LPAR2 receptor activation attenuates radiation-induced disruption of apical junctional complexes and mucosal barrier dysfunction in mouse colon.

The tight junction (TJ) and barrier function of colonic epithelium is highly sensitive to ionizing radiation. We evaluated the effect of lysophosphatidic acid (LPA) and its analog, Radioprotein-1, on γ-radiation-induced colonic epithelial barrier dysfunction using Caco-2 and m-ICC12 cell monolayers in vitro and mice in vivo. Mice were subjected to either total body irradiation (TBI) or partial body irradiation (PBI-BM5). Intestinal barrier function was assessed by analyzing immunofluorescence localization of TJ proteins, mucosal inulin permeability, and plasma lipopolysaccharide (LPS) levels. Oxidative stress was analyzed by measuring protein thiol oxidation and antioxidant mRNA. In Caco-2 and m-ICC12 cell monolayers, LPA attenuated radiation-induced redistribution of TJ proteins, which was blocked by a Rho-kinase inhibitor. In mice, TBI and PBI-BM5 disrupted colonic epithelial tight junction and adherens junction, increased mucosal permeability, and elevated plasma LPS; TJ disruption by TBI was more severe in Lpar2-/- mice compared to wild-type mice. RP1, administered before or after irradiation, alleviated TBI and PBI-BM5-induced TJ disruption, barrier dysfunction, and endotoxemia accompanied by protein thiol oxidation and downregulation of antioxidant gene expression, cofilin activation, and remodeling of the actin cytoskeleton. These data demonstrate that LPAR2 receptor activation prevents and mitigates γ-irradiation-induced colonic mucosal barrier dysfunction and endotoxemia.

Botulinum neurotoxin type B uses a distinct entry pathway mediated by CDC42 into intestinal cells versus neuronal cells.

Botulinum neurotoxins (BoNTs) are responsible for severe flaccid paralysis by inhibiting the release of acetylcholine at the neuromuscular junctions. BoNT type B (BoNT/B) most often induces mild forms of botulism with predominant dysautonomic symptoms. In food borne botulism and botulism by intestinal colonisation such as infant botulism, which are the most frequent naturally acquired forms of botulism, the digestive tract is the main entry route of BoNTs into the organism. We previously showed that BoNT/B translocates through mouse intestinal barrier by an endocytosis-dependent mechanism and subsequently targets neuronal cells, mainly cholinergic neurons, in the intestinal mucosa and musculosa. Here, we investigated the entry pathway of BoNT/B using fluorescent C-terminal domain of the heavy chain (HcB), which is involved in the binding to specific receptor(s) and entry process into target cells. While the combination of gangliosides GD1a /GD1b /GT1b and synaptotagmin I and to a greater extent synaptotagmin II constitutes the functional HcB receptor on NG108-15 neuronal cells, HcB only uses the gangliosides GD1a /GD1b /GT1b to efficiently bind to m-ICcl2 intestinal cells. HcB enters both cell types by a dynamin-dependent endocytosis, which is efficiently prevented by Dynasore, a dynamin inhibitor, and reaches a common early endosomal compartment labeled by early endosome antigen (EEA1). In contrast to neuronal cells, HcB uses a Cdc42-dependent pathway to enter intestinal cells. Then, HcB is transported to late endosomes in neuronal cells, whereas it exploits a nonacidified pathway from apical to basal lateral side of m-ICcl2 cells supporting a transcytotic route in epithelial intestinal cells.

Wnt5a induces renal AQP2 expression by activating calcineurin signalling pathway.

Heritable nephrogenic diabetes insipidus (NDI) is characterized by defective urine concentration mechanisms in the kidney, which are mainly caused by loss-of-function mutations in the vasopressin type 2 receptor. For the treatment of heritable NDI, novel strategies that bypass the defective vasopressin type 2 receptor are required to activate the aquaporin-2 (AQP2) water channel. Here we show that Wnt5a regulates AQP2 protein expression, phosphorylation and trafficking, suggesting that Wnt5a is an endogenous ligand that can regulate AQP2 without the activation of the classic vasopressin/cAMP signalling pathway. Wnt5a successfully increases the apical membrane localization of AQP2 and urine osmolality in an NDI mouse model. We also demonstrate that calcineurin is a key regulator of Wnt5a-induced AQP2 activation without affecting intracellular cAMP level and PKA activity. The importance of calcineurin is further confirmed with its activator, arachidonic acid, which shows vasopressin-like effects underlining that calcineurin activators may be potential therapeutic targets for heritable NDI.

