Ammoniagenesis in Renal Cell Culture

• Published in: Kidney & blood pressure research Vol. 16; no. 4; pp. 203 - 211
• Main Authors: Gstraunthaler, Gerhard, Landauer, Franz, Pfaller, Walter
• Format: Journal Article
• Language: English
• Published: Basel, Switzerland 11.11.2008
• Subjects: Original Paper; Phosphate-independent glutaminase; Acivicin (AT-125); γ-Glutamyl transpeptidase; Apical (extracellular) ammoniagenesis; Hippurate
• ISSN: 1420-4096; 1423-0143
• DOI: 10.1159/000173765

In renal ammoniagenesis, two major pathways of glutamine metabolism have been described: (i) intracellular metabolism by phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase and (ii) extracellular metabolism by phosphate-independent glutaminase. The latter has been identified as the hydrolytic activity of the apically membrane-bound γ-glutamyl transpeptidase (γ-GT). The growth properties of cultured renal epithelia enable the study of in vitro extracellular metabolic properties occurring at the apical epithelial surface in the culture dish. Therefore, confluent epithelia of the LLC-PK 1 renal epithelial cell line were used to elucidate the role of extracellular (apical) hydrolysis of glutamine by γ-GT in LLC-PK 1 ammonia production. To distinguish between intra- and extracellular metabolism of glutamine, confluent LLC-PK 1 epithelia were incubated with either D-glutamine as substrate, which cannot be metabolized intracellularly by PDG, or with L-glutamine and hippurate to stimulate, and AT-125 (acivicin) to inhibit γ-GT activity, respectively. In addition, cellular uptake of the glutamate, extracellularly formed by γ-GT, was inhibited by D-aspartate. D-Glutamine (2 mM) did not increase ammonia formation above endogenous production levels, indicating the negligible role of extracellular hydrolysis of glutamine by γ-GT. After modulating γ-GT activity by hippurate or AT-125, almost identical ammonia production rates were found within the various experimental protocols, further confirming that extracellular metabolism of glutamine does not significantly contribute to LLC-PK 1 ammoniagenesis.