SIRT3 deficiency leads to induction of abnormal glycolysis in diabetic kidney with fibrosis

Abstract

The regulation of aberrant glucose metabolism in diabetes associated-kidney fibrosis is not well known. In this study we found the suppression of SIRT3 protein level in diabetic kidney, displays responsibility in fibrogenic programming associated with aberrant glycolysis and such abnormal glycolysis is the therapeutic target in diabetes associated-kidney fibrosis. When analyzing different strains of streptozotocin-induced diabetic mice model (fibrotic model: CD-1, less fibrotic model: C57Bl6), we found SIRT3 suppression was associated with kidney fibrosis in fibrotic CD-1; further SIRT3 suppression by systemic administration of SIRT3 siRNA in the diabetic mice, showed profound fibrogenic phenotype in the kidney. Such suppression in SIRT3 was associated with the induction of transforming growth factor-β (TGF-β)/smad signaling, higher level of HIF1α accumulation and PKM2 dimer formation; these alterations subsequently led to abnormal glycolysis and linked abnormal mesenchymal transformations in vivo and in vitro. Inhibition of such aberrant glycolysis suppressed fibrogenic programming and restored SIRT3 level as well. Such aberrant glycolysis was confirmed in the KK/Ta-Ins2Akita mouse, the mouse model of progressive diabetic kidney disease. These data demonstrate that SIRT3 deficiency promotes abnormal glycolysis which is responsible for the fibrogenic pathway in diabetic kidney. Restoration of SIRT3 could be an alternative strategy in combating diabetes associated-kidney fibrosis via inhibition of aberrant glycolysis.

Fig. 1. SIRT3 deficiency is associated with fibrogenic phenotype in the kidney of diabetic mice.

a Western blot analysis of SIRT1 and SIRT3 protein in the time dependent STZ-induced diabetic kidney of CD-1 mice. Representative blots from three independent experiments are shown. Densitometric quantification by ImageJ program. b Western blot analysis of SIRT1 and SIRT3 protein in the control and streptozotocin (STZ)-induced diabetic kidney of CD-1 and C57Bl6 mice. The representative blots from six independent experiments are shown. c Immuno-histochemical analysis of SIRT1 and SIRT3 protein. The representative pictures are shown. d Immuno-fluorescence analysis was performed by fluorescence microscopy. FITC-labeled SIRT1 and SIRT3, Rhodamine-labeled α-SMA and DAPI blue. Merged images are shown. Scale bar: 50 μm in each panel. Representative pictures are shown. e Western blot analysis of SIRT3, α-SMA and FSP-1 in the diabetic kidney of scramble and sirt3 siRNA-injected mice. Scramble and sirt3 siRNA were injected into the tail vein twice in the week for 4 weeks at a dose of 5 mg/kg body weight. A representative image from five blots is shown. f Western blot analysis of smad3 phosphorylation and TGFβR1 in the diabetic kidney of scramble and sirt3 siRNA-injected mice. A representative image from five blots is shown. g Masson’s trichrome staining (MTS) in the diabetic kidney of scramble and sirt3 siRNA-injected mice and the quantification of the relative area fibrogenesis (RAF) by ImageJ. Scale bar: 50 µM. h Sirius red staining and quantification. Scale bar: 50 µM. N = 6 were analyzed in each group. Data are expressed as the mean ± SEM and are shown in the graph. Tukey test was performed to calculate statistical significance. C for control while DM depicts diabetic group

Fig. 2. SIRT3 deficiency leads to induction of abnormal glycolysis.

a Western blot analysis of HK2, PKM1, PKM2, PDK1, PDK4, and GLUT1, in the control and diabetic kidneys of CD-1 and C57Bl6 mice. Representatives from five blots are shown. Densitometry quantifications were normalized by β-actin. b Western blot analysis of HK2, PKM1, PKM2, PDK1, PDK4, and GLUT1, in the scramble, injected and sirt3 siRNA tail vein injected kidneys of diabetic CD-1 mice. Representative from four blots is shown. Densitometric quantifications were normalized by β-actin. c DAG level was analyzed by Elisa assay. N = 6 were analyzed in the kidneys of control and diabetic CD-1 mice. d Western blot analysis of PKC phophorylation and PKC were analyzed in the kidneys of control and diabetic CD-1 mice. The representative from four blots is shown. e DAG level was measured by Elisa assay. N = 6 were analyzed in the kidneys of scramble siRNA and sirt3 siRNA injected mice. f Western blot analysis of PKC phophorylation and PKC were analyzed in the kidneys of scramble siRNA and sirt3 siRNA injected mice. The representative from four blots is shown. g Western blot analysis of SIRT3, HK-2 and PKM2 in the kidneys of littermate control and KK/Ta-Ins2Akita mice. The representative from four blots is shown. Densitometric quantifications were normalized by β-actin. Data in the graph are expressed as mean ± SEM. Tukey test was performed to calculate statistical significance

Fig. 3. Abnormal glycolysis is linked with myofibroblasts formation, proliferation, and O-linked glycosylation.

a Co-immunolabelling of HK2, PKM2, GLUT1, PDK4, proliferation marker ki67 and O-linked glycosylation with α-SMA, were analyzed by using fluorescence microscope. N = 3 were analyzed in the case of control while n = 5 were analyzed in the diabetic group of both strains. Representative figure in each panel is shown. b Co-immunolabelling of glycolytic enzymes, proliferation marker ki67 and O-linked glycosylation with α-SMA were analyzed using fluorescence microscope in the diabetic kidney of scramble and sirt3 siRNA injected mice. Scramble N = 3, Sirt3 siRNA n = 4 were analyzed. Representative figure in each panel is shown

