Temperature-dependence of ⁸⁶Rb⁺ uptake.

<p>⁸⁶Rb⁺ uptake in the absence or presence of 10 µM bumetanide was assessed following a 60 min pre-incubation in isotonic basic (<i>open bars</i>) or hypotonic, low [Cl⁻] (<i>filled bars</i>) medium at either RT (A) or 37°C (B). Bumetanide-sensitive (B-S) ⁸⁶Rb⁺ uptakes are shown as mean ± s.e.m. (n = 9–14). In experiments designed specifically to assess the effects of increasing the temperature from RT to 37°C, ⁸⁶Rb⁺ uptake increased by: fNKCC2A, 343±13% (n = 4); fNKCC1, 509±22% (n = 3); and HEK-293, 150±49% (n = 3) under basal conditions; and by: 236±36% (n = 4), 106±17% (n = 5), and 145±31% (n = 3) following incubation in a hypotonic, low [Cl⁻] medium. Bumetanide-resistant ⁸⁶Rb⁺ uptakes at 37°C in basic medium were (nmol mg protein⁻¹ min⁻¹): fNKCC2A, 1.86±0.77 (n = 10); fNKCC1, 1.95±0.37 (n = 10); and HEK-293, 1.04±0.39 (n = 8) and did not change substantially under any condition tested.</p

Effects of phosphatase and kinase inhibitors on phosphorylation of fNKCC2A and fNKCC1.

<p>Experiments were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017992#pone-0017992-g004" target="_blank">Figure 4</a>, but without the addition of ⁸⁶Rb⁺. Cell lysates were subjected to SDS-PAGE, immunoblotted, and probed with antibodies against phospho-NKCC1 and 2 (NKCC-P, R5), NKCC2 (anti-NKCC2), NKCC1 and 2 (T4) and NKCC1 (N1). Representative blots are shown with control and drug-treated samples from the same blot. Estimated molecular mass is indicated to the right of each panel. (A) Effect of 0.25 µM calyculin A on monomeric NKCC. An approximate 20 kDa band-shift in calyculin-treated cells is only observed with the phospho-specific antibody R5 (<i>arrow</i>) but not with NKCC-P. (B) High molecular mass bands in calyculin A treated fNKCC2A cells. (C) Example of 20 kDa band-shift (<i>arrow</i>) in HEK-293 cells. Cells had been pre-incubated in basic medium, a Na⁺-free medium or with calyculin. (D) Effect of 50 µM PP1 and 120 µM genistein on monomeric NKCC. (E) High molecular mass bands in PP1 or genistein treated fNKCC2A cells.</p

Effects of inhibitors and changes to the ionic environment on cotransporter phosphorylation.

<p>Phosphorylation levels detected by antibodies R5 and NKCC-P expressed as factors of appropriate controls. Values are given as mean ± s.e.m. (n≥4) or as averages if n = 2.</p>a<p>Cells were exposed to ouabain for 35 min.</p>b<p>Cells were exposed to Na⁺-free medium for 5 min.</p>c<p>134 kDa and 274 kDa bands represent monomeric and dimeric fNKCC2A; 155 kDa bands, endogenous NKCC and fNKCC1. As antibodies R5 and NKCC-P detect similar changes in cotransporter phosphorylation data have been pooled.</p

⁸⁶Rb⁺ uptake after pre-incubation in Na⁺-free medium.

<p>Bumetanide-sensitive (B-S) ⁸⁶Rb⁺ uptake was measured at 37°C in confluent HEK-293 cells (<i>triangles</i>), fNKCC2A cells (<i>squares</i>) or fNKCC1 cells (<i>circles</i>). Cells were pre-incubated in a Na⁺-free (NMDG) medium for the times indicated with 0.1 mM ouabain present for the last 5 min. ⁸⁶Rb⁺ uptake was then measured in the presence of Na⁺. Points represent means ± s.e.m. (n = 5–11).</p

Effect of ouabain on ⁸⁶Rb⁺ uptake and phosphorylation of fNKCC2A and fNKCC1.

