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nephritis and mesangial proliferative glomerulonephritis. [2]. However, the mechanisms controlling cell death that occurs during mesangial cell proliferation have ...

Clinical Science (2001) 101, 11–19 (Printed in Great Britain)

Bimodal effects of platelet-derived growth factor on rat mesangial cell proliferation and death, and the role of lysophosphatidic acid in cell survival Chiyoko N. INOUE*, Isao NAGANO†, Ryo ICHINOHASAMA‡, Natsumi ASATO§, Yoshiaki KONDO* and Kazuie IINUMA* *Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan, †Department of Neurology, Tohoku University School of Medicine, Sendai, Japan, ‡Department of Oral Pathology, Tohoku University School of Dentistry, Sendai, Japan, and §Biomedical Laboratory-Pathology and Cytology Center, Saitama, Japan

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Although mesangial cell death has been shown to be correlated with mesangial cell mitosis in vivo, little is known about how these two apparently opposite events are regulated. We show that the addition of platelet-derived growth factor (PDGF ; 10–50 ng/ml) to primary cultured rat mesangial cells for 24 h caused continuous proliferation along with simultaneous cell death. This process was accompanied by the fragmentation of DNA into nucleosomal oligomers, the development of apoptotic morphological changes in the nucleus, and increased expression of p53. Accumulation of lactate dehydrogenase (LDH) was also observed in the culture medium, suggesting that both apoptosis and necrosis are involved in the cell death mechanisms observed. We also observed that addition of 30 µM lysophosphatidic acid (LPA) to the culture medium greatly suppressed PDGF-induced cell death, leading to synergistically enhanced mesangial cell proliferation. DNA fragmentation, p53 expression and LDH release were all suppressed by LPA. We suggest that PDGF is a bifunctional molecule in mesangial cells that evokes both cell proliferation and cell death simultaneously, whereas LPA is a survival factor. We speculate that PDGF and LPA may play important roles in the progression or exacerbation of proliferative glomerulonephritis.

INTRODUCTION The interrelated occurrence of mesangial cell death during cell proliferation has been identified in kidney tissues in many types of human renal diseases, including post-streptococcal acute glomerulonephritis, IgA nephropathy, lupus nephritis, systemic vasculitis and haemolytic uraemic syndrome [1–3], and in experimental

animals, including those with Thy-1.1 glomerulonephritis [4,5] and uni-nephrectomized rats [6]. In turn, aberration of cell death has been observed to be correlated with protracted, sustained renal diseases, including lupus nephritis and mesangial proliferative glomerulonephritis [2]. However, the mechanisms controlling cell death that occurs during mesangial cell proliferation have yet to be clarified. In addition, the pathophysiological role of the

Key words: apoptosis, glomerulonephritis, lysophospholipid, survival. Abbreviations: Edg, endothelial differentiation gene ; FBS, fetal bovine serum ; LDH, lactate dehydrogenase ; LPA, lysophosphatidic acid ; PDGF, platelet-derived growth factor. Correspondence: Dr Chiyoko N. Inoue, Department of Pediatrics, Japanese Red Cross Sendai Hospital 43–3, Yagiyamahon-cho 2-chome, Taihaku-ku, Sendai 982–8501, Japan (e-mail cnagano!sendai.jrc.or.jp).

