WNK1 is an Ancient Cell Volume Regulator that was Repurposed for Terrestrial Evolution

With No Lysine (WNK) kinases are serine/threonine (S/T) kinases with a uniquely positioned catalytic lysine required for chloride sensing. In mammals, WNK1 is expressed ubiquitously and has an important function in cell chloride and volume regulation. However, WNK1 also regulates blood pressure and potassium homeostasis by controlling salt reabsorption in the renal tubule, suggesting functional evolution over time. Prior work indicates that the renin‐angiotensin‐aldosterone system (RAAS) targets WNK1 through specific sequences harbored in exons 11 and 12, two alternatively spliced cassette exons that are represented in kidney. Additionally, a kidney‐specific kinase‐deficient isoform of WNK1 called KS‐WNK1—defined by the presence of exon 4a—has been shown to regulate renal WNK pathway activity in vitro and in vivo . We hypothesized that the unique kinase domain, alternative promoter usage, and splicing of WNK1 emerged at key points in evolution to facilitate its role in cell volume control and renal electrolyte handling. To test this hypothesis, we employed phylogenetic reconstruction methods to analyze WNK1 gene evolution. Using human WNK1 exons 4a, 11, and 12 and rat WNK1 kinase domain sequences as reference queries, we gathered nucleotide and protein sequences from available genome databases utilizing NCBI and UCSC Genome Browser. WNK1 protein sequences were then analyzed in SeaView to create multiple sequence alignments with Clustal Omega and phylogenetic trees with PhyML 3.0. The known structure of the WNK1 kinase domain (PDB ID 4Q2A) was used as a template for homology modeling of ancestral WNK‐like S/T kinase domain sequences in MODELLER 9.17. Exons 11 and 12 were only detected as far back as amniotes, the earliest evidence of these exons being in reptiles. The relatively recent appearance of these exons in evolution postdates the emergence of the RAAS, suggesting that aldosterone signaling may have applied a selective pressure that forced their incorporation into the WNK1 gene structure. The earliest we detect exon 4a is in coelacanth, a lobe‐finned fish closely related to lungfish, the first vertebrate to crawl on land. Consistent with an ancient role of the kinase domain in cell‐volume regulation, we detected sequences consistent with the WNK‐specific subdomain I catalytic lysine and chloride binding pocket in several protists. This preliminary finding challenges previous work suggesting that WNKs are solely present in multicellular organisms. These observations suggest that WNK kinases were first designed for cellular chloride and volume regulation in unicellular species, and were subsequently repurposed through alternative splicing and promoter usage for whole body salt and volume homeostasis during kidney development in metazoans. Critical elements of the WNK1 gene appear to have emerged during a key point in kidney evolution; namely, during the transition from water to land for temporary and permanent inhabitance. Support or Funding Information NIH R01DK098145, T35DK065521