Hydrogen Sulfide Modulates Endothelial-Mesenchymal Transition in Heart Failure.

BACKGROUND: Hydrogen sulfide is a critical endogenous signaling molecule that exerts protective effects in the setting of heart failure. Cystathionine γ-lyase (CSE), 1 of 3 hydrogen-sulfide-producing enzyme, is predominantly localized in the vascular endothelium. The interaction between the endothelial CSE-hydrogen sulfide axis and endothelial-mesenchymal transition, an important pathological process contributing to the formation of fibrosis, has yet to be investigated. METHODS: Endothelial-cell-specific CSE knockout and Endothelial cell-CSE overexpressing mice were subjected to transverse aortic constriction to induce heart failure with reduced ejection fraction. Cardiac function, vascular reactivity, and treadmill exercise capacity were measured to determine the severity of heart failure. Histological and gene expression analyses were performed to investigate changes in cardiac fibrosis and the activation of endothelial-mesenchymal transition. RESULTS: Endothelial-cell-specific CSE knockout mice exhibited increased endothelial-mesenchymal transition and reduced nitric oxide bioavailability in the myocardium, which was associated with increased cardiac fibrosis, impaired cardiac and vascular function, and worsened exercise performance. In contrast, genetic overexpression of CSE in endothelial cells led to increased myocardial nitric oxide, decreased endothelial-mesenchymal transition and cardiac fibrosis, preserved cardiac and endothelial function, and improved exercise capacity. CONCLUSIONS: Our data demonstrate that endothelial CSE modulates endothelial-mesenchymal transition and ameliorate the severity of pressure-overload-induced heart failure, in part, through nitric oxide-related mechanisms. These data further suggest that endothelium-derived hydrogen sulfide is a potential therapeutic for the treatment of heart failure with reduced ejection fraction.

Gasotransmitter synthesis and signalling in the renal glomerulus. Implications for glomerular diseases.

Glomerular injury is a hallmark of kidney diseases such as diabetic nephropathy, IgA nephropathy or other forms of glomerulonephritis. Glomerular endothelial cells, mesangial cells, glomerular epithelial cells (podocytes) and, in an inflammatory context, infiltrating immune cells crosstalk to mediate signalling processes in the glomerulus. Under physiological conditions, mesangial cells act by the control of extracellular matrix production and degradation, by the synthesis of growth factors and by preserving a well-defined crosstalk with glomerular podocytes and endothelial cells to regulate glomerular structure and function. It is well known that mesangial cells are able to amplify an inflammatory process by the formation of cytokines, reactive oxygen species (ROS) and nitric oxide (NO). This exaggerated reaction may result in a vicious cycle with subsequent damage of neighboured podocytes and endothelial cells, loss of the filtration barrier and, finally destruction of the whole glomerulus. Unfortunately, all efforts to develop new therapies for the treatment of glomerular diseases by controlling unbridled ROS or NO production directly had so far no success. However, on-going research on ROS and NO defined these autacoids more as important signalling molecules than as endogenously produced cytotoxic compounds. New findings on signalling activities of ROS, NO but also hydrogen sulfide (H2S) and carbon monoxide (CO) supported this paradigm shift. Because of their similar chemical properties and their similar signal transduction capacities, NO, H2S and CO are meanwhile designated as the group of gasotransmitters. In this review, we describe the current knowledge of the signalling properties of gasotransmitters with a focus on glomerular cells and their role in glomerular diseases.

Endothelial Cell Cystathionine γ-Lyase Expression Level Modulates Exercise Capacity, Vascular Function, and Myocardial Ischemia Reperfusion Injury.

Background Hydrogen sulfide (H2S) is an important endogenous physiological signaling molecule and exerts protective properties in the cardiovascular system. Cystathionine γ-lyase (CSE), 1 of 3 H2S producing enzyme, is predominantly localized in the vascular endothelium. However, the regulation of CSE in vascular endothelium remains incompletely understood. Methods and Results We generated inducible endothelial cell-specific CSE overexpressed transgenic mice (EC-CSE Tg) and endothelial cell-specific CSE knockout mice (EC-CSE KO), and investigated vascular function in isolated thoracic aorta, treadmill exercise capacity, and myocardial injury following ischemia-reperfusion in these mice. Overexpression of CSE in endothelial cells resulted in increased circulating and myocardial H2S and NO, augmented endothelial-dependent vasorelaxation response in thoracic aorta, improved exercise capacity, and reduced myocardial-reperfusion injury. In contrast, genetic deletion of CSE in endothelial cells led to decreased circulating H2S and cardiac NO production, impaired endothelial dependent vasorelaxation response and reduced exercise capacity. However, myocardial-reperfusion injury was not affected by genetic deletion of endothelial cell CSE. Conclusions CSE-derived H2S production in endothelial cells is critical in maintaining endothelial function, exercise capacity, and protecting against myocardial ischemia/reperfusion injury. Our data suggest that the endothelial NO synthase-NO pathway is likely involved in the beneficial effects of overexpression of CSE in the endothelium.