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inhibition of cell death during mesangial cell proliferation remains unknown. The role of lysophosphatidic acid (LPA) in the pathophysiology of glomerular diseases is now beginning to be appreciated [7]. Generated by activated platelets or by the enzymic cleavage of stores of glycerophospholipids in the membranes of injured cells, LPA has been suggested to act in an autocrine and juxtacrine manner to promote a wide variety of biological responses, including tissue injury, inflammation, neoplasia and wound healing [8]. To date, three isotypes of the LPA receptor, designated endothelial differentiation gene (Edg)-2, Edg4 and Edg-7, have been identified and cloned [9–11]. These receptors are developmentally regulated and differ in tissue distribution, but couple similarly to multiple types of G-proteins, including Gi, Gq and G / [8]. We "# "$ have reported previously novel biological functions for LPA in primary cultured rat mesangial cells, including contraction\relaxation [12], mild mitogenic responses, and co-mitogenic action with platelet-derived growth factor (PDGF) [13]. Since both PDGF and LPA are products of aggregated platelets [14] and are suggested to be localized in injured glomeruli, these observations led us to speculate that LPA may play an important role in the progression of proliferative glomerulonephritis [7]. During that series of experiments [7], we made the surprising observation that, although PDGF induced mesangial cell proliferation, cell numbers did not necessarily increase, because substantial cell death occurred concurrently with cell proliferation. Induction of mesangial cell death by PDGF was greatest when cells were stimulated with a high dose of PDGF, but was nonetheless clearly apparent in the presence of a low dose of PDGF. On the other hand, when mesangial cells were costimulated with LPA and PDGF, even a high concentration of PDGF did not evoke mesangial cell death, so that there was a high degree of cell proliferation. Based on these observations, we hypothesized that PDGF, while stimulating growth signal transduction, may also either cause inappropriate growth stimulation or activate the cell death programme, which could be suppressed by a survival factor. We also speculated that PDGF-induced mesangial cell death might be comparable with that identified histologically in proliferative glomerulonephritis, since PDGF is a major growth factor acting in vivo in pathological situations, as well as in vitro [15]. In the present study, we analysed the relationship between mesangial cell death and proliferation upon treatment with PDGF, and demonstrated for the first time that PDGF could cause apoptosis simultaneously with cellular proliferation. We also showed that LPA acts as a survival factor, protecting cells against PDGF-induced cell death. The relevance of these observations to histologically detected mesangial cell growth and death in renal biopsy specimens from human renal diseases is also discussed. #

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This work was first presented at the American Society of Nephrology meeting on November 5, 1999, in Miami.

MATERIALS AND METHODS Materials 1-α-LPA (1-oleoyl), insulin\transferrin\selenite premix and fatty acid-free BSA were purchased from Sigma (St. Louis, MO, U.S.A.). Recombinant human PDGF-BB was obtained from Pepro Tech EC (London, U.K.). Fetal bovine serum (FBS) was purchased from JRH Biosciences (Lenexa, KS, U.S.A.). RPMI 1640 and other cell culture supplements were obtained from Gibco BRL (Grand Island, NY, U.S.A.). SYBR Green I was purchased from Molecular Probes Inc. (Eugene, OR, U.S.A.). Except where noted, all other reagents were purchased from Wako Pure Chemicals Co. (Tokyo, Japan) or Sigma.

Culture of mesangial cells Isolated glomeruli were prepared from the kidney cortices of male Sprague–Dawley rats (body weight 75–100 g) by consecutive sieving with three different stainless steel meshes (106, 150 and 75 µm), as described previously [16]. The glomeruli were then digested with collagenase (type 1) and cultured in plastic culture flasks. Cells were maintained in RPMI 1640 medium containing 20 % (v\v) FBS, insulin\transferrin\selenite mix, penicillin (50 units\ml) and streptomycin (50 µg\ml) at 37 mC in a 5 % CO atmosphere. Cells from passages 3–6 were # subcultured 1 : 3 at 7-day intervals, and the medium was changed at 2-day intervals. Prior to all experiments, cells at 70 % confluence were growth-arrested with RPMI 1640 medium supplemented with 0.1 % BSA and 0.5 % (v\v) FBS without antibiotics for 2 days, in order to remove factors that may regulate mesangial cell growth and death.

