Functional evidence that vascular endothelial growthfactor may act as an autocr - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:37 |只看该作者 |倒序浏览 |打印

作者：Rebecca R.Foster, RachelHole, KarenAnderson, Simon C.Satchell, Richard J.Coward, Peter W.Mathieson, David A.Gillatt, Moin A.Saleem, David O.Bates,  Steven J.Harper,作者单位：1 Microvascular Research Laboratories, Department ofPhysiology, University of Bristol, Preclinical Veterinary School,Bristol BS2 8EJ; and Department of Pathology, Academic and Children‘s Renal Unit, Universityof Bristol, and Bristol Urological Institute, SouthmeadHospital, Westbury on Trym, Br 4 t! w2 r0 P7 O

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      【摘要】. k$ S, K" v# }  f/ ?8 g
   Vascular endothelial growthfactor (VEGF) is expressed by renal glomerular epithelial cells(podocytes) and is thought to be protective against nephrotoxic agents.VEGF has been shown to be an autocrine survival factor inneuropilin-1-positive, VEGF receptor-negative breast carcinoma cells.Normal human podocytes are also known to express neuropilin-1, VEGF,and are VEGF-R2 negative. Here, we investigated whether a similarVEGF autocrine loop may exist in podocytes. Podocytecytosolic calcium concentration ([Ca 2  ] i ) was analyzed in primary culturedand conditionally immortalized podocytes using ratiometric fluorescencemeasurement. Cytotoxicity was determined by lactate dehydrogenaseassay, proliferation by [ 3 H]-thymidine incorporation, andcell counts by hemocytometric assay. VEGF decreased[Ca 2  ] i in primary podocytes (from 179 ± 36 to 121 ± 25 nM, P andconditionally immortalized podocytes (from 95 ± 10 to 66 ± 8 nM, P III receptor tyrosine-kinase inhibitorPTK787/ZK222584 abolished this reduction. VEGF increased podocyte[ 3 H]-thymidine incorporation (3,349 ± 283 cpm,control 2,364 ± 301 cpm, P 4 /ml, control 2.6 ± 0.5 × 10 4 /ml, P 12 ± 3%, P cytotoxicity. Electron microscopy of normal human glomerulidemonstrated that the glomerular VEGF is mostly podocyte cellmembrane associated. These results indicate that one of the functionsof VEGF secreted from podocytes may be to act as an autocrine factor oncalcium homeostasis and cell survival.
      【关键词】 intracellular calcium apoptosis glomerulus cell survival immunogold
   INTRODUCTION

THE GLOMERULUS IS aunique functional unit characterized by differential permeability, highto water and electrolytes and low to protein. The podocytes (visceralglomerular epithelial cells; GECs) are believed to play a crucial rolein the maintenance of this selective barrier. Podocyte dysfunction,either genetic ( 20 ) or acquired in glomerular disease( 13 ), results in loss of the macromolecular selectivity ofthe glomerular filtration barrier and proteinuria. How podocytes exerttheir influence is poorly understood, but the available data suggestthat podocytes contribute structurally by the provision of slitdiaphragm and glomerular basement membrane ( 20, 22 ) andfunctionally by production of molecules known to affect endothelialpermeability in other vascular beds. Examples of these moleculesinclude vascular endothelial growth factor (VEGF) ( 2 ),known to increase microvascular permeability ( 5, 6 ), andangiopoietin-1 ( 33 ), the only podocyte-secreted moleculethat has been shown to decrease macromolecular extravasation( 38 ).
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Despite the production of VEGF by podocytes at high levels,the detailed role of VEGF in normal glomerular physiology and itspotential contribution to glomerular macromolecular permeability remaincontroversial. Normal VEGF biology is complex. Differential exonsplicing of the VEGF gene results in a number of mRNA species, whichcode for a series of isoforms containing different numbers of aminoacids termed VEGF 189 and VEGF 165 (the mostwidespread isoform and also that found predominantly in the renalglomerulus) and VEGF 121 ( 10 ). VEGF isoformexpression in glomeruli is heterogeneous. Individual human glomeruliexpress one, two, or all three of these main isoforms at the mRNA level( 40 ). Minor VEGF mRNA splice variants(VEGF 206, VEGF 183, VEGF 148, andVEGF 145 ) have also been reported, but they are less wellcharacterized ( 18, 19, 29, 40 ). In addition, evidence fora new set of almost identical sister molecules ofinhibitory VEGF isoforms in the renal cortex has recently beendescribed by this laboratory ( 4 )., I% o) ?5 l7 e7 }6 \3 _, d% x

VEGF signals through two receptors. The primary targets of VEGF onvascular endothelial cells are the class III receptor tyrosine-kinases, VEGFR-1 (flt-1) and VEGFR-2 (KDR), both of which are expressed by theglomerular endothelium ( 9 ). The latter initiatesangiogenesis, cell migration, and permeability changes. VEGFR-1 alsoexists in a soluble form, sVEGFR-1 (sFlt), which is inhibitory whenbound 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), forexample, facilitates the binding of VEGF 165 to VEGFR-2( 12 ) enhancing VEGFR-2-mediated effects.

