Chronic hyperdynamic sepsis in the rat. II. Characterization of liver and muscle energy metabolism.

Sepsis was induced in male rats by injections of live Escherichia coli No. 4 (or E. coli No. 3) and Bacteroides fragilis organisms into a preformed subcutaneous abscess. Body weight, food and water intake, and cardiac output were measured daily. After 1, 2, or 3 weeks, animals were sacrificed, and blood, liver, and muscle were collected for measurements of plasma glucose and carnitine, mitochondrial respiratory activity, mitochondrial cytochrome concentrations, and tissue adenine nucleotides. Compared with sham controls, no significant differences were found in state 3 respiratory activities of liver mitochondria isolated from rats with moderate (no weight loss, cardiac output increased to 150% of control) or severe (0.5% weight loss/day, cardiac output increased to 200% of control) sepsis at any time. After 1 week of severe, but not moderate, sepsis, pyruvate-supported respiration in muscle mitochondria was significantly decreased, while branched-chain ketoacid and beta-hydroxybutyrate-supported respiration remained unchanged. After 2 weeks of severe, but not moderate, sepsis, beta-hydroxybutyrate and branched-chain ketoacid oxidation increased severalfold; pyruvate utilization remained depressed. Severe or moderate sepsis did not uncouple mitochondrial respiration at any time. Total muscle carnitine concentration was significantly decreased after long-term but not short-term severe sepsis. Severe short-term sepsis caused a significant increase in liver short-chain acyl and total carnitines. Muscle energy charge was unaltered by either moderate or severe sepsis. These results represent the first demonstration of sepsis-induced fuel shifts at the mitochondrial level in muscle: Severe hyperdynamic sepsis is characterized by the reduced ability of muscle mitochondria to utilize pyruvate with a simultaneous increase in branched-chain ketoacid and ketone body utilization. These changes were not observed in liver mitochondria.

Effect of peroxide, sodium, and calcium on brain mitochondrial respiration in vitro: potential role in cerebral ischemia and reperfusion.

Mitochondrial pyruvate-supported respiration was studied in vitro under conditions known to exist following ischemia, i.e., elevated extramitochondrial Ca2+, Na+, and peroxide. Ca2+ alone (7-10 nmol/mg) decreased state 3 and increased state 4 respiration to 81 and 141% of control values, respectively. Sodium (15 mM) and/or tert-butyl hydroperoxide (tBOOH; up to 2,000 nmol/mg protein) alone had no effect on respiration; however, Na+ or tBOOH in combination with Ca2+ dramatically altered respiration. Respiratory inhibition induced by Ca2+ and tBOOH does not involve pyruvate dehydrogenase (PDH) inhibition since PDH flux increased linearly with tBOOH concentration (R = 0.96). Calcium potentiated tBOOH-induced mitochondrial NAD(P)H oxidation and shifted the redox state of cytochrome b from 67 to 47% reduced. Calcium (5.5 nmol/mg) plus Na+ (15 mM) decreased state 3 and increased state 4 respiratory rates to 55 and 202% of control values, respectively. Sodium- as well as tBOOH-induced state 3 inhibition required mitochondrial Ca2+ uptake because ruthenium red addition before Ca2+ addition negated the effect. The increase in state 4 respiration involved Ca2+ cycling since ruthenium red immediately returned state 4 rates back to control values. The mechanisms for the observed Ca2(+)-, Na(+)-, and tBOOH-induced alterations in pyruvate-supported respiration in vitro are discussed and a multifactorial etiology for mitochondrial respiratory dysfunction following cerebral ischemia in vivo is proposed.

Renal cortical mitochondrial integrity in experimental cyclosporine nephrotoxicity.

The function of renal cortical mitochondria isolated from rats with cyclosporine nephrotoxicity was studied. Renal cortical mitochondria were isolated from 5 male Fischer rats after 14 days of daily intraperitoneal administration of CsA, 25 mg/kg body wt. Compared with the mitochondrial function of 5 pair-fed control rats receiving vehicle alone, state 3 respiration (ADP-dependent) using several substrates was mildly depressed only with pyruvate-malate supported respiration (27 +/- 3 vs. 36 +/- 2 nmol O2/min/mg protein; P less than 0.05). The Ca2+ accumulation rate was slightly reduced (354 +/- 14 vs. 416 +/- 18 nmol/min/mg protein; P less than 0.025) while the cytochrome enzyme concentrations were not different from controls. Respiratory control ratios were not affected (CsA group: 9.5 +/- 2.8, control group: 8.9 +/- 2.3; glutamate-malate as substrates). These minor alterations in mitochondrial function occurred in the presence of severe depression in the glomerular filtration rate and renal morphologic changes commonly seen with CsA administration. Moreover, there was no increase in enzymuria. These results indicate that CsA has minor effects on the respiratory function of renal cortical mitochondria. The severe depression in the glomerular filtration rate is out of proportion to these minor alterations in mitochondrial function. These findings argue against a prominent role for a direct toxic action of CsA on tubular cells in the pathogenesis of acute cyclosporine-induced renal dysfunction.

Renal mitochondrial integrity during continuous gentamicin treatment.

Rats were given gentamicin over a period of 21 days. At 5, 10, 14 and 21 days renal cortical mitochondria were isolated, and respiratory and Ca2+ transport functions and cytochrome concentrations were determined. The mitochondrial data were correlated with indicators of deteriorating renal function and tissue gentamicin accumulation. During the first 10 days of chronic gentamicin treatment, mitochondrial cytochrome oxidase and cytochrome c concentrations declined significantly. This decline was followed by a partial spontaneous recovery by days 14 and 21. Cytochrome b concentration was not significantly different from normal. Parallel with the cytochrome concentration changes, State 3 respiratory activities with all substrates studied and the rates of Ca2+ accumulation declined during the first 10 days and recovered spontaneously thereafter. It is concluded that chronic gentamicin treatment leading to renal failure inhibits mitochondrial energy-linked functions, which inhibition is induced by rate-limiting synthesis of those mitochondrial respiratory chain enzymes coded outside the mitochondrion.

Verapamil pretreatment preserves mitochondrial function and tissue magnesium in the ischemic kidney.

These studies were designed to test the efficacy and possible mechanisms of the prevention of mitochondrial functional deterioration in renal ischemia by the slow-channel calcium blocker verapamil. Renal ischemia was induced in guinea pigs by a unilateral ligation of the renal artery for 30 or 60 min. In the pretreated animals verapamil was given twice a day over a 5-d period prior to the induction of ischemia. Sham-operated animals were used as normal controls. After 30 and 60 minutes, the kidneys were removed and used for mitochondrial isolation and analyses, total tissue Ca2+ and Mg2+ determinations, or for electron microscopy. Verapamil pretreatment completely blocked the decrease of mitochondrial Ca2+ uptake rate induced by 30 or 60 min of ischemia. The pretreatment delayed by 30 min the ischemic decrease of state 3 respiratory activity. Total tissue Ca2+ concentration was not altered by ischemia or verapamil pretreatment. Total tissue Mg2+ concentration, however, was significantly reduced in the ischemic kidney at 60 min. This reduction was prevented completely by verapamil pretreatment. These data suggest that the mitochondrial functional deterioration induced by 30 min of ischemia is a primary cellular insult secondarily leading to loss of tissue Mg2+. The point of irreversibility in the ischemic cell injury might be initiated by lowered tissue Mg2+/Ca2+ ratios.