Proliferation Of Rat Mesangial Cells Research Articles

A Tyrosine Kinase-Signaling Pathway Involving ERK-2 Activation Links Bradykinin B2 Receptors with AP-1 and is Crucial for Bradykinin-Induced DNA Synthesis in Mesangial Cells. • 1646

We have recently shown that bradykinin (BK), acting via B2 receptors, stimulates DNA synthesis and proliferation of rat mesangial cells and that these responses were preceded by induction of c-fos and AP-1 binding activity (Kidney Int. 50: 1850, 1996). The present study was designed to examine the role of protein tyrosine phosphorylation in coupling the B2 receptors with AP-1 and DNA replication. BK (100 nM) induced tyrosine phosphorylation of distinct substrates of 170, 120-130, 70, 55-60, and 44-42 kDa as detected by antiphosphotyrosine immunoblotting. Immunoblotting with a polyclonal ERK antibody of BK-stimulated cells revealed predominant phosphorylation of the 42-kDa MAP kinase (ERK-2). Inhibition of tyrosine kinase activity with genistein (15-100 μM) abolished the stimulatory effect of BK on tyrosine phosphorylation and ERK-2 activation. Furthermore, genistein attenuated BK-mediated c-fos and c-jun induction and AP-1-DNA binding activity and suppressed BK-stimulated DNA synthesis in a dose-dependent manner. Herbimycin A (50-200 nM), a chemically and mechanistically distinct tyrosine kinase inhibitor, also abolished BK-stimulated DNA synthesis. Thus, tyrosine phosphorylation is an essential requirement in the mitogenic signaling of BK in glomerular mesangial cells. The data implicate the MAP kinase, ERK-2, and the transcription factor, AP-1, as the downstream signals linking BK-B2 receptor stimulation with DNA replication.

Mesangial cell proliferation mediated by PDGF and bFGF is determined by levels of the cyclin kinase inhibitor p27Kip1

Mesangial cell proliferation in vitro is regulated by many cytokines. Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are potent mesangial cell mitogens, whereas transforming growth factor-beta1 (TGF-beta1) reduces their effects. We examined how these cytokines regulate rat mesangial cell proliferation at the level of the cell-cycle. Quiescent mesangial cells in vitro express the cyclin kinase inhibitor, p27Kip1 (p27), and PDGF- and bFGF-induced mesangial cell proliferation is associated with a substantial decrease in p27 levels. Consequently there is a marked increase in expression (Western blot analysis, immunostaining) of cyclin A and CDK2. The decline in p27 levels was prevented by TGF-beta1 during inhibition of PDGF- and bFGF-induced mesangial cell proliferation. To determine the functional role of p27 during cytokine-mediated mesangial cell proliferation, the expression of p27 was reduced with specific p27Kip1 antisense oligodeoxynucleotides. Reducing the levels of p27 resulted in an increased magnitude of mesangial cell proliferation (BrdU and 3H-thymidine incorporation) induced by PDGF and bFGF compared to non-transfected mesangial cells and mesangial cells transfected with control mismatch oligodeoxynucleotides. Furthermore, the onset of maximal proliferation occurred earlier in mesangial cells transfected with antisense compared to control. The reduction in proliferation by TGF-beta1 were not altered by decreased p27 expression. Reducing p27 expression in the absence of mitogens was not associated with entry into the cell-cycle. These results suggest cytokine mediated mesangial cell proliferation is associated with specific cell-cycle proteins, and that the levels of p27 may be important in determining the mesangial cell's proliferative response to PDGF and bFGF in vitro.

Overexpression of cell cycle inhibitors (p16INK4 and p21Cip1) and cyclin D1 using adenovirus vectors regulates proliferation of rat mesangial cells.

