The Role Of MUTYH In Acute Kidney Injury Induced By Ischemia-reperfusion And Its Mechanism

Acute kidney injury?AKI?is a common clinical complication in patients,especially in patients who severely ill.It can be caused by a variety of causes.It often manifests as a sharp decline in acute renal function,with or without oliguria or anuria.The incidence of AKI increased year by year.A cross-sectional study in 2013 showed that the detectable rate of AKI in inpatients in China was 1%?2%according to expand criterion?.AKI has been a dependent risk for predication of death.The mortality of AKI patients is 10%and the mortality rate of AKI with multiple organ dysfunction is up to 50%.When AKI progress to require renal replacement therapy,the mortality rates can reach 80%.Because of the lack of effective treatments,AKI can easily progress to chronic kidney disease or even end-stage renal disease.Severe patients need renal replacement therapy,which brings a great economic burden to families and society.Consequently,we should investigate the mechanism of AKI and provide new therapeutic targets for acute kidney injury.Mitochondria,the body's energy factory,is the main site for intracellular oxidative phosphorylation and synthesis of adenosine triphosphate?ATP?.In addition,mitochondria also play a vital role in physiological functions such as cell differentiation,cell information transmission,and cell apoptosis.Our group's research found that mitochondrial dysfunction was an early event of acute kidney injury.Oxidation stress is an important factor leading to mitochondrial damage.Excessive production of reactive oxygen species?ROS?attacks membrane proteins,lipids and mitochondrial DNA?mitochondrial DNA?in mitochondria,leading to mitochondrial dysfunction.The main manifestations of mitochondrial dysfunction:mitochondrial respiratory chain oxidative phosphorylation?OXPHOS?dysfunction resulting in decreased ATP production,increased mitochondrial ROS production and mtDNA damage?copy number reduction and mutation?.8-oxo-Guanine?8-oxo-G?is produced under oxidative stress by guanine,the most common product of DNA damage.It can mismatched with adenine which induce GC-AT point mutations.Human mutY homolog?hMUTYH?is one of the key enzymes in the repair of 8-oxo-G.It cleaves the adenine mismatched with 8-oxo-G and initiate the basic excision repair?BER?pathways.It has been reported that abnormality of MUTYH gene function can lead to multiple spontaneous tumors.YUSAKU and other reseachers found that the loss of MUTYH can cause mitochondrial dysfunction and neuronal degenerative changes.There are fewer studies relative to MUTYH in kidney disease,and its effect and mechanism in acute kidney injury are still unclear.Whether it can play a protective role by maintaining the stability of genomic DNA and mitochondrial DNA needs further study.Objective:To observe MUTYH expression in mice renal epithelial tubular cells?RETC?treated with hypoxia/reoxygenation and mice ischemia/reperfusion injury model.To investigate the role of MUTYH in acute renal injury induced by ischemia reperfusion.Methods:1.Cultivation of RETC with low oxygen for 12 hours then turn to normoxia for 4 hours.AnnexinV-FITC/PI staining to detect the apopsis of MPTC.CCk-8 kit was used to detect the cell proliferation.Real-time PCR was used to detect the expression of MUTYH mRNA.MUTYH protein expression was detected by Western blotting.2.C57BL/6 wild type mice were assigned to two groups randomly—control group?Sham?and model group?I/R?.The knock-out mice?Knock-out mice,KO mice?were divided into four groups:control group(MUTYH^?+/+?+Sham),KO mice group(MUTYH^?-?/-₊sham),model group and KO mice model group(MUTYH^?-/-?+I/R)which were performed sham surgery or ischemia.The mice were sacrificed 24 hours after I/R treatment.The blood was transferred into heparinized tubes and centrifuged at 3000rpm for 10 min to separate the plasma,which is prepared for detect serum creatinine and BUN.HE and PAS stain were used to observe the pathological morphology of renal tissue,to estimate the injury score of renal tubular injury.The expression and distribution of the 8-oxo-G and MUTYH in renal tissues were detected by immunohistochemistry.Applying immunofluorescence to detect the expression and localization of MUTYH.The mRNA expression of MUTYH,KIM-1,NGAL,and TNF-?,MCP-1 was detected by Real-time PCR;MUTYH protein expression and NGAL were detected by Western blotting.Results:?1?Renal tubular cells treated with hypoxia/reoxygenation,our results shows cell apoptisis and cell proliferation ability decreased obviously.The expression of Bcl-2 protein decreased significantly,only 50%of the control group.The mRNA of MUTYH was increased while MUTYH protein decreased significantly.?2?In I/R model of wild type mice,PAS stain showed the morphology of renal tubular epithelial cells—degeneration,swelling,necrosis,dilatation,loss of brush border and protein cast formation;HE staining showed the damage of tubule tissue and the infiltration of inflammatory cells.Serum creatinine,urea nitrogen,KIM-1 mRNA,and NGAL mRNA increased dramatically.The NAGL protein also increased by 2.55 times.The expression of inflammatory cytokines TNF-?and IL-1?was increased,TNF-?was 6.44 times of the control group,IL-1?was 3.86 times of the control group.Western blotting showed MCP-1 protein was significantly increased.In addition,Bcl-2 protein decreased in model group.Compared with model group,MUTYH mRNA was 1.61 times higher while MUTYH protein decreased by 40%.The immunohistochemically staining showed the MUTYH mainly expressed in the proxiamal tubular cells and MUTYH protein decreased significantly in the model group.The fluorescence of MUTYH showed the MUTYH mainly expressed in cytoplasm and the totol intensity of MUTYH decreased dramatically.The 8-oxo-G increased in the model group and mainly located in the nucleus.?3?Ischemia-reperfusion surgery performed on MUTYH knock-out mice.The result showed that the MUTYH knockout mice pathological damage was more serious—large renal tubular necrosis,loss of brush border,inflammatory infiltration.Its pathological damage score was higher,increased by 40%compared with the model group.After the operation,the serum creatinine and urea nitrogen increased,and the MUTYH knockout mice increased higher.Compared with the model group,CR increased by 4 times and BUN increased by 1.7 times.The result of Real-time PCR showed that KIM-1 and NAGL significantly increased,5.6 times and 3.24 times of the model group respectively.Western blotting showed a significant increase in NGAL protein.By semi-quantitative analysis of NGAL protein,wild type mice and MUTYH knockout mice increased by 5.2 times and 3.9 times respectively.MUTYH knockout mice had a higher level of inflammatory activation after operation,compared with the model group,MCP-1 mRNA was dramatically increased.Conclusions:The MUTYH activity decreased both in vivo and vitro.The I/R model of MUTYH knockout mice shows that the absence of MUTYH aggravated the acute renal injury induced by ischemia-reperfusion,indicating that MUTYH plays an important role in the acute kidney injury.