glutaminase and Potassium-Deficiency

Hypokalemia is associated with increased ammoniagenesis and stimulation of net acid excretion by the kidney in both humans and experimental animals. The molecular mechanisms underlying these effects remain unknown. Toward this end, rats were placed in metabolic cages and fed a control or K(+)-deficient diet (KD) for up to 6 days. Rats subjected to KD showed normal acid-base status and serum electrolytes composition. Interestingly, urinary NH(4)(+) excretion increased significantly and correlated with a parallel decrease in urine K(+) excretion in KD vs. control animals. Molecular studies showed a specific upregulation of the glutamine transporter SN1, which correlated with the upregulation of glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase. These effects occurred as early as day 2 of KD. Rats subjected to a combined KD and 280 mM NH(4)Cl loading (to induce metabolic acidosis) for 2 days showed an additive increase in NH(4)(+) excretion along with an additive increment in the expression levels of ammoniagenic enzymes GA and GDH compared with KD or NH(4)Cl loading alone. The incubation of cultured proximal tubule cells NRK 52E or LLC-PK(1) in low-K(+) medium did not affect NH(4)(+) production and did not alter the expression of SN1, GA, or GDH in NRK cells. These results demonstrate that K(+) deprivation stimulates ammoniagenesis through a coordinated upregulation of glutamine transporter SN1 and ammoniagenesis enzymes. This effect is developed before the onset of hypokalemia. The signaling pathway mediating these events is likely independent of KD-induced intracellular acidosis. Finally, the correlation between increased NH(4)(+) production and decreased K(+) excretion indicate that NH(4)(+) synthesis and transport likely play an important role in renal K(+) conservation during hypokalemia.

Topics: Acids; Amino Acid Transport Systems, Basic; Ammonia; Ammonium Chloride; Animals; Blotting, Northern; Chlorides; Eating; Glutamate Dehydrogenase; Glutaminase; Glutathione Peroxidase; Kidney; Kidney Tubules; LLC-PK1 Cells; Male; Membranes; Potassium; Potassium Deficiency; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; RNA; Signal Transduction; Swine

Kidneys produce ammonium to buffer and excrete acids through metabolism of glutamine. Expression of the glutamine transporter Slc38a3 (SNAT3) increases in kidney during metabolic acidosis (MA), suggesting a role during ammoniagenesis. Potassium depletion and high dietary protein intake are known to elevate renal ammonium excretion. In this study, we examined SNAT3, phosphate-dependent glutaminase (PDG), and phosphoenolpyruvate carboxykinase (PEPCK) regulation during a control (0.36%) or low-K(+) (0.02%) diet for 7 or 14 days or a control (20%) or high-protein (50%) diet for 7 days. MA was induced in control and low-K(+) groups by addition of NH(4)Cl. Urinary ammonium excretion increased during MA, after 14-day K(+) restriction alone, and during high protein intake. SNAT3, PDG, and PEPCK mRNA abundance were elevated during MA and after 14-day K(+) restriction but not during high protein intake. SNAT3 protein abundance was enhanced during MA (both control and low K(+)), after 14-day low-K(+) treatment alone, and during high protein intake. Seven-day dietary K(+) depletion alone had no effect. Immunohistochemistry showed SNAT3 staining in earlier parts of the proximal tubule during 14-day K(+) restriction with and without NH(4)Cl treatment and during high protein intake. In summary, SNAT3, PDG, and PEPCK mRNA expression were congruent with urinary ammonium excretion during MA. Chronic dietary K(+) restriction, high protein intake, and MA enhance ammoniagenesis, an effect that may involve enhanced SNAT3 mRNA and protein expression. Our data suggest that SNAT3 plays an important role as the glutamine uptake mechanism in ammoniagenesis under these conditions.

Topics: Acidosis; Amino Acid Transport Systems, Neutral; Ammonium Chloride; Animals; Caseins; Disease Models, Animal; Glutaminase; Kidney; Kidney Tubules, Proximal; Male; Mice; Phosphoenolpyruvate Carboxykinase (GTP); Potassium Deficiency; Potassium, Dietary; Quaternary Ammonium Compounds; RNA, Messenger; Time Factors; Up-Regulation

