| Background Chronic renal failure(CRF)is a major public health problem worldwide.It is now commonly agreed that the presence of CRF identifies a higher risk state in the middle aged and elderly population.There is no better way to treat other than regular dialysis.Progression of CRF is associated with increased oxygen species production,apoptosis or necrosis,inflammatory cells,events that cause extracellular matrix accumulation and tubulointerstitial fibrosis.Therefore therapeutic interventions aimed at ameliorate of renal function can be highly effective in retarding progression of CRF.Hydrogen sulfide(H2S)has been recognized as an important signalling molecule throughout the body,contributing to many physiological and pathological processes.Many studies suggest that H2 S participates in the control of renal function and increases urinary sodium excretion via both vascular and tubular actions in the kidney.In the model of renal ischemia and reperfusion,diabetic nephropathy and so on,it has been proved that hydrogen sulfide has a certain effect on these diseases.However,whether H2 S could protect against CRF in rats remains unclear.Objective To investigate the effects and mechanisms of hydrogen sulfide on chronic renal failure and its mechanism.Methods 1.In vitro studies NRK-52 E cells were incubated with 3 m M GEN for 24 h to induce nephrotoxicity,The cells were divided into three groups: Control group,GEN group,and GEN+H2S group.The control and GEN groups were treated with phosphate-buffered saline(PBS)and the GEN+H2S group was treated with 100 μM Na HS(an H2 S donor,dissolved in PBS).Treatments with PBS and Na HS were concomitant to GEN-induced nephrotoxicity for 24 h.Cell proliferation and viability test 5-ethynyl-2’-deoxyuridine(Ed U)incorporation assay and MTS assayThe proliferating cells were examined using the Cell-Light Ed U Apollo 567 In Vitro Imaging Kit.Cell proliferation rate =(Ed U-positive cells)/(total number of cells)×100%.Cell growth was also measured using the Cell Titer 96 AQueous One Solution Cell Proliferation MTS Assay.The higher OD value,the greater the level of cell proliferation.1.2 Detection of intracellular reactive oxygen species(ROS)Intracellular ROS generation was measured by using a 2’,7’-dichlorofluorescin diacetate(DCF-DA)-Cellular reactive oxygen species detection Assay.Cells were incubated with 10 μM DCF-DA for 30 min at 37°C and washed three times with serum free cell culture medium.The fluorescence was observed by a fluorescent microscope.1.3 Cellular apoptosis analysis Cellular apoptosis was analyzed by performing a terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL)assay using an in situ cell death detection.Then after a 10 min DAPI(5 mg/ml)counterstain at room temperature,cells were photographed with a fluorescent microscope.The apoptotic index =(positively stained apoptotic cells)/(total number of cells)×100%.2.In vivo studies 2.1 Establishment and grouping of experimental models in rats The experimental has 3 groups: control group,CRF group and CRF+H2S group.CRF was induced with feeding adenine at a concentration of 0.75%w/w for four weeks.The rats from control and CRF groups received an intraperitoneal(i.p.)injection of saline and the rats from the CRF+H2S group received an i.p.injection of Na HS(100 μmol/kg/day,dissolved in saline)Treatments with saline and Na HS were concomitant to adenine-induced CRF for 4 weeks.During the treatment periods,the rats were weighed weekly and the food intake,water intake,and urine volume were measured in 24 h.At the end of experiments,the rats were killed and the plasma and kidneys were collected.2.2 Histological analysis The renal tissues were fixed in formalin,embedded in paraffin,and cut into 5-μm-thick sections which were then stained with hematoxylin and eosin(HE)and Masson’s trichrome(MT).2.3 Examination of blood routine,blood biochemical and inflammatory factors The levels of white blood cell(WBC),red blood cell(RBC),hemoglobin(HGB),and hematocrit(HCT)were detected.Blood urea nitrogen(BUN),creatinine(Cre),and urinary protein(UP)were measured.Monocyte chemoattractant protein(MCP)-1,tumor necrosis factor(TNF)-α,interleukin(IL)-6,and IL-10 in kidney tissues were determined using commercial ELISA methods.2.4 Western blot analysis Expression of related proteins with MAPK pathway: ERK1/2,phosphoated-ERK,JNK,phosphoated-JNK,p38,phosphoated-p38.Expression of related proteins with NF-κB pathway:p50,p65,phosphoated-p65.Proteins associated with apoptosis: Bax,Bcl-2,Caspase-3 and Cleaved Caspase-3.2.5 Measurement of oxidative stress products The levels of malondialdehvde(MDA)and ROS,as well as the activities of glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)were measured.Results In this study,we found that H2 S alleviated gentamicin-induced nephrotoxicity by reducing reactive oxygen species(ROS)-mediated apoptosis in normal rat kidney-52 E cells.In addition,hydrogen sulfide can also improve the proliferation of cells treated with gentamicin.We demonstrated that H2 S significantly improved the kidney structure and function of CRF rats.We found that H2 S decreased the protein levels of Bax,Caspase-3 and Cleaved-caspase-3,but increased the expression of Bcl-2.Treatment with H2 S reduced the levels of malondialdehyde and ROS and increased the activities of superoxide dismutase and glutathione peroxidase.H2 S significantly abolished the phosphorylation of extracellular signal-regulated protein kinase 1/2,c-Jun N-terminal kinase,and p38 in the kidney of CRF rats.Furthermore,H2 S decreased the expression levels of tumor necrosis factor-α,interleukin(IL)-6,IL-10,and monocyte chemoattractant protein-1,as well as the protein levels of p50,p65,and p-p65 in the kidney of CRF rats.Conclusion Hydrogen sulfide can relieve chronic renal failure in rats;H2S inhibits renal cell apoptosis via ROS/MAPK signaling pathway;H2S inhibits cell inflammatory response through nuclear factor-kappa B signaling pathways.Hydrogen sulfide or its releasing compounds may serve as a potential therapeutic molecule for CRF. |
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