Title

Mechanisms and control of sulfate, phosphate, and urate transport by avian renal proximal tubule epithelium

Date of Completion

January 2002

Keywords

Biology, Cell|Biology, Animal Physiology

Degree

Ph.D.

Abstract

Primary monolayer cultures of chick renal proximal tubule cells in Ussing chambers were used to study mechanisms and control of transepithelial sulfate (Si), phosphate (Pi), and urate (UA) transport. ^ The competitive anion, S2O32−, provided evidence for mediated bi-directional Si flux and inhibited active transepithelial Si reabsorption. Carbonic anhydrase II (CAII) protein and activity were detected in isolated chick proximal tubules by immunoblots and biochemical assay, respectively. Inhibition of Si reabsorption by the CA inhibitor, ethoxzolamide, revealed the role of CAII in facilitating proximal tubule Si transport. Cortisol was shown to have a direct action on the epithelium reducing net Si reabsorption in a concentration dependent manner, and thyroid hormone and parathyroid hormone (PTH) acted directly to stimulate net Si reabsorption. ^ Inhibition of Pi reabsorption by direct action of PTH on the epithelium provided evidence for active transepithelial Pi reabsorption. Activation of protein kinase C (PKC) or adenylate cyclase/PKA dramatically decreased net Pi reabsorption, similar to PTH. These inhibitory effects were blocked with bisindolylmaleimide I (BIM), a highly selective PKC inhibitor, and H-89, a potent PKA inhibitor. PTH inhibition of P i reabsorption was also blocked by either BIM or H-89. Additionally, there was a decreased immunoreactivity of luminal Na+:P i cotransporter, NaPiIIa, following PTH treatment.^ Inhibition of active transepithelial UA secretion by p-aminohippuric acid, probenecid, lithium, and glutarate provided evidence for an Oat-like organic anion transport system. Additionally, bromcresol green and ouabain significantly decreased net UA secretion. The Oat3 inhibitor, estrone sulfate, had no inhibitory effect on UA secretion. Cytoskeletal disruptors, cytochalasin-D and nocodazole, used to investigate possible vesicular transcellular UA transport, had no effect. Neither depolarization nor hyperpolarization of the apical membrane affected net UA transport. Also, increasing luminal pH and removing luminal Cl had no effect on net UA secretion. The Mrp2 inhibitor, MK571, substantially decreased net UA secretion. The UA transport data indicate the absence of potential dependent UA transport and anion exchange across the apical membrane, but an Mrp-like mechanism emerged as a possibility for mediating apical UA efflux. ^