Determination of mesangial cell proliferation and death Quiescent mesangial cells in 48-well culture dishes were exposed to mitogenic stimuli for 24 h and pulsed for the last 6 h with 1 µCi\ml [$H]thymidine (Amersham Japan, Tokyo, Japan). The cells were harvested after rinsing with PBS, and the trichloroacetic acid-precipitated material was processed for counting of radioactivity, as described previously [13]. For cell growth and cell death studies, cells were grown in 12-well culture dishes to 70 % confluence, serum-deprived, and treated with mitogens. For counting of viable mesangial cells, adherent mesangial cells were trypsinized with 0.05 % trypsin\0.53 mM EDTA, and an aliquot of this cell suspension was then mixed with an equal volume of 0.08 % Trypan Blue in

Platelet-derived growth factor-induced mesangial cell growth and death

Hanks balanced salt solution ; dye exclusion was assessed visually by microscopy. The number of viable cells was calculated by multiplying the total cell number by the fraction of cells excluding Trypan Blue. For counts of dead cells, cells were harvested from the supernatant by centrifugation (700 g for 5 min at room temperature), and nuclei of dead cells were stained with 1 µg\ml propidium iodide for 30 min and counted under fluorescent microscopy (LEICA DMRXA ; Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany).

Morphological observation of cultured mesangial cells Electron microscopical morphological examination was performed on pellets of PDGF-stimulated and detached, as well as quiescent and adherent, mesangial cells, as described previously [17]. Briefly, cells were harvested, washed with PBS, fixed in 2.5 % glutaraldehyde for 2 h at 4 mC, and post-fixed in 2 % osmium tetroxide for 2 h at 4 mC. Fixed cells were dehydrated in a graded series of ethanol, embedded in epoxy resin, and polymerized at 60 mC for 2 days. Ultrathin sections were cut with an ultramicrotom diamond knife, stained with uranyl acetate and lead citrate, and examined using an Hitachi H-7000 electron microscope.

Measurement of the release of lactate dehydrogenase (LDH) Following incubation, media from 48-well culture dishes were collected and the floating cells were spun down by gentle centrifugation (700 g for 5 min at 4 mC). The cells attached to the culture dishes and the detached cells were combined, lysed with 0.2 % Triton X-100 (in PBS) for 4 h at 4 mC, and left to stand as whole-cell lysates. Utilizing a commercially available test kit (Kyokuto Pharmaceutical Co., Tokyo, Japan), LDH activity was assayed spectrophotometrically in the culture medium and in whole-cell lysates. LDH release was expressed as a percentage of the total LDH activity of the corresponding culture dish.

Cell lysis and Western blotting Expression of p53 was determined by Western blot analysis. Quiescent mesangial cells in 60 mm dishes were stimulated with LPA (30 µM), PDGF (50 ng\ml) or LPA (30 µM)\PDGF (50 ng\ml) for 2, 4 and 12 h, and cell lysates were prepared using ice-cold cell solubilization buffer (50 mM Tris\HCl, pH 8.0, 150 mM NaCl, 0.1 % SDS, 1 % Nonidet P40, 100 µg\ml PMSF, 1 µg\ml leupeptin and 1 µg\ml aprotinin). For Western blotting, 5 µg of each protein sample was electrophoresed using a 12.5 % (w\v) polyacrylamide SDS\PAGE gel (Bio-Rad Laboratories, Hercules, CA, U.S.A.), and then transferred to a PVDF membrane and blocked with 5 % (w\v) non-fat dry milk. p53 protein was detected with anti-p53

monoclonal antibody (Transduction Lab, Lexington, KY, U.S.A.) at 1 : 500 dilution, followed by a 1 : 2000 dilution of goat anti-(mouse IgG) conjugated to horseradish peroxidase (Amersham, Arlington Heights, IL, U.S.A.). The blots were developed by enhanced chemiluminescence reagent (ULTRA kit ; Pierce, Rockford, IL, U.S.A.) according to the manufacturer’s instructions.

DNA fragmentation assay Quiescent rat mesangial cells in 12-well plates were stimulated with LPA (30 µM), PDGF (50 ng\ml) or LPA (30 µM)\PDGF (50 ng\ml) for 12 h. Both attached and floating cells were collected and washed with cold PBS, and then the DNA was isolated using a DNA isolation kit (Wako Pure Chemicals Co.). A 1 µg sample of DNA was loaded on to a 3 % (w\v) agarose gel. After electrophoresis was performed in 1iTAE buffer (40 mM Tris\acetate and 1 mM EDTA, pH 8.0), DNA bands were stained with SYBR Green I for 45 min at room temperature, excited at 470 nm and visualized with a yellow filter using a Fluoroimage analyser (FLA 2000 ; Fujifilm, Tokyo, Japan).

