A SPAK Isoform Switch Modulates Renal Salt Transport and Blood Pressure

SummaryThe renal thick ascending limb (TAL) and distal convoluted tubule (DCT) play central roles in salt homeostasis and blood pressure regulation. An emerging model suggests that bumetanide- and thiazide-sensitive NaCl transporters (NKCC2 and NCC) along these segments are phosphorylated and activated by WNK kinases, via SPAK and OSR1. Here, we show that a kidney-specific SPAK isoform, which lacks the kinase domain, inhibits phosphorylation of NCC and NKCC2 by full-length SPAK in vitro. Kidney-specific SPAK is highly expressed along the TAL, whereas full-length SPAK is more highly expressed along the DCT. As predicted from the differential expression, SPAK knockout in animals has divergent effects along TAL and DCT, with increased phosphorylated NKCC2 along TAL and decreased phosphorylated NCC along DCT. In mice, extracellular fluid volume depletion shifts SPAK isoform abundance to favor NaCl retention along both segments, indicating that a SPAK isoform switch modulates sodium avidity along the distal nephron

Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter

Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms1, 2. Interruption of IL18 action reduces atherosclerosis in mice3, 4. Here, we show that absence of the IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E–deficient (Apoe−/−) mice, nor does it affect IL18 cell surface binding to or signaling in endothelial cells. As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney5. NCC is expressed in atherosclerotic lesions, where it colocalizes with IL18r. In Apoe−/− mice, combined deficiency of IL18r and NCC, but not single deficiency of either protein, protects mice from atherosclerosis. Peritoneal macrophages from Apoe−/− mice or from Apoe−/− mice lacking IL18r or NCC show IL18 binding and induction of cell signaling and cytokine and chemokine expression, but macrophages from Apoe−/− mice with combined deficiency of IL18r and NCC have a blunted response. An interaction between NCC and IL18r on macrophages was detected by co-immunoprecipitation. IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression. This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis

Sputum biomarkers and the prediction of clinical outcomes in patients with cystic fibrosis.

Lung function, acute pulmonary exacerbations (APE), and weight are the best clinical predictors of survival in cystic fibrosis (CF); however, underlying mechanisms are incompletely understood. Biomarkers of current disease state predictive of future outcomes might identify mechanisms and provide treatment targets, trial endpoints and objective clinical monitoring tools. Such CF-specific biomarkers have previously been elusive. Using observational and validation cohorts comprising 97 non-transplanted consecutively-recruited adult CF patients at the Intermountain Adult CF Center, University of Utah, we identified biomarkers informative of current disease and predictive of future clinical outcomes. Patients represented the majority of sputum producers. They were recruited March 2004-April 2007 and followed through May 2011. Sputum biomarker concentrations were measured and clinical outcomes meticulously recorded for a median 5.9 (interquartile range 5.0 to 6.6) years to study associations between biomarkers and future APE and time-to-lung transplantation or death. After multivariate modeling, only high mobility group box-1 protein (HMGB-1, mean=5.84 [log ng/ml], standard deviation [SD] =1.75) predicted time-to-first APE (hazard ratio [HR] per log-unit HMGB-1=1.56, p-value=0.005), number of future APE within 5 years (0.338 APE per log-unit HMGB-1, p<0.001 by quasi-Poisson regression) and time-to-lung transplantation or death (HR=1.59, p=0.02). At APE onset, sputum granulocyte macrophage colony stimulating factor (GM-CSF, mean 4.8 [log pg/ml], SD=1.26) was significantly associated with APE-associated declines in lung function (-10.8 FEV(1)% points per log-unit GM-CSF, p<0.001 by linear regression). Evaluation of validation cohorts produced similar results that passed tests of mutual consistency. In CF sputum, high HMGB-1 predicts incidence and recurrence of APE and survival, plausibly because it mediates long-term airway inflammation. High APE-associated GM-CSF identifies patients with large acute declines in FEV(1)%, possibly providing a laboratory-based objective decision-support tool for determination of an APE diagnosis. These biomarkers are potential CF reporting tools and treatment targets for slowing long-term progression and reducing short-term severity

Sympathetic stimulation of thiazide-sensitive sodium chloride cotransport in the generation of salt-sensitive hypertension

