Reduced Hydrogen Sulfide Bioavailability Contributes to Cardiometabolic Heart Failure with Preserved Ejection Fraction.

Background: Heart failure with preserved ejection fraction (HFpEF) is a significant public health concern with limited treatment options. Dysregulated nitric oxide-mediated signaling has been implicated in HFpEF pathophysiology, however, little is known about the role of endogenous hydrogen sulfide (H2S). Objectives: This study evaluated H2S bioavailability in patients and two animal models of cardiometabolic HFpEF and assessed the impact of H2S on HFpEF severity through alterations in endogenous H2S production and pharmacological supplementation. Methods: HFpEF patients and two rodent models of HFpEF ("two-hit" L-NAME + HFD mouse and ZSF1 obese rat) were evaluated for H2S bioavailability. Two cohorts of two-hit mice were investigated for changes in HFpEF pathophysiology: (1) endothelial cell cystathionine-γ-lyase (EC-CSE) knockout; (2) H2S donor, JK-1, supplementation. Results: H2S levels were significantly reduced (i.e., 81%) in human HFpEF patients and in both preclinical HFpEF models. This depletion was associated with reduced CSE expression and activity, and increased SQR expression. Genetic knockout of H2S -generating enzyme, CSE, worsened HFpEF characteristics, including elevated E/e' ratio and LVEDP, impaired aortic vasorelaxation and increased mortality. Pharmacologic H2S supplementation restored H2S bioavailability, improved diastolic function and attenuated cardiac fibrosis corroborating an improved HFpEF phenotype. Conclusions: H2S deficiency is evident in HFpEF patients and conserved across multiple HFpEF models. Increasing H2S bioavailability improved cardiovascular function, while knockout of endogenous H2S production exacerbated HFpEF pathology and mortality. These results suggest H2S dysregulation contributes to HFpEF and increasing H2S bioavailability may represent a novel therapeutic strategy for HFpEF. Highlights: H2S deficiency is evident in both human HFpEF patients and two clinically relevant models.Reduced H2S production by CSE and increased metabolism by SQR impair H2S bioavailability in HFpEF.Pharmacological H2S supplementation improves diastolic function and reduces cardiac fibrosis in HFpEF models.Targeting H2S dysregulation presents a novel therapeutic strategy for managing HFpEF.

Cross-Regulation of the Cellular Redox System, Oxygen, and Sphingolipid Signalling.

Redox-active mediators are now appreciated as powerful molecules to regulate cellular dynamics such as viability, proliferation, migration, cell contraction, and relaxation, as well as gene expression under physiological and pathophysiological conditions. These molecules include the various reactive oxygen species (ROS), and the gasotransmitters nitric oxide (NO∙), carbon monoxide (CO), and hydrogen sulfide (H2S). For each of these molecules, direct targets have been identified which transmit the signal from the cellular redox state to a cellular response. Besides these redox mediators, various sphingolipid species have turned out as highly bioactive with strong signalling potential. Recent data suggest that there is a cross-regulation existing between the redox mediators and sphingolipid molecules that have a fundamental impact on a cell's fate and organ function. This review will summarize the effects of the different redox-active mediators on sphingolipid signalling and metabolism, and the impact of this cross-talk on pathophysiological processes. The relevance of therapeutic approaches will be highlighted.

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.

The Pathophysiology of H2S in Renal Glomerular Diseases.

Renal glomerular diseases such as glomerulosclerosis and diabetic nephropathy often result in the loss of glomerular function and consequently end-stage renal disease. The glomerulus consists of endothelial cells, mesangial cells and glomerular epithelial cells also referred to as podocytes. A fine-tuned crosstalk between glomerular cells warrants control of growth factor synthesis and of matrix production and degradation, preserving glomerular structure and function. Hydrogen sulfide (H2S) belongs together with nitric oxide (NO) and carbon monoxide (CO) to the group of gasotransmitters. During the last three decades, these higher concentration toxic gases have been found to be produced in mammalian cells in a well-coordinated manner. Recently, it became evident that H2S and the other gasotransmitters share common targets as signalling devices that trigger mainly protective pathways. In several animal models, H2S has been demonstrated as a protective factor in the context of kidney disorders, in particular of diabetic nephropathy. Here, we focus on the synthesis and action of H2S in glomerular cells, its beneficial effects in the glomerulus and its action in the context of the other gaseous signalling molecules NO and CO.

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.

H2S-induced thiol-based redox switches: Biochemistry and functional relevance for inflammatory diseases.

During the last decades, small inorganic molecules like reactive oxygen species (ROS), nitric oxide (NO), carbon monoxide (CO) and even the highly toxic hydrogen sulfide (H2S) have been evolved as important signaling molecules that trigger crucial cellular processes by regulating the activity of kinases, phosphatases and transcription factors. These redox molecules use similar target structures and therefore, the composition of the complex "redox environment" determines the final outcome of signaling processes and may subsequently also affect the behavior of a cell in an inflammatory environment. Here, we discuss the role of H2S in this complex interplay with a focus on the transcription factors Nrf2 and NFκB.

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.

Expression of ubiquitin carboxy-terminal hydrolase-l1 in photocoagulated human retinal pigment epithelial cells.

