Abstract: FR-PO1112
Developing an Imaging Mass Cytometry-Based Injury Panel to Define the Pathogenesis of Delayed Graft Function
Session Information
- Transplantation: Basic
November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Transplantation
- 1901 Transplantation: Basic
Authors
- Avigan, Zachary, Yale University School of Medicine, New Haven, Connecticut, United States
- Singh, Nikhil, Yale University School of Medicine, New Haven, Connecticut, United States
- Kliegel, Judith, Yale University School of Medicine, New Haven, Connecticut, United States
- Weiss, Marlene, Yale University School of Medicine, New Haven, Connecticut, United States
- Moeckel, Gilbert W., Yale University School of Medicine, New Haven, Connecticut, United States
- Cantley, Lloyd G., Yale University School of Medicine, New Haven, Connecticut, United States
Background
Delayed Graft Function (DGF) is a significant source of morbidity in transplant patients and is associated with decreased graft survival and increased mortality; however, treatment is restricted by limited mechanistic understanding of its pathogenesis. To better define the cellular architecture of the human kidney, we developed an imaging analysis pipeline using Imaging Mass Cytometry, a novel approach for simultaneous mass spectrometry-based analysis of up to 42 protein targets on formalin-fixed tissue. While antibodies to resident and infiltrating cell populations are in our panel, antibodies to identify cell injury are needed.
Methods
In vitro models were developed for inducing programmed cell death (apoptosis—TNFa, ATP depletion; necroptosis—TNFa, ATP depletion, zVAD) and cell stress (autophagy—serum starvation, TGFb; ER stress—serum starvation, tunicamycin) pathway activation in human proximal tubule HK-2 cells. Antibodies showing specificity by IF were conjugated to heavy metals to be used for IMC.
Results
HK-2 cells showed reduced viability under cell death conditions (control 95.0+/-0.9%; apoptosis 46.3 +/- 5.9%; necroptosis 66.6 +/- 5.9%). Validated antibodies showed significant differences under injury vs control conditions, including: apoptosis (anti-cPARP: 9.5 +/- 2.5% vs 0.8 +/- 0.3%); necroptosis (anti-pMLKL: 17.9 +/- 5.1% vs 2.8 +/- 0.7%); autophagy (anti-P62: 29.8 +/- 7.4% vs 7.1 +/- 1.9%); and ER stress (anti-GRP94: 21.0 +/- 0.6% vs 2.0 +/- 0.7%). All 4 antibodies retained staining by IMC in a tumor-associated interstitial nephritis kidney biopsy.
Conclusion
We now have 35 validated antibodies, including the 4 described above, to simultaneously quantify injured tubular and vascular cell populations and their frequency of interaction with immune subtypes. We will perform IMC with this expanded panel using our cohort of pre-implantation transplant biopsies from 16 deceased donors (7 with subsequent DGF) and 15 living donors as healthy controls.
Funding
- NIDDK Support