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

AIMS: 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. METHODS AND RESULTS: A total of 104 genetically proven FD patients were annually followed at the University Hospitals Zurich and Bern. The main outcome was a composite of incident renal replacement therapy (RRT), hospitalisation due to decompensated Heart Failure, new onset atrial fibrillation, pacemaker/ICD implantation, stroke/TIA and death. Estimated glomerular filtration rate (eGFR) and left ventricular myocardial mass index (LVMMI) where explored as the primary exposure variables. During the median follow-up of 103 [59-155] months, events occurred in 27 patients. In a Cox regression analysis, both higher LVMMI and lower eGFR were independently associated with a greater risk of developing adverse events after adjustment for multiple confounders (HR 1.67 [1.04-2.73] P=0.03 per SD increase in LVMMI, HR 0.45 [0.25-0.83], P=0.01 per SD decrease in eGFR). In patients with CRS, the risk to develop events was significantly increased if adjusted for demographics and RRT (HR 4.46 [1.07-18.62], P=0.04), approaching significance if additionally adjusted for hypertension (HR 4.05 [0.95-17.29], P=0.06). In Kaplan-Meier-Analysis, the poorest event-free survival was observed among patients with CRS. CONCLUSIONS: CRS was associated with a high risk to develop cardiovascular complications and death, emphasizing the importance of its prevention and early recognition. A focus on cardio-reno-protective therapies is crucial.

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 Ca(2+) 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 Ca(2+) transients and significantly reduced ATP/UTP-induced decrease of NCC expression. To test whether local variations in the intracellular Ca(2+) concentration ([Ca(2+)]i) may control NCC transcription, we overexpressed the Ca(2+)-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 Ca(2+). Using a firefly luciferase reporter gene strategy, we observed that the activity of NCC promoter region from -1 to -2,200 bp was not regulated by changes in [Ca(2+)]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 Ca(2+) signaling induced by ATP/UTP stimulation; (2) P2Y2-mediated increase of cytoplasmic Ca(2+) concentration down-regulates the expression of NCC; (3) the decrease of NCC expression occurs, at least in part, via destabilization of its mRNA.

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.

Consequence of parvalbumin deficiency in the mdx mouse: histological, biochemical and mechanical phenotype of a new double mutant.

We tested the hypothesis whether the mild dystrophy in mdx mice could result from the contribution of the cytosolic calcium buffer parvalbumin in maintaining a normal cytosolic [Ca2+]i, in spite of an increased passive Ca2+ influx. By crossing mdx mice with parvalbumin-deficient mice, double mutant mice, lacking both dystrophin and parvalbumin, were obtained. Though resting cytosolic [Ca2+]i and total calcium content were similar to that of mdx muscles, this new animal model presented a slightly more severe phenotype than the mdx mouse. Muscle pseudo-hypertrophy, the density of myotubes and of centronucleated fibres as well as the loss of IIB fibres were all increased in parvalbumin-deficient mdx mice. Many of these deficits were overcome in late adulthood, albeit fibrosis was clearly more pronounced than in mdx muscles. At 90 days, parvalbumin-deficient mdx mice showed higher levels of creatine phosphokinase and lower muscle strength, in vivo, than mdx mice. Isometric tension of isolated muscle was reduced, but the susceptibility to eccentric contraction was not increased. The slight aggravation of muscle dystrophy observed in mdx mice deprived of parvalbumin cannot explain the severity of the affection observed in xmd dogs and Duchenne dystrophy patients where parvalbumin is constitutively not expressed.

Intracellular pH regulation in isolated fast-twitch skeletal muscle from dystrophin-deficient mouse.

In muscles from anaesthetized dystrophin-deficient mdx mice, exercise results in a stronger acidification and a slower intracellular pH recovery compared to control mice. We examined whether this observation could be attributed to defective H+-carriers in dystrophin-lacking muscles. Immunohistochemistry and Western blots revealed no defect in mdx muscles for the presence of the lactate-/H+co-transporter MCT4 and of the Na+/H+ antiporter NHE1, the main H+-carriers active in fast-twitch skeletal muscle after exercise. Functional tests of the H+-transporters, on isolated muscles submitted to identical flow of superfusion, were performed in conditions meant to lower intracellular pH: repetitive electrical stimulation or NH4Cl pre-pulse. These revealed no defect in intracellular pH recovery in mdx muscles. Therefore, we conclude that impaired intracellular pH regulation in anaesthetized mdx mice is not attributable to a reduced presence or activity of H+-extruders. We propose that CO2 washout might be slowed down in vivo in mdx muscles because of the defective vascular response in contracting muscles from these mice.