الفهرس 
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Abstract
Renal mesangial cells liberate high amounts of reactive oxygen species (ROS), nitric oxide and other proinflammatory mediators after stimulation with cytokines or growth factors. There is increasing evidence that hydrogen sulfide that is endogenously produced in several cell types serves as a potent gasotransmitter in inflammatory diseases. Therefore, the regulation of the hydrogen sulfide synthesizing enzyme cystathionine yylyase (CSE) in cultured rat and human renal mesangial cells was analysed. As demonstrated by qPCR and Western blot experiments, mesangial cells showed a marked time- and dose-dependent upregulation of CSE mRNA and protein levels after treatment with platelet-derived growth factor-BB (PDGF-BB) in rat mesangial cells. The JAK2 inhibitor, AG-490 and the extracellular signal-regulated kinases (ERK) inhibitor Uo126 markedly reduced PDGF-BB-induced CSE mRNA and protein levels. Interestingly, co-administration of the ROS scavengers N-acety1cysteine (NAC), the glutathione peroxidase mimetic ebselen and the NADPH oxidase inhibitor diphenylen iodonium chloride (DPI) drastically reduced PDGFinduced CSE expression, indicating a crucial role for endogenously produced ROS in the regulation of CSE. Moreover, ROS measurement revealed that PDGF-BB induced ROS generation is paralleled by an induction of CSE. As shown by electrophoretic mobility shift (EMSA) experiments, PDGF-BB induced binding of NF-E2-related factor 2 (Nrf2) to a respective consensus antioxidant element (AREcons) or a promoter sequence (AREcse) and this effect was nearly abolished by cooadministration of the antioxidants mentioned above. In addition, the binding of nuclear Nrf2 protein to wild-type AREcse induced by PDGF-BB was abolished, when a mutant-type of AREcse was used as a probe for the EMSA studies. LPS-induced CSE expression in mouse spleen macrophages was completely abolished in Nrf2 knock-out mice compared to wild-type
mice corroborating a role for Nrf2 in CSE expression. In addition, the activator of Nrf2, namely tBHQ, upregulated CSE mRNA in a comparable manner to PDGF-BB. PDGF-BB and tBHQ regulated HO-l mRNA or protein expression in a similar manner to that of CSE, further suggesting a role for Nrf2 in CSE regulation. Using luciferase reporter gene analysis of the CSE promoter, it was demonstrated that tBHQ drastically increased the luciferase activity conferred by a genomic CSE DNA fragment spanning 3060 bp of the 5’ -flanking region of CSE. This effect was completely abolished, when a point mutation was introduced in the binding motif of a putative ARE in the CSE promoter. However, PDGF-BB did not affect CSE promoter activity suggesting further regulatory elements upstream of the cloned genomic CSE DNA to be essential for PDGF-BB-induced CSE transcription. Besides the detailed analyis of Nrf2, additional experiments were performed to clarify a possible role ofNFKB, another redox-sensitive transcription factor in CSE expression. As demonstrated by qPCR, the IkBBkinase inhibitor III and transfection of a siRNA targeted to the NFKB subunit p65 clearly reduced PDGF-BB-induced CSE mRNA expression. Analysis of nuclear protein revealed that PDGF-BB clearly translocated p65 into the nucleus after 8 hour indicating that PDGF-BB drives NF-KB activation in rat mesangial cells. Some additional experiments were also performed with human mesangial cells. In cooperation with the group of Prof. Liliana Schaefer (University of Frankfurt am Main), CSE and Nrf2 protein expression were analyzed in a rat model of anti- Thy-I-induced proliferative glomerulonephritis. The observation established a marked upregulation of CSE protein paralleled by a stabilization of the Nrf2 protein at day 3 after the injection of the anti- Thy. I-antibody. from these data, the current study concludes and hypothesizes that, PDGF induces CSE mRNA and protein expression with subsequent H2S formation in a time and dose dependent manner in rMC. Induction of CSE expression by PDGF .