INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE GLOMERULUS IS a unique functional unit characterized by differential permeability, high to water and electrolytes and low to protein. The podocytes (visceral glomerular epithelial cells; GECs) are believed to play a crucial role in the maintenance of this selective barrier. Podocyte dysfunction, either genetic (20) or acquired in glomerular disease (13), results in loss of the macromolecular selectivity of the glomerular filtration barrier and proteinuria. How podocytes exert their influence is poorly understood, but the available data suggest that podocytes contribute structurally by the provision of slit diaphragm and glomerular basement membrane (20, 22) and functionally by production of molecules known to affect endothelial permeability in other vascular beds. Examples of these molecules include vascular endothelial growth factor (VEGF) (2), known to increase microvascular permeability (5, 6), and angiopoietin-1 (33), the only podocyte-secreted molecule that has been shown to decrease macromolecular extravasation (38).

Despite the production of VEGF by podocytes at high levels, the detailed role of VEGF in normal glomerular physiology and its potential contribution to glomerular macromolecular permeability remain controversial. Normal VEGF biology is complex. Differential exon splicing of the VEGF gene results in a number of mRNA species, which code for a series of isoforms containing different numbers of amino acids termed VEGF₁₈₉ and VEGF₁₆₅ (the most widespread isoform and also that found predominantly in the renal glomerulus) and VEGF₁₂₁ (10). VEGF isoform expression in glomeruli is heterogeneous. Individual human glomeruli express one, two, or all three of these main isoforms at the mRNA level (40). Minor VEGF mRNA splice variants (VEGF₂₀₆, VEGF₁₈₃, VEGF₁₄₈, and VEGF₁₄₅) have also been reported, but they are less well characterized (18, 19, 29, 40). In addition, evidence for a new set of almost identical sister molecules of inhibitory VEGF isoforms in the renal cortex has recently been described by this laboratory (4).

VEGF signals through two receptors. The primary targets of VEGF on vascular endothelial cells are the class III receptor tyrosine-kinases, VEGFR-1 (flt-1) and VEGFR-2 (KDR), both of which are expressed by the glomerular endothelium (9). The latter initiates angiogenesis, cell migration, and permeability changes. VEGFR-1 also exists in a soluble form, sVEGFR-1 (sFlt), which is inhibitory when bound to free VEGF. In addition, the neuropilins have been shown to bind specific isoforms of VEGF, although their signaling properties remain unknown (14, 16, 35, 36). Neuropilin-1 (Np-1), for example, facilitates the binding of VEGF₁₆₅ to VEGFR-2 (12) enhancing VEGFR-2-mediated effects.

VEGF, one of the most potent mediators of angiogenesis and endothelial permeability known, is produced at a high level by the podocytes 200-300 nm from its receptors on the glomerular endothelial cells. A paracrine action for VEGF would therefore appear clear (7). For this to occur, however, VEGF needs to act against a significant filtration of fluid across the glomerular barrier. It has therefore been suggested that VEGF might act on cells other than the glomerular endothelium. This led us previously to investigate VEGF-R expression by human podocytes themselves. Although we were unable to detect tyrosine-kinase VEGFR-2 expression, we demonstrated the expression of Np-1 by normal human podocytes in vitro and in vivo (17). These results suggest that podocytes may have the potential to bind the VEGF they secrete. We therefore hypothesized that the potential VEGF-Np-1 interaction may be important in terms of an autocrine loop or in VEGF sequestration in podocytes (17).

Although Np-1 has been considered as a nonsignaling VEGF coreceptor, VEGF has more recently been identified as an autocrine survival factor for Np-1-positive, VEGF tyrosine-kinase receptor-negative breast carcinoma cells (1). Because VEGF has been shown to stimulate increases in cytosolic calcium concentration [Ca²⁺]_i in endothelial cells, we were prompted to investigate intracellular cytosolic calcium responses of cultured human podocytes to exogenous VEGF to address the hypothesis that VEGF may play a role as a podocyte autocrine factor. We studied these potential functional responses in proliferating dedifferentiated primary culture podocytes and nonproliferating differentiated podocytes in vitro. In addition, we investigated the potential of exogenous VEGF to act as a survival factor for dedifferentiated proliferating podocytes. Finally, we determined the distribution of VEGF within the region of the glomerular filtration barrier using transmission electron microscopic (TEM) analysis of colloidal gold immunohistochemistry on normal human glomeruli.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

VEGF used in these experiments was recombinant VEGF₁₆₅, a kind gift of N. Ferrara (Genentech). All chemicals/solutions were from Sigma unless otherwise stated.

