Ofloxacin: new applications for the prevention of urinary tract infections in renal graft recipients.

BACKGROUND: Urinary tract infections (UTIs), the most common form of bacterial infection in kidney transplant recipients, recently have been demonstrated to be detrimental for long-term graft outcome. Therefore, reinforcing antibiotic prophylaxis might be vital, in addition to basic hygiene recommendations, surgical care, and prophylaxis by trimethoprim-sulfamethoxazole. METHODS: In 2006, a Legionella pneumophila contamination of our department's water pipes meant that all the patients undergoing renal transplantation underwent a 1-month regimen of ofloxacin (OFLO) (200 mg every other day). We took this opportunity to measure the incidence of UTI, including acute pyelonephritis (APN), in 100 consecutive patients transplanted before (n = 50) and after (n = 50) this treatment decision was reached. We also studied the antimicrobial resistance profiles in our department and in the rest of the hospital. RESULTS: No patient developed Legionnaire's disease. A dramatic decrease in the incidence of UTI (-63%) was also seen in patients undergoing OFLO treatment. Logistic regression analysis demonstrated that the use of OFLO was independently associated with a reduction in UTI (odd ratio [OR] = 0.31%, 95% confidence interval [CI] 0.11-0.84, P = 0.02) and APN (OR = 0.21%, 95% CI 0.07-0.98, P = 0.045). This protection was sustained during the whole first year post transplantation. As for resistance rates, we observed a decrease in the susceptibility of Pseudomonas aeruginosa to ciprofloxacin in our nephrology department, compared with that observed in the rest of the hospital. The incidence of multi-resistant bacteria was stable. DISCUSSION: Our unintentional extension of prophylactic antibiotherapy with OFLO gave rise to a dramatic decrease in the 1-year incidence of UTI and APN in kidney recipients. Emergence of resistant strains is, however, a major concern.

Heat shock protein gp96 and NAD(P)H oxidase 4 play key roles in Toll-like receptor 4-activated apoptosis during renal ischemia/reperfusion injury.

Ischemia/reperfusion injury (IRI) causes inflammation and cell injury as a result of activating innate immune signaling. Toll-like receptor 4 (TLR4) has a key role in mediating kidney damages during IRI, but the downstream signaling pathway(s) stimulating apoptosis remains debated. In this study we show that TLR4 mediates MyD88-dependent activation of TNF receptor-associated factor 2, apoptosis signal-regulating kinase 1 (ASK1), and Jun N-terminal kinase (JNK) and p38 MAP kinases in ischemic-reperfused kidneys and posthypoxic renal tubule epithelial cells (RTECs). Hypoxia stimulated the expression of the endoplasmic-resident gp96, which co-immunoprecipitated TLR4, whereas silencing gp96 mRNA expression impaired hypoxia-induced apoptosis in TLR4-expressing RTECs. NAD(P)H oxidase 4 (NOX4) was shown to interact with TLR4 and to be required in lipopolysaccharide-induced production of reactive oxygen species (ROS). IRI stimulated the expression of a 28-kDa NOX4 spliced isoform abundantly expressed in wild-type RTECs, which co-immunoprecipitated with TLR4, but not with gp96 in TLR4-deficient RTECs. Silencing NOX4 mRNA expression impaired hypoxia-induced activation of ASK1 and both JNK and p38, leading to the inhibition of ROS production and apoptosis in posthypoxic TLR4-expressing RTECs. These findings show that, concomitantly to the activation of p38, the gp96/TLR4 interaction is required for activation of ASK1/JNK signaling in posthypoxic mouse RTECs, and that the 28-kDa NOX4 has a key role in TLR4-mediated apoptosis during renal IRI.

Cell models for studying renal physiology.

The development over the past 20 years of a variety of cultured renal tubule cell lines derived from different parts of the renal tubule has provided invaluable powerful cell systems for in vitro analyses of the various tubule segment-specific biochemical functions and ion transport processes. Immortalized cell lines have been established using different hybrid gene constructs, most of them carrying the immortalizing simian virus 40 large T antigen (Tag) gene. The development of transgenic mice carrying unregulated Tag, and of others in which the expression of Tag remains controlled, has made it possible to establish permanent cell lines derived from microdissected or immunoselected renal proximal, distal, and collecting duct tubules. This review summarizes the different strategies of cellular immortalization used and the most frequently used human, rabbit, rat, and mouse tubule cell lines. This review provides an overview of the use of immortalized mouse tubule cell lines for in vitro analyses of various tubule cell-specific functions and the regulation of ion transporters and membranous channels. The advantages of using primary cultures of isolated tubules dissected from physiopathological models of transgenic mice are also discussed.