Fig. 4. Glycolysis inhibitions suppress fibrogenic phenotypes and aberrant glycolysis associated injury.

a MTS, b, Sirius red, c PAS in the control, diabetic, dichloroacetate (DCA) and 2-deoxyglucose (2-DG) treated diabetic kidney. Quantification of the relative area fibrogenesis (RAF), relative collagen deposition (RCD) and surface area (SA) was performed by ImageJ. Scale bar: 50 µM Control n = 5, Diabetic n = 6, DCA n = 7 and 2-DG n = 3, were analyzed. The representative picture in each panel is shown. d Western blot analysis of Colla1A, α-SMA, Vimentin, and PGC-1α was analyzed. Representative blots from five independent sets have been shown. N = 5 were analyzed in each group. e Western blot analysis of HK2, PKM2, PDK4, and SIRT3 was analyzed in control diabetes and DCA and 2-DG treated diabetic kidney. Representative blots from five independent sets are shown. N = 5 were analyzed in each group. f, g Co-immunolabelling of HK2, PKM2, PDK4, and GLUT1, ki67, O-linked glucosylation and SIRT3 with α-SMA were analyzed using fluorescence microscope. Control n = 3, DM n = 4, DCA n = 5 and 2-DG n = 3 were analyzed. Representative figure in each panel is shown. Data in the graph are expressed as the mean ± SEM. Tukey test was performed to calculate statistical significance

Fig. 5. Glycolysis inhibition suppresses the fibrogenic characteristics and associated abnormal glycolysis in vitro.

a–c Western blot analysis of FSP-1, α-SMA, TGFβR1, smad3 phosphorylation, total smad3, HK2, PKM2, PDK4, GLUT1, SIRT3, and PGC-1α was performed in the DCA treated cells which were incubated with (10 ng/ml) or without TGFβ1 treatment in HK-2 cells. d Western blots analysis of α-SMA, TGFβR1, HK2, PKM2, SIRT3 and HIF1α in the hk2 siRNA, pkm2 siRNA and hif1α siRNA transfected HK-2 cells. Representative blots from five independent experiments are shown. e Western blots analysis of SIRT3, FSP-1, α-SMA, TGFβR1, smad3 phosphorylation and total smad3 in the scramble siRNA and sirt3 siRNA transfected HK-2 cells. Representative blots from four independent experiments are shown. f BrdU cell proliferation assays were performed in the DCA treated (at 0.5 and 5 mM concentration) scramble siRNA and sirt3 siRNA transfected HK-2 cells, in the growth media and diluted media. Samples in triplicate were analyzed. g Western blot analysis of SIRT3, HK2, PKM2 PDK4 and GLUT1 in the scramble and sirt3 siRNA transfected HK-2 cells. The representative from four blots is shown here. h Western blot analysis of α-SMA, HK2, PKM2, PDK4, and HIF1α in the DCA treated scramble and sirt3 siRNA transfected HK-2 cells. Three independent sets were analyzed. Data in the graph are expressed as mean ± SEM. Tukey test was performed to calculate statistical significance

Fig. 6. Over-expression of SIRT3 protein leads to suppression of TGFβ2-associated fibrogenic features in HK2 cells.

a, b Western blot analysis of SIRT3, α-SMA, TGFβR1, HK2, PKM2 and PDK4 in the empty vector and SIRT3 vector transfected HK-2 cells-treated with or without TGFβ1. The representative from four blots is shown. Densitometric quantifications were normalized by β-actin. Data in the graph are expressed as mean ± SEM. Tukey test was performed to calculate statistical significance

Fig. 7. Cumulative effects of HIF1α accumulation and PKM2 dimer formation lead to aberrant glycolysis during fibrotic events.

a Immunostaining of HIF1α. b Western blot analysis of HIF1α in the control and diabetic kidney of CD-1 and C57Bl6 mice strains. Representative blot from four blots is shown. c Co-immunolabelling of HIF1α with αSMA was analyzed by fluorescence microscope. Green FITC-HIF1α, Red Rhodamine-αSMA. Representative pictures are shown. Scale bar 50 µM. d Immunostaining of HIF1α in the scramble and sirt3 siRNA injected diabetic kidney. Representative pictures are shown. e Western blot analysis of HIF1α in scramble and sirt3 siRNA injected diabetic kidney. Representative blot from three blots is shown. f Western blot analysis of HIF1α in kidney of control, diabetic, DCA and 2-DG treated diabetic CD-1 mice. Representative blot from four blots is shown. g Co-immunolabelling of HIF1α with αSMA in the kidney of control, diabetic, DCA and 2-DG treatment in diabetic CD-1 mice. Scale bar 50 µM. Green FITC-HIF1α, Red Rhodamine-αSMA. Representative pictures are shown. h Glutaryldehyde chemical cross-linking experiment was performed in the kidney of control and diabetes of CD-1 and C57Bl6 mice strains. The representative from four blots is shown. N = 4 were evaluated in each group. i In the scramble and sirt3 siRNA transfected diabetic CD-1 mice. N = 5 were evaluated. j In the kidneys of littermate control and diabetic KK/Ta-Ins2Akita mice. k In control, diabetes, and DCA treated diabetic CD-1 mice. N = 5 were analyzed in each group. l In the scramble, sirt3 siRNA transfected and TGFβ alone treated HK-2 cells. N = 4 were analyzed in each group. Data in the each graph are shown as mean ± SEM. Tukey test was performed for statistical significance

Fig. 8

The working hypothesis showing SIRT3 suppression associated activation of the mesenchymal program and renal fibrosis through induction of abnormal glycolysis by modulating the HIF1α accumulation and increase PKM2 dimer formation