The Na+/H+ antiport in renal mitochondria.

In isolated renal mitochondria, Na+ and Li+ stimulated H+ efflux from the mitochondrial matrix. In submitochondrial particles (SMP), Na+ flux was also coupled to H+ transport in the opposite direction. The overshoot of Na+ uptake in SMP with an outwardly directed H+ gradient indicated that downhill efflux of H+ through the mitochondrial membrane induced uphill transport of Na+. Similar to the Na+/H+ antiport in other types of mitochondria, the antiport in renal mitochondria was more sensitive to amiloride derivatives than to amiloride itself. Benzamil and ethylisopropylamiloride (EIPA), but not amiloride, inhibited the antiport, with 50% inhibition of 10(-4) M for both benzamil in mitochondria and EIPA in SMP. The Na+/H+ antiport in renal mitochondria had simple saturation kinetics for external Na+ [Michaelis-Menten constant (Km) = 3.27 +/- 0.63 mM; maximal velocity (Vmax) = 0.022 +/- 0.002 pH units/s] and Li+ (Km = 3.62 +/- 0.75 mM; Vmax = 0.022 +/- 0.002 pH units/s). NH4Cl and NH4 acetate stimulated Na+ efflux and inhibited Na+ uptake in SMP. Comparable results with NH4 acetate and chloride suggested that NH4+ modified Na+ transport through its direct interaction with the Na+/H+ antiport, rather than through the alkalinization of intra-SMP space from non-ionic diffusion of NH3. These results suggested that the Na+/H+ antiport may be a factor in the exit of NH4+ from renal mitochondria.

Effect of acute pH change on mitochondrial glutamine transport.

The uptake of [3H]glutamine in submitochondrial particles (SMP) was measured at varying medium pH. Glutamine transport, but not glutamine binding, was inversely related to medium pH (range 6.5-8.5). Glutamine uptake was highest at medium pH of 6.5 (4.59 +/- 0.4 pmol.mg-1.30 s-1) and lowest at medium pH of 8.0 (1.99 +/- 0.3 pmol.mg-1.30 s-1). The effect of medium pH on glutamine transport was rapidly reversible. Changes in pH gradient (delta pH) had no influence on the rate of glutamine transport. Kinetics of mitochondrial glutamine transport was studied at pH 6.5 and 8.5 to further elucidate the mechanism by which pH alters glutamine transport. Glutamine concentration at half-maximal velocity ([S]0.5) was 9.07 and 13.32 mM at pH 6.5 and 8.5, respectively (P less than 0.02). The maximal velocity (Vmax) was 1,417.72 +/- 185.69 pmol.mg-1.15 s-1 at pH 6.5 and 910.95 +/- 192.85 pmol.mg-1.15 s-1 at pH 8.5 (P less than 0.01). Thus both the affinity and the Vmax of the transport system were enhanced in acidic pH. These data imply that intracellular pH has influence over glutamine transport into the mitochondrial matrix and, consequently, renal NH3 production. Glutamine delivery to the matrix via changes in cytosolic pH may be one of the regulators for ammoniagenesis during acute acid-base disorders.

Bioavailability of clindamycin during peritoneal dialysis.

Clindamycin phosphate becomes biologically active only with cleavage of the phosphate ester bond. A rat model was used to examine the amount of biologically active clindamycin attainable in the dialysate and in the blood during peritoneal dialysis. Intravenous administration of 10 mg/kg clindamycin phosphate alone without peritoneal dialysis produced peak blood levels of 15-20 micrograms/ml. With peritoneal dialysis, blood levels of less than 5 micrograms/ml were achieved. When clindamycin phosphate was added to the dialysis fluid at an initial concentration of 10 mg/ml, less than 5 micrograms/ml of the active antibiotic can be detected in the dialysis return fluids. Even in rats with induced peritonitis, less than 15 micrograms/ml could be found in the dialysis returns. With or without peritonitis, less than 5 micrograms/ml of active clindamycin was attained in blood from peritoneal installation alone. The conversion of the ester to the active compound appears to be the major problem. It is recommended that in those clinical situations in which the patient requires peritoneal dialysis, an alternate antimicrobial agent be used in place of clindamycin to avoid infections in the abdominal cavity or the blood while under therapy.

Glutamine transport in submitochondrial particles.

