P2Y2 receptor activation inhibits the expression of the sodium-chloride cotransporter NCC in distal convoluted tubule cells

Luminal nucleotide stimulation is known to reduce Na+ transport in the distal nephron. Previous studies suggest that this mechanism may involve the thiazide-sensitive Na+-Cl− cotransporter (NCC), which plays an essential role in NaCl reabsorption in the cells lining the distal convoluted tubule (DCT). Here we show that stimulation of mouse DCT (mDCT) cells with ATP or UTP promoted Ca2+ transients and decreased the expression of NCC at both mRNA and protein levels. Specific siRNA-mediated silencing of P2Y2 receptors almost completely abolished ATP/UTP-induced Ca2+ transients and significantly reduced ATP/UTP-induced decrease of NCC expression. To test whether local variations in the intracellular Ca2+ concentration ([Ca2+]i) may control NCC transcription, we overexpressed the Ca2+-binding protein parvalbumin selectively in the cytosol or in the nucleus of mDCT cells. The decrease in NCC mRNA upon nucleotide stimulation was abolished in cells overexpressing cytosolic PV but not in cells overexpressing either a nuclear-targeted PV or a mutated PV unable to bind Ca2+. Using a firefly luciferase reporter gene strategy, we observed that the activity of NCC promoter region from −1 to −2,200bp was not regulated by changes in [Ca2+]i. In contrast, high cytosolic calcium level induced instability of NCC mRNA. We conclude that in mDCT cells: (1) P2Y2 receptor is essential for the intracellular Ca2+ signaling induced by ATP/UTP stimulation; (2) P2Y2-mediated increase of cytoplasmic Ca2+ concentration down-regulates the expression of NCC; (3) the decrease of NCC expression occurs, at least in part, via destabilization of its mRNA

Regulation of Macrophage Motility by the Water Channel Aquaporin-1: Crucial Role of M0/M2 Phenotype Switch

The water channel aquaporin-1 (AQP1) promotes migration of many cell types. Although AQP1 is expressed in macrophages, its potential role in macrophage motility, particularly in relation with phenotype polarization, remains unknown. We here addressed these issues in peritoneal macrophages isolated from AQP1-deficient mice, either undifferentiated (M0) or stimulated with LPS to orientate towards pro-inflammatory phenotype (classical macrophage activation; M1). In non-stimulated macrophages, ablation of AQP1 (like inhibition by HgCl2) increased by 2-3 fold spontaneous migration in a Src/PI3K/Rac-dependent manner. This correlated with cell elongation and formation of lamellipodia/ruffles, resulting in membrane lipid and F4/80 recruitment to the leading edge. This indicated that AQP1 normally suppresses migration of resting macrophages, as opposed to other cell types. Resting Aqp1-/- macrophages exhibited CD206 redistribution into ruffles and increased arginase activity like IL4/IL13 (alternative macrophage activation; M2), indicating a M0-M2 shift. In contrast, upon M1 orientation by LPS in vitro or peritoneal inflammation in vivo , migration of Aqp1-/- macrophages was reduced. Taken together, these data indicate that AQP1 oppositely regulates macrophage migration, depending on stimulation or not by LPS, and that macrophage phenotypic and migratory changes may be regulated independently of external cues

Impaired autophagy bridges lysosomal storage disease and epithelial dysfunction in the kidney

The endolysosomal system sustains the reabsorptive activity of specialized epithelial cells. Lysosomal storage diseases such as nephropathic cystinosis cause a major dysfunction of epithelial cells lining the kidney tubule, resulting in massive losses of vital solutes in the urine. The mechanisms linking lysosomal defects and epithelial dysfunction remain unknown, preventing the development of disease-modifying therapies. Here we demonstrate, by combining genetic and pharmacologic approaches, that lysosomal dysfunction in cystinosis results in defective autophagy-mediated clearance of damaged mitochondria. This promotes the generation of oxidative stress that stimulates Gα12/Src-mediated phosphorylation of tight junction ZO-1 and triggers a signaling cascade involving ZO-1-associated Y-box factor ZONAB, which leads to cell proliferation and transport defects. Correction of the primary lysosomal defect, neutralization of mitochondrial oxidative stress, and blockage of tight junction-associated ZONAB signaling rescue the epithelial function. We suggest a link between defective lysosome-autophagy degradation pathways and epithelial dysfunction, providing new therapeutic perspectives for lysosomal storage disorders

A novel renal carbonic anhydrase type III plays a role in proximal tubule dysfunction.

Dysfunction of the proximal tubule (PT) is associated with variable degrees of solute wasting and low-molecular-weight proteinuria. We measured metabolic consequences and adaptation mechanisms in a model of inherited PT disorders using PT cells of ClC-5-deficient (Clcn5Y/-) mice, a well-established model of Dent's disease. Compared to cells taken from control mice, those from the mutant mice had increased expression of markers of proliferation (Ki67, proliferative cell nuclear antigen (PCNA), and cyclin E) and oxidative scavengers (superoxide dismutase I and thioredoxin). Transcriptome and protein analyses showed fourfold induction of type III carbonic anhydrase in a kidney-specific manner in the knockout mice located in scattered PT cells. Kidney-specific carbonic anhydrase type III (CAIII) upregulation was confirmed in other mice lacking the multiligand receptor megalin and in a patient with Dent's disease due to an inactivating CLCN5 mutation. The type III enzyme was specifically detected in the urine of mice lacking ClC-5 or megalin, patients with Dent's disease, and in PT cell lines exposed to oxidative stress. Our study shows that lack of PT ClC-5 in mice and men is associated with CAIII induction, increased cell proliferation, and oxidative stress

Renal expression of parvalbumin is critical for NaCl handling and response to diuretics.

