The angiotensin receptor-associated protein Atrap is a stimulator of the cardiac Ca2+-ATPase SERCA2a.

AIMS: The angiotensin II type 1 receptor-associated protein (Atrap) is highly expressed in the heart, but its function in the heart is unknown. We hypothesized that cardiac Atrap may interact with proteins other than the AT1 receptor. METHODS AND RESULTS: To identify potential novel interacting partners of Atrap, pull-down assays were performed. Sequencing by MALDI-MS of the isolated complexes showed that Atrap interacts with the cardiac Ca(2+)-ATPase SERCA2a. The interaction between Atrap and SERCA2a was confirmed by co-immunoprecipitation and by surface plasmon resonance (SPR) spectroscopy. Atrap enhanced the SERCA-dependent Ca(2+) uptake in isolated SR membrane vesicles. Furthermore, sarcomere shortenings and [Ca(2+)]i transients (CaTs) were determined in ventricular myocytes isolated from Atrap-/- and wild-type (WT) mice. The amplitudes of CaTs and sarcomere shortenings were similar in Atrap-/- and WT myocytes. However, the CaT decay and sarcomere re-lengthening were prolonged in Atrap-/- myocytes. To further evaluate the functional relevance of the Atrap-SERCA2a interaction in vivo, left-ventricular function was assessed in WT and Atrap-/- mice. The heart rates (564 ± 10 b.p.m. vs. 560 ± 11 b.p.m.; P = 0.80) and ejection fractions (71.3 ± 1.3 vs. 72 ± 1.8%; P = 0.79) were similar in WT and Atrap-/- mice, respectively (n = 15 for each genotype). However, the maximum filling rate (dV/dtmax) was markedly decreased in Atrap-/- (725 ± 48 µL/s) compared with WT mice (1065 ± 122 µL/s; P = 0.01; n = 15). CONCLUSION: We identified Atrap as a novel regulatory protein of the cardiac Ca(2+)-ATPase SERCA2a. We suggest that Atrap enhances the activity of SERCA2a and, consequently, facilitates ventricular relaxation.

Inhibition of COX-1 attenuates the formation of thromboxane A2 and ameliorates the acute decrease in glomerular filtration rate in endotoxemic mice.

Thromboxane (Tx) A2 has been suggested to be involved in the development of sepsis-induced acute kidney injury (AKI). Therefore, we investigated the impact of cyclooxygenase (COX)-1 and COX-2 activity on lipopolysaccharide (LPS)-induced renal TxA2 formation, and on endotoxemia-induced AKI in mice. Injection of LPS (3 mg/kg ip) decreased glomerular filtration rate (GFR) and the amount of thrombocytes to ∼50% of basal values after 4 h. Plasma and renocortical tissue levels of TxB2 were increased ∼10- and 1.7-fold in response to LPS, respectively. The COX-1 inhibitor SC-560 attenuated the LPS-induced fall in GFR and in platelet count to ∼75% of basal levels. Furthermore, SC-560 abolished the increase in plasma and renocortical tissue levels of TxB2 in response to LPS. The COX-2 inhibitor SC-236 further enhanced the LPS-induced decrease in GFR to ∼40% of basal values. SC-236 did not alter thrombocyte levels nor the LPS-induced increase in plasma and renocortical tissue levels of TxB2. Pretreatment with clopidogrel inhibited the LPS-induced drop in thrombocyte count, but did not attenuate the LPS-induced decrease in GFR and the increase in plasma TxB2 levels. This study demonstrates that endotoxemia-induced TxA2 formation depends on the activity of COX-1. Our study further indicates that the COX-1 inhibitor SC-560 has a protective effect on the decrease in renal function in response to endotoxin. Therefore, our data support a role for TxA2 in the development of AKI in response to LPS.

The angiotensin II AT1 receptor-associated protein Arap1 is involved in sepsis-induced hypotension.

INTRODUCTION: Hypotension in septic patients results from hypovolemia, vasodilatation and hyporeactivity to vasoconstrictors, such as angiotensin II. The AT1 receptor-associated protein 1 (Arap1) is expressed in vascular smooth muscle cells and increases the surface expression of the AT1-receptor in vitro. We hypothesized that dysregulation of Arap1 may contribute to vascular hyporeactivity to angiotensin II during endotoxemia. METHODS: Arap1-deficient mice were used to assess the role of Arap1 in sepsis-induced hypotension. The isolated perfused kidney was used as an in vitro model to determine the relevance of Arap1 for vascular resistance and sensitivity to angiotensin II. RESULTS: During endotoxemia, mean arterial blood pressure (MAP) decreased in both genotypes, with the time course of sepsis-induced hypotension being markedly accelerated in Arap1-/- compared to +/+ mice. However, baseline MAP was similar in Arap1-/- and wildtype mice (102 ± 2 vs.103 ± 2 mmHg; telemetry measurements; n = 10; P = 0.66). Following lipopolysaccharide (LPS) injections (3 mg/kg), Arap1 expression was successively down-regulated in the wildtype mice, reaching levels below 10% of baseline expression. The endotoxemia-related decline in Arap1 expression could be recapitulated in cultured mesangial cells by incubation with pro-inflammatory cytokines, such as tumor necrosis factor α and interferon γ. Plasma renin concentration was increased in Arap1-/- mice compared to wildtype mice (66 ± 6 vs. 41 ± 4 ng AngI/ml/h; n = 23; P = 0.001), presumably contributing to preserved MAP under baseline conditions. The sensitivity of the vasculature to angiotensin II was reduced in Arap1-/- compared to +/+ mice, as determined in the isolated perfused kidney. CONCLUSIONS: Our data suggest that down-regulation of Arap1 expression during sepsis contributes to the development of hypotension by causing reduced vascular sensitivity to angiotensin II.

