Transepithelial urate transport by avian renal proximal tubule epithelium in primary culture

Abstract

SUMMARY Birds are uricotelic, and because they excrete urate by renal tubular secretion, they provide a convenient model for examination of this process. Primary monolayer cultures of the isolated renal proximal tubule epithelium from the domestic chicken, Gallus gallus L., were mounted in Ussing chambers where several substrates/inhibitors of renal organic anion transporters were tested for the sidedness and specificity of their effects on transepithelial urate transport. Transepithelial electrical resistance,electrical potential and sodium-dependent glucose current were monitored to detect nonspecific effects. Under control short-circuited conditions the ratio of unidirectional fluxes of [14C]urate was found to be 3:1. Active net secretion was specifically inhibited by 1 mmol l–1probenecid and 10 mmol l–1para-aminohippuric acid(PAH). Bromocresol Green, cimetidine, nocodozole, cytochalasin D and ouabain also inhibited secretion but were toxic. Interstitial-side lithium (5 mmol l–1) and glutarate (1 mmol l–1) specifically blocked transport, but 10–100 μmol l–1 glutarate had no effect. Interstitial estrone sulfate (ES) stimulated urate secretion at 10μmol l–1 but was inhibitory at 500 μmol l–1. Active PAH secretion (5:1 flux ratio) was inhibited 34%by 330 μmol l–1 urate. ES (500 μmol l–1) blocked the remainder. From the lumen side,glucose-free, Cl--free and high K+ (30 mmol l–1) solutions, or an alkaline pH of 7.7 had no effect on urate transport and neither did several compounds known to be uricosuric. Lumen-side methotrexate (500 μmol l–1) and MK571 (20μmol l–1) strongly inhibited urate secretion. MK571 had no effect from the interstitial side. RT-PCR revealed mRNA for OAT1-, OAT3-,MRP2- and MRP4-like organic anion transporters in chicken proximal epithelium.

Diclofenac toxicity in Gyps vulture is associated with decreased uric acid excretion and not renal portal vasoconstriction

“…Since the mammalian kidney is influenced by the uricosuric effects of DF and MLX through the inhibition of the epithelial cell's basolateral UA-excreting organic anionic transporters (OATs) (also referred to as a PAH transporter), multi-drug resistance protein (MRP) and the apical UA reabsorptive channel known as URAT1, it is possible that a similar effect occurs in the vulture. The latter is plausible as similar PAH and MRP channels are involved with UA excretion in the chicken (birds do not reabsorbed UA) (Khamdang et al, 2002, Rafey et al, 2003, Dudas et al, 2005, Enomoto and Endou, 2005and Mount et al, 2006. In addition both diclofenac and phenylbutazone have been shown to induce uricemia in the chicken (Berger et al, 1960 andNaidoo et al, 2007).…”

Avian renal proximal tubule epithelium urate secretion is mediated by Mrp4

“…Urate is the major nitrogenous waste in birds, and, unlike in great apes (21), there is no significant facilitated reabsorption of urate detected in the perfused chicken renal proximal tubule (9) or in primary monolayer cultures of chicken renal proximal tubule epithelial cells (cPTCs) (13), only active secretion. URAT1 is believed by many to be the main contributor to urate reabsorption in humans; however, no similar sequence is present in the chicken genome, consistent with the lack of reabsorption in the avian system.…”

“…Urate secretion has been directly measured in the perfused renal proximal tubule of adult chicken (9). Our previous characterization study of renal proximal tubule monolayer cultures from the neonate chicken indicated equivalent transport and inhibitor effects as seen in the adult perfused tubule (13).…”

“…Proposed secretion models suggest that urate is transported across the BLM by organic anion transporters 1 and 3 (Oat1 and Oat3; both expressed in chicken kidney) (13). Urate exchange for intracellular ␣-ketoglutarate (␣-KG) by these organic anion transporters has been implicated as the mechanism for BLM urate transport (43) and has been noted in avian-perfused tubules (9) as well as in isolated BLM vesicles from pig (59) and turkey kidney (18).…”

Uric acid transporters hiding in the intestine

“…In cultured chicken proximal tubules, RT-PCR revealed mRNA for MRP2-and MRP4-like organic anion transporters in avian proximal epithelium. Luminal application of MK-571 (a known substrate for MRP2 and MRP4) dramatically reduced both uric acid secretory and re-absorptive flux (Dudas et al 2005). …”