Contains fulltext : 51558.pdf (publisher's version ) (Closed access)The distal convoluted tubule (DCT) plays an essential role in the reabsorption of NaCl by the kidney, a process that can be inhibited by thiazide diuretics. Parvalbumin (PV), a Ca(2+)-binding protein that plays a role in muscle fibers and neurons, is selectively expressed in the DCT, where its role remains unknown. We therefore investigated the renal phenotype of PV knockout mice (Pvalb(-/-)) vs. wild-type (Pvalb(+/+)) littermates. PV colocalized with the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) in the early DCT. The Pvalb(-/-) mice showed increased diuresis and kaliuresis at baseline with higher aldosterone levels and lower lithium clearance. Acute furosemide administration increased diuresis and natriuresis/kaliuresis, but, surprisingly, did not increase calciuria in Pvalb(-/-) mice. NaCl supplementation of Pvalb(-/-) mice increased calciuria at baseline and after furosemide. The Pvalb(-/-) mice showed no significant diuretic response to hydrochlorothiazide, but an accentuated hypocalciuria. A decreased expression of NCC was detected in the early DCT of Pvalb(-/-) kidneys in the absence of ultrastructural and apoptotic changes. The PV-deficient mice had a positive Ca(2+) balance and increased bone mineral density. Studies in mouse DCT cells showed that endogenous NCC expression is Ca(2+)-dependent and can be modulated by the levels of PV expression. These results suggest that PV regulates the expression of NCC by modulating intracellular Ca(2+) signaling in response to ATP in DCT cells. They also provide insights into the Ca(2+)-sparing action of thiazides and the pathophysiology of distal tubulopathies

Impact of cardio-renal syndrome on adverse outcomes in patients with Fabry disease in a long-term follow-up

Fabry disease (FD) is a rare X-linked lysosomal storage disease with a deficiency of α-galactosidase A leading to progressive sphingolipid accumulation in different organs, among them heart and kidney. We evaluated the impact of cardio-renal syndrome (CRS) on the incidence of major cardiovascular complications and death in a prospective FD cohort

Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide

Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal AQP1 is a major isoform. AQP1 expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (H2O2), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H2O2 through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by H2O2 Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H2O2 transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell-derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of H2O2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies.Virginie Montiel, Ramona Bella, Lauriane Y. M. Michel, Hrag Esfahani, Delphine De Mulder ... et al

Supplementary Material for: Cystic Gene Dosage Influences Kidney Lesions After Nephron Reduction

<p>Cystic kidney disease is characterized by the progressive development of multiple fluid-filled cysts. Cysts can be acquired, or they may appear during development or in postnatal life due to specific gene defects and lead to renal failure. The most frequent form of this disease is the inherited polycystic kidney disease (PKD). Experimental models of PKD showed that an increase of cellular proliferation and apoptosis as well as defects in apico-basal and planar cell polarity or cilia play a critical role in cyst development. However, little is known about the mechanisms and the mediators involved in acquired cystic kidney diseases (ACKD). In this study, we used the nephron reduction as a model to study the mechanisms underlying cyst development in ACKD. We found that tubular dilations after nephron reduction recapitulated most of the morphological features of ACKD. The development of tubular dilations was associated with a dramatic increase of cell proliferation. In contrast, the apico-basal polarity and cilia did not seem to be affected. Interestingly, polycystin 1 and fibrocystin were markedly increased and polycystin 2 was decreased in cells lining the dilated tubules, whereas the expression of several other cystic genes did not change. More importantly, <i>Pkd1</i> haploinsufficiency accelerated the development of tubular dilations after nephron reduction, a phenotype that was associated to a further increase of cell proliferation. These data were relevant to humans ACKD, as cystic genes expression and the rate of cell proliferation were also increased. In conclusion, our study suggests that the nephron reduction can be considered a suitable model to study ACKD and that dosage of genes involved in PKD is also important in ACKD.</p

Impaired mitophagy links mitochondrial disease to epithelial stress in methylmalonyl-CoA mutase deficiency

Deregulation of mitochondrial network in terminally differentiated cells contributes to a broad spectrum of disorders. Methylmalonic acidemia (MMA) is one of the most common inherited metabolic disorders, due to deficiency of the mitochondrial methylmalonyl-coenzyme A mutase (MMUT). How MMUT deficiency triggers cell damage remains unknown, preventing the development of disease–modifying therapies. Here we combine genetic and pharmacological approaches to demonstrate that MMUT deficiency induces metabolic and mitochondrial alterations that are exacerbated by anomalies in PINK1/Parkin–mediated mitophagy, causing the accumulation of dysfunctional mitochondria that trigger epithelial stress and ultimately cell damage. Using drug–disease network perturbation modelling, we predict targetable pathways, whose modulation repairs mitochondrial dysfunctions in patient–derived cells and alleviate phenotype changes in mmut–deficient zebrafish. These results suggest a link between primary MMUT deficiency, diseased mitochondria, mitophagy dysfunction and epithelial stress, and provide potential therapeutic perspectives for MMA