A Focus on the Proximal Tubule Dysfunction in Dent Disease Type 1

Dent disease type 1 is a rare X-linked recessive inherited renal disorder affecting mainly young males, generally leading to end-stage renal failure and for which there is no cure. It is caused by inactivating mutations in the gene encoding ClC-5, a 2Cl$^{-}$/H$^{+}$ exchanger found on endosomes in the renal proximal tubule. This transporter participates in reabsorbing all filtered plasma proteins, which justifies why proteinuria is commonly observed when ClC-5 is defective. In the context of Dent disease type 1, a proximal tubule dedifferentiation was shown to be accompanied by a dysfunctional cell metabolism. However, the exact mechanisms linking such alterations to chronic kidney disease are still unclear. In this review, we gather knowledge from several Dent disease type 1 models to summarize the current hypotheses generated to understand the progression of this disorder. We also highlight some urinary biomarkers for Dent disease type 1 suggested in different studies

A Chloride Channel at the Basolateral Membrane of the Distal-convoluted Tubule: a Candidate ClC-K Channel

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na+-Cl− cotransporter located at the apical membrane, and Na+, K+ ATPase at the basolateral side. Cl− transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl− channels on microdissected mouse DCTs using the patch-clamp technique. A channel of ∼9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NPo in cell-attached patches was not modified when tubules were preincubated in the presence of 10−5 M forskolin, but the channel was inhibited by phorbol ester (10−6 M). In addition, NPo was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM (NPo increased threefold), or pH increased from 6.4 to 8.0 (NPo increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na+ to Cl− relative permeability was 0.09, and the anion selectivity sequence Cl− ∼ I−> Br− ∼ NO3− > F−. Intracellular NPPB (10−4 M) and DPC (10−3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl− channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family

Heterogeneity in the processing of ClC-5 mutants related to Dent disease

International audienceMutations in the electrogenic Cl-/H+ exchanger ClC-5 gene CLCN5 are frequently associated with Dent disease, an X-linked recessive disorder affecting the proximal tubules. Here, we investigate the consequences in X. laevis oocytes and in HEK293 cells of 9 previously reported, pathogenic, missense mutations of ClC-5, most of them which are located in regions forming the subunit interface. Two mutants trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface like wild-type ClC 5, but exhibited reduced currents. Three mutants displayed improper N-glycosylation, and were non-functional due to being retained and degraded at the endoplasmic reticulum. Functional characterization of four mutants allowed us to identify a novel mechanism leading to ClC-5 dysfunction in Dent disease. We report that these mutant proteins were delayed in their processing and that the stability of their complex glycosylated form was reduced, causing lower cell surface expression. The early endosome distribution of these mutants was normal. Half of these mutants displayed reduced currents, whereas the other half showed abolished currents. Our study revealed distinct cellular mechanisms accounting for ClC-5 loss-of-function in Dent disease

Etude des canaux potassium et chlorure de la membrane basolatérale du tubule distal de souris

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Characterization of the mouse ClC-K1/Barttin chloride channel.

International audienceClC-Kb, a member of the ClC family of Cl(-) channels/transporters, plays a major role in the absorption of NaCl in the distal nephron. CLCNKB mutations cause Bartter syndrome type 3, a hereditary renal salt-wasting tubulopathy. Here, we investigate the functional consequences of a Val to Met substitution at position 170 (V170M, α helix F), which was detected in eight patients displaying a mild phenotype. Conductance and surface expression were reduced by ~40-50 %. The regulation of channel activity by external H(+) and Ca(2+) is a characteristic property of ClC-Kb. Inhibition by external H(+) was dramatically altered, with pKH shifting from 7.6 to 6.0. Stimulation by external Ca(2+) on the other hand was no longer detectable at pH 7.4, but was still present at acidic pH values. Functionally, these regulatory modifications partly counterbalance the reduced surface expression by rendering V170M hyperactive. Pathogenic Met170 seems to interact with another methionine on α helix H (Met227) since diverse mutations at this site partly removed pH sensitivity alterations of V170M ClC-Kb. Exploring other disease-associated mutations, we found that a Pro to Leu substitution at position 124 (α helix D, Simon et al., Nat Genet 1997, 17:171-178) had functional consequences similar to those of V170M. In conclusion, we report here for the first time that ClC-Kb disease-causing mutations located around the selectivity filter can result in both reduced surface expression and hyperactivity in heterologous expression systems. This interplay must be considered when analyzing the mild phenotype of patients with type 3 Bartter syndrome