VEGF, one of the most potent mediators of angiogenesis and endothelialpermeability known, is produced at a high level by the podocytes200-300 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 againsta significant filtration of fluid across the glomerular barrier. It hastherefore been suggested that VEGF might act on cells other than theglomerular endothelium. This led us previously to investigate VEGF-Rexpression by human podocytes themselves. Although we were unable todetect tyrosine-kinase VEGFR-2 expression, we demonstrated theexpression of Np-1 by normal human podocytes in vitro and in vivo( 17 ). These results suggest that podocytes may have thepotential to bind the VEGF they secrete. We therefore hypothesized thatthe potential VEGF-Np-1 interaction may be important in terms of anautocrine loop or in VEGF sequestration in podocytes ( 17 ).
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Although Np-1 has been considered as a nonsignaling VEGF coreceptor,VEGF has more recently been identified as an autocrine survivalfactor 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[Ca 2  ] i in endothelial cells, we wereprompted to investigate intracellular cytosolic calcium responses ofcultured human podocytes to exogenous VEGF to address the hypothesisthat VEGF may play a role as a podocyte autocrine factor. We studiedthese potential functional responses in proliferating dedifferentiatedprimary culture podocytes and nonproliferating differentiated podocytesin vitro. In addition, we investigated the potential of exogenous VEGFto act as a survival factor for dedifferentiated proliferatingpodocytes. Finally, we determined the distribution of VEGF within theregion of the glomerular filtration barrier using transmission electronmicroscopic (TEM) analysis of colloidal gold immunohistochemistry onnormal human glomeruli.  Z+ V) C- K- e& e7 a/ g
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MATERIALS AND METHODS3 J) ]) c8 [: u* }$ v

VEGF used in these experiments was recombinantVEGF 165, a kind gift of N. Ferrara (Genentech). Allchemicals/solutions were from Sigma unless otherwise stated.1 S* T6 a. h/ M4 }% v5 p$ z9 _

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

Podocytes were isolated from the nonmalignant histologically normalpole of renal cell carcinoma nephrectomy specimens by sieving andcultured under standard conditions as previously described ( 26 ). Cells grown by this method demonstrate a typicalpolyhedral shape with a cobblestone appearance on confluence and havebeen characterized as positive for cytokeratin and Wilms tumorprotein-1 (WT-1) by immunofluorescence; positive for VEGF, WT-1, andsynaptopodin by RT-PCR; and negative by RT-PCR for von Willebrandfactor, CD45, and smooth muscle myosin, excluding contamination byendothelial cells, leukocytes, or mesangial cells, respectively, aspreviously described by ourselves and co-workers ( 17, 26 ).This phenotype was confirmed by regular sampling of cells studied.7 I$ E1 v* R& N" a: s' p
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Conditionally immortalized podocytes. This cell line has been conditionally immortalized from normal humanpodocytes with a temperature-sensitive mutant of immortalized SV40 Tantigen. These cells have been previously characterized in detailelsewhere ( 32 ). At the "permissive" temperature of 33°C, the SV40 T antigen is active and allows the cells toproliferate rapidly. Thermoswitching the cells to the"nonpermissive" temperature of 37°C silences the transgene andthe cells become growth arrested and differentiated. Under theseconditions, they express antigens appropriate to in vivo arborizedpodocytes. Cells were grown on coverslips for a period of 14 days toensure growth arrest and differentiation.) W% x- K* l7 n# }2 A6 l- P+ P9 E
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Intracellular calcium studies. Podocytes were grown on coverslips to confluence. Cells were incubatedwith fura 2-AM (10 µM) for 90 min in DMEM at room temperature, andthe coverslip was then placed in a holder. The holder was then mountedon a rig consisting of an inverted fluorescence microscope (DM IRB,Leica) equipped with a UV source (Cairn Instruments, World PrecisionInstruments) with filters for excitation at 340 and 380 nm. Fastswitching was achieved using a rotary filter wheel at 50 Hz and aspectrophotometer for photometric measurement (Cairn Instruments). Thespectrophotometer received emitted light via a 400-nm dichroic filterand a 510- to 530-nm barrier filter in front of the photometer.Powerlab software was used for analysis and graphic display.$ }9 }: }" u4 y. k* k

Experiments were conducted in HBSS media containing 1.3 mM calcium(i.e., normal extracellular calcium concentration,[Ca 2  ] o ) and in nominally calcium-free HBSS(Gibco BRL). Test samples of 1 nM VEGF, 30 µM ATP, used as a positivecontrol, and HBSS, used as a negative control, were left to wash andrecord for 5 min. To ensure that changes in[Ca 2  ] i were effectively detected, 5 µMionomycin were added to stimulate Ca 2  entry into thecells. One millimolar manganese chloride (MnCl 2 ) in thecontinued presence of 5 µM ionomycin was then used to quench thecalcium-sensitive fura to determine the background (Ca 2  independent) fluorescence signal. Three washes with appropriate HBSSwere used between stimuli, and cells were allowed to rest for 20 min.VEGF was used at 1 nM, because this concentration has been shown toproduce physiological responses in our previous in vivo experiments( 3, 6, 27 ).