Increase of cystathionine-γ-lyase (CSE) during late wound repair: Hydrogen sulfide triggers cytokeratin 10 expression in keratinocytes.

The gaseous mediators nitric oxide (NO), carbon monoxide (CO) and lately also hydrogen sulfide (H2S) have been described to contribute to the interplay of protein type- and lipid mediators in the regulation of wound healing. In particular, the recently reported role of H2S in skin repair remains largely unresolved. Therefore we assessed the expressional kinetics of potential H2S-producing enzymes during undisturbed skin repair: the cystathionine-γ-lyase (CSE), the cystathionine-ß-synthase (CBS) and the 3-mercaptopyruvate sulfurtransferase (MPST). All three enzymes were not transcriptionally induced upon wounding and remained silent through the acute inflammatory and proliferative phase of skin repair. By contrast, CSE expression started to increase significantly at the later stages of healing, when cellular proliferation ceases within the granulation tissue and neoepidermis. The importance of H2S production in late healing phases was supported by a strong induction of otherwise not-induced CBS to complement the loss of CSE function in CSE-deficient mice. Immunohistochemistry revealed hair follicle keratinocytes and basal keratinocytes of the neo-epidermis covering the wound area as sources of CSE expression. Subsequent in vitro studies implicated a role of CSE-derived H2S for keratinocyte differentiation: the H2S-donor GYY4137 markedly increased the Ca2+-triggered expression of the early keratinocyte differentiation markers cytokeratin 10 (CK10) and involucrin (IVN) in cultured human keratinocytes. Here, GYY4137-derived H2S strongly enhanced CK10 expression by increasing the binding of RNA polymerase II to the CK10 promoter.

Nitric oxide mediates prolyl hydroxylase 3 expression in mesangial cells and in glomerulonephritis.

Renal mesangial cells are regarded as main players in glomerular inflammatory diseases. To investigate a possible crosstalk between inflammatory and hypoxia-driven signaling processes, we stimulated cultured mouse mesangial cells with different inflammatory agents and analyzed the expression of prolyl hydroxylase domain containing proteins (PHDs), the main regulators of hypoxia-inducible factor (HIF) stability. Administration of IL-1ß (1 nM) and TNF-α (1 nM), a combination further referred to as cytokine mix (CM), resulted in a fivefold increase in PHD3 but not PHD1 and PHD2 mRNA expression compared to untreated controls. In contrast, a combination of IL-1ß, TNF-α with lipopolysaccharide (10 µg/ml), and interferon-γ (20 ng/ml) designated as CM+ showed a high (60-fold) induction of PHD3 and a moderate (twofold) induction of PHD2 mRNA expression. Interestingly, CM+ but not CM induced the expression of inducible NO synthase and endogenously produced NO was responsible for the immense induction of PHD3 in mesangial cells treated with CM+. We found that CM+ affected PHD3 expression mainly via the NO/HIF axis, whereas PHD3 regulation by CM occurred in a NF-κB-dependent manner. In turn, silencing of PHD3 expression resulted in a decrease in the mRNA expression of ICAM-1, MIP-2, MCP-1, and CXCL-10, which are under control of NF-κB. In a rat model of mesangio-proliferative glomerulonephritis, PHD3 mRNA and protein expression was markedly induced and this effect was nearly abolished when rats were treated with the iNOS-specific inhibitor L-NIL, thus confirming our findings also in vivo. KEY MESSAGE: PHD3 expression induced by cytokines is NF-κB dependent in mesangial cells. Endogenously produced NO further augments PHD3 expression via HIF-1α. PHD3 expression is induced by NO in anti-Thy-1 glomerulonephritis.