Translocation and dissemination to target neurons of botulinum neurotoxin type B in the mouse intestinal wall.

Botulinum neurotoxins (BoNTs) are responsible for severe flaccid paralysis (botulism), which in most cases enter the organism via the digestive tract and then disseminate into the blood or lymph circulation to target autonomic and motor nerve endings. The passage way of BoNTs alone or in complex forms with associated nontoxic proteins through the epithelial barrier of the digestive tract still remains unclear. Here, we show using an in vivo model of mouse ligated intestinal loop that BoNT/B alone or the BoNT/B C-terminal domain of the heavy chain (HCcB), which interacts with cell surface receptors, translocates across the intestinal barrier. The BoNT/B or HCcB translocation through the intestinal barrier occurred via an endocytosis-dependent mechanism within 10-20 min, because Dynasore, a potent endocytosis inhibitor, significantly prevented BoNT/B as well as HCcB translocation. We also show that HCcB or BoNT/B specifically targets neuronal cells and neuronal extensions in the intestinal submucosa and musculosa expressing synaptotagmin, preferentially cholinergic neurons and to a lower extent other neuronal cell types, notably serotonergic neurons. Interestingly, rare intestinal epithelial cells accumulated HCcB suggesting that distinct cell types of the intestinal epithelium, still undefined, might mediate efficient translocation of BoNT/B.

A case report of reninoma: radiological and pathological features of the tumour and characterization of tumour-derived juxtaglomerular cells in culture.

CASE REPORT: A 20-year-old woman presented with malignant hypertension associated with hypokalemia, metabolic alkalosis and elevated plasma renin and aldosterone levels. Computed tomography angiography (CTA) evidenced a 22 mm tissular mass in the posterior cortex of the left kidney, and 18F-flurodeoxyglucose PET (18-FDG PET) imaging showed no hypermetabolism of the tumour. Following nephron-sparing surgery, blood pressure and potassium levels rapidly normalized, allowing interruption of all treatments within 2 weeks. DISCUSSION: Reninoma is a rare juxtaglomerular cell tumour (JGCT) producing excessive amounts of renin resulting in severe hypertension. Pathological studies revealed that tumoural cells highly expressed renin and contained electron-dense structures, typical of renin-containing granules. Tumoural cells also exhibited the vascular cell surface marker CD34, but, in contrast with previous reports, did not express the tyrosine-protein kinase Kit (cKit or CD117). Dissociation of the tumour allowed to obtain confluent cultures of elongated smooth muscle actin (SMA)-positive cells producing high amounts of renin. However, after the first passage, subcultured human juxtaglomerular cells rapidly lost renin and CD34 expressions and their ability to produce renin. CONCLUSION: The present case of reninoma emphasizes the need for CTA in the etiologic work up of otherwise unexplained severe hypertension. 18-FDG PET imaging showed no hypermetabolism of the tumour, in accordance with its reported benignity. Pathological studies further emphasized that high expressions of renin and CD34 are typical hallmarks of reninoma. Although CD117 has been proposed to represent a reliable marker of JGCT, the present findings indicate that reninomas may not always express this marker.

Flagellin/TLR5 signalling activates renal collecting duct cells and facilitates invasion and cellular translocation of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) colonizing kidneys is the main cause of acute pyelonephritis. TLR5 that senses flagellin was shown to be highly expressed in the bladder and to participate in host defence against flagellated UPEC, although its role in kidneys still remains elusive. Here we show that TLR5 is expressed in renal medullary collecting duct (MCD) cells, which represent a preferential site of UPEC adhesion. Flagellin, like lipopolysaccharide, stimulated the production of the chemoattractant chemokines CXCL1 and CXCL2, and subsequent migration capacity of neutrophils in cultured wild-type (WT) and Tlr4(-/-) MCDs, but not in Tlr5(-/-) MCDs. UPEC can translocate across intact MCD layers without altering tight junctions. Strikingly, the invasion capacity and transcellular translocation of the UPEC strain HT7 were significantly lower in Tlr5(-/-) than in WT MCDs. The non-motile HT7ΔfliC mutant lacking flagellin also exhibited much lower translocation capacities than the HT7 isolates. Finally, Tlr5(-/-) kidneys exhibited less infiltrating neutrophils than WT kidneys one day after the transurethral inoculation of HT7, and greater delayed renal bacterial loads in the day 4 post-infected Tlr5(-/-) kidneys. Overall, these findings indicate that the epithelial TLR5 participates to renal antibacterial defence, but paradoxically favours the translocation of UPEC across intact MCD cell layers.

Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients.

Acute pyelonephritis (APN), which is mainly caused by uropathogenic Escherichia coli (UPEC), is the most common bacterial complication in renal transplant recipients receiving immunosuppressive treatment. However, it remains unclear how immunosuppressive drugs, such as the calcineurin inhibitor cyclosporine A (CsA), decrease renal resistance to UPEC. Here, we investigated the effects of CsA in host defense against UPEC in an experimental model of APN. We show that CsA-treated mice exhibit impaired production of the chemoattractant chemokines CXCL2 and CXCL1, decreased intrarenal recruitment of neutrophils, and greater susceptibility to UPEC than vehicle-treated mice. Strikingly, renal expression of Toll-like receptor 4 (Tlr4) and nucleotide-binding oligomerization domain 1 (Nod1), neutrophil migration capacity, and phagocytic killing of E. coli were significantly reduced in CsA-treated mice. CsA inhibited lipopolysaccharide (LPS)-induced, Tlr4-mediated production of CXCL2 by epithelial collecting duct cells. In addition, CsA markedly inhibited Nod1 expression in neutrophils, macrophages, and renal dendritic cells. CsA, acting through inhibition of the nuclear factor of activated T-cells (NFATs), also markedly downregulated Nod1 in neutrophils and macrophages. Silencing the NFATc1 isoform mRNA, similar to CsA, downregulated Nod1 expression in macrophages, and administration of the 11R-VIVIT peptide inhibitor of NFATs to mice also reduced neutrophil bacterial phagocytosis and renal resistance to UPEC. Conversely, synthetic Nod1 stimulating agonists given to CsA-treated mice significantly increased renal resistance to UPEC. Renal transplant recipients receiving CsA exhibited similar decrease in NOD1 expression and neutrophil phagocytosis of E. coli. The findings suggest that such mechanism of NFATc1-dependent inhibition of Nod1-mediated innate immune response together with the decrease in Tlr4-mediated production of chemoattractant chemokines caused by CsA may contribute to sensitizing kidney grafts to APN.

Downregulation of the Na/K-ATPase pump by leptospiral glycolipoprotein activates the NLRP3 inflammasome.

Leptospira interrogans is responsible for a zoonotic disease known to induce severe kidney dysfunction and inflammation. In this work, we demonstrate that L. interrogans induces NLRP3 inflammasome-dependent secretion of IL-1ß through the alteration of potassium transport in bone marrow-derived macrophages. Lysosome destabilization also contributed to the IL-1ß production upon stimulation with live, but not dead, bacteria. Using bone marrow-derived macrophages from various TLRs and nucleotide-binding oligomerization domain-deficient mice, we further determined that IL-1ß production was dependent on TLR2 and TLR4, suggesting a participation of the leptospiral LPS to this process. Hypokaliemia in leptospirosis has been linked to the presence of glycolipoprotein, a cell wall component of L. interrogans that is known to inhibit the expression and functions of the Na/K-ATPase pump. We show in this study that glycolipoprotein activates the inflammasome and synergizes with leptospiral LPS to produce IL-1ß, mimicking the effect of whole bacteria. These results were confirmed in vivo, as wild-type mice expressed more IL-1ß in the kidney than TLR2/4-deficient mice 3 d postinfection with L. interrogans. Collectively, these findings provide the first characterization, to our knowledge, of bacteria-induced activation of the NLRP3 inflammasome through the downregulation of a specific host potassium transporter.

A role for collecting duct epithelial cells in renal antibacterial defences.

