I 046-6673/0905-0729$03.(X)/() Journal of the American Society of Nephrology ARTICLES Long-Term Regulation of Renal Urea Transporter Protein Expression in Rat JAMES TERRIS,*t CAROLYN A. ECELBARGER,* JEFF M. SANDS,’r and MARK A. KNEPPER* of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes (t Health, Bethesda, Maryland; TDivision of Nephrologv, Emory University School of Iviedicine, Atlanta, Georgia; and Departinent of Physiology, Uniformed Services Unit’e,-sitv of the Health Sciences, Bethesda, Maryland. Abstract. To test the hypothesis that the abundance of the apical urea transporter of the inner medullary collecting duct (IMCD) is regulated in viva by factors associated with altered water balance, immunoblots of rat inner medullary membrane fractions were probed with rabbit polyclonal antibodies against the renal urea transporter (RUT) gene product. In inner me- dullas of Brattleboro rats, which manifest severe chronic water diuresis, a I 17-kD band was seen, in addition to the previously described 97-kD band. These two bands were detectable by antibodies directed against two different regions of the RUT sequence. When Brattleboro rats were treated with a 5-d infu- sion of arginine vasopressin (AVP) by osmotic minipump, the 1 l7-kD band was markedly diminished, whereas the 97-kD band was unchanged. Simultaneous infusion of the diuretic agent furosemide prevented the AVP-induced decrease in the I 17-kD band. In AVP-infused Sprague Dawley rats, the 1 l7-kD band was barely perceptible. However, when AVP- treated rats were infused with furosemide for 5 d, the 1 17-kD band was markedly accentuated, whereas the 97-kD band was unchanged. The abundance of the I l7-kD RUT protein in the renal papilla was inversely correlated with dietary protein intake. Further immunoblotting studies revealed that the 1 17-kD protein is heavily expressed in IMCD cells and not in non-collecting duct components of the inner medulla, and is present in low-density microsome fractions from inner me- dulla. From this study, the following conclusions can be made: (1) The collecting duct urea transporter is present in at least two forms (97 and 1 17 kD) in the IMCD. (2) The expression level of the 1 1 7-kD urea transporter protein is regulated and is inversely correlated with medullary osmolality and urea con- centration, but does not correlate with circulating AVP level. (3) Although AVP regulates RUT function on a short-term basis, long-term changes in AVP levels do not increase RUT abundance. (J Am Soc Nephrol 9: 729-736, 1998) Within a few minutes of exposure of the renal inner medullary collecting duct (IMCD) to arginine vasopressin (AVP), both urea permeability and osmotic water permeability undergo severalfold increases ( I ,2). The short-term water permeability response is augmented by a long-term regulatory process in which an elevation in circulating AVP for many hours or days results in an increase in the maximally attainable osmotic water permeability of the IMCD epithelium (3-5). This long-term regulation of osmotic water permeability is largely a result of an increase in the expression level of the AVP-regulated water channel aquaporin-2 (5,6). Whether a similar long-term regu- latory process exists for urea transport in the IMCD is still unclear, however. The target for short-term regulation of urea permeability by AVP is believed to be a renal urea transporter Received July 16, 1997. Accepted October 22, 1997. Dr. Jared J. Grantham served as Guest Editor and supervised the review and final disposition of this manuscript. Correspondence to Dr. Mark A. Knepper, National Institutes of Health. Build- ing 10, Room 6N260, 10 Center Drive. MSC 1603, Bethesda, MD 20892- 0951. (RUT) expressed predominantly, if not exclusively, in the IMCD (7,8). This urea transporter, cloned by Shayakul et a!. (7), is a 929-amino acid integral membrane protein. Recently, we have developed a peptide-directed rabbit polyclonal anti- body to this transporter (9). Immunoblots using this antibody demonstrated a predominant band at 97 kD in the inner me- dulla, consistent with the size of the open-reading frame of the cloned transporter. Furthermore, immunocytochemistry and immuno-electron microscopy showed labeling of the apical plasma membrane and of intracellular vesicles, consistent with the view that this transporter is the target for short-term regu- lation of urea transport by AVP. In this article, we report studies using this anti-RUT antibody to test whether the RUT is a target for a long-term regulatory process such as that seen for aquaporin-2. More specifically, we test the hypothesis that the abundance of this urea transporter protein is regulated in the IMCD by factors associated with altered water balance. Materials and Methods Experimental Animals Pathogen-free male Sprague Dawley (Taconic Farms. German- town, NY: National Cancer Institute, Frederick Cancer Research Facility. Frederick, MD) or male Brattleboro homozygous (di/di)