Quantitative Persulfide Site Identification (qPerS-SID) Reveals Protein Targets of H2S Releasing Donors in Mammalian Cells.

H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents.

The H2S-producing enzyme CSE is dispensable for the processing of inflammatory and neuropathic pain.

Accumulating lines of evidence indicate that hydrogen sulfide (H2S) contributes to the processing of chronic pain. However, the sources of H2S production in the nociceptive system are poorly understood. Here we investigated the expression of the H2S releasing enzyme cystathionine γ-lyase (CSE) in the nociceptive system and characterized its role in chronic pain signaling using CSE deficient mice. We show that paw inflammation and peripheral nerve injury led to upregulation of CSE expression in dorsal root ganglia. However, conditional knockout mice lacking CSE in sensory neurons as well as global CSE knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to WT littermates. Thus, our results suggest that CSE is not critically involved in chronic pain signaling in mice and that sources different from CSE mediate the pain relevant effects of H2S.

Cytokines induce protein kinase A-mediated signalling by a redox-dependent mechanism in rat renal mesangial cells.

Glomerular mesangial cells are smooth muscle cell-like pericytes and are regarded as key players in kidney diseases. In an inflammatory setting, these cells produce high amounts of inflammatory cytokines, chemokines and redox mediators such as reactive oxygen species or nitric oxide (NO). The temporal production of ROS, NO and other redox mediators markedly contributes to the final outcome of inflammatory diseases. Recently, we reported that platelet-derived growth factor forced mesangial cells to activate the regulatory subunit of protein kinase A (PKA RI) by a redox-dependent mechanism but independent from changes in cyclic AMP. This prompted us to further analyze the dimerization of PKA RI and activation of PKA-driven signalling in an inflammatory context. Stimulation of rat mesangial cells with interleukin-1ß and tumour necrosis factor-α [2 nM] induced the formation of PKA RI heterodimers in a time-dependent manner. PKA RI dimerization was accompanied with the formation of ROS, NO and peroxynitrite as well as a depletion of reduced glutathione. Furthermore, dimerization of PKA RI was paralleled by enhanced activity of PKA as shown by the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at serine 157 that was independent of the formation of cyclic AMP. Remarkably, exogenously administered peroxynitrite potently induced dimerization of PKA RI, whereas pharmacologic inhibition of inducible NO synthase (iNOS) and scavenging of peroxynitrite reduced PKA RI dimerization and VASP phosphorylation to control levels thus clearly indicating a causal role for endogenously formed peroxynitrite on PKA signalling. Consequently, the treatment of inflammatory diseases with anti-oxidants or NOS inhibitors may alter PKA activity.

Platelet-derived growth factor triggers PKA-mediated signalling by a redox-dependent mechanism in rat renal mesangial cells.

Inflammatory glomerular kidney diseases are often accompanied with a massive production of reactive oxygen species (ROS) that affect the function of the glomerular filtration barrier and contribute to mesangiolysis via the induction of cell death in mesangial cells. Intriguingly, ROS also trigger fine-tuned signalling processes that affect gene expression and cell proliferation or migration. To define such redox-driven signalling devices, a proteomics approach was performed to identify the formation of protein complexes induced by ROS. To this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 µM). Thereafter cell lysates were subjected to diagonal 2D gel electrophoresis and putative redox-affected proteins were analysed by MS/MS analysis. Among others, the regulatory subunit of protein kinase A (PKA) could be identified that forms homodimers under oxidative conditions. To evaluate whether ROS dependent dimerization of PKA also occurs in a more physiological setting, rat mesangial cells were treated with platelet-derived growth factor-BB (PDGF-BB) to induce ROS formation. This regimen resulted in a redox dependent dimerization of the R-subunits of PKA. To demonstrate whether PDGF-BB induced ROS formation affects PKA dependent pathways, the effects of PDGF-BB on phosphorylation of serine 157 of vasodilator stimulated protein (VASP) a classical target of PKA were analysed. Interestingly PDGF-BB induced VASP phosphorylation in a ROS dependent manner but independent of changes in cAMP levels. Taken together, we demonstrate a redox-mediated activation of PKA by PDGF-BB thus highlighting a physiological role of ROS as regulator of PKA activity in rat mesangial cells.

Platelet-derived growth factor-BB induces cystathionine γ-lyase expression in rat mesangial cells via a redox-dependent mechanism.

