Impaired phosphorylation of Na ⁺ -K ⁺ -2Cl ⁻ cotransporter by oxidative stress-responsive kinase-1 deficiency manifests hypotension and Bartter-like syndrome

Abstract

Na + -K + -2Cl − cotransporters (NKCCs), including NKCC1 and renal-specific NKCC2, and the Na + -Cl − cotransporter (NCC) play pivotal roles in the regulation of blood pressure (BP) and renal NaCl reabsorption. Oxidative stress-responsive kinase-1 (OSR1) is a known upstream regulator of N(K)CCs. We generated and analyzed global and kidney tubule-specific (KSP) OSR1 KO mice to elucidate the physiological role of OSR1 in vivo, particularly on BP and kidney function. Although global OSR1 −/− mice were embryonically lethal, OSR1 +/− mice had low BP associated with reduced phosphorylated (p) STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase (SPAK) and p-NKCC1 abundance in aortic tissue and attenuated p-NKCC2 abundance with increased total and p-NCC expression in the kidney. KSP-OSR1 −/− mice had normal BP and hypercalciuria and maintained significant hypokalemia on a low-K + diet. KSP-OSR1 −/− mice exhibited impaired Na + reabsorption in the thick ascending loop on a low-Na + diet accompanied by remarkably decreased expression of p-NKCC2 and a blunted response to furosemide, an NKCC2 inhibitor. The expression of total SPAK and p-SPAK was significantly increased in parallel to that of total NCC and p-NCC despite unchanged total NKCC2 expression. These results suggest that, globally, OSR1 is involved in the regulation of BP and renal tubular Na + reabsorption mainly via the activation of NKCC1 and NKCC2. In the kidneys, NKCC2 but not NCC is the main target of OSR1 and the reduced p-NKCC2 in KSP-OSR1 −/− mice may lead to a Bartter-like syndrome.Bartter syndrome | electrolytes | hormone | knockout mice | volume R ecent studies have shown that Na + -K + -2Cl − cotransporters (NKCCs) and the Na + -Cl − cotransporter (NCC) play very important roles in the regulation of blood pressure (BP) and extracellular volume. NKCCs consist of ubiquitous NKCC1 and renal-specific NKCC2. NKCC1 can modulate BP through vascular and renal effects (1-4). NKCC2 and NCC are two renal Na + cotransporters expressed in the thick ascending limbs (TALs) and distal convoluted tubules (DCTs) of the kidney, respectively, accounting for 20% and 10% of filtered Na + reabsorption (5). In human essential hypertension and salt-sensitive or spontaneously hypertensive animal models, activation of NKCC1 and NKCC2 has also been reported to play a pivotal role in the pathogenesis of hypertension (6, 7). In addition, activation of NCC by gene mutations in WNK1 and WNK4 leads to an autosomal dominant salt-sensitive hypertension known as pseudohypoaldosteronism type II (PAHII) (8). On the other hand, loss-of-function mutations in the SLC12A1 and SLC12A3 genes encoding NKCC2 and NCC can lead to renal salt-wasting hypotension with hypokalemic metabolic alkalosis, known as Bartter syndrome (BS) (9) and Gitelman syndrome (GS) (10), respectively.In vitro studies have shown that posttranscriptional phosphorylation of NKCC1/2 and NCC plays a crucial role in the regulation of normal transport activity. Oxidative stress-responsive kinase-1 (OSR1) (11) and STE20 (s...

Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

“…Although in sensory neurons the two kinases seem to fulfill a redundant function (12) based on the phenotypes of SPAK and kidney-specific OSR1 knock-out mice, the two kinases in kidney seem to have very distinct roles. Indeed, although the SPAK knock-out mouse reveals a primary role for SPAK in regulating the Na-Cl cotransporter in the distal convoluted tubule (13)(14)(15)(16), the kidney-specific OSR1 knock-out mouse displays a phenotype that is consistent with disruption of NKCC2 function in the TAL (17). An intriguing observation from the SPAK knock-out mouse is that NKCC2 in the TAL is hyperphosphorylated (13,14,16), which would be inconsistent if the kinase was phosphorylating this cotransporter under normal conditions.…”

Physiological and molecular mechanisms of salt and water homeostasis in the nematodeCaenorhabditis elegans

“…pressure (70,94). Recent studies of C. elegans gck-3 mutants have broadened GCK-VI function to include multiple developmental processes, including epithelial tube morphogenesis and spermatogenesis (37,58).…”

SPAK Isoforms and OSR1 Regulate Sodium-Chloride Co-transporters in a Nephron-specific Manner

“…Consistent with the unanticipated highly focused phenotype, phosphorylation of NCC, but not NKCC2, is attenuated in these SPAK knock-out mice. Remarkably, SPAK gene ablation actually leads to an increase in NKCC2 phosphorylation (2), a phenotype that seems to be absent in the catalytically inactive SPAK knock-in mouse (4). Examining a different SPAK null strain, which has been reported to be surprisingly normotensive and have normal levels of aldosterone, McCormick et al (3) suggested that removal of a TAL-specific inhibitory SPAK isoform, KS-SPAK, in the SPAK Ϫ/Ϫ mice would allow OSR1 to hyperphosphorylate NKCC2.…”

“…Both have been implicated in controlling the activity of NKCC2 and NCC, the co-transporters responsible for salt reabsorption in the thick ascending limb and distal convoluted tubule (1)(2)(3)(4)(5)(6)(7). As the two individual members of the germinal center kinase VI subfamily of STE20 kinases (8), SPAK and OSR1 exhibit a high degree of structural homology, and share similar common domain architectures.…”

“…Two closely related kinases, Ste20-related proline-alaninerich kinase (SPAK) 2 and the oxidative stress-responsive kinase 1 (OSR1), are well appreciated to phosphoregulate cation-dependent chloride transporters. Both have been implicated in controlling the activity of NKCC2 and NCC, the co-transporters responsible for salt reabsorption in the thick ascending limb and distal convoluted tubule (1)(2)(3)(4)(5)(6)(7).…”