Hydrochlorothiazide and Prevention of Kidney-Stone Recurrence.

BACKGROUND: Nephrolithiasis is one of the most common conditions affecting the kidney and is characterized by a high risk of recurrence. Thiazide diuretic agents are widely used for prevention of the recurrence of kidney stones, but data regarding the efficacy of such agents as compared with placebo are limited. Furthermore, dose-response data are also limited. METHODS: In this double-blind trial, we randomly assigned patients with recurrent calcium-containing kidney stones to receive hydrochlorothiazide at a dose of 12.5 mg, 25 mg, or 50 mg once daily or placebo once daily. The main objective was to investigate the dose-response effect for the primary end point, a composite of symptomatic or radiologic recurrence of kidney stones. Radiologic recurrence was defined as the appearance of new stones on imaging or the enlargement of preexisting stones that had been observed on the baseline image. Safety was also assessed. RESULTS: In all, 416 patients underwent randomization and were followed for a median of 2.9 years. A primary end-point event occurred in 60 of 102 patients (59%) in the placebo group, in 62 of 105 patients (59%) in the 12.5-mg hydrochlorothiazide group (rate ratio vs. placebo, 1.33; 95% confidence interval [CI], 0.92 to 1.93), in 61 of 108 patients (56%) in the 25-mg group (rate ratio, 1.24; 95% CI, 0.86 to 1.79), and in 49 of 101 patients (49%) in the 50-mg group (rate ratio, 0.92; 95% CI, 0.63 to 1.36). There was no relation between the hydrochlorothiazide dose and the occurrence of a primary end-point event (P = 0.66). Hypokalemia, gout, new-onset diabetes mellitus, skin allergy, and a plasma creatinine level exceeding 150% of the baseline level were more common among patients who received hydrochlorothiazide than among those who received placebo. CONCLUSIONS: Among patients with recurrent kidney stones, the incidence of recurrence did not appear to differ substantially among patients receiving hydrochlorothiazide once daily at a dose of 12.5 mg, 25 mg, or 50 mg or placebo once daily. (Funded by the Swiss National Science Foundation and Inselspital; NOSTONE ClinicalTrials.gov number, NCT03057431.).

Resistance of Dynamin-related Protein 1 Oligomers to Disassembly Impairs Mitophagy, Resulting in Myocardial Inflammation and Heart Failure.

We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.

Phagocytosis: coupling of mitochondrial uncoupling and engulfment.

Clearance of apoptotic cells by phagocytes avoids triggering an inflammatory response. A new study reveals that phagocytes dissipate their mitochondrial proton electrochemical gradient to allow for the ingestion of more apoptotic corpses. Mitochondria are therefore involved in all aspects of apoptosis, from its activation through to the phagocytosis of dead cells.

LACTB is a filament-forming protein localized in mitochondria.

LACTB is a mammalian active-site serine protein that has evolved from a bacterial penicillin-binding protein. Penicillin-binding proteins are involved in the metabolism of peptidoglycan, the major bacterial cell wall constituent, implying that LACTB has been endowed with novel biochemical properties during eukaryote evolution. Here we demonstrate that LACTB is localized in the mitochondrial intermembrane space, where it is polymerized into stable filaments with a length extending more than a hundred nanometers. We infer that LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization. These findings have implications for our understanding of mitochondrial evolution and function.

Intermediate conductance Ca2+-activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells.

Patch-clamping mitoplasts isolated from human colon carcinoma 116 cells has allowed the identification and characterization of the intermediate conductance Ca(2+)-activated K(+)-selective channel K(Ca)3.1, previously studied only in the plasma membrane of various cell types. Its identity has been established by its biophysical and pharmacological properties. Its localisation in the inner membrane of mitochondria is indicated by Western blots of subcellular fractions, by recording of its activity in mitochondria made fluorescent by a mitochondria-targeted fluorescent protein and by the co-presence of channels considered to be markers of the inner membrane. Moderate increases of mitochondrial matrix [Ca(2+)] will cause mtK(Ca)3.1 opening, thus linking inner membrane K(+) permeability and transmembrane potential to Ca(2+) signalling.

