Doctor of Philosophy
Microbiology and Immunology
Dr. Lakshman Gunaratnam
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.
Ismail, Ola Z., "Kidney Injury Molecule-1 Signalling in Ischemic Acute Kidney Injury and Phagocytosis" (2015). Electronic Thesis and Dissertation Repository. 3098.
Available for download on Friday, August 31, 2018