Renal Function in Mice with Targeted Disruption of the A Isoform of the Na-K-2Cl Co-Transporter

Abstract

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...

The impact of alternative splicing in vivo: Mouse models show the way

“…Two minor isoforms, A and B, have been targeted in mice using loxP-Cre strategies, introducing premature Stop codons in the respective exons. In contrast to the complete disruption of the entire NKCC2 gene that led to early postnatal death (Takahashi et al 2000), mice deficient in the A or B isoform showed only mild phenotypes (Oppermann et al 2006(Oppermann et al , 2007. Mice deficient in the A isoform showed symptoms consistent with disturbed salt sensing at high Cl concentrations and the B isoform appears to be more active at lower salt concentrations, arguing for specialized functions of the two isoforms.…”

“…The Na-K-2Cl cotransporter (NKCC2) plays a major role in the uptake of ions in the kidney. Four different isoforms are known that differ in their ion-binding capabilities and are differentially expressed in different kidney regions, probably to ensure proper function in areas with varying ion concentrations (Oppermann et al 2007). Two minor isoforms, A and B, have been targeted in mice using loxP-Cre strategies, introducing premature Stop codons in the respective exons.…”

NKCC2A and NFAT5 regulate renal TNF production induced by hypertonic NaCl intake

“…The essential role of NKCC2 in establishing the medullary interstitial gradient is evident in studies using NKCC2 Ϫ/Ϫ mice, which exhibit a lower ambient urinary osmolality than their WT littermates (56). A reduction in ambient urine osmolality also was observed in NKCC2A-deficient mice (46). We recently demonstrated a selective elevation of NKCC2A mRNA accumulation in TNF Ϫ/Ϫ mice, which was accompanied by an increase in NKCC2 activity in mTAL tubules and an increase in ambient urine osmolality, and suggested that the absence of TNF contributes to the increased interstitial gradient generated by elevated NKCC2A expression and activity (2).…”

“…NKCC2 is expressed specifically in the apical membrane of TAL and macula densa cells, and several studies have described the distribution and function of three major alternatively spliced isoforms termed NKCC2A, NKCC2B, and NKCC2F (4,29,43,45,46). As the NKCC2A and F isoforms are expressed in the mTAL, the effect of hypertonic NaCl intake was evaluated on these isoforms.…”

Everything we always wanted to know about furosemide but were afraid to ask

“…Each isoform is expressed differently along the thick ascending limb of Henle. Although the functional significance of these three isoforms is uncertain (117,136), and factors affecting the level of expression are not fully understood, differential splicing of NKCC2 seems to be modulated by dietary salt intake. Dietary sodium restriction enhanced the expression of the high-affinity NKCC2B isoform and reduced the low-affinity NKCC2A isoform (136).…”