Novel endonuclease-targeted approaches to nephroprotection

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ABSTRACT The kidney is an excreting organ that is affected by many toxic chemicals metabolized in the body, including products of rhabdomyolysis (skeletal muscle degradation), which are known to induce acute renal injury (AKI) through the production of reactive oxygen species (ROS). Resulting AKI is a life-threatening condition that requires hemodialysis or kidney transplantation. This proposal is a continuation of the previous research funded by VA Merit Review grant, which was focused on renal apoptotic endonucleases induced or activated by ROS during rhabdomyolysis. The aims in the previous project have been accomplished and the new goals described below are the logical extension of those aims. The present proposal is based on our recent observations that rhabdomyolysis and other tissue/cell injuries mediated by ROS are several times greater mainly because of the activation of apoptotic enzyme, endonuclease G (EndoG), which is induced by ROS. Non-toxic inhibitors of EndoG or other apoptotic endonucleases are not known, and there is virtually no protection against activated EndoG or other endonucleases during this period of time. This gap in knowledge can be explained, in part, by the fact that prior to our studies, the endonuclease activation was not recognized as essential for ROS injury and was considered as secondary postmortem event. We showed that knockout mice deficient in EndoG are protected against rhabdomyolysis-induced AKI, suggesting that EndoG acts premortem. However from a translational/therapeutic point of view a gene knockout is not an option. Therefore we focus this proposal on developing a set of new approaches to inhibit EndoG for nephroprotection during rhabdomyolysis. These approaches include: (a) targeting the broad spectrum of endonucleases and ROS, (b) targeting EndoG expression, and (c) targeting epigenetic regulation of EndoG and its trafficking. All these strategies have a likelihood of translational application to veterans and general public. We hypothesize that acute kidney injury due to rhabdomyolysis can be prevented by inhibiting EndoG expression, trafficking and activity before or, in some cases, after injury. We plan to use complimentary in vivo (glycerol-induced rhabdomyolysis) and in vitro (hemin or myoglobin) approaches to induce tubular epithelial cells injury. In vitro approaches will also be used for mechanistic studies, or to control the in vivo approach if the toxicity or stability of the inhibitor is unknown. Our specific aims are as follows. Specific Aim 1. Determining the efficiency of broad spectrum ROS/endonuclease- targeted kidney protection by zinc-aminothiol chelates. (1A) Testing antioxidant Zn- aminothiol chelates in vitro. (1B) Testing antioxidant Zn-aminothiol chelates in vivo. Specific Aim 2. Testing EndoG-targeted protection by specific shRNA delivery by AAV or naked DNA. (2A) Kidney protection from injury by shRNA in vitro. (2B) Kidney protection from injury by shRNA in vivo. Specific Aim 3. Defining mechanistic anti-EndoG approaches to nephroprotection. (3A) Epigenetic downregulation of EndoG. (3B) Interfering with EndoG trafficking. Potential Impact on Veterans Health Care. Successful completion of these studies can potentially lead to the development of new therapeutic tools to prevent AKI. Some of them (zinc-chelates) have strong translational value because they act even if administered after kidney injury, while others can become therapeutic options of the future. When applied to humans, the results of this study may allow saving human lives, improving the health of veterans, and decreasing the number of disabilities in the veteran population.