Study Of Inhibitive Effects Of Hydrogen Sulfide On Renal Fibrosis And Related Mechanisms

Part I Hydrogen sulfide (H2S) is associated with the pathogenesisof unilateral uretheral obstruction.Objective: To examined the expression of two H2S-producing enzymes-cystathionine-synthase (CBS) and cystathionine γ-lyase(CSE) in the real cortex ofUUO rats, a well-known animal model of renal fibrosis.Methods: Rats were received with sham or UUO operation，and were sacrificed14days after the operation. The expression of CBS and CSE were determined by westernblot and the concentration of serum hydrogen sulfide was examined using a novelhydrogen sulfide fluorescent probe (DNZ-As).Results: Compared with the sham operation rats, the expression of CBS in UUOrats was considerably reduced, whereas CSE expression was enhanced. In spite of this,the serum concentration of H2S in UUO rats was decreased by1/3compared with thesham ones.Conclusions: These results indicated that H2S was involved in the pathogenesis ofrenal fibrosis induced by UUO. It also implied that supplementation with H2S mayreduce renal fibrosis. Part II Low-dose NaHS improved the pathology of UUOObjective: To study the effect of NaHS, a H2S donor on UUO, and to determinethe safe dose range.Methods: NaHS was given at a dose range from5.6to560g/kg/d in UUO rats. The effects of CBS inhibitor, aminooxyacetate (AOAA) and CSE inhibitor,propargylglycine (PAG) on UUO were also explored. Enalapril was set as the positivecontrol. Rats were divided into eight groups as follows: sham, UUO, UUO+10mg/kg/dEnalapril, UUO+5.6g/kg/d NaHS, UUO+56g/kg/d, UUO+560g/kg/d, UUO+5mg/kg/d AOAA, UUO+25mg/kg/d PAG. NaHS, PAG and AOAA were givenintraperitoneally once daily three days before surgery and continued for two weeks afteroperation. Enalapril was given via intragastria. UUO rats received vehicle (saline)treatment.Results: Compared with the UUO group, Enalapril,5.6g/kg/d NaHS and56g/kg/d NaHS attenuated the renal fibrosis of UUO rats by decreasing the volumes ofthe obstructed kidneys and increased the thickness of the renal cortex. Renaltubularinterstitial injury scores in HE staining were also decreased accompanied withreduced deposition of fibers in renal intersitium as shown by Masson staining. Furthermore, Immunohistochemisty and western blot analysis demonstrated that expression ofFN and SMA were reduced by the above mentioned drugs. In contrast,560g/kg/dNaHS、AOAA and PAG were unable to exhibit anti-fibrotic effects in UUO rats.Conclusions: These data suggested that low-dose could attenuate the renal fibrosisinduced by UUO injury. This protective effect started at5.6g/kg/d, culminated at56g/kg/d, and may be reversed at560g/kg/d. Part III Hydrogen sulfide (H2S) suppressed the activation of renalfibroblastObjective: To study whether H2S was able to block the proliferation anddifferentiation of renal fibroblast.Methods:(1) Serum-starved renal fibroblasts were incubated withTGF-2ng/ml) with or without NaHS (100mol/L) for12and24h respectively.RT-PCR and western blot were used to detect the protein of FN and SMA mRNA inthe cells.(2) Serum-starved renal fibroblasts were stimulated with10%FBS with orwithout NaHS (100mol/L) for24h. MTT assay was used to evaluate the number of thesurvived cells. Cell DNA synthesis was assessed by Brdu incorporation and the proliferation-related proteins were detected with western blot.Rsults: Stimulation with TGF-2ng/ml) significantly enhanced the expressionof collagen-I、FN and SMA mRNA by the end of12h and up-regulated FN and SMA proteins (P<0.05). Such effects were reversed by the pretreatment with aHS(100mol/L) for0.5h (P<0.05). MTT assay demonstrated that cell numbers wereconsiderably increased by stimulation with10%FBS. Preincubation with NaHS(10-100μM) for0.5h decreased the cell number induced by FBS. Such an inhibitoryeffect was most prominent at100μM NaHS. Pretreatment with NaHS (100μM) alsodecreased the BrdU incorporation into the cell DNA compared with the stimulation withFBS alone. Further, NaHS100μM suppressed the FBS-induced up-regulation ofproliferation-related proteins, including PCNA and C-myc (P<0.05).Conclusions: aHS is able to inhibit the cell differentiation and proliferation ofrenal fibroblasts. Part IV The mechanisms of the anti-fibrotic effect of HydrogensulfideObjective: To examine the mechanisms of the anti-fibrotic effect of Hydrogensulfide.Methods:(1) The effects of H2S on the TGF-1-induced phorsporylation of ERK,p38, JNK and Smad3with western blotting analysis. TGF-1(2ng/ml) was used tostimulate the renal fibroblast at various time points(0.5-2h). TGF-1(2ng/ml) triggereddistinct expression patterns in these proteins, as indicated by enhanced phosphorylationof these proteins.(2) Cells were incubated with TGF-1for1h with or without NaHS(100mol/L). DCFH-DA was used to detect the ROS production induced by TGF-1inthe renal fibrobalsts.(3) Immunohistochemistry was used to detect the CD68positivemacrophages in renal cortex of different animal groups as indicated in part II.Results:(1) The induction of p38phosphorylation, which reached its peak at0.5hafter TGF-1stimulation, was quicker than that of the other molecules. Specifically,phosphorylation of ERK and JNK peaked at2h after TGF-1exposure whereas Smad3 at1h. Pretreatment with100mol/L NaHS abolished the phosphorylations of MAPKsand Smad3induced by TGF-1stimulation for1h.(2) NaHS attenuated thefluorescence of ROS trigged by TGF-1.(3) Low-dose NaHS inhibited the infiltrationof macrophage in the renal cortex of UUO animals.Conclusions: These data suggested that aHS inhibited renal fibrosis by blockingthe TGF-1and MAPK signal pathway as well as attenuating oxidative stress andinflammation.