Experiments on the rat model
Experiments on the rat model of E. coli pyelonephritis, performed to investigate the relationship between the acute inflammatory processes and the permanent kidney damage (Brooks et al., 1974), have demonstrated a positive correlation between the degree of free radical-mediated renal injury and the extent of chronic renal scarring (Meylan et al., 1989). Pyelonephritis induces an oxidative inflammatory process in the renal parenchyma, which may occur as a result of excessive reactive oxygen radical generation and/or impaired antioxidant capacity (Andreoli, 1991, Gupta et al., 1996). Experimental data confirm that intensity of chemiluminescence was increased, whereas the antioxidant capacity was decreased in patients with active parenchymal kidney disorders (Andreoli, 1991, Siems et al., 2002, Mircescu et al., 2005). Given that free radicals have very short half-lives, it is difficult to detect reactive oxygen species directly. However, luminol- and lucigenin-enhanced chemiluminescence is a simple and reproducible tool used to elucidate the involvement of free radicals. In the present study, it is apparent that luminol and lucigenin chemiluminescence levels were significantly increased at both early and late phases of pyelonephritis, demonstrating increased generation of oxidant metabolites. Since luminol detects H2O2, OH−, hypochlorite, peroxynitrite, and lipid peroxyl radicals, whereas lucigenin is particularly sensitive to superoxide radical (Davies et al., 1994, Ohara et al., 1993), it may be concluded that tissue injury induced by E. coli infection involves toxic oxygen metabolites without any selectivity. On the other hand, it was previously reported that if the early phase of E. coli-induced exudative pyelonephritis involving reactive oxygen species generation, is not interrupted, progressive injury leads to cellular proliferation and sclerosis, where pyelonephritic scarring is inevitable (Glauser et al., 1978, Glauser et al., 1986). In the present study, montelukast reduced the pyelonephritis-induced elevations in chemiluminescence levels detected by both probes. However, the oxygen-derived radicals detected by lucigenin were still higher with respect to those of the control group, implicating that montelukast has a more 17 aag effect on the generation of H2O2, OH−, hypochlorite, peroxynitrite, and lipid peroxyl radicals. In the present study, collagen contents of the kidney tissues, indicating the presence of tissue fibrotic activity, were significantly increased at the first week of E. coli infection, while montelukast treatment attenuated the fibrotic activity that would proceed with renal scarring. Free radicals and reactive oxygen molecules are generated by activated neutrophils, monocytes and mesangial cells during inflammatory processes. By the induction of pyelonephritis, the rapid bacterial multiplication in the kidney parenchyma leads to migration of polymorphonuclear leukocytes and increases the MPO content to several hundred times of the baseline level (Meylan and Glauser, 1988). Moreover, studies have demonstrated that both the acute phase of pyelonephritis and the subsequent chronic scarring are dependent on the activation of the MPO enzyme (Bille and Glauser, 1982, Meylan et al., 1989). During acute inflammation, activated polymorphonuclear leukocytes release lysosomal hydrolytic enzymes, lipid mediators, and reactive oxygen species that protect against invading microorganisms and help to remove the dead cells but also damage the surrounding viable tissues (Noiri et al., 2000). In the present study, MPO levels were significantly increased following E. coli injection, suggesting that oxidant-generated tissue injury involves the extracellular release of the MPO by activated polymorphonuclear leukocytes during the acute suppurative pyelonephritis. On the other hand, montelukast attenuated neutrophil recruitment and promoted the resolution of inflammation by antagonizing the effects of leukotrienes, which are potent stimuli for leukocyte infiltration, intrarenal vasoconstriction and mesangial cell contraction (Clarkson et al., 1998). In accordance with our present results, we have previously shown that montelukast acts through the inhibition of neutrophils in several organs targeted by various inflammatory challenges, including the kidneys (Sener et al., 2005a, Sener et al., 2005b, Sener et al., 2006). There is also evidence that a potential counterregulatory interaction between leukotrienes and lipoxins may be relevant during glomerular inflammation (Badr et al., 1989), where the lipoxins could play a role in the attenuation of injury. Although our results clearly verify the impact of cysteinyl leukotrienes in the pathogenesis of renal inflammation via the inhibition of neutrophil recruitment, they do not exclude the role of non-cysteinyl leukotrienes and lipoxins.