Mitochondrial bioenergetics during the initiation of mercuric chloride-induced renal injury. I. Direct effects of in vitro mercuric chloride on renal mitochondrial function

Increasing data suggest that mitochondrial dysfunction may be an important early component of nephrotoxin-induced changes in renal cell function and viability. This study was designed to obtain more detailed information about the effects on several basic bioenergetic parameters of the direct interaction of Hg2+ with renal cortical mitochondria in vitro as a necessary prelude to studies of mitochondrial functional changes after treatment with mercuric chloride in vivo. Beginning at a threshold level of 2 nmol of Hg2+/mg of mitochondrial protein, Hg2+ induced marked stimulation of State 4 respiration, mild inhibition of State 3 respiration, and 2,4-dinitrophenol uncoupled respiration, a striking increase in atractyloside-insensitive ADP uptake and stimulation of both basal- and Mg2+-activated oligomycin-sensitive mitochondrial ATPase activity. These effects of Hg2+ could be prevented and reversed by the sulfhydryl reagent dithioerythritol and by albumin but were not affected by Mg2+. Detailed studies on the addition of HgCl2 to the preparation at different stages of the mitochondrial isolation procedure demonstrated that the presence of other proteins decreased mitochondrial Hg2+ binding, that the Hg2+ was not readily washed off the mitochondria by nonprotein-containing solutions, and that prolonged exposure of mitochondria to Hg2+ during the isolation procedure did not markedly alter its functional effects or their reversibility as assessed on the final mitochondrial preparation. These data provide an important basis for critically assessing the changes in function of mitochondria isolated after in vivo treatment with mercuric chloride.     

Regeneration of renal proximal tubules after mercuric chloride injury is accompanied by increased binding of aminoacyl-transfer ribonucleic acid.

The liver of the foetal guinea pig accumulates a large quantity of triacyglycerol late in gestation at the same time that adipose-tissue mass grows at its maximum rate and foetal adipose-tissue lipoprotein lipase activity and sensitivity to lipolytic hormones has substantially declined. The fatty acid for triacyglycerol synthesis is not synthesized in the foetal liver and it is unlikely that it originates from any of the foetal tissues. Before the accumulation of hepatic triacyglycerol the concentration of free fatty acids increases in both the umbilical vein and the maternal inferior vena cava. This occurs at a time when the triacyglycerol lipase activity in maternal adipose tissue is elevated and the rate of lipolysis, but not of fatty acid esterification, is higher than earlier in gestation or than in the non-pregnant state. It is proposed that the increase in lipolysis in maternal adipose tissue, brought about by an increase in circulating lipolytic hormones, mobilizes fatty acid, which passes to the foetus and is partly stored as hepatic triacylglycerol. The foetal liver effectively removes both long-and short-chain fatty acids from umbilical-vein blood. The rate of placental fatty acid transfer is more than adequate to account for the triacylglycerol accumulation.     

Mitochondrial dysfunction in Huntington's disease: the bioenergetics of isolated and in situ mitochondria from transgenic mice

Mitochondrial dysfunction is believed to participate in Huntington's disease (HD) pathogenesis. Here we compare the bioenergetic behavior of forebrain mitochondria isolated from different transgenic HD mice (R6/2, YAC128 and Hdh150 knock-in) and wild-type littermates with the first determination of in situ respiratory parameters in intact HD striatal neurons. We assess the Ca2+-loading capacity of isolated mitochondria by steady Ca2+-infusion. Mitochondria from R6/2 mice (12-13 weeks) and 12 months YAC128, but not homozygous or heterozygous Hdh150 knock-in mice (15-17 weeks), exhibit increased Ca2+-loading capacity when compared with respective wild-type littermates. In situ mitochondria in intact striatal neurons show high respiratory control. Moreover, moderate expression of full-length mutant huntingtin (in Hdh150 knock-in heterozygotes) does not significantly impair mitochondrial respiration in unstimulated neurons. However, when challenged with energy-demanding stimuli (NMDA-receptor activation in pyruvate-based media to accentuate the mitochondria role in Ca2+-handling), Hdh150 neurons are more vulnerable to Ca2+-deregulation than neurons from their wild-type littermates. These results stress the importance of assessing HD mitochondrial function in the cellular context.     