BACKGROUND AND PURPOSE: So far, there is only limited information about the regulation of the endogenous synthesis of hydrogen sulfide (H(2) S), an important gaseous signalling molecule. This study was done to evaluate the redox-dependent signalling events that regulate the expression of the H(2) S synthesising enzyme cystathionine-γ-lyase (CSE) in rat mesangial cells. EXPERIMENTAL APPROACH: The effects of platelet-derived growth factor (PDGF)-BB and antioxidants on CSE expression and activity in cultured rat renal mesangial cells were assessed. Activity of nuclear factor erythroid-2-related factor-2 (Nrf2) was measured as the binding capacity to a radiolabelled consensus element by electrophoretic mobility shift assay (EMSA). Furthermore, CSE and Nrf2 expression was analysed in a rat model of anti-Thy-1-induced glomerulonephritis by immunohistochemistry. KEY RESULTS: Treatment of mesangial cells with PDGF-BB resulted in a marked time- and dose-dependent up-regulation of CSE mRNA and protein levels, as well as CSE activity accompanied with increased formation of reactive oxygen species. Remarkably, co-administration of antioxidants, such as N-acetylcysteine, ebselen or diphenylene iodonium chloride, drastically reduced PDGF-BB-induced CSE expression. PDGF-BB induced binding of Nrf2 to a corresponding consensus antioxidant element in a redox-dependent manner. Furthermore, PDGF-BB-induced CSE expression in mouse mesangial cells was completely abolished in Nrf2 knockout mice compared with wild-type mice. In a rat model of anti-Thy-1-induced proliferative glomerulonephritis, we observed a marked up-regulation of CSE protein paralleled by a stabilization of Nrf2 protein. CONCLUSIONS AND IMPLICATIONS: PDGF-BB regulated CSE via a redox-mediated activation of Nrf2. Such action would aid the resolution of glomerular inflammatory diseases. LINKED ARTICLE: This article is commented on by Gallyas, pp. 2228-2230 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01976.x.

Nitric oxide inhibits glomerular TGF-beta signaling via SMOC-1.

Cytokines and nitric oxide (NO) stimulate rat mesangial cells to synthesize and secrete inflammatory mediators. To understand better the signaling pathways that contribute to this response, we exposed rat mesangial cells to the prototypic inflammatory cytokine IL-1beta and analyzed the changes in the pattern of gene expression. IL-1beta downregulated the gene encoding the matricellular glycoprotein secreted modular calcium-binding protein 1 (SMOC-1) in mesangial cells. Inflammatory cytokines attenuated SMOC-1 mRNA and protein expression through endogenous production of NO, which activated the soluble guanylyl cyclase. Silencing SMOC-1 expression with small interfering RNA decreased the formation of TGF-beta, reduced SMAD binding to DNA, and decreased mRNA expression of genes regulated by TGF-beta. In a rat model of anti-Thy-1 glomerulonephritis, glomerular SMOC-1 mRNA and protein decreased and inducible NO synthase expression increased simultaneously. Treatment of nephritic rats with the inducible NO synthase-specific inhibitor l-N(6)-(1-iminoethyl)-lysine prevented SMOC-1 downregulation. In summary, these data suggest that NO attenuates SMOC-1 expression in acute glomerular inflammation, thereby limiting TGF-beta-mediated profibrotic signaling.

PPARalpha activation upregulates nephrin expression in human embryonic kidney epithelial cells and podocytes by a dual mechanism.

Nephrin is an important member of the glomerular ultrafiltration complex and changes in its expression are associated with severe proteinuria. In this study, we show that synthetic PPARalpha agonists, but not PPARgamma agonists, stimulate an increased nephrin mRNA and protein expression in cultures of human podocytes and A293 human embryonic kidney epithelial cells which are blocked by the PPARalpha antagonist Ru486. Furthermore, the PPARalpha agonists have an additive effect on the interleukin-1beta (IL-1beta)-induced nephrin upregulation. Luciferase-reporter assays reveal that human nephrin promoter activity is stimulated by the PPARalpha agonists. Neither IL-1beta nor TNFalpha alone has an effect on nephrin promoter activity suggesting that additional posttranscriptional regulatory events might be operative. The role of nephrin mRNA stability regulation was evaluated in cells treated with actinomycin D to stop further RNA transcription. In the presence of PPARalpha agonists, IL-1beta or TNFalpha, the decay of nephrin mRNA was drastically reduced thus arguing for an additional posttranscriptional mode of action. In summary, these data show that PPARalpha activation causes an increased nephrin expression by a dual action, on the one hand by stimulating nephrin promoter activity and on the other hand by reducing nephrin mRNA degradation. These findings may have importance for treatment strategies of renal diseases affecting the expression of nephrin and subsequently the proper action of the glomerular filtration apparatus.

