Mutation Update of the CLCN5 Gene Responsible for Dent Disease 1.
; ; et al
in Human mutation (2015), 36(8), 743-52
Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable ... [more ▼]
Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies. [less ▲]Detailed reference viewed: 17 (0 ULg)
The Role of the Renal Ammonia Transporter Rhcg in Metabolic Responses to Dietary Protein
; Ruffoni, Davide ; et al
in JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY (2014), 25(9), 2040-2052
High dietary protein imposes a metabolic acid load requiring excretion and buffering by the kidney. Impaired acid excretion in CKD, with potential metabolic acidosis, may contribute to the progression of ... [more ▼]
High dietary protein imposes a metabolic acid load requiring excretion and buffering by the kidney. Impaired acid excretion in CKD, with potential metabolic acidosis, may contribute to the progression of CKD. Here, we investigated the renal adaptive response of acid excretory pathways in mice to high-protein diets containing normal or low amounts of acid-producing sulfur amino acids (SAA) and examined how this adaption requires the RhCG ammonia transporter. Diets rich in SAA stimulated expression of enzymes and transporters involved in mediating NH4+ reabsorption in the thick ascending limb of the loop of Henle. The SAA-rich diet increased diuresis paralleled by downregulation of aquaporin-2 (AQP2) water channels. The absence of Rhcg transiently reduced NH4+ excretion, stimulated the ammoniagenic pathway more strongly, and further enhanced diuresis by exacerbating the downregulation of the Na+/K+/2Cl(-) cotransporter (NKCC2) and AQP2, with less phosphorylation of AQP2 at serine 256. The high protein acid load affected bone turnover, as indicated by higher Ca2+ and deoxypyridinoline excretion, phenomena exaggerated in the absence of Rhcg. In animals receiving a high-protein diet with low SAA content, the kidney excreted alkaline urine, with low levels of NH4+ and no change in bone metabolism. Thus, the acid load associated with high-protein diets causes a concerted response of various nephron segments to excrete acid, mostly in the form of NH4+, that requires Rhcg. Furthermore, bone metabolism is altered by a high-protein acidogenic diet, presumably to buffer the acid load. [less ▲]Detailed reference viewed: 21 (1 ULg)
Diagnosis of cyst infection in patients with autosomal dominant polycystic kidney disease: attributes and limitations of the current modalities.
JOURET, François ; ; et al
in Nephrology Dialysis Transplantation (2012), 27(10), 3746-51
Cyst infection is a diagnostic challenge in patients with autosomal dominant polycystic kidney disease (ADPKD) because of the lack of specific manifestations and limitations of conventional imaging ... [more ▼]
Cyst infection is a diagnostic challenge in patients with autosomal dominant polycystic kidney disease (ADPKD) because of the lack of specific manifestations and limitations of conventional imaging procedures. Still, recent clinical observations and series have highlighted common criteria for this condition. Cyst infection is diagnosed if confirmed by cyst fluid analysis showing bacteria and neutrophils, and as a probable diagnosis if all four of the following criteria are concomitantly met: temperature of >38 degrees C for >3 days, loin or liver tenderness, C-reactive protein plasma level of >5 mg/dL and no evidence for intracystic bleeding on computed tomography (CT). In addition, the elevation of serum carbohydrate antigen 19-9 (CA19-9) has been proposed as a biomarker for hepatic cyst infection. Positron-emission tomography after intravenous injection of 18-fluorodeoxyglucose, combined with CT, proved superior to radiological imaging techniques for the identification and localization of kidney and liver pyocyst. This review summarizes the attributes and limitations of these recent clinical, biological and imaging advances in the diagnosis of cyst infection in patients with ADPKD. [less ▲]Detailed reference viewed: 43 (17 ULg)
Segmental and subcellular distribution of CFTR in the kidney.
