American Veterans are disproportionately affected by kidney-damaging agents during deployment operations, and failing health and medical countermeasures add long-term health effects. Studies admit the urgent need to develop novel protective or preventive strategies to overcome the short therapeutic window for kidney protection. Thus, a therapy based on the delayed effects of acute kidney injury (AKI) would be clinically relevant because it gives a wider therapeutic window for intervention. The study of kidney nucleases initiated by our group several years ago has determined that of nine known endonucleases, DNase I and EndoG are the most active and mechanistically involved in tubular epithelial cell death during AKI. Our unexpected finding was that one of the endonucleases, EndoG, that has RNase activity, induced alternative splicing (AS) of DNase I pre-mRNA, thus inactivating DNase I and mitigating other DNases’ expression. Our data also showed that EndoG indices AS of other genes. Therefore, this proposal is focused on EndoG-mediated pathways through its RNase and DNase activities. We hypothesize that during AKI, EndoG induces tubular epithelial cell death by fragmenting DNA and causing alternative pre-mRNA splicing of several proteins including DNase I, while endonuclease inhibitors will mitigate this injury. In Aim 1 we will determine EndoG RNase-mediated mechanisms in normal kidney and during myoglobinuric AKI. This aim will determine (a) the protective mechanisms of EndoG against DNase I expression, and (b) the mechanism of AS mediated by EndoG. Aim 2 will define the role of EndoG DNase activity, the mechanisms of EndoG and DNase I leakage to the nucleus, and the role of ROS in the induction of endonucleases during myoglobinuric AKI. Finally, Aim 3 will evaluate the therapeutic modulation of EndoG for kidney tissue protection and limiting AS in myoglobinuric and other AKI models. Inhibition of individual endonucleases with the use of our one-of-a-kind chemical inhibitors will be tested. Potential Impact on Veterans Health Care. The results of this study will benefit American Veterans and the general population by providing critical knowledge of endonuclease-mediated mechanisms of AKI and their therapeutic targeting. These studies can potentially lead to the development of new therapeutic tools (endonuclease inhibitors) to prevent or ameliorate myoglobinuric AKI. Some of them will have strong translational value because they act even if administered after kidney injury, while others can become future therapeutic options. When applied to humans, the results of this study may allow for saving human lives, improving the health of Veterans, and decreasing the number of disabilities in the Veteran population.