DNA Synthesis In Mesangial Cells Research Articles

Inhibitory effect of Perilla frutescens and its phenolic constituents on cultured murine mesangial cell proliferation.

The inhibitory effects of Perilla frutescens and its phenolic constituents on cytokine-induced proliferation of murine cultured mesangial cells were investigated. DNA synthesis of mesangial cells stimulated by platelet derived growth factor (PDGF, 10 ng/ml) or tumor necrosis factor (TNF)-alpha (100 U/ml) was inhibited by the extract of P. frutescens (IC50 values, 3.3 and 1.4 micrograms/ml, respectively). The strength of the anti-proliferative activity was nearly equal in various chemotypes of P. frutescens. Caffeic acid, methyl caffeate, rosmarinic acid, and luteolin 7-O-glucuronide-6"-methyl ester were isolated as active constituents from the extract of the typical strain of P. frutescens, and their IC50 values for PDGF-induced mesangial cell proliferation were estimated as 26 microM, 2.6 microM, 1.8 microM, and 4.1 microM, respectively. We also compared the activities of related flavonoids previously isolated from P. frutescens, and luteolin had the highest anti-proliferative activity.

Atherogenic lipoproteins enhance mesangial cell expression of platelet-derived growth factor: Role of protein tyrosine kinase and cyclic amp-dependent protein kinase A

Mesangial cell proliferation and extracellular matrix accumulation are fundamental in the pathogenesis of glomerulosclerosis. Platelet-derived growth factor (PDGF) is a major cytokine involved in mesangial cell proliferation, and its increased expression is seen in glomerular injury. Atherogenic lipoproteins stimulate mesangial cell proliferation and induce glomerular injury in experimental animals. We examined the effect of low-density lipoprotein (LDL) and its more atherogenic oxidized forms, minimally modified LDL (mm-LDL) and oxidized LDL (ox-LDL) on mesangial cell PDGF mRNA expression. Incubation with 2.5 to 25 μg/ml LDL or mm-LDL for 1 to 4 hours stimulated mesangial cell PDGF mRNA expression (mm-LDL 2 to 3 times greater than LDL); ox-LDL had no effect. Similarly, both LDL and mm-LDL induced mesangial cell DNA synthesis (mm-LDL 1.5 to 2 times greater). In further studies evaluating key associated intracellular signal transduction mechanisms, the protein tyrosine kinase (PTK) inhibitors herbimycin and genistein markedly decreased basal and lipoprotein-induced PDGF mRNA expression. Both pertussis toxin and isoproterenol, cyclic AMP-generating substances, stimulated PDGF mRNA expression. Preincubation with H-8 or H-89, cyclic AMP-dependent protein kinase A (PKA) inhibitors, blocked the lipoprotein-induced PDGF message, whereas preincubation with calphostin C, a protein kinase C inhibitor, did not alter LDL- or mm-LDL-mediated PDGF mRNA expression. These data suggest that the accumulation of atherogenic lipoproteins and their endogenous oxidized forms within the glomerulus may regulate mesangial cell PDGF expression and related cellular responses. These events appear to be modulated by signal transduction pathways involving PTK and PKA.

HDL causes mesangial cell mitogenesis through a tyrosine kinase-dependent receptor mechanism.