Emission fluorescent measurements (I f ) were taken 50 timesa second. The ratio of the I f measured during 340-nmexcitation to that during 380-nm excitation (R), proportional to thecalcium concentration, was calculated from = R exp /R min
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where R exp = (I f340 B 340 )/(I f380 B 380 ).I f340 is the I f measured during excitation at340 nm, I f380 is the I f measured duringexcitation at 380 nm, and B 340 and B 380 are thebackground I f values measured during excitations at 340 and380 nm, respectively (measured as the I f afterMn 2  quenching). R min is the in vitro ratio forzero [Ca 2  ][Ca 2  ] i wascalculated from the following formula 2 + ] i =K d&bgr; × [(R − 0.85) / (0.85 · R max −( i# Y! `* g3 [3 f

Where K d (the product of the fura dissociationconstant from bound-to-free calcium and the ratio of maximal-to-minimalI f380 ) was calculated from an in vitro calibration curve.

The order in which the test samples were added was varied betweenexperiments. Inhibition studies were conducted in which conditionallyimmortalized cells were challenged with 1 nM VEGF after preincubationfor 10 min with the class III tyrosine-kinase receptor inhibitorPTK787/ZK222584 (100 nM) (a kind gift from J. Wood, Novartis, Basle,Switzerland), a response recorded, the cells washed three times withHBSS (minimal calcium) and allowed to rest for 20 min.3 d5 O/ Y0 A- c6 A/ F& L% v
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[ 3 H]-thymidine assays and cell count. A 24-well plate was seeded with primary cultured podocytes, which wereincubated in RPMI media containing 1% penicillin/streptomycin, 1% L -glutamine, 1% insulin transferrin selenite (all LifeTechnologies), and 20% FBS. One well was set aside for 1 ml serum-freemedia and one for FBS-free media plus VEGF. These were used as negative controls. Podocytes were left for 48 h until established, and thenmedia were removed and replaced with FBS-free media. One nanomolarVEGF 165 was added to half the wells and one-half were leftuntreated. Twenty-four hours later, 37 kBq methyl-[ 3 H]thymidine (Amersham Pharmacia) were added to each well. Four hourslater, media were removed and 0.2 ml trypsin was added to each well andleft for 2 min. Two hundred microliters of RPMI were added and ahemocytometer was used to determine cell number in each well (Weber).Remaining cells were pipetted into 1.5-ml tubes (Eppendorf) and spun at300 g for 10 min (Biofuge, Heraeus). The supernatant wasremoved, and 0.2 ml NaOH was added and left at room temperature for 30 min. Cells were then pipetted into scintillation vials (Fisher), 5 mlof 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 withprimary cultured podocytes and 100 µl 20% FBS-RPMI. Podocytes wereleft 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 BichrometricMultiscan plate reader (Labsystems). These samples were used forbackground LDH measurement (T x min). The media of half ofthe wells were replaced with 100 µl FBS-free media, the other halfwith FBS-free media containing 1 nM VEGF 165. Twenty-four hours later, 100 µl media were again removed from each well and cytotoxicity was assayed and quantified. These samples were used todetermine the cytotoxicity (T x exp). Finally, 100 µl of2% Triton X-100/1 × PBS (final concentration 1%) were added toeach 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 x max). Percent cytotoxicity (T x ) wascalculated for each well as x exp − T x min) / (T x max − T x min)] ×
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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) ].
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RT-PCR. Total RNA was extracted as previously described ( 8 ) usingthe TRIzol method from the conditionally immortalized human podocyte cell line, human thyroid, brain, and kidney. Reverse transcription wascarried out using 1 µg RNA and 5 µM oligo dT (Promega) in 10 µlRNAse-free water (Sigma). This mixture was incubated at 65°C for 5 min and immediately placed on ice. The reaction mixture was thenaltered to 1× first-strand synthesis buffer (Roche), 10 mM DTT(Roche), 2.5 mM dNTPs (Promega), 1 U RNA guard (Amersham), and 2.5 Uexpand RT (Roche) in a total of 20 µl RNAse-free water (Sigma). Thiswas incubated at 42°C for 2 h. PCR was performed using theprimers as detailed in Table 1. The PCRmixture consisted of 1× PCR buffer (Abgene), 1.25 mM MgCl 2 (Abgene), 375 µM dNTPs, 10 µM forward primer, 10 µM reverseprimer (except for GAPDH where 5 µM of each primer were used), 1 µlcDNA, and 1 U Taq (Abgene) in 20 µl RNAse-free water. Astandard PCR cycle was used, i.e., 55°C, 35 cycles (Hybaid). RT-PCRproducts were run on 2% agarose (Roche) gels in the presence of 0.5 µg/ml ethidium bromide (Invitrogen). Gels were photographed under UVtransillumination (Gibco). A 100-bp ladder (Sigma) was used tovisualize bands from 100 to 1,000 bp.
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Table 1. Primers and primer sequences& o  _( Z# @6 H/ {; m+ y  @

Western blot analysis. Confluent primary cultured podocytes from a T75 flask were leftuntreated or treated with VEGF (1 nM) for 30 min. The cells were thentrypsinized, rinsed in PBS, pelleted, and the protein was extracted in0.2% (vol/vol) SDS, 300 mM NaCl, 20 mM Tris, 10 mM ethyldiaminetetraacetic acid, 2 mM Na 3 VO 4, 1 mMphenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µM E64, 2 µg/ml aprotinin, 62.5 mM -glycerophosphate, and 1 µM pepstatinA. Protein quantification was performed spectrophotometrically usingBio-Rad dye. Equal amounts of protein were electrophoresed against aprestained protein size marker using 10% SDS-polyacrylamide gelelectrophoresis. Proteins were electroblotted to polyvinylidenedifluoride membrane. Membranes were blocked in 10% (wt/vol) nonfat drymilk (Marvel) in 1× PBS-Tween 20 0.1% (vol/vol) (PBST) for 1 hand 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. Unboundprimary 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 byenhanced chemiluminescence.

