Quiescent Mesangial Cells Research Articles

High glucose activates PKC-ζ and NADPH oxidase through autocrine TGF-β1 signaling in mesangial cells

Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-beta(1) is central to the pathogenesis of diabetic nephropathy. Previously, we reported that mesangial cells respond to high glucose by generating reactive oxygen species (ROS) from NADPH oxidase dependent on protein kinase C (PKC) -zeta activation. We investigated the role of TGF-beta(1) in this action of high glucose on primary rat mesangial cells within 1-48 h. Both high glucose and exogenous TGF-beta(1) stimulated PKC-zeta kinase activity, as measured by an immune complex kinase assay and immunofluorescence confocal cellular imaging. In high glucose, Akt Ser473 phosphorylation appeared within 1 h and Smad2/3 nuclear translocation was prevented with neutralizing TGF-beta(1) antibodies. Neutralizing TGF-beta(1) antibodies, or a TGF-beta receptor kinase inhibitor (LY364947), or a phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC-zeta activation by high glucose. TGF-beta(1) also stimulated cellular membrane translocation of PKC-alpha, -beta(1), -delta, and -epsilon, similar to high glucose. High glucose and TGF-beta(1) enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-beta(1) antibodies, LY364947, or a specific PKC-zeta pseudosubstrate peptide inhibitor. Expression of constitutively active PKC-zeta in normal glucose caused upregulation of p22(phox), a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-beta(1) stimulation of PKC isozymes including PI3 kinase activation of PKC-zeta and consequent generation of ROS by NADPH oxidase.

Lovastatin inhibits oxidized low-density lipoprotein-induced plasminogen activator inhibitor and transforming growth factor-β1 expression via a decrease in Ras/extracellular signal-regulated kinase activity in mesangial cells

Oxidized low-density lipoprotein (Ox-LDL) might be involved in the progression of renal disease. Ox-LDL stimulation of plasminogen activator inhibitor-1 (PAI-1) expression via transforming growth factor-beta (TGF-beta)/Smad signaling in mesangial cells required activation of extracellular signal-regulated kinase (ERK). Mevalonate depletion by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, or statins, decreases the levels of farnesyl pyrophosphate (FPP) for isoprenylation of Ras. We postulate that statins may ameliorate the Ox-LDL-induced mesangial matrix accumulation by inhibiting Ras/ERK activation with subsequent downregulation of TGF-beta target genes. Quiescent mesangial cells were incubated for 18 h with and without the presence of lovastatin before 50 microg/mL of Ox-LDL treatment for 1 h. Lovastatin inhibited markedly the stimulatory effects of Ox-LDL on ERK1/2 activation, nuclear Smad3 expression, TGF-beta1 and PAI-1 mRNA and protein expression, and PAI-1 luciferase activity. These inhibitory effects of lovastatin were reversed almost completely by mevalonate or FPP. Similar to lovastatin, FTI-277, which is an inhibitor of Ras farnesylation, decreased the Ox-LDL-induced activation of ERK/Smad3 and induction of TGF-beta1/PAI-1. These results indicate that lovastatin prevents the Ox-LDL-induced Ras/ERK activation that results in inhibition of Smad3 activation in mesangial cells with subsequent downregulation of TGF-beta target genes. Thus, statins seem to have antifibrotic effects through their anti-TGF-beta response that are relevant in the treatment of chronic renal disease with dyslipidemia.

Transferrin up-regulates chemokine synthesis by human proximal tubular epithelial cells: Implication on mechanism of tubuloglomerular communication in glomerulopathic proteinura

