Master of Science
Physiology and Pharmacology
Kidney injury molecule-1 (KIM-1) is a transmembrane glycoprotein expressed apically on proximal tubule epithelia during acute kidney injury (AKI). KIM-1, as a phagocytic receptor, facilitates clearance of apoptotic cells (efferocytosis) from tubular lumen, thereby reducing inflammation and promoting repair. Human KIM-1 undergoes spontaneous and accelerated ectodomain shedding into urine and blood via metalloproteases; tumour necrosis factor-a converting enzyme (TACE/ADAM17) and a disintegrin and metalloprotease 10 (ADAM10). Blood and urine KIM-1 are clinical AKI biomarkers, however, biological significance of KIM-1 shedding is unknown. To study this in vivo in mice, I first aimed to identify murine KIM-1 cleavage site and study its functional relevance in vitro.
I202 was mapped out as the potential cleavage site and mutated to Q or A. Mutants showed significantly reduced ability to accelerate KIM-1 shedding and efferocytosis. ADAM10 mediated both baseline and accelerated shedding of murine KIM-1. Surprisingly, ADAM10-mediated KIM-1 shedding was required for efficient efferocytosis.
Summary for Lay Audience
Acute kidney injury (AKI) affects about 5-20% of hospitalized patients and is associated with increased mortality and risk of future chronic kidney disease. Severe AKI results in massive death of kidney epithelial cells. Rapid removal of dead cells occurs via "phagocytosis" which is vital for preventing further inflammation and hastening tissue repair. Recently, we discovered that injured kidney epithelial cells express KIM-1, a gene product not normally found in healthy kidney, which transforms surviving kidney epithelial cells into phagocytes that clear dead or dying cells and protect from AKI. KIM-1 is expressed on the cell membrane and “cut” by an enzyme and shed into urine and blood from kidney cells. In humans, KIM-1 is known to be primarily shed by tumour necrosis factor-a converting enzyme (TACE/ADAM17) and a disintegrin and metalloprotease 10 (ADAM10). Blood and urinary KIM-1 are used in clinics to identify kidney injury, but the importance of this shedding mechanism in biology is yet to be understood. We therefore attempted to identify and alter the cut site on mouse KIM-1 (mKIM-1) to study any changes in its shedding and phagocytic function, in hopes of eventually generating a KIM-1 shedding defective mutant mouse. This will be an invaluable tool to study the importance of shedding in the context of the animal as a whole.
The cleavage site of mKIM-1 is in a region near the cell membrane outside the cell (T194 to I202) and altering nearby amino acids will render it shedding defective but maintain phagocytic function.
Mapped out potential cut site based on patterns observed on other proteins cut by TACE and/or ADAM10. Alter relevant amino acids at the predicted cut site of mKIM-1 to make mutants for testing effect on shedding and phagocytosis.
Interestingly, it was observed that ADAM10 is the major enzyme cutting mKIM-1. Position I202 was determined to be a potential cut site. Reducing mKIM-1 shedding unexpectedly resulted in reduced phagocytosis levels.
Sriranganathan, Saranga, "Mapping and functional characterization of proteolytic cleavage of murine kidney injury molecule-1" (2019). Electronic Thesis and Dissertation Repository. 6474.