Renal cell death in urinary tract infections : role of E. coli toxins

Febrile urinary tract infections (UTI) are common bacterial illnesses in children. Several follow up studies have shown that 10-15 % of infants with pyelonephritis will develop permanent renal damage, i.e., renal scars, which in turn may lead to chronic diseases such as hypertension and renal insufficiency. During the last decades, as a result of the tremendous improvement of diagnostic and therapeutic approaches, the incidence of severe complications after UTI has dramatically decreased. However, young age seems to be a risk factor for the development of renal scars. Thus, all children with UTI are followed-up for several years, resulting in high costs for the health care system. In a long run, it might be therefore a benefit to identify early those children with high risk to develop renal scars, a pathological process associated with cell death. This study, mainly by studying the interaction between the toxins from uropathogenic E.coli strains and renal cells, was aimed to provide information about the mechanisms that regulated cell survival or death in renal proximal tubular cells. By using a renal proximal tubular cell line (LLC-PK1 cells), we found that soluble toxins from the uropathogenic E.coli ARD6 strain (O6) induce apoptotic cell death in a dose- and time-dependent manner. The expression of Fas receptors and the phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) were significantly upregulated by E.coli soluble toxins. Cell death could be completely inhibited by two specific ERK1/2 inhibitors, but not by a broad caspase inhibitor, zVAD-fmk, implicating a caspase-independent pathway via ERK. We also found that lysophosphatidic acid (LPA) could trigger a survival signal through G-proteins and phosphatidylinositol 3-kinase (PI3K). Moreover, this study demonstrated that inducible nitric oxide synthase (iNOS) protein expression was up regulated in renal tubular cells undergoing E. coli toxins-mediated death, whereas endothelial nitric oxide synthase (eNOS) was down regulated. When NOS activity is inhibited by the specific inhibitor of NOS or MEK1, cells were rescued from death. Up-regulation of heme oxygenase-1 (HO-1) by nitric oxide (NO) donor, sodium nitroprusside (SNP) or by the specific activator (hemin) inhibited O6-toxin(s)-induced cell death. Furthermore, incubation with a metabolite of HO-1, carbon monoxide, could counteract the effects of E.coli-toxins. The protection by carbon monoxide was associated with up-regulation of p21 protein expression. We also found that 21% of E.coli strains isolated from children admitted to our emergency unit with acute UTI show hemolytic activity. All of these strains induced apoptosis in renal tubular cells. In the non-hemolytic strains, only 45% induced apoptosis in cultured cells. It is therefore likely that hemolysin is one of the main toxins being responsible for the pro-apoptotic activity. The pro-apoptotic cascade activated by hemolysin can be inhibited by HO-1 activation. We also used an animal model of fetal growth retardation induced by antenatal exposure to excess dexamethasone (DEX). We isolated renal proximal tubular cells (PTCs) from 20-day-old offspring in DEX-exposed-group (DEX-cells) and control-group (CON-cells). After 4 days in culture, cells were exposed to uropathogenic E.coli toxins. We found that cell death rate was higher in DEX-cells than that in CON-cells. Cell death exhibited morphological and biochemical features of apoptosis, such as shrinkage, membrane blebbing, nucleus fragmentation and caspase activation. Conversely, the activity of the antioxidant enzyme catalase was significantly increased in renal cortex homogenate from 20-day-old DEX-rats. Furthermore, we observed that cell death rate induced by hydrogen peroxide was not significantly different between DEX- and CON-cells and that the antioxidant vitamin E could not prevent E.coli-induced death. Therefore, oxidative stress does not seem to be the crucial factor for E.coli toxins-induced cell death. In conclusion, activation of ERK mediates E.coli toxins-induced renal cell death via the iNOS activation. Caspases, although being activated, are not necessary for cell death, and they act after the ERK signaling at which point cells become committed to cell death or can be rescued. Hemolysin is one of secreted toxins that are involved in apoptosis during UTI. Intrinsic and extrinsic factors such as HO-1 and LPA may protect cells against death. There are long-lasting effects of prenatal glucocorticoids that may predispose renal cells to apoptosis after urinary tract infection