Defining molecular target of arsine gas-induced acute kidney injury and associated morbidity - Arsine (SA), the most toxic form of inorganic arsenic is a potent chemical warfare (CW) agent. Although arsine was never used in the battlefield, concerns still exist that it may be used as a small-scale weapon during indoor public meetings or concerts. None-the-less arsine exposure in occupational settings due to accidental release is known among the industrial workers. Pathophysiology associated with inhaled arsine gas involves hemolysis of massive red blood cells that often leads to kidney failure and associated morbidity/mortality. Erythrocyte destruction leads to the release of hemoglobin (Hb) and heme into circulation, which subsequently accumulates in the tubular epithelium, causing acute kidney injury (AKI). However, the detailed molecular mechanisms of its toxic action are not fully defined and represent a major knowledge gap. Our preliminary data demonstrate that arsine gas exposure at 15 ppm for 1 h to C57BL/6 mice induced intravascular hemolysis as evidenced by bloody urine and dark red plasma, associated with a significant decrease in RBCs, hematocrit, and hemoglobin. Additionally, a significant induction of NGAL and KIM-1, the early and late respective biomarker of AKI in urine and kidneys were prominent. Utilizing an arsine-unrelated surrogate murine model of hemolysis, we show that mice treated with phenylhydrazine (PHZ), a potent hemolytic agent, significantly causes hemolysis along with an induction of AKI biomarkers, which is identical to that we observed in arsine gas-exposed mice. Thus, PHZ mouse model provides critical utility to determine the pathogenic molecular underpinnings of arsine-mediated hemolysis and associated kidney injury. Our preliminary data identified NOD1/2 as a key target in hemolysis- mediated kidney injury in both of these mouse models. NOD1/NOD2 are immune response sensors and can modulate inflammation and cellular injury. Two specific aims are proposed. In Aim-1, we propose to characterize a mouse model of arsine toxicity. Both dose-and time-dependent responses will be characterized. Biomarkers of AKI will be examined in urine, plasma/serum and kidney. Pro-inflammatory mediators will be examined in serum and kidney. In addition, lung and liver damage will also be examined cursorily by histopathological analysis to evaluate multi-organ damage by arsine exposure. This aim will help in selection of an optimum dose-and time- point to develop medical countermeasures (MCM) for arsine toxicity. Aim-2 will define the therapeutic targets of arsine toxicity. Here, we will first screen three NOD inhibitors for identifying the most efficacious nontoxic dose employing PHZ mouse model. Then, one best NOD inhibitor (identified from PHZ model) will be tested for its efficacy against arsine-mediated AKI and associated morbidity. Successful completion of studies proposed in this R21 grant will provide a fully characterized mouse model and a novel molecular target-based therapeutic drug agent as MCM against arsine. The efficacy of these MCMs against other surrogate hemolysis-related disorders such as autoimmune hemolytic anemia, sickle cell disease, β-thalassemia, paroxysmal nocturnal hemoglobinuria, hemolytic uremic syndrome etc. will help in getting faster FDA approval.
