Rat Renal Mesangial Cells Research Articles

Endothelin signalling and regulation of protein kinases in glomerular mesangial cells.

The molecular mechanisms of endothelin (ET)-dependent activation of extracellular signal-regulated kinase (ERK)and p38 mitogen-activated protein (MAP) kinase were studied in rat and human renal glomerular mesangial cells. ET-1 induced a rapid and transient activation of Ras in renal mesangial cells, which was dependent upon the formation of the Shc/Grb2/Sos1 signalling complex and resulted in transient ERK activation. We have observed that Pyk2, a calcium-dependent cytoplasmic tyrosine kinase, was expressed in human renal mesangial cells and was tyrosine phosphorylated after ET-1 treatment. ET-1-induced activation of p38 MAPK pathway (but not ERK pathway) was inhibited in human and in rat glomerular mesangial cells expressing dominant-negative form of Pyk2, suggesting the engagement of Pyk2 in ET-1-mediated activation of p38 MAP kinase cascade. Contractive responsiveness of renal mesangial cells was shown to depend on activation of the p38 MAP kinases. Thus, p38 MAP kinase stimulation could perhaps partially account for ET-1 contractive properties, whereas ET-1-induced cell proliferation occurs primarily via Ras-dependent activation of the ERK.

Inhibition of Cytokine-induced Matrix Metalloproteinase 9 Expression by Peroxisome Proliferator-activated Receptor α Agonists Is Indirect and Due to a NO-mediated Reduction of mRNA Stability

Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.

Role of oxidative stress in advanced glycation end product-induced mesangial cell activation

Levels of advanced glycation end products (AGE) are elevated in individuals with advancing age, renal failure, and diabetes, and accumulation of these molecules may contribute to disease progression. The mechanism by which AGE proteins alter glomerular mesangial cell function, however, is not completely understood. The present study assessed the involvement of oxidative stress in AGE-dependent mesangial cell signaling events. Primary cultures of rat renal mesangial cells were exposed to in vitro AGE-BSA and H2O2. Nuclear factor-kappaB (NF-kappaB) and protein kinase C (PKC) isoform activation were studied using confocal microscopy and Western blotting. Quantitative polymerase chain reaction (PCR) was used to measure transforming growth factor-beta1 (TGF-beta1) levels. The involvement of oxidative stress was assessed by supplementing or compromising cellular antioxidant capacity. NF-kappaB was dose-dependently activated by AGE. PKC activation was not involved in this response, but analysis of PKC-beta1 activation showed a stimulatory effect of AGE proteins on this isoform. Transcription of TGF-beta1 was stimulated by AGE and was prevented by PKC inhibition. Challenge with H2O2 had similar downstream effects on mesangial cell signaling. Antioxidants, vitamin E and nitecapone, prevented AGE-dependent NF-kappaB activation and normalized PKC activity and associated TGF-beta1 transcription. Depletion of the intracellular antioxidant, glutathione, effectively lowered the AGE concentration needed for mesangial cell activation of NF-kappaB and PKC-beta1. Treatment with a suboptimal AGE dose, under glutathione-depleted conditions, revealed a synergistic effect on both parameters. The results support a central role for oxidative stress in AGE-dependent mesangial cell signaling and emphasize the importance of ROS in determining cell responsiveness.

Interleukin-1 inhibits angiotensin II-stimulated protein kinase B pathway in renal mesangial cells via the inducible nitric oxide synthase

Exposure of rat renal mesangial cells to angiotensin II and angiotensin III leads to a rapid phosphorylation and activation of the protein kinase B (PKB) pathway. The angiotensin II analogs angiotensin-(1–7), angiotensin-(1–6) and angiotensin-(3–8) were unable to activate PKB. The angiotensin II and III effects are mediated by the angiotensin type 1 receptor as documented by the inhibitory action of valsartan. Furthermore, angiotensin II-induced activation of PKB involves neither a pertussis toxin-sensitive pathway nor the small G proteins of the Rho/Rac/cdc42 family, but is completely blocked by inhibitors of the PI 3-kinase. Moreover, angiotensin II-stimulated PKB activation is inhibited by long-term pretreatment with interleukin-1β, an effect that is reversed by the NO synthase inhibitor, N G-monomethyl- l-arginine ( l-NMMA). Similarly, the nitric oxide donor ( Z)-1-[2-Aminoethyl)- N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (Deta-NO) blocks the angiotensin II-induced PKB activation. The NO-mediated inhibition of PKB activation in turn is reversed by the phosphatase inhibitor calyculin A but not by ocadaic acid, implying the induction of a protein phosphatase 1 activity by NO.