Statistical analysis All data are expressed as meanspS.E.M. Data sets were compared with multivariate analysis of variance (MANOVA). Differences between groups were compared with a Mann–Whitney U test. Values of P 0.05 were considered significant.

RESULTS Effects of PDGF on the proliferation and survival of rat mesangial cells PDGF-induced mitogenic action was monitored by measuring [$H]thymidine uptake 18–24 h after stimulation with PDGF under serum-deprived culture conditions. Cell viability was assessed by measuring the amount of LDH released into the culture medium as a result of leakage from mesangial cells. As shown in Figure 1, PDGF-BB at concentrations of 10–50 ng\ml dose-dependently induced DNA synthesis in cultured rat mesangial cells, as measured by [$H]thymidine uptake. At concentrations above 50 ng\ml, the mitogenic effect of PDGF reached a plateau. In parallel experiments, leakage of cytosolic LDH into the cell culture supernatant was also found to be dose-dependently (5–50 ng\ml) increased in PDGF-treated cells. At a saturating concentration of PDGF (50 ng\ml), mesangial DNA synthesis was stimulated to a level approx. 14.8 times higher than that of quiescent cells, and LDH release was calculated to be 8.69p0.10 % (n l 3 wells), which was significantly elevated compared with that in dishes of #

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Figure 1 Dose-dependent stimulation of DNA synthesis and LDH release by PDGF in cultured mesangial cells

Cultured rat mesangial cells were grown to subconfluence and rendered quiescent in RPMI 1640/0.5 % FBS/0.1 % BSA for 2 days. Mesangial cells were stimulated with the indicated doses of PDGF-BB for 24 h, and then DNA synthesis was assessed by measuring [3H]thymidine uptake for the last 6 h (n l 3 wells). In parallel, LDH activities in the cell culture supernatant were measured, and expressed as a percentage of total LDH activity in the corresponding culture dish (n l 3 wells). Significance of differences : *P 0.05 compared with quiescent mesangial cells. quiescent cells (4.45p0.22 % ; n l 3 wells ; P 0.05). At a low concentration (5 ng\ml), PDGF caused LDH release only, in the absence of a significant increase in DNA synthesis. These results suggest that PDGF exerts both mitogenic and cytotoxic effects at concentrations between 10 and 50 ng\ml, whereas at a lower concentration (5 ng\ml) PDGF acts like a cytotoxic factor in rat mesangial cells.

Figure 2

The proportion of LDH released into the culture medium has been reported to be quantitatively correlated with the number of dead cells [18,18a]. We therefore counted the actual number of dead cells following treatment with a saturating dose (50 ng\ml) of PDGF. In our preliminary experiments, we were unable to observe most of the dead mesangial cells under the microscope, because they quickly detached from the culture dishes. Therefore we assessed mesangial cell death by collecting detached cells from the culture supernatant by gentle centrifugation 1 and 2 days after treatment with PDGF, followed by staining of the cell nuclei with propidium iodide. As shown in Figure 2(A), PDGF caused substantial cell death during the 2-day incubation period ; the number of dead cells in the culture medium increased to 12 125p1208 cells\well (day 2 ; n l 3 wells), which was significantly higher than that in quiescent culture conditions (1933p375 cells\well at day 2 ; n l 3 wells ; P 0.05). We also observed that, when mesangial cells were stimulated with PDGF (50 ng\ml) in combination with LPA (30 µM), the occurrence of mesangial cell death was significantly suppressed during the 2-day incubation period (PDGF alone, 12 125p1208 cells\well ; PDGFj LPA, 1700p242 cells\well at day 2 ; n l 3 wells ; P 0.05). In addition, LPA was found to significantly reduce mesangial cell death under quiescent culture conditions (no LPA, 1933p376 cells\well ; jLPA, 925p113 cells\ well at day 2 ; n l 3 wells ; P 0.05). These results suggest that the observed LDH release stimulated by PDGF indeed reflected mesangial cell death, and that LPA can inhibit mesangial cell death that occurs follow-

