Genistein attenuates renal fibrosis in streptozotocin‑induced diabetic rats

Abstract

The present study aimed to investigate the antifibrogenic effects of genistein (GEN) on the kidney in streptozotocin (STZ)‑induced diabetic rats and to determine the associated mechanisms. Rats were randomized into four groups: Normal control (N), STZ (S), L (STZ + low‑dose GEN) and H (STZ + high‑dose GEN). After 8 weeks, the fasting blood glucose (FBG) level, the ratio of kidney weight to body weight (renal index), 24‑h urine protein, blood urea nitrogen (BUN), serum creatinine (SCr), renal total antioxidant capacity (T‑AOC), superoxide dismutase (SOD), lipid peroxidation (LPO), malondialdehyde (MDA) and hydroxyproline (Hyp) contents were measured. The histomorphology and ultrastructure of the kidney were also assessed. In addition, mRNA expression levels of transforming growth factor‑β1 (TGF‑β1) and protein expression levels of nuclear factor erythroid 2‑related factor 2 (Nrf2), heme oxygenase‑1 (HO‑1), NAD(P)H:quinone oxidoreductase 1 (NQO1), TGF‑β1, mothers against decapentaplegic homolog 3 (Smad3), phosphorylated (p)‑Smad3 and collagen IV were estimated. Compared with group N, the levels of FBG, renal index, 24‑h urine protein, BUN, SCr, LPO, MDA and Hyp were increased, whereas the levels of T‑AOC and SOD were decreased in group S. The structure of renal tissue was damaged, and the expression of Nrf2, HO‑1 and NQO1 were reduced, whereas the expression of TGF‑β1, Smad3, p‑Smad3 and collagen IV were increased in group S. Compared with group S, the aforementioned indices were improved in groups L and H. In conclusion, GEN exhibited reno‑protective effects in diabetic rats and its mechanisms may be associated with the inhibition of oxidative stress by activating the Nrf2‑HO‑1/NQO1 pathway, and the alleviation of renal fibrosis by suppressing the TGF‑β1/Smad3 pathway.

Keywords: genistein; diabetes mellitus; rat; renal fibrosis; oxidative stress; ransforming growth factor‑β1/mothers against decapentaplegic homolog 3 pathway.

Figure 1.

Levels of kidney function markers in rats. (A) FBG, (B) 24-h urine protein, (C) BUN and (D) SCr level in rats. Data are presented as the mean ± standard deviation; n=6; **P<0.01 vs. N; ^#P<0.05 and ^##P<0.01 vs. S. BUN, blood urea nitrogen; FBG, fasting blood glucose; H, streptozotocin + high-dose genistein group; L, streptozotocin + low-dose genistein group; N, normal control group; S, streptozotocin group; SCr, serum creatinine.

Figure 2.

GEN improved the pathological state of renal tissue from diabetic rats. Morphology of renal tissues by hematoxylin and eosin staining; magnification, ×400. N group: The glomerular structure was normal. S group: The glomerular structure was damaged. L group: Damage to the glomerular structure was ameliorated. H group: Damage to the glomerular structure was notably ameliorated. Arrows indicate the glomerular structure. H, streptozotocin + high-dose genistein group; L, streptozotocin + low-dose genistein group; N, normal control group; S, streptozotocin group.

Figure 3.

Effects of GEN on renal fibrosis. (A) Representative Masson's trichrome stained tissues from each group; magnification, ×200. Arrows indicate collagen. (B) Semi-quantitative analysis of CVF, based on (A). Data are presented as the mean ± standard deviation; n=6; **P<0.01 vs. N; ^#P<0.05 and ^##P<0.01 vs. S. CVF, collagen volume fraction; GEN, genistein; H, streptozotocin + high-dose GEN group; L, streptozotocin + low-dose GEN group; N, normal control group; S, streptozotocin group.

Figure 4.

GEN improved the ultrastructure of renal tissue from diabetic rats. Alterations of renal ultrastructure under transmission electron microscopy in rats; magnification, ×15,000. Red arrows indicate the basement membrane and black arrows indicate the podocyte foot processes. N group: The normal glomerular basement membrane and the podocyte foot processes were neatly arranged. S group: The basement membrane was thickened and foot processes were fused. L group: The fusion of foot processes were ameliorated. H group: The fusion of foot processes were ameliorated notably. H, streptozotocin + high-dose genistein group; L, streptozotocin + low-dose genistein group; N, control group; S, streptozotocin group.

Figure 5.

Expression levels of renal TGF-β1 mRNA in the different groups. Representative images and semi-quantitative analysis of TGF-β1 mRNA expression levels, as determined by reverse transcription-polymerase chain reaction, in renal tissues of rats. Data are presented as the mean ± standard deviation; n=6; **P<0.01 vs. N; ^#P<0.05 and ^##P<0.01 vs. S. H, streptozotocin + high-dose genistein group; L, streptozotocin + low-dose genistein group; N, normal control group; S, streptozotocin group; TGF-β1, transforming growth factor-β1.

Figure 6.

Protein expression levels of Nrf2, HO-1 and NQO1 in the different groups. Representative western blotting images and semi-quantitative analysis of Nrf2, HO-1 and NQO1 proteins; GAPDH was used as a loading control. Data are presented as the mean ± standard deviation; n=6; **P<0.01 vs. N; ^#P<0.05 and ^##P<0.01 vs. S. H, STZ + high-dose GEN group; L, STZ + low-dose GEN group; HO-1, heme oxygenase-1; N, normal control group; Nrf2, nuclear factor erythroid 2-related factor 2; NQO1, NAD(P)H:quinone oxidoreductase 1; S, STZ group.

Figure 7.

Protein expression of TGF-β1, Smad3, p-Smad3 and collagen IV in the different groups. Representative western blotting images and semi-quantitative analysis of TGF-β1, Smad3, p-Smad3, p-Smad3/Smad3 and collagen IV proteins; GAPDH was used as a loading control. Data are presented as the mean ± standard deviation; n=6; **P<0.01 vs. N; ^#P<0.05 and ^##P<0.01 vs. S. H, streptozotocin + high-dose genistein group; L, streptozotocin + low-dose genistein group; N, normal control group; p, phosphorylated; S, streptozotocin group; Smad3, mothers against decapentaplegic homolog 3; TGF-β1, transforming growth factor-β1.