JOURET, François ; ;
in Methods in Molecular Biology (Clifton, N.J.) (2011), 741
Besides its location at the plasma membrane, CFTR is present in intracellular vesicles along both the exocytic and the endocytic pathways. Immunostaining and subcellular fractionation studies of mouse ... [more ▼]
Besides its location at the plasma membrane, CFTR is present in intracellular vesicles along both the exocytic and the endocytic pathways. Immunostaining and subcellular fractionation studies of mouse kidney demonstrate that CFTR is located in endosomes of the cells lining the terminal part of the proximal tubule (PT). The PT cells efficiently reabsorb the ultrafiltered low molecular weight (LMW) proteins by apical endocytosis involving the multiligand receptors megalin and cubilin. The progression from early endosomes to lysosomes depends on the integrity of the cytoskeleton, as well as on vesicular acidification. The latter is mediated by the vacuolar H+-ATPase (V-ATPase) and requires an anionic conductance to dissipate the transmembrane potential gradient. CFTR might ensure such chloride conductance, thereby participating to endosomal acidification and protein uptake by PT cells. Immunostaining with well-characterized antibodies shows that CFTR is located in the terminal segment of PT, where it co-distributes with megalin and cubilin. Subcellular fractionation of total mouse kidneys through Percoll gradients demonstrates the co-localization of CFTR with the V-ATPase and early endosome markers including the Cl-/H+ exchanger, ClC-5, and the small GTPase, Rab5a. Deglycosylation studies and immunoblotting show a distinct glycosylation pattern for CFTR in mouse kidney and lung. The segmental and subcellular distribution of CFTR in mouse kidney supports a role for CFTR in PT receptor-mediated endocytosis of ultrafiltered LMW proteins. [less ▲]Detailed reference viewed: 32 (1 ULg)
Positron-emission computed tomography in cyst infection diagnosis in patients with autosomal dominant polycystic kidney disease.
JOURET, François ; ; et al
in Clinical Journal of the American Society of Nephrology (2011), 6(7), 1644-50
BACKGROUND: Cyst infection remains a challenging issue in patients with autosomal dominant polycystic kidney disease (ADPKD). In most patients, conventional imaging techniques are inconclusive. Isolated ... [more ▼]
BACKGROUND: Cyst infection remains a challenging issue in patients with autosomal dominant polycystic kidney disease (ADPKD). In most patients, conventional imaging techniques are inconclusive. Isolated observations suggest that (18)fluorodeoxyglucose ((1)(8)FDG) positron-emission computed tomography (PET/CT) might help detect cyst infection in ADPKD patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Comparative assessment of administrative databases from January 2005 to December 2009 identified 27 PET/CT scans performed in 24 ADPKD patients for suspicion of abdominal infection. Cyst infection was definite if confirmed by cyst fluid analysis. Cyst infection was probable if all four of the following criteria were met: temperature of >38 degrees C for >3 days, loin or liver tenderness, C-reactive protein plasma level of >5 mg/dl, and no CT evidence for intracystic bleeding. Episodes with only two or three criteria were grouped as "fever of unknown origin". RESULTS: Thirteen infectious events in 11 patients met all criteria for kidney (n = 3) or liver (n = 10) cyst infection. CT was contributive in only one patient, whereas PET/CT proved cyst infection in 11 patients (84.6%). In addition, 14 episodes of "fever of unknown origin" in 13 patients were recorded. PET/CT identified the source of infection in nine patients (64.3%), including 2 renal cyst infections. Conversely, PET/CT showed no abnormal (1)(8)FDG uptake in 5 patients, including 2 intracystic bleeding. The median delay between the onset of symptoms and PET/CT procedure was 9 days. CONCLUSIONS: This retrospective series underscores the usefulness of PET/CT to confirm and locate cyst infection and identify alternative sources of abdominal infection in ADPKD patients. [less ▲]Detailed reference viewed: 72 (16 ULg)
Decreased renal accumulation of aminoglycoside reflects defective receptor-mediated endocytosis in cystic fibrosis and Dent's disease.
