Astragaloside IV improves renal function and fibrosis via inhibition of miR-21-induced podocyte dedifferentiation and mesangial cell activation in diabetic mice

Abstract

Background: Podocyte dedifferentiation and mesangial cell (MC) activation play an important role in many glomerular diseases associated with fibrosis. MicroRNA-21 (miR-21) is closely linked to renal fibrosis, but it is unknown whether and how miR-21 promotes podocyte dedifferentiation and MC activation and whether astragaloside IV (AS-IV) improves renal function and fibrosis through the regulation of miR-21.

Materials and methods: Cultured MCs, primary mouse podocytes, and diabetic KK-Ay mice were treated with AS-IV. Cell transfection, Western blot, real-time PCR, immunofluorescence assay, immunohistochemical assay, and electronic microscopy were used to detect the markers of podocyte dedifferentiation and MC activation and to observe the renal morphology.

Results: Our data showed that miR-21 expression was increased and that AS-IV decreased miR-21 levels in cells, serum, and kidney. Overexpressed miR-21 promoted podocyte dedifferentiation and MC activation, and treatment with AS-IV reversed this effect. Furthermore, the overexpression of miR-21 activated the β-catenin pathway and the transforming growth factor (TGF)-β1/Smads pathway in the process of podocyte dedifferentiation and MC activation, which was abolished by AS-IV treatment. In addition, both the Wnt/β-catenin pathway inhibitor XAV-939 and the TGF-β1/Smads pathway inhibitor SB431542 reversed the effect of AS-IV. Furthermore, AS-IV improved renal function and fibrosis in diabetic KK-Ay mice.

Conclusion: Our results indicated that AS-IV ameliorates renal function and renal fibrosis by inhibiting miR-21 overexpression-induced podocyte dedifferentiation and MC activation in diabetic kidney disease. These findings pave way for future studies investigating AS-IV as a potential therapeutic agent in the management of glomerular diseases.

Keywords: TGF-β1/Smads pathway; astragaloside IV; mesangial cell activation; miR-21; podocyte dedifferentiation; renal fibrosis; β-catenin pathway.

Figure 1

Chemical structure of AS-IV. Abbreviation: AS-IV, astragaloside IV.

Figure 2

Effect of AS-IV on miR-21 expression in vivo and in vitro. (A) The cell viability of podocyte was detected by CCK-8 assay. (B) The relative proliferation rate of MC was detected by CCK-8 assay. (C and D) Real-time PCR results showed that AS-IV decreased the relative miR-21 levels in both MCs and podocyte exposed to hyperglycemia for 48 hours in a dose-dependent manner. (E and F) Real-time PCR results showed that AS-IV decreased the levels of miR-21 in serum and kidney compared with untreated DKD group mice. Data are presented as mean ± SD. n=3. ^*Compared with NC group, P<0.05; ^#compared with HG or DKD group, P<0.05. Abbreviations: AS-IV, astragaloside IV; CCK-8, Cell Counting Kit-8; DKD, diabetic kidney disease; HG, high glucose; MC, mesangial cell; miR-21, microRNA-21; NC, normal control; PCR, polymerase chain reaction.

Figure 3

Effect of AS-IV on miR-21 overexpression-induced podocyte dedifferentiation and MC activation. (A–D) Real-time PCR and Western blot results showed that miR-21 overexpression decreased nephrin expression and increased α-SMA expression in podocyte. AS-IV treatment reversed this effect. (E and F) Real-time PCR and Western blot results showed that AS-IV treatment decreased the levels of α-SMA in miR-21-overexpression-stimulated MCs. Data are presented as mean ± SD. n=3. ^*Compared with NC, P<0.05; ^#compared with miR-21 mimics group, P<0.05. Abbreviations: α-SMA, α-smooth muscle actin; AS-IV, astragaloside IV; DAPI, 4′,6-diamidino-2-phenylindole; DKD, diabetic kidney disease; HG, high glucose; MC, mesangial cell; miR-21, microRNA-21; NC, normal control.