Cumulative death of mesangial cells following treatment with PDGF

(A) Quiescent rat mesangial cells were stimulated with LPA (30 µM), PDGF (50 ng/ml) or LPA (30 µM)/PDGF (50 ng/ml) for 2 days. The whole medium was removed daily and centrifuged gently to separate the cell pellet. Dead cell nuclei were stained with propidium iodide and counted under fluorescent microscopy (n l 3 wells). Cumulative death was plotted against time after treatment (days). (B) LDH leakage was measured in the culture medium obtained 1 day after treatment, and was expressed as a percentage of the total LDH activity of the corresponding dish (n l 3 wells). Significance of differences : *P 0.05. Q, quiescent mesangial cells. #

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Platelet-derived growth factor-induced mesangial cell growth and death

Figure 3 Synergistic growth stimulation of mesangial cells by LPA and PDGF

Quiescent rat mesangial cells were stimulated with LPA (30 µM), PDGF (50 ng/ml) or LPA (30 µM)/PDGF (50 ng/ml) for 4 days, and viable cells were counted in a Trypan Blue exclusion assay. Significance of differences : *P 0.05. Q, quiescent mesangial cells. ing treatment by PDGF, or even under quiescent culture conditions. The survival effect of LPA against PDGF-induced mesangial cell death was confirmed by an LDH release assay. In this series of experiments, LDH release following PDGF treatment was suppressed significantly by LPA (PDGF alone, 5.74p0.31 % ; PDGFjLPA, 3.09p 0.37 % ; n l 3 wells ; P 0.05) (Figure 2B). The amount of LDH released under quiescent culture conditions was also significantly inhibited by LPA (no LPA, 2.53p 0.11 % ; jLPA, 1.385p0.135 % ; n l 3 wells ; P 0.05). In addition, the survival action of LPA against PDGFinduced cell death was totally abrogated by 100 µM LY294002, a selective inhibitor of phosphatidylinositol 3-kinase [19] (PDGFjLPA, 3.09p0.37 % LDH release ; PDGFjLPAjLY294002, 11.67p0.46 % release ; n l 3 wells ; P 0.05). To elucidate further the effect of the inhibition of cell death by LPA on the mesangial cell mitogenic response to PDGF, we stimulated mesangial cells with PDGF (50 ng\ml), LPA (30 µM) or a combination of LPA (30 µM) and PDGF (50 ng\ml) for 4 days, and counted the increase in mesangial cell number (Figure 3). The cell number in PDGF-treated dishes was 128 833p4262 cells\well (n l 3 wells), which was significantly higher than that in dishes of quiescent cells (78 000p7635 cells\well ; n l 3 wells ; P 0.05) or LPA-treated cells (109 667p3106 cells\well ; n l 3 wells ; P 0.05). On the other hand, exposure of mesangial cells to both LPA and PDGF resulted in a synergistic stimulation of cell growth, with a final cell number of 181 067p5084 cells\well, (n l 3 wells), which was approx. 1.4-fold higher than that in PDGF-treated dishes (P 0.05). This suggests that

Figure 4 DNA degradation in cells undergoing PDGFmediated cell death

DNA extracts from quiescent cells (Q) or from cells stimulated with LPA (30 µM), PDGF (50 ng/ml) or LPA (30 µM)/PDGF (50 ng/ml) were analysed as described in the Materials and methods section. A sample of 1 µg of DNA was loaded in each lane, electrophoresed and visualized using SYBR Green I dye. Internucleosomal fragmentation of genomic DNA was noted in PDGF-treated cells, but not in quiescent or in LPA-treated cells. LPA suppressed DNA fragmentation by PDGF. M, 100 bp DNA ladder marker. PDGF-induced mesangial cell proliferation is partially counterbalanced by accompanying cell death, while suppression of mesangial cell death by LPA leads to synergistically augmented cell growth.

