A New Methodology for Quantification of Alternatively Spliced Exons Reveals a Highly Tissue-Specific Expression Pattern of WNK1 Isoforms

Abstract

Mutations in the WNK1 gene, encoding a serine-threonine kinase of the WNK (With No lysine (K)) family, have been implicated in two rare human diseases, Familial Hyperkalemic Hypertension (FHHt) and Hereditary Sensory and Autonomic Neuropathy type 2 (HSAN2). Alternative promoters give rise to a ubiquitous isoform, L-WNK1, and a kidney-specific isoform, KS-WNK1. Several other isoforms are generated through alternative splicing of exons 9, 11 and 12 but their precise tissue distribution is not known. Two additional exons, 8b and HSN2, involved in HSAN2, are thought to be specifically expressed in the nervous system. The purpose of this study was to establish an exhaustive description of all WNK1 isoforms and to quantify their relative level of expression in a panel of human and mouse tissues and in mouse nephron segments. For the latter purpose, we developed a new methodology allowing the determination of the proportions of the different isoforms generated by alternative splicing. Our results evidenced a striking tissue-specific distribution of the different isoforms and the unexpected presence of exon HSN2 in many tissues other than the nervous system. We also found exon 26 to be alternatively spliced in human and identified two new exons, 26a and 26b, within intron 26, specifically expressed in nervous tissues both in humans and mice. WNK1 should therefore no longer be designated as a 28- but as a 32-exon gene, with 8 of them - 8b, HSN2, 9, 11, 12, 26, 26a and 26b - alternatively spliced in a tissue-specific manner. These tissue-specific isoforms must be considered when studying the different roles of this ubiquitous kinase.

WNK1 -related Familial Hyperkalemic Hypertension results from an increased expression of L-WNK1 specifically in the distal nephron

“…The long isoform, long WNK1 (L-WNK1), is expressed ubiquitously, whereas the shorter isoform, kidney-specific WNK1 (KS-WNK1), which lacks a functional kinase domain, is expressed specifically in the kidney (5). In the kidney, L-WNK1 is expressed at a low level in all nephron segments, whereas KS-WNK1 is expressed only in the distal nephron (6). We previously generated a transgenic mouse model that exhibited an ectopic expression of KS-WNK1 and an increased expression of L-WNK1 in the distal nephron on deletion of the first intron (7).…”

“…Total RNA extraction, reverse transcription, and RT-qPCR were performed as described in ref. 6. Primers sequences are indicated in Table S2.…”

With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl− cotransporters

“…The full-length KCCs (39,40,60), as well as the wild-type and mutants of human L-WNK1 kinase cDNAs used in this work, were previously described (7,46,63). L-WNK1 gives rise to several distinct isoforms.…”

Réabsorption du sel et sécrétion du potassium par le néphron distal

“…Huit exons de WNK1 (exons 4a, 8b, HSN2, 9, 11, 12, 26a et 26b) sont soumis à un épissage alternatif [16]. Chez l'homme et la souris, le variant L-WNK1-∆11, qui ne contient pas l'exon 11, est le variant majoritaire dans le néphron (il représente environ 70 % de tous les variants de L-WNK1) [16].…”

“…Huit exons de WNK1 (exons 4a, 8b, HSN2, 9, 11, 12, 26a et 26b) sont soumis à un épissage alternatif [16]. Chez l'homme et la souris, le variant L-WNK1-∆11, qui ne contient pas l'exon 11, est le variant majoritaire dans le néphron (il représente environ 70 % de tous les variants de L-WNK1) [16]. La grande majorité des études concernant la régulation de NCC par L-WNK1 in vitro a paradoxalement été réalisée en utilisant le variant L-WNK1-∆11-12, qui ne représente en fait, dans le rein, que 20 % des transcrits L-WNK1.…”