Calcineurin and Sorting-Related Receptor with A-Type Repeats Interact to Regulate the Renal Na+-K+-2Cl− Cotransporter

Abstract

The furosemide-sensitive Na + -K + -2Cl 2 -cotransporter (NKCC2) is crucial for NaCl reabsorption in kidney thick ascending limb (TAL) and drives the urine concentrating mechanism. NKCC2 activity is modulated by N-terminal phosphorylation and dephosphorylation. Serine-threonine kinases that activate NKCC2 have been identified, but less is known about phosphatases that deactivate NKCC2. Inhibition of calcineurin phosphatase has been shown to stimulate transport in the TAL and the distal convoluted tubule. Here, we identified NKCC2 as a target of the calcineurin Ab isoform. Short-term cyclosporine administration in mice augmented the abundance of phospho-NKCC2, and treatment of isolated TAL with cyclosporine increased the chloride affinity and transport activity of NKCC2. Because sorting-related receptor with Atype repeats (SORLA) may affect NKCC2 phosphoregulation, we used SORLA-knockout mice to test whether SORLA is involved in calcineurin-dependent modulation of NKCC2. SORLA-deficient mice showed more calcineurin Ab in the apical region of TAL cells and less NKCC2 phosphorylation and activity compared with littermate controls. In contrast, overexpression of SORLA in cultured cells reduced the abundance of endogenous calcineurin Ab. Cyclosporine administration rapidly normalized the abundance of phospho-NKCC2 in SORLA-deficient mice, and a functional interaction between calcineurin Ab and SORLA was further corroborated by binding assays in rat kidney extracts. In summary, we have shown that calcineurin Ab and SORLA are key components in the phosphoregulation of NKCC2. These results may have clinical implications for immunosuppressive therapy using calcineurin inhibitors. The furosemide-sensitive renal Na + -K + -2Cl 2 -cotransporter (NKCC2) mediates the transepithelial NaCl reabsorption in the thick ascending limb (TAL) and plays an essential role in the urinary concentration and volume regulation. 1 The transport activity of NKCC2 depends on its phosphorylation at several N-terminal, conserved threonine and serine residues, including T96, T101, and T114. 2,3 These threonines have been identified as targets for the two closely related STE20-like kinases, SPAK (SPS-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), 4,5 whereas much less information is known on the identity of the respective phosphatases. Calcineurin, a ubiquitously expressed calcium-/calmodulin-

Extracellular K+ rapidly controls NaCl cotransporter phosphorylation in the native distal convoluted tubule by Cl−‐dependent and independent mechanisms

“… b ; Borschewski et al . ). Similarly, a kidney‐specific deletion of FK506 binding protein, crucial for the effects of tacrolimus on PP3, attenuates tacrolimus‐induced arterial hypertension (Lazelle et al .…”

Heterogeneity of tight junctions in the thick ascending limb

“…Along this path, they face different functional requirements. On the one hand, tubular fluid has to be depleted from NaCl down to the transport equilibrium of the Na + 2Cl – K + cotransporter NKCC2 . On the other hand, Ca 2+ and Mg 2+ absorption have to be organized in a way to not exceed a certain transport rate in comparison with the limited interstitial clearance and buffering capacity.…”

“…V te, transepithelial voltage; R te , transepithelial resistance; 10+, additional basolateral claudin‐10 staining; P(x)/P(y), permeability ratio for ion x/y. Luminal osmolality depends on luminal flow rate and NKCC2 activity . V te values are taken from measurements under symmetrical isotonic NaCl concentrations.…”

“…Additionally, the luminal Cl – concentration is higher compared with the cytosol concentration and only becomes limiting for the cotransporter when the concentration decreases downstream along the TAL. This limitation originates from the reduced driving force as well as from the affinity of the cotransporter for Cl – . Luminal recycling of K + by the luminal K + channel is a prerequisite for the continuous absorption of large amounts of NaCl at comparably low luminal K + availability.…”

Vasopressin lowers renal epoxyeicosatrienoic acid levels by activating soluble epoxide hydrolase

“…To corroborate these results we studied the effects of the 14,15-EET regioisomer in greater detail and also included the inactive metabolite 14,15-DHET. Phosphorylation of NKCC2 functionally changes the transport current by changing NKCC2 membrane trafficking as well as by changing its Cl -affinity, rate-limiting in the cortical TAL with already dilute luminal fluid (9,22). To investigate the effect of 14,15-EET on TAL tubular transport we therefore measured the equivalent short circuit current I' sc under two Cl -concentrations, at 147 mmol/l to as assess maximal transport velocity ("V max" ) of the transporter and at 30 mmol/l, a concentration close to the described EC50 of NKCC2 for Cl - (22).…”