Blockade of the mineralocorticoid receptor improves markers of human endothelial cell dysfunction and hematological indices in a mouse model of sickle cell disease.

Increased endothelin-1 (ET-1) levels in patients with sickle cell disease (SCD) and transgenic mouse models of SCD contribute to disordered hematological, vascular, and inflammatory responses. Mineralocorticoid receptor (MR) activation by aldosterone, a critical component of the Renin-Angiotensin-Aldosterone-System, modulates inflammation and vascular reactivity, partly through increased ET-1 expression. However, the role of MR in SCD remains unclear. We hypothesized that MR blockade in transgenic SCD mice would reduce ET-1 levels, improve hematological parameters, and reduce inflammation. Berkeley SCD (BERK) mice, a model of severe SCD, were randomized to either sickle standard chow or chow containing the MR antagonist (MRA), eplerenone (156 mg/Kg), for 14 days. We found that MRA treatment reduced ET-1 plasma levels (p = .04), improved red cell density gradient profile (D50 ; p < .002), and increased mean corpuscular volume in both erythrocytes (p < .02) and reticulocytes (p < .024). MRA treatment also reduced the activity of the erythroid intermediate-conductance Ca2+ -activated K+ channel - KCa 3.1 (Gardos channel, KCNN4), reduced cardiac levels of mRNAs encoding ET-1, Tumor Necrosis Factor Receptor-1, and protein disulfide isomerase (PDI) (p < .01), and decreased plasma PDI and myeloperoxidase activity. Aldosterone (10-8 M for 24 h in vitro) also increased PDI mRNA levels (p < .01) and activity (p < .003) in EA.hy926 human endothelial cells, in a manner blocked by pre-incubation with the MRA canrenoic acid (1 µM; p < .001). Our results suggest a novel role for MR activation in SCD that may exacerbate SCD pathophysiology and clinical complications.

Magnesium homeostasis in deoxygenated sickle erythrocytes is modulated by endothelin-1 via Na+ /Mg2+ exchange.

Painful crises in sickle cell disease (SCD) are associated with increased plasma cytokines levels, including endothelin-1 (ET-1). Reduced red cell magnesium content, mediated in part by increased Na+ /Mg2+ exchanger (NME) activity, contributes to erythrocyte K+ loss, dehydration and sickling in SCD. However, the relationship between ET-1 and the NME in SCD has remained unexamined. We observed increased NME activity in sickle red cells incubated in the presence of 500 nM ET-1. Deoxygenation of sickle red cells, in contrast, led to decreased red cell NME activity and cellular dehydration that was reversed by the NME inhibitor, imipramine. Increased NME activity in sickle red cells was significantly blocked by pre-incubation with 100 nM BQ788, a selective blocker of ET-1 type B receptors. These results suggest an important role for ET-1 and for cellular magnesium homeostasis in SCD. Consistent with these results, we observed increased NME activity in sickle red cells of three mouse models of sickle cell disease greater than that in red cells of C57BL/J6 mice. In vivo treatment of BERK sickle transgenic mice with ET-1 receptor antagonists reduced red cell NME activity. Our results suggest that ET-1 receptor blockade may be a promising therapeutic approach to control erythrocyte volume and magnesium homeostasis in SCD and may thus attenuate or retard the associated chronic inflammatory and vascular complications of SCD.

The erythroid K-Cl cotransport inhibitor [(dihydroindenyl)oxy]acetic acid blocks erythroid Ca2+-activated K+ channel KCNN4.

