How are the properties of the paracellular pathway defined and regulated in the thick ascending limb of Henle's loop ?

L'étude des transports ioniques transépithéliaux a longtemps concerné principalement les transports transcellulaires. Néanmoins, une asymétrie de composition des milieux de part et d'autre d'un épithélium ne peut être maintenue que si les propriétés de perméabilité et d'imperméabilité des jonctions serrées intercellulaires le permettent. La découverte récente de maladies génétiques rares affectant les claudines (Cldn), des protéines des jonctions serrées qui déterminent la perméabilité et la sélectivité de la voie paracellulaire, a amené à une prise de conscience de leur importance : l'hypomagnésémie familiale avec hypercalciurie et néphrocalcinose, liée à des mutations des Cldn 16 et 19 ; le syndrome HELIX, lié à des mutations de Cldn 10, caractérisé par une perte rénale de chlorure de sodium (NaCl). Ces Cldn sont exprimées dans la branche large ascendante de l'anse de Henle (BLAH), un site majeur de transport tubulaire de NaCl, de calcium (Ca) et de magnésium (Mg). La réabsorption de Na est en partie paracellulaire. Celle du Ca et du Mg est exclusivement paracellulaire et nécessite d'une part une perméabilité à ces ions et d'autre part une force motrice, le voltage transépithélial lumière positif, généré par le transport actif de NaCl et à la fin de la BLAH corticale (BLAHC), par la diffusion paracellulaire de NaCl (la perméabilité paracellulaire au Na étant supérieure à celle du Cl (PNa/PCl>1)). Dans la BLAH murine, il existe une expression « mosaïque » des Cldn, certaines jonctions serrées exprimant Cldn 10b et d'autres exprimant Cldn 3, 16 et 19. Les études menées pour élucider les fonctions des Cldn et leurs déterminants ont fourni des résultats discordants. De plus, nous savons que la perméabilité paracellulaire peut être régulée, par exemple par l'hormone parathyroïdienne (PTH), mais nous ne savons pas si les Cldn sont nécessaires à ses effets. Nos objectifs étaient de décrire l'expression rénale des Cldn 10, 16 et 19 chez l'homme et le rongeur, d'établir les conséquences fonctionnelles de leur expression en mosaïque, de déterminer si les Cldn sont impliquées dans les variations de la perméabilité paracellulaire, et de développer une technique permettant de mesurer des quantités picomolaires de Mg. Nous montrons que la même mosaïque d'expression des Cldn existe dans la BLAHC humaine. Nous démontrons que cette hétérogénéité d'expression est associée à une hétérogénéité fonctionnelle avec une variation parallèle de l'expression de la Cldn 10 et des propriétés de perméabilité paracellulaire (PNa/PCl) le long de la BLAHC murine. Nous remettons en cause le modèle admis dans lequel la Cldn 10b confèrerait une perméabilité au Na, en montrant qu'en absence de Cldn 10, PCl est significativement augmentée dans la BLAHC. L'hétérogénéité d'expression et de fonction de la BLAHC pourrait être particulièrement importante pour le contrôle du transport ionique. Nous montrons que les propriétés des jonctions serrées peuvent être régulées à court terme par des voies de signalisation intracellulaire et que la Cldn 10b est une cible de la PTH. Enfin, nous avons développé une méthode de chromatographie liquide à ultra haute performance pour mesurer des concentrations de l'ordre du millimolaire de Mg dans des échantillons ayant un volume d'environ 30 nanolitres, permettant de mesurer des flux de Mg lors des expériences de microperfusion ex vivo. Nous concluons que les modèles issus des données fournies par des lignées cellulaires ne décrivent pas fidèlement la réalité. Nos résultats robustes, puisque réalisés sur tissu natif et utilisant des méthodes de référence, permettent d'identifier des cibles thérapeutiques potentielles pouvant être utilisées pour modifier les propriétés