Cilostazol ameliorates diabetic nephropathy by inhibiting highglucose- induced apoptosis

Abstract

Diabetic nephropathy (DN) is a hyperglycemia-induced progressive development of renal insufficiency. Excessive glucose can increase mitochondrial reactive oxygen species (ROS) and induce cell damage, causing mitochondrial dysfunction. Our previous study indicated that cilostazol (CTZ) can reduce ROS levels and decelerate DN progression in streptozotocin (STZ)-induced type 1 diabetes. This study investigated the potential mechanisms of CTZ in rats with DN and in high glucose-treated mesangial cells. Male Sprague-Dawley rats were fed 5 mg/kg/day of CTZ after developing STZ-induced diabetes mellitus. Electron microscopy revealed that CTZ reduced the thickness of the glomerular basement membrane and improved mitochondrial morphology in mesangial cells of diabetic kidney. CTZ treatment reduced excessive kidney mitochondrial DNA copy numbers induced by hyperglycemia and interacted with the intrinsic pathway for regulating cell apoptosis as an antiapoptotic mechanism. In high-glucose-treated mesangial cells, CTZ reduced ROS production, altered the apoptotic status, and down-regulated transforming growth factor beta (TGF-β) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB). Base on the results of our previous and current studies, CTZ deceleration of hyperglycemia-induced DN is attributable to ROS reduction and thereby maintenance of the mitochondrial function and reduction in TGF-β and NF-κB levels.

Keywords: Cilostazol; Diabetic nephropathy; Mesangial cell; Mitochondrial DNA; Oxidative stress.

Fig. 1. Electron micrographs of representative kidney sections from the experimental groups.

(A) Panel A’, control group; Panel B’, STZ-induced diabetic group; and Panel C’, STZ + 5 mg/kg/day of CTZ group treating for 12 weeks (6 rats per group); (B) quantification of thickness of glomerular basement membrane (GBM). All the figures are 20,000× magnification. Mean values within each column with different labels (a, b) are significantly different (p < 0.05). CTZ, cilostazol; STZ, streptozotocin; P, podocyte; G, GBM; M, mesangium; R, red blood cell; DR, diabetic rats.

Fig. 2. Mitochondrion examination under electron micrographs of kidney sections.

(A) control group, (B) STZ-induced diabetic group, (C) STZ + 5 mg/kg/day of CTZ group (6 rats per group). All the photoes were taken at 50,000× magnification. CTZ, cilostazol; STZ, streptozotocin.

Fig. 3. Effects of CTZ on the mtDNA copy number in diabetic nephropathy.

mtDNA obtained from rat kidneys were detected in RT-PCR of C, control; DR, diabetic rats; CTZ, cilostazol treated for 6 weeks (6 rats per group). The results were expressed as the mean ± standard deviation of three determinations. Mean values within each column with different labels (a, b) are significantly different (p < 0.05).

Fig. 4. CTZ improves the expression of mitochondrial apoptotic proteins in diabetic nephropathy.

Immunoblot examination of Bax, Bcl-2, and caspase-3 in rat kidneys of C, control; DR, diabetic rats; CTZ, cilostazol treated for 12 weeks.

Fig. 5. CTZ reduces the ROS in RMC caused by high glucose.

RMC was treated with 25 mM glucose along with various concentrations of CTZ for 24 h. The NADPH oxidase activity (A) and DCFDA assay (B) was conducted to detect the level of ROS. Mean values within each column with different labels (a–e) are significantly different (p < 0.05). The results were expressed as the mean ± standard deviation of three determinations. All data are normalized to the group of high glucose. CTZ, cilostazol; ROS, reactive oxygen species; RMC, rat mesangial cell; NADPH, nicotinamide adenine dinucleotide phosphate.

Fig. 6. CTZ improves the expression of mitochondrial apoptotic proteins in high glucose treated RMC.

Immunoblot examination of Bax and Bcl-2 in RMC was performed after treating with high glucose and CTZ for 24 h. All quantitative determination were normalized to the group of 5 mM glucose. CTZ, cilostazol; RMC, rat mesangial cell.

Fig. 7. CTZ decreases the level of NF-κB, TGF-β and TNF-α.

RMC cell was exposed to high glucose and variable concentration of CTZ. (A) The protein level of cellular TNF-α, TGF-β, IkB and NF-κB p65 were analyzed by immunoblot, the quantitative determination were normalized to the group of 5 mM glucose. The level of secreted (B) TGF-β and (C) TNF-α were determined by ELISA; the results were expressed as the mean ± standard deviation of three determinations and all data were normalized to the group of high glucose represented as 100%. Mean values within each column with different labels (a–d) are significantly different (p < 0.05). CTZ, cilostazol; RMC, rat mesangial cell.