Downregulation of angiotensin II type 1 receptors during sepsis.

Our study aimed to characterize the mechanisms underlying the attenuated cardiovascular responsiveness toward the renin-angiotensin system during sepsis. For this purpose, we determined the effects of experimental Gram-negative and Gram-positive sepsis in rats. We found that sepsis led to a ubiquitous upregulation of NO synthase isoform II expression and to pronounced hypotension. Despite increased plasma renin activity and plasma angiotensin (Ang) II levels, plasma aldosterone concentrations were normal, and the blood pressure response to exogenous Ang II was markedly diminished in septic rats. Mimicking the fall of blood pressure during sepsis by short-term infusion of the NO donor sodium nitroprusside in normal rats did not alter their blood pressure response to exogenous Ang II. Therefore, we considered the possibility of an altered expression of Ang II receptors during sepsis. It turned out that Ang II type 1 receptor expression was markedly downregulated in all organs of septic rats. Further in vitro studies with rat renal mesangial cells showed that NO and a combination of proinflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) downregulated Ang II type 1 receptor expression in a synergistic fashion. In summary, our data suggest that sepsis causes a systemic downregulation of Ang II type 1 receptors that is likely mediated by proinflammatory cytokines and NO. We suggest that this downregulation of Ang II type 1 receptors is the main reason for the attenuated responsiveness of blood pressure and of aldosterone formation to Ang II and, therefore, contributes to the characteristic septic shock.

G Protein-coupled Receptors Desensitize and Down-regulate Epidermal Growth Factor Receptors in Renal Mesangial Cells

Different types of plasma membrane receptors engage in various forms of cross-talk. We used cultures of rat renal mesangial cells to study the regulation of EGF receptors (EGFRs) by various endogenous G protein-coupled receptors (GPCRs). GPCRs (5-hydroxytryptamine(2A), lysophosphatidic acid, angiotensin AT(1), bradykinin B(2)) were shown to transactivate EGFRs through a protein kinase C-dependent pathway. This transactivation resulted in the initiation of multiple cellular signals (phosphorylation of the EGFRs and ERK and activation of cAMP-responsive element-binding protein (CREB), NF-kappaB, and E2F), as well as subsequent rapid down-regulation of cell-surface EGFRs and internalization and desensitization of the EGFRs without change in the total cellular complement of EGFRs. Internalization of the EGFRs and the down-regulation of cell-surface receptors in mesangial cells were blocked by pharmacological inhibitors of clathrin-mediated endocytosis and in HEK293 cells by transfection of cDNA constructs that encode dominant negative beta-arrestin-1 or dynamin. Whereas all of the effects of GPCRs on EGFRs were dependent to a great extent on protein kinase C, those initiated by EGF were not. These studies demonstrate that GPCRs can induce multiple signals through protein kinase C-dependent transactivation of EGFRs. Moreover, GPCRs induce profound desensitization of EGFRs by a process associated with the loss of cell-surface EGFRs through clathrin-mediated endocytosis.

Nitric oxide induces MIP-2 transcription in rat renal mesangial cells and in a rat model of glomerulonephritis.

