br Materials and methods Male
Materials and methods Male Sprague–Dawley rats (Faculty of Pharmacy, Pharos University, Alexandria, Egypt) weighing 180 to 200g were used in this study. All experiments were performed in strict accordance with institutional animal care and use guidelines.
Discussion The current study is the first to report on cellular and molecular mechanisms of the protection offered by the selective COX-2 inhibitor, celecoxib, against chronic nephrotoxicity induced by CSA. Simultaneous treatment with celecoxib virtually abolished the manifestations of CSA nephrotoxicity including functional (serum urea and creatinine), inflammatory (IL-2), fibrotic (TGF-β1), and structural (tubular atrophy and interstitial fibrosis) profiles. The upregulation of the COX-2/ETB receptor axis by celecoxib might evidently contribute to its counterbalancing effect against CSA nephrotoxicity because (i) celecoxib abrogated the CSA-evoked reductions in the protein NBI 27914 hydrochloride of medullary COX-2 as well as medullary and cortical ETB receptors in renal tissues, (ii) the selective blockade of ETB receptors by BQ788 negatively influenced the biochemical and structural features of the kidney in a way that mimicked those caused by CSA, and (iii) the detrimental renal effects that followed ETB receptor blockade were largely improved by celecoxib. In agreement with reported studies (El-Mas et al., 2003, Herlitz and Lindelöw, 2000), the present findings that CSA caused significant rises in serum creatinine and BUN are consistent with impaired renal function. Elevated levels of these parameters are usually taken as early signs of renal insult (Klintmalm et al., 1981). Although, the underlying mechanisms of these deleterious renal effects of CSA are not fully understood, both ischemic and histopathological renal changes induced by CSA such as renal cortical interstitial fibrosis and the atrophy in proximal tubules contribute to a great extent to the inability of the kidney to filter creatinine and nitrogenous wastes (Khan et al., 2010, Paul and de Fijter, 2004). Remarkably, our results also suggest a nephroprotective effect for the COX-2 celecoxib because its use along with CSA blunted the detrimental biochemical and histopathological effects evoked by CSA. The current study establishes the first experimental evidence that celecoxib abrogates CSA nephrotoxicity via favorably affecting the renal TGF-β1/IL-2/COX-2 axis. Consistent with previous studies (Roos-van Groningen et al., 2006, Stacchiotti et al., 2002), we showed that the mechanism of the CSA-induced renal injury is multifactorial and involves interrelated increases in inflammatory (IL-2) and fibrogenic (TGF-β1) factors and decreases in the renal expression of COX-2 enzyme. The view that IL-2 and TGF-β1 are causally and mutually correlated is supported by cell culture studies, which showed facilitated IL-2 mRNA generation by TGF-β1 (Han et al., 1998). TGF-β1 is a multifunctional cytokine that causes renal fibrosis via decreasing degradation and increasing production of extracellular matrix proteins (Roos-van Groningen et al., 2006, Wolf, 2006). Likewise, IL-2 increases collagen synthesis in hepatic stellate cells, the major cell type involved in liver fibrosis (Zhan et al., 2003). Therefore, that the increase in renal IL-2-coupled TGF-β1 might have participated in the induction of the renal structural damage and fibrosis and hence, the deterioration in renal function caused by CSA. Although COX-2 and its prostanoid products are generally considered potent proinflammatory mediators (Hao and Breyer, 2008), evidence suggests that they possess antifibrotic effects (Vancheri et al., 2004). Further, COX-2-deficient mice show enhanced susceptibility to pulmonary (Bonner et al., 2002) and cardiac fibrosis (Dinchuk et al., 1995). With that in mind, the decreases caused by CSA in cortical and medullary COX-2 expressions observed in the current study might also be presumably correlated to the CSA-evoked facilitation of TGF-β1/IL-2 signaling and consequent fibrotic and nephrotoxic effects. Conventionally, the inhibitory effect of CSA on COX-2 expression is expected to reduce the renal blood flow and enhance fibrosis as a consequence of diminished generation of prostaglandins, which subserve vasodilator and antifibrotic properties (Harris and Breyer, 2001, Liu et al., 2010). Hétu and Riendeau (2005) reported a decrease of 35–50% in the renal prostanoid content following selective COX-2 inhibition. It is notable that contradictory data of positive and negative regulatory actions for TGF-β1 on COX-2 expression have been reported (Matsumura et al., 2009, Singh et al., 2012, Warner et al., 2011). The vasoconstriction and increased tissue stiffness induced by tissue fibrosis can lead to an adaptive reduction in COX-2 expression (Liu et al., 2010, Warner et al., 2011). Together, it is likely that the elevated renal TGF-β1 and subsequent opposite changes in COX-2 (decreases) and IL-2 (increases) might account, in part, for the renal fibrotic and nephrotoxic manifestations caused by CSA. By the same token, the reversal of the detrimental biochemical and structural effects of CSA by simultaneously administered celecoxib implies a key role for the TGF-β1/IL-2/COX-2 axis in renoprotective effect of celecoxib. Notably, the reason as to why celecoxib reduced cortical COX-2 expression when used alone is not clear. One possible mechanism may involve the ability of celecoxib to inhibit the nuclear factor of activated T cells independent of its COX-2 inhibitory effect (Iñiguez et al., 2010). Since the nuclear factor of activated T cells is believed to enhance the COX-2 gene expression via binding to specific functional sites in the COX-2 gene promoter (Duque et al., 2005), its inhibition by celecoxib would plausibly diminish COX-2 expression.