Protective effect of ginsenosides Rk3 and Rh4 on cisplatin-induced acute kidney injury in vitro and in vivo

Abstract

Background: Nephrotoxicity is the major side effect in cisplatin chemotherapy. Previously, we reported that the ginsenosides Rk3 and Rh4 reduced cisplatin toxicity on porcine renal proximal epithelial tubular cells (LLC-PK1). Here, we aimed to evaluate the protective effect of ginsenosides Rk3 and Rh4 on kidney function and elucidate their antioxidant effect using in vitro and in vivo models of cisplatin-induced acute renal failure.

Methods: An enriched mixture of ginsenosides Rk3 and Rh4 (KG-KH; 49.3% and 43.1%, respectively) was purified from sun ginseng (heat processed Panax ginseng). Cytotoxicity was induced by treatment of 20μM cisplatin to LLC-PK1 cells and rat model of acute renal failure was generated by single intraperitoneal injection of 5 mg/kg cisplatin. Protective effects were assessed by determining cell viability, reactive oxygen species generation, blood urea nitrogen, serum creatinine, antioxidant enzyme activity, and histopathological examination.

Results: The in vitro assay demonstrated that KG-KH (50 μg/mL) significantly increased cell viability (4.6-fold), superoxide dismutase activity (2.8-fold), and glutathione reductase activity (1.5-fold), but reduced reactive oxygen species generation (56%) compared to cisplatin control cells. KG-KH (6 mg/kg, per os) also significantly inhibited renal edema (87% kidney index) and dysfunction (71.4% blood urea nitrogen, 67.4% creatinine) compared to cisplatin control rats. Of note, KG-KH significantly recovered the kidney levels of catalase (1.2-fold) and superoxide dismutase (1.5-fold).

Conclusion: Considering the oxidative injury as an early trigger of cisplatin nephrotoxicity, our findings suggest that ginsenosides Rk3 and Rh4 protect the kidney from cisplatin-induced oxidative injury and help to recover renal function by restoring intrinsic antioxidant defenses.

Keywords: Panax ginseng; acute kidney injury; cisplatin; ginsenoside Rh4; ginsenoside Rk3.

Fig. 1

Structure of ginsenosides and chromatogram of enriched mixture of ginsenosides Rk3 and Rh4 (KG-KH). Structures of (A) ginsenoside Rk3 and (B) ginsenoside Rh4. (C) Chromatogram of KG-KH. Solution of KG-KH (0.5 mg/mL) was analyzed using HPLC coupled with an evaporative light scattering detector.

Fig. 2

Ginsenoside Rk3 and Rh4 extract (KG-KH) protects LLC-PK1 cells against cisplatin cytotoxicity. LLC-PK1 cells were treated with cisplatin and/or KG-KH for 24 h. Cell viability was measured using the MTT assay. Bar represents mean ± standard error of the mean (n = 3). Significant differences were determined by the Student t test (^###p < 0.001 compared with normal cells) or ANOVA (*p < 0.05, **p < 0.01 compared with cisplatin control cells). C, cisplatin control cells. N, normal cells.

Fig. 3

Ginsenoside Rk3 and Rh4 extract (KG-KH) restores cisplatin-induced reactive oxygen species (ROS) generation in LLC-PK1 cells. LLC-PK1 cells were treated with cisplatin (20μM) and/or KG-KH (50 μg/mL) for 24 h. ROS were determined using a ROS-sensitive fluorescence probe (2′,7′-dichlorofluorescin diacetate). (A) Stained cells were visualized using a fluorescence microscope. (B) Scale bar represents 50 μm. Quantitation was achieved using a flow cytometer. Bar represents mean ± standard error of the mean (n = 3). Significant difference was determined by the Student t test (*p < 0.05, *** p < 0.001). C, cisplatin control cells; N, normal cells.

Fig. 4

Ginsenoside Rk3 and Rh4 extract (KG-KH) recovers the antioxidant enzyme activity in cisplatin-treated LLC-PK1 cells. LLC-PK1 cells were treated with cisplatin (20μM) and/or KG-KH (50 μg/mL) for 24 h. At the end of incubation, cells were harvested, and cell homogenates were prepared and used for assays. (A) Superoxide dismutase (SOD) and (B) glutathione reductase (GR) activity was measured using a commercial kit. Bar represents mean ± standard error of the mean (n = 3). Significant differences were determined by the Student t test (*p < 0.05, **p < 0.01, ***p < 0.001). C, cisplatin control cells; N, normal cells.

Fig. 5

Ginsenoside Rk3 and Rh4 (KG-KH) recovers the activity of renal antioxidant enzymes in cisplatin-injected rats. Acute kidney injury was induced by single injection of cisplatin (5.0 mg/kg, i.p.). Rats were given KG-KH (0 mg/kg, 2 mg/kg, and 6 mg/kg, per os) once a day for 5 d from the day of cisplatin injection. Six days after cisplatin injection, kidney levels of (A) superoxide dismutase (SOD) and (B) catalase (CAT) activity were determined in kidney homogenate. Bar represents mean ± standard error of the mean (n = 6). Significant differences were determined by the Student t test (^#p < 0.05, ^##p < 0.01 compared with normal rats) and ANOVA (*p < 0.05, **p < 0.01 compared with cisplatin control rats). C, cisplatin control rats; N, normal cells.

Fig. 6

Ginsenoside Rk3 and Rh4 (KG-KH) improves renal histology in cisplatin-injected rats. (A) Representative images of the renal pathology (hematoxylin–eosin staining) on Day 6 after cisplatin injection with/without KG-KH were presented. Black arrow indicates tissue injury with cell death, infiltration, cast formation, exfoliation and shallowing. Scale bar represents 100 μm. Tissue injury in the (B) cortex and (C) corticomedullary regions were evaluated by semiquantitative scoring of each pathological marker and expressed as the sum of all scores. Bar represents mean ± standard error of the mean (n = 6). Significant differences were determined by the Student t test (^###p < 0.001 compared with normal rats) or analysis of variance (**p < 0.01 compared with cisplatin control rats). C, cisplatin control rats; dt, distal tubule; g, glomerulus; N, normal rats; pt, proximal tubule.