Dahuang Fuzi Decoction Attenuates Renal Fibrosis and Ameliorates Mitochondrial Dysfunction in Chronic Aristolochic Acid Nephropathy

Abstract

Objectives. The effects of the traditional formula Dahuang Fuzi Decoction (DFD) on chronic aristolochic acid nephropathy (AAN) in mice and its underlying mechanisms were studied. Methods. Mice were randomly divided into the following six groups: the control group, the model group (AAN), the saline-treated group (AAN + vehicle), the normal dose DFD-treated group (AAN + NDFD), the high dose DFD-treated group (AAN + HDFD), and the rosiglitazone treated group (AAN + Rosi). After treating for 8 weeks, 24 h urine and blood samples were collected and the mice sacrificed to study the biochemical parameters associated with renal function. The samples were analyzed for renal fibrosis and mitochondrial dysfunction (MtD) markers. To achieve that, collagen III, collagen I, mitochondrial DNA copy numbers (mtDNA), mitochondrial membrane potential (MMP), ATP content, and ROS production were evaluated. Results. Our results showed that proteinuria, kidney function, and the renal pathological characteristics were improved by DFD and rosiglitazone. The expression of collagen III and collagen I decreased after treating with either DFD or rosiglitazone. Mitochondrial dysfunction based on the increase in ROS production, decrease in mitochondrial DNA copy numbers, and reduction of MMP and ATP content was improved by DFD and rosiglitazone. Conclusions. DFD could protect against renal impairments and ameliorate mitochondrial dysfunction in chronic AAN mice.

Figure 1

Effects of Dahuang Fuzi Decoction (DFD) on the formation of renal fibrosis in the renal tissues of chronic aristolochic acid nephropathy (AAN) mice (n = 6 mice/group). Representative Masson trichrome staining (400x) was used to analyze renal fibrosis in kidneys from paraformaldehyde-fixed kidney sections taken from each group of mice.

Figure 2

Western blot analysis for detecting the expressions of collagen I and collagen III in renal tissues. (a) Representative immunoblots obtained from control, AAN, AAN + vehicle, AAN + NDFD, AAN + HDFD, and AAN + Rosi are shown. (b, c) Densitometry analysis for Western blot results of collagen I and collagen III protein in extracts prepared from 6 mice in each group. ^∗P < 0.05 versus control. ^#P < 0.05 versus AAN.

Figure 3

Effect of Dahuang Fuzi Decoction (DFD) on mitochondrial function in chronic aristolochic acid nephropathy (AAN) mice. (a) Mitochondrial membrane potential (MMP) analyzed by flow cytometry. (b) Densitometry analysis of MMP. (c) Adenosine-50-triphosphate (ATP) production. (d) Mitochondrial DNA (mtDNA) copy numbers. Data shown as mean ± SD (n = 6). ^∗P < 0.05 versus control; ^#P < 0.05 versus AAN.

Figure 4

Effect of Dahuang Fuzi Decoction (DFD) on kidney ROS production in chronic aristolochic acid nephropathy (AAN) mice. (a) Detection of oxidative products using 2′,7′-dichlorofluorescein (DCF) to measure hydrogen peroxide in the kidney (×200). (b) Quantification of the pixel density of DCF staining in glomeruli. (c) Kidney mitochondrial levels of reactive oxygen species (ROS). Data are shown as mean ± SD (n = 6). ^∗P < 0.01 versus control; ^#P < 0.01 versus AAN mice by analysis of variance.