; ; et al
in Pflügers Archiv : European Journal of Physiology (2011), 462(6), 851-60
The clinical use of aminoglycoside (AG) antibiotics is limited by their renal toxicity, which is caused by drug accumulation in proximal tubule (PT) cells. Clinical studies reported that renal clearance ... [more ▼]
The clinical use of aminoglycoside (AG) antibiotics is limited by their renal toxicity, which is caused by drug accumulation in proximal tubule (PT) cells. Clinical studies reported that renal clearance of AG is enhanced in cystic fibrosis (CF) patients, which might reflect the role of CFTR in PT cell endocytosis. In order to assess the role of chloride transporters on the renal handling of AG, we investigated gentamicin uptake and renal accumulation in mice lacking functional CFTR (Cftr ( F/F)) or knock-out for the Cl(-)/H(+) exchanger ClC-5 (Clcn5 ( Y/- )). The latter represent a paradigm of PT dysfunction and defective receptor-mediated endocytosis. As compared with controls, Cftr ( F/F) and Clcn5 ( Y/- ) mice showed a 15% to 85% decrease in gentamicin accumulation in the kidney, respectively, in absence of renal failure. Studies on primary cultures of Cftr ( F/F) and Clcn5 ( Y/- ) mouse PT cells confirmed the reduction in gentamicin uptake, although colocalization with endosomes and lysosomes was maintained. Quantification of endocytosis in PT cells revealed that gentamicin, similar to albumin, preferentially binds to megalin. The functional loss of ClC-5 or CFTR was reflected by a decrease of the endocytic uptake of gentamicin, with a more pronounced effect in cells lacking ClC-5. These results support the concept that CFTR, as well as ClC-5, plays a relevant role in PT cell endocytosis. They also demonstrate that the functional loss of these two chloride transporters is associated with impaired uptake of AG in PT cells, reflected by a decreased renal accumulation of the drug. [less ▲]Detailed reference viewed: 25 (2 ULg)
CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.
; ; et al
in American Journal of Physiology - Renal Physiology (2010), 298(2), 365-80
Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked ... [more ▼]
Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney. [less ▲]Detailed reference viewed: 27 (0 ULg)
Glomerular and proximal tubule cysts as early manifestations of Pkd1 deletion.
; JOURET, François ; et al
in Nephrology Dialysis Transplantation (2010), 25(4), 1067-78
BACKGROUND: The homozygous deletion of Pkd1 in the mouse results in embryonic lethality with renal cysts and hydrops fetalis, but there is no precise data on the segmental origin of cysts and potential ... [more ▼]
BACKGROUND: The homozygous deletion of Pkd1 in the mouse results in embryonic lethality with renal cysts and hydrops fetalis, but there is no precise data on the segmental origin of cysts and potential changes associated with polyhydramnios. METHODS: We used Pkd1-null mice to investigate cystogenesis and analyze the amniotic fluid composition from embryonic day 12.5 (E12.5) to birth (n = 257 embryos). RESULTS: Polyhydramnios was consistently observed from E13.5 in Pkd1(-/-) embryos, in absence of placental abnormalities but with a significantly higher excretion of sodium and glucose from E13.5 through E16.5, and increased cyclic adenosine 3'5-monophosphate (cAMP) levels at E14.5 and E15.5. The Pkd1(-/-) embryos started to die at E13.5, with lethality peaking at E15.5, corresponding to the onset of cystogenesis. The first cysts in Pkd1(-/-) kidneys emerged at E15.5 in mesenchyme-derived segments at the cortico-medullary junction, with a majority of glomerular cysts and fewer proximal tubule cysts (positive for megalin). The cysts extended to ureteric bud-derived collecting ducts (positive for Dolichos biflorus agglutinin lectin) from E16.5. CONCLUSIONS: These studies indicate that Pkd1 deletion is associated with a massive loss of solutes (from E13.5) and increased cAMP levels (E14.5) associated with polyhydramnios. These abnormalities precede renal cysts (E15.5), first derived from glomeruli and proximal tubules and later from the collecting ducts, reflecting the expression pattern of Pkd1 in maturing epithelial cells. [less ▲]Detailed reference viewed: 18 (0 ULg)
Single photon emission-computed tomography (SPECT) for functional investigation of the proximal tubule in conscious mice.
