Alteration in renal organic anion transporter 1 after ischemia/reperfusion in cadaveric renal allografts.

We have previously shown that postischemic injury to renal allografts results in profound impairment of p-aminohippuric acid (PAH) extraction. To elucidate the cellular integrity of the human organic anion transporter 1 (hOAT1) in postischemic acute renal failure (ARF), immunohistochemical analysis of hOAT1 was performed in cadaveric renal allografts using confocal microscopy for three-dimensional reconstruction of serial optical images. Biopsy samples were obtained from 10 cadaveric renal allografts 1 hr after reperfusion during transplant operation. Control tissues were obtained from four living donors of healthy kidneys immediately before an arterial clamp was applied to the renal artery. Control tissues demonstrated hOAT1 distributed to basolateral membrane of proximal tubule cells. In contrast, maldistribution of hOAT1 to cytoplasm and/or diminution of the protein was noted in cadaveric allografts. Characteristics of maldistribution were variable: disappearance of lateral distribution, diffuse cytoplasmic aggregates, apical cytoplasmic aggregates, and disappearance of the staining. In addition, iothalamate and PAH clearances were performed on posttransplant days 3-7 in 18 recipients of a cadaveric renal allograft. PAH clearance was depressed <250 ml/min in all but three subjects. We conclude that reperfused, transplanted kidneys exhibit maldistribution of hOAT1 in proximal tubule cells, resulting in impairment of PAH clearance. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Immunocytochemical characterization of the 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 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.

Localization of organic anion transporting polypeptides in the rat and human ciliary body epithelium.

PURPOSE: To identify and localize the expression of multispecific organic anion transporting polypeptides (Oatps/OATPs) in the ciliary body epithelium and to investigate their possible involvement in the transport of the antiglaucoma agent unoprostone. METHODS: Oatps/OATPs were detected by immunoblot analysis and by immunofluorescence microscopy in homogenized and fixed rat and human ciliary body samples using specific polyclonal antibodies. Transport of 3H-labelled unoprostone was measured in Oatp/OATP expressing Xenopus laevis oocytes. RESULTS: Immunoblots of ciliary body extracts were positive for rat Oatp1a4, Oatp1a5 and Oatp1b2 and for human OATP1A2, OATP1C1, OATP2B1, OATP3A1 and OATP4A1. Confocal immunofluorescence microscopy localized Oatp1a4 and Oatp1b2 as well as all immunoblot positive human OATPs at the basolateral plasma membrane of the non-pigmented rat and human ciliary body epithelium, respectively. However, for human OATPs additional regional differences in expression were found with OATP1A2 and OATP1C1 being expressed only in the pars plana of human ciliary body epithelium. Furthermore, OATP1C1, OATP3A1 and OATP4A1 were also expressed at the basolateral plasma membrane of the pars plana pigmented epithelium. And finally, deesterified unoprostone carboxylate was found to be transported by OATP1A2, OATP2B1 and OATP4A1 with approximate K(m)-values of 93, 91 and 132 microm, respectively. CONCLUSIONS: Several multispecific organic anion transporting polypeptides are expressed at the basolateral plasma membrane of the non-pigmented, and to a lesser extent also of the pigmented, epithelium in rat and human ciliary body. These Oatps/OATPs can account for the previously suggested 'liver-like' transport functions of mammalian ciliary body epithelium.