Abstract: SA-PO083
Mapping and Functional Characterization of Proteolytic Cleavage of Murine Kidney Injury Molecule 1
Session Information
- AKI: Mechanisms - Primary Injury and Repair - II
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Sriranganathan, Saranga, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada
- Lee, Ji yun, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada
- Gunaratnam, Lakshman, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada
Background
Kidney injury molecule-1 (KIM-1) is a type I transmembrane glycoprotein expressed apically on proximal tubule epithelial cells. KIM-1, absent in healthy kidneys, is upregulated transiently during acute kidney injury (AKI). KIM-1 as a phagocytic receptor plays a protective role during AKI facilitating the clearance of apoptotic cells (efferocytosis) from the tubular lumen, thereby reducing inflammation and promoting repair. Human KIM-1 undergoes spontaneous and accelerated ectodomain shedding (into urine and blood) via metalloproteases including TACE/ADAM17 and ADAM10. Both blood and urine KIM-1 are clinically relevant biomarkers for AKI, however, the biological role of KIM-1 shedding is not known. In order to study significance of KIM-1 shedding in vivo in mice, we first aimed to identify the murine KIM-1 cleavage site and study its functional relevance in vitro.
Methods
Based on common structural motifs involved in TACE and ADAM10 recognition of substrates, we mapped out a potential cleavage site between A201 and I202 within the predicted cleavage region T194 to I202. Site-directed mutagenesis was used to generate various substitution mutants (I202Q and I202A) at the P1’ position of the potential cleavage site. We generated HEK293 cells stably expressing expression vectors encoding either wild type (WT), I202Q or I202A (murine) KIM-1. Expression of KIM-1 was verified by Western blot. Phorbol 12-myristate 13-acetate (PMA) and ionomycin were used to induce general metalloprotease- or ADAM10-mediated shedding, respectively, with or without GI254023X (ADAM10-specific inhibitor). Transfected cells were fed pHrodo stained apoptotic thymocytes and percent efferocytosis was quantified using flow cytometry. ADAM10 siRNA was used to study effect of ADAM10 silencing on KIM-1 shedding and efferocytosis.
Results
Both PMA and ionomycin accelerated shedding were drastically reduced in both (I202Q and I202A) mutants compared to WT. Efferocytosis was significantly reduced with the two mutants compared to WT KIM-1. Cells treated with ADAM10 inhibitor or ADAM10 siRNA exhibited significantly reduced efferocytosis compared to their respective controls.
Conclusion
ADAM10 is involved with both baseline and accelerated shedding of murine KIM-1. ADAM10-mediated KIM-1 shedding is required for efficient efferocytosis.
Funding
- Government Support - Non-U.S.