JOURET, François ; ; et al
in American Journal of Physiology - Renal Physiology (2010), 298(2), 454-60
Noninvasive analysis of renal function in conscious mice is necessary to optimize the use of mouse models. In this study, we evaluated whether single photon emission-computed tomography (SPECT) using ... [more ▼]
Noninvasive analysis of renal function in conscious mice is necessary to optimize the use of mouse models. In this study, we evaluated whether single photon emission-computed tomography (SPECT) using specific radionuclear tracers can be used to analyze changes in renal proximal tubule functions. The tracers included (99m)TC- dimercaptosuccinic acid ((99m)Tc-DMSA), which is used for cortex imaging; (99m)Tc-mercaptoacetyltriglycine ((99m)Tc-MAG3), used for dynamic renography; and (123)I-beta(2)-microglobulin, which monitors receptor-mediated endocytosis. (99m)Tc-DMSA SPECT imaging was shown to delineate the functional renal cortex with a approximately 1-mm spatial resolution and accumulated in the cortex reaching a plateau 5 h after injection. The cortical uptake of (99m)Tc-DMSA was abolished in Clcn5 knockout mice, a model of proximal tubule dysfunction. Dynamic renography with (99m)Tc-MAG3 in conscious mice demonstrated rapid extraction from blood, renal accumulation, and subsequent tubular secretion. Anesthesia induced a significant delay in the (99m)Tc-MAG3 clearance. The tubular reabsorption of (123)I-beta(2)-microglobulin was strongly impaired in the Clcn5 knockout mice, with defective tubular processing and loss of the native tracer in urine, reflecting proximal tubule dysfunction. Longitudinal studies in a model of cisplatin-induced acute tubular injury revealed a correlation between tubular recovery and (123)I-beta(2)-microglobulin uptake. These data show that SPECT imaging with well-validated radiotracers allows in vivo investigations of specific proximal tubule functions in conscious mice. [less ▲]Detailed reference viewed: 23 (1 ULg)
A coherent organization of differentiation proteins is required to maintain an appropriate thyroid function in the Pendred thyroid.
; ; et al
in Journal of Clinical Endocrinology and Metabolism (2010), 95(8), 4021-30
CONTEXT: Pendred syndrome is caused by mutations in the gene coding for pendrin, an apical Cl-/I- exchanger. OBJECTIVE: To analyze intrathyroidal compensatory mechanisms when pendrin is lacking, we ... [more ▼]
CONTEXT: Pendred syndrome is caused by mutations in the gene coding for pendrin, an apical Cl-/I- exchanger. OBJECTIVE: To analyze intrathyroidal compensatory mechanisms when pendrin is lacking, we investigated the thyroid of a patient with Pendred syndrome. The expression of proteins involved in thyroid hormone synthesis, markers of oxidative stress (OS), cell proliferation, apoptosis, and antioxidant enzymes were analyzed. RESULTS: Three morphological zones were identified: nearly normal follicles with iodine-rich thyroglobulin in the colloid (zone 1.a), small follicles without iodine-rich thyroglobulin in lumina (zone 1.b), and destroyed follicles (zone 2). In zones 1.a, dual oxidase (Duox) and thyroid peroxidase (TPO) were localized at the apical pole, OS and cell apoptosis were absent, but ClC-5 expression was strongly increased. In zones 1.b, Duox and TPO were aberrantly present and increased in the cytosol and associated with high OS, apoptosis, cell proliferation, and increased expression of peroxiredoxin-5, catalase, and dehalogenase-1 but moderate ClC-5 expression. CONCLUSION: In conclusion, the absence of pendrin is accompanied by increased ClC-5 expression that may transiently compensate for apical iodide efflux. In more affected follicles, Duox and TPO are relocated in the cytosol, leading to abnormal intracellular thyroid hormone synthesis, which results in cell destruction presumably because intracellular OS cannot be buffered by antioxidant defenses. [less ▲]Detailed reference viewed: 21 (3 ULg)
CFTR and defective endocytosis: new insights in the renal phenotype of cystic fibrosis.