To elucidate the mechanisms of the cell cycle of mesangial cells, adenovirus vectors containing coding sequences of cyclin D1 (AxCAD1), p16INK4 (AxCAp16) and p21Cip1 (AxCAp21) were produced and investigated to determine whether transfer of these genes changes serum- and PDGF-induced proliferation of rat mesangial cells. Efficiency of the transfer of the genes was examined by Northern and Western blot analyses. The cell cycle of mesangial cells was evaluated by measurement of [3H]-thymidine incorporation, flow cytometry, and cyclin-dependent kinase 4 kinase assay. Expression of cyclin D1, p16INK4 and p21Cip1 was observed from 24 h after the infection, and the expression increased up to 48 h. AxCAp16 and AxCAp21 caused a significant inhibition in the [3H]-thymidine incorporation to 47% and 76%, respectively. AxCAp16 and AxCAp21 also inhibited the mitogen-induced increase in cyclin-dependent kinase 4 kinase activity and reduced the percentage of cells in S phase. Coinfection of AxCAp16 with AxCAp21 showed no additive inhibition. Overexpression of cyclin D1 reduced cell size and increased the percentage of the cells in S and G2 phase. These findings suggest that p16INK4 and p21Cip1 function as inhibitors of the proliferation of mesangial cells induced by growth-promoting factors and that deregulated expression of cyclin D1 causes cell cycle disturbances. Adenovirus-mediated gene transfer of p16INK4 and p21Cip1 serves as a potential therapeutic approach to mesangial proliferative diseases.

Inhibitory effect of the intravenous anesthetic, ketamine, on rat mesangial cell proliferation.

We studied the effects of ketamine and propofol on rat cultured mesangial cell (MC) proliferation. The 72-h exposure to ketamine (1-100 microM) inhibited [3H]thymidine incorporation into the MC in a concentration-dependent manner. Propofol, however, was not effective at inhibition of MC proliferation at doses from 1 to 100 microM. Ketamine also decreased the cell number at clinically relevant concentrations and increased adenosine 3',5'-cyclic monophosphate (cAMP) levels in MC. We also studied the effects of ketamine on cytokine-induced [3H]thymidine incorporation into the cells. Ketamine decreased the tumor necrosis factor-alpha (TNF-alpha)-, interleukin 1 (IL-1)-, and interleukin 6 (IL-6)-induced [3H]thymidine incorporation into the cells. It also decreased angiotensin II (Ag II)-induced [3H]thymidine incorporation. These results suggest that ketamine has inhibitory effects on MC proliferation, but that propofol does not. Because ketamine inhibits TNF-alpha-, IL-1-, and IL-6-induced MC proliferation, it may be useful in suppressing the cell growth clinically.

Inhibitory Effect of the Intravenous Anesthetic, Ketamine, on Rat Mesangial Cell Proliferation

Inhibitory Effect of the Intravenous Anesthetic, Ketamine, on Rat Mesangial Cell Proliferation

Inhibitory effect of adrenomedullin on rat mesangial cell mitogenesis.

We investigated the effects of adrenomedullin on rat mesangial cell proliferation. Adrenomedullin (10(-10)-10(-7) M inhibited both [3H]thymidine incorporation into cultured rat mesangial cells and cell proliferation in a concentration-dependent manner. Adrenomedullin (10(-10)-10(-6) M) stimulated cyclic adenosine monophosphate accumulation in rat mesangial cells in a concentration-dependent manner. These findings suggest that adrenomedullin inhibits proliferation of rat mesangial cells probably through a cyclic adenosine monophosphate-dependent mechanism.

Parathyroid hormone-related protein is an autocrine modulator of rabbit proximal tubule cell growth

Parathyroid hormone-related protein (PTHrP), a likely mediator for humoral hypercalcemia of malignancy, is also synthesized in various normal tissues. In the kidney, PTHrP, mainly detected in proximal and distal tubules, has been shown to stimulate proliferation of rat mesangial cells in culture. Experiments were carried out to investigate the possible mitogenic effect of PTHrP in cultures of rabbit proximal tubule cells (PTC). Immunocytochemical analysis, using antihuman (h)PTHrP antibodies to (38-64) and (107-111) epitopes in the PTHrP molecule, showed strong cytoplasmic staining in PTC and proximal tubule-like LLC-PK1 cells. PTC secreted immunoreactive PTHrP (54.8 +/- 7.0 fmol/10(6) cells) into the culture medium. Human PTHrP(1-141) stimulated proliferation in subconfluent cultures of these cells dose-dependently. This effect was similar to that induced by [Tyr34]hPTHrP(1-34) amide (hPTHrP[1-34]), hPTHrP(1-86), and bovine (b)PTH(1-34), while hPTHrP(38-64) amide, hPTHrP9107-111) amide, and hPTHrP(107-139) amide were ineffective. Addition of anti-hPTHrP neutralizing antibodies to (1-34), (38-64), and (107-111) epitopes of PTHrP decreased PTC growth. The mitogenic effect of these agonists was abolished in confluent PTC. In contrast, [Nle8,18, Tyr34]bPTH(3-34)amide (bPTH[3-34]) increased DNA synthesis in either subconfluent or confluent PTC. In LLC-PK1 cells, which also secreted PTHrP and are devoid of PTH receptors, none of these peptides affected proliferation. Forskolin (10 microM) or H-8 (2 microM), a protein kinase A inhibitor, did not affect basal or hPTHrP(1-34)-stimulated DNA synthesis, respectively, in subconfluent PTC. On the other hand, 10 nM staurosporine and 100 nM calphostin C, protein kinase C (PKC) inhibitors, blunted the effects of hPTHrP(1-34) or bPTH(3-34) on DNA synthesis in these cells. These studies suggest that PTHrP may function as an autocrine factor in the regulation of proximal tubule cell growth by a PKC-mediated mechanism.

