2-Methoxyestradiol TPGS Micelles Attenuate Cyclosporine A-Induced Nephrotoxicity in Rats through Inhibition of TGF-β1 and p-ERK1/2 Axis

Abstract

The immunosuppressant cyclosporine A (CSA) has been linked to serious renal toxic effects. Although 2-methoxyestradiol (2ME) possesses a wide range of pharmacological abilities, it suffers poor bioavailability after oral administration. The purpose of this study was to evaluate the potential of 2ME loaded D-ɑ-tocopheryl polyethylene glycol succinate (TPGS) micelles to prevent CSA-induced nephrotoxicity in rats. A 2ME-TPGS was prepared and showed particle size of 44.3 ± 3.5 nm with good entrapment efficiency and spherical structures. Male Wistar rats were divided into 5 groups, namely: Control, Vehicle, CSA, CSA + 2ME-Raw, and CSA + 2ME-Nano. CSA was injected daily at a SC dose of 20 mg/kg. Both 2ME-Raw and 2ME-Nano were given daily at oral doses of 5 mg/kg. Treatments continued for three successive weeks. 2ME-TPGS exerted significant protective effects against CSA nephrotoxicity. This was evidenced in ameliorating deterioration of renal functions, attenuation of pathological changes in kidney tissues, exerting significant anti-fibrotic, antioxidant, and anti-inflammatory effects together with significant anti-apoptotic effects. Western blot analyses showed both 2ME-Raw and 2ME-Nano significantly inhibited protein expression of TGF-β1 and phospho-ERK (p-ERK). It was observed that 2ME-TPGS, in almost all experiments, exerted superior protective effects as compared with 2ME-Raw. In conclusion, 2ME loaded in a TPGS nanocarrier possesses significant protective activities against CSA-induced kidney injury in rats. This is attributable to 2ME anti-fibrotic, antioxidant, anti-inflammatory, and anti-apoptotic activities which are mediated at least partly by inhibition of TGF-β1/p-ERK axis.

Keywords: 2-methoxyestradiol; TGF-β1; TPGS; cyclosporine A; nephrotoxicity; p-ERK.

Figure 1

TEM image of 2ME TPGS micelles.

Figure 2

Effect of 2ME-TPGS micelles on serum markers of kidney function following cyclosporine A (CSA) challenge in rats. (A): creatinine, (B): Urea, (C): Cystatin C. Data are presented as Mean ± SD (n = 6). a: Significantly different from respective Control at p < 0.05; b: significantly different from Vehicle at p < 0.05; c: significantly different from CSA at p < 0.05; d: significantly different from CSA + 2ME-Raw at p < 0.05.

Figure 3

Effects of 2ME-TPGS on CSA-induced kidney injury as visualized using H&E, Masson’s Trichrome (MT), Picrosirius Red (PSR), and Periodic acid–Schiff (PAS) stains. In H&E-stained sections, black arrows indicate atrophy of glomerular capillary tuft and red arrows indicate extensive renal tubular damage. In MT-stained sections, blue coloration indicates collagen deposition. In PSR-stained sections, red coloration indicates collagen deposition. In PAS-stained sections, intense bluish-purple coloration indicates thickening of the glomerular basement membrane.

Figure 4

Effect of 2-ME-TPGS on oxidative stress markers in CSA-treated rats. (A) MDA; (B) SOD; and (C) CAT. Data are displayed as mean ± SD (n = 6). a: Significantly different from respective Control at p < 0.05; b: significantly different from Vehicle at p < 0.05; c: significantly different from CSA at p < 0.05; d: significantly different from CSA + 2ME-Raw at p < 0.05.

Figure 5

Effect of 2ME-TPGS on IL-6, TNF-α, COX-2 and NFκB expression as determined immunohistochemically in kidney tissues of rats challenged with cyclosporine (CSA). Bar charts are mean ± SD (n = 6). a: Significantly different from respective Control at p < 0.05; b: significantly different from Vehicle at p < 0.05; c: significantly different from CSA at p < 0.05; d: significantly different from CSA + 2ME-Raw at p < 0.05.

Figure 6

Effect of 2ME-TPGS on kidney mRNA expression of Bax (A) and Bcl-2 (B) in CSA-treated rats. Data presented in bar charts are expressed as mean ± SD (n = 6). a, b, c, or d: significantly different when compared with respective Control, Vehicle, CSA, or CSA + 2ME-Raw at p < 0.05, respectively.

Figure 7

Effect of 2-ME-TPGS on TGF-β1 and phosphorylated ERK1/2 expression in renal tissues of CSA-challenged rats (A–C). Data in bar charts are shown as mean ± SD (n = 6). a, b, c, or d: significantly different when compared with respective Control, Vehicle, CSA, or CSA + 2ME-Raw at p < 0.05, respectively.