Role Of Mitochondrial Dysfunction In Renal Fibrosis Promoted By Hypochlorite-modified Albumin In A Remnant Kidney Model And Protective Effects Of Antioxidant Peptide SS-31

BackgroundOxidative stress aggravates renal fibrosis,a pathway involved in almost all forms of chronic kidney disease(CKD).Oxidative stress is defined as disequilibrium between the generation and scavenging of reactive oxygen species(ROS).Mitochondria are not only the main source of ROS but also the organelles most sensitive to oxidative stress,lead to mitochondrial dysfunction and closely associated with various kidney disease such as Diabetic Nephropathy,Obstructive Nephropathy and Ischemic Kidney Injury.However,the underlying mechanism involved in the pathogenesis of renal oxidative stress has not been completely elucidated.In this study,we explored the role and mechanism of hypochlorite-modified albumin(HOCl-alb)in mediating oxidative stress and fibrotic response in a remnant-kidney rat model.MethodsFive-sixths nephrectomy(5/6 NX)was performed on the rats(n=32,male Sprague-Dawley)and then the 5/6 NX animals were randomly assigned to one of these four groups(n = 8 in each group):(1)vehicle,5/6 NX rats were given phosphate-buffered saline(PBS)intravenously(i.v.)once every two days at equal volume with Hypochlorite-modified rat serum albumin(HOCl-RSA);(2)HOCl-RSA,20 mg/kg HOCl-RSA i.v.once every two days;(3)SS-31,3 mg/kg i.p.once a day;(4)HOCl-RSA + SS-31,20 mg/kg HOCl-RSA i.v.once every two days and 3 mg/kg i.p.once a day.At the end of 13 weeks,24-hour urine samples were collected using a metabolism cage.Serum samples and the remnant kidneys were collected for further analysis.The levels of serum and urinary creatinine(Scr and Ucr,respectively),blood urea nitrogen(BUN),and 24-hour urinary protein(Up)concentrations were measured and the creatinine clearance(Ccr)was calculated.Pathological changes of renal tissues were observed under light microscope.Mitochondrial and cytosolic fractions were isolated and Mitochondrial Membrane Potential(MMP),ATP content and Mitochondrial reactive oxygen species(mtROS)were detected.HOCl-alb content in plasma and kidney tissue and the enzyme activities of manganese superoxide dismutase(MnSOD)were determined.Macrophage infiltration(ED-1 positive cells)were detected by Immuno-peroxidase staining.Cytochrome C(Cyto C)were measured by western blot in both mitochondria and cytoplasm of the renal cortex,mitochondrial DNA(mtDNA)copy numbers were detected by Real-time PCR.In addiation,levels of transforming growth factor(TGF)-?1,collagen(COL)-I,monocyte chemoattractant protein(MCP)-1,?-smooth muscle actin(?-SMA)were also measured by Immuno-peroxidase staining,western blot and real-time PCR.ResultsCompared with the control group,5/6 NX animals displayed marked mitochondrial(mt)dysfunction,as evidenced by decrease of mitochondrial membrane potential(MMP),ATP production,mtDNA copy number alterations and manganese superoxide dismutase(MnSOD)activity,release of cytochrome C(Cyto C)from mitochondria to the cytoplasm,and increase of mt reactive oxygen species(ROS)in renal tissues.They also displayed increased levels of HOCl-alb in both plasma and renal tissues.These changes were accompanied by accumulation of extracellular matrix,worsened proteinuria,deteriorated renal function,and a marked increase of macrophage infiltration along with up-regulation of monocyte chemoattractant protein(MCP)-1 and transforming growth factor(TGF)-?1 expression.HOCl-alb challenge further exacerbated the above biological effects in 5/6 NX animals,but these adverse effects were prevented by administration of SS-31,a mitochondrial targeted antioxidant peptide.ConclusionThese data suggest that accumulation of HOCl-alb may promote renal inflammation and fibrosis,probably related to mitochondrial oxidative stress and dysfunction and that the mitochondrial targeted peptide SS-31 might be a novel therapy for renal fibrosis and chronic renal failure(CRF).