Disintegrin Modulates Rat Glomerular Mesangial Cell Behavior

Disintegrins are a group of molecules containing Arg-Gly-Asp (RGD) sequence which can interfere with cell-matrix interaction. Rat mesangial cells are known to express an RGD-sensitive integrin receptor. We investigate the effect of a potent disintegrin, triflavin, on rat mesangial cell adhesion and proliferation. While synthetic RGD-containing peptide (GRGDS) inhibits mesangial cell adhesion to fibronectin, type I and type III collagen, triflavin has a similar but much more potent effect. Triflavin, when added to the medium, inhibits serum-stimulated rat mesangial cell proliferation in both growing and growth-arrested cells. In serum-free medium, platelet-derived growth factor promoted mesangial cell growth is also inhibited by triflavin. When coated as a substratum, both GRGDS and triflavin inhibit mesangial cell growth. The inhibition of proliferation by exogenously added triflavin and GRGDS is, at least in part, due to their antiadhesive effect. We conclude that triflavin can modulate both adhesion and proliferation of rat mesangial cells.

The effect of lovastatin on proliferation of cultured rat mesangial and aortic smooth muscle cells.

In order to investigate the anti-proliferative effect of 3-hydroxy-3-methylglutaryl coenzyme. A reductase inhibitor, we evaluated the effects of lovastatin on DNA replication and the proliferation of rat mesangial and aortic smooth muscle cells, both of which were mesenchymal origin cells. Proliferations were determined by measuring [3H]thymidine uptake, and counting the number of cells. Growth-arrested mesangial and aortic smooth muscle cells were exposed to platelet-derived growth factor (PDGF), endothelin (ET) and angiotensin II (Ang II) to stimulate mitogenesis. All agents exhibited dose-dependent stimulation of [3H] thymidine uptake. PDGF was more potent than the others. Ang II increased [3H] thymidine uptake without demonstrable mitogenic activity. Lovastatin inhibited PDGF (10 ng/ml in mesangial cell, 25 ng/ml in smooth muscle cell)-, ET (10(-7)M)- and Ang II (10(-7)M)-induced [3H] thymidine uptake significantly in a dose-dependent manner in both cells. The increase of cell number in response to PDGF and ET treatment were also inhibited at 10 microM of lovastatin. The inhibitory effect of lovastatin was largely overcome in the presence of exogenous mevalonate at 200 microM, with 75.5% restoration from lovastatin-induced inhibition on PDGF-induced [3H] thymidine uptake in mesangial cells (77.8% in aortic smooth muscle cells). However, the addition of cholesterol did not prevent inhibition by lovastatin. In conclusion, lovastatin had an inhibitory effect on mesangial and aortic smooth muscle cell proliferation, and mevalonate was essential for DNA replication in both types of cells. Lovastatin may reduce glomerular and atherosclerotic injury through an anti-proliferative effect on mesangial and vascular smooth muscle cells, in addition to lowering circulating lipids.

Vasodilator agents modulate rat glomerular mesangial cell growth and collagen synthesis.

