SPAK Differentially Mediates Vasopressin Effects on Sodium Cotransporters

Activation of the Na⁺-K⁺-2Cl⁻-cotransporter (NKCC2) and the Na⁺-Cl⁻-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 receptor–specific 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 Na⁺-K⁺-2Cl⁻-cotransporter (NKCC2) of the thick ascending limb (TAL) and the thiazide-sensitive Na⁺-Cl⁻-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 kidney-specific OSR1-deficient mice: deletion of SPAK primarily impairs the function of NCC, whereas deletion of OSR1 negatively affects NKCC2.^9–11 The complex functional properties of a WNK-SPAK/OSR1-N(K)CC interaction cascade are currently being defined.¹² Recently, arginine vasopressin (AVP), signaling via the V2 receptor (V2R), has been identified as an efficient activator of both cotransporters, affecting their luminal trafficking and phosphorylation.^13–18 Because plasma AVP levels may vary not only with the sleep-wake cycle or long-term physiologic challenges, but also with pulsatile changes over the short term, distinct, time-dependent responses may occur.¹⁹ SPAK and OSR1 are well placed to regulate distal NaCl reabsorption in response to AVP. Here we tested the role of SPAK in AVP-induced activation of NKCC2 and NCC, acutely and during long-term treatment. The results suggest that SPAK is an essential kinase that modulates distal nephron function in response to AVP stimulation.     

Hyperkalemic hypertension–associated cullin 3 promotes WNK signaling by degrading KLHL3

Familial hyperkalemic hypertension (FHHt) is a monogenic disease resulting from mutations in genes encoding WNK kinases, the ubiquitin scaffold protein cullin 3 (CUL3), or the substrate adaptor kelch-like 3 (KLHL3). Disease-associated CUL3 mutations abrogate WNK kinase degradation in cells, but it is not clear how mutant forms of CUL3 promote WNK stability. Here, we demonstrated that an FHHt-causing CUL3 mutant (CUL3 Δ403–459) not only retains the ability to bind and ubiquitylate WNK kinases and KLHL3 in cells, but is also more heavily neddylated and activated than WT CUL3. In cells, activated CUL3 Δ403–459 depleted KLHL3, preventing WNK degradation, despite increased CUL3-mediated WNK ubiquitylation; therefore, CUL3 loss in kidney should phenocopy FHHt in murine models. As predicted, nephron-specific deletion of Cul3 in mice did increase WNK kinase levels and the abundance of phosphorylated Na-Cl cotransporter (NCC). Over time, however, Cul3 deletion caused renal dysfunction, including hypochloremic alkalosis, diabetes insipidus, and salt-sensitive hypotension, with depletion of sodium potassium chloride cotransporter 2 and aquaporin 2. Moreover, these animals exhibited renal inflammation, fibrosis, and increased cyclin E. These results indicate that FHHt-associated CUL3 Δ403–459 targets KLHL3 for degradation, thereby preventing WNK degradation, whereas general loss of CUL3 activity — while also impairing WNK degradation — has widespread toxic effects in the kidney.     

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

The furosemide-sensitive Na⁺-K⁺-2Cl⁻-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 Aβ 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 A-type 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 Aβ 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 Aβ. Cyclosporine administration rapidly normalized the abundance of phospho-NKCC2 in SORLA-deficient mice, and a functional interaction between calcineurin Aβ and SORLA was further corroborated by binding assays in rat kidney extracts. In summary, we have shown that calcineurin Aβ and SORLA are key components in the phosphoregulation of NKCC2. These results may have clinical implications for immunosuppressive therapy using calcineurin inhibitors.     

Long-Term Toxicity of Gadolinium to the Freshwater Crustacean Daphnia magna

The lanthanides are considered emerging contaminants but information on their long-term toxicity to aquatic species under environmentally relevant conditions is scarce. We aimed to fill this gap by evaluating the long-term adverse effects of gadolinium on the freshwater model—crustacean Daphnia magna. The exposure of D. magna for up to 39 days to 0.1 mg Gd/L (a 21-days chronic toxicity NOEC value derived by us formerly) in the lake water had no negative effect (p?>?0.05) on vitality, size and reproduction of parent animals as well as their offspring. Thus, assumingly the current Gd contamination levels of surface waters pose no hazard to aquatic crustaceans that in general are very sensitive to various pollutants. Moreover, presence of 0.1 mg Gd/L in the lake water even mitigated the long-term toxic effect of 0.2 mg Ni/L (studied as a model co-contaminant) to D. magna’s vitality and productivity.

     

Molecular analysis of varicella vaccines and varicella-zoster virus from vaccine-related skin lesions

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.