Short-Term Regulation of Basolateral Organic Anion Uptake in Proximal Tubular OK cells

Abstract

Abstract. The organic anion transport system of the kidney is of major importance for the excretion of a variety of endogenous compounds, drugs, and potentially toxic substances. The basolateral uptake into proximal tubular cells is mediated by a tertiary active transport system. Epidermal growth factor (EGF) leads to an increase in the basolateral uptake rate of the model substrate para-aminohippuric acid (PAH) in opossum kidney (OK) cells. This stimulation is mediated by successive activation of the mitogen-activated protein kinases, mitogenactivated/extracellular signal-regulated kinase kinase (MEK) and extracellular regulated kinase isoforms 1 and 2 (ERK1/2). This study investigates the regulatory network of EGF action on PAH uptake downstream ERK1/2 in more detail. EGF stimulation of the basolateral uptake rate of [ 14 C]PAH was abolished by the phospholipase A 2 inhibitor AACOCF3. [ 14 C]PAH uptake was enhanced by arachidonic acid. Furthermore, EGF led to an increase in arachidonic acid release and to the generation of prostaglandins. AACOCF3 did not influence EGF-induced ERK1/2 activation, indicating that ERK1/2 is upstream of PLA 2 . In addition, EGF stimulated the influx of extracellular Ca 2ϩ . However, Ca 2ϩ -influx was not required for the stimulatory action of EGF on [ 14 C]PAH uptake. Inhibitors of COX and lipoxygenases reduced [ 14 C]PAH uptake dosedependently, whereas inhibition of cytochrome P450 did not. In the presence of indomethacin, EGF had no stimulatory effect on [ 14 C]PAH uptake. The inhibitory effect of indomethacin was not due to competitive action on PAH uptake. Furthermore, prostaglandin E 2 (PGE 2 ) increased basolateral [ 14 C]PAH uptake rate dose-dependently, and this increase was also observed in the presence of indomethacin. Selective inhibition of COX2 by indomethacin amid or indomethacin n-heptyl ester did not inhibit [ 14 C]PAH uptake, whereas selective inhibition of COX1 dose-dependently inhibited [ 14 C]PAH uptake. This and previous data lead to the conclusion that EGF successively activates MEK, ERK1/2, and PLA 2 , leading to an increased release of arachidonic acid. Subsequently, arachidonic acid is metabolized to prostaglandins via COX1, which then mediate EGF-induced stimulation of basolateral organic anion uptake rate.The organic anion transport system of the renal proximal tubule plays a crucial role in the excretion of a variety of potentially toxic compounds (1). This system consists of a basolaterally located organic anion exchanger and a less well characterized transport step at the apical membrane (2).The basolateral organic anion exchanger is a tertiary active transport system, dependent on an inward-directed Na ϩ -gradient to drive the uptake of ␣-ketoglutarate, which is then exchanged for organic anions (1-4). The basolateral exchanger for organic anions and dicarboxylates was cloned by three independent groups (5-7) in 1997 and named OAT1 (rat), ROAT1 (rat), or fROAT1 (winter flounder). The homologous protein was cloned from human kidney only recently and wa...

Organic anion transporters: discovery, pharmacology, regulation and roles in pathophysiology

“…To confirm the involvement of eicosanoids, the effect of prostaglandin E 2 (PGE 2 ) was assessed. PGE 2 also increased PAH uptake, but the stimulation was irrespective of COX1 inhibition [108]. This was consistent with the known role of COX1 in the production of prostaglandins.…”

“…Supporting this finding, arachidonic acid (AA), a major eicosanoid precursor liberated by PLA 2 from membrane phospholipids, was also able to increase PAH transport [108]. However, when cyclooxygenase 1 (COX1), but not COX2, was inhibited, PAH transport was decreased and the stimulation mediated by EGF was blunted [108]. However, the involvement of COX2 in OAT regulation cannot be ruled out, as unlike COX1, this enzyme is not constitutively expressed (i.e.…”

“…A subsequent study expanded the investigation to ascertain the involvement of other signaling proteins. It was determined that EGF could stimulate basolateral PAH uptake in OK cells only if phospholipase A2 (PLA 2 ) was active [108]. Supporting this finding, arachidonic acid (AA), a major eicosanoid precursor liberated by PLA 2 from membrane phospholipids, was also able to increase PAH transport [108].…”

“…It was determined that EGF could stimulate basolateral PAH uptake in OK cells only if phospholipase A2 (PLA 2 ) was active [108]. Supporting this finding, arachidonic acid (AA), a major eicosanoid precursor liberated by PLA 2 from membrane phospholipids, was also able to increase PAH transport [108]. However, when cyclooxygenase 1 (COX1), but not COX2, was inhibited, PAH transport was decreased and the stimulation mediated by EGF was blunted [108].…”

Short-Term Regulation of Basolateral Organic Anion Uptake in Proximal Tubular Opossum Kidney Cells

“…Finally, we investigated whether the stimulatory action of EGF on proximal tubular OA uptake described before (25,26) includes the PGE 2 mechanism presented here. Therefore, we investigated the effect of inhibition of PKA (by H89; Figure 9A) or adenylate cyclase (by MDL-12,330A; Figure 9B) on EGF-induced stimulation of basolateral OA uptake in OK cells.…”