The pathogenesis of glomerulosclerosis and tubulointerstitial fibrosis in proteinuric renal disease is obscure. We recently showed that transferrin, a key proteinuric component, mediates proximal tubular epithelial cell (PTEC) C3 synthesis. To further examine whether proteinuric tubular injury may induce glomerular inflammation and to characterize the role of transferrin in activating PTEC, glomerular mesangial cells (MC) were exposed to transferrin-activated PTEC culture supernatant and their proliferative and profibrotic responses analyzed. Human PTEC and MC were obtained by primary culture. Confluent, transferrin-stimulated PTEC were grown in serum-free medium to produce a "conditioned" medium that was incubated with quiescent MC. The proliferative response of MC was then assessed by thymidine uptake, and the expression of fibrogenic factors measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The chemokine profile in PTEC after transferrin treatment was examined by RT-PCR and ELISA. "Conditioned" supernatant from PTEC, which contained the highest amounts of platelet-derived growth factor (PDGF), stimulated MC proliferation compared with serum-free (P = 0.03) or transferrin-containing (P = 0.009) control media. This proliferative response was partially abrogated by treating MC with anti-PDGF. MC expression of PDGF, but not transforming growth factor-beta or intercellular cell adhesion molecule-1, was up-regulated by conditioned PTEC medium. Transferrin up-regulated monocyte chemoattractant peptide-1, interleukin-8, and macrophage migration inhibitory factor expression in a time- and dose-dependent fashion, but had no effect on RANTES expression by PTEC. These results provide experimental evidence suggesting that there is a tubuloglomerular "cross-talk" mechanism in the proteinuric state. PTEC-secreted PDGF, which further induces mesangial PDGF, could partially account for the mesangial proliferation frequently observed in proteinuric renal disease. Transferrin is one of the culprit nephrotic proteins leading to tubular overexpression of various proinflammatory chemokines, which may explain the interstitial changes observed in proteinuric states.

Elastic fiber proteins in the glomerular mesangium in vivo and in cell culture

Glomerular capillaries of the mammalian kidney are exposed to high intraluminal hydrostatic pressures and require elastic constraint to maintain size, shape, and integrity. Previous morphological and functional studies indicated that the extracellular matrices of glomeruli, that is, basement membrane and mesangial matrix, contribute to glomerular resilience and mechanical stability. Immunofluorescence microscopy findings demonstrated elastic fiber components to be located in the renal vasculature, including glomeruli. The aim of this study was to clarify the exact glomerular localization, composition, and cellular production of these proteins. We examined the renal distribution of the elastic fiber proteins fibrillin-1, emilin, microfibril-associated glycoproteins (MAGPs) 1 and 2, latent transforming growth factor-binding protein-1 (LTBP-1), and elastin using immunohistology and immunoelectron microscopy of human, rat, and mouse kidneys. In mesangial cell cultures, we also studied the expression and extracellular deposition of such proteins by use of Northern blotting and immunocytochemistry. Fibrillin-1, emilin, MAGPs 1 and 2, and LTBP-1 were present in glomeruli of mouse, rat, and human kidney, where they were located predominantly in the mesangial extracellular matrix underlying glomerular endothelium and basement membrane. Several of these proteins, as well as elastin, were also expressed in the renal vasculature. While elastin localized to the glomerular vascular pole in afferent and efferent arterioles extending to Bowman's capsule, it was not found in the glomerular capillary tuft. Cultured mesangial cells of rat, mouse, and human kidneys expressed mRNAs of fibrillin-1, emilin, MAGP-2, and elastin, and the respective proteins localized within and outside of mesangial cells, as shown by immunocytochemistry. mRNA expression of fibrillin-1, emilin, and elastin was strong in quiescent mesangial cells; their gene expression was further up-regulated by transforming growth factor-beta1, while it was transiently reduced when cells were exposed to mitogenic 10% fetal calf serum and platelet-derived growth factor. These findings demonstrate that specific elastic fiber proteins are produced and secreted by mesangial cells. This process is regulated by growth factors. Their abundance in the extracellular matrix of the mesangium is in keeping with the concept that elastic fiber proteins contribute to the mechanical stability and elastic strength of the glomerular capillary tuft.

Calcium Channel Blockers Inhibit Proliferation and Matrix Production in Rat Mesangial Cells: Possible Mechanism of Suppression of AP-1 and CREB Activities

