ETA receptor-mediated Ca2+ signaling in thin descending limbs of Henle's loop: Impairment in genetic hypertension

ETA-mediated Ca2+ signaling in thin descending limbs of Henle's loop: Impairment in genetic hypertension.BackgroundEndothelins (ET) have diuretic and natriuretic actions via ETB receptors that are found in most renal tubular segments, although the thin limbs have not been studied. Data also suggest that dysfunction of the renal ET system may be important in the pathogenesis of hypertension. The present study was aimed at determining the presence and nature of ET receptors in the thin limbs of Henle's loop and their ability to activate a Ca2+-dependent signaling pathway, as well as whether ET-induced Ca2+ signals are altered in hypertension.MethodsReverse transcription-polymerase chain reaction (RT-PCR) and Fura 2 fluorescence measurements of [Ca2+]i were made to characterize ET receptors in descending thin limbs (DTL) of Sprague-Dawley rats, spontaneously hypertensive (SH) rats, and control Wistar-Kyoto (WKY) rats, and the three selected strains of Lyon rats with low-normal (LL), normal (LN), and high (LH) blood pressure.ResultsIn SD rats, ET induced Ca2+ signals in DTL of long-looped nephrons, but not in DTL of short loops, or in ascending thin limbs. Ca2+ increases were abolished by BQ123, an antagonist of the ETA receptor, but not by BQ788, an antagonist of the ETB subtype. Endothelin-3 and sarafotoxin 6c, two ETB receptor agonists, were both inactive. RT-PCR showed the presence of both ETA and ETB receptor mRNA. Ca2+ signals measured in DTL of WKY LL and LN rats were similar to those in Sprague-Dawley rats, but were significantly diminished (LH) or abolished (SH) in hypertensive rats.ConclusionA functional ETA receptor activating a Ca2+-dependent pathway is expressed in DTL. This ETA-induced calcium signaling is impaired in two strains of genetically hypertensive rats

Epithelial sodium channel is a key mediator of growth hormone-induced sodium retention in acromegaly.: Antinatriuretic action of growth hormone

International audienceAcromegalic patients present with volume expansion and arterial hypertension, but the renal sites and molecular mechanisms of direct antinatriuretic action of GH remain unclear. Here, we show that acromegalic GC rats, which are chronically exposed to very high levels of GH, exhibited a decrease of furosemide-induced natriuresis and an increase of amiloride-stimulated natriuresis compared with controls. Enhanced Na(+),K(+)-ATPase activity and altered proteolytic maturation of epithelial sodium channel (ENaC) subunits in the cortical collecting ducts (CCDs) of GC rats provided additional evidence for an increased sodium reabsorption in the late distal nephron under chronic GH excess. In vitro experiments on KC3AC1 cells, a murine CCD cell model, revealed the expression of functional GH receptors and IGF-I receptors coupled to activation of Janus kinase 2/signal transducer and activator of transcription 5, ERK, and AKT signaling pathways. That GH directly controls sodium reabsorption in CCD cells is supported by: 1) stimulation of transepithelial sodium transport inhibited by GH receptor antagonist pegvisomant; 2) induction of alpha-ENaC mRNA expression; and 3) identification of signal transducer and activator of transcription 5 binding to a response element located in the alpha-ENaC promoter, indicative of the transcriptional regulation of alpha-ENaC by GH. Our findings provide the first evidence that GH, in concert with IGF-I, stimulates ENaC-mediated sodium transport in the late distal nephron, accounting for the pathogenesis of sodium retention in acromegaly

Heterogeneous distribution of chloride channels along the distal convoluted tubule probed by single-cell RT-PCR and patch clamp

International audienceThe distal convoluted tubule (DCT) is a heterogeneous segment subdivided into early (DCT1) and late (DCT2) parts, depending on the distribution of various transport systems. We do not have an exhaustive picture of the Cl(-) channels on the basolateral side: the presence of ClC-K2 channels is generally accepted, whereas that of ClC-K1 remains controversial. We used here single-cell RT-PCR and patch clamp to probe Cl(-) channel heterogeneity in microdissected mouse DCT at the molecular and functional levels. Our findings show that 63% of the DCT cells express ClC-K2 mRNA, either alone (type 1 cells: 47 and 23% in DCT1 and DCT2, respectively), or combined with ClC-K1, mostly in DCT2 (type 2 cells: 33%), but 37% of DCT1 and DCT2 cells do not express any ClC-K. Patch-clamp experiments revealed that a Cl(-) channel, with 9-pS conductance and Cl(-) > NO(3)(-) = Br(-) anion selectivity sequence, is present in the DCT1 and DCT2 basolateral membranes (87 and 71% of the patches, respectively). This dominant channel is likely to be ClC-K2 in type 1 cells. In type 2 cells, it could be ClC-K2 and/or ClC-K1 homodimers, but also ClC-K1/ClC-K2 heterodimers, or a mixture of all combinations. A second, distinct Cl(-) channel (13% of DCT1 patches, 29% of DCT2 patches) also displayed 9-pS conductance but had a completely different anion selectivity (I(-) > NO(3)(-) > Br(-) > Cl(-)), which was not compatible with that of the ClC-Ks. This indicates that a Cl(-) channel that is unlikely to belong to the ClC family may also be involved in Cl(-) absorption in the DCT2