TEM. One-millimeter cubed pieces of renal cortex were taken from the normalpole of nephrectomy samples taken for unipolar cancer and fixed in0.2% glutaraldehyde (Agar Scientific) and 0.2 M phosphate buffer at pH7.4 at room temperature for 30 min. The tissue was then stored in 0.2 Mphosphate buffer until processed. Specimens were partially dehydratedusing a 10-min wash in 50% IMS followed by three 10-min washes in 70%methylated spirits (IMS). Specimens were infiltrated with LR white hardgrade 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 leftto 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. Thecapsules were then exposed to the air and left to set. Sections werecut at 0.5-0.9 µm on a Leica Reichert Ultracut S ultramicrotomeand placed on a glass slide and stained with 1% toluidine blue in 1%borax to determine whether the tissue was suitable for furtherinvestigation. Appropriate tissue was cut into 90-nm sections andmounted onto 300 mesh hexagonal nickel grids (Agar Scientific) and leftto 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) inPBS (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 inPBS (pH 7.4) and PBS (pH 8.2). The secondary antibody was 15-nmgold-conjugated goat anti-rabbit IgG in Tris (pH 8.2), sodium azide,and 0.6% BSA (BioCell at Agar Scientific) 1:10 dilution, applied for60 min. Grids were washed in PBS (pH 8.2) and deionized H 2 Ofor 1 min and then stained with a saturated solution of uranyl acetatefor 20 min; sections were then washed in deionized water and stainedwith lead citrate for 1 min. The grids were then rinsed with deionizedwater and left to air dry. Grids were viewed under an electronmicroscope (Philips CM10). Podocyte (intracellular or membraneassociated), glomerular basement membrane, and glomerular endothelialcell-associated gold particles were enumerated in 16 random fields fromfour different kidneys. Colloidal gold particles were consideredmembrane associated if they were within two particle widths (i.e., 30 nm) of the membrane on either side.
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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 cellpopulations treated differently. ANOVA was used to compare distributionof gold particles on podocyte foot processes.. v9 U- b* U5 E4 C. X- H5 p/ X
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RESULTS
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Primary cultured podocytes. Figure 1 shows the effect of HBSS on[Ca 2  ] i in primary cultured podocytes (Fig. 1 A ) and human tubular epithelial cells (HK2). HBSS did notchange calcium in either cell line or in human tubular epithelial cells(Fig. 1 B ) in either the presence or absence (not shown) ofextracellular Ca 2 . ATP, on the other hand, caused atransient rapid increase in [Ca 2  ] i on alloccasions (Fig. 1 C ). [Ca 2  ] i increased from 113.2 ± 22.1 to 209.5 ± 41.0 nM( P 1 D ) peaking at 30 ± 10 s and returning to baseline after 3.2 ± 0.5 min.Surprisingly, although VEGF did not alter[Ca 2  ] i in the presence of extracellularcalcium (Fig. 2 A ), VEGFproduced a slow and sustained reduction in[Ca 2  ] i, which was significantlydifferent from baseline in minimal extracellular calcium (Fig. 2 B in primary cultured podocytes). There was a significantreduction in the ratio (R) in minimal, but not normal extracellularcalcium (Fig. 2 C ), which corresponds to a change in[Ca 2  ] i from 178.9 ± 35.6 to 121.1 ± 25.4 nM with VEGF ( P A minimum was reachedafter 5 ± 1 min.' [! y* X4 b9 P/ Q& A
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Fig. 1. Effect of HBSS and ATP on podocyte and HK2 cell calcium. A : example of no significant change of cytosolicCa 2  concentration ([Ca 2  ] i ) inprimary culture human podocytes in response to 1 µl HBSS in thepresence of 1.3 mM extracellular Ca 2  concentration([Ca 2  ] o ). B : means ± SE of[Ca 2  ] i change in podocytes and human tubularepithelial cells in response to 1 µl HBSS in the presence of 1.3 mM[Ca 2  ] o. C : transient increase in[Ca 2  ] i in primary culture human podocytes inresponse to 30 µM ATP. D : means ± SE ofATP-stimulated response compared with baseline values( n = 6, ** P t -test).6 P1 `/ _4 u8 I
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Fig. 2. Effect of vascular endothelial growth factor (VEGF) on[Ca 2  ] i of primary cultured and conditionallyimmortalized podocytes. A : example of[Ca 2  ] i change in primary culture humanpodocytes in response to 1 nM VEGF in the presence of 1.3 mM[Ca 2  ] o. B : example of[Ca 2  ] i change in primary culture podocytesin response to 1 nM VEGF in the absence of[Ca 2  ] o. C : means ± SE ratiobefore (open bars) and after (filled bars) VEGF treatment. D : example of [Ca 2  ] i change inresponse to 1 nM VEGF in the presence of 1.3 mM[Ca 2  ] o. E : example of effect ofVEGF on [Ca 2  ] i change in the presence ofminimal [Ca 2  ] o. F : means ± SE ratio before (open bars) and after (filled bars) VEGF treatment.* P P# x8 c) K  K. J: @! e
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Conditionally immortalized podocytes. ATP administration stimulated a transient increase in[Ca 2  ] i in conditionally immortalized cellsfrom 102.2 ± 34.5 to 142.1 ± 35.8 nM postexposure, P seen in primaryculture podocytes, VEGF did not alter [Ca 2  ] i in differentiated podocytes in the presence of[Ca 2  ] o (Fig. 2 D ) but againproduced a slow but sustained significant reduction in[Ca 2  ] i when these differentiated podocyteswere incubated in minimal extracellular calcium (Fig. 2 E ).There was a significant reduction in the ratio (Fig. 2 F ),which corresponds to a reduction in [Ca 2  ] i from 94.7 ± 9.8 to 66.1 ± 8.4 nM ( P after preincubatingthe cells for 10 min with the type III tyrosine-kinase receptorinhibitor PTK787/ZK222584. To our surprise, the addition of VEGF tocells preincubated in PTK787/ZK222584 resulted in a small butsignificant increase in [Ca 2  ] i (Fig. 3 A ), whereas addition ofPTK787/ZK222584 to cells did not result in any change in intracellularcalcium by itself (Fig. 3 B ). This VEGF-induced increase inthe presence of PTK787/ZK222584 was consistent in all six sets ofexperiments, with a mean ± SE increase from 78.8 ± 35 to115.2 ± 50.6 nM ( P t -test; Fig. 3 C ). Therefore, the reduction inCa 2  stimulated by VEGF (0.69 ± 0.06-fold) wasreversed by this inhibitor (1.47 ± 0.15-fold, P 3 D ), suggesting that the VEGF-dependent reduction in [Ca 2  ] i may be a constitutiveevent in podocytes, mediated by one or more type III receptor tyrosinekinases.