Background: Calcium channel blockers (CCBs) are reported to attenuate the loss of renal function in various glomerulonephritides. Methods: To determine the mechanism of action of these drugs, we investigated the effects of CCBs on cell proliferation and extracellular matrix (ECM) production in cultured rat mesangial cells. Results: While stimulation with 5% fetal calf serum (FCS) increased [³H]thymidine and [³H]proline incorporation into quiescent mesangial cells, incubation with nifedipine and cilnidipine inhibited the increase in a dose-dependent manner. Northern blot analysis demonstrated that 5% FCS increased the expression of transforming growth factor beta (TGF-β) and fibronectin (FN) mRNA and that CCBs significantly reduced this induction, indicating that CCBs may reduce ECM production through inhibiting TGF-β and FN. Since activator protein 1 (AP-1) regulates cell proliferation and TGF-β expression, we evaluated the AP-1 activity by gel mobility shift analysis. Nuclear extracts of FCS-treated cells showed a strong binding to AP-1-specific oligonucleotides which was suppressed by CCBs, suggesting that these agents may inhibit cell proliferation by suppressing AP-1. CCBs also inhibited the binding activity of cyclic adenosine monophosphate responsive element binding protein which regulates FN gene expression. However, neither CCBs nor FCS affected the NFĸB activity. Conclusion: These results suggest that CCBs may, in part, inhibit the progression of glomerulonephritis through non-hemodynamic actions that include the suppression of mesangial cell proliferation and the production of ECM.

Cholesterol feeding activates macrophages to upregulate rat mesangial cell fibronectin production.

Cholesterol feeding has been shown to accelerate the development of glomerulosclerosis in many experimental renal diseases, possibly by promoting the infiltration of macrophages into the glomerulus. In order to assess whether hyperlipidaemia could directly modulate macrophage function to promote glomerulosclerosis, confluent quiescent mesangial cells were exposed to resident (r) or elicited (e) macrophages, from either control (C) or cholesterol-fed (HC) rats or the conditioned media derived from the various macrophage preparations. All macrophage preparations stimulated mesangial cell fibronectin accumulation over medium alone, but eHC macrophages stimulated significantly greater fibronectin levels. Similarly, all macrophage conditioned media (MPCM) stimulated mesangial cell fibronectin production over medium alone and again the effect was greatest with MPCM derived from eHC macrophages. Proliferation studies using [(3)H]thymidine incorporation demonstrated that all conditioned media, with the exception of rC, stimulated significant mesangial cell proliferation over control levels. TGF-beta and PDGF, pro-fibrogenic growth factors known to be associated with macrophage infiltration, could not be detected in the MPCMs per se. However, they were detected in the culture supernatants of mesangial cells exposed to MPCMs and again secretion was greatest from mesangial cells exposed to eHC-MCPM. Monocytes are systemically activated by high serum cholesterol levels so that following maturation to macrophages they elaborate soluble factors that can stimulate mesangial cell fibronectin production, cell proliferation, and growth factor secretion. Hypercholesterolaemia may therefore accelerate glomerulosclerosis not only by increasing macrophage number, but also by upregulating the ability of macrophages to induce pro-sclerotic responses in glomerular mesangial cells.

Radiation-Induced Alterations in Rat Mesangial Cell Tgfb1 and Tgfb3 Gene Expression Are Not Associated with Altered Secretion of Active Tgfb Isoforms

Despite evidence of selective radiation-induced modulation of expression of rat mesangial cell Tgfb gene isoforms, it is unclear whether these changes in gene expression are accompanied by changes in protein secretion. To address this issue, primary cultures of rat mesangial cells (passage number 6- 11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy of (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 24 h. Irradiation of quiescent mesangial cells resulted in a significant (P </= 0.05) dose-independent increase in steady-state levels of Tgfb1 mRNA 24 h postirradiation. In contrast, steady-state levels of Tgfb3 mRNA exhibited a dose-dependent reduction after irradiation; this reduction was statistically significant after doses of 5 and 10 Gy compared to control cells (P </= 0.05). These radiation-induced changes in Tgfb gene expression were associated with modest increases in Tgfb protein as determined using mink lung epithelial cells transfected with the Pai1 promoter-luciferase construct. Twenty-four hours after a single dose of 5 Gy, the total Tgfb protein secreted by the mesangial cells was 181 +/- 2.0% of that secreted by unirradiated control cells (P </= 0.01). However, this increase was seen in terms of latent Tgfb protein; radiation failed to increase significantly the amount of active Tgfb protein secreted by mesangial cells. Both quiescent and irradiated rat mesangial cells secreted active Tgfb as primarily the Tgfb3 isoform. These data reinforce the need to interpret changes in Tgfb gene expression with caution.