Exploration of the Basolateral Chloride Channels in the Renal Tubule using

International audienceChloride channels located on the basolateral membrane are known to be involved in chloride absorption in several parts of the renal tubule, and particularly in the thick ascending limb and distal convoluted tubule. The data available suggest that the ClC-K channels play the major role in this process. We provide here a description of the electrophysiological properties of these channels, still very incomplete at this stage, and we attempt to compare ClC-Ks to three chloride channels that we have identified in the basolateral membrane of microdissected fragments of the mouse renal tubule using the patch-clamp technique. Based on anion selectivity and dependence on external pH and calcium shown by the ClC-Ks, we propose candidate ClC-K1 and ClC-K2 in native tissue. We also discuss the possibility that chloride channels that do not belong to the ClC family may also be involved in the absorption of chloride across the cortical thick ascending limb

Membrane progestin receptors α and γ in renal epithelium

AbstractSex hormones have broader effects than regulating reproductive functions. Recent identification of membrane progestin receptors expressed in kidney prompted us to investigate their putative involvement in the renal effects of this hormone. We first focused our investigations on mPRα and γ by analyzing three parameters 1/ their distribution along the mouse nephron and their subcellular location in native kidney, 2/ the ability of progesterone to stimulate ERK pathway and/or Ca2+ release from internal stores in native kidney structures and 3/ the cellular localization of mPRα and its molecular determinants in heterologous expression system. We observed that 1/ mPRα expression is restricted to proximal tubules of both male and female mice whereas mPRγ exhibits a much broader expression all along the nephron except the glomerulus, 2/ mPRα and γ are not localized at the plasma membrane in native kidney, 3/ this expression does not permit either progesterone-induced ERK phosphorylation or Ca2+ release and 4/ in HEK transfected cells, mPRα localizes in the endoplasmic reticulum (ER) due to a C-terminal ER retention motif (−KXX). Therefore, we have characterized mPRs in kidney but their role in renal physiology remains to be elucidated

How Bartter’s and Gitelman’s Syndromes, and Dent’s Disease Have Provided Important Insights into the Function of Three Renal Chloride Channels: ClC-Ka/b and ClC-5

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

Renal Proteinase-activated Receptor 2, a New Actor in the Control of Blood Pressure and Plasma Potassium Level

International audienceProteinase-activated receptor 2 (PAR2) is a G protein-coupled membrane receptor that is activated upon cleavage of its extracellular N-terminal domain by trypsin and related proteases. PAR2 is expressed in kidney collecting ducts, a main site of control of Na(+) and K(+) homeostasis, but its function remains unknown. We evaluated whether and how PAR2 might control electrolyte transport in collecting ducts, and thereby participate in the regulation of blood pressure and plasma K(+) concentration. PAR2 is expressed at the basolateral border of principal and intercalated cells of the collecting duct where it inhibits K(+) secretion and stimulates Na(+) reabsorption, respectively. Invalidation of PAR2 gene impairs the ability of the kidney to control Na(+) and K(+) balance and promotes hypotension and hypokalemia in response to Na(+) and K(+) depletion, respectively. This study not only reveals a new role of proteases in the control of blood pressure and plasma potassium level, but it also identifies a second membrane receptor, after angiotensin 2 receptor, that differentially controls sodium reabsorption and potassium secretion in the late distal tubule. Conversely to angiotensin 2 receptor, PAR2 is involved in the regulation of sodium and potassium balance in the context of either stimulation or nonstimulation of the renin/angiotensin/aldosterone system. Therefore PAR2 appears not only as a new actor of the aldosterone paradox, but also as an aldosterone-independent modulator of blood pressure and plasma potassium