Role Of Mitochondrial And Non-mitochondrial Mechanisms Of MUTYH In Protecting Against Acute Renal Injury

Acute kidney injury(AKI)refers to the sharp deterioration in renal function resulted from various insults,leading to a group of severe clinical syndrome involving many systems and presenting as acute renal tubular epithelial cell necrosis in pathology.Our previous studies have confirmed that the mitochondrial dysfunction may play an important role in kidney diseases.Renal tubular epithelial cells are rich in mitochondria.dysfunction of mitochondria can cause the reduction of energy synthesis and increase of reactive oxygen species(ROS).ROS is a kind of typical mutagenic inducers which can oxidize the guanine in DNA chain to 8-hydroxy guanine(8-OHdG),leading to mutation from G:C to T:A,and resulting in DNA mutations causing disease.There are many repair mechanisms in human body,and base excision repair(BER)is the more conservative and common way.Human mutY homologue(MUTYH)gene,human mutT homologue 1(hMTH1)and 8-hydroxy human guanine glycosylation enzyme(hOGGl)collaborate to the repairment of 8-OHdG and the maintainance of the stability of DNA.MUTYH gene plays a major role in the BER system,and its mutation will lead to a decline of body repair function,participating in a variety of diseases.The proteins encoded by human MUTYH gene have two different subcellular localization.The type ? protein locates in the mitochondria to maintain the stability of mitochondrial DNA(mtDNA),while the type ? protein locates in the nucleus to maintain the stability of the nuclear DNA(nDNA)Based on above research background,it is suggested that MUTYH can alleviate acute renal injury by protecting mitochondrial and nuclear DNA.In this study,the role of MUTYH in AKI was studied by using MUTYH gene KO or overexpression in cisplatin-induced acute renal injury model,and the mitochondrial inhibitor rotenone was used to explore the role of mitochondria in folic acid-induced acute renal injury We found that:(1)MUTYH gene knockout reduced the mitochondrial DNA copy number in the mouse kidney and aggravated the renal dysfunction,mitochondrial injury,apoptosis and inflammation in cisplatin induced AKI.(2)Overexpression of type ? MUTYH in mice can effectively improve the renal function,renal tubular injury,apoptosis and inflammatory response in cisplatin-induced AKI.Meanwhile,overexpression of type ? MUTYH in renal tubular cells can significantly alleviate apoptosis and oxidative stressinduced by cisplatin.However,overexpression of mitochondrial(type ?)MUTYH only showed moderate effect in protecting against apoptosis in vitro and inflammation in vivo without significantly affecting the renal function in cisplatin-induced AKI.(3)Mitochondrial respiratory chain complex ?inhibitor rotenone aggravated folic acid-induced renal dysfunction,renal tubular injury,mitochondrial damage,oxidative stress,apoptosis and inflammatory response The findings from current study suggest that MUTYH could protect against the acute kidney injury induced by cisplatin though nonmitochondrial and mitochondrial mechanisms;and mitochondrial dysfunction contributes to the pathogenesis of acute kidney injury.Targeting mitochondria might be a promising strategy for the treatment of acute kidney injuryPart ? MUTYH deletion aggravates cisplatin-induced acute renal injuryObjective:To explore the role of MUTYH gene knockout in the acute renal injury induced by cisplatin and its effect on mitochondriaMethods:The acute renal injury model was established by intraperitoneal injection with 20mg/kg cisplatin(Cis.)in C57BL/6 mice.MUTYH expression was detected by qRT-PCR and Western blot,and the accumulation of 8-hydroxyl-2'-deoxyguanosine(8-OHdG)in kidney was measured by immune fluorescent confocal microscopy Then male MUTYH+/+,MUTYH+/-,and MUTYH-/-mice with ages at 8-10 weeks were divided into 6 groups.The control groups include MUTYH+/+group,MUTYH+/-group