Ameliorative Effect of Daidzein on Cisplatin-Induced Nephrotoxicity in Mice via Modulation of Inflammation, Oxidative Stress, and Cell Death

Abstract

Oxidative stress and inflammation are part and parcel of cisplatin-induced nephrotoxicity. The purpose of this work is to study the role of soy isoflavone constituent, daidzein, in cisplatin-induced renal damage. Cisplatin-induced nephrotoxicity was evident by the histological damage in proximal tubular cells and by the increase in serum neutrophil gelatinase-associated lipocalin (NGAL), blood urea nitrogen (BUN), creatinine, and urinary kidney injury molecule-1 (KIM-1). Cisplatin-induced cell death was shown by TUNEL staining and caspase-3/7 activity. Daidzin treatment reduced all kidney injury markers (NGAL, BUN, creatinine, and KIM-1) and attenuated cell death (apoptotic markers). In cisplatin-induced kidney injury, renal oxidative/nitrative stress was manifested by the increase in lipid peroxidation and protein nitration. Cisplatin induced the reactive oxygen species-generating enzyme NOX-2 and impaired antioxidant defense enzyme activities such as glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities. Cisplatin-induced oxidative/nitrative stress was attenuated by daidzein treatment. Cisplatin induced CD11b-positive macrophages in kidneys and daidzein attenuated CD11b-positive cells. Daidzein attenuated cisplatin-induced inflammatory cytokines tumor necrosis factor α (TNFα), interleukin 10 (IL-10), interleukin 18 (IL-18), and monocyte chemoattractant protein-1 (MCP-1). Daidzein attenuated cell death in vitro. Our data suggested that daidzein attenuated cisplatin-induced kidney injury through the downregulation of oxidative/nitrative stress, immune cells, inflammatory cytokines, and apoptotic cell death, thus improving kidney regeneration.

Figure 1

Effect of daidzein on cisplatin-induced kidney tubular damage in mice. (a) Cisplatin induced tubular damage as shown by PAS staining. The damage was attenuated by daidzein (daidz) treatment at dose 200 mg/kg. (b) Quantification of the tubular damage score from PAS-stained slide. Results are mean ± SEM (n = 6/group). ^∗p < 0.05 versus vehicle and ^#p < 0.05 versus cisplatin.

Figure 2

Effect of daidzein on cisplatin-induced renal dysfunction in mice. Cisplatin caused significant renal dysfunction as determined by the levels of NGAL (a), BUN (b), creatinine (c), and urinary KIM-1 at 72 hours (d). Cisplatin induced kidney injury which was attenuated by daidzein treatment. Results are mean ± SEM (n = 6/group). ^∗p < 0.05 versus vehicle and ^#p < 0.05 versus cisplatin.

Figure 3

Effects of daidzein on cisplatin-induced cell death. Histological examination (a) demonstrated cisplatin-induced TUNEL staining (green) in the kidney and TUNEL staining was significantly attenuated with daidzein administration. Nuclei were stained with Hoechst 33342 (blue). (b) Caspase-3 activities were determined and daidzein attenuated cisplatin-induced caspase-3 activity. Results are mean ± SEM (n = 6/group). ^∗p < 0.05 versus vehicle and ^#p < 0.05 versus cisplatin.

Figure 4

Effect of daidzein on cisplatin-induced oxidative/nitrative stress. (a) Quantitative measurement of HNE adducts and protein nitration by ELISA demonstrated cisplatin-induced lipid peroxidation and protein nitration. Daidzein attenuated both cisplatin-induced oxidative/nitrative stress markers. (b) Histological staining of protein nitration. A trend similar to quantitative protein nitration was observed. Results are mean ± SEM (n = 6/group). ^∗p < 0.05 versus vehicle and ^#p < 0.05 versus cisplatin.

Figure 5

Effect of daidzein on cisplatin-induced changes in the ROS-generating enzyme NOX2 and antioxidant defense in mice. (a) Cisplatin induced the ROS-generating enzyme NOX2 mRNA as determined by real-time PCR, and daidzein attenuated cisplatin-reduced reduced glutathione reserve, glutathione peroxidase activity, and SOD activity. Daidzein administration restored those antioxidant defenses close to the control group. Results are mean ± SEM (n = 6/group). ^∗p < 0.05 versus vehicle and ^#p < 0.05 versus cisplatin.

Figure 6

Effect of daidzein on cisplatin-induced CD11b-positive monocyte/macrophage in mice. Immunofluorescence examination revealed significant CD11b-positive cells (yellow) of the cisplatin-treated group. Nuclear staining (blue) was carried out using Hoechst 33342. In the cisplatin group, a zoom image of single cells was provided as an inset to demonstrate that staining covers surface staining and a larger area than nuclear staining. Daidzein treatment reduced the number of CD11b-positive cells. Either vehicle (Veh) or daidzein (daidz) control group does not have any CD11b-positive cells.

Figure 7

Effect of daidzein on cisplatin-induced proinflammatory cytokines in mice. (a) Real-time PCR-based analyses of proinflammatory cytokines TNFα, IL-10, IL-18, and MCP-1 indicated a profound increase in cisplatin-treated mice. Daidzein treatment attenuated cisplatin-induced cytokine MRNA expression. Results are mean ± SEM (n = 6/group). ^∗p < 0.05 versus vehicle and ^#p < 0.05 versus cisplatin. (b) Western blot analyses of TNFα and control GAPDH.

Figure 8

Effect of daidzein on cisplatin-induced cell death in vitro. (a) Representative dot plot of flow cytometric data of an HK-2 cell treated with either saline or daidzein in the presence or absence of cisplatin. x-axis represented the apoptotic cell death marker Annexin V whereas y-axis represented the dead cell marker SYTOX Green. (b) Quantitative determination of cell death (combined Q2 and Q3) among different groups. Results are mean ± SEM (n = 3/group). ^∗p < 0.05 versus vehicle and ^#p < 0.05 versus cisplatin.

Figure 9

Schematic diagram of the protection mechanism of daidzein in cisplatin-induced kidney injury. EGCG inhibit cisplatin-induced ROS by attenuating ROS-generating enzymes and improving cisplatin-impaired antioxidant defense mechanisms in the renal tubular cells which caused cell death. Cell death also leads to proinflammatory response with cytokines and infiltrating leukocytes with the additional release of ROS. Daidzein attenuates cell death directly. Daidzein also neutralize cytokines and infiltrating leukocytes. Both antioxidant and anti-inflammatory effects leads to reduced cell death, thus protecting against cisplatin-induced kidney injury.