A maxi-chloride channel in the inner membrane of mammalian mitochondria.

Patch-clamp experiments on swollen mitochondria of human, mouse and rat origins have revealed activity by an approximately 400 pS (in 150 mM KCl), voltage-dependent and anion-selective channel. This channel is located in the inner membrane, as shown by experiments with mitochondria from cells expressing a fluorescent mitochondrial tag protein and by the co-presence of the 107 pS channel and of the permeability transition pore (PTP). The frequency of appearance was inversely related to the presence of the PTP. This and the comparison of its electrophysiological characteristics with those of the PTP indicate that it is closely related to the latter, possibly corresponding to a monomeric unit whose dimer constitutes the full PTP. The channel is similar but not identical to isolated-and-reconstituted mitochondrial porin, and it is present also in mitochondria from cells lacking porin isoforms. Its identification with porin is therefore to be excluded. It most likely coincides instead with the "maxi-chloride channel" characterized in the plasma membrane of various cell types.

Copper(II) binding to the human Doppel protein may mark its functional diversity from the prion protein.

Doppel (Dpl) is the first described homologue of the prion protein, the main constituent of the agent responsible for prion diseases. The cellular prion protein (PrP(C)) is predominantly present in the central nervous system. Although its role is not yet completely clarified, PrP(C) seems to be involved in Cu(2+) recycling from synaptic clefts and in preventing neuronal oxidative damage. Conversely, Dpl is expressed in heart and testis and has been shown to regulate male fertility by intervening in gametogenesis and sperm-egg interactions. Therefore, despite a high sequence homology and a similar three-dimensional fold, the functions of PrP(C) and Dpl appear unrelated. Here we show by electron paramagnetic resonance and fluorescence spectroscopy that the in vitro binding of copper(II) to human recombinant Dpl occurs with a different pattern from that observed for recombinant PrP. At physiological pH values, two copper(II)-binding sites with different affinities were found in Dpl. At lower pH values, two additional copper(II)-binding sites can be identified as follows: one complex is present only at pH 4, and the other is observed in the pH range 5-6. As derived from the electron paramagnetic resonance characteristics, all Dpl-copper(II) complexes have a different coordination sphere from those present in PrP. Furthermore, in contrast to the effect shown previously for PrP(C), addition of Cu(2+) to Dpl-expressing cells does not cause Dpl internalization. These results suggest that binding of the ion to PrP(C) and Dpl may contribute to the different functional roles ascribed to these highly homologous proteins.

Stability and Cu(II) binding of prion protein variants related to inherited human prion diseases.

All inherited forms of human prion diseases are linked with mutations in the prion protein (PrP) gene. Here we have investigated the stability and Cu(II) binding properties of three recombinant variants of murine full-length PrP(23-231)-containing destabilizing point mutations that are associated with human Gerstmann-Sträussler-Scheinker disease (F198S), Creutzfeld-Jakob disease (E200K), and fatal familial insomnia (D178N) by electron paramagnetic resonance and circular dichroism spectroscopy. Furthermore, we analyzed the variants H140S, H177S, and H187S of the isolated C-terminal domain of murine PrP, mPrP(121-231), to test a role of the histidine residues in Cu(II) binding. The F198S and E200K variants of PrP(23-231) differed in Cu(II) binding from the wild-type mPrP(23-231). However, circular dichroism spectroscopy indicated that the variants and the wild type did not undergo conformational changes in the presence of Cu(II). The D178N variant showed a high tendency to aggregate at pH 7.4 both with and without Cu(II). At lower pH values, it showed the same Cu(II) binding behavior as the wild type. The analysis allowed for a better location of the Cu(II) binding sites in the C-terminal part of the protein. Our present data indicate that hereditary forms of prion diseases cannot be rationalized on the basis of altered Cu(II) binding or mutation-induced protein destabilization alone.