Primary culture podocytes. Nephrectomy tissue was supplied by the Department of Urology, Southmead Hospital, from patients undergoing nephrectomy for unipolar renal tumor (age range 43-68 yr). All patients were nondiabetic, normotensive with normal excretory renal function and no urinary sediment. Cells and mRNA from human tissue were derived from material removed at surgery and the excess to diagnostic requirements or postmortem. Informed consent was obtained from patients or relatives as appropriate.

Conditionally immortalized podocytes. This cell line has been conditionally immortalized from normal human podocytes with a temperature-sensitive mutant of immortalized SV40 T antigen. These cells have been previously characterized in detail elsewhere (32). At the "permissive" temperature of 33°C, the SV40 T antigen is active and allows the cells to proliferate rapidly. Thermoswitching the cells to the "nonpermissive" temperature of 37°C silences the transgene and the cells become growth arrested and differentiated. Under these conditions, they express antigens appropriate to in vivo arborized podocytes. Cells were grown on coverslips for a period of 14 days to ensure growth arrest and differentiation.

Intracellular calcium studies. Podocytes were grown on coverslips to confluence. Cells were incubated with fura 2-AM (10 µM) for 90 min in DMEM at room temperature, and the coverslip was then placed in a holder. The holder was then mounted on a rig consisting of an inverted fluorescence microscope (DM IRB, Leica) equipped with a UV source (Cairn Instruments, World Precision Instruments) with filters for excitation at 340 and 380 nm. Fast switching was achieved using a rotary filter wheel at 50 Hz and a spectrophotometer for photometric measurement (Cairn Instruments). The spectrophotometer received emitted light via a 400-nm dichroic filter and a 510- to 530-nm barrier filter in front of the photometer. Powerlab software was used for analysis and graphic display.

[³H]-thymidine assays and cell count. A 24-well plate was seeded with primary cultured podocytes, which were incubated in RPMI media containing 1% penicillin/streptomycin, 1% L-glutamine, 1% insulin transferrin selenite (all Life Technologies), and 20% FBS. One well was set aside for 1 ml serum-free media and one for FBS-free media plus VEGF. These were used as negative controls. Podocytes were left for 48 h until established, and then media were removed and replaced with FBS-free media. One nanomolar VEGF₁₆₅ was added to half the wells and one-half were left untreated. Twenty-four hours later, 37 kBq methyl-[³H] thymidine (Amersham Pharmacia) were added to each well. Four hours later, media were removed and 0.2 ml trypsin was added to each well and left for 2 min. Two hundred microliters of RPMI were added and a hemocytometer was used to determine cell number in each well (Weber). Remaining cells were pipetted into 1.5-ml tubes (Eppendorf) and spun at 300 g for 10 min (Biofuge, Heraeus). The supernatant was removed, and 0.2 ml NaOH was added and left at room temperature for 30 min. Cells were then pipetted into scintillation vials (Fisher), 5 ml of biodegradable scintillation fluid (Amersham Pharmacia) were added, and counts per minute were read by a scintillation counter (1217 Rachbeta, LKB Wallac).

