| Objective: To observe the effects of recombinant adeno-associatedvirus-mediated klotho gene delivery on type2diabetes mellitus ratshypernephrotrophy、kidney fibrosis and its mechanisms.Methods: Using high-fat diet STZ-induced type2diabetes SD ratsmodel, constructed recombinant adeno-associated virus mice klothofull-length (mKL) cDNA (rAAV.mKL) and the GFP empty plasmid reportgene (rAAV.GFP). Male SD rats were randomly divided into four groups,three groups were randomly selected as the type2diabetes mellitus modeland transfected rAAV.mKL(T2DM-mKL group)、rAAV.GFP(T2DM-GFPgroup) and PBS(T2DM-PBS group), respectively. The normal controlgroup SD rats were injected with PBS (SD-PBS group). After12weeks,sacrificed all animal and collected blood、tissue samples. The GFPfluorescent expression of renal was observed by cryo-sectioning technique; recorded the bilateral kidney weight and body weight to calculate thekidney hypertrophy index. The pathological changes of kidney、hypernephrotrophy and the expression of collagen fiber were observed byPAS、Masson staining. The mKL、ROCKI mRNA expression of kidney wasdetected by RT-PCR. ELISA detected the level of serum KL protein.immunohistochemistry analysed kidney KL、fibronectin and vimentinexpression. ROCKI protein activity was tested by Western blot.Results:(1) Strongly GFP expression was found in kidneys of theT2DM-GFP and T2DM-mKL groups;(2) Compared with T2DM-PBSgroup, specific expression of mouse klotho was seen only in T2DM-mKLgroup, the expression of serum and kidney KL protein were significantlyincreased in T2DM-mKL group(P<0.05);(3) The kidney hypertrophy indexin T2DM-PBS group was significantly higher than that of T2DM-mKLgroup(P<0.05);(4) Compared with T2DM-PBS group, the glomerularvolume and total glomerular cell number in T2DM-mKL group weresignificantly decreased(P<0.05);(5) The collagen fibers of the glomerularmesangial area and tubular interstitial area in T2DM-mKL group wassignificantly reduced compared with T2DM-PBS group(P<0.05);(6)Compared with T2DM-PBS group, the renal pathological changes inT2DM-mKL group was significantly improved;(7) Compared toT2DM-PBS group, the FN and VIM protein expression were significantlydecreased in T2DM-mKL group(P<0.05);(8) The ROCKI mRNA expression and protein activity in T2DM-mKL group were significantlylower than those of T2DM-PBS group(P<0.05).Conclusion: rAAV.mKL transfection significantly increased the KLexpression in type2diabetic rats kidney, and the over expression KL cansignificantly alleviat the progression of hypernephrotrophy and renalfibrosis in diabetes to protect kidney function, its mechanism may beassociated with KL suppresse ROCK signaling pathway. |
PDF Full Text Request