Red cell volume is a major determinant of HbS concentration in sickle cell disease. Cellular deoxy-HbS concentration determines the delay time, the interval between HbS deoxygenation and deoxy-HbS polymerization. Major membrane transporter protein determinants of sickle red cell volume include the SLC12/KCC K-Cl cotransporters KCC3/SLC12A6 and KCC1/SLC12A4, and the KCNN4/KCa3.1 Ca2+-activated K+ channel (Gardos channel). Among standard inhibitors of KCC-mediated K-Cl cotransport, only [(dihydroindenyl)oxy]acetic acid (DIOA) has been reported to lack inhibitory activity against the related bumetanide-sensitive erythroid Na-K-2Cl cotransporter NKCC1/SLC12A2. DIOA has been often used to inhibit K-Cl cotransport when studying the expression and regulation of other K+ transporters and K+ channels. We report here that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also abrogate activity of the KCNN4/KCa3.1 Gardos channel in human and mouse red cells and in human sickle red cells. DIOA inhibition of A23187-stimulated erythroid K+ uptake (Gardos channel activity) was chloride-independent and persisted in mouse red cells genetically devoid of the principal K-Cl cotransporters KCC3 and KCC1. DIOA also inhibited YODA1-stimulated, chloride-independent erythroid K+ uptake. In contrast, DIOA exhibited no inhibitory effect on K+ influx into A23187-treated red cells of Kcnn4-/- mice. DIOA inhibition of human KCa3.1 was validated (IC50 42 µM) by whole cell patch clamp in HEK-293 cells. RosettaLigand docking experiments identified a potential binding site for DIOA in the fenestration region of human KCa3.1. We conclude that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also block the KCNN4/KCa3.1 Gardos channel in normal and sickle red cells.

Activation of 2-oxoglutarate receptor 1 (OXGR1) by α-ketoglutarate (αKG) does not detectably stimulate Pendrin-mediated anion exchange in Xenopus oocytes.

SLC26A4/Pendrin is the major electroneutral Cl- /HCO3- exchanger of the apical membrane of the Type B intercalated cell (IC) of the connecting segment (CNT) and cortical collecting duct (CCD). Pendrin mediates both base secretion in response to systemic base load and Cl- reabsorption in response to systemic volume depletion, manifested as decreased nephron salt and water delivery to the distal nephron. Pendrin-mediated Cl- /HCO3- exchange in the apical membrane is upregulated through stimulation of the ß-IC apical membrane G protein-coupled receptor, 2-oxoglutarate receptor 1 (OXGR1/GPR99), by its ligand α-ketoglutarate (αKG). αKG is both filtered by the glomerulus and lumenally secreted by proximal tubule apical membrane organic anion transporters (OATs). OXGR1-mediated regulation of Pendrin by αKG has been documented in transgenic mice and in isolated perfused CCD. However, aspects of the OXGR1 signaling pathway have remained little investigated since its original discovery in lymphocytes. Moreover, no ex vivo cellular system has been reported in which to study the OXGR1 signaling pathway of Type B-IC, a cell type refractory to survival in culture in its differentiated state. As Xenopus oocytes express robust heterologous Pendrin activity, we investigated OXGR1 regulation of Pendrin in oocytes. Despite functional expression of OXGR1 in oocytes, co-expression of Pendrin and OXGR1 failed to exhibit αKG-sensitive stimulation of Pendrin-mediated Cl- /anion exchange under a wide range of conditions. We conclude that Xenopus oocytes lack one or more essential molecular components or physical conditions required for OXGR1 to regulate Pendrin activity.

Dysregulated Erythroid Mg2+ Efflux in Type 2 Diabetes.

Hyperglycemia is associated with decreased Mg2+ content in red blood cells (RBC), but mechanisms remain unclear. We characterized the regulation of Mg2+ efflux by glucose in ex vivo human RBC. We observed that hemoglobin A1C (HbA1C) values correlated with Na+-dependent Mg2+ efflux (Na+/Mg2+ exchange) and inversely correlated with cellular Mg content. Treatment of cells with 50 mM D-glucose, but not with sorbitol, lowered total cellular Mg (2.2 ± 0.1 to 2.0 ± 0.1 mM, p < 0.01) and enhanced Na+/Mg2+ exchange activity [0.60 ± 0.09 to 1.12 ± 0.09 mmol/1013 cell × h (flux units, FU), p < 0.05]. In contrast, incubation with selective Src family kinase inhibitors PP2 or SU6656 reduced glucose-stimulated exchange activation (p < 0.01). Na+/Mg2+ exchange activity was also higher in RBC from individuals with type 2 diabetes (T2D, 1.19 ± 0.13 FU) than from non-diabetic individuals (0.58 ± 0.05 FU, p < 0.01). Increased Na+/Mg2+ exchange activity in RBC from T2D subjects was associated with lower intracellular Mg content. Similarly increased exchange activity was evident in RBC from the diabetic db/db mouse model as compared to its non-diabetic control (p < 0.03). Extracellular exposure of intact RBC from T2D subjects to recombinant peptidyl-N-glycosidase F (PNGase F) reduced Na+/Mg2+ exchange activity from 0.98 ± 0.14 to 0.59 ± 0.13 FU (p < 0.05) and increased baseline intracellular Mg content (1.8 ± 0.1 mM) to normal values (2.1 ± 0.1 mM, p < 0.05). These data suggest that the reduced RBC Mg content of T2D RBC reflects enhanced RBC Na+/Mg2+ exchange subject to regulation by Src family kinases and by the N-glycosylation state of one or more membrane proteins. The data extend our understanding of dysregulated RBC Mg2+ homeostasis in T2D.