Urinary tract infections (UTIs), which are mainly due to uropathogenic Escherichia coli (UPEC), occur via the retrograde ascent of the bacteria along the urinary tract system. The adhesion and invasion mechanisms of UPEC have been extensively studied in bladder epithelial cells, but less is known about the role of renal tubule epithelial cells (RTEC) in renal antibacterial defences. This review considers recent advances in the understanding of the role of RTECs in inducing an innate immune response mediated by Toll-like receptors (TLRs) in experimental UTI. Collecting duct cells are a preferential site of adhesion of UPEC colonizing the kidneys. Epithelial TLR4 activation induces an inflammatory response and the recruitment of lipid rafts to the plasma membrane, both of which facilitate the transcytosis of non-cytolytic UPEC strains across intact collecting duct cell layers to invade the renal interstitium. Arginine vasopressin, which regulates water absorption in the collecting duct, also acts as a potent modulator of the TLR4-mediated intrarenal innate response caused by UPEC. The role of epithelial TLR5 in renal host defences is also discussed. These findings highlight the role of RTECs in triggering the innate immune response in the context of ascending UTIs.

Potentiation of epithelial innate host responses by intercellular communication.

The epithelium efficiently attracts immune cells upon infection despite the low number of pathogenic microbes and moderate levels of secreted chemokines per cell. Here we examined whether horizontal intercellular communication between cells may contribute to a coordinated response of the epithelium. Listeria monocytogenes infection, transfection, and microinjection of individual cells within a polarized intestinal epithelial cell layer were performed and activation was determined at the single cell level by fluorescence microscopy and flow cytometry. Surprisingly, chemokine production after L. monocytogenes infection was primarily observed in non-infected epithelial cells despite invasion-dependent cell activation. Whereas horizontal communication was independent of gap junction formation, cytokine secretion, ion fluxes, or nitric oxide synthesis, NADPH oxidase (Nox) 4-dependent oxygen radical formation was required and sufficient to induce indirect epithelial cell activation. This is the first report to describe epithelial cell-cell communication in response to innate immune activation. Epithelial communication facilitates a coordinated infectious host defence at the very early stage of microbial infection.

Decreased renal corin expression contributes to sodium retention in proteinuric kidney diseases.

Patients with proteinuric kidney diseases often have symptoms of salt and water retention. It has been hypothesized that dysregulated sodium absorption is due to increased proteolytic cleavage of epithelial sodium channels (ENaCs) and increased Na,K-ATPase expression. Microarray analysis identified a reduction in kidney corin mRNA expression in rat models of puromycin aminonucleoside-induced nephrotic syndrome and acute anti-Thy1 glomerulonephritis (GN). As atrial natriuretic peptide (ANP) resistance is a mechanism accounting for volume retention, we analyzed the renal expression and function of corin; a type II transmembrane serine protease that converts pro-ANP to active ANP. Immunohistochemical analysis found that corin colocalized with ANP. The nephrotic and glomerulonephritic models exhibited concomitant increased pro-ANP and decreased ANP protein levels in the kidney consistent with low amounts of corin. Importantly, kidneys from corin knockout mice had increased amounts of renal ß-ENaC and its activators, phosphodiesterase (PDE) 5 and protein kinase G II, when compared to wild-type mice. A similar expression profile was also found in cell culture suggesting the increase in PDE5 and kinase G II could account for the increase in ß-ENaC seen in nephrotic syndrome and GN. Thus, we suggest that corin might be involved in the salt retention seen in glomerular diseases.

Contribution of renal tubule epithelial cells in the innate immune response during renal bacterial infections and ischemia-reperfusion injury.