Nitric oxide is a crucial mediator of several forms of glomerulonephritis. We examined the effects of NO on the mRNA expression pattern in glomerular mesangial cells by using a low-stringency reverse transcriptase-polymerase chain reaction method and detected a cDNA fragment that was induced by interleukin 1b (IL-1b) and further up-regulated by the NO donor diethylenetriamine-nitric oxide (DETA-NO). Each respective cDNA fragment was found to match with the cDNAs of rat macrophage inflammatory protein 2 (MIP-2) and GRO/cytokine-induced neutrophil chemoattractant 2b (CINC-2b). Further characterization of MIP-2 regulation by Northern blot analysis confirmed an NO- and IL-1b-dependent increase in MIP-2 mRNA levels. Moreover, inhibition of IL-1b-induced endogenous NO formation by the NO-synthase (NOS) inhibitor L-NMMA markedly attenuated MIP-2 protein expression. We cloned 770 bp of the 5'-flanking region of rat MIP-2 and fused this fragment to a luciferase reporter gene. Transfection of the construct into mesangial cells resulted in a 3.5-fold increase in luciferase activity in cells treated with DETA-NO when compared to controls, suggesting a transcriptional mechanism for NO-induced MIP-2 expression. Deletion and mutational analysis identified critical nuclear factor (NF)-kB and NF-IL-6 binding sites required for NO regulation of MIP-2. In vivo, inhibition of NO synthesis in the Thy-1.1 model of mesangioproliferative glomerulonephritis by the specific inducible-NOS inhibitor L-NIL resulted in a marked reduction of MIP-2 mRNA expression. Furthermore, infiltration of neutrophils into the glomerulus was dramatically attenuated in L-NIL-treated rats.

Cox-2 and osteopontin in cocultured platelets and mesangial cells: Role of glucocorticoids

Glomerular inflammation is characterized by a consecutive infiltration of immunoreactive cells. To mimic the early phase of glomerular injury, a coculture system of platelets and rat renal mesangial cells was established. As prototypes, the inflammation-related proteins cyclooxygenase-2 (Cox-2) and the chemotactic protein osteopontin (OPN) were investigated. The expression of OPN and Cox-2 mRNA and protein was determined by Northern and Western blot analyses. Coincubation of platelets and mesangial cells led to a rapid, transient induction of Cox-2 mRNA, which peaked at two hours, whereas OPN and monocyte chemoattractant protein-1 (MCP-1) were induced at later time points. The induction of Cox-2 mRNA was concentration dependent and highly reproducible when platelets of different donors were investigated. Partial Cox-2 induction was observed when supernatants of preactivated platelets were incubated with mesangial cells. The inhibition of the signaling pathways of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) or interference with Gi-protein signaling partially inhibited platelet-induced Cox-2 expression. Down-regulation of protein kinase C (PKC), which is a common signaling module in many pathways leading to Cox-2 induction, almost completely abrogated platelet-induced Cox-2 expression. The time pattern of Cox-2 and OPN expression suggested that Cox-2 might play a role in OPN induction. The up-regulation of OPN was dependent on de novo protein synthesis and was induced by high levels of exogenous prostaglandin E2 (PGE2; 10 micromol/L). Endogenous PGE2, however, proved not to be essential for OPN mRNA expression, because inhibition of Cox activity did not change OPN mRNA levels. Dexamethasone inhibited Cox-2 mRNA induction but increased OPN mRNA and protein expression. These data indicate that Cox-2 and OPN are independently up-regulated upon interaction of platelets and mesangial cells.

Induction of cyclooxygenase-2 by platelet-derived growth factor (PDGF) and its inhibition by dexamethasone are independent of NF-kappaB/IkappaB transcription factors.

Glucocorticoids are potent inhibitors of cyclooxygenase-2 (prostaglandin G/H synthase-2, COX-2) expression. The focus of this work was to investigate the molecular mechanisms, by which glucocorticoids interfere with platelet-derived growth factor (PDGF)-mediated induction of COX-2 with special emphasis on the role of the transcription factors NF-kappaB/IkappaB alpha. In rat renal mesangial cells, PDGF induced a rapid and transient increase of COX-2 mRNA and protein, which reached maximal levels after 1-2 and 4 h, respectively. The in vivo half-life of COX-2 mRNA, which was estimated to be less than 1 h, was reduced by dexamethasone. Kinetic studies and COX-2 promoter activity assays indicated that dexamethasone also interfered with COX-2 transcription. Inhibition of COX-2 induction by dexamethasone was abrogated by cycloheximide, an inhibitor of translation, indicating dependence on de novo protein synthesis. As a possible mediator of dexamethasone action, the NF-kappaB/IkappaB alpha system of transcription factors was investigated. Dexamethasone doubled IkappaB alpha protein levels within 1 h and reduced complex formation of nuclear NF-kappaB proteins with DNA. Newly synthesized IkappaB alpha may thus bind to NF-kappaB and interfere with gene activation. PDGF-induced signalling, however, barely affected the NF-kappaB/IkappaB alpha system: IkappaB alpha protein remained unaltered for 30 min after treatment of mesangial cells with PDGF and was only reduced by 30% after 1 h. Concomitantly, binding of NF-kappaB proteins to DNA, detected by electrophoretic mobility shift assays, was slightly increased by 30%. Furthermore, stably transfected COX-2 promoter constructs with and without the NF-KB binding site were comparably activated by PDGF (2.5-fold increase of luciferase activity). Taken together, these data indicate that although dexamethasone interferes with the NF-kappaB/IkappaB alpha system of transcription factors, this mechanism is not essential for the inhibition of PDGF-induced COX-2 expression.

