The Dual Endothelin Receptor Antagonist Bosentan Prevents the Acute Sickle Cell-Related Hypoxic Lung and Kidney Injury in Transgenic SAD Mice.

Vaso-occlusive (VOC) crisis is a major manifestation of sickle cell disease (SCD) leading to acute organ damages. Increased levels of endothelin-1 (ET-1), a potent vasoconstrictor peptide, have been reported in SCD patients. In addition, ETB receptor has been identified in red blood cells (RBCs) membrane and in vitro, ET-1 increases the activity of Gardos channel in sickle RBCs, which is important in generation of dehydrated sickle RBCs. First, we evaluated the effects of the dual-endothelin-receptor (ETR) antagonist bosentan in vitro on mouse RBCs from transgenic SAD mice and in vivo on hypoxic model of acute sickle VOCs. ET-1 (500 nM) significantly increased Gardos channel activity in SAD mouse RBCs, this was prevented by bosentan (10 μM). We next evaluated the in vivo effects of bosentan (100 mg/kg/d by gavage for 14 days) in both control and SAD mice. Under normoxic condition, bosentan did not modify either RBCs density profiles or RBCs K⁺ content in SAD mice, suggesting that ET-1 may not contribute to sickle cell dehydration in the steady state. The bosentan-treated mice were then exposed to hypoxia (8%, 18 hrs). We first evaluated the cardiac output and renal artery blood flow (RBF) by ultrasound color imaging. In SAD mice only, hypoxia induced a marked reduction in RBF (− 40%; p<0.01, n=10) and in the time-average mean blood velocities (p<0.01) compared to basal values. Such changes were not explained by changes in cardiac output. Infusion of bosentan reversed the increase in renal vascular resistance by 50% (p<0.01) within 5 minutes, suggesting an ET-1-dependent vasoconstriction on the SCD renal vasculature. In a third set of experiments, we evaluated the effect of bosentan on experimental lung and kidney VOC-induced damages. Normal and SAD mice were exposed to 46 hrs hypoxia (8%) followed by 2 hrs recovery under normoxia. Mice were treated with either vehicle or bosentan (100 mg/kg/d, starting one week before hypoxia). We evaluated RBF and cardiac output, complete blood counts, RBCs density profiles and cation content, bronchoalveolar fluid leukocyte counts, kidney and lung histopathology. In SAD mice exposed to hypoxia, bosentan ameliorated

• RBCs dehydration;

• the reduction in RBF;

• congestion of the renal microcirculation and injury;

• lung injury and pulmonary vessel constriction;

• renal and pulmonary leukocytic infiltrates.

In conclusion, ETR blockade was beneficial by preventing RBC dehydration in vivo with protection from kidney and lung injury. In addition, we provide the first evidence that VOC is due not only to cell entrapment within the microvasculature but also to marked ET-dependent vasoconstriction. These data provide new insights into the possible use of bosentan in the treatment of acute VOCs in SCD.