JOURET, François ;
in Pflügers Archiv : European Journal of Physiology (2009), 457(6), 1227-36
Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype is ... [more ▼]
Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype is associated with CF. Recent studies have shown that the level of CFTR mRNA in mouse kidney approaches that found in lung. CFTR is particularly abundant in the apical area of proximal tubule cells, where it co-distributes with the Cl(-)/H(+) exchanger ClC-5 and Rab5a in endosomes. The biological relevance of CFTR in proximal tubule endocytosis has been tested in CF mouse models and CF patients. Mice lacking CFTR show a defective receptor-mediated endocytosis, as evidenced by impaired uptake of (125)I-beta(2)-microglobulin, a decreased expression of the cubilin receptor in the kidney, and a significant excretion of cubilin and its low-molecular-weight ligands into the urine. Low-molecular-weight proteinuria (and particularly transferrinuria) is similarly detected in CF patients in comparison with normal controls or patients with chronic lung inflammation. These studies suggest that the functional loss of CFTR impairs the handling of low-molecular-weight proteins by the kidney, supporting a role of CFTR in receptor-mediated endocytosis in proximal tubule cells. The selective proteinuria should be integrated in the pathophysiology of multi-systemic complications increasingly observed in CF patients. [less ▲]Detailed reference viewed: 24 (2 ULg)
A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility
Biver, Sophie ; ; et al
in Nature (2008), 456(7220), 339-343Detailed reference viewed: 22 (1 ULg)
Cystic fibrosis is associated with a defect in apical receptor-mediated endocytosis in mouse and human kidney.
JOURET, François ; ; et al
in Journal of the American Society of Nephrology [=JASN] (2007), 18(3), 707-18
Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype has ... [more ▼]
Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype has been documented in patients with CF. This study investigated the expression, subcellular distribution, and processing of CFTR in the kidney; used various mouse models to assess the role of CFTR in proximal tubule (PT) endocytosis; and tested the relevance of these findings in patients with CF. The level of CFTR mRNA in mouse kidney approached that found in lung. CFTR was located in the apical area of PT cells, with a maximal intensity in the straight part (S3) of the PT. Fractionation showed that CFTR co-distributed with the chloride/proton exchanger ClC-5 in PT endosomes. Cftr(-/-) mice showed impaired (125)I-beta(2)-microglobulin uptake, together with a decreased amount of the multiligand receptor cubilin in the S3 segment and a significant loss of cubilin and its low molecular weight (LMW) ligands into the urine. Defective receptor-mediated endocytosis was found less consistently in Cftr(DeltaF/DeltaF) mice, characterized by a large phenotypic heterogeneity and moderate versus mice that lacked ClC-5. A significant LMW proteinuria (and particularly transferrinuria) also was documented in a cohort of patients with CF but not in patients with asthma and chronic lung inflammation. In conclusion, CFTR inactivation leads to a moderate defect in receptor-mediated PT endocytosis, associated with a cubilin defect and a significant LMW proteinuria in mouse and human. The magnitude of the endocytosis defect that is caused by CFTR versus ClC-5 loss likely reflects functional heterogeneity along the PT. [less ▲]Detailed reference viewed: 10 (4 ULg)
A primary culture of mouse proximal tubular cells, established on collagen-coated membranes.
