Targeting T lymphocyte Keap1 for acute kidney injury

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PROJECT SUMMARY /ABSTRACT Acute kidney Injury (AKI)-associated morbidity and mortality is a major clinical problem that involves multiple overlapping pathophysiological mechanisms. Our lab and others have established the modulatory roles of T lymphocytes as well as the protective role of transcription factor Nrf2 in ischemia reperfusion (IR) and cisplatin induced AKI. We recently demonstrated that T cell deletion of Keap1 which augments specific Nrf2 activity provides significant protection from IR induced AKI in knockout mice, and that adoptive transfer of T cells with augmented Nrf2 activity improves kidney function and survival following AKI in wild type mice. These findings were accompanied by a significant increase in regulatory T (Treg) cell frequency and numbers, and reduced proinflammatory cytokine production by T cells in the kidneys of mice with Keap1 deficient T cells. These novel observations reveal an unexpected relationship between Keap1 and T cell homeostasis and function in AKI, however the underlying mechanisms are unknown. The overarching aim of this proposed study is to test the hypothesis that Keap1 regulates the expansion of Treg cell population that subsequently suppresses harmful inflammatory responses during AKI. In order to test our hypotheses, we will determine whether Treg cells with Keap1 deletion have enhanced proliferation or decreased apoptosis as compared to Treg cells from wild type (WT) mice. We will also evaluate if Keap1 deletion increases the suppressive function of Treg cells, enhances TCR sensitivity in T cells and regulates response to IL-2 under steady state and in two different models (IR and cisplatin) of AKI in mice. To further establish the specificity of Keap1 deletion and to investigate any Nrf2 independent effects of Keap1 in T cells we will generate mice lacking Keap1 specifically in Treg, CD4 and CD8 T cells and determine whether Treg specific Keap1 deletion is sufficient to prevent ischemic and nephrotoxic AKI. We will further generate mice with T cell specific deletion of either Nrf2 alone or Keap1 and Nrf2 together (double KO) to delineate Nrf2 dependent and independent effects. In the final aim we will transition from mechanistic studies to experiments that will set the stage for clinical translation. We will explore therapeutic approaches including pharmacologic Nrf2 activators to activate Nrf2 and Keap1 specific CRISPR/Cas9 technology, siRNAs and miRNAs to delete Keap1 in mouse T cells ex vivo. We will then examine the effect of these Nrf2 activation/Keap1 deletion strategies on phenotypic and functional T cell responses as well as AKI outcome in IR and cisplatin AKI models. We will test the most promising Nrf2 activation/Keap1 deletion strategy in human primary T lymphocytes and examine its effects on Nrf2 regulated antioxidant response, proinflammatory cytokine production, and T cell dynamics. Successful completion of these studies should result in major advances regarding our understanding on the role of Keap1/Nrf2 in T cell homeostasis and function, and also set the stage for future clinical interventions targeting Keap1-Nrf2 pathway for AKI and other inflammatory diseases.