Irradiation of Rat Mesangial Cells Alters the Expression of Gene Products Associated with the Development of Renal Fibrosis

To determine the ability of radiation to modulate mesangial cell expression of various molecules involved in promoting extracellular matrix (ECM) accumulation [fibronectin, plasminogen activator-inhibitor 1 (Pai1), and tissue inhibitor of metalloproteinase-2 (Timp2)] and degradation (Tgfb, plasminogen activators u-PA or t-PA, matrix metalloproteinases Mmp2 and Mmp9), primary cultures of rat mesangial cells (passage number 6-11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 48 h. Irradiation of quiescent mesangial cells resulted in significant (P < 0.05) time- and dose-dependent increases in Fn and Pai1 mRNA and/or immunoreactive protein. Despite an increase in Tgfb1 mRNA, there was little evidence for an increase in total Tgfb protein. Indeed, active levels remained unaltered after irradiation. Irradiation led to differential changes in MMP expression; active Mmp2 levels increased, while Mmp9 levels appeared unaltered. In addition, secretion of plasminogen activators into the medium was unchanged after irradiation, while secretion of Timp2 increased. We conclude that irradiating mesangial cells leads to altered production of various molecules involved in accumulation and degradation of extracellular matrix.

Angiotensin II-Induced Modulation of Rat Mesangial Cell Phenotype

Inhibition of angiotensin II (AII) can ameliorate the severity of experimental radiation nephropathy. To determine the ability of AII to modulate mesangial cell phenotype, primary cultures of rat mesangial cells (passage number 6-11) were placed in serum-free medium 24 h prior to addition of AII (10(-9)-10(-5) M); control cells received serum-free medium alone. Cells were maintained in serum-free medium for a further 48 h. Addition of AII to quiescent mesangial cells resulted in significant (P < 0.05) time- and/or dose-dependent increases in Fn and Pail mRNA and/or immunoreactive protein. No significant change was observed in terms of Tgfb1 mRNA. A significant increase in total Tgfb1 protein (P < 0.01) secreted by AII-treated mesangial cells was noted; however, this increase was primarily in terms of latent TGF-beta; the relative proportion of active TGF-beta secreted decreased after AII incubation. AII had no effect on the activity of Mmp2 or Mmp9. However, AII-treated mesangial cells did show an increase in the amount of tissue inhibitor of metalloproteinase-2 (Timp2) immunoreactive protein secreted into the medium. The AII-mediated increase in Pail mRNA levels appeared due in part to activation of the AT1 receptor and was independent of TGF-beta; co-incubation with TGF-beta-neutralizing antibody failed to inhibit the AII-mediated increase in Pail mRNA. Thus mesangial cells treated with AII exhibit a pro-fibrosis phenotype.

Inhibitory effects of the herbal medicine Saiboku‐to on the proliferation of cultured murine mesangial cells

SUMMARY: Patients with certain types of glomerulonephritis often present with upper respiratory tract infection (URTI). In this in vitro study, we have examined several Japanese herbal medicines, Shosaiko‐to (TJ‐9), Saiboku‐to (TJ‐96), Sairei‐to (TJ‐114) and Ryokankyomi‐singenin‐to (TJ‐119), which are occasionally used for collagen diseases, chronic inflammatory diseases or renal diseases. In these drugs, TJ‐96 is used to treat bronchial asthma or URTI, which has anti‐inflammatory effects. In this study, we demonstrate that TJ‐9, TJ‐96 or TJ‐114 inhibits the proliferation of murine mesangial cells induced by platelet derived growth factor (PDGF) or tumour necrosis factor (TNF)‐α. Quiescent mesangial cells were stimulated with 10 ng/mL of PDGF or 100 U/mL of TNF‐α together with various concentrations of the medicines for 20 h. After incubation with 50μ/mL of TJ‐9, TJ‐96 or TJ‐114, 3H‐thymidine incorporation induced by PDGF was reduced to 26, 19 and 29% of the control; and 66, 56 and 75% on TNF‐α stimulation, respectively. TJ‐96 had a more intense inhibitory effect than TJ‐9 or TJ‐114. TJ‐119 had no significant inhibitory effect at the same dose. Northern blot analysis for PDGF‐B transcript after addition of TJ‐96 revealed a 24% reduction in PDGF‐B mRNA levels stimulated by recombinant PDGF‐BB, suggesting inhibitory mechanisms of autocrine production of PDGF. No cytotoxic effect against mesangial cells in culture was observed up to a concentration of 100 μg/mL of TJ‐9, TJ‐96 or TJ‐114 by lactic dehydrogenase release assay. By another cell viability assay, after medium with 50 μg/mL of TJ‐96 was replaced with only PDGF‐BB‐containing medium, 3H‐thymidine incorporation was found to exceed the control, suggesting rebound proliferation. the present findings suggest that administration of TJ‐96 is beneficial for patients with mesangioproliferative glomerulonephritis accompanied by URTI, serving to regulate the effect on mesangial cell proliferation.

