title: SPAK isoforms and OSR1 regulate sodium-chloride co-transporters in a nephron-specific manner.
abstract: STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress-related kinase (OSR1) activate the potassium-dependent sodium-chloride co-transporter, NKCC2, and thiazide-sensitive sodium-chloride cotransporter, NCC, in vitro, and both co-localize with a kinase regulatory molecule, Cab39/MO25α, at the apical membrane of the thick ascending limb (TAL) and distal convoluted tubule (DCT). Yet genetic ablation of SPAK in mice causes a selective loss of NCC function, whereas NKCC2 becomes hyperphosphorylated. Here, we explore the underlying mechanisms in wild-type and SPAK-null mice. Unlike in the DCT, OSR1 remains at the TAL apical membrane of KO mice where it is accompanied by an increase in the active, phosphorylated form of AMP-activated kinase. We found an alterative SPAK isoform (putative SPAK2 form), which modestly inhibits co-transporter activity in vitro, is more abundant in the medulla than the cortex. Thus, enhanced NKCC2 phosphorylation in the SPAK knock-out may be explained by removal of inhibitory SPAK2, sustained activity of OSR1, and activation of other kinases. By contrast, the OSR1/SPAK/M025α signaling apparatus is disrupted in the DCT. OSR1 becomes largely inactive and displaced from M025α and NCC at the apical membrane, and redistributes to dense punctate structures, containing WNK1, within the cytoplasm. These changes are paralleled by a decrease in NCC phosphorylation and a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the dependent nature of OSR1 on SPAK in the DCT, NCC is unable to be activated. Consequently, SPAK(-/-) mice are highly sensitive to dietary salt restriction, displaying prolonged negative sodium balance and hypotension.
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: P Richard
authors.initials: PR
authors.lastname: Grimm
authors.forename: Tarvinder K
authors.initials: TK
authors.lastname: Taneja
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.forename: Richard
authors.initials: R
authors.lastname: Coleman
authors.forename: Yang-Yi
authors.initials: YY
authors.lastname: Chen
authors.forename: Eric
authors.initials: E
authors.lastname: Delpire
authors.forename: James B
authors.initials: JB
authors.lastname: Wade
authors.forename: Paul A
authors.initials: PA
authors.lastname: Welling
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D015870:Gene Expression; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007672:Kidney Cortex / Q000378:metabolism; D007679:Kidney Medulla / Q000378:metabolism; D007686:Kidney Tubules, Distal / Q000378:metabolism; D008138:Loop of Henle / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009399:Nephrons / Q000378:metabolism*; D010766:Phosphorylation; D017419:Potassium, Dietary / Q000008:administration & dosage / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D049992:Sodium Chloride Symporters / Q000235:genetics / Q000378:metabolism; D017673:Sodium Chloride, Dietary / Q000008:administration & dosage / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism; D064466:Solute Carrier Family 12, Member 1; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C000629659:Cab39 protein, mouse; D002135:Calcium-Binding Proteins; D007527:Isoenzymes; D017419:Potassium, Dietary; D011955:Receptors, Drug; C578715:Slc12a1 protein, mouse; C578875:Slc12a3 protein, mouse; D049992:Sodium Chloride Symporters; D017673:Sodium Chloride, Dietary; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; C486407:Stk39 protein, mouse; C534421:OXSR1 protein, mouse; D017346:Protein Serine-Threonine Kinases