PURPOSE: To date, the exact mechanisms involved in laser-induced remission of ocular neovascular disorders are still poorly understood. Recent studies suggest that the expression of various antiangiogenic cytokines is upregulated after thermal photocoagulation. In the current study, we sought to identify novel laser-regulated proteins in cultured human retinal pigment epithelial (HRPE) cells. METHODS: Protein extracts from photocoagulated HRPE cells were subjected to 2D-gel electrophoresis. Potentially regulated protein spots were identified by mass spectroscopy. Regulation of protein and mRNA was determined by Western blot analysis and reverse transcription-polymerase chain reaction, respectively. RESULTS: 2D-Gel electrophoresis of HRPE whole-cell extracts exposed to photocoagulation revealed a reproducible increase in a protein band identified as ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) compared with untreated controls. Protein levels showed a time-dependent upregulation over 24 hr. UCH-L1 mRNA was maximally increased after 8 hr. CONCLUSIONS: Our findings indicate that the ubiquitin-proteasome system contributes to the effects seen clinically after thermal photocoagulation in eyes with neovascular diseases of the retina or choroid. Because ubiquitin carboxy-terminal hydrolase-L1 has been shown to be involved in the regulation of cell cycle proteins, it may be speculated that deubiquitinating enzymes have a role in the regeneration and proliferation of retinal pigment epithelial cells.

Nitric oxide down-regulates the expression of the catalytic NADPH oxidase subunit Nox1 in rat renal mesangial cells.

Glomerular mesangial cells can produce high amounts of nitric oxide (NO) and reactive oxygen species (ROS). Here we analyzed the impact of NO on the ROS-generating system, particularly on the NADPH oxidase Nox1. Nox1 mRNA and protein levels were markedly decreased by treatment of mesangial cells with the NO-releasing compound DETA-NO in a concentration- and time-dependent fashion. By altering the cGMP signaling system with different inhibitors or activators, we revealed that the effect of NO on Nox1 expression is at least in part mediated by cGMP. Analysis of a reporter construct comprising the 2547 bp of the nox1 promoter region revealed that a stimulatory effect of IL-1beta on nox1 transcription is counteracted by an inhibitory effect of IL-1beta-evoked endogenous NO formation. Moreover, pretreatment of mesangial cells with DETA-NO attenuated platelet-derived growth factor (PDGF)-BB or serum stimulated production of superoxide as assessed by real-time EPR spectroscopy and dichlorofluorescein formation. Transfection of mesangial cells with siRNAs directed against Nox1 and Nox4 revealed that inhibition of Nox1, but not Nox4 expression, is responsible for the reduced ROS formation by NO. Obviously, there exists a fine-tuned crosstalk between NO and ROS generating systems in the course of inflammatory diseases.

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.

Pigment-epithelium-derived factor is upregulated in photocoagulated human retinal pigment epithelial cells.

There is much evidence that pigment-epithelium-derived factor (PEDF) is a potent antiangiogenic cytokine which inhibits retinal and choroidal neovascularization by inducing apoptosis in activated vascular endothelial cells. Furthermore, the regulation of PEDF appears to be linked to the regulation of vascular endothelial growth factor (VEGF), one of the most potent inducers of intraocular neovascularization. Previous studies have established that thermal photocoagulation, the mainstay in the therapy of various neovascular diseases of the posterior segment, results in a decrease in intraocular concentrations of VEGF and other angiogenic growth factors, thereby inhibiting active retinal neovascularization. In the current study, we sought to determine whether thermal photocoagulation has the potential to regulate the expression of PEDF in human retinal pigment epithelial (RPE) cells. Cultures of RPE cells were photocoagulated with a 532-nm diode laser. Subsequently, RNA was isolated for RT-PCR, and whole-cell extracts and precipitated cell culture supernatant were subjected to Western blot analysis. According to our results, PEDF mRNA and protein are significantly upregulated after photocoagulation. Moreover, PEDF protein was found to be secreted in the cell culture medium.

Nitric oxide upregulates induction of PDGF receptor-alpha expression in rat renal mesangial cells and in anti-Thy-1 glomerulonephritis.

PDGF and nitric oxide (NO) have been shown to participate in the progression of several forms of glomerulonephritis. A potential influence of NO on PDGF-mediated signaling cascades was therefore examined. Treatment of rat mesangial cells (MC) with the NO donors diethylenetriamine NO (DETA-NO) or spermine-NONOate resulted in a time- and dose-dependent upregulation of PDGF receptor alpha (PDGFRalpha) but not PDGFRbeta mRNA levels. Administration of DETA-NO also induced PDGFRalpha protein expression that was paralleled also by an enhanced receptor phosphorylation. Further experiments using 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1), an activator of the soluble guanylyl cyclase (sGC), the membrane-soluble cyclic GMP (cGMP) analog 8-Bromo-PET-cGMP, and the inhibitors of sGC ODQ and NS2028 suggest that elevated cGMP levels are responsible for the effects of NO. Importantly, NO-dependent autophosphorylation of PDGFRalpha drastically augmented PDGF-AA-evoked phosphorylation of PKB/Akt, a classical downstream target of PDGFRalpha signaling. Furthermore, in a rat model of anti-Thy-1 glomerulonephritis, expression and phosphorylation of PDGFRalpha but not PDGFRbeta expression was markedly reduced in nephritic animals that were treated with the inducible NO synthase inhibitor L-N6(1-iminoethyl)lysine(dihydrochloride) (L-NIL) compared with non-L-NIL-treated nephritic rats as demonstrated by Western blotting and immunohistochemistry. Taken together, the data suggest that NO modulates PDGFRalpha-triggered signaling in a cGMP-dependent manner by induction of PDGFRalpha expression in MC and in a rat model of mesangioproliferative glomerulonephritis. The mechanistic details of this regulation have to be elucidated in further experiments.