Biglycan, a nitric oxide-regulated gene, affects adhesion, growth, and survival of mesangial cells.

During glomerular inflammation mesangial cells are the major source and target of nitric oxide that pro-foundly influences proliferation, adhesion, and death of mesangial cells. The effect of nitric oxide on the mRNA expression pattern of cultured rat mesangial cells was therefore investigated by RNA-arbitrarily-primed polymerase chain reaction. Employing this approach, biglycan expression turned out to be down-regulated time- and dose-dependently either by interleukin-1beta-stimulated endogenous nitric oxide production or by direct application of the exogenous nitric oxide donor, diethylenetriamine nitric oxide. There was a corresponding decline in the rate of biglycan biosynthesis and in the steady state level of this proteoglycan. In vivo, in a model of mesangioproliferative glomerulonephritis up-regulation of inducible nitric-oxide synthase mRNA was associated with reduced expression of biglycan in isolated glomeruli. Biglycan expression could be normalized, both in vitro and in vivo, by using a specific inhibitor of the inducible nitric-oxide synthase, l-N6-(l-iminoethyl)-l-lysine dihydrochloride. Further studies showed that biglycan inhibited cell adhesion on type I collagen and fibronectin because of its binding to these substrates. More importantly, biglycan protected mesangial cells from apoptosis by decreasing caspase-3 activity, and it counteracted the proliferative effects of platelet-derived growth factor-BB. These findings indicate a signaling role of biglycan and describe a novel pathomechanism by which nitric oxide modulates the course of renal glomerular disease through regulation of biglycan expression.

Arginase 1 overexpression in psoriasis: limitation of inducible nitric oxide synthase activity as a molecular mechanism for keratinocyte hyperproliferation.

Abnormal proliferation of keratinocytes in the skin appears crucial to the pathogenesis of psoriasis, but the underlying mechanisms remain unknown. Nitric oxide (NO), released from keratinocytes at high concentrations, is considered a key inhibitor of cellular proliferation and inducer of differentiation in vitro. Although high-output NO synthesis is suggested by the expression of inducible NO synthase (iNOS) mRNA and protein in psoriasis lesions, the pronounced hyperproliferation of psoriatic keratinocytes may indicate that iNOS activity is too low to effectively deliver anti-proliferative NO concentrations. Here we show that arginase 1 (ARG1), which substantially participates in the regulation of iNOS activity by competing for the common substrate L-arginine, is highly overexpressed in the hyperproliferative psoriatic epidermis and is co-expressed with iNOS. Expression of L-arginine transporter molecules is found to be normal. Treatment of primary cultured keratinocytes with Th1-cytokines, as present in a psoriatic environment, leads to de novo expression of iNOS but concomitantly a significant down-regulation of ARG1. Persistent ARG1 overexpression in psoriasis lesions, therefore, may represent a disease-associated deviation from normal expression patterns. Furthermore, the culturing of activated keratinocytes in the presence of an ARG inhibitor results in a twofold increase in nitrite accumulation providing evidence for an L-arginine substrate competition in human keratinocytes. High-output NO synthesis is indeed associated with a significant decrease in cellular proliferation as shown by down-regulation of Ki67 expression in cultured keratinocytes but also in short-term organ cultures of normal human skin. In summary, our data demonstrate for the first time a link between a human inflammatory skin disease, limited iNOS activity, and ARG1 overexpression. This link may have substantial implications for the pathophysiology of psoriasis and the development of new treatment strategies.

Effects of high glucose on cytokine-induced nerve growth factor (NGF) expression in rat renal mesangial cells.