5-HT(2A) receptors stimulate mitogen-activated protein kinase via H(2)O(2) generation in rat renal mesangial cells.

Serotonin (5-HT) stimulates mitogenesis in rat renal mesangial cells through a G protein-coupled 5-HT(2A) receptor. We tested the hypothesis that oxidants might be involved in the signal transduction pathway linking the receptor to extracellular signal-regulated protein kinase (ERK). 5-HT rapidly increased the activity and phosphorylation of ERK. These effects were blocked by the 5-HT(2A) receptor antagonist ketanserin. The peak effect was noted at 5-10 min, and half-maximal stimulation was achieved at 10-30 nM 5-HT. Chemical inhibitor and activator studies supported the involvement of phospholipase C, protein kinase C (PKC), and reactive oxygen species (ROS, i.e., H(2)O(2) and superoxide) generated by an NAD(P)H oxidase-like enzyme in the ERK activation cascade. Mapping studies supported a location for the NAD(P)H oxidase enzyme and the ROS downstream from PKC. Our studies are most consistent with an ERK activation pathway as follows: 5-HT(2A) receptor --> G(q) protein --> phospholipase C --> diacylglycerol --> classical PKC --> NAD(P)H oxidase --> superoxide --> superoxide dismutase --> H(2)O(2) --> mitogen-activated extracellular signal-regulated kinase --> ERK. These studies demonstrate a role for the 5-HT(2A) receptor in rapid, potent, and efficacious activation of ERK in rat renal mesangial cells. They support a role for oxidants in conveying the stimulatory signal from 5-HT, because 1) chemical antioxidants attenuate the 5-HT signal, 2) oxidants and 5-HT selectively activate ERK to a similar degree, 3) 5-HT produces superoxide and H(2)O(2) in these cells, and 4) a specific enzyme [NAD(P)H oxidase] has been implicated as the source of the ROS, which react selectively downstream of classical PKC.

Transcriptional Regulation of Steroid Receptor Coactivator-1 (SRC-1) in Glucocorticoid Action

Diverse mechanisms of steroid receptor action have been clarified in recent years, as a consequence of the discovery of multiple coactivators. Among them, steroid receptor coactivator-1 (SRC-1) is a member of the p160 coactivator families, which are 160 kDa proteins that interact with steroid receptors in a hormone-sensitive manner. Since coactivators function as transcriptional power boosters, subtle changes in coactivator expression levels in certain cells markedly change of receptor-mediated transcriptional activity. Expression of the glucocorticoid receptor (GR) has been shown to be autoregulated in glucocorticoid action, i.e., GR is downregulated by its cognate ligand, indicating that this autoregulation of GR may protect target cells against excessive hormone action. In the present study, we examined whether coactivator expression levels are also regulated by glucocorticoids. Among several coactivators, the SRC-1 mRNA level was downregulated by dexamethasone treatment in rat tissues, such as liver, heart, kidney, stomach, and cerebrum, in vivo We also demonstrated dexamethasone-mediated downregulation of SRC-1 mRNA and its protein levels in rat renal mesangial cells in vitro These results suggest that ligand-mediated downregulation of SRC-1 is crucial in the physiology of glucocorticoid action.