Mesangial cell activation and extracellular matrix accumulation are hallmarks of many forms of glomerulonephropathy. We investigated the effect of several agents possessing vasodilating activities on rat mesangial cell growth and collagen synthesis. Using cell counting and a modified MTT assay, it was shown that dipyridamole, pentoxifylline, dicentrine, prazosin and doxazosin all caused a dose-dependent inhibition of serum-stimulated rat mesangial cell proliferation. Platelet-derived growth factor-induced cell proliferation was also inhibited by doxazosin and pentoxifylline. Dipyridamole and pentoxifylline inhibited collagen synthesis in confluent mesangial cells while dicentrine and doxazosin did not. The procollagen alpha 1 (I) mRNA expression was also decreased by dipyridamole and pentoxifylline. These results suggested that, in addition to dipyridamole, pentoxifylline and alpha 1-adrenoceptor blockers may have a potential to delay the progression of chronic glomerulopathy associated with mesangial proliferation.

Effects of endogenously produced arachidonic acid metabolites on rat mesangial cell proliferation

The role of endogenously produced arachidonic acid metabolites on glomerulonephritis was investigated using cultured rat mesangial cells. The cultured mesangial cells could produce prostaglandin (PG) E 2 and F 2α and 12-hydroxyeicosatetraenoic acid (12-HETE). The treatment of the mesangial cells with indomethacin enhanced the cell growth stimulated by 10% fetal calf serum (FCS). This stimulatory effect was significantly attenuated by concomitant treatment with PGE 2, but not with PGF 2α. To test whether the mechanism by which PGE 2 inhibited the mesangial cell growth is related to in the increment of cyclic AMP (cAMP), we examined the effect of dibutyryl cAMP on mesangial cell growth. As expected, treatment with dibutyryl cAMP decreased the cell proliferation. Moreover treatment with KT-5720, a protein kinase A (PKA) inhibitor, stimulated the cell growth as well as indomethacin. These data strongly suggest that the inhibition of mesangial cell growth by PGE 2 involves an activation of PKA. In contrast, treatment with baicalein, a specific inhibitor of 12-lipoxygenase, inhibited the mesangial cell growth. The up and down regulations by arachidonic acid metabolites were also observed in the growth induced by platelet-derived growth factor (PDGF). These results suggest that endogenously produced arachidonic acid metabolites are involved in the regulation of mesangial cell growth.

Growth modulation and proteoglycan turnover in cultured mesangial cells.

Proliferation of mesangial cells is a common feature of renal disease, and conditioned media from glomerular epithelial and endothelial cells have been found to contain heparin-like molecules that suppress proliferation of rat mesangial cells (RMC). We have partially characterized the glycosaminoglycans that are labeled with 35SO4(2-) by RMC in culture at early passage and examined their ability to inhibit mitogenic stimulation of the cells. Four chondroitin/dermatan sulfate proteoglycans (CS/DSPG) were identified, the largest and smallest of which (Kd of 0.04 and 0.26 on Superose 6) were retained in the cell layer while the other two (Kd = 0.17 and 0.22) were secreted into the medium. Heparan sulfate proteoglycans (HSPG) with Kd values of 0.09, 0.13, and 0.39 were minor components of the cell layer, while a single heparan sulfate (Kd = 0.17) was recovered from the medium. After 16 h of labeling in serum-free medium, about 60% of macromolecular 35S was cell-associated and 40% was in the medium. Cell-associated label consisted of 7% CS/DSPG, 9% HSPG, and 84% free glycosaminoglycan chains (mostly CS/DS), whereas the medium contained 52% CS/DSPG, 17% HSPG, and approximately equal amounts of free HS and CS/DS chains. Bovine lung heparin (1 microgram/ml) decreased by 45% the incorporation of [3H]-thymidine into DNA after release of serum-starved RMC from growth arrest. Heparin acted prior to the G1/S interface; arrest of the cells in early S phase with aphidicolin abrogated the heparin response. The endogenous HSPGs had a slight antimitogenic effect on the RMC, but heparan sulfate chains from both the medium and cell layer had a potent effect. On an equivalent mass basis, only the free glycosaminoglycan chains were more potent than heparin in this regard, decreasing thymidine incorporation by over 90% when present at 1 microgram/ml. These results demonstrate that heparan sulfate glycosaminoglycans derived from mesangial proteoglycans are potential negative autocrine growth regulators. Proteoglycan metabolism releases these soluble heparan sulfate chains, determining the level of this activity.