Ammonia production and excretion are elevated in potassium depletion alkalosis, although normally they are reduced in alkalosis and elevated in acidosis. Studies were conducted with or without acute acid loading in normokalemic rats or rats made chronically hypokalemic with deoxycorticosterone acetate and a potassium-deficient diet to examine the role of phosphate-dependent glutaminase (PDG) in regulating ammonia excretion. Renal cortical PDG rose fourfold, and urinary ammonia excretion (UAE) doubled in potassium depletion compared to potassium-repleted controls. Following acid challenge PDG and urinary ammonia increased four- to sevenfold in both normokalemic and hypokalemic animals, but the rise in UAE did not correspond to the increase in PDG. Thus, PDG levels in acidotic normokalemic rats were one half those seen in potassium-depleted rats, but UAE in the acidotic rats was six times greater. These results could not be explained solely by changes in blood pH. The poor correlation between PDG and UAE also could not be explained by limited substrate availability, since blood glutamine levels were unaffected by potassium depletion. The disparity between UAE and PDG in potassium depletion was studied further during 9 days of potassium repletion of depleted rats. UAE was again increased by depletion but, after only 3 days of potassium repletion, UAE fell to levels found in normokalemic rats. The renal PDG activity, however, remained three times normal. Indeed, PDG remained significantly elevated even after 9 days of potassium replacement. Other enzymes involved in renal ammoniagenesis, including delta glutamyl transferase, glutamine transferase and omega deamidase, were assayed, and alterations in their activities could not account for the changes in UAE.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acidosis; Ammonia; Animals; Citrates; Glutaminase; Kidney; Male; Phosphates; Potassium Chloride; Potassium Deficiency; Rats; Rats, Inbred Strains

Ammonia is quantitatively the major buffer for hydrogen ion in the urine. Further, the excretion of ammonia can be varied by acid base status and is therefore of homeostatic importance. Acid base status exerts its effect on ammonia excretion both directly and also via an effect on renal ammonia production from glutamine. The mechanism of the effect of acid base status on glutamine deamidation and deamination is uncertain. Apart from its homeostatic role in health and disease alterations in renal ammonia production may assume pathological importance in potassium depletion and uric acid urolithiasis.

Topics: Acid-Base Equilibrium; Acidosis; Ammonia; Animals; Dogs; Glutaminase; Glutamine; Humans; Hydrogen-Ion Concentration; Kidney; Kidney Calculi; Potassium Deficiency; Rats; Uric Acid

Topics: Ammonia; Animals; Carbon Dioxide; Chlorides; Disease Models, Animal; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Hydrogen-Ion Concentration; Kidney; Liver; Male; Muscles; Organ Size; Potassium; Potassium Deficiency; Rats; Renal Veins; Sodium

Glutamate is an inhibitor of phosphate dependent glutaminase (PDG), and renal cortical glutamate is decreased in metabolic acidosis. It has been postulated previously that the rise in renal production of ammonia from glutamine in metabolic acidosis is due primarily to activation of cortical PDG as a consequence of the fall in glutamate. The decrease in cortical glutamate has been attributed to the increase in the capacity of cortex to convert glutamate to glucose in acidosis. In the present study, administration of ammonium chloride to rats in an amount inadequate to decrease cortical glutamate increased the capacity of cortex to produce ammonia from glutamine in vitro and increased cortical PDG. Similarly, cortex from potassium-depleted rats had an increased capacity to produce ammonia and an increase in PDG, but glutamate content was normal. The glutamate content of cortical slices incubated at pH 7.1 was decreased, and that at 7.7 was increased, compared to slices incubated at 7.4, yet ammonia production was the same at all three pH levels. These observations suggest that cortical glutamate concentration is not the major determinant of ammonia production. In potassium-depleted rats there was a 90% increase in the capacity of cortex to convert glutamate to glucose, yet cortical glutamate was not decreased. In vitro, calcium more than doubled conversion of glutamate to glucose by cortical slices without affecting the glutamate content of the slices, and theophylline suppressed conversion of glutamate to glucose yet decreased glutamate content. These observations indicate that the rate of cortical gluconeogenesis is not the sole determinant of cortical glutamate concentration. The increase in cortical gluconeogenesis in acidosis and potassium depletion probably is not the primary cause of the increase in ammonia production in these states, but the rise in gluconeogenesis may contribute importantly to the maintenance of increased ammoniagenesis by accelerating removal of the products of glutamine degradation.

Topics: Acid-Base Equilibrium; Acidosis; Adenine Nucleotides; Alkalosis; Ammonia; Animals; Calcium; Culture Media; Cyclic AMP; Food Deprivation; Gluconeogenesis; Glutamates; Glutaminase; Glutamine; Kidney; Male; Methods; Nucleosides; Potassium Deficiency; Rats; Theophylline

Topics: Acidosis; Amidohydrolases; Ammonia; Ammonium Chloride; Body Fluids; Glutaminase; Kidney; Pharmacology; Potassium Deficiency; Rats; Research; Urine

Topics: Amidohydrolases; Animals; Carbonic Anhydrases; Glutaminase; Hypokalemia; Kidney; Potassium; Potassium Deficiency; Rats

Topics: Amidohydrolases; Animals; Blood; Carbonic Anhydrases; Dogs; Electrolytes; Glutaminase; Hydro-Lyases; Hypokalemia; Kidney; Muscles; Potassium; Potassium Deficiency