Renal gluconeogenesis and ammoniagenesis studied in tissue culture in vitro. Investigation of putative signaling pathways in the regulation and acid-base response of renal gene expression

A major task in the regulatory function of the kidney is its ability to excrete excess acid, mainly in form of ammonium salts, thereby maintaining acid-base homeostasis. The coordinate adaptive modulation of gluconeogenesis and ammoniagenesis by the acid-base status of mammalian organisms allows proximal tubular epithelial cells to increase the extraction and metabolism of plasma glutamine in metabolic acidosis, resulting in enhanced excretion of acid equivalents. In renal proximal tubule cells gluconeogenesis and ammoniagenesis are tightly coupled, especially under enhanced metabolic rates, which occur in acidosis. Renal glutamine metabolism leads to the generation of NH 4 + and alpha- ketoglutarate. for net acid secretion, the two-fold negatively charged alpha-ketoglutarate must be neutralized either by complete oxidation, or by conversion to glucose. The biochemical basis for this adaptive change in proximal tubular cell metabolism is an increase in the catalytic activity of the key enzymes phosphoenolpyruvate carboxykinase (PEPCK) and mitochondrial phosphate-dependent glutaminase (PDG). Both the increase in renal PDG and PEPCK activity under acidotic conditions are mediated by an increase in renal PDG and PEPCK activity under acidotic conditions are mediated by an increase in the relative abundance of the respective messenger RNAs. Whereas the increased levels of PEPCK mRNA result from increased rates of transcription, the increased PDG mRNA during metabolic acidosis occur through increased stability of the PDG mRNA. However, the signal transduction pathways which initiates and mediates this acid- induced response leading to increased gene expression and which accounts for its cell specificity in unknown. During the past decade, renal epithelial cell and tissue cultures have emerged as a powerful tool to study in vitro several aspects of renal metabolism, epithelial transport, and renal cell growth and differentiation. Modern cell and tissue culture techniques enable renal epithelial cells to grow and be maintained at a state of differentiation, comparable with the in vivo tissue. The use of continuos renal epithelial cell lines in studies of renal metabolism has already been described extensively. In addition, a vast amount of literature has accumulated on the use of renal proximal tubular primary cultures from a variety of mammalian species including human. However, the previous absence has greatly limited the approaches which could be used to characterize in tissue culture the mechanism(s) by which acidosis causes a cell-specific induction of renal gene expression. In this context, we recently succeeded in isolating a gluconeogenic strain from a continuous renal cell line. This isolated cell strain was shown to express high levels of PEPCK and PDG mRNA, which are regulated in these cells by culture medium pH. The observation that the renal adaptation in PEPCK and PDG expression can be reproduced in these cultured cells transferred to acidic medium strongly indicates that this response is not mediated by a circulating humor factor. Instead, the renal cells must possess the full biochemical machinery to directly sense changes in extracellular and/or intracellular pH and to transduce this information to initiate separate mechanisms that lead to the coordinate induction of PDG and PEPCK enzyme activities. There are certain candidates of signaling cascades which transduce extracellular signals into the cell nucleus resulting in a specific cell response by altering the expression of specific enzymes. The primary objective of the present grant proposal is to elaborate, and possibly to identify, putative signaling pathways in the transduction of the adaptive response of cultured proximal tubular epithelial cells to changes in extracellular pH, thereby triggering the acid-induction of PEPCK gene transcription and the stabilization of PDG mRNA, respectively.