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


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Kim
Gheun-Ho
GH
Department of Internal Medicine, Hallym University Hangang Sacred Heart Hospital, Seoul National University, Seoul, South Korea. gheunho@hanmail.net
Na
Ki Young
KY
Kim
So-Young
SY
Joo
Kwon Wook
KW
Oh
Yoon Kyu
YK
Chae
Seoung-Wan
SW
Endou
Hitoshi
H
Han
Jin Suk
JS
D000818:Animals; D004232:Diuretics / Q000494:pharmacology*; D005665:Furosemide / Q000494:pharmacology*; D006852:Hydrochlorothiazide / Q000494:pharmacology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism*; D007672:Kidney Cortex / Q000378:metabolism*; D008297:Male; D027362:Organic Anion Transport Protein 1 / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects*
D004232:Diuretics; D027362:Organic Anion Transport Protein 1; D049993:Sodium Chloride Symporter Inhibitors; D006852:Hydrochlorothiazide; D005665:Furosemide