Fig. 3. Effect of class III receptor tyrosine-kinase inhibitorPTK787/ZK222584 on VEGF-mediated [Ca 2  ] i changes in transformed human podocytes. A : example of[Ca 2  ] i change in response to 1 nM VEGF aftertreatment with PTK787/ZK222584 incubated in minimal calcium. B : means ± SE ratio before (open bars) and after (graybars) treatment with PTK787/ZK222584 and 1 nM VEGF. C : example of [Ca 2  ] i measurement in response to treatment with PTK787/ZK222584 alone. D : comparison of the response of vGEC[Ca 2  ] i to 1 nM VEGF in the absence andpresence of PTK787/ZK222584. Values are the relative change in R frombaseline (1 = no change). * P P/ Z8 a- I0 ^% q9 O) J

Proliferation and cytotoxicity. In primary cultured podocytes, addition of 1 nM VEGF to culturemedium resulted in a significant increase in[ 3 H]thymidine incorporation (from 2,364 ± 301 to3,349 ± 283 cpm, P 4 A ). Assuming that the[ 3 H]thymidine incorporation is the same for each cell foreach division, then [ 3 H]thymidine incorporation gives thenumber of cells dividing within a defined time. If more cells aresurviving, then there will be more cells present to undergo the normalrate of division. Therefore, to determine whether this increase in[ 3 H]thymidine incorporation was due to increasedproliferation rate or due to an increase in the survival ofVEGF-treated cells (and hence increased cell number), we measured thenumber of cells in each well. The cell number also increased from2.6 ± 0.5 to 4.5 ± 0.7 × 10 4 /ml( P 4 B ) with VEGF treatment.[ 3 H]thymidine incorporation calculated per cell wastherefore not affected by VEGF (untreated 0.1 ± 0.015 cpm/cell, treated 0.125 ± 0.029 cpm/cell, not significant; Fig. 4 C ), suggesting that VEGF was acting not by increasingproliferation rate but by reducing cell death. To assess independentlywhether VEGF could reduce cytotoxicity, the effect of VEGF on LDHrelease into the media (which occurs when cells lyse) was carried out.VEGF stimulated a reduction in cytotoxicity from 12.5 ± 3.0 to5.9 ± 0.67% ( P 4 D ).

Fig. 4. Effect of VEGF on primary cultured podocytes proliferation. A : means ± SE 3 H-thymidine incorporationwithout (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 C : means ± SE proliferation rate (measured as thymidineincorporation per cell) without (open bars) and with (closed bars) 1 nMVEGF. D : means ± SE cytotoxicity without (open bars) andwith (closed bars) 1 nM VEGF. * P

To determine whether this decrease in cytotoxicity was also broughtabout by endogenous VEGF, proliferating primary cultured podocytes wereincubated with a neutralizing antibody to VEGF. This resulted in asignificant increase in cell death, which was abolished by addition ofVEGF. The effect of exogenous VEGF, furthermore, was abolished by theaddition of PTK787, although this concentration of PTK787 alone did notsignificantly increase endogenous cytotoxicity (Fig. 5 ). The reduction in cytotoxicityappeared to occur through phosphatidylinositol (PI3)-kinase activation,because the reduction in cytotoxicity was blocked by treatment with thePI3 kinase inhibitor Wortmannin (Fig. 6 ).

