Three different full-length splice isoforms of the Na-K-2Cl co-transporter (NKCC2/BSC1) are expressed along the thick ascending limb of Henle (TAL), designated NKCC2A, NKCC2B, and NKCC2F. NKCC2F is expressed in the medullary, NKCC2B mainly in the cortical, and NKCC2A in medullary and cortical portions of the TAL. NKCC2B and NKCC2A were shown to be coexpressed in the macula densa (MD) segment of the mouse TAL. The functional consequences of the existence of three different isoforms of NKCC2 are unclear. For studying the specific role of NKCC2A in kidney function, NKCC2A؊/؊ mice were generated by homologous recombination. NKCC2A؊/؊ mice were viable and showed no gross abnormalities. Ambient urine osmolarity was reduced significantly in NKCC2A؊/؊ compared with wild-type mice, but water deprivation elevated urine osmolarity to similar levels in both genotypes. Baseline plasma renin concentration and the effects of a high-and a low-salt diet on plasma renin concentration were similar in NKCC2A؉/؉ and ؊/؊ mice. However, suppression of renin secretion by acute intravenous saline loading (5% of body weight), a measure of MD-dependent inhibition of renin secretion, was reduced markedly in NKCC2A؊/؊ mice compared with wild-type mice. Cl and water absorption along microperfused loops of Henle of NKCC2A؊/؊ mice were unchanged at normal flow rates but significantly reduced at supranormal flow. Tubuloglomerular feedback function curve as determined by stop flow pressure measurements was left-shifted in NKCC2A؊/؊ compared with wild-type mice, with maximum responses being significantly diminished. In summary, NKCC2A activity seems to be required for MD salt sensing in the high Cl concentration range. Coexpression of both high-and low-affinity isoforms of NKCC2 may permit transport and Cl-dependent tubuloglomerular feedback regulation to occur over a wider Cl concentration range. T he Na-K-2Cl co-transporter (NKCC2), located in the apical membrane of the epithelial cells of the thick ascending limb of Henle (TAL), constitutes the major cellular uptake pathway in this portion of the nephron. In addition, NKCC2 transport activity in macula densa (MD) cells is considered to be the initial step in the signaling chain that links tubular epithelial cells of the TAL with vascular cells of the afferent arteriole (1-5). At least four different full-length splice isoforms of NKCC2 are expressed along the TAL, designated NKCC2A, NKCC2B, NKCC2F, and NKCC2AF (6 -10). These isoforms of the transporter are derived from differential splicing of the variable exon 4 of the NKCC2 gene (7,8,11). Exon 4 encodes for amino acids of the second transmembrane domain and the adjacent intracellular loop of NKCC2, a region that has been shown to be involved in the ion-binding characteristics of the co-transporter (11). Cell-specific splicing results in heterogeneous expression of the various NKCC2 isoforms along the TAL. NKCC2F, the most abundant NKCC2 isoform, is expressed in the medullary TAL, NKCC2B mainly in the cortical TAL, and NKCC2A both in medullar...

Na,K,2Cl co-transporter (NKCC2), the primary NaCl uptake pathway in the thick ascending limb of Henle, is expressed in three different full-length splice variants, called NKCC2F, NKCC2A, and NKCC2B. These variants, derived by differential splicing of the variable exon 4, show a distinct distribution pattern along the loop of Henle, but the functional significance of this organization is unclear. By introduction of premature stop codons into exon 4B, specific for the B isoform, mice with an exclusive NKCC2B deficiency were generated. Relative expression levels and distribution patterns of NKCC2A and NKCC2F were not altered in the NKCC2B-deficient mice. NKCC2B-deficient mice did not display a salt-losing phenotype; basal plasma renin and aldosterone levels were not different from those of wild-type mice. Ambient urine osmolarities, however, were slightly but significantly reduced. Distal Cl concentration was significantly elevated and loop of Henle Cl absorption was reduced in microperfused superficial loops of Henle of NKCC2B-deficient mice. Because of the presence of NKCC2A in the macula densa, maximum tubuloglomerular feedback responses were normal, but tubuloglomerular feedback function curves were right-shifted, indicating reduced sensitivity in the subnormal flow range. Plasma renin concentration in NKCC2B-deficient mice was reduced under conditions of salt loading compared with that in wild-type mice. This study shows the feasibility of generating mice with specific deletions of single splice variants. The mild phenotype of mice that are deficient in the B isoform of NKCC2 indicates a limited role for NKCC2B for overall salt retrieval. Nevertheless, the high-affinity NKCC2B contributes to salt absorption and macula densa function in the low NaCl concentration range.

By crossing mice with expression of Cre recombinase under control of the endogenous renin promoter (Sequeira Lopez ML, Pentz ES, Nomasa T, Smithies O, Gomez RA. Dev Cell 6: 719–728, 2004) with mice in which exon 1 of the Gnas gene was flanked by loxP sites (Chen M, Gavrilova O, Liu J, Xie T, Deng C, Nguyen AT, Nackers LM, Lorenzo J, Shen L, Weinstein LS. Proc Natl Acad Sci USA), we generated animals with preferential and nearly complete excision of Gsα in juxtaglomerular granular (JG) cells. Compared with wild-type animals, mice with conditional Gsα deficiency had markedly reduced basal levels of renin expression and very low plasma renin concentrations. Furthermore, the acute release responses to furosemide, hydralazine, and isoproterenol were virtually abolished. Consistent with a state of primary renin depletion, Gsα-deficient mice had reduced arterial blood pressure, reduced levels of aldosterone, and a low glomerular filtration rate. Renin content and renin secretion of JG cells in primary culture were drastically reduced, and the stimulatory response to the addition of PGE2 or isoproterenol was eliminated. Unexpectedly, Gsα recombination was also observed in the renal medulla, and this was associated with a vasopressin-resistant concentrating defect. Our study shows that Cre recombinase under control of the renin promoter can be used for the excision of floxed targets from JG cells. We conclude that Gsα-mediated signal transduction is essential and nonredundant in the control of renin synthesis and release.