MicroRNA-630 alleviates inflammatory reactions in rats with diabetic kidney disease by targeting toll-like receptor 4

Figure 1 The expression of miR-630 in diabetic kidney disease rats. A: Differential expression of miR-630, renal weight index, and blood glucose in diabetic kidney disease rats; B: The correlations between the expression of miR-630 and clinical parameters, including body weight, serum albumin, renal weight index, blood urea nitrogen, serum creatinine and proteinuria, were analysed by Pearson correlation analysis and linear regression analysis; C: Pathological changes in renal tissue in rats under a high glucose environment (hematoxylin and eosin, × 400). n = 15. Data are presented as mean ± SD, ^aP < 0.001, ^bP < 0.001, ^cP < 0.001, ^dP < 0.001. DKD: Diabetic kidney disease.

Figure 2 Overexpression of miR-630 inhibits renal tubular epithelial cell damage induced by high glucose in vitro. A: Quantitative reverse transcription polymerase chain reaction detection of miR-630 and toll-like receptor 4 (TLR4) expression in rat renal tubular epithelial cells in a high glucose environment (NRK-52E); B: Detection of tumor necrosis factor-α, interleukin (IL)-1β and IL-6 expression in rat renal tubular epithelial cells (NRK-52e) under a high glucose environment by enzyme-linked immunosorbent assay; C: Western blot detection of NRK-52E TLR4 in rats under a high glucose environment α-smooth muscle actin and collagen IV protein expression. Data are presented as mean ± SD, ^aP < 0.01 vs normal glucose, and ^bP < 0.01 vs high glucose + mimic normal glucose. NG: Normal glucose, 5.6 mmol/L; HG: High glucose, 20 mmol/L; HM: High mannitol (5.6 mmol/L glucose + 14.4 mmol/L mannitol); TLR4: Toll-like receptor 4; α-SMA: α-smooth muscle actin; IL: Interleukin; TNF: Tumor necrosis factor.

Figure 3 Toll-like receptor 4 is the target gene of miR-630. A: Bioinformatic analysis showed the putative miR-630 target sites in the toll-like receptor 4 (TLR4) 3’-untranslated regions (UTR). The mutated nucleotides are underlined; B: The WT-TLR4 3’-UTR and the MUT-TLR4 3’-UTR reporters were cotransfected with miR-630 mimic or negative control into NRK-52Es. Forty-eight hours after transfection, the luciferase activities were measured; C: Quantitative reverse transcription polymerase chain reaction detection of miR-630-targeted downregulation of TLR4 mRNA expression levels; D: Enzyme-linked immunosorbent assay detection of tumor necrosis factor in miR-630-targeted downregulation of TLR4, α-smooth muscle actin (SMA), interleukin (IL)-1β, and IL-6 content; E: Western blotting was used to detect the protein expression of TLR4, α-SMA, collagen IV and E-cadherin downregulated by miR-630. Data are presented as mean ± SD, ^aP < 0.01 vs high glucose + mimic normal glucose, ^bP < 0.01 vs high glucose + miR-630 mimic + oe normal glucose. NG: Normal glucose, 5.6 mmol/L; HG: High glucose, 20 mmol/L; HM: High mannitol (5.6 mmol/L glucose + 14.4 mmol/L mannitol); TLR4: Toll-like receptor 4; α-SMA: α-smooth muscle actin; IL: Interleukin; TNF: Tumor necrosis factor; NC: Negative control.

Figure 4 Overexpression of miR-630 improves the biochemical changes in diabetic kidney disease model rats. A: Effects of miR-630 overexpression on body weight, renal weight index, blood glucose, fasting blood glucose, 24-h urinary protein, blood urea nitrogen and serum creatinine in rats; B: Effect of overexpression of miR-630 on the expression of toll-like receptor 4 (TLR4) mRNA in diabetic kidney disease rats; C: MiR-630 was overexpressed, and the levels of interleukin (IL)-6, IL-1β and tumor necrosis factor-α were detected by enzyme-linked immunosorbent assay; D: MiR-630 was overexpressed, and the expression levels of TLR4, E-cadherin, α-smooth muscle actin and collagen IV were detected by western blotting. Data are presented as mean ± SD, ^aP < 0.01 vs diabetic kidney disease + normal glucose agomir. DKD: Diabetic kidney disease; FBG: Fasting blood glucose; 24 h UP: 24-h urinary protein; BUN: Blood urea nitrogen; Scr: Serum creatinine; TLR4: Toll-like receptor 4; α-SMA: α-smooth muscle actin; IL: Interleukin; TNF: Tumor necrosis factor; NC agomir: Negative control of lentivirus.

Figure 5 Effects of overexpression of miR-630 on glomerular morphology in diabetic kidney disease rats. A: Effect of overexpression of miR-630 on glomerular morphology in diabetic kidney disease (DKD) rats (hematoxylin and eosin, × 200); B: Effect of overexpression of miR-630 on glomerular morphology in DKD rats (Masson, × 200). DKD: Diabetic kidney disease; NC agomir: Negative control of lentivirus.