Date of Award
Medical Doctor (MD)
Lloyd G. Cantley
DEVELOPMENT OF IMAGING MASS CYTOMETRY-BASED CELLULAR PROFILING TO INVESTIGATE DELAYED GRAFT FUNCTION.
Zachary M. Avigan, Nikhil Singh, Judith A. Kliegel, Marlene Weiss, Gilbert W. Moeckel, and Lloyd G. Cantley. Section of Nephrology, Department of Internal Medicine, Yale University, School of Medicine, New Haven, CT.
Delayed graft function (DGF) is a form of ischemic acute kidney injury affecting over a quarter of deceased donor kidney transplant (DDKT) patients that results in increased short-term morbidity and cost and is associated with worsened graft outcomes and mortality. There is no current treatment for DGF, and identifying therapeutic targets is limited by our incomplete understanding of the mechanisms of ischemic kidney injury. Imaging Mass Cytometry (IMC) is a recently developed, highly-multiplexed, and spatially-preserved methodology for immunolabeling of formalin-fixed, paraffin- embedded biopsy sections using metal-tagged antibodies. We recently presented an application of IMC to human kidney biopsies to profile the healthy kidney; however, IMC has not yet been implemented in human kidney disease. We here describe the development and validation of a robust, automated single-cell data analysis pipeline for IMC devised specifically for the complex cellular architecture of the human kidney. We additionally present our in vitro approach to screen and validate antibodies for IMC directed toward renal cell death and injury. To investigate DGF, we assembled a panel of pre-implantation kidney biopsies from a cohort of high-risk DDKT recipients and living donor kidney transplant (LDKT) patients. We then applied our IMC imaging and analysis system to study biopsies from grafts which progressed to DGF or immediate graft function (IGF) compared to LDKT biopsies. Our pipeline was effective in cellular profiling of biopsies across a range of image quality and validated the LDKT control
group as consistent with prior healthy kidney data. We found that a statistically significant reduction in tubular cell mass distributed proportionally across all tubular segments was predictive of DGF versus both IGF (p=.02) and LDKT kidneys (p<.0001). This methodology was more sensitive in identifying tubular cell loss than histopathologic evaluation alone. Additionally, we showed an increase in macrophage infiltration in deceased donor kidneys compared to the LDKT biopsies (p=.004), though this did not differentiate the DGF and IGF groups. In summary, the current study identifies tubular cell loss as a precursor of DGF. Further, this analysis demonstrates that our IMC platform improves detection of cellular changes in tubular injury and is predictive of DGF among high-risk deceased donor transplants.
Avigan, Zachary, "Development Of Imaging Mass Cytometry-Based Cellular Profiling To Investigate Delayed Graft Function" (2020). Yale Medicine Thesis Digital Library. 3882.