Acute kidney injury (AKI) is a significant and increasing problem. Medical management currently consists of supportive care, with dialysis implemented for the most severe cases; however, morbidity and mortality remain very high. A major reason for the lack of available treatments for AKI is a gap in the knowledge of how kidney tubule cells recover from AKI, which has, therefore, limited possible approaches for treatment. Identifying, a therapeutic target and pathway would meet a major unmet need by allowing for rational drug design. The goal here is to determine whether the highly conserved eight-protein exocyst trafficking complex, and particularly the central Sec10 (aka Exoc5) component, can be used to enhance recovery, and/or prevent injury, following AKI. After renal tubule cell injury, there is initial loss of cell polarity, followed by cell death and sloughing of cells into the lumen, then spreading and dedifferentiation of viable cells to cover the denuded area, with proliferation, differentiation, and reestablishment of cell polarity. The polarity, or secretory, pathway is crucial for AKI recovery, and cell function, and the exocyst is known for mediating the targeting and docking of secretory vesicles carrying membrane proteins. Over the past twenty years, we showed that the mitogen-activated protein kinase (MAPK) pathway regulates tubulogenesis. We also showed that the exocyst, especially the Sec10 component, is centrally involved in renal ciliogenesis and tubulogenesis. Specifically, Sec10 knockdown inhibited, and Sec10 overexpression increased, ciliogenesis and tubulogenesis. These distinct research areas recently converged, as we showed that Sec10 speeded recovery from oxidative damage, an ischemia-like injury, by activating MAPK. We have now generated Sec10fl/fl mice, and have preliminary data showing Sec10 deletion in murine proximal tubules worsens ischemia and reperfusion (I/R) injury, and inhibits repair. Furthermore, site-specific mutation of the highly-conserved VxPx ciliary targeting sequence in human SEC10 inhibits tubulogenesis in cells grown in 3D collagen gels, and prevents the rescue of sec10 mutant zebrafish. The proposed experiments will test the overall hypothesis that Sec10 activates the MAPK pathway, through the EGF receptor, to prevent injury and/or enhance renal recovery following AKI, that this effect is mediated via primary cilia, and that Sec10 is a therapeutic target. Accordingly, we will investigate how Sec10 increases EGF receptor sensitivity, which activates MAPK to enhance recovery from injury (Aim 1.1). We will then investigate how Sec10 and the exocyst are involved in mitochondrial function. A critical pathway that has been identified in AKI is alterations in primary tubular metabolism, which secondarily affect the regional circulation through decreased levels of ATP and mitochondrial dysfunction. Mitochondria are also involved in ADPKD, the most common ciliopathy, suggesting a possible link between cilia and mitochondria. Here we will investigate this novel pathway, and the possibility that the exocyst could be the mediator between cilia and mitochondria function, possibly by differential protein trafficking regulated by different small GTPases (Aim 1.2). We will test if Sec10 protection following AKI is mediated via primary cilia by determining in mice if proximal tubule-specific knockout of Ift88, a protein necessary for ciliogenesis, worsens injury and prevents recovery following I/R (Aim 2.1). If cilia appear to be centrally involved, we will confirm this using our newly-generated Sec10 ciliary targeting sequence mutant mice and I/R injury (Aim 2.2). Regardless of ciliary involvement, we will confirm that the MAPK pathway is involved in Sec10- mediated protection from I/R injury in mice using small molecule inhibitors. We will then obtain proof of principle that Sec10 can enhance recovery, and/or prevent injury, by using our newly-generated inducible Sec10- overexpressing mice and performing I/R injury (Aim 3.1). Finally, we will determine if Sec10 gene delivery can prevent injury, and/or enhance recovery, using viral and non-viral delivery of Sec10 in vivo prior to, and after, I/R (Aim 3.2). Successful completion of these experiments will provide novel mechanistic insights into AKI pathogenesis and recovery, and have a major impact on the development of new approaches to treat AKI.
Acute kidney injury (AKI) occurs in 1-7% of hospitalizations and up to 25% of intensive care unit (ICU) admissions. Mortality rates in AKI patients, especially in ICUs, can be as high as 50-70%. AKI is also a significant contributor to the progression of chronic kidney disease. Given that in FY17 there were 683,185 acute admissions to VA Hospitals, AKI affected up to 47,823 Veterans. There were also 38,991 ICU admissions to VA Hospitals in FY17, so AKI affected up to 9,748 Veterans. As a kidney doctor at the VA, and in the South Carolina Army National Guard, I frequently see patients with AKI, and it is difficult to tell them there are no approved treatments other than supportive care. One of the most common forms of AKI is acute tubular necrosis (ATN). We linked an organelle called the primary cilium to ATN, and showed that the exocyst complex and cilia are necessary for tubule formation in cell culture. Here, we will determine how the exocyst protects against injury and enhances repair in mice, and test possible treatments for AKI.
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