Huangkui capsule in combination with metformin ameliorates diabetic nephropathy via the Klotho/TGF-β1/p38MAPK signaling pathway

Ethnopharmacological relevance: Huangkui capsule (HKC), extracted from Abelmoschus manihot (L.) medic (AM), as a patent proprietary Chinese medicine on the market for approximately 20 years, has been clinically used to treat chronic glomerulonephritis. Renal fibrosis has been implicated in the onset and development of diabetic nephropathy (DN). However, the potential application of HKC for preventing DN has not been evaluated. Aim of the study: This study was designed to investigate the efficacy and underlying mechanisms of HKC combined with metformin (MET), the first-line medication for treating type 2 diabetes, in the treatment of renal interstitial fibrosis. Materials and methods: A rat model of diabetes-associated renal fibrosis was established by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg) combined with a high-fat and high-glucose diet. The rats were randomly divided into five groups: normal control, DN, HKC (1.0 g/kg/day), MET (100 mg/kg/d), and HKC plus MET (1.0 g/kg/day + 100 mg/kg/d). Following drug administration for 8 weeks, we collected blood, urine, and kidney tissue for analysis. Biochemical markers and metabolic parameters were detected using commercial kits. Histopathological staining was performed to monitor morphological changes in the rat kidney. High-glucose induced human kidney HK-2 cells were used to evaluate the renal protective effects of HKC combined with MET (100 mu g/mL+10 mmol/L). MTT assay and acridine orange/ethidium bromide were used to examine cell proliferation inhibition rates and apoptosis. Immunofluorescence assay and Western blot analysis were performed to detect renal fibrosis-related proteins including Klotho, TGF-beta 1, and phosphorylated (p)-p38. Results: Combination therapy (HKC plus MET) significantly improved the weight, reduced blood glucose (BG), blood urea nitrogen (BUN), total cholesterol (T-CHO), triglycerides (TG), low-density lipoprotein (LDL) and increased the level of high-density lipoprotein (HDL) of DN rats. Combination therapy also significantly reduced urine serum creatinine (SCR) and urine protein (UP) levels as well as reduced the degrees of renal tubule damage and glomerulopathy in DN rats. Combination therapy ameliorated renal fibrosis, as evidenced by reduced levels of alpha-smooth muscle actin and fibronectin and increased expression of E-cadherin in the kidneys. Moreover, HKC plus MET alleviated the degree of DN in part via the Klotho/TGF-beta 1/p38MAPK signaling pathway. In vitro experiments showed that combination therapy significantly inhibited cell proliferation and apoptosis and regulated fibrosis-related proteins in high-glucose (HG)-induced HK-2 cells. Further studies revealed that combination therapy suppressed cell proliferation and fibrosis by inhibiting the Klotho-dependent TGF-beta 1/p38MAPK pathway. Conclusions: HKC plus MET in combination suppressed abnormal renal cell proliferation and fibrosis by inhibiting the Klotho-dependent TGF-beta 1/p38MAPK pathway. Collectively, HKC combined with MET effectively improved DN by inhibiting renal fibrosis-associated proteins and blocking the Klotho/TGF-beta 1/p38MAPK signaling pathway. These findings improve the understanding of the pathogenesis of diabetes-associated complications and support that HKC plus MET combination therapy is a promising strategy for preventing DN.