de perméabilité paracellulaire. La description de l'hétérogénéité d'expression et de fonction de la BLAH doit être poursuivie afin d'identifier différents types cellulaires ayant des propriétés de transport transcellulaire et paracellulaire particulières.Until recently, studies of electrolyte transport across epithelia have focused on transcellular transport mechanisms. However, an asymmetry in the composition of apical and basolateral compartments of an epithelium can only be sustained if the permeability and impermeability properties of the tight junction allow it. The recent finding that rare genetic diseases affect claudins (Cldn), proteins located at the tight junction that determine the selectivity and the permeability of the paracellular pathway, led to an awareness of their importance: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is caused by a loss of function of Cldn 16 or 19. Cldn 10 mutation causes HELIX syndrome with a severe renal loss of sodium chloride (NaCl). These Cldn are expressed in the thick ascending limb (TAL) of Henle's loop, which play a key role in the tubular transport of NaCl, calcium (Ca) and magnesium (Mg). The reabsorption of Na is partly paracellular. That of Ca and Mg is exclusively paracellular: it requires both a paracellular permeability to these ions and a driving force, the lumen positive transepithelial voltage, generated by the active transport of NaCl and, at the end of the cortical TAL (CTAL), by the paracellular diffusion of NaCl (the paracellular permeability to Na being higher than that to Cl (PNa/PCl> 1)). Murine TAL tight junctions show a mosaic expression of either Cldn 10b or Cldn 3/Cldn 16/Cldn 19. Studies conducted to elucidate the functions of Cldn and their determinants have provided conflicting results. In addition, we know that paracellular permeability can be regulated, for example by parathyroid hormone (PTH), but we do not know whether Cldn are involved in its effects. Our objectives were to describe the expression of Cldn 10, 16 and 19 in human and rodent kidney, to establish the functional consequences of their mosaic expression, if any, to determine whether Cldn are involved in the changes of paracellular permeability, and to develop a technique for measuring picomolar amounts of Mg. We show that the same mosaic expression of Cldn as that in mouse exists in human CTAL. We demonstrate that this heterogeneity in expression is associated with a heterogeneity in function, changes in Cldn 10 expression being paralleled by changes in paracellular permeability (PNa/PCl) along murine CTAL. Our results challenge the accepted model in which Cldn 10b confers permeability to Na because the absence of Cldn 10 significanly increases PCl. The heterogeneity of expression and function of CTAL may be particularly important for the control of ion transport. We show that the properties of tight junction can be altered on short term by intracellular signaling pathways and that Cldn 10b is a target of PTH. Finally, we have developed an ultra-high performance liquid chromatography method to measure Mg concentrations of the millimolar range in samples with a volume of about 30 nanoliters, making it possible to measure Mg fluxes during ex vivo microperfusion experiments. We conclude that the models developed from data collected in studies conducted on cell lines do not accurately describe the real life. Our robust results, since carried out on native tissue and using reference methods, allow the identification of potential therapeutic targets that can be used to alter the properties of paracellular permeability. The description of the heterogeneity of expression and function of TAL should be pursued in order to better understand its basis and to identify distinct cell types with specific transcellular and paracellular transport properties