Fig. 5. Effect of VEGF on primary culture podocyte cytotoxicity. VEGFsignificantly reduced the cytotoxicity of primary culturedpodocytes. A monoclonal antibody to VEGF increased cytotoxicity(VEGFMab) and blocked the VEGF-mediated decrease (VEGF VEGFMab). Thereduction in VEGF-mediated cytotoxicity was reversed byinhibition with the type III receptor tyrosine kinase PTK787(PTK787  VEGF), although PTK787 alone did not stimulate an increasein cytotoxicity. P P: G5 s# m* |7 z: f

Fig. 6. VEGF-mediated reduction in cytotoxicity is PI3 kinasedependent. Pretreatment of conditionally immortalized podocytes (CIP;gray bars) and primary cultured podocytes (PCP; black bars) by thephosphatidylinositol 3-kinase inhibitor Wortmannin abolished thereduction in cytotoxicity induced by exposure to 1 nM VEGF( P P
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VEGF receptor expression. mRNA for VEGF-R1 (333 bp), R3 (381 bp), and Np-1 (504 bp) was detectedin the conditionally immortalized human podocyte cell line, but VEGF-R2(332 bp) was not (Fig. 7 A ).Brain, thyroid (not shown), and kidney cDNA (Fig. 7 B ) wereused as a positive control for the primers and GAPDH (364 bp) forintegrity of the cDNA. Each came up positive (results not shown). Allnegative controls (water and RNA without reverse transcription) wereblank (results not shown). Furthermore, expression of VEGF-R1 but notVEGF-R2 protein was detected in Western blot analysis of proteinextracted from conditionally immortalized podocytes (Fig. 7 C ).
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Fig. 7. A : expression of VEGR-R1, VEGF-R3, and NP-1 mRNA inhuman conditionally immortalized podocyte cell line. VEGF-R2 was notdetected. B : VEGF R2 and GAPDH mRNA expression in humankidney tissue. C : Western blot for VEGF-R1 with and withouttreatment with VEGF.

Transmission electron microscopy. Immunogold transmission electron microscopy was carried out to detectthe subcellular localization of VEGF in isolated human glomeruliderived from the normal pole of nephrectomy specimens. The protocoldescribed to detect VEGF by colloidal gold resulted from a compromisebetween fixation, morphology, and antigen detection, optimized finallyfor antigen detection. A short fixation with 0.2% glutaraldehyde wasthe only fixation protocol to result in antigen detection.
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Colloidal gold particles were seen throughout the glomerularfiltration barrier, within the podocyte foot processes (77.9 ± 1.81%), glomerular basement membrane (11.9 ± 1.2%), and what were taken to be glomerular endothelial cells (10.2 ± 1.6%)(Figs. 8 and 9 A ). Unfortunately, theendothelial morphology was poor with this technique, despite goodmorphological preservation of podocytes and basement membrane, so wewere unable to determine whether the staining was predominantly luminalor abluminal. Of the podocyte foot process-bound VEGF, 63.15 ± 3.29% was membrane associated in contrast to 36.85 ± 3.29%( P intracellular (Fig. 9 B ). Particles were seen throughout the glomerular basementmembrane and on both luminal and abluminal surfaces of the glomerularendothelial cells. The differential expression of VEGF at progressivelyfurther distances from the membrane was highly significant( P 9 C ). Colloidalgold particles were only identified within Bowman's space whenassociated with podocyte cell debris. Negative controls (no primaryantibody included) revealed no gold particles at all within theglomeruli, although occasional scattered particles were seen in tubularcells (not shown)./ m  x2 r( \8 E+ |$ g- Y" B
3 C' H+ R+ x6 O( t( V" s* q) C  R) D
Fig. 8. Identification of VEGF expression by immunogold TEM in anormal human glomerulus. Gold particles can be clearly seen on the edgeof the podocyte foot processes. BS, Bowman's space; GBM, glomerularbasement membrane; PFP, podocyte foot process.
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Fig. 9. Distribution of VEGF expression by immunogold in a normalhuman glomerulus. A : means ± SE% of gold particledistribution within the 3 components of the glomerular filtrationbarrier. B : gold particle distribution within podocyte footprocesses, either membrane associated or intracellular. C :means ± SE% of gold particle distribution at 25-nm intervalsfrom the membrane, with results significanly different using ANOVA.
9 ~2 c( u, q5 h9 b( H' B
DISCUSSION, q% y# |4 p4 s/ q( o5 o

Characteristics of primary cultured and conditionally immortalizedpodocytes. Despite the extensive use of primary cultured podocytes for renalresearch, two longstanding criticisms remain. First, there is theobservation that podocytes alter their phenotype in culture, becomingdedifferentiated and proliferative. This contrasts with thegrowth-arrested, differentiated podocytes in vivo. We therefore studiedboth phenotypes. Second, there is the issue of purity. We did ourutmost to ensure podocyte purity of primary cultures. There has beensome debate over the origin of epithelial cells grown using the sievingmethod particularly with regard to the effective removal of parietalepithelial cells. These issues are fully covered by ourselves andco-workers elsewhere ( 17, 26 ). We showed that the vastmajority of cells isolated using this technique have identicalexpression characteristics of visceral GECs but cannot exclude aminority of cells being parietal in origin. For both of thesereasons, we also chose to study a conditionally immortalized podocytecell line in addition to primary cultured podocytes. The conditionallyimmortalized cell line has a differentiated phenotype and is a purepopulation ( 32 ). Both primary cultured podocytes andconditionally immortalized, differentiated podocytes demonstrated asimilar fall in [Ca 2  ] i in response toVEGF 165 when intra- and extracellular calcium concentrations are similar. Furthermore, in the event of significant mesangial or glomerular endothelial cell contamination of primary cellcultures, an increase in [Ca 2  ] i in responseto VEGF 165 would be expected rather than a decrease. Bothmesangial and glomerular endothelial cells have been shown to respondto a variety of agents with increased [Ca 2  ] 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[Ca 2  ] i ( 6, 28 ).1 `; Z: T8 f! J8 U* |* A3 `" o

