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

Abstract

Background: Full-length SPAK is thought to be necessary and sufficient to activate NCC in the distal convoluted tubule (DCT). Results: SPAK knock-out disrupts a signaling network, involving OSR1, in the DCT but not the TAL, preventing NCC activation. Conclusion: SPAK and OSR1 function interdependently in the DCT to positively regulate NCC. Significance: This study provides insights into the mechanisms whereby SPAK/OSR1 regulates renal salt transport.

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

“…In contrast, co-injection of the two smaller fragments led to a significant decrease in NKCC2 func- tion consistent with dominant negative effect of SPAK fragments on NKCC2 function. This observation is in agreement with the dominant negative effect of other fragments previously studied (16,18).…”

“…In fact two sites yield C-terminal fragments with sizes close to the native fragments that are observed by Western blot analyses of kidney samples. The predicted molecular weights of 40.8 (V2LEG) and 36.7 (F2LAT) are close to the size of the inhibitory KS fragments measured previously (14,16). We created the cDNA encoding these fragments (Fig.…”

“…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.…”

“…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. In the kidney medulla several smaller SPAK fragments are observed by Western blot analysis, and these fragments have been postulated to act as negative regulators of OSR1 function in the thick ascending limb of Henle (14,16,18).…”

“…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. In the kidney medulla several smaller SPAK fragments are observed by Western blot analysis, and these fragments have been postulated to act as negative regulators of OSR1 function in the thick ascending limb of Henle (14,16,18). Indeed, if these fragments were to inhibit OSR1 function, their absence in the knock-out would lead to increased OSR1 and hyperphosphorylation of NKCC2.…”