Protective effects of polyphenols against cadmium-induced glomerular mesangial cell myocontracture.

The main objective of this work was to determine the ability of polyphenols (procyanidoloic oligomers; PCO) to diminish the contracture of CdCl2-induced mesangial cells (smooth muscle cell type). Glomeruli were isolated by passing rat renal cortex pulp through calibrated sieves followed by a culture step for outgrowth of cells. PCO lethality was measured by microassay (Neutral Red uptake). This study has revealed an absence of PCO toxicity during exposure for 24 h and for concentrations ranging from 0.031 to 1% (w/v) on rat renal mesangial cells. We observed a lack of cytotoxicity response for the PCO mixture dissolved in medium. Quantitative assessments of the planar cell surface area (PCSA) were performed with an accurate automatized image analyser. The use of isolated cultured mesangial cells permits us to evaluate by quantitative morphometric analysis the contracture elicited either with CdCl2 salts alone or by previous incubation with a non-lethal dose of PCO. When renal mesangial cells were exposed for 10 min to the PCO mixture, the Cd-mediated myocontracturant response of the mesangial cells was totally abolished. These results suggest that polyphenols could be effective against renal damages induced by cadmium.

Modulation of cytokine-induced expression of secretory phospholipase A2-type IIA by protein kinase C in rat renal mesangial cells.

Renal mesangial cells express the 14 kDa secretory phospholipase A2-type IIA (sPLA2-IIA) in response to interleukin-1beta (IL-1beta). In order to understand the regulation of cytokine-induced sPLA2-IIA induction in more detail, we investigated whether phorbol ester-activated protein kinase C (PKC) has an influence on the IL-1beta-induced expression of sPLA2-IIA. We found that treatment of mesangial cells with the biologically active phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate inhibited IL-1beta induction of sPLA2-IIA mRNA, protein, and activity, whereas the inactive compound 4alpha-phorbol 12,13-didecanoate was without effect. An 8-hr pretreatment with PMA, which led to down-regulation of PKC-alpha and -delta isoenzymes, still inhibited sPLA2-IIA induction. Only after down-regulation of PKC-epsilon isoenzyme by 24-hr preincubation with PMA were we able to reconstitute the IL-1beta-induced sPLA2-IIA expression. Thrombin as a physiological activator of PKC in mesangial cells exerted similar effects as PMA and inhibited sPLA2-IIA expression. The selective PKC inhibitor calphostin C potentiated IL-1beta induction of sPLA2-IIA mRNA levels and partially reconstituted the thrombin-induced inhibition of sPLA2-IIA mRNA and activity. These data show that IL-1beta induction of sPLA2-IIA can be modulated by PKC and that the epsilon-isoenzyme of the PKC family is the most likely candidate mediating the suppression of cytokine-induced sPLA2-IIA expression in mesangial cells.

Binding affinity of sarpogrelate to 5-HT 2A receptor ligand recognition sites in rat renal cortical and mesangial cells in culture

We detected specific binding of 3H-ketanserin (0.6 nM) in rat renal cortical membrane preparations (4.70 ± 0.57 fmol/mg protein) and mesangial cells (7.55 ± 0.92 fmole/10 6 cells). Thus, the value in the renal cortical membrane corresponded to 15% of that in the cerebral cortical membranes (30.0 ± 2.9 fmole/mg protein). The affinity of 3H-ketanserin binding displacement activities by sarpogrelate, a selective 5-HT 2A receptor antagonist, in the renal cortical membrane (IC 50; 0.448 ± 0.061 μM) and mesangial cells (IC 50; 0.656 ± 0.187 μM) were almost 100-fold less than that in the cerebral cortical membrane (IC 50; 4.62 ± 1.02 nM). In the renal cortical membranes and mesangial cells, methysergide displaced a tiny fraction of 3H-ketanserin binding at concentrations up to 10 μM. These results did not explain the functional activity of 5-HT in the mesangial cells, and we conclude that specific 3H-ketanserin binding sites in the mesangial cells consisted of methysergide-resistant and non-serotonergic sites with low affinity for sarpogrelate.

Effect of trace levels of nigericin on intracellular pH and acid-base transport in rat renal mesangial cells.

Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes. In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere with intracellular pH (pHi) changes in the next. Using the pH-sensitive fluorescent dye 2', 7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured pHi in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pHi decrease elicited by removing extracellular Na+, (ii) an increase in acid extrusion during the pHi increase caused by elevating extracellular [K+], and (iii) an acid shift in the pHi dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride (EIPA). However, contamination had no effect on the pHi dependence of Na-H exchange, computed by adding the pHi dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each experiment.

Cross-talk between group IIA-phospholipase A2 and inducible NO-synthase in rat renal mesangial cells.

Features of glomerulonephritis are expression of the inducible form of NO synthase (iNOS) as well as expression of the secretory group IIA-phospholipase A2 (sPLA2) in mesangial cells. Interleukin 1beta (IL-1beta) induces both enzymes with a similar time course resulting in an increase in nitrite production and sPLA2-IIA activity. In this study we investigated the relationship between the formation of NO and sPLA2-IIA induction in rat renal mesangial cells. Incubation of mesangial cells with the NO-donor, spermine-NONOate, for 24 h induced sPLA2-IIA mRNA expression and activity, whereas S-nitroso glutathione alone had only a small stimulatory effect. Stimulation of cells with IL-1beta caused a marked increase in sPLA2-IIA mRNA and activity that were potentiated 3 fold by both NO donors. Coincubation of cells with IL-1beta and the NOS inhibitor, L-N(G) monomethylarginine (L-NMMA), caused a dose-dependent inhibition of cytokine-induced sPLA2-IIA mRNA expression and activity. sPLA2-IIA activity was not stimulated by 8-bromo-cyclic GMP indicating that NO-induced sPLA2-IIA induction is independent of cyclic GMP-mediated signal transduction. These data show that NO contributes to the expression by cytokines of sPLA2-IIA and establishes a novel type of interaction between iNOS and sPLA2-IIA in mesangial cells. This cross-talk between inflammatory mediators may help to promote and sustain an inflammatory state in the kidney.

Nitric oxide stimulates the stress-activated protein kinase p38 in rat renal mesangial cells.

Nitric oxide (NO) has gained increased attention as a diffusible universal messenger that plays a crucial role in the pathogenesis of inflammatory and autoimmune diseases. Recently, we reported that exogenous NO is able to activate the stress-activated protein kinase (SAPK) cascade in mesangial cells. Here, we demonstrate that exposure of glomerular mesangial cells to compounds releasing NO, including spermine-NO and (Z)-1- (N-methyl-N-[6-(N-methylammoniohexyl)amino]diazen)-1-ium-1,2-diolate (MAHMA-NO), results in an activation of the stress-activated p38-mitogen-activated protein kinase (p38-MAPK) cascade as measured by the phosphorylation of the activator of transcription factor-2 (ATF2) in an immunocomplex kinase assay. Activation of the p38-MAPK cascade by a short stimulation (10 min) with the NO donor MAHMA-NO causes a large increase in ATF2 phosphorylation that is several times greater than that observed after stimulation with interleukin-1beta, a well-known activator of the p38-MAPK pathway. Time course studies reveal that MAHMA-NO causes rapid and maximal activation of p38-MAPK after 10 min of stimulation and that activation declines to basal levels within 60 min. The longer-lived NO donor spermine-NO causes a comparable rapid activation of the p38-MAPK pathway; however, the increased activation state of p38-MAPK was maintained for several hours before control values were reattained after 24 h of stimulation. Furthermore, the NO donors also activated the classical extracellular signal-regulated kinase (ERK) p44-MAPK cascade as shown by phosphorylation of the specific substrate cytosolic phospholipase A2 in an immunocomplex kinase reaction. Both MAHMA-NO and spermine-NO cause a rapid activation of p44-MAPK after 10 min of stimulation. Interestingly, there is a second delayed peak of p44-MAPK activation after 4-24 h of stimulation with NO donors. These results suggest that there is a differential activation pattern for stress-activated and mitogen-activated protein kinases by NO and that the integration of these signals may lead to specific cell responses.

Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells.