<p>(A) time-dependent activation of ⁸⁶Rb⁺ uptake by ouabain. Bumetanide-sensitive (B-S) ⁸⁶Rb⁺ uptakes were measured at 37°C following pre-incubation of cells in an isotonic medium containing 0.1 mM ouabain for the times indicated. Values are means ± s.e.m. (n = 4–7). <i>Circles</i> represent fNKCC1; <i>squares</i>, fNKCC2A; and <i>triangles</i>, control HEK-293 cells. Lines, fitted to the data by non-linear regression analysis, are of the form: y = R+S(1−exp(−kt)), where R is the initial, and R+S the maximum uptake, k is the rate constant and t, the time in min. (B) Effect of omitting Ca²⁺ from the medium during exposure to ouabain. Cells were exposed to 0.1 mM ouabain and ⁸⁶Rb⁺ uptake was measured in the presence (<i>open symbols</i>) or absence (<i>filled symbols</i>) of 1 mM Ca²⁺ in the medium. Ca²⁺-chelators were not used. (C) Lysates from cells exposed to ouabain (in the presence of Ca²⁺) for 30 min were immunoblotted and probed with antibodies against phospho-NKCC1 and 2 (NKCC-P, R5), NKCC2 (anti-NKCC2), NKCC1 and 2 (T4) and NKCC1 (N1). An indication of molecular mass is shown to the right of each panel. Similar results were obtained when the experiment was repeated.</p

Circulating Angiopoietin-2 and Its Soluble Receptor Tie-2 Concentrations Are Related to Renal Function in Two Population-Based Cohorts

<div><p>Background</p><p>An intact angiopoietin/Tie-2 ligand receptor system is indispensable for life. High circulating angiopoietin-2 (Ang-2) concentrations are strongly associated with kidney disease involving the progressive loss of glomerular filtration. The aim of our study was to investigate the associations between renal function and serum Ang-2 or serum Tie-2 concentrations in the general population.</p><p>Methods</p><p>Data of 3081 and 4088 subjects from two population-based studies, the Study of Health in Pomerania (SHIP-1) and SHIP-Trend, were used. Renal function was assessed by serum creatinine, cystatin C concentration, creatinine-based estimated glomerular filtration rate [eGFR(crea)], cystatin C-based eGFR [eGFR(cys)] and urinary albumin-to-creatinine ratio (uACR). Analyses of variance and linear regression models were calculated.</p><p>Results</p><p>In both cohorts, strong positive associations between serum cystatin C concentrations and serum Ang-2 or Tie-2 concentrations as well as inverse associations between eGFR(cys) and serum Ang-2 or Tie-2 concentrations were found. These relations were also present in a subpopulation without hypertension or diabetes mellitus type 2. Furthermore, we detected weak U-shaped associations between serum creatinine concentrations or eGFR(crea) and serum Ang-2 concentrations. With respect to uACR a strong positive association with serum Ang-2 concentrations was revealed.</p><p>Conclusion</p><p>Serum Ang-2 concentrations are strongly associated with sensitive parameters of renal impairment like serum cystatin C, uACR and eGFR(cys). These findings persisted even after exclusion of subjects with hypertension or diabetes mellitus type 2, conditions that predispose to chronic renal disease and are associated with increased Ang-2 concentrations. Interestingly, we did not detect the same strong relations between serum creatinine and eGFR(crea) with serum Ang-2 concentration. Additionally, significant association of serum Tie-2 concentrations with cystatin C and eGFR(cys) were detected.</p></div

Associations between markers of renal function and serum angiopoietin-2 concentration or serum Tie-2 concentration.

<p>Associations between markers of renal function and serum angiopoietin-2 concentration or serum Tie-2 concentration.</p

Baseline characteristics stratified by study population.

<p>Baseline characteristics stratified by study population.</p

Associations between serum creatinine concentration, creatinine-based estimated glomerular filtration rate [eGFR(crea)], serum cystatin C concentration or cystatin C-based eGFR [eGFR(cys)] and serum angiopoietin-2 (Ang-2) concentration in the SHIP-1 population.

<p>For each exposure left side: Estimated mean serum Ang-2 with 95% confidence intervals (CI) by sex-specific quartiles of exposure calculated by analysis of variance adjusted for age, sex and waist circumference. Right side: linear regression line with 95% CI (grey shaded area). Linear regression models with restricted cubic splines were adjusted for age, sex, waist circumference, smoking, total cholesterol, systolic and diastolic blood pressure and additionally in the whole population for diabetes mellitus type 2 and use of antihypertensive medication.</p

Histograms and boxplots of serum angiopoietin-2 and Tie-2 receptor concentrations by study sample (SHIP-1 and SHIP-Trend).

<p>Histograms and boxplots of serum angiopoietin-2 and Tie-2 receptor concentrations by study sample (SHIP-1 and SHIP-Trend).</p