The epithelial cells that line the renal tubule are sometimes severely injured in the course of inflammatory kidney diseases. These renal tubule epithelial cells (RTECs) express some of the Toll-like receptors (TLRs) of the innate immune system. A number of studies have implicated RTECs, together with bone marrow-derived cells, in triggering an innate immune response to bacterial infection and/or ischemic stress. RTECs expressing TLR4, which recognizes lipopolysaccharide (LPS), contribute to defending the host against ascending urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPECs). Activation of TLR2 and TLR4 signaling by endogenous damage-associated molecular patterns controls the inflammatory responses of RTECs and cell apoptosis in kidneys subjected to ischemia/reperfusion (I/R) injury. This review will consider some recent advances in understanding of the role of RTECs in inducing the innate immune response in experimental models of ascending UTIs and renal I/R injury. Arginine vasopressin, which regulates renal water absorption, has been shown to act as a potent modulator of the innate response in collecting duct cells, a preferred intrarenal site for UPEC adhesion. The activation of the mitogen-associated protein kinase ERK1/2 in post-hypoxic RTECs has also been shown to be selectively regulated by TLR2 via the serine-threonine protein phosphatase 5, which is associated with the endoplasmic reticulum resident heat shock protein, gp96, which acts as a master chaperone of TLRs. These findings provide further support for the concept that RTECs are actively involved in triggering the innate immune response, at least in the context of ascending UTIs and I/R injury.

Nedd4-2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia.

Nedd4-2, a E3 ubiquitin ligase, regulates epithelial sodium channel-mediated transcellular Na(+) transport in the collecting duct. We investigated the effect of Nedd4-2 on the junctional complex and paracellular conductance in mpkCCD(c14) cells, a collecting duct cell line. We demonstrate that Nedd4-2 coimmunoprecipitated with and reduced the expression of transfected occludin in HEK293 cells. This interaction was mediated via a conserved PY motif in the COOH terminus of occludin and mutation of this PY motif increased the half-life of transfected occludin in HEK293 cells from 6.4 to 11.4 h. We demonstrate that Nedd4-2 ubiquitinates occludin, which was not seen when a catalytically inactive form of Nedd4-2 was used. Overexpression of Nedd4-2 in mpkCCD(c14) cells reduced occludin at the tight junction and transiently increased paracellular conductance in a Ca(2+) switch assay consistent with a delay in the formation of tight junctions. Conversely, siRNA-mediated knockdown of Nedd4-2 increased occludin levels and reduced paracellular conductance. In summary, we demonstrate that Nedd4-2 plays a role in tight junction assembly and the regulation of paracellular conductance in the collecting duct.

EGF and its related growth factors mediate sodium transport in mpkCCDc14 cells via ErbB2 (neu/HER-2) receptor.

Amiloride-sensitive sodium entry, via the epithelial sodium channel (ENaC), is the rate-limiting step for Na(+) absorption. Epidermal growth factor (EGF) is involved in the regulation of Na(+) transport and ENaC activity. However it is still controversial exactly how EGF regulates ENaC and Na(+) absorption. The aim of the present study was to characterize the EGF regulation of Na(+) transport in cultured mouse renal collecting duct principal mpkCCD(c14) cells, a highly differentiated cell line which retains many characteristics of the cortical collecting duct (CCD). EGF dose dependently regulates basal transepithelial Na(+) transport in two phases: an acute phase (<4 h) and a chronic phase (>8 h). Similar effects were observed with TGF-alpha, HB-EGF, and amphiregulin which also belong to the EGF-related peptide growth factor family. Inhibition of MEK1/2 by PD98059 or U0126 increased acute effects and disrupted chronic effects of EGF on Na(+) reabsorption. Inhibition of PI3-kinase with LY294002 abolished acute effect of EGF. As assessed by Western blotting, ErbB2 is the most predominant member of the ErbB family detected in mpkCCD(c14) cells. Immunohistochemistry analysis revealed localization of ErbB2 in the CCD in Sprague-Dawley rat kidneys. Both acute and long-term effects of EGF were abolished when cells were treated with tyrphostin AG-825 and ErbB2 inhibitor II, chemically dissimilar selective inhibitors of the ErbB2 receptor. Thus, we conclude that EGF and its related growth factors are important for maintaining transepithelial Na(+) transport and that EGF biphasically modulates sodium transport in mpkCCD(c14) cells via the ErbB2 receptor.

Clostridium septicum alpha-toxin forms pores and induces rapid cell necrosis.