Mechanism of PDGF-induced mesangial cell death To investigate whether rat mesangial cell death following PDGF treatment is mediated by apoptosis, we evaluated cell nuclei for the presence of internucleosomal cleavage by monitoring DNA laddering, a hallmark of apoptosis, in agarose gels. DNA fragmentation in cultured mesangial cells was observed 24 h after treatment with PDGF, whereas little DNA laddering was observed in quiescent or LPA-treated cells (Figure 4). On the other hand, DNA fragmentation induced by PDGF was greatly suppressed by LPA. This result suggests that PDGF-induced mesangial cell death may be mediated by apoptosis. The morphological changes of mesangial apoptosis were assessed further at the single-cell level by electron microscopy (Figure 5). In contrast with quiescent mesangial cells (Figure 5A), PDGF-treated mesangial cells exhibited changes in cell structure typical of apoptosis, including nuclear condensation, chromatin fragmentation, cellular shrinkage and the formation of multiple cytoplasmic vacuoles (Figure 5B). Increased expression of the tumour suppressor protein p53 has been shown to be involved in the process of apoptosis in cultured mesangial cells [20]. We therefore #

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Figure 5

Electron microscopic observation of mesangial cells after PDGF treatment

Serum-starved rat mesangial cells were stimulated with PDGF (50 ng/ml for 24 h) and then processed for electron microscopy. In PDGF-stimulated and detached cells (B), but not in quiescent and adherent cells (A), typical apoptotic morphological changes, including cellular shrinkage, nuclear chromatin condensation and fragmentation and multiple cytoplasmic vacuole formation, are prominent. Magnification i2690. To determine whether the effect of LPA in promoting cell survival was mediated by inhibition of mesangial cell apoptosis, we examined the effects of LPA on DNA ladder formation and p53 expression. LPA was effective in preventing PDGF-induced apoptotic DNA fragmentation in mesangial cells (Figure 4). PDGF-induced increases in p53 levels were also inhibited when LPA was present during the 12 h incubation period (Figure 6). These results suggest that LPA is a survival factor that prevents mesangial apoptosis induced by PDGF.

DISCUSSION Figure 6

Western blot analysis of p53 in mesangial cells

Quiescent rat mesangial cells were stimulated with LPA (30 µM), PDGF (50 ng/ml) or LPA (30 µM)/PDGF (50 ng/ml) for 2, 4 or 12 h. Samples of 5 µg of cell lysate were fractionated by SDS/PAGE, transferred to a PVDF membrane and probed with the murine p53-specific monoclonal antibody. Q, quiescent mesangial cells.

examined the change in the content of p53 protein following stimulation by PDGF using Western blotting, to strengthen further the hypothesis that apoptosis may be involved in PDGF-induced cell death. As shown in Figure 6, p53 protein was barely detectable in quiescent mesangial cells, whereas a significant increase in p53 protein levels was observed 2–12 h after stimulation with PDGF. At 12 h, p53 protein expression in PDGF-treated cells was 2.2 times higher than that of quiescent cells. #

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A significant finding in the present study was that PDGF could drive two apparently opposite biological events, i.e. proliferation and death, simultaneously in cultured rat mesangial cells. At a concentration of 50 ng\ml PDGF, it was shown that approx. 5–10 % of total mesangial cells were committed to die when calculated at one time based on live\dead cell number counting, as well as measurements of LDH content released into the culture medium. Although this calculated death frequency appeared relatively low, cell death continued to occur asynchronously for at least 2 days after PDGF treatment, resulting in a significantly greater accumulation of dead cells in the culture medium. Both morphological and biochemical criteria appeared to suggest that PDGF-induced cell death was mediated

