Alain Vandewalle

Toll-like Receptor 4 Resides in the Golgi Apparatus and Colocalizes with Internalized Lipopolysaccharide in Intestinal Epithelial Cells

Toll-like receptor (TLR) 4 is mainly found on cells of the myelopoietic lineage. It recognizes lipopolysaccharide (LPS) and mediates cellular activation and production of proinflammatory cytokines. Less is known about the distribution and role of TLR4 in epithelial cells that are continuously exposed to microbes and microbial products. Here we show that the murine small intestinal epithelial cell line m-ICcl2 is highly responsive to LPS and expresses both CD14 and TLR4. Transcription and surface membrane staining for CD14 were up-regulated upon LPS exposure. Surprisingly, TLR4 immunostaining revealed a strictly cytoplasmic paranuclear distribution. This paranuclear compartment could be identified as the Golgi apparatus. LPS added to the supernatant was internalized by m-ICcl2 cells and colocalized with TLR4. Continuous exposure to LPS led to a tolerant phenotype but did not alter TLR4 expression nor cellular distribution. Thus, intestinal epithelial cells might be able to provide the initial proinflammatory signal to attract professional immune cells to the side of infection. The cytoplasmic location of TLR4, which is identical to the final location of internalized LPS, further indicates an important role of cellular internalization and cytoplasmic traffic in the process of innate immune recognition.

NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells

ABSTRACT The mechanisms involved in the cytotoxic action of oxysterols in the pathogenesis of atherosclerosis still remain poorly understood. Among the major oxysterols present in oxidized low-density lipoprotein, we show here that 7-ketocholesterol (7-Kchol) induces oxidative stress and/or apoptotic events in human aortic smooth muscle cells (SMCs). This specific effect of 7-Kchol is mediated by a robust upregulation (threefold from the basal level) of Nox-4, a reactive oxygen species (ROS)-generating NAD(P)H oxidase homologue. This effect was highlighted by silencing Nox-4 expression with a specific small interfering RNA, which significantly reduced the 7-Kchol-induced production of ROS and abolished apoptotic events. Furthermore, the 7-Kchol activating pathway included an early triggering of endoplasmic reticulum stress, as assessed by transient intracellular Ca2+ oscillations, and the induction of the expression of the cell death effector CHOP and of GRP78/Bip chaperone via the activation of IRE-1, all hallmarks of the unfolded protein response (UPR). We also showed that 7-Kchol activated the IRE-1/Jun-NH2-terminal kinase (JNK)/AP-1 signaling pathway to promote Nox-4 expression. Silencing of IRE-1 and JNK inhibition downregulated Nox-4 expression and subsequently prevented the UPR-dependent cell death induced by 7-Kchol. These findings demonstrate that Nox-4 plays a key role in 7-Kchol-induced SMC death, which is consistent with the hypothesis that Nox-4/oxysterols are involved in the pathogenesis of atherosclerosis.

Intracellular Recognition of Lipopolysaccharide by Toll-like Receptor 4 in Intestinal Epithelial Cells

Toll-like receptor (TLR)4 has recently been shown to reside in the Golgi apparatus of intestinal crypt epithelial m-ICcl2 cells, colocalizing with internalized lipopolysaccharide (LPS). Here we demonstrate that disruption of the integrity of the Golgi apparatus significantly reduced LPS-mediated nuclear factor κB activation. Also, the TLR4 adaptor protein MyD88 and the serine/threonine kinase IRAK-1 were rapidly recruited to the Golgi apparatus upon stimulation. LPS-mediated activation required lipid raft formation and intact clathrin-dependent internalization. In contrast to macrophages, prevention of ligand internalization by use of LPS-coated beads significantly impaired recognition by epithelial cells. The localization of TLR4 to the Golgi apparatus was abrogated by expression of a genetically modified form of the TLR4 binding chaperone gp96. Thus, our data provide evidence that in contrast to the situation in macrophages, LPS recognition in intestinal epithelial cells may occur in the Golgi apparatus and require LPS internalization.

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

Liddles 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 ammo‐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.—Kamynina, E., Debonneville, C., Bens, M., Vandewalle, A., Staub, O. A novel mouse Nedd4 protein suppresses the activity of the epithelial Na+ channel. . FASEB J. 15, 204–214 (2001)

Early development of polycystic kidney disease in transgenic mice expressing an activated mutant of the β-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 β-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 β-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/β-catenin signaling pathway causes polycystic kidney disease and support the view that dysregulation of the Wnt/β-catenin signaling is involved in its pathogenesis.

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 (mpkCCDcl4) 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 mpkCCDcl4 cell line either by Northern blot hybridization or reverse transcription–PCR. The hepatocyte nuclear transcription factor HNF-3-α (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.

