Heightened susceptibility of sickle cell trait mice to chlorine-induced multiorgan injury and the protective role of haptoglobin

Abstract Background: Sickle cell trait (SCT), found in 1 in 13 African Americans, is a carrier state of sickle cell disease marked by heterozygous hemoglobin S (AS). Though often asymptomatic, SCT individuals face increased risks of sudden death and multiorgan injury under environmental stressors such as excessive heat, high altitude, or strenuous exercise. These complications stem from acute hemolysis and rhabdomyolysis, which release free hemoglobin and myoglobin, overwhelming scavenging systems and causing oxidative damage. Humanized SCT mouse models replicate these vulnerabilities, evidenced by increased vascular injury and organ failure under stress. Given the global prevalence of SCT and the frequency of chlorine (Cl₂)-related industrial accidents, it is crucial to recognize SCT vulnerability, understand the mechanisms of organ injury, and identify protective strategies. We hypothesized that humanized SCT mice are more susceptible to multiorgan injury following Cl2inhalation, and that haptoglobin improves the outcomes. Methods: Humanized mice with SCT (AS) and normal hemoglobin (AA) were exposed to air or Cl₂ (500 ppm for 45 minutes) and returned to room air. Survival, organ injury, and physiological responses were measured at various intervals up to 24 hours post-exposure. Measurements included arterial blood gases, plasma markers (free heme, hemoglobin, KIM-1, NGAL, myoglobin), kidney function measurement as glomerular filtration rate (GFR), bronchoalveolar fluid (BALF) protein levels, and lung histology scored for acute lung injury (ALI) per ATS criteria. Haptoglobin (100 mg/kg) or vehicle was administered intramuscularly 30 minutes post-exposure. Statistical significance was set at p < 0.05. Results: Following Cl₂ exposure, humanized SCT mice exhibited significantly higher mortality (40.9%) compared to controls (13.6%), with a hazard ratio of 3.8 (95% CI of 1.2 to 11.7; p<0.05). SCT mice also experienced greater weight loss, averaging 4.4 ± 0.4 grams versus 3.3 ± 0.3 grams in controls (p<0.05). Acute lung injury was more severe in SCT mice, with higher BALF protein levels (p<0.05)and ALI scores (p<0.01) in the surviving SCT mice compared to the controls. Although both groups developed respiratory acidosis, only SCT mice showed significant hypoxemia (p<0.01). In addition to pulmonary complications, SCT mice developed more pronounced acute kidney injury 24 hours after Cl2 exposure. While baseline GFRs were similar, post-exposure GFR declined significantly in SCT mice (p<0.01) but remained stable in controls (p=0.92). KIM-1 and NGAL increased more substantially in SCT mice (p<0.05 and p<0.001, respectively). Following Cl₂ exposure, humanized SCT mice exhibited significantly greater hemolysis and rhabdomyolysis compared to control mice. Hemoglobin and hematocrit levels dropped only in the SCT group (p<0.05 for hemoglobin drop in the SCT vs p=0.99 in the controls) (p<0.05 for hematocrit drop in the SCT vs p=0.99 in the controls), along with a significant rise in potassium (SCT p<0.01, vs AA: p=0.99), a marker of hemolysis. Free heme levels increased in both groups post-exposure (SCT p<0.0001 vs AA: 0.01) but were significantly higher in SCT mice (p<0.05). Creatine kinase levels increased in both (SCT: p<0.0001 vs AA: p<0.05), but more significantly in SCT mice (p<0.05), confirming more severe rhabdomyolysis. Post-exposure haptoglobin therapy significantly increased survival by 93%, from 40% to 77% (p<0.05), and reduced weight loss compared to vehicle-treated mice (p<0.05). It also preserved oxygen saturation at both 4 (p<0.0001) and 24 (p<0.001) hours post-exposure. Furthermore, haptoglobin-treated mice had significantly lower BALF protein levels (p<0.01), indicating reduced lung injury. Conclusions:These findings highlight the heightened vulnerability of humanized SCT mice to Cl₂-induced multiorgan injury, including respiratory and renal systems, compared to controls, driven by hemolysis and rhabdomyolysis. Haptoglobin therapy significantly improved survival and reduced lung injury, supporting its potential as a targeted treatment for hemolysis-related complications in environmentally vulnerable populations.