Cytotoxicity assays. Ninety-four wells of a 96-well plate (Costar) were seeded with primary cultured podocytes and 100 µl 20% FBS-RPMI. Podocytes were left for 48 h, and then media were removed and replaced with FBS-free media. After 24 h, 100 µl media were removed from each well and cytotoxicity was assayed using a lactate dehydrogenase (LDH) cytotoxicity detection kit (Roche) and quantified using a Bichrometric Multiscan plate reader (Labsystems). These samples were used for background LDH measurement (T_xmin). The media of half of the wells were replaced with 100 µl FBS-free media, the other half with FBS-free media containing 1 nM VEGF₁₆₅. Twenty-four hours later, 100 µl media were again removed from each well and cytotoxicity was assayed and quantified. These samples were used to determine the cytotoxicity (T_xexp). Finally, 100 µl of 2% Triton X-100/1 × PBS (final concentration 1%) were added to each well and left for 10 min to completely lyse the cells. One hundred microliters were removed and cytotoxicity was again assayed and quantified. This enabled determination of the maximum LDH from the well (T_xmax). Percent cytotoxicity (T_x) was calculated for each well as
The fold-increase in cytotoxicity was calculated as [T_x(treatment)]/[T_x(control)]. The percent reduction in cytotoxicity was calculated as [T_x(VEGF) × 100]/[T_{x(No VEGF)}].

RT-PCR. Total RNA was extracted as previously described (8) using the TRIzol method from the conditionally immortalized human podocyte cell line, human thyroid, brain, and kidney. Reverse transcription was carried out using 1 µg RNA and 5 µM oligo dT (Promega) in 10 µl RNAse-free water (Sigma). This mixture was incubated at 65°C for 5 min and immediately placed on ice. The reaction mixture was then altered to 1× first-strand synthesis buffer (Roche), 10 mM DTT (Roche), 2.5 mM dNTPs (Promega), 1 U RNA guard (Amersham), and 2.5 U expand RT (Roche) in a total of 20 µl RNAse-free water (Sigma). This was incubated at 42°C for 2 h. PCR was performed using the primers as detailed in Table 1. The PCR mixture consisted of 1× PCR buffer (Abgene), 1.25 mM MgCl₂ (Abgene), 375 µM dNTPs, 10 µM forward primer, 10 µM reverse primer (except for GAPDH where 5 µM of each primer were used), 1 µl cDNA, and 1 U Taq (Abgene) in 20 µl RNAse-free water. A standard PCR cycle was used, i.e., 55°C, 35 cycles (Hybaid). RT-PCR products were run on 2% agarose (Roche) gels in the presence of 0.5 µg/ml ethidium bromide (Invitrogen). Gels were photographed under UV transillumination (Gibco). A 100-bp ladder (Sigma) was used to visualize bands from 100 to 1,000 bp.

Western blot analysis. Confluent primary cultured podocytes from a T75 flask were left untreated or treated with VEGF (1 nM) for 30 min. The cells were then trypsinized, rinsed in PBS, pelleted, and the protein was extracted in 0.2% (vol/vol) SDS, 300 mM NaCl, 20 mM Tris, 10 mM ethyldiamine tetraacetic acid, 2 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µM E64, 2 µg/ml aprotinin, 62.5 mM -glycerophosphate, and 1 µM pepstatin A. Protein quantification was performed spectrophotometrically using Bio-Rad dye. Equal amounts of protein were electrophoresed against a prestained protein size marker using 10% SDS-polyacrylamide gel electrophoresis. Proteins were electroblotted to polyvinylidene difluoride membrane. Membranes were blocked in 10% (wt/vol) nonfat dry milk (Marvel) in 1× PBS-Tween 20 0.1% (vol/vol) (PBST) for 1 h and incubated with primary antibody (1:300 goat anti-VEGF-R1, SC-316, Santa Cruz) for 1 h in 1× PBST plus 5% (wt/vol) Marvel. Unbound primary antibody was removed by five washes in 1× PBST (5 min/wash). The membrane was incubated for 1 h with 1× PBST plus 5% (wt/vol) Marvel and secondary antibody (1:3,000 donkey anti-goat IgG). Washes were performed as previously described, and the protein was detected by enhanced chemiluminescence.