Altered nitric oxide production and exogenous nitric oxide do not affect the proliferation of rat mesangial cells

To examine the hypothesis that nitric oxide (NO) acts as an autocrine, antiproliferative regulator and that exogenous NO donor inhibitors the proliferation of cultured rat mesangial cells. The cellular effects of endogenous and exogenous NO were studied in rat mesangial cells in a two-dimensional culture of early mesangial cell passages. The proliferation of mesangial cells was determined by cell-counting and uptake of [3H]-thymidine. NO generation was induced by incubation with interleukin-1 beta (5 u/ml) or bacterial lipopolysaccharide (10 micrograms/ml) for 24 h. NO release by mesangial cells was assessed by measuring the accumulation of nitrite, the major stable end-product of NO, in mesangial cell supernates. In addition, cyclic GMP (cGMP) formation was measured by radioimmunoassay as an indicator for NO generation. The formation of nitrite and cGMP was significantly increased after incubation of mesangial cells with interleukin-1 beta or lipopolysaccharide. This effect was greatly reduced by an inhibitor of NO synthesis. NG-monomethyl-L-arginine (L-NMMA; 0.1 mmol/l). The NO donor 3-morpholino-sydnonimine-HCl also increased the cGMP concentrations in the mesangial cells. The proliferation of mesangial cells was analysed in growth-arrested and mitogen-stimulated (platelet-derived growth factor, platelet-derived growth factor plus ATP and fetal calf serum) mesangial cells in the presence and absence of L-NMMA and the NO synthase substrate L-arginine (1 mmol/l). At 48 h platelet-derived growth factor (50 ng/ml), and platelet-derived growth factor (50 ng/ml) plus ATP (0.1 mmol/l) and fetal calf serum 5% each significantly increased the uptake of [3H]-thymidine in mesangial cells. These effects were not altered in the presence of L-NMMA or L-arginine. Pretreatment with interleukin-1 beta or with lipopolysaccharide also failed to affect the uptake of [3H]-thymidine in resting or proliferating mesangial cells. 3-Morpholino-sydnonimine-HCl (10(-3) to 10(-6) mol/l) did not suppress the mitogen-induced proliferation of mesangial cells, even when it was administered three times a day. The present findings support recent observations that interleukin-1 beta and lipopolysaccharide strongly induce NO production in mesangial cells, as is shown indirectly by the greatly increased formation of nitrite and cGMP. However, these effects were not associated with antiproliferative action on mitogen-stimulated mesangial cells. Similarly, the exogenous NO donor 3-morpholino-sydnonimine-HCl induced cGMP formation but failed to inhibit proliferation of mesangial cells when used at a non-toxic dose. Our observations do not support the contention that the formation of NO and cGMP constitutes an autocrine downregulating mechanism in the control of the growth of mesangial cells. It remains to be seen what pathophysiological role the induction of NO release plays in the regulation of the behaviour of mesangial cells, e.g. during an inflammatory response to glomerular injury.

Effect of serum from patients with mesangial proliferative glomerulonephritis on cultured rat mesangial cells.

We studied the effect of serum from patients with renal diseases characterized by mesangial proliferation on the proliferation of rat mesangial cells (MC) in culture. Indirect immunofluorescence technique with human anti-proliferating cell nuclear antigen (PCNA) antibody was utilized to detect proliferating MC in S-phase on microscope slides. MC from serum-starved culture medium containing 0.5% fetal calf serum (FCS) showed 6 +/- 2% of PCNA-positive MC, whereas those from complete medium containing 20% FCS showed 45 +/- 7%. This was confirmed by flow cytometry for MC from both conditions, in which 10.9% and 55.2% of S-phase MC were detected, respectively. Serum from 17 patients with IgA nephropathy as a prototype of mesangial proliferative glomerulonephritis produced more PCNA-positive MC than serum from 18 healthy controls (mean +/- S.D. = 21.5 +/- 9.9% vs. 4.4 +/- 5.9%, p < 0.001). In IgA nephropathy, this effect correlated tentatively with active histologic lesion in the glomeruli, but not with severity of urinary abnormalities. Serum from membrano-proliferative glomerulonephritis (MPGN) also produced significant PCNA-positive MC (mean +/- S.D. = 15.6 +/- 9.3%), compared to controls (p < 0.01), independent of the severity of urinary abnormalities. These findings suggest that serum effect on the proliferation of cultured rat MC may reflect an active histologic lesion in the glomeruli, and that anti-PCNA antibody to detect proliferating MC is, if not quantitative, relevant methodology to enumerate proliferating MC in culture.