Using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+ concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between approximately 6.7 and approximately 7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (approximately 300 mosmol/kgH2O). In the nominal absence of CO2/HCO-3, exposing cell populations to a HEPES-buffered solution supplemented with approximately 300 mM mannitol (600 mosmol/kgH2O) causes steady-state pHi to increase by approximately 0.4. The pHi increase is due to activation of Na+/H+ exchange because, in single cells, it is blocked in the absence of external Na+ or in the presence of 50 microM ethylisopropylamiloride (EIPA). Preincubating cells in a Cl--free solution for at least 14 min inhibits the shrinkage-induced pHi increase by 80%. We calculated the pHi dependence of the Na+/H+ exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pHi dependence of the total acid-extrusion rate and 2) the pHi dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pHi dependence of Na+/H+ exchange by approximately 0.7 pH units.

Nitric Oxide Donors Induce Stress Signaling via Ceramide Formation in Rat Renal Mesangial Cells

Exogenous NO is able to trigger apoptosis of renal mesangial cells, and thus may contribute to acute lytic phases as well as to resolution of glomerulonephritis. However, the mechanism involved in these events is still unclear. We report here that chronic exposure of renal mesangial cells for 24 h to compounds releasing NO, including spermine-NO, (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1, 2-diolate (MAHMA-NO), S-nitrosoglutathione (GS-NO), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) results in a potent and dose-dependent increase in the lipid signaling molecule ceramide. Time courses reveal that significant effects occur after 2-4 h of stimulation with NO donors and reach maximal levels after 24 h of stimulation. No acute (within minutes) ceramide production can be detected. When cells were stimulated with NO donors in the presence of phorbol ester, a direct activator of protein kinase C, both ceramide production and DNA fragmentation are completely abolished. Furthermore, addition of exogenous ceramide partially reversed the inhibitory effect of phorbol ester on apoptosis, thus suggesting a negative regulation of protein kinase C on ceramide formation and apoptosis. In contrast to exogenous NO, tumor necrosis factor (TNF)-alpha stimulates a very rapid and transient increase in ceramide levels within minutes but fails to induce the late-phase ceramide formation. Moreover, TNF fails to induce apoptosis in mesangial cells. Interestingly, NO and TNFalpha cause a chronic activation of acidic and neutral sphingomyelinases, the ceramide-generating enzymes, whereas acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNFalpha. Furthermore, in the presence of an acidic ceramidase inhibitor, N-oleoylethanolamine, TNFalpha leads to a sustained accumulation of ceramide and in parallel induces DNA fragmentation. In summary, our data demonstrate that exogenous NO causes a chronic up-regulation of ceramide levels in mesangial cells by activating sphingomyelinases and concomitantly inhibiting ceramidases, and that particularly the late-phase of ceramide generation may be responsible for the further processing of a proapoptotic signal.

Differential regulation of vascular endothelial growth factor and its receptor fms-like-tyrosine kinase is mediated by nitric oxide in rat renal mesangial cells

Under conditions associated with local and systemic inflammation, mesangial cells and invading immune cells are likely to be responsible for the release of large amounts of nitric oxide (NO) in the glomerulus. To further define the mechanisms of NO action in the glomerulus, we attempted to identify genes which are regulated by NO in rat glomerular mesangial cells. We identified vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase (FLT-1) to be under the regulatory control of exogenously applied NO in these cells. Using S-nitroso-glutathione (GSNO) as an NO-donating agent, VEGF expression was strongly induced, whereas expression of its FLT-1 receptor simultaneously decreased. Expressional regulation of VEGF and FLT-1 mRNA was transient and occurred rapidly within 1–3 h after GSNO treatment. Expression of a second VEGF-specific receptor, fetal liver kinase-1 (FLK-1/KDR), could not be detected. The inflammatory cytokine interleukin-1β mediated a moderate increase in VEGF expression after 24 h and had no influence on FLT-1 expression. In contrast, platelet-derived growth factor–BB and basic fibroblast growth factor had no effect on VEGF expression, but strongly induced FLT-1 mRNA levels. Obviously, there is a differential regulation of VEGF and its receptor FLT-1 by NO, cytokines and growth factors in rat mesangial cells.