Blockade of the Mineralocorticoid Receptor in Sickle Transgenic Mice Reduces Circulating Protein Disulfide Isomerase and Gardos Channel Activity

Abstract 999

Endothelin-1 (ET-1), erythrocyte sickling and endothelial cell activation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have provided evidence for the use of ET-1 receptor antagonists in improving hematological parameters in two transgenic mouse models of SCD (Rivera A., 2008, Amer J Physiol). However, the mechanisms that mediate the interplay between red blood cells (RBC) and the endothelium in SCD remain unresolved. Activation of endothelial cells leads to, among other factors, increased levels of protein disulfide isomerase (PDI). PDI catalyzes disulfide interchange reactions, mediates redox modifications and has been observed to be up-regulated under hypoxic conditions. We now report that circulating PDI activity is increased in BERK sickle transgenic mice when compared to wild-type controls. The mineralocorticoid receptor (MR) is a member of the steroid family of nuclear receptors that function as a transcription factor that upon binding to the hormone responsive element of genes such as edn1, the gene for ET-1, leads to increased ET-1 expression. In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and kidney ET-1 mRNA expression. We hypothesized that MR blockade of BERK sickle transgenic mice would lead to reduced PDI activity and improved hematological parameters. Sickle mice were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR receptor antagonist, and tap water ad libitum for 14 days at which time the mice were sacrificed and tissues and blood collected. Plasma PDI activity was calculated by optimization of fluorescently labeled GSSG conversion to GSH. We observed that mice on eplerenone had significantly lower plasma PDI activity than mice on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units; P<0.005, n=6 and 9, respectively). We also studied RBC Gardos channel activity in these mice and observed a significant reduction in clotrimazole-sensitive K⁺ efflux following MR blockade (2.49±0.5 control and 1.37±0.3 mmol/10¹³ cells × hr; P<0.04 n= 5 and 7 respectively). MR blockade was associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7) as determined by an ADVIA 120 hematology analyzer. In contrast no significant effects on MCHC levels were observed under these conditions. We then studied ET-1 gene expression using quantitative RT-PCR with ABI Taqman chemistries and GAPDH and β-actin as endogenous controls. We observed that MR blockade was associated with reduced expression of ET-1 mRNA in heart tissue (0.654 ± 0.233, ΔΔCT, relative to mice on regular chow, P<0.04, n=5 and n=7) but not lung tissue. Western blot analyses in membranes from human and mouse sickle erythrocytes and endothelial cells revealed the presence of both MR and PDI proteins. We then studied the effects of ET-1 in early cultures of BERK mouse aortic endothelial cells (MAEC). We observed that stimulation of MAEC cells with 100nM ET-1 for 4 hr was associated with increased mRNA expression of PDI levels that was 1.71 fold greater than vehicle treated cells (n=4, P<0.05). Thus, our results suggest that MR blockade reduces ET-1 levels leading to reduced Gardos and PDI activity in Sickle mice. These effects on PDI activity and Gardos channel regulation may represent a novel mechanism for protective effects of MR blockade aimed at ameliorating vascular complications of Sickle Cell Disease. Supported by NIH R01HL090632 to AR.