engleski

Immunocytochemical characterization of the in vitro incubated rat renal cortical slices

Rat renal cortical slices (RRCS) have been a useful experimental model for the in vitro studies of cellular transport, metabolism, and toxicity of various compounds relevant to basic science and pharmaceutical industry. Their use 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 tubules (PT) during incubation in vitro. However, detailed studies of the tubule integrity, that would warrant such questions, have not been performed. To correlate functional and structural viability of RRCS, incubated in vitro in optimally conditioned media for up to 25 hours, we studied a 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 the general integrity of the tubule epithelium and cytoskeleton (actin filaments, microtubules). The PAH uptake in the slices was liner with time within 1 hour of incubation, and gradually declined thereafter. However, the immunostaining experiments pointed to a fast, time-dependent loss of basolateral transporters in PT, with a rate: OAT1>Na/K-ATPase>OAT3. In the brush-border membrane, the loss of megalin was faster than of NHE3, and a time-dependent increase in redistribution of NHE3 into the basolateral membrane was observed, indicating the loss of cell polarity. Fragmentation and loss of intracellular actin and tubulin cytoskeleton in PT started already after 15 min of incubation, and gradually increased with time, whereas a partial redistribution of actin to the basolateral domain indicated compromised polarity of the cells. The data also revealed very early (after 15 min) necrotic events in PT epithelium, with sloughter of brush-border and cell debris into the tubule lumen, detachment of the cells from the basal membrane, 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 RRCS incubated in vitro.

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

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