Study On The Antioxidation Of Klotho In The Formation Of Calcium Oxalate Stone In Kidney

Objective:Based on the clinicaldata of patients,cellular and animal facet,this study revealed the role of Klotho in the formation of calcium oxalate（Ca Ox）kidney stones and its regulation of intracellular signal transduction mechanism.To explore whether the antioxidant damage mechanism of Klotho is realized by Keap1-Nrf2-ARE signal pathway when oxidative damage is caused by Ca Ox crystals.Methods:（1）The clinical data and blood samples of 148patients with Ca Ox kidney stones and 151healthy people were collected.The levels of s KL,Nrf2,NQO-1,HO-1 and GSK3βin serum were determined by enzyme-linked immuno sorbent assay（ELISA）.Independent sample t test or Rank sum test of two independent samples was used to compare the two groups of data;Spearman correlation analysis was used to evaluate the correlation between serum s KL and Nrf2,NQO-1,HO-1 and GSK3βlevels in patients with Ca Ox kidney stones;Logistic regression analysis was used to determine the influencing factors of Ca Ox kidney stones;ROC curve was used to evaluate the value of oxidative stress markers in the diagnosis of Ca Ox kidney stones.（2）The overexpression of Klotho gene was constructed by injection of lentivirus,and the HKC cell model of silencing sh Klohto gene was constructed by si RNA.In addition,the model of Ca Ox kidney stone cell（COM）was established by incubating HKC cells with COM.HKC was randomly divided into Ctrl group,NC+COM group,COM group,sh Klotho+COM group and Klotho+COM group.The expression of Klotho,Bax,Caspase3,Bcl2 protein and m RNA was detected by Western blot and RT-PCR;the levels of NQO-1,αGST,GSH,GPx,SOD,CAT,HO-1 and MDA were detected by ELISA;apoptosis of HKC cells was detected by flow cytometry;COM crystals were labeled with Alexa Fluor-488-and adhesion test was performed.（3）C57BL/6 mice were injected intraperitoneally with 100mg/kg glyoxalate to establish Ca Ox kidney stone model group（model）,and mice in Vector group were injected with the same volume of normal saline.Mice in Model group were injected with 5×10⁸IU knockout/overexpressed lentivirus（100μL）via tail vein to construct sh Klotho/Klotho group.The model was randomly dividedinto Ctrl group,model group,model+vector group,sh Klotho+model group and Klotho+model group.Western blot and RT-PCR techniques were used to detect the expression of Bax,Caspase3,Bcl2,Keap1,Nrf2,Klotho,HO-1,Nrf2 protein and m RNA,ELISA was used to detect the levels of blood MDA,GPx,SOD,LDH,CAT,GSH and HO-1,Hematoxylin-eosin（HE）staining was used to observe the pathological changes of kidney,and immunohistochemistry（IHC）was used to detect the protein expression in renal tissue.Fonkusa staining was used to observe the adhesion of Ca Ox crystals in kidney,and TUNEL method was used to detect apoptosis in renal tissue.Results:（1）There were statistically significant differences in age,BMI,serum s KL,Nrf2,HO-1,NQO-1,GSK3β,potassium,sodium and magnesium levels between the healthy group and the Ca Ox kidney stone group（P<0.05）,while there were no statistically significant differences among other variables（P>0.05）.Correlation analysis showed that serum s KL level was positively correlated with NQO-1（r=0.207,P=0.011）and serum Ca²⁺（r=0.17,P=0.13）,and negatively correlated with GSK3β（r=-0.206,P=0.012）.Logistic regression showed that increased serum HO-1 and NQO-1 levels were protective factors for the occurrence of Ca Ox kidney stones（P<0.05）,and increased BMI and serum GSK3βlevels were risk factors for the occurrence of Ca Ox kidney stones（P<0.05）.ROC curve analysis of single index showed that serum HO-1 sensitivity（0.77）,specificity（0.58）,area under the curve AUC（0.75）,and optimal cut-off value（9.11）showed better diagnostic efficiency.The combined ROC curve analysis of the two indexes showed the combined sensitivity（0.80）,specificity（0.68）and AUC（0.82）of serum HO-1+NQO-1.The combined ROC curve analysis of the three indexes showed that the combined sensitivity（0.85）,specificity（0.70）and AUC（0.84）of serum HO-1+NQO-1+GSK3βwere significantly higher than the combined detection of single or two indexes,and the difference was statistically significant（P<0.05）.