data were corrected for total protein concentration and reported as picograms of VEGF per microgram of protein.
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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.
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Table 1. Primer sequences, product sizes, and annealing temperatures
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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-Tyr 397 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). s4 |6 \( E! J' N+ S7 V" x
2 ~6 ^" }* @& N* E* e _" VApoptosis. Differentiated podocytes were starved for 36 h, exposed to rVEGF 165 (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.; ?" q) L. z9 g# ]! D4 S
% K8 J' X$ z& c/ r* zStatistical 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 + n! F6 Z; q0 b; {; f. {" f/ Y
( ~- X2 B8 a; x$ H/ Z2 D4 {8 t, JRESULTS
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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. 1 A ), as previously described ( 20 ). Accordingly, SV40-T antigen was abundantly expressed at 33°C in the presence of -IFN, whereas it was barely detected at 37°C in the absence of -IFN ( Fig. 1 B ). Synaptopodin, a marker of podocyte differentiation, was not detected under permissive conditions and abundantly expressed in nonpermissive conditions ( Fig. 1 B ).: Y- a( q/ _7 B' Q4 X
1 x- Q& t& O3 n) {: ?, Z3 SFig. 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.( J; Q* J# z; Q. l. R* Z
. {# |1 N$ g$ R4 GVEGF-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. 2 A ). Upon podocyte differentiation, VEGF-A expression increased threefold ( Fig. 2 B ). Podocyte VEGF-A secretion to the supernatant also increased threefold on cell differentiation, as indicated by ELISA ( Fig. 2 C ). Supernatant VEGF-A was normalized for total protein content. Secreted VEGF-A isoforms were undetectable by immunoblotting in the supernatant of undifferentiated podocytes, whereas VEGF 164 and VEGF 120 were secreted into the supernatant by differentiated podocytes at 2:1 ratio ( Fig. 2 D ). 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.
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5 o( w. r+ w7 Z/ m9 J5 B9 VFig. 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 ' m, z$ Z" A" P* b4 \
3 V- S$ d3 D- V$ A" IVEGF-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. 3 A ). 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. 3 D ).
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) ]. k! ^% F& s1 V% W1 |2 {# IFig. 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 . H3 e, Q. E9 f' Z
" z& f2 Z; L# g# U6 p4 w R0 L/ RTo determine whether VEGF-A receptors are regulated by their ligand, differentiated podocytes were exposed to recombinant VEGF 165 (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 ). VEGF 165 induced a twofold VEGFR2 protein upregulation and significantly increased VEGFR2 tyrosine phosphorylation (2.1 ± 0.85-fold, n = 3; Fig. 3, G and H ).2 f% L) q( ]$ o' _* M0 u; j
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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. 4 A ). 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. 4 A ). 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.& \5 r3 @7 S5 W
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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 + x$ T, \" q5 A9 Z% Y
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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.) }0 m5 g* D1 F+ m! ?
; q8 s$ v) C: u8 p( \! LVEGF regulates slit diaphragm proteins. We determined that VEGF-A signaling regulates the expression of slit diaphragm proteins. In cultured podocytes, VEGF 165 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 VEGF 165, CD2AP expression did not change ( Fig. 5 C ), but its interaction with podocin increased significantly by 8 h, as shown by coimmunoprecipitation ( Fig. 5 D ). Podocin immunoblotting shows equal loading and confirms the enhanced interaction between podocin and CD2AP ( Fig. 5 D ).
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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 VEGF 165 -induced podocin upregulation (means ± SE, n = 4, * P " g. Z& Z) @# X, b) D
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DISCUSSION
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. ?. } q' W! G4 Z6 y: mIn 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.
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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, 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-2K b -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.
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0 f1 o! Q( k6 ]$ lVEGF-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 ). VEGF 120, VEGF 164, and VEGF 188 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 ).5 d+ \1 V. {" F9 v
& r, r: J& g1 f, v! Z5 o" f& EIn 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 VEGF 120 is expressed at a low level and constitutively secreted, because VEGF 120 164 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 VEGF 120 and VEGF 164, 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.
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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.
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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 VEGF 165 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 VEGF 165 -induced VEGFR2 tyrosine phosphorylation in murine podocytes. Podocyte VEGFR2 was the only VEGF-A receptor regulated by differentiation and ligand availability.
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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.
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9 W$ T; I/ g* K% @" d: t! D: WSoluble 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 VEGF 165. 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.+ O0 }) w* j3 ^8 p+ T
0 I5 T* M7 W$ C9 i4 U; pOur 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. VEGF 165 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.
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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.) }6 C& B% U [* P! C
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% \, L6 Y2 F u' V0 kThis 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).
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P+ o' G4 ?/ m8 B; tACKNOWLEDGMENTS: r. ^7 s. o8 U; J
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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.
$ _4 i4 y' t4 w9 | 【参考文献】
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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.
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