Excessive renal efferent sympathetic nerve activity contributes to hypertension in many circumstances. Although both hemodynamic and tubular effects likely participate, most evidence supports a major role for α-adrenergic receptors in mediating the direct epithelial stimulation of sodium retention. Recently, it was reported, however, that norepinephrine activates the thiazide-sensitive NaCl cotransporter (NCC) by stimulating β-adrenergic receptors. Here, we confirmed this effect and developed an acute adrenergic stimulation model to study the signaling cascade. The results show that norepinephrine increases the abundance of phosphorylated NCC rapidly (161% increase), an effect largely dependent on β-adrenergic receptors. This effect is not mediated by the activation of angiotensin II receptors. We used immunodissected mouse distal convoluted tubule to show that distal convoluted tubule cells are especially enriched for β₁-adrenergic receptors, and that the effects of adrenergic stimulation can occur ex vivo (79% increase), suggesting they are direct. Because the 2 protein kinases, STE20p-related proline- and alanine-rich kinase (encoded by STK39) and oxidative stress-response kinase 1, phosphorylate and activate NCC, we examined their roles in norepinephrine effects. Surprisingly, norepinephrine did not affect STE20p-related proline- and alanine-rich kinase abundance or its localization in the distal convoluted tubule; instead, we observed a striking activation of oxidative stress-response kinase 1. We confirmed that STE20p-related proline- and alanine-rich kinase is not required for NCC activation, using STK39 knockout mice. Together, the data provide strong support for a signaling system involving β₁-receptors in the distal convoluted tubule that activates NCC, at least in part via oxidative stress-response kinase 1. The results have implications about device- and drug-based treatment of hypertension

A SPAK isoform switch modulates renal salt transport and blood pressure

The renal thick ascending limb (TAL) and distal convoluted tubule (DCT) play central roles in salt homeostasis and blood pressure regulation. An emerging model suggests that bumetanide- and thiazide-sensitive NaCl transporters (NKCC2 and NCC) along these segments are phosphorylated and activated by WNK kinases, via SPAK and OSR1. Here, we show that a kidney-specific SPAK isoform, which lacks the kinase domain, inhibits phosphorylation of NCC and NKCC2 by full-length SPAK in vitro. Kidney-specific SPAK is highly expressed along the TAL, whereas full-length SPAK is more highly expressed along the DCT. As predicted from the differential expression, SPAK knockout in animals has divergent effects along TAL and DCT, with increased phosphorylated NKCC2 along TAL and decreased phosphorylated NCC along DCT. In mice, extracellular fluid volume depletion shifts SPAK isoform abundance to favor NaCl retention along both segments, indicating that a SPAK isoform switch modulates sodium avidity along the distal nephron

Patient Characteristics^a.

a<p>Results are median (interquartile range) unless noted.</p>b<p>This group of patients provided two samples each, one from a stable state and one from an APE state at admission for a hospitalization. Data shown here are derived from the time point of the stable sample collection for each individual.</p>c<p>The 26 patients that gave paired samples necessarily suffered an APE during the study in order to give the necessary APE state sputums. This criterion selected patients with significantly lower lung function, <i>t</i>-test <i>p</i> = 0.005, increased incidence of CF-related diabetes, χ-square <i>p</i><0.001, decreased 5-year predicted survival, <i>t</i>-test <i>p</i> = 0.01 and more frequent APE (differences not tested due to confounding) than the other patients in the study.</p>d<p>Patients in Validation Group 1 had higher FEV₁% and 5-year predicted survival and remarkably no incidence of CF-related diabetes. Despite these differences, the coefficients for HMGB-1 reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone-0042748-t007" target="_blank">Table 7</a> for Validation Groups 1 and 2 are quite similar to those for Study Group 1 patients and pass testing for mutual consistency.</p>e<p>The 5-year predicted survival is a clinically useful composite estimate of overall disease state in CF but may be difficult to use in interpretation of inflammatory states. Similar to lung function and other clinical markers of disease, it may require years to see a change <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone.0042748-Liou1" target="_blank">[2]</a>.</p

Kaplan-Meier Curves for the Time from Stable Sputum Collection to First Event.

<p>The curves illustrate the difference in time to A) first APE and B) death or censoring by listing for lung transplantation for patients with HMGB-1 measurements higher and lower than the value of 6.0 (log ng/ml). The value is the rounded median of the actual HMGB-1 data for both the 26 patients in A and the 76 patients in B. P-values shown are the results of log rank testing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone.0042748-Peto1" target="_blank">[52]</a>. These graphs show the results of evaluation of HMGB-1 simplified to high or low values, which are consistent with the proportional hazards modeling <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone.0042748-Cox1" target="_blank">[29]</a> of the effects of HMGB-1 as a continuous variable. Models were tested for consistency with proportionality <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone.0042748-Grambsch1" target="_blank">[31]</a> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone-0042748-t006" target="_blank">Table 6</a>).</p