Hyperhomocysteinemia Aggravates Cisplatin-induced Acute Kidney Injury By Downregulating Heme Oxygenase-1

BackgroundAcute kidney injury(AKI)is defined as a clinical syndrome.Cisplatin nephrotoxicity is an ideal model to study the early pathophysiological features of all types of AKI.Homocysteine(Hcy)is a thiol-containing amino acid formed in the metabolic conversion of methionine to cysteine.Recently,the prevalence of HHcy in China is increasing rapidly.HO-1 was subsequently shown to be a protectant in various types of AKI.HuR is best known for binding to a subset of mRNAs and for influencing their stability and/or translation.As it is said that HuR protein can bind to the 3-untranslated region(UTR)of HO-1.So to explore the mechanism of the effect of hyperhomocysteinemia on cisplatin-induced AKI,we designed the following experiment.1.Hyperhomocysteinemia aggravates cisplatin-induced acute kidney injury in mice.2.Hyperhomocysteinemia aggravates cisplatin-induced acute kidney injury by downregulating heme oxygenase-1.3.The mechanism of homocysteine downregulating heme oxygenase-1 expression.Chapter IHyperhomocysteinemia aggravates cisplatin-induced acute kidney injury in miceObjectiveTo investigate the effect of hyperhomocysteinemia on cisplatin-induced AKI.Methods1.8 weeks male C57BL/6 micewere randomly divided into two groups.2.Serum Hey levels were measured.3.Serum creatinine and BUN were measured.4.HE staining.5.TUNEL assay.6.Kidney tissues were used to determine expression of cleaved-caspase 3 and p53.7.The mice kidney paraffin section was applied to detect the number of renal interstitial CD3 positive and F4/80 positive cells and y-H2AX by immunohistochemistry.Results1.The level of serum Hey in H-Met Diet group was significant increased compared with regular diet group.2.The levels of serum creatinine,BUN and renal tubular injury score in H-Met diet group received cisplatin injection was higher than that in regular diet group received cisplatin injection.3.The number of renal tubular epithelial apoptotic cells and the level of renal cleaved-caspase 3 and p53 in H-Met diet group received cisplatin injection was higher than that in regular diet group received cisplatin injection.4.The level of renal y-H2AX in H-Met diet group received cisplatin injection was higher than that in regular diet group received cisplatin injection.5.No positive number of CD3 and F4/80 cells were found in four groups.Conclusions1.Increasing methionine content of the diet can induce hyperhomocysteinemia.2.Hyperhomocysteinemia aggravates cisplatin-induced acute kidney injury.Chapter ?Hyperhomocysteinemia aggravates cisplatin-induced acute kidney injury by down regulating heme oxygenase-1ObjectiveTo explored the mechanism of hyperhomocysteinemia aggravating cisplatin-induced AKI by downregulating HO-1.Methods1.Hcy induces HO-1 expression in NRK52E cells.2.The mice frozen kidney tissue from cisplatin-induced AKI was used to determine expression of HO-1.3.After receiving HO-1 activator,the blood and the frozen kidney tissues were used for detection of the serum creatinine,BUN and the renal tubular injury score.And also measure the substrates of HO-1,such as the ferritin and bilirubin and also the reactive oxygen species(ROS)content in the kidney,and the detection of TUNEL.Results1.Hcy downregulates the expression of HO-1 in NRK52E cells.2.The level of renal HO-1 in H-Met diet group received cisplatin injection was lower than that in regular diet group received cisplatin injection.3.After receiving HO-1 inducer,the levels of serum creatinine,BUN,renal tubular injury score were lower than that of no receiving of HO-1 inducer group.The number of renal tubular epithelial apoptotic cells and the level of renal cleaved-caspase 3 and p53 was lower.Conclusions1.Homocysteine downregulates the expression of heme oxygenase-1.2.It is demonstrate that HO-1 has protectant function in acute kidney injury.Chapter IIIThe mechanism of homocysteine downregulating heme oxygenase-1 expressionObjectiveIn this section we want to explore the mechanism of how homocysteine downregulate HO-1 expression.Methods1.The mice frozen kidney tissue from cisplatin-induced AKI was used to determine expression of Nrf2 and HuR.2.Hey induces HuR expression in NRK52E cells.3.EMSA assay detects the binding of HuR protein to 3-UTR of HO-1 mRNA.Results1.Compared with the corresponding control group,different diet groups receiving high methionine had significant lower levels of renal HuR,no difference were found in the expression of Nrf2.2.Hey downregulates the expression of HuR in NRK52E cells.3.EMSA assay demonstrates that HuR protein specifically binds to the 3-UTR of HO-1 mRNA.ConclusionsHuR protein binds to the 3-UTR region of HO-1 mRNA.Paper Summaries1.Increasing methionine content of the diet can induce hyperhomocysteinemia.2.Hyperhomocysteinemia aggravates cisplatin-induced acute kidney injury.3.Hyperhomocysteinemia aggravates cisplatin-induced acute kidney injury by downregulating heme oxygenase-1.4.The mechanism of homocysteine downregulating HO-1 was through the binding of HuR protein to the 3-UTR of HO-1 mRNA.