Pioglitazone ameliorates methotrexate-induced renal endothelial dysfunction via amending detrimental changes in some antioxidant parameters, systemic cytokines and Fas production

Abstract

Methotrexate (MTX) is widely used in treatment of cancers and autoimmune diseases. However, nephrotoxicity is one of its most important side effects. The peroxisome proliferator-activated receptor gamma agonist, pioglitazone, is known to exert antiinflammatory and reno-protective effects in various kidney injuries. The purpose of this study was to investigate the potential involvement of endothelial damage in MTX-induced renal injury and to elaborate the possible protective effect of pioglitazone against MTX-induced endothelial impairment. Compared with saline-treated rats, treatment with MTX (7 mg/kg for 3 days) caused significant elevations in serum levels of urea and creatinine, increased renal nitrate/nitrite level and impaired renovascular responsiveness of isolated perfused kidney to endothelium-dependent vasodilations induced by acetylcholine (0.01–2.43 nmol) and isoprenaline (1 μmol). These effects were abolished by concurrent treatment with pioglitazone (2.5 mg/kg, for 5 days starting 2 days before MTX). Alternatively, MTX treatment did not affect endothelium-independent renovascular relaxation induced by sodium nitroprusside (0.001–10 μmole). The possibility that alterations in renal antioxidants, circulating cytokine and apoptotic factor (Fas) levels contributed to MTX-pioglitazone interaction was assessed. Pioglitazone treatment abrogated renal oxidative stress (decreased reduced glutathione and catalase activity and increased malondialdehyde), elevated serum cytokine (interleukin-6, interleukin-10, tumor necrosis factor-alpha and transforming growth factor-beta1) and Fas induced by MTX. Histologically, MTX caused defused tubular cells swelling and vacuolization associated with endothelial damage in renal arterioles. These effects disappeared upon co-treated with pioglitazone. Collectively, pioglitazone abolished MTX-induced endothelium dysfunction and nephrotoxicity via ameliorating oxidative stress and rectifying cytokines and Fas abnormalities caused by MTX.

Introduction

Methotrexate (MTX) is widely used antifolate for treatment of different types of cancer [61] and autoimmune diseases [66]. However, its negative nephrotoxic impact continues to present serious challenges in medicine restricting its use [11], [79]. MTX is excreted mainly via the kidney [13]; therefore, renal dysfunction affects MTX clearance leading to its accumulation [49]. Alternatively, the buildup of MTX in plasma due renal injury further compromises renal functions [11]. The pathogenesis of MTX-induced renal dysfunction is not clear, although it is believed to be mediated via the precipitation of MTX in the kidney plugging renal tubules [97] and by direct toxic effect of MTX on tubular cells [43]. This eventually causes renal tubular necrosis and acute renal failure [32]. Remarkably, increased reactive oxygen species (ROS) production is considered major cause for MTX-related renal toxicity [2], [14]. Also, in 2006, Güler and co-workers showed that MTX elevated plasma nitrate/nitrite level, the stable products of the endogenous endothelial vasodilator nitric oxide (NO), which was accompanied by decline in renal functions. Accumulating evidences indicate that endothelium-derived NO is tonically synthesized within the kidney, where it plays a major role in the regulation of renal blood flow. Under pathologic conditions, renal NO generation is markedly enhanced, which implicates NO in renal dysfunction [8]. Although few experimental studies have shown that endothelial injury is involved in MTX toxic effects in brain leading to stroke [22], or in liver where MTX administered causes damage to sinusoidal endothelium [4], to our knowledge, no study was performed to assess the involvement of endothelial damage in MTX nephrotoxic action and its possible mechanism.

Pioglitazone is peroxisome proliferator-activated receptor gamma (PPARγ) agonist that lowers blood glucose level through improving insulin resistance [30]. Studies demonstrated that PPARγ are expressed in the kidneys. Furthermore, pioglitazone exhibits reno-protective properties [33] via (i) facilitation of endothelium-dependent vasodilation through rectifying abnormalities in NO production [1], [23], (ii) improving the antioxidant profile [26], and (iii) regulation of the expression of inflammatory mediators [42], [93] and apoptotic factors [18].

The endothelium is the largest organ in body; hence, endothelial damage is implicated in many diseases including renal failure and its associated cardiovascular complications [58]. Therefore, the rationale of this study was to verify whether endothelial damage is involved in MTX-induced renal injury and its possible correction by concurrent administration of pioglitazone. This interesting possibility was investigated at functional level using isolated perfused rat kidney to test the involvement of endothelium in such interaction as well as at biochemical (serum urea and creatinine levels, oxidative stress and cytokine production) and histopathological levels to gain insight of the mechanism of MTX-pioglitazone interaction.