Hypercholesterolemia and mesangial cell proliferation have been proposed to play a role in the progression of glomerulosclerosis in diabetic nephropathy and other renal diseases. Although LDL is mitogenic for and cytotoxic to mesangial cells, the effect of HDL on these cells is unknown. HDL stimulates fibroblast mitogenesis and is the principal cholesterol-bearing lipoprotein in the rat, the experimental model for studying the effect of hyperlipidemia on renal disease. Insulin is mitogenic in several cell systems, and its levels are increased in serum in non-insulin-dependent diabetes mellitus. This study investigates whether HDL acts as a growth factor in mesangial cells and whether it functions in parallel with insulin. It was found that HDL at protein concentrations between 10 and 500 microg/ml, both alone and in the presence of 100 nM insulin, increased DNA synthesis in mesangial cells (129 to 165% of control for HDL alone; 140 to 235% for HDL plus insulin), whereas HDL at 1000 microg/ml and greater inhibited mesangial cell proliferation. Insulin alone at 100 nM stimulated [3H]thymidine incorporation in the same cell system (145% of control); the mitogenic effect of insulin was additive to that of HDL. Purified apo A-I had a similar effect, but at significantly lower concentrations. Specific binding of HDL to mesangial cells was demonstrated (B(max) [binding constant] of 5.19 +/- 0.70 x 10(-7) micromol of HDL bound/mg cell protein and K(b) of 2.83 +/- 0.22 nM). Tetranitromethane alters apo A-I, preventing binding to its cognate receptor. Tetranitromethane-modified HDL did not bind to mesangial cells and had no effect on [3H]thymidine incorporation. Addition of HDL to mesangial cells caused an immediate transient increase in free intracellular calcium in several representative mesangial cells, similar to the response seen with platelet-derived growth factor. The mitogenic effect of HDL was not altered after attenuation of cellular protein kinase C activity, but the stimulatory effect of HDL alone and in combination with insulin on DNA synthesis was completely eliminated after inhibition of cellular tyrosine kinases by 24-h pretreatment with 0.25 microM herbimycin A. Thus, HDL binds to a specific apo A-I-dependent receptor, promotes DNA synthesis, and initiates second-messenger events by a tyrosine kinase-dependent and protein kinase C-independent mechanism.

Angiotensin II requires PDGF-BB to induce DNA synthesis in rat mesangial cells cultured in an exogenous insulin-free medium.

Mesangial cell proliferation is an important feature of chronic glomerular disease. Different cytokines and vasoactive substances have been implicated in mesangial cell proliferation. However, the role of angiotensin II remains controversial. Furthermore, all the studies made to date have been performed using a medium supplemented with high concentrations of insulin (5 micrograms/ml) in order to facilitate mesangial cell proliferation in vitro. As has recently been reported, insulin causes a change both in extracellular matrix composition and in mesangial cell phenotype, thus mimicking disease conditions. We examined the effects of Ang II in synchronized and proliferating mesangial cells with and without growth factor (PDGF-BB, bFGF and/or insulin) supplementation and the effects of exogenous administration of insulin on mesangial proliferation by measuring the bromodeoxyuridine (BrdU) uptake. Angiotensin II itself caused no increase in BrdU incorporation. However, it exerted a significant synergistic effect on PDGF-BB-induced DNA synthesis (P < 0.05) in quiescent medium and tended to stimulate BrdU uptake by proliferating cells (P = 0.09). BrdU incorporation significantly correlated with direct cell count (r = 0.95). PDGF-BB had the maximal stimulatory effect on DNA synthesis both in quiescent and in proliferative culture conditions. The insulin dose (5 micrograms/ml) which has been shown to cause mesangial cell proliferation in vitro, caused an increase in BrdU incorporation by itself in quiescent medium. We conclude that in an insulin-free medium, mimicking in vivo glomerular conditions, PDGF-BB or proliferative medium are needed to allow Ang II-induced DNA synthesis in mesangial cell culture.

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.

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.

Modifying influence of enalaprilat on mesangial cell DNA synthesis induced by hydrogen peroxide

This study examined the effect of the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, on mesangial cell (MC) DNA synthesis induced by H 2O 2, IL-6 and PDGF. MC were incubated with enalaprilat (2.5–100 μmol/l) alone and together with combinations of H 2O 2 (3 daily pulses of 10 −6 mol/l), IL-6 (5 ng/ml) and PDGF (10 ng/ml). DNA synthesis was assessed after 72 h using [ 3H]thymidine ( 3H-TdR) incorporation. Enalaprilat alone had no effect on MC DNA synthesis. Stimulation of MC by H 2O 2, PDGF and IL-6 alone resulted in increases in 3H-TdR of 4936.6 ± 1147.5, 5640.5 ± 1537.6 and 4413.5 ± 998.4 cpm, respectively ( P < 0.05 above control). Only 2.5 μmol/l enalaprilat effected a significant reduction in IL-6 and PDGF-induced DNA synthesis. Incubation of MC with H 2O 2 + PDGF or H 2O 2 + IL-6 resulted in increases of 3H-TdR of 6471.9 ± 1785.1 and 5507.2 ± 1270 cpm, respectively ( P < 0.05 above control). Addition of enalaprilat with either H 2O 2 + PDGF or H 2O 2 + IL-6 effected significant reductions in DNA synthesis over the range 2.5–100 μmol/l. These data demonstrate that ACE inhibitors modulate MC DNA synthesis induced by reactive oxygen species.