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.

Upregulation of chemokine CXCL1/KC by leptospiral membrane lipoprotein preparation in renal tubule epithelial cells.

We have previously shown that leptospiral membrane lipoprotein preparation (LMLP) extracted from pathogenic Leptospira santarosai serovar Shermani stimulates the secretion of pro-inflammatory mediators in renal tubule epithelial cells, and implicated its role in the initiation of tubulointerstitial nephritis. Renal tubulointerstitial injury is characterized by inflammatory cell infiltrate; however, the stimuli for leukocyte recruitment are not fully understood. Initial studies by cytokine protein array analysis revealed significant upregulation of neutrophil-chemoattractant keratinocyte-derived chemokine (CXCL1/KC) at nanogram range of LMLP stimulation in cultured murine proximal tubule cells (PTCs). As PTCs express Toll-like receptors (TLRs), this study investigated the roles of TLR signaling pathways in PTCs stimulated by LMLP and its relation to CXCL1/KC secretion. The LMLP stimulated the early secretion of CXCL1/KC and enhanced the level of TLR2 mRNA expression in PTCs through time- and dose-dependent effect. The LMLP-stimulated secretion of human growth-related oncogene alpha, a functional homolog to murine KC, in TLR-defective human embryonic kidney 293 cells transiently transfected with TLR2-expressing plasmids and the response was augmented by coexpression of TLR1 and TLR2. Moreover, silencing of TLR2, myeloid differentiation factor 88, and TNF receptor-associated factor 6 with specific small interfering RNA significantly reduces the response caused by LMLP in PTCs. The LMLP-stimulated CXCL1/KC secretion was also significantly reduced by pre-incubating PTCs with a specific p38 inhibitor. These results indicate that LMLP stimulates the production of CXCL1/KC to recruit polymorphonuclear neutrophils at the site of inflammation through a TLR2-mediated pathway in renal tubule cells.

Toll-like receptor 2 mediates early inflammation by leptospiral outer membrane proteins in proximal tubule cells.

Tubulointerstitial nephritis is a cardinal renal manifestation in leptospirosis and LipL32, the major lipoprotein component of leptospiral outer membrane proteins (OMPs), induces a robust inflammatory response in cultured renal proximal tubule cells through a nuclear factor-kappaB-related pathway. Here, we investigated whether Toll-like receptor (TLR), known to play a pivotal role in innate immunity, could mediate the inflammatory response induced by leptospiral OMPs in renal proximal tubule cells. TLR expression was analyzed by flow cytometry and indirect immunofluorescence in cultured mouse proximal tubule (pyruvate kinase simian virus 40-proximal straight (PKSV-PR)) cells. Reverse transcription-competitive polymerase chain reaction and enzyme-linked immunosorbent assay were undertaken to analyze the inducible effects of inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1 also termed CCL2) by pathogenic and non-pathogenic leptospiral OMPs and recombinant lipoproteins in either PKSV-PR cells or TLR-transfected human embryonic kidney (HEK) 293 cells. Anti-TLR antibodies were used for blocking experiments. Leptospira santarosai serovar Shermani OMPs and LipL32 induced a significant increase in TLR2 but not TLR4 expression in PKSV-PR cells. The increase in iNOS and CCL2/MCP-1 mRNA expressions could be prevented by an anti-TLR2 antibody, but not by an anti-TLR4 antibody. Furthermore, leptospiral OMPs stimulated both CCL2/MCP-1 mRNA and secreted protein in transfected HEK 293 cells with a TLR2-expressing plasmid, but had no effect in cells with a TLR4-expressing plasmid. In conclusion, these findings indicate that the stimulation of iNOS and CCL2/MCP-1 caused by pathogenic leptospiral OMPs, in particular LipL32, in proximal tubule cells requires TLR2 for the early inflammatory response.

Epithelial Na+ channel mutants causing Liddle's syndrome retain ability to respond to aldosterone and vasopressin.

