MicroRNA-130b improves renal tubulointerstitial fibrosis via repression of Snail-induced epithelial-mesenchymal transition in diabetic nephropathy

Abstract

MicroRNA-130b (miR-130b) downregulation has been identified in diabetes, but the role and mechanisms for miR-130b in mediating renal tubulointerstitial fibrosis in diabetic nephropathy (DN) remain unknown. We demonstrated that plasma miR-130b downregulation exhibited clinical and biological relevance as it was linked to increased serum creatinine, β2-microglobulin and proteinuria, increased Snail expression and tubulointerstitial fibrosis in renal biopsies of DN patients. MiR-130b inhibitor caused Snail upregulation and enhanced molecular features of epithelial-to-mesenchymal transition (EMT) in high glucose (30 mM) cultured NRK-52E cells. In contrast, miR-130b mimic downregulated Snail expression and increased epithelial hallmarks. Notably, Snail was identified as an miR-130b direct target and inversely correlated with E-CADHERIN expression. Furthermore, the miR-130b-dependent effects were due to Snail suppression that in turn deregulated E-CADHERIN, VIMENTIN, COLLAGEN IV and α-smooth muscle actin (α-SMA), key mediators of EMT. These effects were reproduced in streptozotocin-induced diabetic rats. Thus, we propose a novel role of the miR-130b-SNAIL axis in fostering EMT and progression toward increased tubulointerstitial fibrosis in DN. Detection of plasma miR-130b and its association with SNAIL can be extrapolated to quantifying the severity of renal tubulointerstitial fibrosis. Targeting miR-130b could be evaluated as a potential therapeutic approach for DN.

Figure 1. Plasma miR-130b downregulation contributes to increased tubulointerstitial fibrosis and unfavorable renal function in diabetic nephropathy (DN) patients.

(a) Decreased plasma miR-130b in 27 DN patients compared with 20 healthy controls. (b) Increased BUN, serum creatinine, β2-microglobulin, blood glucose, blood pressure and urine albumin in 27 DN patients compared with 20 normal controls using Mann-Whitney U-test. (c) Pearson correlation analysis demonstrated the inverse correlations of plasma miR-130b with serum creatinine, (d) β2-microglobulin and (e) proteinuria in 27 DN patients, respectively. (f) Upregulation of SNAIL, VIMENTIN, COLLAGEN IV and α-SMA but downregulation of E-CADHERIN by immunohistochemistry and quantification of the staining intensity in 86 renal biopsy samples compared with 20 normal controls. (g) Increased renal tubulointerstitial fibrosis in DN patients by MTS and the quantification analysis. Results are presented as mean ± SD of three independent experiments. ^#P < 0.0001. NC, normal control; DN, diabetic nephropathy; MTS, Masson’ s trichrome stain.

Figure 2. SNAIL negatively correlates with E-CADHERIN and corresponded with increased renal tubulointerstitial fibrosis in DN.

(a) Immunofluorescence staining of SNAIL and E-CADHERIN in renal tubules of 20 normal controls and (b) 42 snap-frozen DN samples. (c) Magnified images of the boxed area showing increased E-CADHERIN expression. (d) Yellow color represents the co-localization between SNAIL and E-CADHERIN (arrows). (e) Increased renal tubulointerstitial fibrosis and increased interstitial injury score in DN compared with NC by MTS. Results are presented as mean ± SD of three independent experiments. ^#P < 0.0001. NC, normal control; DN, diabetic nephropathy; MTS, Masson’ s trichrome stain.

Figure 3. MiR-130b ablation enhances Snail-induced EMT in vitro.

(a) MiR-130b inhibitor increased the expression of SNAIL and co-localized with E-CADHERIN (double arrows) (immunofluorescence). (b) Decreased E-CADHERIN by quantification of the staining intensity. (c) MiR-130b inhibitor upregulated the mRNA level of Snail, Vimentin and Collagen IV, but downregulated E-cadherin by qRT-PCR; miR-130b inhibitor increased SNAIL, VIMENTIN and COLLAGEN IV but decreased E-CADHERIN by (d) Western blot analysis. (e) MiR-130b inhibitor induced phenotypic changes of NRK-52E cells with elongated spindle-shaped cell bodies like fibroblasts by SEM. (f) Increased migrated cells treated with miR-130b inhibitor by transwell assay. (g) Longer invaded distances in NRK-52E cells treated with miR-130b inhibitor by wound healing assay. Results are presented as mean ± SD of three independent experiments. **P < 0.01; ^#P < 0.0001. miR-iNC: miRNA inhibitor negative control; miR-130bi: miR-130b inhibitor; SEM, Scanning Electron Microscope.

Figure 4. MiR-130b mimic suppresses Snail-induced EMT in vitro.