Modulation by angiotensin II of endothelial cell control of DNA synthesis in human mesangial cells.

To determine whether angiotensin II (Ang II) may modulate the control by endothelial cells of DNA synthesis in mesangial cells, we cocultured human umbilical vein endothelial cells (HUVEC) and human mesangial cells. HUVEC released endothelin-1 (ET-1), and prostaglandin I(2) (PGI(2)). Ang II stimulated the production of PGI(2), but did not modify that of ET-1. DNA synthesis in mesangial cells estimated by [3H]-thymidine incorporation was increased when mesangial cells and endothelial cells were cocultured. The mitogenic effect of endothelial cells in coculture resulted from ET-1 production since it was suppressed by bosentan, a mixed specific ET-1 receptor antagonist. The effects of Ang II on mesangial cell proliferation varied according to the protocol. Under control conditions, Ang II was inactive. In the presence of HUVEC-conditioned medium, Ang II exerted a mitogenic effect. By contrast, Ang II inhibited DNA synthesis by mesangial cells in the experiments of coculture. The latter effect could be attributed to HUVEC-released PGI(2) via the increase of cyclic AMP in mesangial cells since it was abolished by indomethacin, a cyclooxygenase inhibitor. In conclusion, endothelial cells may stimulate DNA synthesis in mesangial cells via ET-1 production and Ang II inhibits this effect by stimulating PGI(2) production in endothelial cells. In contrast, Ang II reinforces the mitogenic effect of the endothelial cell-conditioned medium when it acts directly on mesangial cells.

Interaction of native and oxidized lipoprotein(a) with human mesangial cells and matrix

The trapping of apolipoprotein(a) and apolipoprotein B-100 in glomeruli of patients with the nephrotic syndrome seems to be linked to a less favorable course of renal disease. To evaluate the potential role of lipoprotein(a) as a mediator of glomerular injury, we measured uptake of native lipoprotein(a) [Lp(a)] and oxidatively modified Lp(a) by cultured human mesangial cells and matrix and studied the effects of Lp(a) on mesangial cell DNA-synthesis and cellular proliferation. Uptake of Lp(a) by mesangial cells occurred at a significantly lower affinity (KD 16 micrograms/ml vs. 39 micrograms/ml) and a lower maximum degradative capacity (6.7-fold) than for LDL. Specificity of receptor mediated uptake was 50% for Lp(a) compared to 84% for LDL. Oxidative modification of both Lp(a) and LDL was accompanied by a significant decrease in uptake and degradative capacities. Due to the limited uptake, native and oxidatively modified Lp(a) had only marginal effects on intracellular cholesterol metabolism, which was measured as inhibition of sterol synthesis and stimulation of cholesterol esterification. However, binding of Lp(a), oxidized Lp(a) and oxidized LDL to extracellular mesangial matrix was enhanced compared to LDL. Furthermore, incubation of mesangial cells with Lp(a) and oxLp(a) in concentrations of 2.5 micrograms/ml and higher resulted in a decrease of DNA synthesis. Regardless of the oxidative status, a maximal suppression of DNA synthesis was observed at 20 micrograms/ml Lp(a). Native Lp(a) also blunted the stimulatory effects of PDGF on mesangial cell DNA-synthesis. Lp(a) did not alter basal TGF-beta transcription in human mesangial cells. The avid interaction of Lp(a) and modified lipoproteins with mesangial matrix provides a concept for the enhanced entrapment of these lipoproteins in the diseased glomerulum. Native Lp(a) is a poor ligand for the LDL receptor; oxidation of Lp(a) even lowers the affinity towards this receptor. Further studies must be carried out to clarify the pathophysiological significance of Lp(a) trapping in the mesangial matrix.

