Kidney transplantation offers the greatest survival advantage to patients with end stage kidney disease and is vastly more cost effective than dialysis. Long-term survival of kidney transplants has not improved in recent decades. Antibody mediated rejection (AMR) has been identified as a major cause of transplant failure. Currently, management of patients with AMR is inconsistent among centers and frequently fails. We hypothesize that the cell types and cell states unique to AMR can be resolved by single cell RNA sequencing (scRNA-seq) of biopsy samples taken from patients at the time of diagnosis. This approach uses an unsupervised framework for dissecting transcriptional heterogeneity within complex tissues such as the kidney. This allows for the interrogation of cell states and subpopulations using an unbiased clustering approach that is independent of previous knowledge and can provide unprecedented resolution. To test our hypothesis we propose the following aims:
In Aim 1, we will perform scRNA-seq of 40 research biopsy cores (20 AMR and 20 non-AMR). From this data we will identify genes expressed in endothelial cells and antibody secreting cells that define AMR specific phenotypes. We have demonstrated feasibility of this approach by generating single cell data from human biopsy samples as outlined in the research plan.
For Aim 2, we will use immunohistochemistry to validate these AMR specific markers on a set of independent tissue samples.
For Aim 3, we will use publicly available Affymetrix microarray datasets from kidney transplant biopsies with associated outcomes data to determine which cell types are associated with allograft outcome. This proposal logically builds on the principal investigator?s previous research experience and clinical training. To date he has been working full time in clinical transplantation and continuing his research endeavors on an ?out of hours? basis. Despite this his research output and experience continue to grow and he has recently published a report on the first successful application of this technology to human kidney biopsy tissue (co-first author). This proposal now focuses on expanding his scientific skills by attaining additional knowledge and practical research experience in single cell methods, bioinformatics and immunology. The career development goals will be achieved through a multi-faceted approach involving mentoring by Dr. Benjamin Humphreys and an advisory committee consisting of Drs. Barbara Murphy (transplant genomics), Phil Payne (biomedical informatics and translational science), Rob Mitra (single cell applications) and Paul Allen (translational immunology), didactic coursework, scientific investigation, and training in scientific communication and research ethics. This work will take place in Washington University which has a rich history of mentoring successful physician-scientists. Successful completion of this career development award will result in a better understanding of AMR, result in the principal investigator?s transition to an independent physician- scientist, and provide a solid foundation from which he will apply for RO1-level funding.
Antibody mediated rejection is a major cause of kidney transplant failure. The pathophysiology of antibody mediated rejection is poorly understood and current treatment strategies frequently fail. Successful completion of this project has the potential to identify new markers of disease and novel targets for treatment.