Platelet-derived growth factor-induced mesangial cell growth and death

mainly via apoptosis. This was based on the observation that both DNA fragmentation and chromatin condensation (as seen by electron microscopy) were observed in PDGF-treated cells (Figures 4 and 5B). p53 expression, which may comprise a component of the apoptotic signalling pathway [21] and has been shown to be related to apoptotic cell death in mesangial cells [22], was also elevated in PDGF-treated cells (Figure 6). In addition, PDGF-induced cell death was suppressed by exogenously added LPA, which also inhibited both DNA fragmentation and the up-regulation of p53 expression (Figures 4 and 6). Since cell death estimated by LDH release was also significantly suppressed by cycloheximide at 10 µg\ml, a concentration that completely blocks protein synthesis [23] (8.69p0.10 % in PDGFtreated cells compared with 4.26p0.20 % in PDGF\ cycloheximide-treated cells ; n l 3 wells ; P 0.05), and given that sensitivity to cycloheximide is one of the characteristic features of apoptosis [24], all of these observations support the view that apoptosis may be the major mechanism whereby mesangial cell death occurs upon PDGF treatment. However, there is increasing evidence that apoptotic cell death cannot be distinguished clearly from necrotic cell death, and that these two types of cell death occasionally co-exist in various types of cells, including mesangial cells [25–27]. We also observed that LDH in the culture medium, a marker of necrosis due to the loss of cell membrane integrity [18,18a], increased following PDGF treatment of mesangial cells (Figure 1). Therefore it is more than likely that necrosis occurs secondarily to, or at the same time as, apoptosis in the presence of PDGF under our experimental conditions. Although PDGF has been utilized as a mitogen for cultured mesangial cells for more than a decade [15], there have been no reports that describe the induction by PDGF of mesangial apoptosis. Since dying mesangial cells tend to detach from culture dishes and are unable to be observed via microscopy, as was our experience, cell death may previously have been overlooked. Although the low frequency and asynchronous nature of cell death observed upon PDGF treatment are characteristic features during apoptosis in a variety of cells, including PC12 cells [28], there appear to be other obstacles to detecting the death of mesangial cells. In this regard, another well characterized experimental system, the activation of apoptotic cell death by Fas\APO-1, shows a distinct pattern, whereby caspase family members are activated at the level of the receptor, leading to almost immediate and synchronized massive apoptotic cell death [29]. We have also established a principal action of LPA in cultured mesangial cells, as a survival factor. Thus LPA suppressed PDGF-induced cell death. LPA could also inhibit the low level of mesangial cell death that was observed under quiescent culture conditions

(Figure 2). Finally, by inhibiting cell death, LPA could synergize with PDGF, substantially augmenting PDGF’s mitogenic action, leading to exaggerated mesangial cell proliferation (Figure 3). As we have demonstrated previously, LPA on its own is poorly mitogenic for mesangial cells [13]. Given that the net doubling times of living cells in PDGF-treated and LPA\PDGF-treated dishes were similar (4.2 and 4.5 days respectively), it is reasonable to assume that LPA cannot promote or halt the progression of the cell cycle. Moreover, the effect of LPA in inhibiting PDGF-treated cell death, as measured by the LDH release assay, was abolished by a selective inhibitor of phosphatidylinositol 3-kinase, one of the most important regulatory proteins involved in antiapoptotic signalling pathways [19]. This suggests that phosphatidylinositol 3-kinase may be crucial for the cell survival action of LPA. Our data may permit us to draw some conclusions as to the importance of mesangial cell apoptosis and its inhibition during the course of proliferative glomerulonephritis. Studies during the past decade have established the important role of PDGF in glomerulonephritis, acting directly or as an intermediary for other growth factors to mediate mesangial cell proliferation [30,31]. In contrast, the present study has shown that PDGF has paradoxical actions, including the induction of mesangial cell death simultaneously with mesangial cell growth. Under our experimental conditions, mesangial cells died even in the presence of low concentrations (5 ng\ml) of PDGF (Figure 1). These phenomena may be interpreted as indicating that a mechanism exists whereby the hyperplasia of mesangial cells is counterbalanced by their death. Tashiro et al. [1] observed that the apoptotic response is paralleled by the intensity of the mesangial proliferative reaction in IgA nephropathy. RodriguezLopez et al. [6] also detected parallel increases in proliferation and apoptotic rates in glomerular cells in uni-nephrectomized spontaneously hypertensive rats. These reports appear to provide clear clinical evidence that simultaneous mesangial cell growth and death do indeed occur in vivo. In contrast, the induction of cell death by PDGF could be suppressed in the presence of LPA, resulting in magnified or persistent mesangial hypercellularity. Such powerful and uncontrolled mesangial cell growth might occur in situations in which LPA is produced within glomeruli in injured or inflamed endothelial cells, as well as in mesangial cells themselves when group II phospholipase A is activated [8]. Hyper# coagulability within diseased glomeruli may also contribute to an increase in the local production of LPA [14]. A low incidence of apoptotic mesangial cell death has been shown to be correlated with a clinically protracted resolution of renal diseases in lupus nephritis and IgA nephropathy [2]. We have recently found LPA receptor (Edg-4 isotype) mRNA to be highly expressed in renal glomeruli isolated from biopsy samples of patients with #

