. Action of EGF and PGE2 on basolateral organic anion uptake in rabbit proximal renal tubules and hOAT1 expressed in human kidney epithelial cells. Am J Physiol Renal Physiol 286: F774-F783, 2004. First published November 25, 2003 10.1152/ajprenal.00326.2003We recently showed that, in a proximal tubule cell line (opossum kidney cells), epithelial growth factor (EGF) stimulates basolateral organic anion transport (OAT) via ERK1/2, arachidonic acid, phospholipase A 2, and generation of prostaglandins. PGE2 binds the prostanoid receptor and, thus, activates adenylate cyclase and PKA, which stimulate basolateral organic anion uptake. In the present study, we investigated whether this regulatory cascade is also true 1) for ex vivo conditions in isolated renal proximal (S2) tubules from rabbit and 2) in a human renal epithelial cell line stably expressing human OAT1 (IHKE-hOAT1). EGF activated ERK1/2 in S2 tubules and IHKE-hOAT1, and, in both cases, inhibition of ERK activation (by U-0126) abolished this stimulation. In S2 tubules and IHKE-hOAT1, EGF led to an increase of organic anion uptake, which again was inhibited by U-0126. PGE 2 stimulated basolateral organic anion uptake in rabbit S2 tubules and IHKEhOAT1. EGF-and PGE 2-mediated stimulation of organic anion uptake was abolished by inhibition of PKA in rabbit S2 tubules and IHKE-hOAT1, respectively. We conclude that 1) stimulation of basolateral organic anion uptake by EGF or PGE 2 is a widespread (if not general) regulatory mechanism, 2) the signal transduction pathway involved seems to be general, 3) stimulation of basolateral organic anion uptake by EGF or PGE 2 is also present under ex vivo conditions and, thus, is not a cell culture artifact, 4) activation of OAT1 is sufficient to explain the stimulatory effects of EGF and PGE 2 in opossum kidney cells and rabbit S2 segments, and 5) stimulation of basolateral OAT1 by EGF or PGE 2 is also important in humans and, thus, may have clinical implications. basolateral transport; epithelial growth factor; extracellular signalregulated kinase 1/2; rabbit isolated proximal tubule; mitogen-activated protein kinase; mitogen-activated protein kinase kinase; IHKE cells; organic anion transport; phospholipase A 2; prostaglandin E2; protein kinase A; regulation THE ORGANIC ANION TRANSPORT (OAT) system of the renal proximal tubule plays a crucial role in the excretion of a variety of potentially toxic compounds (37,56). This system consists of organic anion exchanger(s) located at the basolateral membrane and a less well-characterized transport step at the apical membrane (18).The classical basolateral organic anion exchanger is the terminal step in a tertiary active transport system that is dependent on an inward-directed Na ϩ gradient to drive the uptake of ␣-ketoglutarate (␣-KG), which is then exchanged for organic anions (17,23,38,55). The basolateral exchanger for organic anions and dicarboxylates was cloned by three independent groups (46, 51, 58) in 1997 and named OAT1 (rat), ROAT1 (rat), and fROAT1 (winter flounde...

Physiologically, OAT1 is located in the basolateral membrane of proximal tubular cells. During renal damage loss of polarity occurs in renal epithelial cells, leading to missorting of proteins or complete loss of polarity. Missorting or loss of polarity generally leads to disturbance of vectorial transport. In the present study, hOAT1 was expressed in human renal epithelial IHKE cells (IHKE-hOAT1) and in non polarized CHO cells (CHO-hOAT1). Because EGF and its receptor is described to play on important role in recovery from renal damage, we compared the regulation of hOAT1 by EGF in the (a) basolateral and (b) apical membrane of epithelial cells, and in (c) non polarized cells, resembling the above mentioned pathophysiological situations. Expression of hOAT1 was verified by determination of the kinetic parameters (using fluorescein as a substrate) and western blot (CHO-hOAT1) or RT-PCR (IHKE-hOAT1). To investigate the EGF effect on hOAT1, CHO-hOAT1 cells were additionally co-transfected with the human EGF receptor HER1. In agreement with previous publications, incubation of IHKE-hOAT1 cells with EGF increased fluorescein uptake via basolateral hOAT1. In opposite, EGF inhibited hOAT1 mediated fluorescein uptake across the apical membrane of IHKE-hOAT1 cells. Additionally EGF inhibited hOAT1 mediated fluorescein uptake into non polarized CHO-hOAT1-HER1 cells, too. In summary, we confirmed that EGF stimulates basolateral uptake of organic anions (a) in proximal tubular cells mediated by hOAT1. However, EGF inhibits hOAT1 located in the apical membrane (b) or in non polarized cells (c). Renal failure is associated with successive loss of epithelial polarity. Therefore, inverted regulation of hOAT1 falsely located in the apical membrane of proximal tubular cells may be part of a mechanism stabilizing organic anion secretion in pathophysiological situations.