; JOURET, François ; et al
in American Journal of Physiology - Renal Physiology (2007), 293(2), 476-85
A simple method is described to establish primary cultures of kidney proximal tubule cells (PTC) on membranes. The permeable membranes represent a unique culture surface, allowing a high degree of ... [more ▼]
A simple method is described to establish primary cultures of kidney proximal tubule cells (PTC) on membranes. The permeable membranes represent a unique culture surface, allowing a high degree of differentiation since both apical and basolateral membranes are accessible for medium. Proximal tubule (PT) segments from collagenase-digested mouse renal cortices were grown for 7 days, by which time cells were organized as a confluent monolayer. Electron microscopic evaluation revealed structurally polarized epithelial cells with numerous microvilli, basolateral invaginations, and apical tight junctions. Immunoblotting for markers of distinct parts of the nephron demonstrated that these primary cultures only expressed PT-specific proteins. Moreover immunodetection of distinct components of the receptor-mediated endocytic pathway and uptake of FITC-albumin indicated that these cells expressed a functional endocytotic apparatus. In addition, primary cultures possessed the PT brush-border enzymes, alkaline phosphatase, and gamma-glutamyl-transferase, and a phloridzin-sensitive sodium-dependent glucose transport at their apical side. Electrophysiological measurements show that the primary cultured cells have a low transepithelial resistance and high short-circuit current that was completely carried by Na(+) similar to a leaky epithelium like proximal tubule cells. This novel method established well-differentiated PTC cultures. [less ▲]Detailed reference viewed: 30 (2 ULg)
Topiramate induces type 3 renal tubular acidosis by inhibiting renal carbonic anhydrase.
; JOURET, François ; et al
in Nephrology Dialysis Transplantation (2006), 21(10), 2995-6Detailed reference viewed: 11 (1 ULg)
The loss of the chloride channel, ClC-5, delays apical iodide efflux and induces a euthyroid goiter in the mouse thyroid gland.
; ; JOURET, François et al
in Endocrinology (2006), 147(3), 1287-96
Genetic inactivation of ClC-5, a voltage-gated chloride channel prominently expressed in the kidney, leads to proteinuria because of defective apical endocytosis in proximal tubular cells. Because thyroid ... [more ▼]
Genetic inactivation of ClC-5, a voltage-gated chloride channel prominently expressed in the kidney, leads to proteinuria because of defective apical endocytosis in proximal tubular cells. Because thyroid hormone secretion depends on apical endocytosis of thyroglobulin (Tg), we investigated whether ClC-5 is expressed in the thyroid and affects its function, using Clcn5-deficient knockout (KO) mice. We found that ClC-5 is highly expressed in wild-type mouse thyroid ( approximately 40% of mRNA kidney level). The protein was immunolocalized at the apical pole of thyrocytes. In Percoll gradients, ClC-5 overlapped with plasma membrane and early endosome markers, but best codistributed with the late endosomal marker, Rab7. ClC-5 KO mice were euthyroid (normal T4 and TSH serum levels) but developed a goiter with parallel iodine and Tg accumulation (i.e. normal Tg iodination level). When comparing ClC-5 KO with wild-type mice, thyroid 125I uptake after 1 h was doubled, incorporation into Tg was decreased by approximately 2-fold, so that trichloroacetic acid-soluble 125I increased approximately 4-fold. Enhanced 125I- efflux upon perchlorate and presence of 125I-Tg as autoradiographic rings at follicle periphery demonstrated delayed iodide organification. Endocytic trafficking of 125I-Tg toward lysosomes was not inhibited. Expression of pendrin, an I-/Cl- exchanger involved in apical iodide efflux, was selectively decreased by 60% in KO mice at mRNA and protein levels. Thus, ClC-5 is well expressed in the thyroid but is not critical for apical endocytosis, contrary to the kidney. Instead, the goiter associated with ClC-5 KO results from impaired rate of apical iodide efflux by down-regulation of pendrin expression. [less ▲]Detailed reference viewed: 20 (0 ULg)
Ubiquitous and kidney-specific subunits of vacuolar H+-ATPase are differentially expressed during nephrogenesis.