Purinergic signaling is essential for full Psickle activation by hypoxia and by normoxic acid pH in mature human sickle red cells and in vitro-differentiated cultured human sickle reticulocytes.

Paracrine ATP release by erythrocytes has been shown to regulate endothelial cell function via purinergic signaling, and this erythoid-endothelial signaling network is pathologically dysregulated in sickle cell disease. We tested the role of extracellular ATP-mediated purinergic signaling in the activation of Psickle, the mechanosensitive Ca2+-permeable cation channel of human sickle erythrocytes (SS RBC). Psickle activation increases intracellular [Ca2+] to stimulate activity of the RBC Gardos channel, KCNN4/KCa3.1, leading to cell shrinkage and accelerated deoxygenation-activated sickling.We found that hypoxic activation of Psickle recorded by cell-attached patch clamp in SS RBC is inhibited by extracellular apyrase, which hydrolyzes extracellular ATP. Hypoxic activation of Psickle was also inhibited by the pannexin-1 inhibitor, probenecid, and by the P2 antagonist, suramin. A Psickle-like activity was also activated in normoxic SS RBC (but not in control red cells) by bath pH 6.0. Acid-activated Psickle-like activity was similarly blocked by apyrase, probenecid, and suramin, as well as by the Psickle inhibitor, Grammastola spatulata mechanotoxin-4 (GsMTx-4).In vitro-differentiated cultured human sickle reticulocytes (SS cRBC), but not control cultured reticulocytes, also exhibited hypoxia-activated Psickle activity that was abrogated by GsMTx-4. Psickle-like activity in SS cRBC was similarly elicited by normoxic exposure to acid pH, and this acid-stimulated activity was nearly completely blocked by apyrase, probenecid, and suramin, as well as by GsMTx-4.Thus, hypoxia-activated and normoxic acid-activated cation channel activities are expressed in both SS RBC and SS cRBC, and both types of activation appear to be mediated or greatly amplified by autocrine or paracrine purinergic signaling.

Trpv1 and Trpa1 are not essential for Psickle-like activity in red cells of the SAD mouse model of sickle cell disease.

The molecular identity of Psickle, the deoxygenation-activated cation conductance of the human sickle erythrocyte, remains unknown. We observed in human sickle red cells that inhibitors of TRPA1 and TRPV1 inhibited Psickle, whereas a TRPV1 agonist activated a Psickle-like cation current. These observations prompted us to test the roles of TRPV1 and TRPA1 in Psickle in red cells of the SAD mouse model of sickle cell disease. We generated SAD mice genetically deficient in either TRPV1 or TRPA1. SAD;Trpv1-/- and SAD;Trpa1-/- mice were indistinguishable in appearance, hematological indices, and osmotic fragility from SAD mice. We found that deoxygenation-activated cation currents remained robust in SAD;Trpa1-/- and SAD;Trpv1-/- mice. In addition, 45Ca2+ influx into SAD mouse red cells during prolonged deoxygenation was not reduced in red cells from SAD;Trpa1-/- and SAD;Trpv1-/- mice. We conclude that the nonspecific cation channels TRPA1 and TRPV1 are not required for deoxygenation to stimulate Psickle-like activity in red cells of the SAD mouse model of sickle cell disease. (159).

Genetic disruption of KCC cotransporters in a mouse model of thalassemia intermedia.