What are the physiological roles for VEGF in the glomerulus? The physiological role of podocyte-derived VEGF is still poorlyunderstood. It has, however, been hypothesized that glomerular VEGF mayhave an important function in the maintenance of the glomerularendothelium (including maintaining fenestration) and/or selectivepermeability to macromolecules ( 7, 30 ). In fact, wepreviously showed in vivo that VEGF can effectively increase hydraulicconductivity (permeability to water) without reducing macromolecularselectivity in the mesenteric microcirculation, exactly that scenariopresent in the glomerular endothelial barrier ( 3 ).Continued controversy over the role of VEGF in normal glomerularphysiology stems from a number of anomalies, however, not least ofwhich is the apparent minimal disruption that results from the in vivoadministration or inhibition of VEGF 165 in normal animals( 25, 39 ). Although both of these reports only administered or inhibited VEGF 165 (one species among many potentialisoforms), neither study demonstrated any abnormality save forVEGF-associated hypotension ( 39 ).

In addition, despite a high level of podocyte VEGF production andassociated high permeability of the glomerular filtration barrier towater, the glomerulus is not a site of new vessel formation in healthysubjects. It is clear then that under normal circumstances, theproangiogenic properties of VEGF must be modified by other factors.Angiopoietin-1, VEGF 165 b, and other members of theinhibitory family of isoforms are good candidate molecules ( 5, 33 ). It has therefore been suggested that VEGF may have nophysiological role in health but, in contrast, may only be important inglomerular disease (stimulating endothelial cell proliferation).Evidence for this, however, is only apparent from animal models rather than in human pathology ( 37 ), and it does not explain whyVEGF is so strongly expressed in normal human glomeruli. Although VEGF decreases cytotoxicity of podocytes grown in culture, the relevance ofthis finding to healthy kidneys in vivo is still unclear. Future studies addressing the amounts of specific isoforms of VEGF including the inhibitory isoforms of VEGF (VEGF 165 b) produced bypodocytes in vivo will help clarify this issue.1 i& f( {6 a2 ^
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Furthermore, the microanatomic positioning of glomerular VEGFproduction and receptor expression suggests that VEGF has to diffuseagainst a significant filtration gradient to bind to its targetmolecules [receptor binding studies would suggest there are no VEGFRsin the distal nephron ( 34 )]. Therefore, the synthesis ofsome isoforms of VEGF, for example VEGF 121, which haslittle or no heparin-binding properties (and therefore no ability to sequestrate into the glomerular basement membrane), would appear redundant since the glomerular filtration would tend to wash such molecules into Bowman's space. Our colloidal gold TEM finding ofsignificant localization to endothelial cells would suggest that atleast some VEGF isoforms are able to travel against the gradient ofglomerular filtration down a concentration gradient.; [" y5 @. G; E, {; a/ P0 z9 w

The above paradox, in conjunction with the identification of Np-1podocyte expression, led us to study potential VEGF-podocyte autocrineresponses. In this report, we provide the first functional data tosupport the notion that podocyte-derived VEGF may have autocrinepotential in addition to its other putative roles. Not only have weshown that exogenous VEGF acts directly on cultured human podocytes,but inhibition of endogenous VEGF, by a neutralizing monoclonalantibody, increases cytotoxicity of podocytes, an effect that isovercome by exogenous VEGF. Interestingly, however, this response isnot mimicked by VEGF receptor inhibitors, suggesting that theendogenous effect may circumvent receptor inhibition [possibly byactivation of an internal autocrine loop ( 15 )]. Furthermore, we provide the first TEM studies of VEGF expression inhuman renal glomerulus. These indicate that most VEGF within theglomerular filtration barrier is podocyte cell membrane associated. This phenomenon could be explained either by an accumulation of VEGFprotein before secretion or by the sequestration of VEGF onto thepodocyte cell surface, via chemical or receptor binding. Therefore, wehave evidence that supports the hypothesis that one of the roles ofVEGF in the glomerulus is to act as an autocrine survival factor for podocytes.3 P( ^4 y# j# L+ q" V& e
6 ?- U% `; e; H  u
How does VEGF act on podocytes? [Ca 2  ] i is an important second messenger inmost cells and, certainly in endothelial cells, plays an important rolein VEGF-mediated permeability, mitogenesis, and vasodilatation. Thenature of the [Ca 2  ] i response wedemonstrated in podocytes, however, is atypical. This is the firstevidence that VEGF can stimulate a reduction in[Ca 2  ] i under any circumstances in any cells.The conditions under which reductions in[Ca 2  ] i are seen in podocytes in response toVEGF are nonphysiological (i.e., minimal calcium). The fact that VEGFcan stimulate a reduction in calcium under low external calciumconditions, however, suggests that VEGF is activating calcium extrusionor sequestering mechanisms. It is possible that VEGF stimulatessarcoendoplasmic reticulum calcium or plasmalemmal calcium ATPases(SERCA or PMCA). Activation of either of these pumps would reduce[Ca 2  ] i, but this effect would normally bemasked by normal calcium homeostasis. The functional significance ofthis VEGF-mediated reduction in [Ca 2  ] i inpodocytes is not clear, but there are a number of possibilities. VEGFis known to act as an autocrine survival factor in breast carcinomacells that express the same VEGF receptor profile as do podocytes.Bachelder et al. ( 1 ) showed that VEGF inhibits theapoptosis 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 thein vitro inhibition of cyclosporin A-induced podocyte apoptosisvia Bcl-X ( 11 ). The evidence described above is consistentwith a role of PI3-kinase in this VEGF-mediated reduction incytotoxicity. It is well recognized that intracellular subcellularCa 2  localization plays an important role in regulatingapoptosis ( 21 ). Our findings, that VEGF acts as asurvival factor for podocytes when dedifferentiated and proliferative,support this hypothesis. Further details of the signaling mechanismsthat underlie this mechanism await further investigation.