Dysfunction of the Epithelial Sodium Channel Expressed in the Kidney of a Mouse Model for Liddle Syndrome

The Liddle syndrome is a dominant form of salt-sensitive hypertension resulting from mutations in the beta or gamma subunit of ENaC. A previous study established a mouse model carrying a premature Stop codon corresponding to the R(566stop) mutation (L) found in the original pedigree that recapitulates to a large extent the human disease. This study investigated the renal Na(+) transport in vivo, ex vivo (intact perfused tubules), and in vitro (primary cultured cortical collecting ducts [CCD]). In vivo, upon 6 to 12 h of salt repletion, after 1 week of low-salt diet, the L/L mice showed a delayed urinary sodium excretion, despite a lower aldosterone secretion as compared with controls. After 6 h salt of repletion, ENaC gamma subunit is rapidly removed from the apical plasma membrane in wild-type mice, whereas it is retained at the apical membrane in L/L mice. Ex vivo, isolated perfused CCD from L/L mice exhibited higher transepithelial potential differences than perfused CCD isolated from +/+ mice. In vitro, confluent primary cultures of CCD microdissected from L/L kidneys grown on permeable filters exhibited significant lower transepithelial electrical resistance and higher negative potential differences than their cultured L/+ and +/+ CCD counterparts. The equivalent short-circuit current (I(eq)) and the amiloride-sensitive I(eq) was approximately twofold higher in cultured L/L CCD than in +/+ CCD. Aldosterone (5 x 10(-7)M for 3 h) further increased I(eq) from cultured L/L CCD. Thus, this study brings three independent lines of evidence for the constitutive hyperactivity of ENaC in CCD from mice harboring the Liddle mutation.

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 m2, p < 0.01) and still persistent (∼− 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.

A novel role for glucocorticoid-induced leucine zipper protein in epithelial sodium channel-mediated sodium transport

The steroid hormone aldosterone stimulates sodium (Na+) transport in tight epithelia by altering the expression of target genes that regulate the activity and trafficking of the epithelial sodium channel (ENaC). We performed microarray analysis to identify aldosterone-regulated transcripts in mammalian kidney epithelial cells (mpkC-CDc14). One target, glucocorticoid-induced leucine zipper protein (GILZ), was previously identified by serial analysis of gene expression (SAGE); however, its function in epithelial ion transport was unknown. Here we show that GILZ expression is rapidly stimulated by aldosterone in mpkCCDc14 and that GILZ, in turn, strongly stimulates ENaC-mediated Na+ transport by inhibiting extracellular signal-regulated kinase (ERK) signaling. In Xenopus oocytes with activated ERK, heterologous GILZ expression consistently inhibited phospho-ERK expression and markedly stimulated ENaC-mediated Na+ current, in a manner similar to that of U0126 (a pharmacologic inhibitor of ERK signaling). In mpkCCDc14 cells, GILZ transfection similarly consistently inhibited phospho-ERK expression and stimulated transepithelial Na+ transport. Furthermore, aldosterone treatment of mpkCCDc14 cells suppressed phospho-ERK levels with a time course that paralleled their increase of Na+ transport. Finally, GILZ expression markedly increased cell surface ENaC expression in epidermal growth factor-treated mammalian kidney epithelial cells, HEK 293. These observations suggest a novel link between GILZ and regulation of epithelial sodium transport through modulation of ERK signaling and could represent an important pathway for mediating aldosterone actions in health and disease.

Long Term Regulation of Aquaporin-2 Expression in Vasopressin-responsive Renal Collecting Duct Principal Cells

Fine regulation of water reabsorption by the antidiuretic hormone [8-arginine]vasopressin (AVP) occurs in principal cells of the collecting duct and is largely dependent on regulation of the aquaporin-2 (AQP2) water channel. AVP-inducible long term AQP2 expression was investigated in immortalized mouse cortical collecting duct principal cells. Combined RNase protection assay, Western blot, and immunofluorescence analyses revealed that physiological concentrations of AVP added to the basal side, but not to the apical side, of cells grown on filters induced both AQP2 mRNA and apical protein expression. The stimulatory effect of AVP on AQP2 expression followed a V2receptor-dependent pathway because [deamino-8-d-arginine]vasopressin (dDAVP), a specific V2 receptor agonist, produced the same effect as AVP, whereas the V2 antagonist SR121463B antagonized action of both AVP and dDAVP. Moreover, forskolin and cyclic 8-bromo-AMP fully reproduced the effects of AVP on AQP2 expression. Analysis of protein degradation pathways showed that inhibition of proteasomal activity prevented synthesis of AVP-inducible AQP2 mRNA and protein. Once synthesized, AQP2 protein was quickly degraded, a process that involves both the proteasomal and lysosomal pathways. This is the first study that delineates induction and degradation mechanisms of AQP2 endogenously expressed by a renal collecting duct principal cell line.