Pore-forming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells.

Clostridium perfringens epsilon toxin (ET) is a potent pore-forming cytotoxin causing fatal enterotoxemia in livestock. ET accumulates in brain and kidney, particularly in the renal distal-collecting ducts. ET binds and oligomerizes in detergent-resistant membranes (DRMs) microdomains and causes cell death. However, the causal linkage between membrane permeabilization and cell death is not clear. Here, we show that ET binds and forms 220-kDa insoluble complexes in plasma membrane DRMs of renal mpkCCD(cl4) collecting duct cells. Phosphatidylinositol-specific phospholipase C did not impair binding or the formation of ET complexes, suggesting that the receptor for ET is not GPI anchored. ET induced a dose-dependent fall in the transepithelial resistance and potential in confluent cells grown on filters, transiently stimulated Na+ absorption, and induced an inward ionic current and a sustained rise in [Ca2+]i. ET also induced rapid depletion of cellular ATP, and stimulated the AMP-activated protein kinase, a metabolic-sensing Ser/Thr kinase. ET also induced mitochondrial membrane permeabilization and mitochondrial-nuclear translocation of apoptosis-inducing factor, a potent caspase-independent cell death effector. Finally, ET induced cell necrosis characterized by a marked reduction in nucleus size without DNA fragmentation. DRM disruption by methyl-beta-cyclodextrin impaired ET oligomerization, and significantly reduced the influx of Na+ and [Ca2+]i, but did not impair ATP depletion and cell death caused by the toxin. These findings indicate that ET causes rapid necrosis of renal collecting duct cells and establish that ATP depletion-mediated cell death is not strictly correlated with the plasma membrane permeabilization and ion diffusion caused by the toxin.

Acute pyelonephritis represents a risk factor impairing long-term kidney graft function.

Urinary tract infections (UTIs) and acute pyelonephritis (APN) often occur after renal transplantation, but their impact on graft outcome is unclear. One hundred and seventy-seven consecutive renal transplantations were investigated to evaluate the impact of UTIs and APN on graft function. The cumulative incidence of UTIs was 75.1% and that of APN was 18.7%. UTIs occurred mainly during the first year after transplantation and Escherichia coli, Pseudomonas aeruginosa and Enteroccocus sp. were the most frequent pathogens identified. The risk of developing APN was higher in female (64%) than in male recipients, and was correlated with the frequency of recurrent UTIs (p < 0.0001) and rejection episodes (p = 0.0003). APN did not alter graft or recipient survival, however, compared to patients with uncomplicated UTIs, patients with APN exhibited both a significant increase in serum creatinine and a decrease in creatinine clearance, already detected after 1 year (aMDRD-GFR: APN: 39.5 +/- 12.5; uncomplicated UTI: 54.6 +/- 21.7 mL/min/1.73 m(2), p < 0.01) and still persistent ( approximately - 50%) 4 years after transplantation. Multivariate analysis revealed that APN represents an independent risk factor associated with the decline of renal function (p = 0.034). Therefore, APN may be associated with an enduring decrease in renal graft function.

Transimmortalized proximal tubule and collecting duct cell lines derived from the kidneys of transgenic mice.

This review summarizes the strategy of cellular immortalization based on the principle of targeted oncogenesis in transgenic mice, used to establish models of transimmortalized renal proximal tubule cells, referred to as PKSV-PCT and PKSV-PR-cells, and collecting duct principal cells, referred to as mpkCCD(cl4) cells. These cell lines have maintained for long-term passages the main biochemical and functional properties of the parental cells from which they were derived. Proximal tubule PKSV-PCT and PKSV-PR cells have been proved to be suitable cell systems for toxicological and pharmacological studies. They also permitted the establishment of a model of multidrug-resistant (MDR) renal epithelial tubule cells, PKSV-PR(col50), which have served for the study of both MDR-dependent extrusion of chemotherapeutic drugs and inappropriate accumulation of weak base anthracyclines in intracellular acidic organelles. The novel collecting duct cell line mpkCCD(cl4), which has maintained the characteristics of tight epithelial cells, in particular Na(+) absorption stimulated by aldosterone, has been extensively used for pharmacological studies related to the regulation of ion transport. These cells have permitted the identification of several aldosterone-induced proteins playing a key role in the regulation of Na(+) absorption mediated by the epithelial Na(+) channel ENaC. Recent studies have also provided evidence that these cell lines represent valuable cell systems for the study of host-pathogen interactions and the analysis of the role of renal tubule epithelial cells in the induction of inflammatory response caused by uropathogens that may lead to severe renal damage.

Regulation of NaCl transport in the renal collecting duct: lessons from cultured cells.

The fine control of NaCl absorption regulated by hormones takes place in the distal nephron of the kidney. In collecting duct principal cells, the epithelial sodium channel (ENaC) mediates the apical entry of Na(+), which is extruded by the basolateral Na(+),K(+)-ATPase. Simian virus 40-transformed and "transimmortalized" collecting duct cell lines, derived from transgenic mice carrying a constitutive, conditionally, or tissue-specific promoter-regulated large T antigen, have been proven to be valuable tools for studying the mechanisms controlling the cell surface expression and trafficking of ENaC and Na(+),K(+)-ATPase. These cell lines have made it possible to identify sets of aldosterone- and vasopressin-stimulated proteins, and have provided new insights into the concerted mechanism of action of serum- and glucocorticoid-inducible kinase 1 (Sgk1), ubiquitin ligase Nedd4-2 (neural precursor cell-expressed, developmentally down-regulated protein 4-2), and 14-3-3 regulatory proteins in modulating ENaC-mediated Na(+) currents. Epidermal growth factor and induced leucine zipper protein have also been shown to repress and stimulate ENaC-dependent Na(+) absorption, respectively, by activating or repressing the mitogen-activated protein kinase externally regulated kinase(1/2). Overall, these findings have provided evidence suggesting that multiple pathways are involved in regulating NaCl absorption in the distal nephron.