The cellular origin and proliferative status of regenerating renal parenchyma after mercuric chloride damage and erythropoietin treatment

OBJECTIVES: In this study, we have sought to establish the cellular origin and proliferative status of the renal parenchyma as it regenerates after damage induced by mercuric chloride, with or without erythropoietin treatments, that might alter the response. MATERIALS AND METHODS: Female mice were irradiated and male whole bone marrow was transplanted into them. Six weeks later recipient mice were assigned to one of four groups: control, mercuric chloride treated, erythropoietin treated and treated with mercuric chloride plus erythropoietin. RESULTS: Tubular injury scores were high 3 days after mercuric chloride and had recovered partially after 14 days, in line with serum urea nitrogen levels. Confocal microscopy confirmed the tubular location of bone marrow-derived cells. A 'four-in-one' analytical technique (identifying cell origin, tubular phenotype, tubular basement membranes and S-phase status) revealed that tubular necrosis increased bone marrow derivation of renal tubular epithelium from a baseline of approximately 1.3% to approximately 4.0%. Erythropoietin increased the haematocrit, but no other effects were detected. CONCLUSION: As 1 in 12 proximal tubular cells in S-phase was derived from bone marrow, we conclude that in the kidney, the presence of bone marrow-derived cells makes a minor but important regenerative contribution after tubular necrosis.     

Changes in rat renal cortex, isolated plasma membranes and urinary enzymes following the injection of mercuric chloride.

Some of the biochemical changes in rat kidney following the administration of mercuric chloride have been determined. Mercuric chloride had an immediate effect on the renal brush border resulting in rapid loss of the microvilli. Plasma membranes were isolated and characterised at various stages in the necrotic process, mircovilli were absent from these preparations and the activities of marker enzymes for the brush border were significantly decreased. In contrast the basal plasma membranes were unaffected by the nephrotoxin during the early stages and no change occurred in the activity of (Na+ + K+)-ATPase, a marker enzyme for the basal membranes. The change in the pattern of urinary enzyme excertion closely paralleled the ultrastructural changes in the tubular cells. The sequence of subcellular change following the administration of mercuric chloride is discussed in relation to the known mechanism of action of this agent.     

Mechanisms of gentamicin-induced dysfunction of renal cortical mitochondria: II. Effects on mitochondrial monovalent cation transport

Mitochondrial swelling techniques were used to evaluate the effects of the aminoglycoside antibiotic gentamicin on renal cortical mitochondrial monovalent cation permeability. Gentamicin behaved like EDTA to enhance energy-dependent Na⁺- and K⁺-acetate uptake with a relatively greater effect on Na⁺-acetate uptake. Mg²⁺ prevented and reversed the effects of both EDTA and gentamicin. Neither agent affected energy-independent uptake of Na⁺ and K⁺-acetate. Gentamicin did not enhance energy-independent uptake of K⁺- and Na⁺-nitrate. Gentamicin enhanced energy-dependent swelling in a chloride- and phosphate-containing medium as a function of the medium Na⁺ and K⁺ concentration. This effect occurred simultaneously with gentamicin-induced stimulation of State 4 respiration and was blocked by Mg²⁺. Gentamicin did not affect phosphate transport. The results are taken to indicate a specific action of gentamicin to enhance mitochondrial monovalent cation permeability at an Mg²⁺-sensitive site and it is proposed that this accounts for the effects of gentamicin on mitochondrial respiration.     

Studies on the pathophysiology of acute renal failure. II. A histochemical study of the proximal tubule of the rat following administration of mercuric chloride.

Acute renal failure was induced in male rats by the subcutaneous injectioon of 4 mg HgC12 per kg body weight. Enzyme activities of the proximal tubule were studied histochemically at six time intervals from 15 min to 24 h. The enzyme studied were alkaline phosphatase, 5'-nucleotidase, acid phosphatase, alpha-glycerophosphate dehydrogenase (NAD-independent), malic dehydrogenase, succinic dehydrogenase, latic dehydrogenase, glucose-6-phosphate dehydrogenase and glucose-6-phosphatase. Decreases in activity were observed for alkaline phosphatase and 5'-nucleotidase after 15 min. Acid phosphatase was decreased after 30 min. These three enzymes returned to control levels after 3 h, but malic dehydrogenase and alpha-glycerophosphate dehydrogenase were decreased at this time interval. Succinic dehydrogenase was first decreased after 6 h. The earliest morphological changes detectable by light microscopy were observed in pars recta tubules in the medullary rays after 6 h, a time when all enzymes studied showed widespread decreased activity throughout the proximal tubule. After 24 h, the pars convoluta appeared morphologically normal but the pars recta was necrotic and exhibited calcification, whereas enzyme activity was decreased (absent in some cases) in both pars convoluta and pars recta. These results support the hypothesis that Hg++, when given in a sublethal dose, is associated with early histochemical changes in the brush border of the proximal tubule, which may be related to early changes in sodium reabsorption and to the subsequent development of acute renal failure. The observation that changes in plasma membrane-associated enzymes occur early and prior to alterations in enzymes of mitochondria and the endoplasmic reticulum suggests that Hg++ interacts initially with the plasma membrane.     