JOURET, François ; ; et al
in Journal of the American Society of Nephrology (2005), 16(11), 3235-46
The vacuolar H(+)-ATPase (V-ATPase) is a ubiquitous multisubunit pump that is responsible for acidification of intracellular organelles. In the kidney, a particular form of V-ATPase, made of specific ... [more ▼]
The vacuolar H(+)-ATPase (V-ATPase) is a ubiquitous multisubunit pump that is responsible for acidification of intracellular organelles. In the kidney, a particular form of V-ATPase, made of specific subunits isoforms, has been located at the plasma membrane of intercalated cells (IC). Mutations in genes encoding IC-specific subunits cause infant distal renal tubular acidosis (dRTA), suggesting that the segmental distribution of these subunits is acquired at birth or during early infancy. However, the comparative ontogeny of the IC-specific versus the ubiquitous subunits of V-ATPase and the mechanisms involved in their segmental expression remain unknown. Real-time reverse transcription-PCR, in situ hybridization, immunoblotting, immunostaining, and subcellular fractionation analyses characterized the expression and distribution of V-ATPase subunits, transcription factors, and differentiation markers during mouse nephrogenesis. Ubiquitous A, E1, B2, G1, and C1 subunits showed an early (embryonic day 13.5 [E13.5]) and stable expression throughout nephrogenesis, followed by a slight increase around birth. The developmental pattern of a1 was bimodal, with early induction, gradual decrease during organogenesis, and neonatal increase. These patterns contrasted with the later (from E15.5) and progressive expression of IC-specific a4, B1, G3, and C2 subunits, after the induction of the forkhead transcription factor Foxi1. From E15.5, Foxi1 mRNA was detected in IC, where it co-distributed with B1 in late nephrogenesis. Immunostaining showed that the distribution of ubiquitous E1 and B2 was acquired from E15.5, whereas a4 was located in IC during late nephrogenesis. Subcellular fractionation showed that in both fetal and mature (cortex and medulla) kidneys, E1 and a4 were located in endosomes. These data demonstrate a differential expression and a coordinate regulation of IC-specific versus ubiquitous V-ATPase subunits during nephrogenesis. They provide new insights into the complex regulation of V-ATPase subunits, the maturation of IC along the nephron, and the pathophysiology of hereditary dRTA. [less ▲]Detailed reference viewed: 36 (9 ULg)
Chloride channels and endocytosis: new insights from Dent's disease and ClC-5 knockout mice.
; JOURET, François ; et al
in Nephron. Clinical Practice (2005), 99(3), 69-73
Dent's disease is a hereditary renal tubular disorder characterized by low-molecular weight (LMW) proteinuria, hypercalciuria and nephrolithiasis. The disease is due to mutations of ClC-5, a member of the ... [more ▼]
Dent's disease is a hereditary renal tubular disorder characterized by low-molecular weight (LMW) proteinuria, hypercalciuria and nephrolithiasis. The disease is due to mutations of ClC-5, a member of the family of voltage-gated CLC chloride channels. ClC-5 is expressed in part in cells lining the proximal tubule (PT) of the kidney, where it colocalizes with albumin-containing endocytic vesicles belonging to the receptor-mediated endocytic pathway that ensures efficient reabsorption of ultrafiltrated LMW proteins. Since progression along the endocytic apparatus requires endosomal acidification, it has been suggested that dysfunction of ClC-5 in endosomes may lead to inefficient reabsorption of LMW proteins and dysfunction of PT cells. Analysis of a ClC-5 knockout (KO) mouse model, displaying all the characteristic renal tubular defects of Dent's disease, showed evidence of a severe LMW proteinuria. Cytochemical studies with the endocytic tracer, peroxidase, showed poor transfer into early endocytic vesicles, suggesting that impairment of receptor-mediated endocytosis in PT cells is the basis for the defective uptake of LMW proteins in patients with Dent's disease. Endocytosis and processing of LMW proteins involve the multiligand tandem receptors, megalin and cubilin, that are abundantly expressed at the brush border of PT cells. Characterization of the endocytic defect in ClC-5 KO mice revealed that ligands of both megalin and cubilin were affected. The total kidney content of megalin and especially cubilin at the protein level was decreased but, more importantly, using analytical subcellular fractionation and quantitative immunogold labelling we demonstrated a selective disappearance of megalin and cubilin at the brush border of PT cells. These observations allowed us to conclude that defective protein endocytosis linked to ClC-5 inactivation is due at least in part to a major and selective loss of megalin and cubilin at the brush border, reflecting a trafficking defect in renal PT cells. These results improve our understanding of Dent's disease, taken as a paradigm for renal Fanconi syndrome and nephrolithiasis, and demonstrate multiple roles for ClC-5 in the kidney. These studies also provided insights into important functions such as apical endocytosis, handling of proteins by renal tubular cells, calcium metabolism, and urinary acidification. [less ▲]Detailed reference viewed: 28 (2 ULg)
Vacuolar H+-ATPase d2 subunit: molecular characterization, developmental regulation, and localization to specialized proton pumps in kidney and bone.