Claudins in Renal Physiology and Pathology

International audienceClaudins are integral proteins expressed at the tight junctions of epithelial and endothelial cells. In the mammalian kidney, every tubular segment express a specific set of claudins that give to that segment unique properties regarding permeability and selectivity of the paracellular pathway. So far, 3 claudins (10b, 16 and 19) have been causally traced to rare human syndromes: variants of CLDN10b cause HELIX syndrome and variants of CLDN16 or CLDN19 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis. The review summarizes our current knowledge on the physiology of mammalian tight junctions and paracellular ion transport, as well as on the role of the 3 above-mentioned claudins in health and disease. Claudin 14, although not having been causally linked to any rare renal disease, is also considered, because available evidence suggests that it may interact with claudin 16. Some single-nucleotide polymorphisms of CLDN14 are associated with urinary calcium excretion and/or kidney stones. For each claudin considered, the pattern of expression, the function and the human syndrome caused by pathogenic variants are described

How to Monitor Hydration Status and Urine Dilution in Patients with Nephrolithiasis

Maintenance of hydration status requires a tight balance between fluid input and output. An increase in water loss or a decrease in fluid intake is responsible for dehydration status, leading to kidney water reabsorption. Thus, urine volume decreases and concentration of the different solutes increases. Urine dilution is the main recommendation to prevent kidney stone recurrence. Monitoring hydration status and urine dilution is key to preventing stone recurrence. This monitoring could either be performed via spot urine or 24 h urine collection with corresponding interpretation criteria. In laboratory conditions, urine osmolality measurement is the best tool to evaluate urine dilution, with less interference than urine-specific gravity measurement. However, this evaluation is only available during time lab examination. To improve urine dilution in nephrolithiasis patients in daily life, such monitoring should also be available at home. Urine color is of poor interest, but reagent strips with urine-specific gravity estimation are currently the only available tool, even with well-known interferences. Finally, at home, fluid intake monitoring could be an alternative to urine dilution monitoring. Eventually, the use of a connected device seems to be the most promising solution

MEDULLARY AND CORTICAL THICK ASCENDING LIMB: SIMILARITIES AND DIFFERENCES

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Performance of ion chromatography to measure picomole amounts of magnesium in nanolitre samples

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Hydration for Adult Patients with Nephrolithiasis: Specificities and Current Recommendations

Nephrolithiasis affects around 10% of the population and is frequently associated with impaired dietary factors. The first one is insufficient fluid intake inducing reduced urine volume, urine supersaturation, and subsequently urinary lithiasis. Kidneys regulate 24 h urine volume, which, under physiological conditions, approximately reflects daily fluid intake. The aim of this study is to synthesize and highlight the role of hydration in the treatment of nephrolithiasis. Increasing fluid intake has a preventive effect on the risk of developing a first kidney stone (primary prevention) and also decreases the risk of stone recurrence (secondary prevention). Current guidelines recommend increasing fluid intake to at least at 2.5 L/day to prevent stone formation, and even to 3.5–4 L in some severe forms of nephrolithiasis (primary or enteric hyperoxaluria or cystinuria). Fluid intake must also be balanced between day and night, to avoid urinary supersaturation during the night. Patients should be informed and supported in this difficult process of increasing urine dilution, with practical ways and daily routines to increase their fluid intake. The liquid of choice is water, which should be chosen depending on its composition (such as calcium, bicarbonate, or magnesium content). Finally, some additional advice has to be given to avoid certain beverages such as those containing fructose or phosphoric acid, which are susceptible to increase the risk of nephrolithiasis

Differential localization patterns of claudin 10, 16, and 19 in human, mouse, and rat renal tubular epithelia

International audienceFunctional properties of the paracellular pathway depend critically on the set of claudins (CLDN) expressed at the tight junction. Two syndromes are causally linked to loss-of-function mutations of claudins: hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia (HELIX) syndrome caused by genetic variations in the CLDN10 gene and familial hypomagnesemia with hypercalciuria and nephrocalcinosis caused by genetic variations in the CLDN16 or CLDN19 genes. All three genes are expressed in the kidney, particularly in the thick ascending limb (TAL). However, localization of these claudins in humans and rodents remains to be delineated in detail. We studied the segmental and subcellular expression of CLDN10, CLDN16, and CLDN19 in both paraffin-embedded and frozen kidney sections from the adult human, mouse, and rat using immunohistochemistry and immunofluorescence, respectively. Here, CLDN10 was present in a subset of medullary and cortical TAL cells, localizing to basolateral domains and tight junctions in human and rodent kidneys. Weak expression was detected at the tight junction of proximal tubular cells. CLDN16 was primarily expressed in a subset of TAL cells in the cortex and outer stripe of outer medulla, restricted to basolateral domains and tight junctional structures in both human and rodent kidneys. CLDN19 predominantly colocalized with CLDN16 in tight junctions and basolateral domains of the TAL but was also found in basolateral and junctional domains in more distal sites. CLDN10 expression at tight junctions almost never overlapped with that of CLND16 and CLDN19, consistent with distinct junctional pathways with different permeation profiles in both human and rodent kidneys.NEW & NOTEWORTHY This study used immunohistochemistry and immunofluorescence to investigate the distribution of claudin 10, 16, and 19 in the human, mouse, and rat kidney. The findings showed distinct junctional pathways in both human and rodent kidneys, supporting the existence of different permeation profiles in all species investigated

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