Liddle's syndrome is a monogenic form of hypertension caused by mutations in the PY motif of the COOH terminus of beta- and gamma-epithelial Na+ channel (ENaC) subunits. These mutations lead to retention of active channels at the cell surface. Because of the critical role of this PY motif in the stability of ENaCs at the cell surface, we have investigated its contribution to the ENaC response to aldosterone and vasopressin. Mutants of the PY motif in beta- and gamma-ENaC subunits (beta-Y618A, beta-P616L, beta-R564stop, and gamma-K570stop) were stably expressed by retroviral gene transfer in a renal cortical collecting duct cell line (mpkCCDcl4), and transepithelial Na+ transport was assessed by measurements of the benzamil-sensitive short-circuit current (Isc). Cells that express ENaC mutants of the PY motif showed a five- to sixfold higher basal Isc compared with control cells and responded to stimulation by aldosterone (10(-6) M) or vasopressin (10(-9) M) with a further increase in Isc. The rates of the initial increases in Isc after aldosterone or vasopressin stimulation were comparable in cells transduced with wild-type and mutant ENaCs, but reversal of the effects of aldosterone and vasopressin was slower in cells that expressed the ENaC mutants. The conserved sensitivity of ENaC mutants to stimulation by aldosterone and vasopressin together with the prolonged activity at the cell surface likely contribute to the increased Na+ absorption in the distal nephron of patients with Liddle's syndrome.

The serine/threonine kinases SGK2 and SGK3 are potent stimulators of the epithelial Na+ channel alpha,beta,gamma-ENaC.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) has been identified as a signalling molecule up-regulated by aldosterone, which stimulates the renal epithelial Na(+) channel ENaC. It is therefore thought to participate in the antinatriuretic action of this hormone. More recently, two isoforms, SGK2 and SGK3, have been cloned. The present study was performed to establish whether SGK2 and SGK3 influence ENaC activity similarly to SGK1. Dual-electrode voltage-clamp experiments in Xenopus laevis oocytes expressing alpha,ss,gamma-ENaC with or without SGK1, SGK2 or SGK3 revealed a stimulatory effect of all three kinases on the amiloride-sensitive current (I(Na)). To establish whether the SGK isoforms exert their effects through direct phosphorylation, we replaced the serine at the SGK consensus site of alphaENaC (alpha(S622A)ENaC) by site-directed mutagenesis. alpha(S622A),beta,gamma-ENaC was up-regulated similar to wild-type ENaC, suggesting that SGK isoforms do not act via direct phosphorylation of the transport proteins. In conclusion, SGK2 and SGK3 mimic the function of SGK1 and are likely to participate in the regulation of ENaC activity.

Immortalized renal proximal and collecting duct cell lines derived from transgenic mice harboring L-type pyruvate kinase promoters as tools for pharmacological and toxicological studies.

Targeted oncogenesis in transgenic mice, where an oncogene is placed under the control of the regulatory sequences of a cell-specific gene, has been used to derive lines of differentiated kidney epithelial cells derived from proximal or distal tubules or from the collecting duct. These renal cell lines were obtained from kidneys of transgenic mice harboring the large-T and little-t antigens placed under the control of regulatory sequences of the L-type pyruvate kinase gene. This review summarizes the main properties of these differentiated cell lines, which are useful ex vivo cell systems for pharmacological and toxicological studies.

[Function of the CLC chloride channels and their implication in human pathology]. / Fonction des canaux chlorures de la famille CLC et leur implication en pathologie humaine.

To date, nine chloride channels belonging to the family of CLC chloride channels have been identified. They are localized either in plasma membranes or in intracellular vesicles (endosomes or lysosomes) and can have an ubiquitus or a more restrained tissue distribution. Recent studies on ClC-K1, ClC-2, ClC-3, ClC-5 and ClC-7 knockout mice and the identification of human inherited diseases caused by mutations of some of these chloride channels (myotonia congenita for ClC-1, Bartter disease for ClC-Kb, Dent's disease for ClC-5 and osteopetrose for ClC-7) have provided lines of direct evidence of the physiological relevance and importance of these chloride channels in the transport of chloride and in the endocytosis and transcytosis of proteins in specialized cells from the kidney and other tissues.

Early development of polycystic kidney disease in transgenic mice expressing an activated mutant of the beta-catenin gene.