Protective effect of sodium molybdate on the acute toxicity of mercuric chloride. V. Enhancement of renal regeneration after exposure to HgCl2

Pretreatment with Na2MoO4 protected rats from HgCl2-induced decreases in the renal concentration of amino acids, RNA, DNA, ATP and dry matter. It also reduced the mercury-induced increases in renal water, Ca and serum creatinine. Ma2MoO4 considerably elevated the RNA/DNA ratio in the renal cortex after treatment with HgCl2. In addition, subcellular distribution of mercury was markedly altered by pretreatment with Na2MoO4, specifically Na2MoO4 pretreatment decreased the mercury content in the particulate fractions such as the nuclei and mitochondria while increasing the mercury content of the cytosol. Sephadex G-75 gel filtration showed that the increase in mercury content in the cytosol of Na2MoO4-pretreated rats is due to an increase in the metal content of a metallothionein-like fraction. These results suggest that Na2MoO4-pretreatment protects against HgCl2 renal toxicity by stimulating mercury-mediated metallothionein induction in the renal cortex and renal regenerative processes.     

Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 ± 0.1, 0.6 mM NaHCO₃, had an electrophoretic mobility of - 2.88 ± 0.01 µ/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 ± 0.02, of rat liver mitoplasts was - 1.22 ± 0.07, and of rat cerebral cortex mitoplasts - 0.91 ± 0.04 µ/sec per v per cm. Treatment of these particles with 50 µg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in µ/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 µg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pK_a ∼ 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.     

The effects of ammonium chloride and bicarbonate on the activity of glutaminase in isolated liver mitochondria.

1. Glutamine hydrolysis in liver mitochondria was studied by measuring the production of glutamate under conditions where this compound could not be further metabolized. 2. Glutaminase activity in intact mitochondria was very low in the absence of activators. 3. Glutamine hydrolysis was markedly stimulated by NH4Cl and also by HCO3- ions. 4. The stimulation by each of these compounds was much decreased if the mitochondria were uncoupled. 5. Maximum rates of glutamine hydrolysis required the addition of phosphate. A correlation was observed between the activity of glutaminase in the presence of NH4Cl plus HCO3- and the intramitochondrial content of ATP. 6. In disrupted mitochondria, NH4Cl stimulated glutaminase to a much smaller extent than in intact mitochondria. The NH4Cl stimulation in disrupted mitochondria was much increased by the addition of ATP. KHCO3 also stimulated glutaminase activity in disrupted mitochondria, and ATP increased the magnitude of this stimulation. 7. It was concluded that maximum rates of glutaminase activity in liver mitochondria require the presence of phosphate, ATP and either HCO3- or NH4+. A comparison of the results obtained on intact and broken mitochondria indicates that these effectors have a direct effect on the glutaminase enzyme system rather than an indirect effect mediated by changes in transmembrane ion gradients or in the concentrations of intramitochondrial metabolites.     

Modifications of oxidative phosphorylations in mitochondria isolated from a mutant of Saccharomyces cerevisiae. Possible alterations of the phosphate transport

Mutants of Saccharomyces cerevisiae were isolated which supported two unlinked nuclear mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial one conferring paromomycin sensitivity. Mitochondria isolated from such a mutant exhibited modifications of several phosphate-requiring functions: (a) kinetic parameters of the phosphate dependence of ATP synthesis were modified; (b) in the absence of phosphate the inner mitochondrial membrane exhibited a high proton leakage; (c) mutant mitochondria always exhibited a poor respiratory control and required tenfold more phosphate to reach a maximal state 3 of respiration; (d) phosphate transport, as measured by swelling experiments, was mersalyl-insensitive and, consequently, state 3 of the respiration and ATP synthesis remained less mersalyl-sensitive than in wild-type mitochondria. Analysis of the mitochondrial metabolism of diploid and segregant strains indicates that these modifications are related to the cryosensitive phenotype; however, at present, a cooperative effect of the mitochondrial mutation cannot be eliminated. It is proposed that the phosphate carrier itself or a regulatory element was modified.