Nicardipine hydrochloride suppresses DNA synthesis in human mesangial cells stimulated with recombinant human (rh) platelet-derived growth factor AA, rh interleukin-1 alpha, or rh tumor necrosis factor-alpha.

In order to define the role of cytokines in mesangial cell pathophysiology, we measured the mitogenic activity of recombinant human platelet-derived growth factor AA (rhPDGF-AA), rh interleukin-1 alpha (rhIL-1 alpha) and rh tumor necrosis factor-alpha (rhTNF-alpha) in cultured human mesangial cells, and investigated the effect of the calcium channel blocker nicardipine hydrochloride on their cell mitogenic activity. DNA synthesis in mesangial cells, stimulated by rhPDGF-AA, rhIL-1 alpha or rhTNF-alpha, was measured using [3H]TdR up take and similar investigations, with nicardipine hydrochloride added to the above, were conducted. The results showed DNA synthesis in cultured human mesangial cells were stimulated by rhPDGF-AA, rhIL-1 alpha and rhTNF-alpha, and this effect was reduced by the addition of nicardipine hydrochloride. Since rhPDGF-AA, rhIL-1 alpha and rhTNF-alpha are released by the inflammatory cells which infiltrate glomeruli, these cytokines may be involved in the mechanisms of mesangial cell proliferation observed in immune-mediated mesangial proliferative glomerulonephritis. Nicardipine hydrochloride reduced DNA synthesis in cultured human mesangial cells in response to these cytokines, suggesting possible application in medical therapy for immune-mediated mesangial proliferative glomerulonephritis.

Mesangial cell DNA synthesis induced by hydrogen peroxide, interleukin-6, and platelet-derived growth factor: effects of indomethacin and dazmegrel.

This study indirectly examined the role of prostanoids (PG) in mediating rat mesangial cell (MC) DNA synthesis induced by hydrogen peroxide (H2O2), interleukin 6 (IL-6), and platelet-derived growth factor (PDGF). MC were exposed to three daily pulses of 10(-6) mol/l H2O2 alone or in combination with IL-6 (5 ng/ml) or PDGF (10 ng/ml). In order to examine (indirectly) the role of PG in mediating changes in MC DNA synthesis, indomethacin (1.5 x 10(-5) mol/l) or the thromboxane A2 synthetase inhibitor Dazmegrel (10(-5) mol/l) was added to the medium and DNA synthesis assessed after 72 h using 3H-thymidine incorporation (3H-TdR). Stimulation of MC by H2O2 alone resulted in an increase in 3H-TdR of 34.7 +/- 5.5% (p < 0.01). H2O2 enhanced the mitogenic effects of IL-6 and PDGF, 3H-TdR increasing by 52 +/- 12.1% (p < 0.01) and 100 +/- 21% (p < 0.001), respectively. Indomethacin suppressed the DNA synthesis induced by H2O2 alone, 3H-TdR decreasing by 33 +/- 12% (p < 0.05). Indomethacin also reduced the mitogenic response to H2O2 plus IL-6 and H2O2 plus PDGF by 91 +/- 17 and 97 +/- 12%, respectively (p < 0.05). Dazmegrel reduced 3H-TdR when MC were exposed to H2O2 alone by 31.8 +/- 16% (p < 0.05) and when combined with IL-6 or PDGF by 80 +/- 26 and 120 +/- 13%, respectively (p < 0.05). These data suggest that the pathways through which H2O2-induced growth of MC is mediated appear, at least in part, to involve PG, particularly thromboxane A2.