Alpha-toxin is the unique lethal virulent factor produced by Clostridium septicum, which causes traumatic or non-traumatic gas gangrene and necrotizing enterocolitis in humans. Here, we analyzed channel formation of the recombinant septicum alpha-toxin and characterized its activity on living cells. Recombinant septicum alpha-toxin induces the formation of ion-permeable channels with a single-channel conductance of about 175pS in 0.1M KCl in lipid bilayer membranes, which is typical for a large diffusion pore. Septicum alpha-toxin channels remained mostly in the open configuration, displayed no lipid specificity, and exhibited slight anion selectivity. Septicum alpha-toxin caused a rapid decrease in the transepithelial electrical resistance of MDCK cell monolayers grown on filters, and induced a rapid cell necrosis in a variety of cell lines, characterized by cell permeabilization to propidium iodide without DNA fragmentation and activation of caspase-3. Septicum alpha-toxin also induced a rapid K(+) efflux and ATP depletion. Incubation of the cells in K(+)-enriched medium delayed cell death caused by septicum alpha-toxin or epsilon-toxin, another potent pore-forming toxin, suggesting that the rapid loss of intracellular K(+) represents an early signal of pore-forming toxins-mediated cell necrosis.

Tumor necrosis factor and interferon-gamma down-regulate Klotho in mice with colitis.

BACKGROUND & AIMS: Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis. METHODS: We studied 3 mouse models of IBD: colitis induced by trinitrobenzene sulfonic acid, colitis induced by microflora (in gnotobiotic interleukin-10(-/-)), and colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells. Effects of the tumor necrosis factor (TNF) and interferon (IFN)-gamma on Kl expression and the activity of its promoter were examined in renal epithelial cells (mpkDCT4 and mIMCD3). RESULTS: Renal expression of Kl messenger RNA (mRNA) and protein was reduced in all 3 models of IBD. Reduced level of KL correlated with the severity of colitis; the effect was reversed by neutralizing antibodies against TNF. In vitro, TNF inhibited Kl expression, an effect potentiated by IFN-gamma. The combination of TNF and IFN-gamma increased expression of inducible nitric oxide synthase (iNOS) and increased NO production. The effect of IFN-gamma was reproduced by exposure to an NO donor and reversed by the iNOS inhibitor. In cells incubated with TNF and/or IFN-gamma, Kl mRNA stability was unaffected, whereas Kl promoter activity was reduced, indicating that these cytokines regulate Kl at the transcriptional level. CONCLUSIONS: The down-regulation of KL that occurs during inflammation might account for the extraintestinal complications such as abnormalities in bone homeostasis that occur in patients with IBD.

TLR4- and TLR2-mediated B cell responses control the clearance of the bacterial pathogen, Leptospira interrogans.

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira interrogans that are transmitted by asymptomatic infected rodents. Leptospiral lipoproteins and LPS have been shown to stimulate murine cells via TLRs 2 and 4. Host defense mechanisms remain obscure, although TLR4 has been shown to be involved in clearing Leptospira. In this study, we show that double (TLR2 and TLR4) knockout (DKO) mice rapidly died from severe hepatic and renal failure following Leptospira inoculation. Strikingly, the severe proinflammatory response detected in the liver and kidney from Leptospira-infected DKO mice appears to be independent of MyD88, the main adaptor of TLRs. Infection of chimeric mice constructed with wild-type and DKO mice, and infection of several lines of transgenic mice devoid of T and/or B lymphocytes, identified B cells as the crucial lymphocyte subset responsible for the clearance of Leptospira, through the early production of specific TLR4-dependent anti-Leptospira IgMs elicited against the leptospiral LPS. We also found a protective tissue compartmentalized TLR2/TLR4-mediated production of IFN-gamma by B and T lymphocytes, in the liver and kidney, respectively. In contrast, the tissue inflammation observed in Leptospira-infected DKO mice was further characterized to be mostly due to B lymphocytes in the liver and T cells in the kidney. Altogether these findings demonstrate that TLR2 and TLR4 play a key role in the early control of leptospirosis, but do not directly trigger the inflammation induced by pathogenic Leptospira.

Heat shock protein gp96 interacts with protein phosphatase 5 and controls toll-like receptor 2 (TLR2)-mediated activation of extracellular signal-regulated kinase (ERK) 1/2 in post-hypoxic kidney cells.

