Klotho Deficiency Aggravates Tacrolimus-Induced Renal Injury Via The Phosphatidylinositol 3-Kinase-Akt-Forkhead Box Protein O Pathway

Objective:Whether reduced Klotho expression affects tacrolimus(Tac)-induced renal injury in an experimental model of chronic Tac nephropathy.Methods:Step 1:First select wild-type(WT)healthy mice and feed them with low-salt feed.One week later,body weight matched animals were randomly assigned to 4 groups,each containing 8 mice.The specific groupings were as follows:(1)VH group:5 ml/kg olive oil was injected subcutaneously(s.c.)every day for 4 weeks;(2)Tac 0.25 group:daily subcutaneous injection of tacrolimus(0.25 mg/kg/d)for 4 weeks;(3)Tac 0.5 group:daily subcutaneous injection of Tac(0.5 mg/kg/d)for 4 weeks;(4)Tac 1.0 group:Tac(1.0 mg/kg/d)was injected subcutaneously daily for 4 weeks.We evaluated the association between Tac dose and Klotho expression by administering different doses of Tac(0.25,0.5,and 1 mg/kg/d)to WT mice.Step2:The experiment was divided into two groups.Eight-week-old male Klotho Heterozygous(HT)mice were purchased from CLEA in Japan.Eight WT and eight HT mice were randomly selected.The specific groups were as follows:(1)WT group:Tac(1.0 mg/kg/d)was injected subcutaneously daily for 4 weeks;(2)HT group:Tac(1.0 mg/kg/d)was injected subcutaneously daily for 4 weeks.We compared Klotho levels,kidney function,fibrosis,and apoptosis-related markers in WT mice and Klotho HT mice in an experimental model of chronic Tac kidney disease.In step 3,we examined whether oxidative stress and the Phosphatidylinositol 3-kinase(PI3K)-Akt-Forkhead box protein O(FoxO)signaling pathway are involved in Klotho's protection against tacrolimus-induced renal injury.Testing items include:8-Hydroxy-2'-deoxyguanosine(8-OHdG),4-HHE,PI3K,Akt,FoxO1,FoxO3a,Manganese speroxide dismutase(MnSOD),and Bim.Results:(1)In experimental step one,different doses of Tac-treated WT mice,Blood urea nitrogen(BUN)and Serum creatinin(Scr)concentrations increased in a dose-dependent manner compared to control mice.The excretion of Collagen I and urinary 8-OHdG also increased in a dose-dependent manner,and Klotho levels in kidney tissue and urine decreased in a dose-dependent manner.(2)In experimental step two,Klotho knock out HT mice exhibited lower Klotho levels in the renal cortex and urine compared to WT mice,and Scr levels increased,fibrosis and apoptosis were also higher.(3)As a result of the measurement of oxidative stress,compared with WT mice,the excretion of 8-OHdG in urine of Tac-treated HT mice and the expression in serum were significantly increased;4-HHE also observed higher levels in histological sections of HT mice compared to WT mice.(4)Intervention of Tac in WT mice increased oxidative stress markers,such as phosphorylation of PI3K,Akt,and Fox03a.However,dephosphorylation of FoxOl was reduced and these effects were more severe in HT mice.(5)Immunoblot analyses were performed on the FoxO target genes MnSOD and Bim.Lower expression of MnSOD and higher expression of Bim were detected in WT mice treated with Tac compared to control mice.These effects are more severe in Tac-treated HT mice.Conclusion:Through the above experiments,we draw the following conclusions:(1)Chronic Tac Treatment Reduces Klotho Expression in a Dose-Dependent Manner.(2)Reduced Klotho expression aggravates Tac-induced nephropathy and oxidative stress in Tac-induced nephropathy.(3)The Response to Tac Treatment Involves the PI3K-Akt-FoxO Pathway in Klotho-Deficient Mice.