Nerve growth factor (NGF) accumulates at sites of inflammation and modulates local immune reactions. To characterize the mechanisms of cytokine-induced NGF expression under physiological and pathophysiological conditions, we have used cultured glomerular mesangial cells, which play a key role in glomerular inflammatory diseases such as diabetic nephropathy. To study the effects of high glucose on cytokine-induced NGF expression, rat mesangial cells were treated with the cytokines interleukin-1beta and tumor necrosis factor alpha under normal (1.0 g/L) and high (4.5 g/L) glucose concentrations. In the presence of high glucose concentrations, the cytokines drastically potentiated NGF protein but not mRNA expression when compared to physiological glucose levels. The specific protein kinase C inhibitors Ro31-8220 and CGP41251 suppressed cytokine-induced NGF expression. Moreover, blocking the oxidative activation of the protein kinase C pathway by N-acetylcysteine inhibited glucose effects on NGF synthesis. Neutralizing antibodies against transforming growth factor-beta inhibited cytokine-induced NGF expression under normal glucose concentrations but not under high glucose conditions. Enhanced expression of NGF under high glucose conditions may contribute to kidney diseases such as diabetic nephropathy.

Potentiation of TNF-alpha-stimulated group IIA phospholipase A(2) expression by peroxisome proliferator-activated receptor alpha activators in rat mesangial cells.

Natural activators of peroxisome proliferator-activated receptors (PPAR) are lipid metabolites, including those produced by phospholipases A(2) (PLA(2)). In glomerular mesangial cells, the secreted group IIA PLA(2) (sPLA(2)-IIA), which is thought to be a crucial factor in pathologic processes in the kidney, may provide free fatty acids and eicosanoids directly or indirectly, by activating a cytosolic PLA(2). The scope of this study was to investigate whether synthetic PPAR(alpha) activators have an effect on sPLA(2)-IIA mRNA expression in rat mesangial cells, thus constituting a feedback modulation of sPLA(2)-IIA transcription. In the presence of tumor necrosis factor-alpha (TNF-alpha), the PPAR(alpha) agonists WY14643 and LY171883 as well as the lipid-lowering compound clofibrate potentiated expression, secretion, and activity of group IIA sPLA(2) in mesangial cells. MK886, known as a noncompetitive inhibitor of PPAR(alpha), completely abolished the potentiation of sPLA(2)-IIA secretion and activity by WY14643, thus indicating that the effect of WY14643 is specifically mediated by PPAR(alpha). When cells were transfected with different constructs of the rat sPLA(2)-IIA promoter fused to a luciferase reporter gene, a stimulation with TNF-alpha in the presence of the PPAR(alpha) activators caused an enhanced promoter activity compared with that induced by TNF-alpha alone. Site-directed mutagenesis of a putative PPRE site in the sPLA(2)-IIA promoter abolished the potentiating effect of PPAR(alpha) agonists, thus strongly indicating its contribution to the enhanced promoter activity. In summary, this study shows that the rat sPLA(2)-IIA promoter is sensitive to PPAR(alpha) agonists, which act synergistically with cytokines, resulting in an enhanced expression of sPLA(2)-IIA in rat mesangial cells.

Downregulation of SPARC expression is mediated by nitric oxide in rat mesangial cells and during endotoxemia in the rat.

Nitric oxide (NO) has been implicated in several forms of glomerulonephritis. In this study, a low stringency reversed transcription/PCR protocol was used to evaluate the action of NO on the mRNA expression pattern in rat mesangial cells (MC). To mimic the state of glomerular inflammation, MC were stimulated by exposure to the cytokines interleukin-1beta and tumor necrosis factor-alpha into producing high levels of NO via expression of inducible nitric oxide synthase (NOS). To detect NO-mediated effects, the resulting expression pattern was compared to that of MC stimulated by the cytokines in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA). Computer analysis of a differentially expressed cDNA fragment resulted in a 100% homology to the recently characterized mRNA of SPARC (secreted protein acidic and rich in cysteine). Further characterization of SPARC regulation revealed a cytokine- and cAMP-dependent decrease in SPARC mRNA and protein levels. Blocking NO formation by L-NMMA reversed the effects of cytokines and cAMP on SPARC expression, suggesting an NO-mediated mechanism. The NO donors S-nitroso-N-acetyl-penicillamine and diethylenetriamine/NO further reduced SPARC expression in cytokine-treated MC as well as in controls. Moreover, downregulation of SPARC mRNA and protein expression in whole kidneys obtained from rats treated with endotoxin was observed. This downregulation of SPARC was reversed by treatment with L-N6-l (iminoethyl) lysine dihydrochloride, a potent and highly selective inhibitor of inducible NOS. These data characterize SPARC as an NO-regulated gene. This observation may be important in the context of tissue remodeling in chronic inflammatory kidney diseases.