We have assessed the impact of increasing oxygen availability on cellular phenotype expression of rabbit proximal tubule cells in primary culture developed with variable glucose and/or insulin contents. To mitigate hypoxia at the cell/medium interface, cells were shaken for the whole culture duration and their expressed phenotype was compared with those expressed by static cultures. O2 and CO2 tensions were kept constant in the incubator atmosphere. Glycolysis and gluconeogenesis pathways, detoxication system, and mitochondrial, apical and basolateral membrane marker enzyme activities were assessed. This study showed that the induction of glycolysis which appear in primary cultures of proximal tubule cells may be partially prevented by continuously shaking the cultures. This effect was more marked in the presence of glucose, suggesting better substrate oxidation in shaken cultures.
Glucose and insulin impact on cellular phenotype expression was assessed on rabbit proximal tubule cells in primary culture. Glycolysis, gluconeogenesis pathways and lysosomal, mitochondrial, detoxication system, apical and basolateral membrane marker enzyme activities were assessed. Both insulin and glucose deprivation partially prevented the rise in glycolysis and delayed the drop of gluconeogenesis pathways. Scanning electron microscopy analysis of the apical surface of the monolayer revealed a much higher density of microvilli in glucose-free cultures compared to cultures developed with glucose. In conclusion, culture medium deprivation in both glucose and insulin allowed closer functional, biochemical and morphological properties to those which exist in the in vivo situation for proximal tubule cells.
Compared to prior studies which frequently pinpoint the impairment of one parameter or function, this paper reports for the first time an extensive characterization of the toxic effects of gentamicin in a single model of primary cultured rabbit proximal tubule cells developed without insulin and glucose. Biochemical, functional and morphological approaches were used. Cellular response pattern was examined after a 72-h exposure during either the exponential growth phase or the stationary confluency phase of the culture to 0.2, 1, and 2.5 mM gentamicin. The biochemical study after gentamicin exposure showed increased activities for N-acetyl-beta-D-glucosaminidase and alkaline phosphatase, decreased activities for sphingomyelinase, cathepsin B, Na+/K(+)-ATPase, lactate dehydrogenase and NADPH cytochrome C reductase. Functional evaluation revealed decreased protein synthesis and alpha-methylglucose transport after gentamicin exposure. Morphometric study made it possible to show that the density of lysosomes, the cell fractional volume of the lysosomal compartment, and the mean size of the lysosomal profiles are increased in the cells. Intracellular accumulation of gentamicin in proximal tubular cells was dose dependent and reached high levels in cultured cells. In conclusion, this model compared to others in the literature allowed us to demonstrate in vitro a close response pattern to the in vivo situation after gentamicin exposure.
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