Up-regulation of organic anion transporter 1 protein is induced by chronic furosemide or hydrochlorothiazide infusion in rat kidney.

Abstract

Thiazide and loop diuretics are secreted from the proximal tubule via the organic anion transport system to reach their principal sites of action. Recently, a multispecific organic anion transporter 1 (OAT1) was identified in rat kidney and was localized to the basolateral membrane of the S2 segment in the proximal tubule. We postulated that interactions between thiazide or loop diuretics and OAT1 may play a role in the adaptation to long-term diuretic use, and investigated whether OAT1 is regulated in vivo by chronic administration of diuretics at the protein level. Semi-quantitative immunoblotting and immunohistochemistry were carried out in kidneys from male Sprague-Dawley rats using a polyclonal peptide-derived antibody to OAT1. Furosemide (12 mg/day/rat, n = 6), hydrochlorothiazide (3.75 mg/day/rat, n = 6) or vehicle (1.7% ethanolamine, n = 6) were infused subcutaneously for 7 days using osmotic minipumps. Experimental and vehicle-control rats were pair-fed, and two bottles of drinking water were provided, one containing tap water and the other containing a solution of 0.8% NaCl with 0.1% KCl. Overt diuretic responses were observed to both furosemide and hydrochlorothiazide infusions. There were no differences in body weight or creatinine clearance between the experimental and control rats. Although OAT1 protein abundance in cortical homogenates was increased by furosemide infusion (271 +/- 35 vs 100 +/- 15%, P < 0.05), Na-K-ATPase alpha1 subunit protein abundance was not affected (113 +/- 14 vs 100 +/- 8%, P = 0.42). Immunohistochemical localization in tissue sections confirmed a strong increase in OAT1 expression in the basolateral membrane of the S2 segment of proximal tubule. OAT1 protein abundance in cortical homogenates was also increased by hydrochlorothiazide infusion (181 +/- 25 vs 100 +/- 7%, P < 0.01), whereas Na-K-ATPase alpha1 subunit protein abundance was not affected (105 +/- 4 vs 100 +/- 4%, P = 0.34). Chronic furosemide or hydrochlorothiazide infusion caused increases in OAT1 protein abundance in rat kidney. These results suggest that OAT1 may be up-regulated in vivo by substrate stimulation at the protein level.