ß-thalassemia (ß-Thal) is caused by defective ß-globin production leading to globin chain imbalance, aggregation of free alpha chain in developing erythroblasts, reticulocytes, and mature circulating red blood cells. The hypochromic thalassemic red cells exhibit increased cell dehydration in association with elevated K+ leak and increased K-Cl cotransport activity, each of which has been linked to globin chain imbalance and related oxidative stress. We therefore tested the effect of genetic inactivation of K-Cl cotransporters KCC1 and KCC3 in a mouse model of ß-thalassemia intermedia. In the absence of these transporters, the anemia of ß-Thal mice was ameliorated, in association with increased MCV and reductions in CHCM and hyperdense cells, as well as in spleen size. The resting K+ content of ß-Thal red cells was greatly increased, and Thal-associated splenomegaly slightly decreased. Lack of KCC1 and KCC3 activity in Thal red cells reduced red cell density and improved ß-Thal-associated osmotic fragility. We conclude that genetic inactivation of K-Cl cotransport can reverse red cell dehydration and partially attenuate the hematologic phenotype in a mouse model of ß-thalassemia.

Erythrocyte ion content and dehydration modulate maximal Gardos channel activity in KCNN4 V282M/+ hereditary xerocytosis red cells.

Hereditary xerocytosis (HX) is caused by missense mutations in either the mechanosensitive cation channel PIEZO1 or the Ca2+-activated K+ channel KCNN4. All HX-associated KCNN4 mutants studied to date have revealed increased current magnitude and red cell dehydration. Baseline KCNN4 activity was increased in HX red cells heterozygous for KCNN4 mutant V282M. However, HX red cells maximally stimulated by Ca2+ ionophore A23187 or by PMCA Ca2+-ATPase inhibitor orthovanadate displayed paradoxically reduced KCNN4 activity. This reduced Ca2+-stimulated mutant KCNN4 activity in HX red cells was associated with unchanged sensitivity to KCNN4 inhibitor senicapoc and KCNN4 activator Ca2+, with slightly elevated Ca2+ uptake and reduced PMCA activity, and with decreased KCNN4 activation by calpain inhibitor PD150606. The altered intracellular monovalent cation content of HX red cells prompted experimental nystatin manipulation of red cell Na and K contents. Nystatin-mediated reduction of intracellular K+ with corresponding increase in intracellular Na+ in wild-type cells to mimic conditions of HX greatly suppressed vanadate-stimulated and A23187-stimulated KCNN4 activity in those wild-type cells. However, conferral of wild-type cation contents on HX red cells failed to restore wild-type-stimulated KCNN4 activity to those HX cells. The phenotype of reduced, maximally stimulated KCNN4 activity was shared by HX erythrocytes expressing heterozygous PIEZO1 mutants R2488Q and V598M, but not by HX erythrocytes expressing heterozygous KCNN4 mutant R352H or PIEZO1 mutant R2456H. Our data suggest that chronic KCNN4-driven red cell dehydration and intracellular cation imbalance can lead to reduced KCNN4 activity in HX and wild-type red cells.

Increased Red Cell KCNN4 Activity in Sporadic Hereditary Xerocytosis Associated With Enhanced Single Channel Pressure Sensitivity of PIEZO1 Mutant V598M.

Supplemental Digital Content is available in the text.

An Intact Dissecting Baker's Cyst Mimicking Recurrent Deep Vein Thrombosis.

We report a case of a 75-year-old female with a history of acute deep vein thrombosis (DVT) 6 years ago who presented with unilateral calf swelling and pain. D-dimer was normal, and compression ultrasound revealed findings typical of DVT, including an incompressible dilated and hypoechoic peroneal vein. Despite 4 months of anticoagulation for supposed recurrent DVT, pain symptoms persisted and repeat D-dimer and compression ultrasound were unchanged. A magnetic resonance imaging scan to investigate the leg demonstrated a 6-cm dissecting Baker's cyst extending posterolaterally resulting in venous compression and distal dilation, which appeared to have been confused with a DVT. Ultrasound-guided aspiration of the cyst provided immediate and sustained relief. Herein we provide a review of the literature for the management of this rare scenario.

Novel Gardos channel mutations linked to dehydrated hereditary stomatocytosis (xerocytosis).