Tumor necrosis factor alpha from peripheral blood mononuclear cells of IgA nephropathy and mesangial cell proliferation.

BackgroundTo investigate the role of mononuclear cells and their products on rat mesangial cell proliferation, we evaluated the effect of peripheral blood mononuclear cells (PBMC) culture supernatant of patients with IgA nephropathy on rat mesangial cells 3[H]-thymidine uptake, and measured the concentration of cytokines in the supernatant to find out which cytokine in PBMC culture supernatant has a major influence on mesangial cell 3[H]-thymidine uptake.Methods:In ten patients with primary IgA nephopathy and 10 normal controls, PBMC were cultured with PHA (20 μg/ml) and con-A (10 μg/ml) for 24 hours, and supernatants were collected and stored at −70°C until tested. Mesangial cell was cultured for 48 hours at a concentration of 104 cells/well with 1:2 or 1:10 diluted PBMC culture supernatant. 3[H]-thymidine uptake into rat mesangial cell was assayed after 18 hour culture. The cytokine concentrations of PBMC culture supernatant were measured by radioimmunoassay.Results:When 1:2 diluted PBMC culture supernatant was added, the supernatant of IgA nephropathy induced higher 3[H]-thymidine uptake into mesangial cell compared with that of normal controls. 3[H]-thymidine uptake induced by 1:2 diluted PBMC culture supernatant was significantly higher than that induced by 1:10 diluted supernatant of IgA nephropathy. The concentration of TNF-α in PBMC culture supernatant of the patients with IgA nephropathy was higher than that of normal controls and showed a good correlation with the mesangial cell 3[H]-thymidine uptake.Conclusion:It is suggested that the mononuclear cell of IgA nephropathy may have the intrinsic property to produce more TNF-α which may be mitogenic to mesangial cells when stimulated with mitogens, and it may be related to the mesangial cell proliferation in IgA nephropathy.

Interactions of Hydrogen Peroxide with Interleukin-6 and Platelet-Derived Growth Factor in Determining Mesangial Cell Growth: Effect of Repeated Oxidant Stress

1. This study examined the influence of H2O2, interleukin-6 and platelet-derived growth factor on the proliferation of rat mesangial cells. Mesangial cells were exposed to either a single pulse or three daily pulses of H2O2 (10(-8)-10(-4) mol/l), alone or in combination with interleukin-6 (5 ng/ml) and/or platelet-derived growth factor (10 ng/ml). Proliferation was assessed after 24 h and 72 h of incubation using [3H]thymidine incorporation and cell counts. 2. Although one pulse of H2O2 had no significant effect on mesangial cell proliferation, three daily pulses of 10(-6) mol/l H2O2 resulted in a significant increase in [3H]thymidine incorporation of 31 (52.6, 10.3)% (median and 75th-25th interquartile range) (P < 0.001). Both interleukin-6 and platelet-derived growth factor were also mitogenic to mesangial cells, [3H]thymidine incorporation increasing by 19 (36.7, -6.7)% (P < 0.05) and 53.5 (107, 21.9)% (P < 0.001), respectively. The mitogenic effect of interleukin-6 was enhanced by 10(-6) mol/l H2O2 [49.9 (77.7, 12.3)%] (P < 0.01), whereas the addition of 10(-6) mol/l H2O2 to platelet-derived growth factor resulted in a summated increase in [3H]thymidine incorporation of 82.7 (113, 57.4)% (P < 0.001). Incubation with all three substances simultaneously resulted in down-regulation of growth compared with H2O2 plus platelet-derived growth factor by 55.4 (77.7, 10.3)% (P < 0.05). 3. These findings suggest that reactive oxygen species may play a major role in determining the mesangial cell proliferation that occurs in certain forms of glomerulonephritis, acting either alone or in combination with other growth factors.