Extracellular ATP augments mesangial cell growth induced by multiple growth factors

ATP is released into the extracellular milieu during tissue injury and platelet aggregation and has a modest mitogenic effect on cultured rat glomerular mesangial cells (MCs). In this study we investigated the interaction of ATP with multiple growth factors during MC mitogenesis. Replication of MCs was determined by direct cell counting and DNA synthesis was measured by 3H-thymidine uptake. Activity of phosphatidylinositol-specific phospholipase C (PI-PLC) was assessed by measuring formation of inositol phosphates from 3H-labelled precursors. Extracellular ATP (1-100 microM) exerted a powerful synergistic effect on DNA synthesis of MCs when used simultaneously with various mitogens, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), endothelin-1 (ET-1), arginine vasopressin (AVP), thrombin (Thr), serotonin (5-HT), and interleukin-1 beta (IL-1 beta). ATP synergized with these factors by increasing the maximum of DNA synthesis, without changing the half-maximal concentration of the growth factor. In addition cell counts revealed that ATP significantly augments MC growth induced by EGF, bFGF, PDGF-BB, ET-1, AVP, Thr, 5-HT, and IL-1 beta. ATP caused activation of PI-PLC, but did not synergize with any of the tested growth factors in this respect. ATP-induced PI-PLC activation was inhibited by preincubation with pertussis toxin by 40-93%. This treatment did not suppress DNA synthesis induced by ATP alone, by PDGF, or by PDGF plus ATP. The calcium antagonists, TMB-8 and verapamil, as well as the inhibitors of protein kinase C, calphostin C and H7, had no effect on DNA synthesis induced by ATP or PDGF plus ATP. Also, ATP synergistically stimulated DNA synthesis in combination with the direct activator of protein kinase C, phorbol myristate acetate (PMA), and with the reaction product of phospholipase D, phosphatidic acid. Finally, ATP was mitogenic in an MC line of higher passage which did not respond with PI-PLC activation. Extracellular ATP synergistically augments MC growth induced by multiple growth factors. While the ATP-induced mitogenic signal is presently unclear, our observations support the concept that ATP may play a role during the course of glomerulonephritis when multiple growth factors are released from glomerular and inflammatory cells.

P2U-purinergic receptor activation mediates inhibition of cAMP accumulation in cultured renal mesangial cells.

Extracellular ATP has been reported to exert mitogenic and contractile effects on cultured renal mesangial cells (MCs). Since it is possible that these actions involve changes in the cAMP second messenger system, we examined the effect of extracellular nucleotides on the accumulation of cAMP in rat MCs. ATP, UTP and adenosine 5'-0-(3-thio)triphosphate (ATP gamma S) (100 microM) had no significant effects on baseline cAMP levels, but inhibited forskolin-stimulated accumulation of cAMP by 21-75% in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Maximal inhibitory effects were observed at 100 microM of ATP gamma S with a threshold dose of 1 microM. ATP gamma S, ATP and UTP were the most potent inhibitors indicating stimulation of the P2u receptor. The P2x agonists adenosine 5'-(alpha, beta-methylene) triphosphate and adenosine 5'-(beta, gamma-methylene) triphosphate, and the P2y agonist 2-methylthio-ATP did not affect cAMP accumulation. Treatment with the P2 receptor antagonist suramin (200 microM) reduced the inhibition by 58%. The inhibitory effects of the nucleotides were significantly attenuated by preincubation with pertussis toxin (10-100 ng/ml). Inhibition of phospholipase C and protein kinase C did not prevent the inhibitory effect of the nucleotides. Inhibitors of forskolin-stimulated cAMP accumulation had different effects on DNA synthesis in cultured MCs as measured by 3H-thymidine uptake at 48 h: ATP, ATP gamma S and the inhibitor of adenylyl cyclase, SQ 22536, stimulated DNA synthesis in MCs, while UTP showed no significant mitogenic effect. Agents which increased baseline levels of intracellular cAMP (forskolin, IBMX, dibutyryl-cAMP) significantly diminished DNA synthesis in MCs. The results indicate that the P2u-purinergic receptor mediates inhibition of forskolin-induced cAMP accumulation which is likely due to inhibition of adenylyl cyclase. This effect appears to be partially mediated by PTX-sensitive G proteins. While the increase in cAMP accumulation is anti-mitogenic, inhibition of cAMP accumulation by P2u receptors is not correlated with MC growth control. Thus, additional mechanisms other than inhibition of cAMP accumulation by P2u receptors are likely to be involved in the mitogenesis of extracellular ATP.

Expression of parathyroid hormone-related protein in the rat glomerulus and tubule during recovery from renal ischemia.

