engleski

Immunocytochemical characterization of the in vitro incubated rat renal cortical slices

The use of renal cortical slices in vitro and the data obtained in these studies have been subjects of controversy, largely due to uncertain viability, e.g. structural and functional integrity of proximal (PT) and other tubules. However, detailed studies of the tubule integrity, that would warrant such questions, have not been reported. To correlate functional and structural viability of the rat renal cortical slices, incubated in vitro in optimally conditioned media for up to 25 hours, time course of p-aminohippurate (PAH) uptake and immunocytochemical distribution of several transporters that reside in the PT basolateral (Na+/K+-ATPase, OAT1, OAT3) or brush-border membrane (megalin, NHE3), as well as a general integrity of the tubule epithelium and its cytoskeleton (actin filaments, microtubules) were studied. The PAH uptake in slices was proportional to the time within one hour of incubation, and gradually declined thereafter. The immunostaining experiments indicated a time-dependent loss of basolateral transporters (OAT1> Na+/K+-ATPase > OAT3). In brush-border membranes, the loss of megalin was faster that that of NHE3. A partial redistribution of NHE3 into basolateral membranes indicated the loss of cell polarity. The loss of intracellular actin and tubulin cytoskeleton in PT was visible already after 15 min of incubation, and gradually increased with time. A partial redistribution of actin to the basolateral domain pointed at a compromised polarity of the cells. The data also revealed very early (after 15 min) necrotic events in PT epithelium, with slaughter of brush-border and cell debris into the tubule lumen, detachment of the cells from the basal membrane, and opening and widening of the tubule lumen. We conclude that the loss of cellular structure, cytoskeleton, and cell membrane transporters in the nephron epithelium is a very early event in the in vivo incubated rat renal cortical slices. Nevertheless, this model can be used to study in rats and other mammals effects of age, sex, and hormones on the PAH transport in renal PT.

tissue slices; Na/K-ATPase; OAT1; OAT3; megalin; NHE3

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