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advanced mesangial proliferative glomerulonephritis (C. N. Inoue, unpublished work). All of the evidence appears to suggest that LPA is generated by, and indeed acts as a survival factor involved in, protracted progressive glomerulonephritis. In conclusion, we have demonstrated, for the first time, that PDGF can function as a bi-modal molecule, regulating both mesangial cell growth and death, thereby determining the degree of mesangial cell proliferation in vitro. On the other hand, LPA is a survival factor, suppressing PDGF-induced cell death, which greatly exaggerates the abnormally sustained proliferation of mesangial cells. Here we have shown an experimental system in which mesangial cell growth and apoptosis can be regulated and analysed in the presence of well characterized growth molecules. We believe that future investigations utilizing a more detailed approach, such as investigating the molecular mechanisms of interaction of PDGF and LPA, may help to increase our understanding of the pathophysiology of the initiation and intractable progression of renal diseases.

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ACKNOWLEDGMENTS This work was supported by a Grand-in-Aid for Scientific Projects from the Ministry of Education, Japan. We thank Ms Naoko Sato for secretarial assistance.

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REFERENCES Tashiro, K., Kodera, S., Takahashi, Y., Horikoshi, S., Shirato, I. and Tomino, Y. (1998) Detection of apoptotic cells in glomeruli of patients with IgA nephropathy. Nephron 79, 21–27 2 Soto, H., Mosquera, J., Rodriguez-Iturbe, B., Henriquez La Roche, C. and Pinto, A. (1997) Apoptosis in proliferative glomerulonephritis : decreased apoptosis expression in lupus nephritis. Nephrol. Dial. Transplant. 12, 273–280 3 Arends, M. J. and Harrison, D. J. (1989) Novel histopathologic findings in a surviving case of hemolytic uremic syndrome after bone marrow transplantation. Hum. Pathol. 20, 89–91 4 Shimizu, A., Kitamura, H., Masuda, Y., Ishizaki, M., Sugisaki, Y. and Yamanaka, N. (1995) Apoptosis in the repair process of experimental proliferative glomerulonephritis. Kidney Int. 47, 114–121 5 Baker, A. J., Mooney, A., Hughes, J., Lombardi, D., Johnson, R. J. and Savill, J. (1994) Mesangial cell apoptosis : the major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis. J. Clin. Invest. 94, 2105–2116 6 Rodriguez-Lopez, A. M., Flores, O., Arevalo, M. A. and Lopez-Novoa, J. M. (1998) Glomerular cell proliferation and apoptosis in uninephrectomized spontaneously hypertensive rats. Kidney Int. Suppl. 68, S36–S40 7 Inoue, C. N., Epstein, M., Forster, H. G., Hotta, O., Kondo, Y. and Iinuma, K. (1999) Lysophosphatidic acid and mesangial cells : implications for renal diseases. Clin. Sci. 96, 431–436

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Received 2 November 2000/30 January 2001; accepted 21 March 2001

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2001 The Biochemical Society and the Medical Research Society

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