Glutamine transport was studied in submitochondrial particles (SMP) to avoid interference from glutamine metabolism. Phosphate-dependent glutaminase activity in SMP was only 0.04% of that in intact mitochondria. The uptake of glutamine in SMP represented both the transport into vesicles and membrane binding (about one-third of total uptake). Sulfhydryl reagents inhibited glutamine uptake in SMP. The uptake of L-[3H]glutamine increased more than twofold in SMP preloaded with 1 mM L-glutamine, an effect that was not seen with 1 mM D-glutamine. The uptake of L-[3H]glutamine was inhibited in the presence of either L-glutamine or L-alanine in the incubation medium. Other amino acids did not inhibit glutamine uptake. Alanine was also shown to trans-stimulate glutamine transport in SMP and cis-inhibit glutamine transport in both SMP and intact mitochondria. Glutamine transport showed a positive cooperativity effect with a Hill coefficient of 1.45. Metabolic acidosis increased the affinity of the transporter for glutamine without any change in other kinetic parameters. These data indicated that mitochondrial glutamine transport occurs via a specific carrier with multiple binding sites and that the transport of glutamine into mitochondria has an important role in increased ammoniagenesis during metabolic acidosis.

Light chain effects on alanine and glucose uptake by renal brush border membranes.

Effect of varying concentrations (0 to 800 microM) of three different light chains on sodium-dependent L-(14C)alanine and D-(14C)glucose uptake by rat renal brush border membrane vesicles were studied. One kappa and two lambda type light chains (lambda-1 and lambda-2) were isolated from urines of patients with multiple myeloma. At maximal inhibitory concentrations the kappa chain reduced alanine uptake from 206 +/- 18 to 77 +/- 18 pmole/mg protein (P less than 0.005) and glucose uptake from 357 +/- 22 to 146 +/- 8 pmole/mg protein (P less than 0.001). lambda-1 reduced alanine uptake from 136 +/- 17 to 60 +/- 8 pmole/mg protein (P less than 0.005) and glucose uptake from 354 +/- 17 to 77 +/- 14 pmole/mg protein (P less than 0.001). lambda-2 reduced alanine uptake from 105 +/- 9 to 28 +/- 5 pmole/mg protein (P less than 0.001) and glucose uptake from 194 +/- 7 to 66 +/- 7 pmole/mg protein (P less than 0.001). The half maximal inhibitory concentrations (I50) of kappa, lambda-1 and lambda-2 light chains were 68, 76 and 140 microM for alanine uptake and 120, 70 and 105 microM for glucose uptake. Control experiments using bovine serum albumin and beta-lactoglobulin showed no inhibitory effect on alanine and glucose uptake by either protein. These data reveal brush border membrane effects by myeloma light chains and confirm that direct Bence Jones protein nephrotoxicity may play an important role in the pathogenesis of kidney dysfunction associated with multiple myeloma.

Effect of acivicin on glutamine transport by rat renal brush border membrane vesicles.

Acivicin (L-[alpha S,5S]-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid) reversibly and competitively inhibits glutamine transport in rat renal brush border membrane (BBM) vesicles. Acivicin alters the affinity of the low-Km high-affinity glutamine transport system, but has minimal effect on the high-Km low-affinity system. The drug interferes with glutamine transport by a mechanism that does not involve the maintenance of a Na+-chemical gradient as shown by its uniform effect on glutamine uptake rate, regardless of the NaCl gradient imposed (0 to 150 mmol/L). The effect of acivicin on membrane potential was ruled out by the finding that acivicin modified glutamine transport even when the membrane potential was minimized either by substituting Cl- with an impermeant anion, isethionate, or by short-circuiting the membrane potential with carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. In these experiments the activity of the enzyme gamma-glutamyltranspeptidase (gamma-GT), a component of the putative amino acid transport system, was not suppressed because the BBM was not preincubated with acivicin. Acivicin also interferes with the uptake of other solutes tested (alanine, proline, glutamate, and glucose). But with the exception of L-alanine, the transport of these solutes is less sensitive to the inhibitory effect of acivicin than is that of glutamine. Our results indicate that acivicin profoundly affects the metabolism of renal tubular cells by its influence on the metabolite transport systems. Acivicin can reduce renal NH3 production independently of its effect on gamma-GT by interfering directly with renal cellular uptake of glutamine.

Renal mitochondrial glutamine metabolism during K+ depletion.