Inactivation of kinase cascades in mesangial cells grown on collagen type I.

Growth on collagen type I gels is known to suppress the mitogenic responsiveness of mesangial cells. Because these cells proliferate in some renal diseases and themselves synthesize collagen type I, we examined the influence of growth on collagen upon several kinase signaling cascades involved in mesangial cell proliferation. Quiescent mesangial cells grown on collagen type I and then stimulated with serum showed a markedly diminished induction of the protooncogene c-fos, compared with their counterparts on plastic or fibronectin. This effect was accompanied by decreased activation of mitogen-activated (Erk family) and Ca2+/calmodulin-dependent protein kinases. Cells on collagen showed lower basal protein kinase C (PKC) activity and diminished levels of PKC-alpha and -zeta isoforms. Global phosphorylation of tyrosine residues was diminished on collagen, and tyrosine phosphorylation of Erk and focal adhesion kinase in response to serum was not detected, in contrast to cells on plastic. We conclude that attachment of mesangial cells to collagen type I results in a broad suppression of protein phosphorylation that is reflected in diminished induction of the c-fos gene and probably underlies the conversion of cultured mesangial cells to a nonproliferative phenotype.

Association and direct activation of signal transducer and activator of transcription1alpha by platelet-derived growth factor receptor.

PDGF stimulates tyrosine phosphorylation of Janus kinase 1 (JAK1) and the signal transducer and activator of transcription 1 (STAT1alpha). However, it is not known whether JAKs are required for STAT1alpha phosphorylation or if the PDGF receptor itself can directly tyrosine phosphorylate and activate STAT1alpha. In vitro immunecomplex kinase assay of PDGF beta receptor (PDGFR) or STAT1alpha immunoprecipitates from lysates of mesangial cells treated with PDGF showed phosphorylation of a 91- and an 185-kD protein. Incubation of lysates prepared from quiescent mesangial cells with purified PDGFR resulted in STAT1alpha activation. Immunodepletion of Janus kinases from the cell lysate before incubation with the purified PDGFR showed no effect on STAT1alpha activation. Moreover, lysates from mesangial cells treated with JAK2 inhibitor, retained significant STAT1alpha activity. To confirm that STAT1alpha is a substrate for PDGFR, STAT1alpha protein was prepared by in vitro transcription and translation. The addition of purified PDGFR to the translated STAT1alpha resulted in its phosphorylation. This in vitro phosphorylated and activated protein also forms a specific protein-DNA complex. Dimerization of the translated STAT1alpha protein was also required for its DNA binding. Incubation of pure STAT1alpha with autophosphorylated PDGFR resulted in physical association of the two proteins. These data indicate that activated PDGFR may be sufficient to tyrosine phosphorylate and thus directly activate STAT1alpha.

Microlocalization and effects of adrenomedullin in nephron segments and in mesangial cells of the rat.

We examined microlocalization of mRNA coding for adrenomedullin (AM), using reverse transcription-polymerase chain reaction (RT-PCR), and the effects of AM on adenosine 3',5'-cyclic monophosphate (cAMP) generation and water transport in microdissected rat nephron segments. We also examined intraglomerular site of the expression of AM and AM-stimulated cAMP generation in cultured rat mesangial cells (MC). RT-PCR demonstrated the signals for AM mRNA in glomerulus (Glm), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD) but not in proximal convoluted tubule (PCT) or medullary thick ascending limb (MTAL). AM (10(-7) M) stimulated cAMP generation in Glm >> CCD = IMCD > OMCD but not in PCT or MTAL, which corresponded to the results of the expression of AM mRNA. AM (10(-8) M) slightly increased osmotic water permeability by 24% in perfused terminal IMCD. Northern blot analysis revealed high expression of AM mRNA in MC. AM (10(-7) M) stimulated cAMP generation in MC both in the presence and absence of fetal calf serum, suggesting that AM-dependent cAMP generation was evident both in cycling MC and in quiescent MC. AM may work as a diuretic peptide mainly by increasing glomerular filtration rate via cAMP in MC.

