Real-time assessment of organic anion secretion in isolated, perfused rabbit renal proximal tubules

Abstract

A newly developed epifluorescense microscopy system has been employed to measure net transepithelial secretion of fluorescein (FL) in real time in isolated perfused S2 segments of rabbit renal proximal tubules. Net FL secretion (K(t), ∼4 μM, and J(max), ∼280 fmol·min⁻¹·mm⁻¹) shares the same transport system with that of para -aminohippurate (PAH). The basolateral Na-DC cotransporter supports ∼25% of the "basal" FL secretion in the absence of exogenous αKG via recycling of αKG that has been exchanged for FL. Physiological αKG concentrations in the bath (∼10 μM) or in the perfusate (∼50 μM) stimulated net secretion of FL by ∼30 or ∼20%, respectively. These data indicate that the basolateral Na-DC cotransporter supports ∼42% of the net FL secretion. The luminal and basolateral effects of physiological concentrations of αKG were additive. Together, the basolateral and luminal Na-DC cotransporters can directly support ∼50% of the net FL secretion, apparently, by their establishing and maintaining the outwardly directed αKG gradient responsible for driving basolateral FL/αKG exchange. The remaining ∼50% would be maintained by metabolic production of αKG in the cells. Adding of 100 nM phorbol 12-myristate 13-acetate (PMA), a known PKC activator, to the bath decreased steady-state secretion of FL by ∼30% after 25 min incubation. This inhibition was irreversible and increased to ∼60% 25 min following removal of PMA. The inhibition produced by PMA was blocked when 100 nM of either staurosporine (ST) or bisindolylmaleimide I (BIM), both known PKC inhibitors, was added to the bath. ST or BIM alone had no significant effect on FL secretion, suggesting that the basal FL secretion was not under the influence of PKC. Adding of 1 μM of either the peptide hormone bradykinin (BK) or the α₁-receptor agonist phenylephrine (PE), to the bath both of which stimulate PKC via a ligand-receptor-PKC coupling reaction, inhibited FL secretion by ∼22% and ∼27%, respectively. The inhibition was completely reversible after removal of BK or PE. In conclusion, PKC negatively regulates the net secretion of FL in rabbit renal proximal tubules. The data indicate that BK or catecholamines can play a physiological role in regulating organic anion secretion via PKC activation.