; JOURET, François ; et al
in Journal of the American Society of Nephrology [=JASN] (2005), 16(5), 1245-56
The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane ... [more ▼]
The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane of intercalated cells in the distal nephron, where it is required for urine acidification, and in osteoclasts, playing an important role in bone resorption by acid secretion across the ruffled border membrane. It was reported previously that, in human, several of the renal pump's constituent subunits are encoded by genes that are different from those that are ubiquitously expressed. These paralogous proteins may be important in differential functions, targeting or regulation of H+-ATPases. They include the d subunit, where d1 is ubiquitous whereas d2 has a limited tissue expression. This article reports on an investigation of d2. It was first confirmed that in mouse, as in human, kidney and bone are two of the main sites of d2 mRNA expression. d2 mRNA and protein appear later during nephrogenesis than does the ubiquitously expressed E1 subunit. Mouse nephron-segment reverse transcription-PCR revealed detectable mRNA in all segments except thin limb of Henle's loop and distal convoluted tubule. However, with the use of a novel d2-specific antibody, high-intensity d2 staining was observed only in intercalated cells of the collecting duct in fresh-frozen human kidney, where it co-localized with the a4 subunit in the characteristic plasma membrane-enhanced pattern. In human bone, d2 co-localized with the a3 subunit in osteoclasts. This different subunit association in different tissues emphasizes the possibility of the H+-ATPase as a future therapeutic target. [less ▲]Detailed reference viewed: 15 (0 ULg)
Comparative ontogeny, processing, and segmental distribution of the renal chloride channel, ClC-5.
JOURET, François ; ; et al
in Kidney International (2004), 65(1), 198-208
BACKGROUND: The renal chloride channel ClC-5, which is responsible for Dent's disease, is coexpressed with the vacuolar H+-ATPase in proximal tubules (PT) and alpha-type intercalated cells (IC) of the ... [more ▼]
BACKGROUND: The renal chloride channel ClC-5, which is responsible for Dent's disease, is coexpressed with the vacuolar H+-ATPase in proximal tubules (PT) and alpha-type intercalated cells (IC) of the mature kidney. Neonatal cases of Dent's disease suggest that ClC-5 distribution must be acquired before birth. However, the ontogeny of ClC-5, and its processing and segmental distribution with respect to related proteins during nephrogenesis remain unknown. METHODS: Immunoblotting, real-time polymerase chain reaction (RT-PCR), immunostaining, and deglycosylation studies were used to investigate the expression, distribution, and maturation of ClC-5 during mouse and human nephrogenesis, in comparison with H+-ATPase, type II carbonic anhydrase (CAII), and aquaporin-1 (AQP1). RESULTS: An early induction (E13.5-E14.5) of ClC-5 was observed in mouse kidney, with persistence at high levels through late nephrogenesis. This pattern contrasted with the progressive expression of H+-ATPase and AQP1, and the postnatal upregulation of CAII. Immunostaining showed expression of ClC-5 in ureteric buds and, from E14.5, its location in developing PT. From E15.5, ClC-5 codistributed with H+-ATPase in PT cells and alpha-type IC. In the human kidney, ClC-5 was detected from 12 gestation weeks; its distribution was similar to that observed in mouse, except for a later detection in IC. Although mouse and human ClC-5 proteins are glycosylated, biochemical differences between fetal and adult proteins were observed in both species. CONCLUSION: The segmental expression of ClC-5 and H+-ATPase is essentially achieved during early nephrogenesis, in parallel with the onset of glomerular filtration. These data give insight into PT and IC maturation, and explain early phenotypic variants of Dent's disease. [less ▲]Detailed reference viewed: 27 (7 ULg)