Impact of culture conditions, culture media volumes, and glucose content on metabolic properties of renal epithelial cell cultures - Are renal cells in tissue culture hypoxic?

When renal proximal tubular cells are brought into tissue culture, they rev ert from oxidative metabolism and gluconeogenesis to high rates of glycolys is. Among the factors possibly responsible for this metabolic conversion, l imited oxygen availability and/or substrate supply are discussed. In order to study the role of these factors on long-term cultures, the impact of gro wth conditions, culture media volume, and glucose content on carbohydrate m etabolism of the continuous renal cell lines LLC-PK1 (porcine kidney) and O K (opossum kidney) was investigated. The impact of culture media volumes an d glucose content, respectively, was determined by overlaying confluent mon olayer cultures of LLC-PK1 and OK cells (i) with increasing volumes of cult ure medium and thus increasing amounts of glucose, and (ii) with increasing culture medium volumes at constant absolute amounts of glucose by adding g lucose-free medium, in order to increase volume at a constant glucose suppl y. Alternatively, and in order to improve cell oxygenation, LLC-PK1 cells w ere also cultured in roller bottles. Cell carbohydrate metabolism was asses sed by measuring rates of glucose consumption and lactate production, respe ctively, and by determination of specific activities of the key glycolytic enzymes hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), a nd lactate dehydrogenase (LDH). Mitochondrial phosphate-dependent glutamina se (PDG) was assayed as marker enzyme of oxidative metabolism of glutamine. In LLC-PK1 and OK cells, rates of glucose consumption were independent of the initial glucose concentrations and/or the culture media volumes used. G lucose was quantitatively converted to lactate, which accumulated in a 1:2 molar ratio. Lactate in culture media reached a maximum content after 24h, and was reutilized by the cell lines thereafter. Interestingly, the rates o f lactate reuptake strictly depended on culture medium volume, indicating a volume-induced stimulation of oxidative lactate metabolism. Marked changes were found for the specific activities of glycolytic enzymes. In LLC-PK1 c ells, increased glucose supply caused increases in HK, PFK, PK and LDH acti vities, which were superimposed to the stimulatory effects of increased med ia volumes. Enzyme activity showed a biphasic response, indicating that bot h glucose supply and culture media volumes covering the cell monolayer are factors determining glycolytic rates of LLCPK1 renal cells. Conversely, in OK cells glycolytic enzyme activities decreased with increasing culture med ia volumes at constant glucose levels. As expected, under conditions of enh anced oxygenation of LLC-PK1 cells in roller bottle culture, glycolytic enz yme activities decreased, whereas PDG activity increased, which was paralle led by increased rates of ammonia generation. Thus, changes in nutrient sup ply and oxygenation of renal epithelial cell cultures by altered culture me dia volumes dramatically influence metabolic rates and levels of enzyme act ivities, respectively.