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Articles in PresS, published online ahead of print September 24, 2002
Am J Physiol Renal Physiol, 10.1152/ajprenal.00227.2002
Submitted on June 17, 2002
Accepted on September 10, 2002
1 Department of Internal Medicine, Seoul National University, Seoul, Korea, Republic of
2 Department of Internal Medicine, Eulji Medical College, Seoul, Korea, Republic of
3 Department of Internal Medicine, Chungbuk National University, Cheongju, Korea, Republic of
4 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, MD, USA
5 Department of Internal Medicine, Hallym University Hangang Sacred Heart Hospital, Seoul, Korea, Republic of
* To whom correspondence should be addressed. E-mail: jshan{at}snu.ac.kr.
Furosemide and hydrochlorothiazide (HCTZ) exert their diuretic actions by binding to apical Na transporters, viz. the Na-K-2Cl cotransporter in the thick ascending limb and the Na-Cl cotransporter in the distal convoluted tubule, respectively. We carried out semiquantitative immunoblotting and immunohistochemistry of rat kidneys to investigate whether chronic administration of furosemide or HCTZ is associated with compensatory changes in the abundance of sodium transporters downstream from the primary site of action. Osmotic minipumps were implanted into Sprague-Dawley rats to deliver furosemide (12 mg/d) or HCTZ (3.75 mg/d) for 7 days. To prevent volume depletion all animals were offered tap water and a solution containing 0.8 percent NaCl and 0.1 percent KCl as drinking fluid. The diuretic/natriuretic response was quantified in response to both agents using quantitative urine collections. Semiquantitative immunoblotting revealed that the abundances of thick ascending limb Na-K-2Cl cotransporter and all three subunits of the epithelial Na channel (ENaC) were increased by furosemide infusion. HCTZ infusion increased the abundances of thiazide-sensitive Na-Cl cotransporter and ENaC ß-subunit in the cortex and ENaC ß- and 
-subunit in the outer medulla. Consistent with these results, ENaC ß-subunit immunohistochemistry showed a remarkable increase in immunoreactivity in the principal cells of collecting ducts with either diuretic treatment. These increases in the abundance of Na+ transporters in response to chronic diuretic treatment may account for the generation of diuretic tolerance associated with long-term diuretic use.
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