TEM. One-millimeter cubed pieces of renal cortex were taken from the normal pole of nephrectomy samples taken for unipolar cancer and fixed in 0.2% glutaraldehyde (Agar Scientific) and 0.2 M phosphate buffer at pH 7.4 at room temperature for 30 min. The tissue was then stored in 0.2 M phosphate buffer until processed. Specimens were partially dehydrated using a 10-min wash in 50% IMS followed by three 10-min washes in 70% methylated spirits (IMS). Specimens were infiltrated with LR white hard grade resin (London Resin) in a 2:1 ratio with 70% IMS for 30 min. Specimens were infiltrated with LR white resin for four 30-min periods. The specimens were then embedded in LR white resin plus an accelerator (London Resin) in size 00 gelatin capsules (Agar Scientific) and left to polymerize via a cold catalytic process at 4°C for at least 2 h. The blocks were then transferred to a 50°C oven for 2 h. The capsules were then exposed to the air and left to set. Sections were cut at 0.5-0.9 µm on a Leica Reichert Ultracut S ultramicrotome and placed on a glass slide and stained with 1% toluidine blue in 1% borax to determine whether the tissue was suitable for further investigation. Appropriate tissue was cut into 90-nm sections and mounted onto 300 mesh hexagonal nickel grids (Agar Scientific) and left to air dry. Grids were washed in 0.01 M PBS (pH 7.4) for 10 min and then incubated in polyclonal rabbit anti-VEGF antibody (A. Menarini) in PBS (pH 7.4) and 0.6% BSA at 1:10 in Antibody diluent (A. Menarini) for 60 min at room temperature. Sections were then washed for 1 min in PBS (pH 7.4) and PBS (pH 8.2). The secondary antibody was 15-nm gold-conjugated goat anti-rabbit IgG in Tris (pH 8.2), sodium azide, and 0.6% BSA (BioCell at Agar Scientific) 1:10 dilution, applied for 60 min. Grids were washed in PBS (pH 8.2) and deionized H₂O for 1 min and then stained with a saturated solution of uranyl acetate for 20 min; sections were then washed in deionized water and stained with lead citrate for 1 min. The grids were then rinsed with deionized water and left to air dry. Grids were viewed under an electron microscope (Philips CM10). Podocyte (intracellular or membrane associated), glomerular basement membrane, and glomerular endothelial cell-associated gold particles were enumerated in 16 random fields from four different kidneys. Colloidal gold particles were considered membrane associated if they were within two particle widths (i.e., 30 nm) of the membrane on either side.

Statistics. Data are presented as means ± SE. Two-tailed, paired t-tests were used to compare paired data on the same cells, and unpaired t-tests were used to compare separate cell populations treated differently. ANOVA was used to compare distribution of gold particles on podocyte foot processes.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Primary cultured podocytes. Figure 1 shows the effect of HBSS on [Ca²⁺]_i in primary cultured podocytes (Fig. 1A) and human tubular epithelial cells (HK2). HBSS did not change calcium in either cell line or in human tubular epithelial cells (Fig. 1B) in either the presence or absence (not shown) of extracellular Ca²⁺. ATP, on the other hand, caused a transient rapid increase in [Ca²⁺]_i on all occasions (Fig. 1C). [Ca²⁺]_i increased from 113.2 ± 22.1 to 209.5 ± 41.0 nM (P < 0.01; Fig. 1D) peaking at 30 ± 10 s and returning to baseline after 3.2 ± 0.5 min. Surprisingly, although VEGF did not alter [Ca²⁺]_i in the presence of extracellular calcium (Fig. 2A), VEGF produced a slow and sustained reduction in [Ca²⁺]_i, which was significantly different from baseline in minimal extracellular calcium (Fig. 2B in primary cultured podocytes). There was a significant reduction in the ratio (R) in minimal, but not normal extracellular calcium (Fig. 2C), which corresponds to a change in [Ca²⁺]_i from 178.9 ± 35.6 to 121.1 ± 25.4 nM with VEGF (P < 0.05). A minimum was reached after 5 ± 1 min.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effect of HBSS and ATP on podocyte and HK2 cell calcium. A: example of no significant change of cytosolic Ca2+ concentration ([Ca2+]i) in primary culture human podocytes in response to 1 µl HBSS in the presence of 1.3 mM extracellular Ca2+ concentration ([Ca2+]o). B: means ± SE of [Ca2+]i change in podocytes and human tubular epithelial cells in response to 1 µl HBSS in the presence of 1.3 mM [Ca2+]o. C: transient increase in [Ca2+]i in primary culture human podocytes in response to 30 µM ATP. D: means ± SE of ATP-stimulated response compared with baseline values (n = 6, **P < 0.005, paired t-test).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of vascular endothelial growth factor (VEGF) on [Ca2+]i of primary cultured and conditionally immortalized podocytes. A: example of [Ca2+]i change in primary culture human podocytes in response to 1 nM VEGF in the presence of 1.3 mM [Ca2+]o. B: example of [Ca2+]i change in primary culture podocytes in response to 1 nM VEGF in the absence of [Ca2+]o. C: means ± SE ratio before (open bars) and after (filled bars) VEGF treatment. D: example of [Ca2+]i change in response to 1 nM VEGF in the presence of 1.3 mM [Ca2+]o. E: example of effect of VEGF on [Ca2+]i change in the presence of minimal [Ca2+]o. F: means ± SE ratio before (open bars) and after (filled bars) VEGF treatment. *P < 0.05, **P < 0.02 compared with before treatment.

