Rhein lysinate protects renal function in diabetic nephropathy of KK/HlJ mice

Abstract

The purpose of the present study was to assess the protective effects of rhein lysinate (RHL) in a KK/HlJ mouse model of diabetic nephropathy (DN) and to explore its mechanism of action. A total of 4 groups were established: C57BL/J control, the KK/HlJ model and 25 and 50 mg/kg/day RHL-treated KK/HlJ groups. The KK/HlJ mouse model of DN was established by streptozotocin injection, followed by maintenance on a specific diet. The albumin-to-creatinine ratio (ACR) was determined at 5 weeks and at 16 weeks, the kidneys were harvested, and morphological examination and immunohistochemical analysis were performed. The levels of malondialdehyde (MDA), as well as superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) activities in the kidneys were measured using appropriate assay kits. The expression of inflammatory factors and associated proteins was analyzed using western blot analysis. At 5 weeks, the levels of ACR in KK/HlJ mice were increased, which was inhibited by treatment with RHL. Treatment with RHL (50 mg/kg/day) decreased the body weight of KK/HlJ mice. Compared with the C57BL/J control, the KK/HlJ model mice had a significantly lower activity of SOD and GSH-px in the kidneys, but had significantly higher levels of MDA. Treatment of KK/HlJ mice with RHL significantly increased the activities SOD and GSH-px, and reduced the MAD level in the kidneys. Renal tubular epithelial cell edema was observed in KK/HlJ mice but not in C57BL/J mice. RHL decreased the incidence of renal tubular epithelial cell edema and significantly decreased the expression of TNF-α and IL-6 as well as the expression and phosphorylation of NF-κB in the kidneys. Therefore, DN is associated with the expression of inflammatory factors, renal tubular epithelial cell edema and renal dysfunction in KK/HlJ mice. RHL improves renal function by decreasing kidney inflammation.

Keywords: KK/HlJ mice; inflammatory factors; kidney; oxidative stress; rhein lysinate.

Figure 1.

Kidney and body weights of the mice and the kidney weight to body weight ratio. The diabetic nephropathy model group mice were intraperitoneally injected with streptozotocin for 5 consecutive days and received a specific diet for 16 weeks. Values are expressed as the mean ± standard deviation. *P<0.05 vs. C57BL/J control group; ^#P<0.05 vs. KK/HlJ model group.

Figure 2.

Effect of RHL on changed of the histopathological profile of the kidneys of mice, as determined by hematoxylin and eosin staining (magnification, ×200). Renal sections from (A) the C57BL/6J control group, (B) the KK/HlJ model group, (C) the 25 mg/kg/day RHL-treated KK/HlJ group and (D) the 50 mg/kg/day RHL-treated KK/HlJ group. Renal tubular epithelial cell edema was present in the KK/HlJ control mice. RHL, rhein lysinate.

Figure 3.

Effects of RHL on the MDA content and on the activity of SOD and GSH-Px in the kidney tissues of mice in each group. (A) MDA content and the activities of (B) SOD and (C) GSH-px were determined using test kits. *P<0.05 vs. C57BL/6J control group; ^#P<0.05 vs. KK/HlJ model group. RHL, rhein lysinate; SOD, superoxide dismutase; MDA, malondialdehyde; GSH-Px, glutathione peroxidase.

Figure 4.

Effects of RHL on the expression of TNF-α in mouse kidney tissue. Representative immunohistochemically stained renal tissue sections from (A) the C57BL/6J control group, (B) the KK/HlJ model group, (C) the 25 mg/kg/day RHL-treated KK/HlJ group and (D) the 50 mg/kg/day RHL-treated KK/HlJ group (magnification, ×200). TNF-α expression was indicated by brown staining. TNF, tumor necrosis factor; RHL, rhein lysinate.

Figure 5.

Effects of RHL on the expression of IL-6 in the mouse kidney tissue. Representative immunohistochemically stained renal tissue sections from (A) the C57BL/6J control group, (B) the KK/HlJ model group, (C) the 25 mg/kg/day RHL-treated KK/HlJ group and (D) the 50 mg/kg/day RHL-treated KK/HlJ group (magnification, ×200). IL-6 expression was indicated by brown staining. IL, interleukin; RHL, rhein lysinate.

Figure 6.

Effects of RHL on the expression of proteins associated with inflammation-associated genes in the kidney were assessed by western blot analysis. β-actin was used as the internal control. Values are expressed as the mean ± standard deviation. *P<0.05 vs. C57BL/6J control group; ^#P<0.05 vs. KK/HlJ model group. RHL, rhein lysinate.