Dehydrated hereditary stomatocytosis (DHSt) is an autosomal dominant congenital hemolytic anemia with moderate splenomegaly and often compensated hemolysis. Affected red cells are characterized by a nonspecific cation leak of the red cell membrane, reflected in elevated sodium content, decreased potassium content, elevated MCHC and MCV, and decreased osmotic fragility. The majority of symptomatic DHSt cases reported to date have been associated with gain-of-function mutations in the mechanosensitive cation channel gene, PIEZO1. A recent study has identified two families with DHSt associated with a single mutation in the KCNN4 gene encoding the Gardos channel (KCa3.1), the erythroid Ca(2+) -sensitive K(+) channel of intermediate conductance, also expressed in many other cell types. We present here, in the second report of DHSt associated with KCNN4 mutations, two previously undiagnosed DHSt families. Family NA exhibited the same de novo missense mutation as that recently described, suggesting a hot spot codon for DHSt mutations. Family WO carried a novel, inherited missense mutation in the ion transport domain of the channel. The patients' mild hemolytic anemia did not improve post-splenectomy, but splenectomy led to no serious thromboembolic events. We further characterized the expression of KCNN4 in the mutated patients and during erythroid differentiation of CD34+ cells and K562 cells. We also analyzed KCNN4 expression during mouse embryonic development.

Evaluation of an automated digital imaging system, Nextslide Digital Review Network, for examination of peripheral blood smears.

CONTEXT: Several automated digital imaging systems have been introduced in recent years to improve turnaround time and proficiency in examining peripheral blood smears in hematology laboratories. OBJECTIVE: To evaluate a new automated digital imaging system, Nextslide Digital Review Network (Nextslide), for examination of peripheral blood smears. DESIGN: We evaluated 479 peripheral blood smears, of which 247 (51.6%) were included for comparison of Nextslide and manual white blood cell differential counts and morphology evaluation, 204 (42.6%) were included for comparison of Nextslide and CellaVision (DM96) differential counts, and 28 (5.8%) were neonatal samples examined for enumeration of nucleated red blood cells. RESULTS: Results from both method comparisons showed excellent correlation for all major white blood cell classes with correlation coefficients ranging from 0.70 to 0.99. Evaluation of white blood cell, red blood cell, and platelet morphology also showed good correlation among methods. White blood cell preclassification capability in the system was evaluated for rate and accuracy. Leukopenic samples demonstrated markedly decreased review time with Nextslide. Enumeration of nucleated red blood cells showed good correlation among methods. CONCLUSIONS: Our evaluation of Nextslide shows excellent correlation when compared with conventional manual differentials and CellaVision (DM96) differentials for evaluation of peripheral blood smears.

Early verification of myocardial ischemia with a novel biomarker of acute tissue damage: C-reactive protein fractional forms.

BACKGROUND: We evaluated the utility of an independent biomarker of early ischemic cellular damage-circulating fractional forms of C-reactive protein (fracCRP), to verify the diagnostic relevance of low Troponin I (TnI) values within the context of a workup for Acute Coronary Syndrome (ACS). METHODS: On a semi-preparative scale, the molecular characteristics of fracCRP were established by electron microscopy and Western Blot, using isolates captured from patient serum on phosphorylcholine beads and purified by size exclusion high-pressure liquid chromatography (SE-HPLC). Captured on an analytical scale, the diagnostic utility of fracCRP was evaluated in first-draw plasma specimens (total CRP not exceeding 6 mg/l) recovered from 300 cardiac emergency patients with final discharge diagnoses of ACS ruled out (N=132) or ruled in (N=168). RESULTS: At a cutoff value chosen for 97.7% test specificity, the test metric (fracCRP×TnI) identified in the first blood draw 39.9% of all emergency patients ultimately diagnosed with ACS, and 17.9% of ultimately diagnosed patients who arrived with TnI values within the normal reference range (0.01-0.04 ng/ml). CONCLUSIONS: These findings suggest that the fracCRP test metric could serve as a rule-in test for ACS in a significant proportion of low to moderate risk emergency patients.

Analytical validation of an HPLC assay for urinary albumin.