Conditionally immortalized podocytes. ATP administration stimulated a transient increase in [Ca²⁺]_i in conditionally immortalized cells from 102.2 ± 34.5 to 142.1 ± 35.8 nM postexposure, P < 0.05. In a similar pattern to that seen in primary culture podocytes, VEGF did not alter [Ca²⁺]_i in differentiated podocytes in the presence of [Ca²⁺]_o (Fig. 2D) but again produced a slow but sustained significant reduction in [Ca²⁺]_i when these differentiated podocytes were incubated in minimal extracellular calcium (Fig. 2E). There was a significant reduction in the ratio (Fig. 2F), which corresponds to a reduction in [Ca²⁺]_i from 94.7 ± 9.8 to 66.1 ± 8.4 nM (P < 0.02).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effect of class III receptor tyrosine-kinase inhibitor PTK787/ZK222584 on VEGF-mediated [Ca2+]i changes in transformed human podocytes. A: example of [Ca2+]i change in response to 1 nM VEGF after treatment with PTK787/ZK222584 incubated in minimal calcium. B: means ± SE ratio before (open bars) and after (gray bars) treatment with PTK787/ZK222584 and 1 nM VEGF. C: example of [Ca2+]i measurement in response to treatment with PTK787/ZK222584 alone. D: comparison of the response of vGEC [Ca2+]i to 1 nM VEGF in the absence and presence of PTK787/ZK222584. Values are the relative change in R from baseline (1 = no change). *P < 0.05, ***P < 0.001.

Proliferation and cytotoxicity. In primary cultured podocytes, addition of 1 nM VEGF to culture medium resulted in a significant increase in [³H]thymidine incorporation (from 2,364 ± 301 to 3,349 ± 283 cpm, P < 0.05; Fig. 4A). Assuming that the [³H]thymidine incorporation is the same for each cell for each division, then [³H]thymidine incorporation gives the number of cells dividing within a defined time. If more cells are surviving, then there will be more cells present to undergo the normal rate of division. Therefore, to determine whether this increase in [³H]thymidine incorporation was due to increased proliferation rate or due to an increase in the survival of VEGF-treated cells (and hence increased cell number), we measured the number of cells in each well. The cell number also increased from 2.6 ± 0.5 to 4.5 ± 0.7 × 10⁴/ml (P < 0.05; Fig. 4B) with VEGF treatment. [³H]thymidine incorporation calculated per cell was therefore not affected by VEGF (untreated 0.1 ± 0.015 cpm/cell, treated 0.125 ± 0.029 cpm/cell, not significant; Fig. 4C), suggesting that VEGF was acting not by increasing proliferation rate but by reducing cell death. To assess independently whether VEGF could reduce cytotoxicity, the effect of VEGF on LDH release into the media (which occurs when cells lyse) was carried out. VEGF stimulated a reduction in cytotoxicity from 12.5 ± 3.0 to 5.9 ± 0.67% (P < 0.05; Fig. 4D).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of VEGF on primary cultured podocytes proliferation. A: means ± SE 3H-thymidine incorporation without (open bars) and with (filled bars) 1 nM VEGF. B: means ± SE cell number without (open bars) and with (filled bars) 1 nM VEGF. *P < 0.05 compared with 1 nM VEGF. C: means ± SE proliferation rate (measured as thymidine incorporation per cell) without (open bars) and with (closed bars) 1 nM VEGF. D: means ± SE cytotoxicity without (open bars) and with (closed bars) 1 nM VEGF. * P < 0.05 compared with or without VEGF.

