Isolation, growth, and characterization of a gluconeogenic strain of renal cells

Abstract

LLC-PK1 cells, derived from pig kidney, retain several properties of the proximal tubule, but are incapable of gluconeogenesis, due to the lack of fructose-1,6-bisphosphatase (FBPase) [Am. J. Physiol. 248 (Cell Physiol. 17): C181-185, 1985]. Cells incapable of gluconeogenesis require a hexose, pentose, or nucleoside to provide ribose-5-phosphate for RNA biosynthesis. To induce or select cells that express FBPase activity, we cultured LLC-PK1 cells in glucose-free medium. We obtained cells (designated LLC-PK1-FBPase+) that express FBPase activity and are capable of growing in the complete absence of sugars or nucleosides. The cells have apical membrane enzyme activities that differ from those of wildtype cells. Tests of metabolic flow through the gluconeogenic pathway, using 3-mercaptopicolinic acid, a specific inhibitor of phosphoenolpyruvate carboxykinase, confirmed that the cells are gluconeogenic. LLC-PK1-FBPase+ cells grown in medium containing 5 mM glucose for five weekly passages continued to express FBPase activity and apical membrane enzyme activities characteristic of the FBPase+ strain. When switched back to glucose-free medium, they proliferated well. The strain appears to be stable. It should provide a model for studying the relationship between gluconeogenesis and other proximal tubule functions. An incidental finding is that in both strains, the activity of lactate dehydrogenase varied directly with the concentration of glucose in the growth medium, indicating that the expression of lactate dehydrogenase may be regulated by glucose or a metabolite of glucose.