Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Microbiology and Immunology

Supervisor

Dr. Lakshman Gunaratnam

Abstract

Acute kidney injury (AKI) is defined by the rapid loss of kidney function due to tissue damage. It affects 10-30 % of hospitalized patients and is independently associated with increased morbidity and mortality. Ischemia-reperfusion injury (IRI) is the most common pathoetiological mechanism of AKI, whereby tissue injury is mediated by reactive oxygen species. Ischemic AKI leads to the rapid upregulation of a transmembrane protein, kidney injury molecule-1 (KIM-1) on the apical membrane of proximal tubular epithelial cells (TECs). Previous work from our group and others demonstrated that the extracellular domain of KIM-1 specifically binds to phosphatidylserine on apoptotic cells, thereby transforming KIM-1-expressing TECs into semi-professional phagocytes for apoptotic corpses. The pathophysiological role of KIM-1 in AKI and relevant signalling mechanisms have not yet been elucidated. Using an in vivo model of AKI in mice genetically deficient in Kim-1, we reveal that Kim-1 expression protects mice from tissue damage and renal dysfunction after AKI. To uncover the signalling pathways downstream of KIM-1 in TECs, we performed protein-protein interaction studies to uncover two signalling partners, the alpha subunit of the heterotrimeric G12 protein (Gα12), and the dynein light chain protein (Tctex-1). We provide evidence that KIM-1 interacts with both Gα12 and Tctex-1, using co-immunoprecipitation, GST-pull down and confocal microscopy co-localization studies. We show that KIM-1 upregulation during IRI suppresses endogenous Gα12 activation and down-stream effectors. Further, we reveal that KIM-1-inhibition of Gα12, and the down-stream mediator RhoA, are crucial for TEC-mediated clearance of apoptotic cells. Tctex-1 is bound to KIM-1 during the early stages of phagocytosis becoming dissociated at later stages. Here, siRNA mediated studies informed us that Tctex-1 expression is necessary for phagocytic uptake of apoptotic cells. Finally, given our data showing that TECs isolated from Kim-1-deficient mice were virtually incapable of phagocytosis, it can be inferred that our findings regarding Gα12 and Tctex-1 do not solely apply to phagocytosis by KIM-1, but to all other mechanisms of uptake of apoptotic cells by TECs. In summary, our results suggest an important protective role of KIM-1 in ischemic AKI that is mediated via non-phagocytic and phagocytic mechanisms. The work described in this thesis provides several novel mechanistic insights into the functional role of KIM-1 and suggest potential therapeutic targets for future development.

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