br Materials and methods br Results As
Materials and methods
Results As shown in Table 1, blood urea nitrogen and serum creatinine levels were significantly elevated at both 1st and 7th days of pyelonephritis, when compared with those of the control group (P<0.001). When montelukast was administered following the intrarenal injection of E. coli, elevations in blood urea nitrogen and serum creatinine levels were significantly depressed (P<0.05 and 0.001). Serum TNF-α levels, measured at both the earlier and later periods following intrarenal E. coli injection, were significantly higher than those of the control group (P<0.001; Fig. 1). However, serum LDH level was not altered in the early phase of pyelonephritis, but it was increased on the 7th day of E. coli injection (P<0.001, Fig. 1 inset), verifying generalized tissue damage. However, pyelonephritis-induced rises in serum TNF-α and LDH levels were abolished by montelukast treatment (P<0.001) (Fig. 1). Chemiluminescence levels in the kidney samples of the pyelonephritic rats were significantly increased at both the 1st and 7th days of pyelonephritis, as compared to the renal chemiluminescence levels of the control group (P<0.01–0.001), demonstrating the involvement of reactive oxygen species in the pyelonephritis-induced tissue damage. On the other hand, in the montelukast-treated rats, pyelonephritis-induced elevations in the renal chemiluminescence levels were significantly reduced (P<0.05–0.001, Table 1). The renal malondialdehyde levels, as an index of lipid peroxidation, were increased significantly (P<0.001) in the late phase of pyelonephritis, as compared to the control group. However, when montelukast treatment was applied to the rats that were injected intrarenally with E. coli, malondialdehyde levels were reversed back to the control levels (P<0.01–0.001, Table 1). Furthermore, levels of GSH, a key antioxidant, were depressed significantly (P<0.01–0.001) at both 1st and 7th days of pyelonephritis, while renal GSH content was restored by montelukast treatment (P<0.05, Table 1). Renal MPO activity, an indirect evidence of neutrophil infiltration into the tissue, was increased both in the earlier and later phases of the experimental pyelonephritis (P<0.05–0.001, Fig. 2A). However, administration of montelukast prevented neutrophil recruitment in both periods of renal infection (P<0.05). In the kidney samples of pyelonephritic rats, collagen content was markedly increased (P<0.001), demonstrating E. coli-induced tissue fibrosis, and this increase was attenuated by montelukast treatment (P<0.01, Fig. 2B). Control group showed minor changes, without atomoxetine hcl formation or intratubular neutrophils, while tubules showed mild dilation (Fig. 3A, Table 1). One-day pyelonephritis group demonstrated severe interstitial invasion of inflammatory cells at and near the injection site, with severe dilation of the tubuli (Fig. 3B, Table 1). In the 7-day pyelonephritis group, there was diffuse accumulation of inflammatory cells, mild to moderate fibrosis and tubular dilation (Fig. 3D, Table 1). The glomeruli were not affected, but the tubuli and the interstistium showed significant degenerations. In the montelukast-treated group, similar findings to those of the 1-day pyelonephritis group were observed, but the tubular dilation was significantly reduced (Fig. 3C, Table 1). Some aggregates of lymphocytes were found between the tubular structures, at the regions other than the scarred tissue. Inflammatory cell invasion, mostly composed of lymphocytes, accompanied the fibrosis. In the tubular lumens, groups of neutrophils, indicating active infection, were observed. Some tubules contained desquamated epithelium and granular casts, which pointed the presence of ongoing tubular cell damage. On the other hand, montelukast treatment demonstrated a reduction in inflammation, with tubules having no casts, neutrophils or dilation (Fig. 3E, Table 1).
Discussion The current data demonstrate that intrarenal E. coli injection causes oxidative inflammatory response as evidenced by alterations in serum TNF-α, LDH, urea and creatinine and in renal MPO, malondialdehyde and GSH levels. On the other hand, the results also demonstrate that montelukast, the leukotriene CysLT1 receptor antagonist, prevented the pyelonephritis-induced local and systemic inflammatory responses, as well as histopathological findings. In a study by Patel et al. (2004), the degree of renal dysfunction and inflammation caused by ischemia–reperfusion was significantly reduced in 5-lipoxygenase knockout mice as compared to wild type mice. Moreover, administration of 5-lipoxygenase inhibitor before ischemia–reperfusion, significantly reduced the degree of renal dysfunction and injury. On the other hand, several studies have demonstrated that leukotriene CysLTs increase in inflammatory conditions, such as ischemia/reperfusion or pyelonephritis (Tardif et al., 1994, Lianos and Bresnahan, 1999, Sener et al., 2006). Moreover, in an animal model of human membranous nephropathy, the synthesis of leukotriene CysLTs from macrophages was increased by an ischemic challenge in the renal tissue (Badr, 1992). Similarly, upregulation of the 5-lipoxygenase pathway of the arachidonate cascade in hemodialysis patients have caused oxidative stress, lipid peroxidation and apoptosis in peripheral blood mononuclear cells (Maccarrone et al., 2002). In accordance with the aforementioned studies, our results show that antagonizing the leukotriene CysLT1 receptors with montelukast treatment ameliorates oxidative renal injury and improves renal functions through the mechanisms that involve an inhibitory action on tissue neutrophil infiltration, release of reactive oxygen species and activation of inflammatory cytokines.