Isoforms of renal Na-K-2Cl cotransporter NKCC2: expression and functional significance

Abstract

The renal Na-K-2Cl cotransporter (NKCC2, BSC1) is selectively expressed in the apical membrane of cells of the thick ascending limb of the loop of Henle (TAL) and macula densa. NKCC2-dependent salt transport constitutes the major apical entry pathway for transepithelial salt reabsorption in the TAL. Although NKCC2 is encoded by a single gene (Slc12a1), differential splicing of the NKCC2 pre-mRNA results in the formation of several alternate transcripts. Thus three full-length splice isoforms of NKCC2 differ in their variable exon 4, resulting in transcripts for NKCC2B, NKCC2A, and NKCC2F. In addition to full-length isoforms, variants with truncated COOH-terminal ends have been described. The various splice isoforms of NKCC2 differ in their localization along the TAL and in their transport characteristics. Data in the literature are reviewed to assess the principles of NKCC2 differential splicing, the localization of NKCC2 splice isoforms along the TAL in various species, and the functional characteristics of the splice isoforms. In addition, we discuss the functional significance of NKCC2 isoforms for TAL salt retrieval and for the specific salt sensor function of macula densa cells based on studies using isoform-specific NKCC2-knockout mice. We suggest that different NKCC2 splice variants cooperate in salt retrieval along the TAL and that the coexpression of two splice variants (NKCC2B and NKCC2A) in the macula densa cells facilitates efficient salt sensing over wide ranges of fluctuating salt concentrations.

Fig. 1.

NaCl reabsorption in the thick ascending limb (TAL). Salt reabsorption in the TAL involves apical electroneutral cotransport of Na⁺/K⁺/2Cl⁻ via NKCC2, recycling of K⁺ via apical K⁺ channels (ROMK), efflux of Cl⁻ via basolateral Cl⁻ channels, and basolateral salt extrusion by Na⁺-K⁺-ATPase. Water permeability of the TAL is low; consequently, salt reabsorption results in successively more dilute ion concentrations in the tubular lumen.

Fig. 2.

A: the Slc12a1 gene and differential splicing of exon 4. During splicing of the pre-mRNA, variant B, A, or F of exon 4 is linked to exon 3. The 3 variants of exon 4 are then fused to exon 5, resulting in transcripts for NKCC2B, NKCC2A, and NKCC2F, respectively (A, B, C). In addition, tandem exons have been described consisting of a combination of exon 4B/F and 4A/F (D, E). B: gene targeting of NKCC2B and NKCC2A. A mutation consisting of the coding sequence for the FLAG peptide and in-frame stop codons followed by a floxed neomycin (neo) resistance expression cassette was introduced in exon 4B (top) or exon 4A (bottom) by homologous recombination in embryonic stem cells. The neomycin cassette was subsequently removed by crossing NKCC2B/neo and NKCC2A/neo mice with the Cre-expressing strain EIIa-Cre. After neo removal, correct splicing was preserved in NKCC2B- and NKCC2A-deficient mice. In NKCC2A-deficient mice, however, an additional aberrant transcript was present that incorporated genomic sequences upstream of exon 4A, the genomic sequence between exon 4A and 4F, and the full sequence of exon 4F.

Fig. 3.

A: relative contribution of NKCC2A and NKCC2B isoforms to the tubuloglomerular feedback (TGF) response. Relative response magnitudes for the flow ranges indicated along the x-axis are expressed as % of the total stop-flow pressure (P_SF) response (NKCC2A−/−: 100% = 7.1 ± 1 mmHg; NKCC2B−/−: 100% = 8.4 mmHg). Data are calculated from Refs. and . B: TGF responses in wild-type mice compared with the sum of TGF responses in NKCC2B−/− and NKCC2A−/− mice. Response magnitudes for the flow ranges indicated along the x-axis are expressed as changes in P_SF (mmHg). The enhanced responses in the low flow ranges (0–5 and 5–7.5 nl/min) are most likely due to the increased expression of the B isoform in NKCC2A−/− mice (50).

Fig. 4.

NKCC2 isoforms and macula densa control of renin secretion. Suppression of plasma renin concentration (PRC) after acute salt loading by saline infusion (data are shown as % change compared with baseline). A reduction in PRC after salt loading is significantly more pronounced in NKCC2B-deficient mice compared with control mice, whereas the suppression of renin secretion is markedly reduced in NKCC2A-deficient mice. Combined from Refs. and .