(a) MiR-130b mimic decreased the expression of SNAIL but increased E-CADHERIN (immunofluorescence). (b) Decreased SNAIL but increased E-CADHERIN expression by quantification of the staining intensity. (c) MiR-130b mimic downregulated the mRNA level of Snail, Vimentin and Collagen IV, but upregulated E-cadherin by qRT-PCR. (d) MiR-130b mimic decreased SNAIL, VIMENTIN and COLLAGEN IV but increased E-CADHERIN by Western blot analysis. (e) NRK-52E cells retained the epithelial feature with miR-130b mimic treatment by SEM. (f) Decreased migrated cells with miR-130b mimic treatment by transwell assay. (g) Shorter invaded distance in NRK-52E cells treated with miR-130b mimic by wound healing assay. Results are presented as mean ± SD of three independent experiments. **P < 0.01; ^#P < 0.0001. miR-NC, miRNA mimic negative control; miR-130bm: miR-130b mimic.

Figure 5. High glucose inhibits miR-130b and regulates Snail-induced downstream gene expressions in vitro.

(a) High glucose (30 mM) reduced miR-130b expression in a time-dependent manner. (b) High glucose (30 mM) increased the level of Snail, Vimentin, and Collagen IV, but decreased E-cadherin by qRT-PCR and (c) Western blot analyses. (d) Increasing concentrations of glucose from 5 to 30 mM reduced miR-130b expression in a dose-dependent manner at 72 h. (e) Increased Snail, Vimentin, and Collagen IV, but decreased E-cadherin expression stimulated with increasing concentrations of glucose at 72 h by qRT-PCR and (f) Western blot analyses. Results are presented as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ^#P < 0.0001. HG, high glucose.

Figure 6. Requirement of Snail for the miR-130b antagonism effect on downstream gene expressions in vitro.

(a) MiR-130bi restored SNAIL expression but further downregulated E-CADHERIN induced by Snail siRNA as shown by immunofluorescence and (b) quantification of the staining intensity. Arrowheads indicated co-localized SNAIL and E-CADHERIN. (c) MiR-130b inhibitor attenuated the silencing effect of Snail siRNA on the expression of Snail, Vimentin and Collagen IV, and downregulated E-cadherin by qRT-PCR and (d) Western blot analyses. (e) MiR-130b and its putative binding sequence in the 3′-UTR of Snail. The mutant Snail binding site was generated in the complementary site for the seed region of miR-130b. (f) MiR-130b inhibitor led to a noticeable increase in the luciferase activity of wt 3′-UTR of Snail. Results are presented as mean ± SD of three independent experiments. *P < 0.05; ^#P < 0.0001. NT: non-targeting; siRNA: small interfering RNA; miR-iNC: miRNA inhibitor negative control; miR-130bi: miR-130b inhibitor; wt: wild type; mt: mutant type.

Figure 7. MiR-130b inhibitor increases EMT markers and promotes renal tubulointerstitial fibrosis in vivo.

(a) Decreased miR-130b in plasma and (b) kidney samples of diabetic rats and miR-130b inhibitor further reduced its expression. (c) MiR-130b inhibitor increased Snail, Vimentin, Collagen IV and α-SMA, but decreased E-cadherin by qRT-PCR and (d) Western blot analysis. (e) MiR-130b inhibitor upregulated SNAIL, VIMENTIN, COLLAGEN IV and α-SMA, but decreased E-CADHERIN by immunohistochemistry. (f) Increased renal tubulointerstitial fibrosis in diabetic rats treated with miR-130b inhibitor by MTS. (g) Quantification for immunohistochemistry and (h) interstitial injury score. Results are presented as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ^#P < 0.0001. NC, normal control; DM, diabete mellitus; DM_miR-iNC, diabetic rats treated with miRNA inhibitor negative control; DM_miR-130bi, diabetic rats treated with miR-130b inhibitor; MTS, Masson’ s trichrome stain.

Figure 8. MiR-130b mimic decreases the expression of EMT markers and inhibits renal tubulointerstitial fibrosis in vivo.

(a) Decreased miR-130b in plasma and (b) kidney samples of diabetic rats and miR-130b mimic upregulated its expression. (c) MiR-130b mimic decreased Snail, Vimentin, Collagen IV and α-SMA, but increased E-cadherin by qRT-PCR and (d) Western blot analysis. (e) MiR-130b mimic downregulated the level of SNAIL, VIMENTIN, COLLAGEN IV and α-SMA, but increased E-CADHERIN by immunohistochemistry. (f) Improved renal tubulointerstitial fibrosis in diabetic rats treated with miR-130b mimic by MTS. (g) Quantification of the staining intensity for immunohistochemistry (left panel) and interstitial injury score (right panel). (h) A hypothetical model illustrated that miR-130b regulates renal tubulointerstitial fibrosis through Snail/Ecadherin-mediated EMT process in diabetic nephropathy. Results are presented as mean ± SD of three independent experiments. *P < 0.05; #P < 0.0001. NC, normal control; DM, diabete mellitus; DM_miR-NC, diabetic rats treated with miRNA mimic negative control; DM_miR-130bm, diabetic rats treated with miR-130b mimic; MTS, Masson’ s trichrome stain.