BACKGROUND: Microalbuminuria is the earliest clinical finding for renal disease. Diabetic individuals often produce modified forms of albumin, perhaps due to impaired lysosomal processing, that are undetectable by common immunoassays but accurately measured by HPLC. METHODS: We evaluated the performance of a commercially available, FDA-approved HPLC assay (AusAm Biotechnologies, NY) and compare results to our immunoturbidimetric assay (ITA, Beckman-Coulter, CA) using random urine specimens from 32 nondiabetic and 60 type 1 and 2 diabetic subjects. RESULTS: The HPLC assay was linear to 963 mg/l with a limit of detection of 6.1 mg/l. Within-run and between-run precision was <2% and 7-10%, respectively. Unpreserved urine was stable for at least 3 days at room temperature and 10 days at 4 degrees C. In both diabetic and nondiabetic subjects urinary albumin concentrations were higher by HPLC than by ITA, and many more diabetic and nondiabetic individuals were classified as microalbuminuric by HPLC than by ITA. The HPLC assay showed acceptable performance; however, because urinary albumin concentrations are higher in apparently healthy nondiabetic as well as diabetic subjects, different cutpoints will be necessary to accurately differentiate microalbuminuria. CONCLUSIONS: Prospective studies are necessary to determine whether the HPLC assay can effectively detect microalbuminuria earlier than current assays without a concomitant increase in the false positive rate.

Pulse-pressure-driven neutral lipid accumulation and correlative proinflammatory markers of accelerated atherogenesis.

In athero-prone areas of the human aorta, an accumulation of neutral lipids in the arterial intima precedes the arrival of monocytes and the initiation of the fatty streak. This study continues the investigation of a similar preinflammatory process in the coronary arteries of the sodium-sensitive hypertensive Dahl S rat, focusing on the hemodynamic forces associated with the neutral lipid influx and on concurrent changes in serum levels of certain proinflammatory markers. Animals were conditioned from weaning on a high-saturated fat, high-cholesterol diet until early adolescence (age 12 weeks), at which point dietary sodium was increased, and changes in blood pressure, serum analytes and histologic markers were tracked. The first half of the 12-day induction period was characterized by an immediate rise in pulse pressure that persisted above baseline. Serum levels of free monomeric C-reactive protein (mCRP) (as monomer/dimer ratio by Western blot densitometry) rose concurrently with pulse pressure and preceded the influx of neutral lipid into the arterial intima. Levels of oxidized LDL-cholesterol (oxLDL) closely tracked that of neutral lipid accumulation. In this model, elevated pulse pressure appears to drive the activation of circulating CRP and the influx of neutral lipids into the arterial intima, which leads to increased oxLDL levels.

Maturation-dependent acquired coronary structural alterations and atherogenesis in the Dahl sodium-sensitive hypertensive rat.

BACKGROUND: The Dahl sodium-sensitive hypertensive rat exhibits atherogenic lesions after the initiation of a high-sodium/high-fat diet. This study was designed to gauge the effect of a preadolescent high-fat diet on the postadolescent rate of atherogenesis after supplementation of the diet with sodium. METHODS AND RESULTS: Fifty-three Dahl S male rats were assigned to 2 dietary groups for the postweaning to early adolescence period (3 to 12 weeks): 29 to a standard diet (low-fat/low-sodium) and 24 to a high-fat/low-sodium diet. At age 9 weeks (just after puberty), animals from the high-fat group exhibited a relatively diminished density of coronary elastic fibers. There was no evidence of either lipid or monocytic infiltration of the subendothelial space. At age 12 weeks, most or all of the remaining animals in both groups were switched to a high-sodium/high-fat diet and were sampled through the following 8 weeks for the appearance of arterial lipid. After the switch, the high-fat-conditioned animals developed more extensive atherosclerotic pathological lesions more rapidly than their prepubertal standard-diet counterparts. The importance of the animal's stage of maturation in this effect was underscored by the observation that delaying onset of the high-fat diet to early adolescence resulted in no ultimate difference from the pubertal controls in elastic fiber density. CONCLUSIONS: The maturation-dependent high-fat conditioning of these postweanling rats correlated with an accelerated rate of atherogenesis on the initiation of the high-sodium/high-fat diet, possibly as a direct result of an alteration in arterial elasticity.