title: Immunocytochemical characterization of the incubated rat renal cortical slices.
abstract: 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 the proximal and other tubules. However, detailed studies of tubule integrity have not been reported. To correlate functional and structural viability of the hand-cut rat renal cortical slices, incubated in optimally conditioned media for up to 25 h, we studied the time course of p-aminohippurate (PAH) uptake, the immunocytochemical distribution of several proteins that reside in the proximal tubule basolateral [Na/K-ATPase, organic anion transporters (OAT)1 and OAT3], or brush border [megalin, sodium-proton exchanger (NHE)3] membrane, as well as the general integrity of the tubule epithelium and its cytoskeleton (actin filaments, microtubules). PAH uptake in slices was proportional to time within 1 h of incubation and gradually declined thereafter. The immunostaining experiments indicated a fast, time-dependent loss of basolateral transporters, at a rate of OAT1 > Na/K-ATPase > OAT3. In the brush border membrane, the loss of megalin was faster than that of NHE3, and a partial redistribution of NHE3 into the basolateral domain indicated the loss of cell polarity. The loss of intracellular actin and tubulin cytoskeleton in the proximal tubule was already visible after 15 min of incubation and gradually increased with time, whereas a partial redistribution of actin to the basolateral domain indicated a compromised polarity of the cells. The data also revealed very early (after 15 min) necrotic events in the proximal tubule epithelium, with sloughing of brush border and cell debris into the tubule lumen, detachment of 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 incubated rat renal cortical slices.
authors.affiliation: Croatian Institute for Brain Research and Department of Physiology, School of Medicine, University of Zagreb, Salata 3, 10000, Zagreb, Croatia.
authors.forename: Vladiana
authors.initials: V
authors.lastname: Crljen
authors.forename: Ivana
authors.initials: I
authors.lastname: Sabolić
authors.forename: Jelena
authors.initials: J
authors.lastname: Susac
authors.forename: Dorothea
authors.initials: D
authors.lastname: Appenroth
authors.forename: Carol M
authors.initials: CM
authors.lastname: Herak-Kramberger
authors.forename: Marija
authors.initials: M
authors.lastname: Ljubojević
authors.forename: Naohiko
authors.initials: N
authors.lastname: Anzai
authors.forename: Roberto
authors.initials: R
authors.lastname: Antolović
authors.forename: Gerhard
authors.initials: G
authors.lastname: Burckhardt
authors.forename: Christian
authors.initials: C
authors.lastname: Fleck
authors.forename: Ivan
authors.initials: I
authors.lastname: Sabolić
mesh_terms: D000199:Actins / Q000032:analysis; D000818:Animals; D005260:Female; D007150:Immunohistochemistry; D007672:Kidney Cortex / Q000166:cytology* / Q000502:physiology; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000032:analysis; D027362:Organic Anion Transport Protein 1 / Q000032:analysis; D029362:Organic Anion Transporters, Sodium-Independent / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000032:analysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D014404:Tubulin / Q000032:analysis; D010130:p-Aminohippuric Acid / Q000378:metabolism
chemical_list: D000199:Actins; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D027362:Organic Anion Transport Protein 1; D029362:Organic Anion Transporters, Sodium-Independent; C421043:Slc22a6 protein, rat; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D014404:Tubulin; C114338:organic anion transport protein 3; D000254:Sodium-Potassium-Exchanging ATPase; D010130:p-Aminohippuric Acid