Properties of an inwardly rectifying K + channel in the basolateral membrane of mouse TAL

International audienceWe investigated the properties of K(+) channels in the basolateral membrane of the cortical thick ascending limb (CTAL) using the patch-clamp technique. Approximately 34% of cell-attached patches contained an inwardly rectifying K(+) channel (K(+)-to-Na(+) permeability ratio approximately 22), having an inward conductance (G(in)) of 44 pS and an outward conductance (G(out)) of approximately 10 pS (G(in)/G(out) approximately 4). Channel activity (NP(o)) increased with depolarization. When the cytosolic sides of inside-out patches were exposed to an Mg(2+)-free medium, the channel had a G(in) of 50 pS and was weakly inwardly rectifying (G(in)/G(out) approximately 1). Cytosolic Mg(2+) reduced G(out), yielding a G(in)/G(out) of 3.8 at 1.3 mM Mg(2+). Internal Na(+) also yielded a G(in)/G(out) of 1.6 at 20 mM Na(+). Spermine reduced NP(o) on inside-out membrane patches. Sensitivity to spermine at depolarizing voltages [half-maximal inhibitory concentration (K(i)) = 0.2 microM] was much greater than at hyperpolarizing voltages (K(i) = 26 microM). Half-inactivation by 0.5 microM spermine occurred at a clamp potential of 43 mV, with an effective valence of 1.25. A sigmoid relationship between bath pH and NP(o) of inside-out membrane patches was observed, with a pK of 7.6 and a Hill coefficient of 1.8. Intracellular acidification also reduced the NP(o) of cell-attached patches. This channel is probably a major component of K(+) conductance in the CTAL basolateral membrane

A chloride channel at the basolateral membrane of the distal-convoluted tubule: a candidate ClC-K channel.

International audienceChloride channels are expressed in almost all cell membranes and are potentially involved in a wide variety of functions. The kidney expresses 8 of the 9 chloride channels of the ClC family that have been cloned so far to date in mammals. This review focuses on the pathophysiology of two renal disorders that have contributed recently to our understanding of the physiological role of chloride channels belonging to the ClC family. First are the related syndromes of Bartter's and Gitelman's, in which inactivating mutations of the genes encoding either of the ClC-Ks, or their regulatory beta-subunit barttin, have shown the important contribution of these chloride channels to renal tubular sodium and chloride (NaCl) transport along the loop of Henle and distal tubule. Second is the renal Fanconi syndrome known as Dent's disease, in which ClC-5 disruption has revealed the key role of this endosomal chloride channel in the megalin-mediated endocytotic pathway in the proximal tubule. The underlying pathophysiology of this inherited disorder demonstrates how ClC-5 is directly or indirectly required for the reabsorption of filtered low-molecular-weight proteins and bioactive peptides, also expression of membrane transporters, and clearance of calcium-based stone-forming crystals

Régulation de la pression artérielle

Le Master de Biologie intégrative et physiologie (BIP) de Sorbonne Université recouvre un large champ disciplinaire, allant de la biologie du vieillissement à la physiopathologie, à la nutrition, aux bioressources marines et aux neurosciences, dont les enjeux sont essentiels aussi bien sur le plan fondamental qu’appliqué. L’objectif de la mention est de former des spécialistes de haut niveau aux concepts les plus récents de la biologie intégrative. La mention forme les étudiants à appréhender les mécanismes qui sous-tendent les différentes fonctions physiologiques et pathologiques depuis la cellule jusqu’à l’organisme, chez les animaux ou dans l’espèce humaine

A calcium-permeable non-selective cation channel in the thick ascending limb apical membrane of the mouse kidney

International audienc