Spironolactone Prevents Diabetic Kidney Disease By Targeting HMGCS2 In Proximal Tubules

Part I: The screening and verification of molecular targets of SPR in protecting DKDObjective: To identify the molecular targets of SPR in protecting diabetic kidney disease(DKD)Methods:Db/m mice treated with normal saline(db/m-ns group, n=6), was used as normal control.6-week-old diabetic db/db mice were randomly divided into two groups: one group was treated with normal saline, and the other group was treated with SPR(db/db-ns group, n=6) for 12 weeks. Electronic microscope was applied to observe the ultrastructure of the kidney. The expression of TGF-β1, Fibronectin, CollagenⅠ, E-cadherin was detected by immunohistochemistry. The micro RNA array was used to analyze the expression profiles of the kidneys. The m RNA and protein expression of HMGCS2 were examined by q PCR, immunohistochemisty and western blot. Glomeruli and tubules were isolated from the kidney of streptozocin(STZ)-induced diabetic rats. The m RNA and protein expression of HMGCS2 was detected. Moreover, the expression of HMGCS2 were examined in normal human kidneys and in human kidneys with diabetes by immunohistochemistry.Results: 1. The electron microscope showed swelling of mitochondria and decreased number of mitochondria in the proximal tubular epithelial cells of db/db-ns group compared with db/m-ns group. The expression of TGF-β1, Fibronecti and CollagenⅠwas decreased in db/db-SPR group as compared to that in db/db-ns group. The expression of E-cadherin was increased in db/db-SPR group as compared to that in db/db-ns group. Meanwhile, SPR effectively improved the ultrastructure of tubular epithelial mitochondria as well as decreased fibrosis in db/db mice. 2. Our microarray results showed that 3-hydroxy-3-methylglutaryl-Co A synthase 2(HMGCS2)was differentially expressed in diabetic and control mice. The m RNA and protein expression of HMGCS2 were significantly increased in db/db-ns group as compared to db/m-ns group(P < 0.05), and decreased in db/db-SPR as compared to db/db-ns group(P<0.05). The HMGCS2 protein expression was significantly increased in isolated renal tubules than that in isolated glomeruli. The HMGCS2 expression was increased in STZ-induced type 1 diabetic rats compared with that in wild-type rats. Moreover, HMGCS2 was majorly expressed in renal tubules of human kidney and up-regulated in diabetic kidney than that in normal human kidney. Conclusion: SPR improved diabetic kidney disease, by reducing inflammation and fibrosis as well as epithelial mesenchymal transition(EMT). HMGCS2 mainly expressed in diabetic renal tubules of mice, rats and human, which was decreased by spironolactone.Part II:The molecular mechanism through which HMGCS2 mediated the injury of proximal tubules of DKD.Objective: To explore the mechanism that HMGCS2 mediated proximal tubular injury of DKD.Methods:We cultured the conditionally immortal human proximal tubular epithelial cells(HK-2) under the different experimental conditions, which involved SPR intervention group, high-fat intervention group,aldosterone intervention group and β-HB intervention groups. normal glucose(5.6mmol/L), high glucose(25mmol/L), SPR(25mmol / L glucose + 0.1u M SPR), hypertonic control(5.6mmol / L glucose + 19.4mmol / L mannitol) and solvent control(25mmol / L glucose + 0.001 u M alcohol). SPR intervention group included normal glucose(5.6mmol / L), 0.5% BSA control, 125μmol / L palmitate, 250μmol / L palmitic acid and 500μmol / L palmitic acid; aldosterone intervention group included normal glucose(5.6mmol/L), different concentrations of aldosterone(0.001μM, 0.01μM, 0.1μM) and SPR intervention(0.1 u M aldosterone + 0.1u M SPR); β-hydroxybutyric acid(β-HB) intervention group included normal glucose(5.6mmol / L) and ketones(normal glucose + 0.1u M β-HB). Hmgcs2 expression was examined by real-time quantitative PCR and western blot. The concentration of β-HB was detected by enzyme-linked immunosorbent assay(Elisa) in HK-2.Results: 1. High glucose increased Hmgcs2 expression in renal tubular epithelial cells, while decreased E-cadherin expression, the marker of EMT(P <0.01). SPR down-regulatedthe expression of Hmgcs2 in the high glucose, while up-regulated E-cadherin expression(P <0.01). 2. 500 u M palmitate increased HMGCS2 m RNA expression in tubular epithelial cells(P <0.05). 3.0.1μM aldosterone increased the expression of HMGCS2 compared to NG group, while Hmgcs2 was down-reguled by SPR. 4. β-HB induced the expression of MCP-1, TNF-a and reduced the expression of E-cadherin(p <0.05).Conclusion: High glucose and aldosterone induced the expression of HMGCS2 whichincreased the production of β-HB, the main metabolitesof HMGCS2; β-HB alsoincreased the expression of inflammatory markers and induced EMT in renal tubular epithelial cells.