View larger version (9K): [in this window] [in a new window]   Fig. 5.   Effect of VEGF on primary culture podocyte cytotoxicity. VEGF significantly reduced the cytotoxicity of primary cultured podocytes. A monoclonal antibody to VEGF increased cytotoxicity (VEGFMab) and blocked the VEGF-mediated decrease (VEGF+VEGFMab). The reduction in VEGF-mediated cytotoxicity was reversed by inhibition with the type III receptor tyrosine kinase PTK787 (PTK787+ VEGF), although PTK787 alone did not stimulate an increase in cytotoxicity. P < 0.001 ANOVA. * P < 0.05 compared with control, Student-Newman-Keuls post hoc test.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   VEGF-mediated reduction in cytotoxicity is PI3 kinase dependent. Pretreatment of conditionally immortalized podocytes (CIP; gray bars) and primary cultured podocytes (PCP; black bars) by the phosphatidylinositol 3-kinase inhibitor Wortmannin abolished the reduction in cytotoxicity induced by exposure to 1 nM VEGF (P < 0.001, ANOVA). Cytotoxicity was reduced by 74 ± 3.9% in CIP and 74 ± 2.7% in PCP cells treated with 1 nM VEGF. This was inhibited by 25 nM (PCP) and 100 nM (CIP) Wortmannin. *P < 0.05 compard with 100% Dunnet's post hoc test.

VEGF receptor expression. mRNA for VEGF-R1 (333 bp), R3 (381 bp), and Np-1 (504 bp) was detected in the conditionally immortalized human podocyte cell line, but VEGF-R2 (332 bp) was not (Fig. 7A). Brain, thyroid (not shown), and kidney cDNA (Fig. 7B) were used as a positive control for the primers and GAPDH (364 bp) for integrity of the cDNA. Each came up positive (results not shown). All negative controls (water and RNA without reverse transcription) were blank (results not shown). Furthermore, expression of VEGF-R1 but not VEGF-R2 protein was detected in Western blot analysis of protein extracted from conditionally immortalized podocytes (Fig. 7C).

View larger version (29K): [in this window] [in a new window]   Fig. 7.   A: expression of VEGR-R1, VEGF-R3, and NP-1 mRNA in human conditionally immortalized podocyte cell line. VEGF-R2 was not detected. B: VEGF R2 and GAPDH mRNA expression in human kidney tissue. C: Western blot for VEGF-R1 with and without treatment with VEGF.

Transmission electron microscopy. Immunogold transmission electron microscopy was carried out to detect the subcellular localization of VEGF in isolated human glomeruli derived from the normal pole of nephrectomy specimens. The protocol described to detect VEGF by colloidal gold resulted from a compromise between fixation, morphology, and antigen detection, optimized finally for antigen detection. A short fixation with 0.2% glutaraldehyde was the only fixation protocol to result in antigen detection.

View larger version (131K): [in this window] [in a new window]   Fig. 8.   Identification of VEGF expression by immunogold TEM in a normal human glomerulus. Gold particles can be clearly seen on the edge of the podocyte foot processes. BS, Bowman's space; GBM, glomerular basement membrane; PFP, podocyte foot process.