Ischemia/reperfusion injury (IRI) induces an innate immune response, leading to an inflammatory reaction and tissue damage that have been attributed to engagement of the Toll-like receptor (TLR) 2 and 4. However, the respective roles of TLR2 and/or TLR4 in mediating downstream activation of mitogen-activated protein kinase (MAPK) pathways during IRI have not been fully elucidated. Here we show that extracellular signal-regulated kinase (ERK)1/2 is activated in both intact kidneys and cultured renal tubule epithelial cells (RTECs) from wildtype and Tlr4 knockout mice, but not those from Tlr2 knockout mice subjected to transient ischemia. Geldanamycin (GA), an inhibitor of heat shock protein 90 and reticulum endoplasmic-resident gp96, and gp96 mRNA silencing (siRNA), did not affect ERK1/2 activation in either post-hypoxic wild-type or Tlr4-deficient RTECs, but did restore its activation in post-hypoxic Tlr2-deficient RTECs. Immunoprecipitation studies revealed that gp96 co-immunoprecipitates with the serine-threonine protein phosphatase 5 (PP5), identified as a negative modulator of the mitogen extracellular kinase (MEK)-ERK pathway, in unstressed wild-type and post-hypoxic Tlr2-deficient RTECs. In contrast, PP5 co-immunoprecipitation with gp96 was strikingly reduced in post-hypoxic wild-type RTECs, suggesting that the inactivation of PP5 resulting from the dissociation of PP5 from gp96 allows the activation of ERK1/2 to occur. Inhibition of PP5 by okadaic acid, and Pp5 siRNA also restored TLR2-mediated phosphorylation of ERK1/2, and apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK)-mediated apoptosis in post-hypoxic Tlr2-deficient RTECs. These findings indicate that gp96 interacts with PP5 and controls TLR2-mediated induction of ERK1/2 in post-hypoxic renal tubule cells.

Uropathogenic Escherichia coli AL511 requires flagellum to enter renal collecting duct cells.

Escherichia coli is the leading cause of urinary tract infections, but the mechanisms governing renal colonization by this bacterium remain poorly understood. We investigated the ability of 13 E. coli strains isolated from the urine of patients with pyelonephritis and cystitis and normal stools to invade collecting duct cells, which constitute the first epithelium encountered by bacteria ascending from the bladder. The AL511 clinical isolate adhered to mouse collecting duct mpkCCD(cl4) cells, used as a model of renal cell invasion, and was able to enter and persist within these cells. Previous studies have shown that bacterial flagella play an important role in host urinary tract colonization, but the role of flagella in the interaction of E. coli with renal epithelial cells remains unclear. An analysis of the ability of E. coli AL511 mutants to invade renal cells showed that flagellin played a key role in bacterial entry. Both flagellum filament assembly and the motor proteins MotA and MotB appeared to be required for E. coli AL511 uptake into collecting duct cells. These findings indicate that pyelonephritis-associated E. coli strains may invade renal collecting duct cells and that flagellin may act as an invasin in this process.

Activation of the thiazide-sensitive Na+-Cl- cotransporter by the WNK-regulated kinases SPAK and OSR1.

Mutations increasing WNK1 kinase expression in humans cause the pseudohypoaldosteronism type II hypertension syndrome. This condition is treated effectively by thiazide diuretics, which exert their effects by inhibiting the Na+-Cl(-) cotransporter (NCC), suggesting a link between WNK1 and NCC. Here, we demonstrate that the SPAK and OSR1 kinases that are activated by WNK1 phosphorylate human NCC at three conserved residues (Thr46, Thr55 and Thr60). Activation of the WNK1-SPAK/OSR1 signalling pathway by treatment of HEK293 or mpkDCT kidney distal-convoluted-tubule-derived cells with hypotonic low-chloride conditions induced phosphorylation of NCC at residues phosphorylated by SPAK/OSR1. Efficient phosphorylation of NCC was dependent upon a docking interaction between an RFXI motif in NCC and SPAK/OSR1. Mutation of Thr60 to Ala in NCC markedly inhibited phosphorylation of Thr46 and Thr55 as well as NCC activation induced by hypotonic low-chloride treatment of HEK293 cells. Our results establish that the WNK1-SPAK/OSR1 signalling pathway plays a key role in controlling the phosphorylation and activity of NCC. They also suggest a mechanism by which increased WNK1 overexpression could lead to hypertension and that inhibitors of SPAK/OSR1 might be of use in reducing blood pressure by suppressing phosphorylation and hence activity of NCC.