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.

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.

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.

Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells.

Cyclic AMP (cAMP) stimulates the transport of Na(+) and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCD(c14) collecting duct cells. db-cAMP (10(-3) M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of (86)Rb(+) uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20 degrees C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

Tissue distribution and subcellular localization of the ClC-5 chloride channel in rat intestinal cells.

ClC-5 is the Cl- channel that is mutated in Dent's disease, an X-chromosome-linked disease characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones. It is predominantly expressed in endocytically active renal proximal cells. We investigated whether this Cl- channel could also be expressed in intestinal tissues that have endocytotic machinery. ClC-5 mRNA was detected in the rat duodenum, jejunum, ileum, and colon. Western blot analyses revealed the presence of the 83-kDa ClC-5 protein in these tissues. Indirect immunofluorescence studies showed that ClC-5 was mainly concentrated in the cytoplasm above the nuclei of enterocytes and colon cells. ClC-5 partially colocalized with the transcytosed polymeric immunoglobulin receptor but was not detectable together with the brush-border-anchored sucrase isomaltase. A subfractionation of vesicles obtained by differential centrifugation showed that ClC-5 is associated with the vacuolar 70-kDa H+-ATPase and the small GTPases rab4 and rab5a, two markers of early endosomes. Thus these results indicate that ClC-5 is present in the small intestine and colon of rats and suggest that it plays a role in the endocytotic pathways of intestinal cells.

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCD(cl4)) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCD(cl4) cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-alpha (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

Vasopressin-stimulated chloride transport in transimmortalized mouse cell lines derived from the distal convoluted tubule and cortical and inner medullary collecting ducts.

BACKGROUND: The fine control of NaCl absorption takes place in the distal parts of the renal tubule, but the regulation of Cl(-) transport in this region has not been fully elucidated. We have analysed the effects of dD-arginine vasopressin (dDAVP) on Cl(-) fluxes in cultured mouse distal convoluted tubule (mpkDCT), cortical collecting duct (mpkCCD) and inner medullary collecting duct (mpkIMCD) cell lines. METHODS: RT-PCR and Western blotting were used to detect the amiloride-sensitive sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNAs and protein in cultured mpkDCT, mpkCCD and mpkIMCD cells. Cl(-) fluxes were analysed by measuring the short-circuit current (I(sc)) and bidirectional (36)Cl(-) fluxes on confluent cells grown on filters. RESULTS: All three cell lines expressed ENaC and CFTR and had I(sc) stimulated by dDAVP. The rise in I(sc) caused by dDAVP (10(-8) M) was inhibited by amiloride, and to a lesser extent by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) in all three cell lines. The dDAVP-dependent I(sc) measured under apical Na(+)-free condition was reduced by Cl(-) channel blockers with a profile (NPPB>glibenclamide>DIDS), similar to that for rat CFTR. dDAVP stimulated the apical-to-basal (36)Cl(-) flux and to a lesser extent the basal-to-apical (36)Cl(-) flux under open-circuit condition in all three cultured cell lines. Adding NPPB to the apical side reduced the basal-to-apical (36)Cl(-) flux but not the opposite (36)Cl(-) flux from dDAVP-treated cells. CONCLUSION: These results indicate that dDAVP stimulates the bi-directional flux of Cl(-), resulting in net Cl(-)absorption, in these cultured mouse distal and collecting duct cells. I(sc) experiments also suggest the presence of a minor component of electrogenic Cl(-) secretion, possibly mediated by CFTR.

A novel mouse Nedd4 protein suppresses the activity of the epithelial Na+ channel.

Liddle's syndrome is a form of inherited hypertension linked to mutations in the genes encoding the epithelial Na+ channel (ENaC). These mutations alter or delete PY motifs involved in protein-protein interactions with a ubiquitin-protein ligase, Nedd4. Here we show that Na+ transporting cells, derived from mouse cortical collecting duct, express two Nedd4 proteins with different structural organization and characteristics of ENaC regulation: 1) the classical Nedd4 (herein referred to as Nedd4-1) containing one amino-terminal C2, three WW, and one HECT-ubiquitin protein ligase domain and 2) a novel Nedd4 protein (Nedd4-2), homologous to Xenopus Nedd4 and comprising four WW, one HECT, yet lacking a C2 domain. Nedd4-2, but not Nedd4-1, inhibits ENaC activity when coexpressed in Xenopus oocytes and this property correlates with the ability to bind to ENaC, as only Nedd4-2 coimmunoprecipitates with ENaC. Furthermore, this interaction depends on the presence of at least one PY motif in the ENaC complex and on WW domains 3 and 4 in Nedd4-2. Thus, these results suggest that the novel suppressor protein Nedd4-2 is the regulator of ENaC and hence a potential susceptibility gene for arterial hypertension.

Cyclosporine reduces basolateral, but not apical, nitric oxide secretion in medullary thick ascending limb cells.

