No abstract

Activation of the Na + -K + -2Cl 2 -cotransporter (NKCC2) and the Na + -Cl 2 -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 receptorspecific 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 Na2 -cotransporter (NKCC2) of the thick ascending limb (TAL) and the thiazide-sensitive Na + -Cl 2 -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 kidneyspecific OSR1-deficient mice: deletion of SPAK

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.

To prevent complications that might follow an infection with varicella-zoster virus (VZV), the live attenuated Oka strain (V-Oka) is administered to children in many developed countries. Three vaccine brands (Varivax from Sanofi Pasteur MSD; Varilrix and Priorix-Tetra, both from Glaxo-Smith-Kline) are licensed in Germany and have been associated with both different degrees of vaccine effectiveness and adverse effects. To identify genetic variants in the vaccines that might contribute to rash-associated syndromes, single nucleotide polymorphism (SNP) profiles of variants from the three vaccines and rash-associated vaccine-type VZV from German vaccinees were quantitatively compared by PCR-based pyrosequencing (PSQ). The Varivax vaccine contained an estimated 3-fold higher diversity of VZV variants, with 20% more wild-type (wt) SNPs than Varilrix and Priorix-Tetra. These minor VZV variants in the vaccines were identified by analyzing cloned full-length open reading frame (ORF) orf62 sequences by chain termination sequencing and PSQ. Some of these sequences amplified from vaccine VZV were very similar or identical to those of the rash-associated vaccine-type VZV from vaccinees and were almost exclusively detected in Varivax. Therefore, minorities of rash-associated VZV variants are present in varicella vaccine formulations, and it can be concluded that the analysis of a core set of four SNPs is required as a minimum for a firm diagnostic differentiation of vaccine-type VZV from wt VZV.The varicella-zoster virus (VZV) live attenuated Oka strain (V-Oka) is licensed in developed countries for vaccination against varicella. In Germany, routine vaccination of healthy children Ͼ11 months of age has been performed since 2004. Since then, varicella morbidity has declined (16). The mixtures of V-Oka strains with different single nucleotide polymorphism (SNP) profiles present in vaccine preparations and the reported genomic variation of vaccine VZV strains have prompted investigations of the linkage of certain genomic variants to episodes of vaccine-induced rash (9, 10).We aimed at comparing the SNP profiles of the varicella vaccines used in Germany and of vaccine-type VZV from German vaccinees for several reasons. First, three vaccine brands are distributed in Germany (Varivax from Sanofi Pasteur MSD [SPMSD], Leimen, Germany; Varilrix and PriorixTetra, both from GlaxoSmithKline [GSK], Munich, Germany) and have been associated with different degrees of vaccine effectiveness (17). In addition, data from the German varicella sentinel (16) indicated that the vaccines might be related to different frequencies of rash-associated syndromes, i.e., mild forms of varicella or zoster (Anette Siedler and Bernhard Ehlers, unpublished data). Second, vaccine-type VZV strains causing rash-associated clinical syndromes in vaccinees have been reported to contain a number of wild-type (wt) SNPs. It has been hypothesized that these vaccine-type, wt SNP-containing VZV strains are minor components of the vaccines (1, 9, 10, 13). To substantiate t...

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.

Caveolin-1 (Cav1) is essential for the formation of caveolae. Little is known about their functional role in the kidney. We tested the hypothesis that caveolae modulate renal salt and water reabsorption. Wild-type (WT) and Cav1-deficient (Cav1−/−) mice were studied. Cav1 expression and caveolae formation were present in vascular cells, late distal convoluted tubule and principal connecting tubule and collecting duct cells of WT but not Cav1−/− kidneys. Urinary sodium excretion was increased by 94% and urine flow by 126% in Cav1−/− mice (p < 0.05). A decrease in activating phosphorylation of the Na-Cl cotransporter (NCC) of the distal convoluted tubule was recorded in Cav1−/− compared to WT kidneys (−40%; p < 0.05). Isolated intrarenal arteries from Cav1−/− mice revealed a fourfold reduction in sensitivity to phenylephrine (p < 0.05). A significantly diminished maximal contractile response (−13%; p < 0.05) was suggestive of enhanced nitric oxide (NO) availability. In line with this, the abundance of endothelial NO synthase (eNOS) was increased in Cav1−/− kidneys +213%; p < 0.05) and cultured caveolae-deprived cells showed intracellular accumulation of eNOS, compared to caveolae-intact controls. Our results suggest that renal caveolae help to conserve water and electrolytes via modulation of NCC function and regulation of vascular eNOS.

Calcineurin inhibitors (CnI) such as Cyclosporin A (CsA) are instrumental for immunosuppression after organ transplantation but may cause serious neurologic side effects, including seizures.Neuronal excitability depends on intact Cl− homeostasis. Generation of hyperpolarizing synaptic responses to GABA and glycine requires a Cl− gradient across the cell membrane with a low [Cl−]i, mainly established by K+‐Cl− cotransporter 2 (KCC2) and to a lesser extent Na+‐K+‐Clcotransporter 1 (NKCC1); both are cation‐coupled chloride cotransporters (CCCs). Calcineurin has been implicated in the regulation of CCCs, whereas impaired CCC function is a well‐known condition in human pharmacoresistant epilepsy. Therefore, we hypothesized that CsA affects KCC2 or NKCC1 functions, thus causing neuronal hyperexcitability.In ex vivo intracellular recordings with sharp microelectrodes, Wistar rat pyramidal neurons in neocortical slices responded to CsA (5 μM for 1h) with a less negative GABAA reversal potential (+7.2 mV) and prolonged Cl− extrusion time after iontophoretic Cl− loading (+3.9 s). 2‐photon fluorescence lifetime imaging in presence of Cl− sensitive dye MQAE showed an increased [Cl−]i in layer V neurons (+6.9 mM), suggesting reduced KCC2 activity. Co‐immunoprecipitation studies in rodent brain tissue suggested interactions of calcineurin Aβ (CnAβ) with KCC2 and NKCC1.In vivo, CsA administration to rats (5–25 mg/kg i.p.) enhanced levels of inhibitory tyrosine KCC2 phosphorylation at short term (4h; +172%) and reduced levels of activating S940‐KCC2 phosphorylation in the long term (14d; −61%). Reduced phospho‐S940‐KCC2 levels were observed in further models of calcineurin inhibition such as genetic CnAβ‐deficiency in mice (−78%) or CsA treatment of zebrafish larvae (10 μM in water: −54% after 24h). In contrast, NKCC1 and its activating kinase SPAK were stimulated upon calcineurin inhibition in rodents. Similar data were obtained in Drosophila melanogaster.Our results provide evidence that CnI attenuate KCC2 function but may stimulate NKCC1 leading to elevated [Cl−]i. These effects may increase neuronal excitability and contribute to CnI neurological adverse effects. The benefits of KCC2 activators or SPAK inhibitors for enhancing chloride extrusion in patients experiencing CnI neurotoxicity deserve further characterization.Support or Funding InformationThis study was supported by Deutsche Forschergemeinschaft.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.