Loss of WNK3 is compensated for by the WNK1/SPAK axis in the kidney of the mouse.

WNK3 kinase is expressed throughout the nephron and acts as a positive regulator of NKCC2 and NCC in vitro. Here we addressed the in vivo relevance of WNK3 using WNK3-deficient mice. WNK3-/- mice were viable and showed no gross abnormalities. The net tubular function was similar in wild-type (WT) and WNK3-/- mice as assessed by determination of 24-h urine output (1.63 ± .06 in WT and 1.55 ± .1 ml in WNK3-/-, n=16; P=0.42) and ambient urine osmolarity (1,804 ± 62 in WT vs. 1,819 ± 61 mosmol/kg in WNK3-/-, n=40; P=0.86). Water restriction (48 h) increased urine osmolarity similarly in both genotypes to 3,440 ± 220 and 3,200 ± 180 mosmol/kg in WT and WNK3-/- mice, respectively (n=11; P=0.41). The glomerular filtration rate (343 ± 22 vs. 315 ± 13 ml/min), renal blood flow (1.35 ± 0.1 vs. 1.42 ± 0.04 ml), and plasma renin concentration (94 ± 18 vs. 80 ± 13 ng ANG I·ml(-1)·h(-1)) were similar between WT and WNK3-/- mice (n=13; P=0.54). WNK1 was markedly upregulated in WNK3-deficient mice, whereas the expression of WNK4 was similar in both genotypes. When the mice were fed a salt-restricted diet [0.02% NaCl (wt/wt)] the levels of pSPAK/OSR1, pNKCC2, and pNCC were enhanced in both genotypes compared with the baseline conditions, with the levels in WNK3-/- exceeding those in WT mice. The upregulation of pSPAK/OSR1, pNKCC2, and pNCC in WNK3-/- mice relative to the levels in WT mice when fed a low-salt diet was paralleled by an increased diuresis in response to hydrochlorothiazide. In summary, the overall relevance of WNK3 for the renal reabsorption of NaCl appears to be limited and can be largely compensated for by the activation of WNK3-independent pathways. Consequently, our data suggest that WNK3 may serve as a member of a kinase network that facilitates the fine-tuning of renal transepithelial NaCl transport.

Angiotensin AT1 receptor-associated protein Arap1 in the kidney vasculature is suppressed by angiotensin II.

Arap1 is a protein that interacts with angiotensin II type 1 (AT(1)) receptors and facilitates increased AT(1) receptor surface expression in vitro. In the present study, we assessed the tissue localization and regulation of Arap1 in vivo. Arap1 was found in various mouse organs, with the highest expression in the heart, kidney, aorta, and adrenal gland. Renal Arap1 protein was restricted to the vasculature and to glomerular mesangial cells and was absent from tubular epithelia. A similar localization was found in human kidneys. To test the hypothesis that angiotensin II may control renal Arap1 expression, mice were subjected to various conditions to alter the activity of the renin-angiotensin system. A high-salt diet (4% NaCl, 7 days) upregulated Arap1 expression in mice by 47% compared with controls (0.6% NaCl, P = 0.03). Renal artery stenosis (7 days) or water restriction (48 h) suppressed Arap1 levels compared with controls (-64 and -62% in the clipped and contralateral kidney, respectively; and -50% after water restriction, P < 0.01). Angiotensin II infusion (2 µg·kg(-1)·min(-1), 7 days) reduced Arap1 mRNA levels compared with vehicle by 29% (P < 0.01), whereas AT(1) antagonism (losartan, 30 mg·kg(-1)·day(-1), 7 days) enhanced Arap1 mRNA expression by 52% (P < 0.01); changes in mRNA were paralleled by Arap1 protein abundance. Experiments with hydralazine and epithelial nitric oxide synthase-/- mice further suggested that Arap1 expression changed in parallel with angiotensin II, rather than with blood pressure per se. Similar to in vivo, Arap1 mRNA and protein were suppressed by angiotensin II in a time- and dose-dependent manner in cultured mesangial cells. In summary, Arap1 is highly expressed in the renal vasculature, and its expression is suppressed by angiotensin II. Thus Arap1 may serve as a local modulator of vascular AT(1) receptor function in vivo.