Figure 4

Effect of AS-IV on the expression of nephrin and α-SMA in vivo. Immunofluorescence images showed that AS-IV increased the expression of nephrin and decreased the expression of α-SMA in glomerulus (white arrows) compared with the untreated DKD mice. Abbreviations: α-SMA, α-smooth muscle actin; AS-IV, astragaloside IV; DKD, diabetic kidney disease.

Figure 5

Effect of miR-21 overexpression on the Wnt/β-catenin pathway and the TGF-β1/Smads pathway and the intervention of AS-IV in podocyte. (A–I) Real-time PCR and Western blot results showed that miR-21 overexpression decreased Smad7 expression and increased the expression of TGF-β1, P-Smad3, and activated β-catenin in podocytes. AS-IV treatment abolished this effect. Data are presented as mean ± SD. n=3. ^*Compared with NC, P<0.05; ^#Compared with miR-21 mimics group, P<0.05. Abbreviations: AS-IV, astragaloside IV; MC, mesangial cell; miR-21, microRNA-21; NC, normal control; TGF, transforming growth factor.

Figure 6

Effect of miR-21 overexpression on the Wnt/β-catenin pathway and the TGF-β1/Smads pathway and the intervention of AS-IV in MCs. (A–I) Real-time PCR and Western blot results showed that miR-21 overexpression decreased Smad7 expression and increased the expression of TGF-β1, P-Smad3, and activated β-catenin in MCs. AS-IV treatment reversed this effect. Data are presented as mean ± SD. n=3. ^*Compared with NC, P<0.05; ^#Compared with miR-21 mimics group, P<0.05. Abbreviations: AS-IV, astragaloside IV; MC, mesangial cell; miR-21, microRNA-21; NC, normal control; TGF, transforming growth factor.

Figure 7

Effect of the Wnt/β-catenin pathway and the TGF-β1/Smads pathway on podocyte dedifferentiation and MC activation. (A–C) Western blot results showed that both SB431542 and XAV-939 decreased nephrin expression in podocytes and increased the expression of α-SMA in podocytes and MCs co-treated with miR-21 and AS-IV. Data are presented as mean ± SD. n=3. ^*Compared with co-treatment with miR-21 mimics and AS-IV group, P<0.05. Abbreviations: α-SMA, α-smooth muscle actin; AS-IV, astragaloside IV; MC, mesangial cell; miR-21, microRNA-21; TGF, transforming growth factor.

Figure 8

Effect of AS-IV on Wnt/β-catenin pathway and TGF-β1/Smads pathway in vivo. (A–D) Immunofluorescence images showed that AS-IV increased Smad7 expression and decreased the expression of TGF-β1, P-Smad3, and activated β-catenin in glomerulus (white arrows) compared with untreated DKD group mice. Blue, nuclear staining (DAPI); Green, target protein staining. Abbreviations: AS-IV, astragaloside IV; DAPI, 4′,6-diamidino-2-phenylindole; DKD, diabetic kidney disease; NC, normal control; TGF, transforming growth factor.

Figure 9

Effect of AS-IV on renal function and the expression of FN and Col IV in diabetic KK-Ay mice. (A and B) The data showed that AS-IV treatment decreased ACR and mAlb compared with the untreated DKD group mice. (C) IHC images showed that the levels of Col IV and FN were remarkably decreased in the kidneys of AS-IV-treated mice compared with the untreated DKD mice. Data are presented as mean ± SD. n=3. ^*Compared with NC group, P<0.05; ^#compared with DKD group, P<0.05. Abbreviations: ACR, albumin–creatinine ratio; AS-IV, astragaloside IV; Col IV, Collagen IV; DKD, diabetic kidney disease; FN, fibronectin; mAlb, microalbuminuria; IHC, immunohistochemical; NC, normal control.

Figure 10

Effect of AS-IV on renal morphology in diabetic KK-Ay mice. H&E, Masson and PAS staining images showed that AS-IV treatment improved ECM overproduction and renal fibrosis, compared with the untreated DKD mice. Electron microscope images showed that AS-IV treatment ameliorated foot process fusion and structure disorder of podocyte, and decreased thickness of the GBM. Abbreviations: AS-IV, astragaloside IV; DKD, diabetic kidney disease; ECM, extracellular matrix; H&E, hematoxylin–eosin; NC, normal control; PAS, Periodic acid–Schiff.