The kidneys play pivotal roles in acid-base homeostasis, and the acid-secreting (␣-type) and bicarbonate-secreting (␤-type) intercalated cells in the collecting ducts are major sites for the final modulation of urinary acid secretion. Since the H ؉ -ATPase and anion exchanger activities in these two types of intercalated cells exhibit opposite polarities, it has been suggested that the ␣-and ␤-intercalated cells are interchangeable via a cell polarity change. Immunohistological studies, however, have failed to confirm that the apical anion exchanger of ␤-intercalated cells is the band 3 protein localized to the basolateral membrane of ␣-intercalated cells. In the present study, we show the evidence that a novel member of the anion exchanger and sodium bicarbonate cotransporter superfamily is an apical anion exchanger of ␤-intercalated cells. Cloned cDNA from the ␤-intercalated cells shows about 30% homology with anion exchanger types 1-3, and functional expression of this protein in COS-7 cells and Xenopus oocytes showed sodium-independent and 4,4-diisothiocyanostilbene-2,2-disulfonic acid-insensitive anion exchanger activity. Furthermore, immunohistological studies revealed that this novel anion exchanger is present on the apical membrane of ␤-intercalated cells, although some ␤-intercalated cells were negative for AE4 staining. We conclude that our newly cloned transporter is an apical anion exchanger of the ␤-intercalated cells, whereas our data do not exclude the possibility that there may be another form of anion exchanger in these cells.

To examine whether expression and distribution of aquaporin of collecting duct (AQP-CD) are regulated by vasopressin V2 receptor (V2R), we performed immunohistochemical studies with specific antibody against AQP-CD.Normal Wistar rats were divided into four groups and treated for 3 d; control, dehydration, vasopressin V1 receptor (V1R) antagonist (OPC-21268 120 mg/kg), V2R antagonist (OPC-31260 30 mg/kg). At time of death, urine osmolality (Uosm) in the dehydration group (1884±245 mOsm/ kg) was significantly higher than that in the control (938±91). In the V2R antagonist group, Uosm was significantly decreased to 249±29, whereas V1R antagonist showed no effect on Uosm. In the control and V1R antagonist groups, immunofluorescence studies showed the AQP-CD staining of both apical membrane and subapical cytoplasm of CD cells of the cortex and the inner medulla. Dehydration increased the immunostaining of both apical membrane and subapical cytoplasm of CD cells of the inner medulla, and the degree of increase was dominant in apical membrane. In the V2R antagonist group, only faint staining of apical membrane and weak labeling of cytoplasm of CD cells of the inner medulla were observed. These changes in the localization and protein amount of AQP-CD by dehydration and V2R antagonist were quantitatively confirmed by immunogold studies and immunoblot analysis of the inner medulla. The present results indicate that the distribution and amount of AQP-CD in the CD cells are regulated by vasopressin V2 receptor. (J. Clin. Invest. 1994. 94:1778-1783

These studies suggest that acidosis activates c-Src and MEK/ERK/c-fos. While both pathways are necessary for activation of NHE3, they are activated independently.

The purpose of the present studies was to determine whether acidosis activates protein tyrosine kinase pathways. Incubation of MCT cells, a renal proximal tubule cell line, in acid media caused increased phosphotyrosine content of 60- to 70- and 120-kDa cytosolic proteins. Media acidification induced a twofold increase in c-Src activity that occurred within 30 s. Significant activation occurred with media pH changes as small as 0.07 pH unit accompanied by cell acidification of 0.06 pH unit. Sodium propionate addition, NH4Cl prepulse, and nigericin addition, maneuvers that decrease intracellular pH in the absence of changes in extracellular pH, activated c-Src. Significant activation by sodium propionate was seen with cell pH changes as small as 0.07 pH unit. Sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prevented c-Src activation by media acidification but did not prevent protein tyrosine phosphorylation. In summary, decreased intracellular pH activates c-Src. Acid activation of c-Src represents a novel mechanism of c-Src activation that may be relevant to many cellular responses to acidosis.

These studies demonstrate that c-Src is required for angiotensin II-induced increases in NHE3 activity. Thus, c-Src plays a key role in antiporter activation by acidosis and angiotensin II.