Protective effects of Zhibai Dihuang Wan on renal tubular cells affected with gentamicin-induced apoptosis

Abstract

Ethnopharmacological relevance: Zhibai Dihuang Wan (ZDW) is an ancient traditional Chinese medicine composed of eight herbal ingredients and has been used to treat chronic kidney inflammation and diabetes for thousands of years. Nonetheless, the influence of ZDW on acute kidney injury is still unknown. We intended to identify the influence of ZDW on cell growth and gentamicin-induced apoptotic injury in renal tubular cells.

Materials and methods: We extracted ZDW with artificial intestinal fluid and treated rat renal tubular cells (NRK-52E) with various concentrations of the ZDW extraction. Cell proliferation and gentamicin-induced apoptosis of NRK-52E cells were evaluated using real-time proliferation monitoring and annexin V staining, respectively. Western blotting was used to evaluate the levels of Bcl-2 and caspase-3 expression. The effect of ZDW on gentamicin-induced kidney injury was also monitored in mice using the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay, and the measurement of serum creatinine and blood urea nitrogen.

Results: We found that 30 μg/ml of ZDW promoted cell proliferation of the rat renal tubular cells. ZDW also expressed a dose-dependent protective effect against gentamicin-induced apoptosis in the cells. Pretreatment with 3 μg/ml or 30 μg/ml of ZDW maximally increased Bcl-2 and decreased cleaved caspase-3 in the gentamicin-treated NRK-52E cells. Among the herbal ingredients of ZDW, only Phellodendron amurense Rupr., bark (Cortex Phellodendri), and Anemarrhena asphodeloides Bunge, rhizome inhibited both the gentamicin-induced Bcl-2 decrease and cleaved caspase-3 increase. Phellodendron amurense Rupr., bark and Anemarrhena asphodeloides Bunge, rhizome also inhibited gentamicin-induced apoptosis at particular concentrations; however, these two ingredients were less effective than ZDW. In the mouse model of gentamicin-induced nephropathy, the ZDW treatment significantly reduced apoptotic cells in the renal cortex and improved renal function.

Conclusions: Our results suggest that ZDW at adequate doses attenuates gentamicin-induced apoptotic injury in renal tubular cells and also protects kidneys from gentamicin-induced injury in mice.

Keywords: Acute kidney disease; Apoptosis; Fluorescein isothiocyanate (Pubchem CID: 18730); Gentamicin; Gentamicin (Pubchem CID:3467); Methanol (Pubchem CID: 887); Pentobarbital (Pubchem CID: 4737); Potassium dihydrogen phosphate (Pubchem CID: 516951); Propidium iodide (Pubchem CID: 104981); Renal tubular cell; Zhibai Dihuang Wan.