SPAK Differentially Mediates Vasopressin Effects on Sodium Cotransporters

Activation of the Na⁺-K⁺-2Cl⁻-cotransporter (NKCC2) and the Na⁺-Cl⁻-cotransporter (NCC) by vasopressin includes their phosphorylation at defined, conserved N-terminal threonine and serine residues, but the kinase pathways that mediate this action of vasopressin are not well understood. Two homologous Ste20-like kinases, SPS-related proline/alanine-rich kinase (SPAK) and oxidative stress responsive kinase (OSR1), can phosphorylate the cotransporters directly. In this process, a full-length SPAK variant and OSR1 interact with a truncated SPAK variant, which has inhibitory effects. Here, we tested whether SPAK is an essential component of the vasopressin stimulatory pathway. We administered desmopressin, a V2 receptor–specific agonist, to wild-type mice, SPAK-deficient mice, and vasopressin-deficient rats. Desmopressin induced regulatory changes in SPAK variants, but not in OSR1 to the same degree, and activated NKCC2 and NCC. Furthermore, desmopressin modulated both the full-length and truncated SPAK variants to interact with and phosphorylate NKCC2, whereas only full-length SPAK promoted the activation of NCC. In summary, these results suggest that SPAK mediates the effect of vasopressin on sodium reabsorption along the distal nephron.The furosemide-sensitive Na⁺-K⁺-2Cl⁻-cotransporter (NKCC2) of the thick ascending limb (TAL) and the thiazide-sensitive Na⁺-Cl⁻-cotransporter (NCC) of the distal convoluted tubule (DCT) are key regulators of renal salt handling and therefore participate importantly in BP and extracellular fluid volume homeostasis.¹ Loss-of-function mutants in the SLC12A1/ A3 genes encoding NKCC2 and NCC cause salt-losing hypotension and hypokalemic alkalosis in Bartter’s and Gitelman’s syndromes,^2,3 whereas their overactivity may contribute to essential hypertension.^4,5 Recently, attention has been focused on the two closely related STE20-like kinases, SPS-related proline/alanine-rich kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1), which can phosphorylate NKCC2 and NCC at their N-terminal conserved threonine or serine residues (T96, T101, and T114 of mouse NKCC2 and T53, T58, and S71 of mouse NCC) and thereby activate the transporters.^6–8 Despite the high homology between SPAK and OSR1 and their overlapping renal expression patterns, distinct roles along the nephron have been suggested based on data from SPAK-deficient and kidney-specific OSR1-deficient mice: deletion of SPAK primarily impairs the function of NCC, whereas deletion of OSR1 negatively affects NKCC2.^9–11 The complex functional properties of a WNK-SPAK/OSR1-N(K)CC interaction cascade are currently being defined.¹² Recently, arginine vasopressin (AVP), signaling via the V2 receptor (V2R), has been identified as an efficient activator of both cotransporters, affecting their luminal trafficking and phosphorylation.^13–18 Because plasma AVP levels may vary not only with the sleep-wake cycle or long-term physiologic challenges, but also with pulsatile changes over the short term, distinct, time-dependent responses may occur.¹⁹ SPAK and OSR1 are well placed to regulate distal NaCl reabsorption in response to AVP. Here we tested the role of SPAK in AVP-induced activation of NKCC2 and NCC, acutely and during long-term treatment. The results suggest that SPAK is an essential kinase that modulates distal nephron function in response to AVP stimulation.