（2）Compared with ctrl group and COM group,the expressions of GSH,GPx,SOD,CAT and HO-1 were significantly decreased,while the expressions of MDA and ROS were significantly increased（P<0.05,P<0.01）.Compared with ctrl group,COM group had adhesion reaction（P<0.05）.The crystal adhesion in Klotho+COM group was significantly lower than that in sh Klotho+model group（P<0.05,P<0.01）.Compared with ctrl group,the expression of Bax,Caspase-3,a key apoptotic protein,was significantly up-regulated in COM group,and the expression of Bcl-2 was significantly down-regulated in Bcl-2 group.The expressions of Bax,Caspase-3 in Klotho+COM group were significantly lower than those in sh Klotho+COM group(P<0.05,P<0.01;（3）After successfully constructing Ca Ox kidney stone model mice with glyoxylate,Klotho overexpression and Klotho silence kidney stone models were constructed.ELISA results showed that the expressions of GPx,SOD,CAT,GSH and HO-1 in serum of model group were significantly decreased,and the expressions of MDA and LDH were significantly increased compared with those of ctrl group（P<0.05,P<0.01）.Western Blot and Real-Time PCR results showed that the protein and m RNA expression levels of HO-1,Nrf2 and NQO-1 in model group were significantly down-regulated（P<0.05,P<0.01）,and the m RNA and protein expression levels of Caspase-3,a key egg for apoptosis,were significantly up-regulated.The m RNA and protein expression levels of Bcl-2 were significantly down-regulated,but the opposite was true in Klotho+model group（P<0.05,P<0.01）.TUNEL results showed that the apoptosis of renal tissue cells in model group was significantly increased compared with ctrl group（P<0.05）,and there was statistical significance between Klotho+model group and sh Klotho+model group（P<0.05,P<0.01）.The results of von Kussa showed that compared with Ctrl group,the number of calcium crystals in kidney tissue of sh Klotho+model group was significantly increased,and the atrophy of renal tubules was aggravated（P<0.05）.The number of calcium crystals in renal tissue of Klotho+model group was significantly reduced,and the atrophy and expansion of renal tubules were significantly improved（P<0.01）.The m RNA and protein expression levels of Klotho,Nrf2 and the downstream proteins HO-1 and NQO-1 of Keap1-Nrf2-ARE signaling pathway were significantly down-regulated in the model group,while the levels of HO-1,Nrf2 and NQO-1 were up-regulated in the Klotho+model group.The reverse is true for the sh Klotho+model group.However,Keap1 protein level was significantly up-regulated only in sh Klotho+model group（P<0.05,P<0.01）.Conclusion:（1）The serum levels of s KL,Nrf2,HO-1 and NQO-1 were decreased in patients with Ca Ox kidney stones,while the serum levels of GSK3βwere increased.Serum s KL level was positively correlated with NQO-1 and negatively correlated with GSK3β.The elevated levels of serum HO-1 and NQO-1 are protective factors for the development of Ca Ox kidney stones,while the elevated levels of GSK3βand BMI are risk factors for the development of Ca Ox kidney stones.Serum HO-1+NQO-1+GSK3βis a suitable combination for the serologic diagnosis of Ca Ox kidney stones.（2）Klotho protein inhibited the oxidative damage reaction of HKC cells through Keap1-Nrf2-ARE signaling pathway,alleviating the injury and apoptosis of HKC cells;At the same time,the adhesion of COM to HKC was decreased,and the occurrence of Ca Ox kidney stones was finally inhibited.（3）Animal models further verified that Klotho could inhibit oxidative damage,cell apoptosis and crystal adhesion to stone formation in Ca Ox kidney stone model mice through Keap1-Nrf2-ARE signaling pathway.