Alternatively, the effect of VEGF on the calcium-handling properties ofpodocytes may be to modify the response of podocytes to other agents.Although many molecules (including bradykinin, thrombin, arginine,vasopressin, and serotonin) have been shown to have no effect onpodocytes [Ca 2  ] i ( 24 ), otherstudies have highlighted a number of agents that result in adose-dependent increase in podocyte [Ca 2  ] i.These include polycations (in primary culture and conditionally modified mouse podocytes) ( 31 ) and angiotensin( 24 ). This latter effect is thought to be mediated viaangiotensin receptors because angiotensin receptors signal byincreasing [Ca 2  ] i and the response isinhibited by the ANG II type 1 receptor blocker losartan. Continuedinjury of podocytes participates in the progression of chronic renallesions. It is generally accepted that ANG II accelerates this process,because inhibition of the renin-angiotensin system produces benefits inrenal survival in both animal models and humans. If the VEGF-inducedreduction in [Ca 2  ] i is functionallyimportant in vivo, then it may explain the mechanism by which VEGF actsas a cytoprotective agent in such lesions by counteracting the ANGII-driven increases in podocyte [Ca 2  ] i.% `" |2 q$ O0 i0 V6 E# B0 g. u
# s+ O3 C* ~% g! n
Our results prompt questions concerning which receptor(s) orintracellular pathway(s) mediate the response we identified. Wedemonstrated that human podocytes in vitro (primary culture) and invivo express Np-1. Np-1 has no signaling domain, however, nor does ithave a commercial inhibitor. Podocytes are not believed to expresstyrosine-kinase VEGF receptors (VEGF-R1 or R2) but we demonstrated thatalthough VEGF-R2 could not be identified, VEGF-R1 and VEGF-R3 mRNA andVEGF-R1 protein are in fact expressed in the conditionally immortalizedhuman podocyte cell line. We therefore addressed the possibility thatVEGF may act through a type III tyrosine-kinase receptor (for example,VEGF-R1). The podocyte response to exogenous VEGF was inhibited byPTK787/ZK222584. In fact, the addition of this inhibitor produced asignificant increase in podocyte [Ca 2  ] i.Because PTK787/ZK222584 is a class III receptor tyrosine-kinase inhibitor that has been shown to inhibit all such tyrosine kinases inthe submicromolar range, including PDGF-R, c-kit, VEGF-R2( 41 ), we cannot use our data to show that VEGF acts onpodocytes via VEGF-1 with or without neuropilin [the literaturesuggests that NP-1 acts as a coreceptor for VEGF-R2 but not VEGF-R1( 23 )]. The likelihood is, however, that in podocytes VEGFeither acts on VEGF-R1 and/or VEGF-R3 or via another unidentifiedpodocyte expressed class III tyrosine-kinase receptor. These datasuggest that these receptors may play a role in normal podocytefunction, and this possibility requires further investigation.
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In conclusion, we showed that VEGF can act as an autocrine factor forpodocytes, acting via an alteration in calcium handling of the cellsand reducing cell death. This appears to be true for proliferatingprimary cultured cells and growth-arrested, differentiated cells. Thedetails of the receptor and intracellular regulatory pathways involvedin this phenomenon and any potential effect on cell function orsurvival in vivo remain to be determined. In vivo studies includingconditional knockouts of specific VEGF receptors expressed on thepodocytes will need to be done to clarify further the role of VEGF inan adult glomerulus.0 X) y; B# k7 d8 p) N) C- s4 k" Q
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ACKNOWLEDGEMENTS

This work was supported by the Southmead Hospital ResearchFoundation Grant RF157 and Wellcome Trust Grant 58083. S. J. Harper is supported by The Wellcome Trust (057936/Z/99), D. O. Bates by the British Heart Foundation (BB2000003), and S. C. Satchell by a South West NHS R and D grant.. s, ?7 a  y" ]' }4 T
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