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.

Group II phospholipase A 2 as an autocrine growth factor mediating interleukin-1 action on mesangial cells

The proliferation of mesangial cells plays a central role in the progression of glomerulonephritis. We studied the role of group II phospholipase A 2 in interleukin-1-stimulated proliferation of mesangial cells. Cultured rat mesangial cells secreted 5.3 units group II phospholipase A 2/24 h per 10 5 cells in response to stimulation of 200 U/ml of interleukin-1. Northern hybridization analysis showed that mRNA for group II phospholipase A 2 was induced by exogenously added group II phospholipase A 2 (15 U/ml) as well as interleukin-1. The pretreatment of quiescent mesangial cells with interleukin-1 augmented [ 3H]thymidine incorporation caused by platelet derived growth factor. Exogenous group II phospholipase A 2 (5–36 U/ml) purified homogeneously from rat spleen also increased [ 3H]thymidine incorporation by platelet derived growth factor-stimulated mesangial cells in a dose dependent manner (36 U/ml phospholipase A 2; 1.9-fold). The stimulatory effect of interleukin-1 on DNA synthesis of mesangial cells was specifically blunted by immunoglobulin raised against group II phospholipase A 2. Group II phospholipase A 2 (16 U/ml) amplified a platelet derived growth factor-stimulated increase in the mesangial cell number by 1.5-fold. Among the products of the phospholipase A 2-catalyzed reaction, lysophospholipids including lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidic acid, but not fatty acids, mimicked the stimulatory effect of interleukin-1 and phospholipase A 2. These results suggest that group II phospholipase A 2 acts as a signaling molecule that mediates interleukin-1-induced growth of rat mesangial cells through yielding lysophospholipids.

Heparin Inhibits Mitogen-activated Protein Kinase-dependent and -independent c-fos Induction in Mesangial Cells

Heparin suppresses mitogenic responses in renal mesangial cells, and when quiescent mesangial cells are stimulated with serum, heparin blocks the induction of c-fos seen at 15 min. Because heparin is taken up by cells over a much longer time course, we addressed mechanisms whereby extracellular heparin might suppress c-fos induction at such early times. Quiescent cells were treated with serum, 12-O-tetradecanoylphorbol-13-acetate, or low concentrations of Ca2+ ionophores that produced increases in intracellular Ca2+ concentration ([Ca2+]i) in the physiological range. Each treatment caused an increase in c-fos mRNA, but they did so by different mechanisms. Serum activated mitogen-activated protein kinase (MAPK) and increased [Ca2+]i without affecting protein kinase C. Activation of protein kinase C with phorbol ester activated MAPK without much effect on [Ca2+]i. Ionophores increased [Ca2+]i without affecting basal levels of protein kinase C or MAPK. Heparin (1 microg/ml) suppressed the induction of c-fos initiated by all three treatments. It did not affect the activity of protein kinase C, but inhibited activation of MAPK by either serum or phorbol ester, suggesting a common site of action at or below the probable convergence of the induced signals at Ras/Raf-1 activation. Heparin also inhibited the serum-stimulated entry of extracellular Ca2+ to the same extent as verapamil, consistent with the ability of verapamil to block L-type Ca2+ channels and the known presence of these channels in mesangial cells. However, this effect does not appear to be related to heparin's ability to inhibit induction of c-fos. First, verapamil had no effect on induction of c-fos by serum. Second, heparin had no effect on changes in [Ca2+]i achieved by ionophores. We conclude that heparin suppresses induction of c-fos in mesangial cells by blocking at least two different points in signal transduction cascades, one upstream of MAPK and the other independent of MAPK, but dependent on intracellular Ca2+.

Adrenomedullin Suppresses Mitogenesis in Rat Mesangial Cells via cAMP Pathway

Adrenomedullin (ADM) is a vasoactive peptide that was recently localized in renal glomeruli. In the present study we explored whether ADM stimulates cAMP system in glomerular mesangial cells (MC) and whether it can via "negative-crosstalk" inhibit the mitogen-activated protein kinase (MAPK) and thus suppress proliferation of MC. We found that ADM elicited accumulation of cAMP and in situ activation of protein kinase A (PKA) in cultured MC. Addition of 1 nM ADM to incubation media inhibited the proliferation in both quiescent MC and cells maximally stimulated by PDGF and also decreased the activation of MAPK induced by PDGF. These results indicate that ADM can suppress MC mitogenesis and suggest that it may function as an endogenous paracrine supressor of MC proliferation.