Autosomal dominant polycystic kidney disease (ADPKD) is common and is a major cause of renal failure. Although the genetics of ADPKD are well known and have led to the discovery of polycystins, a new protein family, the pathogenesis of the disease remains largely unknown. Recent studies have indicated that the beta-catenin signaling pathway is one of the targets of the transduction pathway controlled by the polycystins. We have generated transgenic mice that overproduce an oncogenic form of beta-catenin in the epithelial cells of the kidney. These mice developed severe polycystic lesions soon after birth that affected the glomeruli, proximal, distal tubules and collecting ducts. The phenotype of these mice mimicked the human ADPKD phenotype. Cyst formation was associated with an increase in cell proliferation and apoptosis. The cell proliferation and apoptotic indexes was increased 4-5-fold and 3-4-fold, respectively, in cystic tubules of the transgenic mice compared to that of littermate controls. Our findings provide experimental genetic evidence that activation of the Wnt/beta-catenin signaling pathway causes polycystic kidney disease and support the view that dysregulation of the Wnt/beta-catenin signaling is involved in its pathogenesis.

Short term effect of aldosterone on Na,K-ATPase cell surface expression in kidney collecting duct cells.

Aldosterone controls extracellular volume and blood pressure by regulating Na+ reabsorption, in particular by epithelia of the distal nephron. A main regulatory site of this transcellular transport is the epithelial sodium channel (ENaC) that mediates luminal Na+ influx. The Na,K-ATPase (Na+ pump) that coordinately extrudes Na+ across the basolateral membrane is known to be regulated by short term aldosterone as well. We now show that in the cortical collecting duct (CCD) from adrenalectomized rats, the increase in Na,K-ATPase activity (approximately 3-fold in 3 h), induced by a single aldosterone injection, can be fully accounted by the increase in Na,K-ATPase cell surface expression (+ 497 +/- 35%). The short term aldosterone action was further investigated in cultured mouse collecting duct principal cells mpkCCD(cl4). Within 2 h, maximal Na,K-ATPase function assessed by Na+ pump current (I(p)) measurements and Na,K-ATPase cell surface expression were increased by 20-50%. Aldosterone did not modify the Na+ dependence of the Na+ pumps and induced transcription- and translation-dependent actions on pump surface expression and current independently of ENaC-mediated Na+ influx. In summary, short term aldosterone directly increases the cell surface expression of pre-existing Na+ pumps in kidney CCD target cells. Thus, aldosterone controls Na+ reabsorption in the short term not only by regulating the apical cell surface expression of ENaC (Loffing, J., Zecevic, M., Feraille, E., Kaissling, B., Asher, C., Rossier, B. C., Firestone, G. L., Pearce, D., and Verrey, F. (2001) Am. J. Physiol. 280, F675-F682) but also by coordinately acting on the basolateral cell surface expression of the Na,K-ATPase.

CFTR disruption impairs cAMP-dependent Cl(-) secretion in primary cultures of mouse cortical collecting ducts.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the renal cortical collecting duct (CCD) has not yet been fully elucidated. Here, we investigated the effects of deamino-8-D-arginine vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in primary cultured CCDs microdissected from normal [CFTR(+/+)] and CFTR-knockout [CFTR(-/-)] mice. dDAVP stimulated the benzamyl amiloride (BAm)-sensitive transport of Na(+) assessed by the short-circuit current (I(sc)) method in both CFTR(+/+) and CFTR(-/-) CCDs to a very similar degree. Apical addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or glibenclamide partially inhibited the rise in I(sc) induced by dDAVP and ISO in BAm-treated CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter I(sc) in BAm-treated CFTR(-/-) CCDs. dDAVP stimulated the apical-to-basal flux and, to a lesser extent, the basal-to-apical flux of (36)Cl(-) in CFTR(+/+) CCDs. dDAVP also increased the apical-to-basal (36)Cl(-) flux in CFTR(-/-) CCDs but not the basal-to-apical (36)Cl(-) flux. These results demonstrate that CFTR mediates the cAMP-stimulated component of secreted Cl(-) in mouse CCD.

Analysis of the mouse Scnn1a promoter in cortical collecting duct cells and in transgenic mice.

We have isolated and characterised the promoter of the mouse Scnn1a (alpha ENaC) gene. Using transient transfections of serial deletion mutants into Scnn1a-expressing cells, we demonstrate that 1.56 kb of 5' upstream sequence is required for cell-specific expression and corticosteroid-mediated regulation. These 5' sequences are not sufficient to drive expression of a lacZ reporter gene or a rat Scnn1a cDNA in transgenic mice, where they failed to rescue Scnn1a deficiency.