Cyclosporine (CsA) reduces nitric oxide (NO) production in medullary thick ascending limb (mTAL) cells. We postulated that CsA affected NO secretion in a vectorial manner in polarized renal epithelial cells. The experiments were performed in a model of mTAL subcultured cells. The expression of iNOS in mTAL cells was confirmed by RT-PCR. The cells were grown on a non-permeable filter. Nitrite was measured by the modified Griess method. Transepithelial resistance was measured to ensure the integrity of the tight junction. CsA (100 ng/ml) reduced NO production by 22% in mTAL cells. The inhibitory effect was limited to the basolateral side (control: 165 +/- 11; plus CsA: 93 +/- 17 nM/10(6) cells, P < 0.001) without affecting apical NO secretion. The transepithelial resistance through the epithelial monolayer remained unchanged in CsA-treated cells. CsA reduced basolateral NO secretion without affecting apical secretion. The results suggest that CsA might affect intrarenal hemodynamics at the peritubular level.

Cyclosporine stimulates Na+-K+-Cl- cotransport activity in cultured mouse medullary thick ascending limb cells.

BACKGROUND: Cyclosporine (CsA) has been shown to alter the activity of plasma membrane transporters in kidney epithelial cells. In this study, we have investigated the effects of CsA on Na+,K+-ATPase and Na+-K+-Cl- cotransport activities in cultured cells derived from microdissected mouse medullary thick ascending limb (mTAL) cells. METHODS: Experiments were carried out on subcultured confluent mouse TAL cells. Reverse transcription-polymerase chain reaction experiments showed that they expressed the mNKCC2 electroneutral Na+-K+-Cl- cotransporter and ROM-K1 and ROMK2 potassium channel mRNA. Western blotting also revealed the presence of the 40 kD ROMK protein using an anti-ROMK antibody. The effect of CsA (100 ng/mL) on ion transport was assessed by measuring the influx and efflux of rubidium (86Rb+) and 36Cl-, used as tracers of K+ and Cl- movements, on cells grown on Petri dishes or permeable filters. RESULTS: CsA inhibited by 38% the ouabain-sensitive component of 86Rb+ influx mediated by the Na+,K+-ATPase pumps. CsA also increased by 38% the ouabain-resistant furosemide-sensitive component (Or-Fs) of 86Rb+ influx, reflecting the Na+-K+-Cl- cotransport activity and stimulated the basolateral efflux of 36Cl- from mTAL cells grown on filters. The CsA-stimulated basal efflux of Cl- was prevented by the basal addition of the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB, 10-4 mol/L). Apical addition of the K+ channel blocking agent Ba2+ (10-4 mol/L) partially prevented the CsA-stimulated basal efflux of Cl-. Adding Ba2+ to the luminal side of cells grown on Petri dishes also prevented the rise in apical 86Rb+ efflux and the increased Or-Fs component of 86Rb+ influx caused by CsA. CONCLUSION: These results indicated that CsA may stimulate the Na+-K+-Cl- cotransport activity and also suggested that this immunosuppressive agent may interfere in the recycling of apical K+ in this model of cultured mouse TAL cells.

Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission.

To study transmission patterns of Pneumocystis carinii pneumonia (PCP) in persons with AIDS, we evaluated P. carinii isolates from patients in five U.S. cities for variation at two independent genetic loci, the mitochondrial large subunit rRNA and dihydropteroate synthase. Fourteen unique multilocus genotypes were observed in 191 isolates that were examined at both loci. Mixed infections, accounting for 17.8% of cases, were associated with primary PCP. Genotype frequency distribution patterns varied by patients' place of diagnosis but not by place of birth. Genetic variation at the two loci suggests three probable characteristics of transmission: that most cases of PCP do not result from infections acquired early in life, that infections are actively acquired from a relatively common source (humans or the environment), and that humans, while not necessarily involved in direct infection of other humans, are nevertheless important in the transmission cycle of P. carinii f. sp. hominis.

Immortalized kidney epithelial cells as tools for hormonally regulated ion transport studies.

The development of transgenic mice carrying the simian virus-40 large T antigen gene or the temperature-sensitive simian virus-40 large T antigen gene, either alone or placed under the control of the 5'-regulatory regions of tissue-specific or ubiquitous genes, has permitted the production of differentiated, polarized kidney epithelial cells. This review covers the immortalized cell lines issued from the various parts of the renal tubule and, in particular, the recently established collecting duct cell lines that have been used as ex-vivo cell models to analyze the regulation of ion transport processes by hormones.

Mechanisms of altered sequestration and efflux of chemotherapeutic drugs by multidrug-resistant cells.

This review considers the mechanisms associated with the pleiotropic resistance of cancer cells to chemotherapeutic drugs, and more particularly those related to intracellular pH (pHi). The multidrug resistance (MDR) phenomenon responsible for the decreased accumulation and increased efflux of cytotoxic drugs is generally associated with excess levels of P-glycoproteins (Pgps) encoded by MDR genes and/or the multidrug resistance-associated protein (MRP). MDR cell lines, derived from normal or tumor cells, frequently exhibit abnormally elevated pHi and changes in the production of various proteins. Recent studies have suggested that, in addition to the impact of the ATP-dependent membrane transporters Pgp and MRP on drug transport, other mechanisms linked to pHi changes in MDR cells may play an important role in drug resistance. We have shown that alkalinization of the acidic compartments (endosomes and lysosomes) by lysosomotropic agents could stimulate the efflux of vinblastine from drug-resistant mouse renal proximal tubule cells. The fact that weak base chemotherapeutic drugs can be sequestered within the acidic organelles of MDR cells sheds new light on the cellular mechanisms of drug resistance.