We studied changes in renal mitochondrial glutamine metabolism during the development of and recovery from K+ depletion in rats. Significant increase in mitochondrial NH3 production was noted after 3 days of K+-free diet. Ammoniagenesis in K+-depleted animals reached maximal level within 2 wk of K+ deprivation when there was 64% increase in NH3 production. In contrast to the pattern of changes in mitochondrial ammoniagenesis, phosphate-dependent glutaminase (PDG) activity increased within the first 48 h of K+ deprivation, before there was any increase in NH3 production, and did not plateau even after 2 wk of K+-free diet. The disparity between NH3 production and PDG activity cannot be explained by the difference in matrix glutamate level, thus raising the possibility that mitochondrial glutamine entry may be a rate-limiting step for ammoniagenesis during K+ depletion. Recovery from K+ depletion was studied in animals prefed with K+-free diet for 2 wk prior to the initiation of K+-supplemented diet. Muscle K+ content of K+-depleted animals returned to normal after 1 wk of K+ replacement. Mitochondrial NH3 production decreased concomitantly with the attenuation in K+ deficit but did not reach the base-line value even after K+ deficit was completely corrected. Additional experiments with isolated cortical tubules also showed persistent increase in NH3 production after the correction of K+ deficit. Thus, unlike earlier studies in rats during the recovery from metabolic acidosis, which showed only increased ammoniagenesis in isolated mitochondria but not in cortical slices, animals recovered from K+ depletion demonstrated augmented NH3 production both in isolated mitochondria and intact renal tissues.

Effect of potassium on renal NH3 production.

The influence on renal ammoniagenesis of a high potassium diet and also of acute manipulation of ambient potassium concentration was investigated using both the isolated perfused rat kidney and incubated renal cortical tubules. Ingestion of a high potassium diet for 1 wk resulted in potassium adaptation but had no effect on ammonia production by the isolated kidney perfused with physiologic concentrations of glutamine. By contrast, perfusion with a high ambient potassium concentration (8.0-9.3 mM) significantly increased renal tissue potassium levels and concomitantly reduced the rate of ammonia formation by 30% in comparison with perfusions at a normal potassium concentration. NH3 production by tubules incubated with 1 mM glutamine was also decreased at a K+ concentration of 9.0 mM. Ammonia production was unchanged when kidneys were perfused with a potassium concentration of 2.0 mM despite a 16% decrease in renal tissue potassium levels, but ammonia production by tubules incubated in 2 mM K+ was slightly less than in control incubations at 5.0 mM. Thus, unlike earlier in vitro studies with outer medullary slices, these studies do not support the hypothesis that an adaptive change in ammoniagenesis results from a high dietary potassium intake. However, a high ambient and renal intracellular potassium concentration can depress ammonia production. Although potassium depletion causes an adaptive increase in ammonia production, a decrease in ambient potassium concentration does not increase ammoniagenesis. Accordingly, both a potassium surfeit and deficit can modify renal ammonia production, but the mechanisms involved appear to differ.

Mechanism by which enhanced ammonia production reduces urinary potassium excretion.

To determine the mechanism whereby an increase in ammonia production decreases urinary potassium excretion, we perfused isolated rat kidneys at a pH of either 7.0 or 7.4. After 45 min of perfusion at either pH, glutamine or ammonium chloride was added to the perfusate to result in concentration of 5 and 0.8 mM, respectively and observations were continued for 50 min. Control kidneys were perfused at both pH's without further additions to the perfusate. At pH 7.0 glutamine increased ammonia production and increased urinary ammonium excretion strikingly; whereas the addition of ammonium chloride did not change ammonia production but increased urinary ammonium excretion to a comparably degree. Both maneuvers resulted in a reciprocal fall in urinary potassium excretion in comparison with control perfusions. The decreases in potassium excretion could not be accounted for by differences in perfusate or urinary acid-base parameters, or by changes in urinary sodium, water, or chloride excretion. At pH 7.4, glutamine also significantly increased ammonia production and perfusate ammonia concentration. In contrast to the studied at pH 7.0 in which the urine pH was acid (5.9), the urine remained alkaline (pH 7.2), and both urinary ammonium excretion and urinary potassium excretion were unaltered. Thus, potassium sparing is not a nonspecific effect of glutamine, its metabolism to ammonia, or perfusate ammonia concentration but is directly related to an increase in urinary ammonium excretion.