Parathyroid hormone-related protein (PTHrP) is widely expressed in normal adult and fetal tissues, where it acts in an autocrine/paracrine fashion, stimulates growth and differentiation, and shares early response gene characteristics. Since recovery from renal injury is associated with release of local growth factors, we examined the expression and localization of PTHrP in normal and ischemic adult rat kidney. Male Sprague-Dawley rats underwent complete bilateral renal artery occlusion for 45 min, followed by reperfusion for 15 min, and 2, 6, 24, 48, and 72 h. Renal PTHrP mRNA levels, when compared with sham-operated animals, increased twofold after ischemia, and peaked within 6 h after reperfusion. PTH receptor, beta-actin, and cyclophilin mRNA levels all decreased after ischemia. PTHrP immunohistochemical staining intensity increased in proximal tubular cells after ischemia, changing its location from diffusely cytoplasmic to subapical by 24 h after reperfusion. In addition, PTHrP localized to glomerular epithelial cells (visceral and parietal), but not to mesangial cells. PTHrP and PTH stimulated proliferation two- to threefold in cultured mesangial cells. We conclude that PTHrP mRNA and protein production are upregulated after acute renal ischemic injury, that PTHrP is present in glomerulus and in both proximal and distal tubular cells, and that PTHrP stimulates DNA synthesis in mesangial cells. The precise functions of PTHrP in normal and injured kidney remain to be defined.

Nitric oxide and superoxide in cultured cells: limited production and influence on DNA synthesis

The ability of superoxide (O2-) and NO to influence DNA synthesis, as estimated by [3H]thymidine incorporation in logarithmically growing cells, was evaluated in three human cell types: embryonic lung fibroblasts, skin fibroblasts, and mesangial cells. Nontoxic rates of O2- generation (0.2 nM/min-0.2 microM/min) only slightly stimulated DNA synthesis in mesangial cells (10-13% increase) and did not affect fibroblast DNA synthesis. Nontoxic rates of NO generation (0.1-10 microM/min) also had only limited effects, inhibiting DNA synthesis in lung and skin fibroblasts slightly (10-20% decrease) but not affecting mesangial cells. In all three cell types, neither O2- nor NO was generated at high enough endogenous rates to support an autocrinic regulation of DNA synthesis. This and other lines of evidence indicate that a reaction between O2- and NO is unlikely to account for their opposing effects on DNA synthesis.

Lovastatin Retards the Progression of Established Glomerular Disease in Obese Zucker Rats

Considerable experimental evidence indicates that hyperlipidemia can induce glomerular injury. The importance of lipids in the progression of established glomerular disease has not been established and is of clinical relevance because of the frequent association of lipid abnormalities with human renal disease. In the present study, 26-week-old hyperlipidemic obese Zucker rats (OZRs) with established nephropathy were treated for a period of 18 weeks with daily injections of the cholesterol synthesis inhibitor lovastatin (4 mg/kg). Compared with control OZRs treated with vehicle, lovastatin-treated OZRs had significantly (P < 0.05) lower serum cholesterol and triglyceride levels throughout the treatment period. Blood pressure and urine albumin excretion in lovastatin-treated OZRs were reduced over the first 12 weeks of therapy, but increased toward the levels in the control OZRs at the end of the protocol. After 18 weeks of therapy, the incidence of glomerulosclerosis in lovastatin-treated OZRs (23.2% +/- 5.8%) was approximately half of that in vehicle-treated OZRs (44.6% +/- 7.7%) (P < 0.05). The reduction in glomerular injury in lovastatin-treated OZRs was not associated with changes in either glomerular area or glomerular macrophage content. In separate experiments, mesangial cells were cultured from glomeruli isolated from 26-week-old proteinuric OZRs. Lovastatin (5 to 40 mumol/L) caused a significant dose-dependent inhibition of serum-stimulated mesangial cell DNA synthesis. The inhibitory effects of lovastatin were completely prevented in the presence of exogenous mevalonate (100 mumol/L). Thus, lovastatin retarded the progression of established glomerular disease in OZRs.(ABSTRACT TRUNCATED AT 250 WORDS)

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.