Gentamicin activates rat mesangial cells. A role for platelet activating factor

Gentamicin-induced decreases in glomerular filtration rate have been associated with a marked decline in the glomerular capillary ultrafiltration coefficient which could be mediated by mesangial cell contraction or release of vasoactive hormones. We studied the effect of gentamicin on mesangial cells proliferation, contraction and Ca2+ mobilization. Moreover, we attempted to assess a possible role of platelet activating factor (PAF) as a mediator of the observed effects of gentamicin on mesangial cells. Gentamicin induced a reduction of planar surface area of cultured rat mesangial cells that was blunted by the PAF-antagonist, BN-52021. Gentamicin induced an increase in [Ca2+]i that was inhibited by BN-52021. Gentamicin also stimulated [3H]thymidine incorporation into DNA, an effect that was also reduced by BN-52021, and by other two structurally different PAF receptor antagonists: alprazolam and BB-823. Gentamicin induced c-fos mRNA expression in quiescent mesangial cells. Gentamicin stimulated the synthesis and release of PAF in cultured rat mesangial cells. The present studies demonstrate that gentamicin activates mesangial cell function. These actions seem to be mediated, at least in part, by PAF synthesis and release.

Eicosanoid-induced growth and signaling events in rat glomerular mesangial cells

Renal glomerular injury frequently results in proliferation of a specialized supporting cell of the glomerular capillary known as the mesangial cell. In various forms of renal injury there is enhanced glomerular synthesis of specific eicosanoids of the arachidonic cyclooxygenase and lipoxygenase pathways including prostaglandin (PG) F 2α, thromboxane (Tx) A 2, the hydroxyeicosatetraenoic acids 12-HETE and 5-HETE, and the leukotrienes LTB 4 and LTD 4 and attempts have been made to link these eicosanoids with injury-induced mesangial cell growth. In this study, the growth promoting effect of these eicosanoids on glomerular mesangial cells was correlated with activation of two growth regulatory enzymes: phospholipase C (PLC) and protein kinase C (PKC). PGF 2α, and TxA 2 endoperoxide analog U-46619, and LTD 4 significantly enhanced DNA synthesis ((as assessed by [ 3H]thymidine (TdR) incorporation)] in relatively quiescent (0.5% serum) mesangial cells, activated PLC [as assessed by increased 1,4,5-inositol tris-phosphate (IP 3) generation and diacylglycerol (DAG) synthesis], and activated PKC (as assessed by translocation of the enzyme activity from the cytosol to the membrane). The effect of PGF 2α on IP 3 generation was not blocked by the TxA 2 receptor antagonist, SQ-29,548. PGF 2α was the most effective eicosanoid in inducing all three events, and concentrations that enhanced TdR incorporation (1 μM) also activated PLC and PKC. In contrast, concentrations of U-46619 and LTD 4 which enhanced TdR incorporation (1 μM), also activated PLC, but were insufficient to also activate PKC. Our observations indicate that the growth-promoting effect of PGF 2α, U-46619, and LTD 4 can best be correlated with LPC activation. In addition, PGF 2α does not mediate PLC activation through binding to the TxA 2 receptor.

MAP kinase cascade in cultured rat mesangial cells

In order to evaluate the intracellular signaling pathway of endothelin 1 (ET-1), we examined mitogen-activated protein kinase (MAPK) cascade in cultured rat glomerular mesangial cells. Treatment of quiescent mesangial cells with ET-1 increased kinase activities toward bovine myelin basic protein (MBP). Maximal activation was at 10 min and ED50 was about 5 nM. The 44- and 42-kDa kinases were activated in MBP containing gel kinase assay. These kinases were identified as extracellular signal regulated kinase (ERK) 1 and ERK2 with immunoblotting, respectively. MAPK or ERK kinase (MEK), one of the MAPK kinases, was present in rat mesangial cells and ET-1 also activated this MAPK kinase. These results indicate that MAPK kinase and MAP kinase are rapidly activated by ET-1 and may modulate cellular functions in rat mesangial cells.