View larger version (11K): [in this window] [in a new window]   Fig. 9.   Distribution of VEGF expression by immunogold in a normal human glomerulus. A: means ± SE% of gold particle distribution within the 3 components of the glomerular filtration barrier. B: gold particle distribution within podocyte foot processes, either membrane associated or intracellular. C: means ± SE% of gold particle distribution at 25-nm intervals from the membrane, with results significanly different using ANOVA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Characteristics of primary cultured and conditionally immortalized podocytes. Despite the extensive use of primary cultured podocytes for renal research, two longstanding criticisms remain. First, there is the observation that podocytes alter their phenotype in culture, becoming dedifferentiated and proliferative. This contrasts with the growth-arrested, differentiated podocytes in vivo. We therefore studied both phenotypes. Second, there is the issue of purity. We did our utmost to ensure podocyte purity of primary cultures. There has been some debate over the origin of epithelial cells grown using the sieving method particularly with regard to the effective removal of parietal epithelial cells. These issues are fully covered by ourselves and co-workers elsewhere (17, 26). We showed that the vast majority of cells isolated using this technique have identical expression characteristics of visceral GECs but cannot exclude a minority of cells being parietal in origin. For both of these reasons, we also chose to study a conditionally immortalized podocyte cell line in addition to primary cultured podocytes. The conditionally immortalized cell line has a differentiated phenotype and is a pure population (32). Both primary cultured podocytes and conditionally immortalized, differentiated podocytes demonstrated a similar fall in [Ca²⁺]_i in response to VEGF₁₆₅ when intra- and extracellular calcium concentrations are similar. Furthermore, in the event of significant mesangial or glomerular endothelial cell contamination of primary cell cultures, an increase in [Ca²⁺]_i in response to VEGF₁₆₅ would be expected rather than a decrease. Both mesangial and glomerular endothelial cells have been shown to respond to a variety of agents with increased [Ca²⁺]_i (reviewed in Ref. 24). Moreover, it is well characterized that the VEGF-mediated increase in permeability of systemic capillaries in vivo is mediated via an increase in [Ca²⁺]_i (6, 28).

What are the physiological roles for VEGF in the glomerulus? The physiological role of podocyte-derived VEGF is still poorly understood. It has, however, been hypothesized that glomerular VEGF may have an important function in the maintenance of the glomerular endothelium (including maintaining fenestration) and/or selective permeability to macromolecules (7, 30). In fact, we previously showed in vivo that VEGF can effectively increase hydraulic conductivity (permeability to water) without reducing macromolecular selectivity in the mesenteric microcirculation, exactly that scenario present in the glomerular endothelial barrier (3). Continued controversy over the role of VEGF in normal glomerular physiology stems from a number of anomalies, however, not least of which is the apparent minimal disruption that results from the in vivo administration or inhibition of VEGF₁₆₅ in normal animals (25, 39). Although both of these reports only administered or inhibited VEGF₁₆₅ (one species among many potential isoforms), neither study demonstrated any abnormality save for VEGF-associated hypotension (39).

How does VEGF act on podocytes? [Ca²⁺]_i is an important second messenger in most cells and, certainly in endothelial cells, plays an important role in VEGF-mediated permeability, mitogenesis, and vasodilatation. The nature of the [Ca²⁺]_i response we demonstrated in podocytes, however, is atypical. This is the first evidence that VEGF can stimulate a reduction in [Ca²⁺]_i under any circumstances in any cells. The conditions under which reductions in [Ca²⁺]_i are seen in podocytes in response to VEGF are nonphysiological (i.e., minimal calcium). The fact that VEGF can stimulate a reduction in calcium under low external calcium conditions, however, suggests that VEGF is activating calcium extrusion or sequestering mechanisms. It is possible that VEGF stimulates sarcoendoplasmic reticulum calcium or plasmalemmal calcium ATPases (SERCA or PMCA). Activation of either of these pumps would reduce [Ca²⁺]_i, but this effect would normally be masked by normal calcium homeostasis. The functional significance of this VEGF-mediated reduction in [Ca²⁺]_i in podocytes is not clear, but there are a number of possibilities. VEGF is known to act as an autocrine survival factor in breast carcinoma cells that express the same VEGF receptor profile as do podocytes. Bachelder et al. (1) showed that VEGF inhibits the apoptosis of tyrosine-kinase VEGF receptor-negative, neuropilin-positive breast cancer cells via stimulation of PI3-kinase. In addition, other studies showed that PI3-kinase activity mediates the in vitro inhibition of cyclosporin A-induced podocyte apoptosis via Bcl-X (11). The evidence described above is consistent with a role of PI3-kinase in this VEGF-mediated reduction in cytotoxicity. It is well recognized that intracellular subcellular Ca²⁺ localization plays an important role in regulating apoptosis (21). Our findings, that VEGF acts as a survival factor for podocytes when dedifferentiated and proliferative, support this hypothesis. Further details of the signaling mechanisms that underlie this mechanism await further investigation.