Mechanism Of Mitochondrial Kinetic Imbalance And Nrf2 Signaling Pathway-mediated Oxidative Stress In Nickel-and/or Chromium-induced Kidney Injury In Mice

Nickel-chromium is a heavy metal widely present in nature and is usually present in combination to cause damage to the organism in a chronic or subchronic toxic manner,while current studies on nickel-chromium nephrotoxicity have mainly focused on the acute toxicity to a single heavy metal and have not involved the study of specific molecular mechanisms.Therefore,in this study,in order to reveal the effects of nickel and/or chromium exposure on mouse kidney,the molecular mechanisms of renal oxidative damage due to nickel and/or chromium toxicity were investigated from the perspective of mitochondrial kinetic homeostasis and Nrf2-mediated antioxidant pathway.In this experiment,160 C57BL/6J mice were randomly divided into four groups:control group,Ni group（450 mg/L Ni Cl₂·6H₂O）,Cr group（141 mg/L K₂Cr₂O₇）,and Ni+Cr group（141mg/L K₂Cr₂O₇,450 mg/L Ni Cl₂·6H₂O）.In this experiment,the body weight growth rate and kidney coefficient of mice were measured,the renal pathology as well as ultrastructure were observed by H&E staining and transmission electron microscopy,the renal function indexes and antioxidant indexes were measured by biochemical kits,and the expression levels of genes and proteins related to Nrf2 signaling pathway and mitochondrial dynamics were detected by RT-q PCR technique and Western Blot technique.The relevant experimental results were as follows:1.Body weight growth rate was significantly decreased in each exposure group compared with the control group（P<0.01）;it was significantly increased in the Ni+Cr group compared with the Cr group（P<0.01）and not significantly different compared with the Ni group（P>0.05）.The renal coefficients were significantly increased in each exposed group compared with the control group（P<0.01）;and significantly decreased in the Ni+Cr group compared with the Ni and Cr groups（P<0.01）.2.The H&E staining results showed different degrees of structural damage of kidney tissues in each exposed group,with swelling,degeneration,and even rupture of renal tubular epithelial cells.Swelling of the collecting tubule structure occurred and detached cellular debris appeared.The Cr group had the most severe damage,and the Ni+Cr group had less damage compared to the Ni and Cr groups.3.The results of transmission electron microscopy showed different degrees of mitochondrial vacuolization and swelling of renal tubular epithelial cells in each exposed group,and disorganized arrangement of intra-mitochondrial cristae;fusion of peduncles of peduncle cells.The Cr group had the most severe damage,and the Ni+Cr group had less damage compared to the Ni and Cr groups.4.The results of renal function indexes showed that the blood creatinine（CRE）and blood urea nitrogen（BUN）levels were significantly increased in each exposed group compared with the control group（P<0.05 or P<0.01）;the CRE levels were significantly decreased in the Ni+Cr group compared with the Ni and Cr groups（P<0.01）;the BUN levels in the Ni+Cr group were not significantly different compared with the Ni and Cr groups（P>0.05）.5.The results of oxidation and antioxidant indexes showed that compared with the control group,the MDA content of each exposed group was significantly higher（P<0.05or P<0.01）,the CAT content of the Cr and Ni+Cr groups and the T-AOC activity of the Ni and Cr groups were significantly lower（P<0.05 or P<0.01）,and the CAT content of the Ni group and the T-AOC activity of the Ni+Cr group were not significantly different（P>0.05）;compared with the Ni group,there was no significant difference in MDA and T-AOC content in the Ni+Cr group（P>0.05）,and CAT content was significantly decreased（P<0.01）;compared with the Cr group,there was no significant difference in MDA and CAT content in the Ni+Cr group（P>0.05）,and T-AOC content was significantly increased（P<0.05）.6.Compared with the control group,Keap1 m RNA and protein expression levels were significantly decreased（P<0.01）in all exposed groups,Nrf2 m RNA was significantly increased（P<0.01）in all exposed groups and Nrf2 protein expression in the Cr group,NQO1 m RNA and protein expression levels were significantly increased（P<0.01）in all exposed groups,and HO-1 m RNA and protein expression levels were significantly increased in the Ni and Cr groups（P<0.05 or P<0.01）.Compared with the Cr group,Keap1 protein expression level was significantly increased in the Ni+Cr group（P<0.05）,and there was no significant difference in Keap1 m RNA expression level（P>0.05）;Nrf2m RNA expression level,HO-1 m RNA and protein expression level,and NQO1 m RNA and protein expression level were significantly decreased（P<0.05 or P<0.01）.There was no significant difference in Nrf2 protein expression levels（P>0.05）.The relative expression of Keap1,Nrf2,HO-1,and NQO1 genes and proteins in the kidney tissues of Ni+Cr mice were not significantly different compared with the Ni group（P>0.05）.7.Compared with the control group,Mfn2,OPA1,PGC-1α,Sirt1 m RNA and protein expression levels were significantly decreased in all exposed groups（P<0.01）;Drp1m RNA in all exposed groups and Drp1 protein expression levels in the Ni and Cr groups were significantly increased（P<0.05 or P<0.01）,and Drp1 protein expression in the Ni+Cr group There was no significant change in the level of Drp1 expression in the Ni+Cr group（P>0.05）.Compared with the Ni group,the expression levels of PGC-1αm RNA and protein in the Ni+Cr group increased（P<0.05）,and the protein expression level of Mfn2decreased significantly（P<0.05）.Compared with the Cr group,Drp1 m RNA and protein expression levels were significantly decreased in the Ni+Cr group（P<0.01）;Mfn2,Sirt1,PGC-1α,OPA1 m RNA expression levels were significantly increased in the Ni+Cr group（P<0.01）;OPA1,PGC-1αprotein expression levels were significantly increased in the Ni+Cr group（P<0.05 or P<0.01）.CONCLUSIONS:Nickel and/or chromium exposure led to growth inhibition,abnormal kidney tissue structure and function,and imbalance of mitochondrial dynamics homeostasis in kidney cells,resulting in oxidative stress,and a trend of antagonistic effect of nickel-chromium combination on kidney toxicity in mice.In addition,the Nrf2 pathway may play a regulatory role in alleviating oxidative damage caused by oxidative stress in mouse kidney.This study partially elucidated the molecular mechanism of kidney injury induced by nickel and/or chromium exposure in mice and the regulatory role of the Nrf2pathway in oxidative damage,providing some theoretical basis for the specific molecular mechanism of kidney injury caused by nickel and/or chromium heavy metal exposure as well as its prevention and treatment.