Upregulation of the Secretory-Type Na

The functional role and mechanisms of regulation of the Na/K/2Cl-cotransporter NKCC1 in the kidney have not yet been clarified. NKCC1 mRNA and protein expression in control rats, rats with dehydration (2 d), and rats with metabolic acidosis (NH 4Cl in the food for 6 to 7 d) was examined using reverse transcription-PCR and Western blotting. In contrast to the abundant NKCC1 mRNA expression in the terminal inner medullary collecting ducts in mice, expression was found to be most abundant in the outer medullary collecting ducts (OMCD) in rats. Dehydration and metabolic acidosis increased NKCC1 mRNA expression threeto fivefold not only in the OMCD but also in the cortical collecting ducts and inner medullary collecting ducts. Dehydration and metabolic acidosis increased NKCC1 protein expression twofold in the membrane fraction from the outer medulla. NKCC1 protein expression was observed not in the microdissected medullary thick ascending limbs but in the OMCD, and it was stimulated twofold by dehydration and metabolic acidosis. I cubation of OMCD in low-pH medium increased NKCC1 mRNA expression. In summary, NKCC1 mRNA and protein expression is upregulated with dehydration and metabolic acidosis. NKCC1 may play an important role in adaptation to these physiologic conditions. Low pH and possibly hypertonicity stimulate NKCC1 mRNA expression in OMCD. Electroneutral Na /K/2Cl-cotransporters play an important role not only in sodium chloride absorption but also in sodium chloride secretion for the maintenance of body fluid homeostasis (1). Two types of bumetanide-sensitive Na /K/2Clcotransporters and thiazide-sensitive Na /Cl-cotransporters have been cloned to date (2–6). The cotransporters belong to the family of integral membrane transport proteins that mediate electrically neutral transport of sodium, potassium, and chloride across the membrane. The two types of bumetanidesensitive Na/K/2Cl-cotransporters are referred to as NKCC1 (BSC2) and NKCC2 (BSC1). The secretory type, NKCC1, is present in various secretory epithelia, whereas the absorptive type, NKCC2, is located only in the kidney. Immunohistochemical studies have demonstrated the presence of NKCC2 proteins in medullary thick ascending limbs (MAL) and cortical thick ascending limbs (CAL) (7,8). NKCC2 mRNA expression in MAL and CAL is stimulated by sodium loading (9). Because NH 4 1 is absorbed in MAL and CAL by substituting for K via NKCC2 (10–12), NKCC2 expression has been thought to be stimulated by metabolic acidosis. In fact, NH4 1 absorption in rat MAL is increased during meta bolic acidosis (13). Dehydration and metabolic acidosis, however, have not been found to affect NKCC2 mRNA expression in these segments (14,15), which suggests that the increase in NH4 1 absorption may not occur through the changes in NKCC2 protein expression. NKCC1 has been cloned from mouse terminal inner medullary collecting duct (IMCD) cells, as well as the shark rectal gland and human colon (2–4). Northern blot analyses have demonstrated the presence of NKCC1 mRNA not only in the kidney but also in many secretory epithelia, in sites such as the salivary gland, stomach, jejunum, colon, lung, and heart. Immunohistochemical studies in the kidney have demonstrated the presence of NKCC1 proteins in terminal IMCD in mice but not in terminal IMCD in rats (16,17); the NKCC1 protein has been found to be present in the basolateral membrane of intercalated cells in the outer medullary collecting duct (OMCD) in rats. The OMCD is important not only in acid excretion but also in sodium absorption and secretion (18,19). One role of NKCC1 in rat OMCD has been suggested to be ammonium excretion (17). One of the splice variants of NKCC2, i.e., NKCC2F, has been reported to be located also in the OMCD (14). The direction of sodium movement is different for NKCC1 and -2. Recent reports have revealed that mice lacking NKCC1 exhibit signs of deafness and imbalance (20,21). The functional role of NKCC1 in rat OMCD is still unclear, and it is not known whether NKCC1 expression is regulated by pathophysiologic changes such as dehydration and metabolic acidosis. Therefore, we investigated, using competitive PCR and Western blot analysis, whether NKCC1 exReceived May 17, 1999. Accepted August 30, 2000. Correspondence to Dr. Mika Ikebe, Third Department of Internal Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto, Kumamoto 860, Japan. Phone: 81-96-373-5164; Fax: 81-96-366-8458; E-mail: nono@kaiju.medic. kumamoto-u.ac. jp (Hiroshi Nonoguchi)