A role for platelet-activating factor in endothelin-1-induced rat mesangial cell proliferation

Endothelin-1 stimulated the release of bioassayable platelet-activating factor-like material and the incorporation of acetate to PAF in rat mesangial cells, in a dose-dependent manner, with an EC 50 of about 10 −9 M. Endothelin-1 also stimulated dose dependently [ 3H]thymidine incorporation into DNA, protein synthesis and cell growth. The platelet-activating factor antagonists BN-52021 (10 −5 M), and alprazolam (10 −5 M), reduced significantly endothelin-1-stimulated thymidine incorporation into DNA, protein synthesis and cell growth. Platelet-activating factor also stimulated significantly thymidine incorporation into DNA, cell proliferation and protein synthesis. In conclusion, the present results suggest that endothelin-1 induces mesangial cell proliferation through a mechanism involving among others, synthesis and release of platelet-activating factor by these cells.

TGF-beta stimulates rat mesangial cell proliferation in culture: role of PDGF beta-receptor expression

Transforming growth factor (TGF)-beta is known to increase mesangial cell (MC) matrix; however, its possible role on MC proliferation is controversial. We therefore studied the influence of TGF-beta on MC proliferation in culture and evaluated its effect on the platelet-derived growth factor (PDGF) B-chain as well as the expression of the PDGF beta-receptor. TGF-beta (1 ng/ml) increases MC DNA synthesis by approximately threefold after 48 h of incubation. TGF-beta-induced MC proliferation was also confirmed by cell counts. A neutralizing anti-TGF-beta antibody completely blocked this growth promoting activity. The levels of PDGF beta-receptor steady-state mRNA were increased by TGF-beta at 48 h. This was associated with an increase in receptor density per cell as measured by receptor kinetic studies. PDGF B-chain mRNA was also increased by TGF-beta at 48 h. A neutralizing anti-PDGF B-antibody causes no reduction of TGF-beta-induced DNA synthesis; however, suramin completely inhibited the mitogenic effect of TGF-beta. We conclude that TGF-beta stimulates MC growth in long-term culture, a process in which upregulation of the PDGF beta-receptor and enhanced synthesis of PDGF B-chain might be involved.

Lovastatin inhibits proliferation of rat mesangial cells.

Products of intracellular mevalonate metabolism are essential for cell growth and proliferation. Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, blocks the formation of mevalonate and its metabolites, and has been shown to inhibit proliferation of several cell types. In vivo, lovastatin has reduced mesangial cellularity and glomerular injury in experimental renal disease. In this study, we investigated the effects of lovastatin on DNA replication and proliferation in rat glomerular mesangial cells. Growth-arrested mesangial cells were exposed to medium containing 10% fetal bovine serum to stimulate mitogenesis. Lovastatin (1-20 microM) caused a significant (P < 0.05) dose-dependent reduction in DNA synthesis ([3H]thymidine incorporation) which was completely prevented in the presence of exogenous mevalonate (100 microM). Lovastatin (1 microM) inhibited cell proliferation by 90% over a 5-d period, and this was largely overcome by added mevalonate. Exogenous low density lipoprotein (100 micrograms/ml) did not prevent lovastatin inhibition of DNA synthesis. The isoprenoid end product isopentenyl adenine (5 or 50 microM) had little effect on DNA synthesis and cell proliferation in lovastatin-blocked cells. By contrast, the isoprenoid farnesol (5 microM) largely prevented lovastatin inhibition of DNA synthesis. We conclude that mevalonate metabolism is essential for mesangial cell proliferation, possibly through the production of the isoprenoid farnesol. Moreover, the action of lovastatin to reduce experimental glomerular injury may involve a direct effect on mesangial cells.

Effect of ouabain and hypothalamic, hypophysary inhibitory factor on rat mesangial cell proliferation.

The mitogenic effect of a low-molecular-weight, Na+,K(+)-ATPase inhibitory factor isolated from bovine hypothalamus and hypophysis (hypothalamic hypophysary inhibitory factor, HHIF) and ouabain on cultured rat mesangial cells was examined. Ouabain induced a potent stimulation of the [3H]thymidine incorporation to DNA, ranging from threefold for a dose of 10(-7) M to seven times for 10(-5) M. The amount of proteins per well also increased in a dose-dependent way, which was already significant from basal values for the lower dose used. HHIF also induced a dose-dependent stimulation of [3H]thymidine incorporation into DNA, ranging from 1.7 times for a concentration of 0.02 U/ml to 4.5 times for the concentration of 2 U/ml. The amount of protein per well also increased with HHIF, but the increase was significant only for the higher dose used. These data allow us to conclude that HHIF could play a role in the vascular hypertrophy that is associated with the pathogenesis of hypertension.