and MUTYH-/-group,while the experimental groups include MUTYH+/++cisplatin group,MUTYH+/-+cisplatin group and MUTYH-/-"+cisplatin group.The experimental groups were given ip injection of cisplatin at a dose of 20 mg/kg,and the same volume of saline was intraperitoneally injected into the control mice.After 72 hours,the mice were killed and the kidneys and blood were collected We observed the size,color,and texture of kidneys,detected the blood urea nitrogen and serum creatinine,examined the renal histopathological changes through PAS staining,measured expressions of MUTYH,neutrophils enzyme related lipid carrier protein gelatin molecules(NGAL)and kidney injury molecule-1(KIM-1)by Western blot and qRT-PCR.We also extracted genome DNA of kidney and detected mitochondrial DNA copy number through qRT-PCR.Meanwhile,the expressions of mitochondrial genes(TFAM,mt-COX1,mt-COX2,mt-ATP6,mt-ATP8,mt-CYTB,SOD2),apoptosis protein BAX,inflammatory cytokines of TNF-?,IL-6,and MCP-1 were exanmined.Finally,TUNEL staining of renal tissues was performed to evaluate the apoptotic responseResults:(1)In the kidneys of cisplatin-treated mice,Western blot results showed significant down-regulation of MUTYH protein,while the qRT-PCR results showed a significant up-regulation of MUTYH mRNA expression,suggesting the post-transcriptional regulation of MUTYH.Confocal microscope observed that 8-OHdG expression was increased in line with the downregulation of MUTYH determined by Western blot.Interestingly,mitochondrialcopy number in the kidney of MUTYH knockout mice was slightly decreased as compared with the WT mice,suggesting an effect of MUTYH on mitochondria under the basal conditions.(2)By genotyping analysis,the expressions of MUTYH protein and mRNA were significantly reduced in the kidneys of MUTYH knockout mice.Strikingly,after the mice were challenged with cisplatin to induce AKI,the levels of serum creatinine(Scr)and blood urea nitrogen(BUN)in MUTYH+/-[(108.8±20.25)[mol/L,(55.23±4.16)mmol/L]and MUTYH-/-[(112.5±18.82)?mol/L,(67.65±4.02)mmol/L]groups were significantly higher than that in MUTYH+/+group[(33.60±9.14)[?mol/L,(37.83±4.99)mmol/L].The kidney color of MUTYH knockout mice presented much pale in line with more severe tubular injury as determined by a PAS statining and injury scoring compared with the WT mice after cisplatin treatment.Consistently,renal tubular injury marker of NGAL and KIM-1 were also futher enhanced in MUTYH KO mice,indicting that MUTYH deletion aggravated renal tubular injury induced by cisplatin.(3)After cisplatin treatment,the renal expressions of mitochondrial genes including TFAM,mt-COX1,mt-COX2,mt-ATP6,mt-ATP8,mt-CYTB,SOD2 significantly decreased,which was further reduced in MUTYH+/-and MUTYH-/-groups,suggesting decreased level of mitochondrial transcription,reduced energy synthesis,and weakened anti-oxidation ability.These results indicated that MUTYH deletion further aggravated mitochondrial damage.(4)Apoptotic response is one of the known pathological phenomenon in cisplatin-induced AKI.Here we observed that apoptosis-associated protein BAX wasincreased obviously in line with the enhanced apoptotic cells determined by TUNEL staining in WT mice challenged with cisplatin Significantly,MUTYH knockout mice displayed greater apoptotic response in kidneys as compared with the WT AKI mice.