Calcineurin dephosphorylates nuclear factor of activated T cells transcription factors, thereby facilitating T cell-mediated immune responses. Calcineurin inhibitors are instrumental for immunosuppression after organ transplantation but may cause side effects, including hypertension and electrolyte disorders. Kidneys were recently shown to display activation of the furosemide-sensitive Na-K-2Cl cotransporter (NKCC2) of the thick ascending limb and the thiazide-sensitive Na-Cl cotransporter (NCC) of the distal convoluted tubule upon calcineurin inhibition using cyclosporin A (CsA). An involvement of major hormones like angiotensin II or arginine vasopressin (AVP) has been proposed. To resolve this issue, the effects of CsA treatment in normal Wistar rats, AVP-deficient Brattleboro rats, and cultured renal epithelial cells endogenously expressing either NKCC2 or NCC were studied. Acute administration of CsA to Wistar rats rapidly augmented phosphorylation levels of NKCC2, NCC, and their activating kinases suggesting intraepithelial activating effects. Chronic CsA administration caused salt retention and hypertension, along with stimulation of renin and suppression of renal cyclooxygenase 2, pointing to a contribution of endocrine and paracrine mechanisms at long term. In Brattleboro rats, CsA induced activation of NCC, but not NKCC2, and parallel effects were obtained in cultured cells in the absence of AVP. Stimulation of cultured thick ascending limb cells with AVP agonist restored their responsiveness to CsA. Our results suggest that the direct epithelial action of calcineurin inhibition is sufficient for the activation of NCC, whereas its effect on NKCC2 is more complex and requires concomitant stimulation by AVP.
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-
The antidiuretic hormone vasopressin (AVP) regulates renal salt and water reabsorption along the distal nephron and collecting duct system. These effects are mediated by vasopressin 2 receptors (V2R) and release of intracellular Gs-mediated cAMP to activate epithelial transport proteins. Inactivating mutations in the V2R gene lead to the X-linked form of nephrogenic diabetes insipidus (NDI), which has chiefly been related with impaired aquaporin 2-mediated water reabsorption in the collecting ducts. Previous work also suggested the AVP-V2R-mediated activation of Na(+)-K(+)-2Cl(-)-cotransporters (NKCC2) along the thick ascending limb (TAL) in the context of urine concentration, but its individual contribution to NDI or, more generally, to overall renal function was unclear. We hypothesized that V2R-mediated effects in TAL essentially determine its reabsorptive function. To test this, we reevaluated V2R expression. Basolateral membranes of medullary and cortical TAL were clearly stained, whereas cells of the macula densa were unreactive. A dominant-negative, NDI-causing truncated V2R mutant (Ni3-Glu242stop) was then introduced into the rat genome under control of the Tamm-Horsfall protein promoter to cause a tissue-specific AVP-signaling defect exclusively in TAL. Resulting Ni3-V2R transgenic rats revealed decreased basolateral but increased intracellular V2R signal in TAL epithelia, suggesting impaired trafficking of the receptor. Rats displayed significant baseline polyuria, failure to concentrate the urine in response to water deprivation, and hypercalciuria. NKCC2 abundance, phosphorylation, and surface expression were markedly decreased. In summary, these data indicate that suppression of AVP-V2R signaling in TAL causes major impairment in renal fluid and electrolyte handling. Our results may have clinical implications.
Activation of the thick ascending limb (TAL) Na--cotransporter (NKCC2) by the antidiuretic hormone arginine-vasopressin (AVP) is an essential mechanism of renal urine concentration and contributes to extracellular fluid and electrolyte homeostasis. AVP effects in the kidney are modulated by locally and/or by systemically produced epoxyeicosatrienoic acid derivates (EET). The relation between AVP and EET metabolism has not been determined. Here we show that chronic treatment of AVPdeficient Brattleboro rats with the AVP V2 receptor analog desmopressin (dDAVP; 5ng/h, 3d) significantly lowered renal EET levels (-56 ± 3% for 5,6-EET, -50 ± 3.4% for 11,12-EET, and -60 ± 3.7% for 14,15-EET). The abundance of the principal EET-degrading enzyme soluble epoxide hydrolase (sEH) was increased at the mRNA (+160 ± 37%) and protein levels (+120 ± 26%). Immunohistochemistry revealed dDAVP-mediated induction of sEH in connecting tubules and cortical and medullary collecting ducts, suggesting a role of these segments in the regulation of local interstitial EET signals. Incubation of murine kidney cell suspensions with 1 µM 14,15-EET for 30 min reduced phosphorylation of NKCC2 at the AVPsensitive threonine residues T96 and T101 (-66 ± 5%; p<0.05) while 14,15-DHET had no effect.Concomitantly, isolated perfused cTAL pretreated with 14,15-EET showed a 30% lower transport current
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