(5)Consistent with the enhanced renal tubular damage,mitochondrial dysfunction and cell apoptosis,we observed that the levels of inflammatory cytokines including TNF-?,1L-6,and MCP-1 in MUTYH+/and MUTYH-/-mice were also higher than that in WT mice challenged with cisplatinConclusion:MUTYH deletion aggravated the acute renal injury induced by cisplatin Meanwhile,MUTYH deletion also reduced mitochondrial DNA copy number in kidney tissues andfurther enhanced mitochondrial damage,apoptosis and inflammatory response in AKI induced by cisplatinPart ? Overexpression of type ? MUTYH alleviated cisplatin-induced acute kidney injury.Objective:To investigate the role of type ? and type ? MUTYH in acute renal injury induced by cisplatinMethods:(1)Using C57BL/6 male mice in 8-10 weeks old with body weight of 20-25g,the overexpressions exogenous type ? and type ? MUTYH were conducted through rapid tail vein injection(10 s)with 2 ml of sterile saline containing 60?g of plasmids(same amount of vector plasmids were injected in control mice).The gene and protein expressions of MUTYH in kidneys of mice were varified by the qRT-PCR.Then acute renal injury was induced by cisplatin(20mg/kg,i.p.)(same amount of sterile saline is injected into mice in control group)after 36 hours tail vein injection of plasmids.In this study,32 mice were divided into four groups:vector group,vector+cisplatin group,MUTYH I+cisplatin group and MUTYH II+cisplatin group.We killed the mice after cisplatin injection for 72 hours.Then we observed the size,color,and texture of kidneys,detected BUN and Scr,examined the renal histopathology by PAS staining,measured acute renal tubular injury markers of NGAL,KIM-1,the apoptosis-associated proteins BAX,and inflammatory cytokines of TNF-?,IL-6,MCP-1 and IC AM-1 by Western blot,qRT-PCR or immunohistochemistry.Using TUNEL staining,we examined cell apoptosis.(2)In vitro,using cultured mouse renal tubular epithelial cells(mPTC)treated with cisplatin at the concentrations of 1,5,10?g/ml,we detected the MUTYH expression by qRT-PCR and Western blot.Then mPTC were transfected with MUTYH ? and ?plasmids with G418 screening(400 ?g/ml)for 2 weeks to get stable cell lines.Using immunofluorescence and Western blot,we verified the target protein expression.The stable cell lines were divided into three groups:the vector+cisplatin group,the MUTYH I+cisplatin group and MUTYH ?+cisplatin group.Using AnnexinV-PI and flow cytometry,we examined mPTC apoptosis,and using DCFDA fluorescence,we measured cellular reactive oxygen species(ROS)Results:(1)After rapid tail vein injection of the exogenous MUTYH plasmids,MUTYH expression in mice kidney tissues is significantly higher than that in the control group as determined by qRT-PCR.(2)By detecting renal function indexes,over-expressing type ? MUTYH significantly reduced the levels of Scr and BUN in cisplatin-treated mice compared with vector-and type ? MUTYH-overexpressed mice The concentrations of Scr and BUN in the mice after AKI of vector,type ? MUTYH and type ? MUTYH were[(136.5±5.10)vs.(83.26±27.23)vs.(66.88 ±10.55)?mol/L]and[(39.62±2.88)vs.(53.97±7.97)vs.(17.78±3.51)mmol/L]respectively.PAS staining also displayed an ameliorated morphological lesions in mice with overexpression of type ? MUTYH in parallel with the lowered expressions of NGAL and KIM-1.(3)Overexpression of type ? MUTYH significantly reduced the expression of pro-apoptotic protein BAX in cisplatin-treated mouse kidney accompanied with reduced apoptotic cell number determined by TUNEL staining.(4)Overexpression of type ? MUTYH significantly inhibited inflammatory cytokines of TNF-?,IL-6,MCP-1,and ICAM-1 in cisplatin-treated mouse kidney.Also,over-expression of type ? MUTYH down-regulated the expressions of TNF-? and MCP-1 without significant influence on other parameters.(5)Using different concentrations of cisplatin to stimulate renal tubular epithelial cells,MUTYH protein expression was dose-dependently reduced,while MUTYH mRNA expression was increased,suggesting that post-transcriptional regulation mechanism might be existed in modulating MUTYH protein expression.Immunofluorescence analysis confirmed that type ? MUTYH locates in the mitochondria,while type ? MUTYH locates in the nucleus.Because MUTYH plasmids contain FLAG label and neomycin resistance gene,thus,after G418 screening,we detected the FLAG and MUTYH protein expressionsby Western blot to verified the transfection efficiency.(6)After cisplatin stimulation,we used flow cytometry to test cell apoptosis by AnnexinV-PI and found that overexpressions of type ? and type ? MUTYH significantly inhibited cell apoptosis with greater efficacy of type ? MUTYH overexpression.Using fluorescent probe DCHFDA and flow cytometry we observed that overexpression of type ?MUTYH but not type ? MUTYH can significantly suppress the ROS production These data indicated that type ? MUTYH is more potent that type ? MUTYH in suppressing renal tubular cell apoptosis induced by cisplatinConclusion:Overexpression of type ? MUTYH can more effectively improve the renal function,renal tubular injury,cell apoptosis and inflammatory response in cisplatin-induced AKI.Part ? Inhibition of mitochondrial complex I aggravated folic acid-induced acute kidney injuryObjective:To explore the effect of mitochondrial complex I inhibitionon folic acid induced acute kidney injuryMethods:8-10 weeks old male C57BL/6 mice were randomly divided into 3 groups:control group(sodium bicarbonate injection group),model group(folic acid group),the treatment group(Rotenone+folic acid group),Twenty four hours before folic acid(FA)injection,the mice were subjected to the jelly diet with or without rotenone at a dose of 200 ppm.Then mice were injected(i.p.)with FA at 240 mg/kg bodyweight.Sodium bicarbonate(0.3M NaHCO3,the vehicle used for FA administration)alone was injected into the controls.After 72h FA administration,the animals were killed.Using biochemical analyzer we detected the levels of BUN and Scr,observed the renal histopathology by PAS staining,examined the expressions of NGAL,SOD2,HO-1 and BAX by Western blot,measured the level of mitochondrial DNA copy number and mRNA expressions of mt-COX1,mt-ND1,HO-1,IL-6 and ICAM-1 by qRT-PCR.Kiidney tissue MDA content was also examined by using a MDA Assay Kit.Serum TNF-? concentration was measured by ELISA.Using TUNEL staining,we observed cell apoptosis of kidney tissues.In addition,to evaluate the toxicity of rotenone at a dose of 200ppm in food,we observed body weight changes and serum concentrations of ALT,AST,LDH,BUN,and Scr in mice treated with vehicleor rotenone(200ppm in food)aloneResults:(1)Compared with the folic acid alone group,rotenone treatment further increased the levels of BUN and Scr in AKI mice in line with the aggravated morphological damage shown by the PAS staining and enhanced tubular injury marker of NGAL determined by qRT-PCR and Western blot.The concentrations of BUN and Scr in the control group,model group and experimental group were[(10.32±0.68)vs.(18.09±2.04)vs.(121.0±2.91)mmol/L]and[(31.80±3.92)vs.(67.40±9.24)vs.(368.1±21.23)?mol/L]respectively.(2)In folic acid-treated mice,the mtDNA copy number and the expressions of mtND1 and mtCO1 were obviously reduced,which was further worsened in rotenone-treated AKI mice.(3)The MDA content and heme oxygenase-1(HO-1)expression in kidneys were significantly higher in rotenone-treated AKI mice than that in the mice with folic acid alone treatment Meanwhile,the expression of antioxidant SOD2 was further downregulated in rotenone-treated AKI mice.These data suggested that rotenone treatment promoted the oxidative stress.(4)In folic acid-induced AKI,the cell apoptosis was obviously induced as shown by the TUNEL staining BAX expression.Strikingly,such an apoptotic response was further worsened in rotenone-treated AKI mice.(5)As shown by the analyses of qRT-PCR and ELISA,the levels of IL-6,ICAM-1 in kidney tissues and the level of serum TNF-? were further enhanced in rotenone-treated AKI mice.(6)In order to assess the toxicity of rotenone,we observed body weight change and the blood levels of ALT,AST,LDH,BUN,and Scr,and found no significant difference between the groups with or without rotenone treatmentConclusion:Inhibition of mitochondrial complex I activity aggravated renal tubular injury,mitochondrial injury,oxidative stress,cell apoptosis,and inflammation in acute kidney injury induced by folic acid.