Introduction: As of 2020, over 41,000 Canadians are living with end-stage kidney disease, where the kidneys no longer have sufficient function to maintain body homeostasis. While transplantation is the best treatment for end stage kidney disease, 50% of grafts fail within 10 years, due primarily to antibody-mediated rejection (ABMR), where recipient donor-specific antibodies (DSA) trigger immune-driven tissue injury. There are no effective treatments for ABMR and predicting its onset is complex, as 30-60% of DSA+ transplant patients do not develop rejection. The purpose of our study is to identify factors that regulate kidney protein expression in DSA+ ABMR kidney transplant recipients compared to DSA+ recipients without rejection.
Methods: Laser capture microdissection was used to extract glomeruli and tubulointerstitium from kidney biopsies from DSA+ kidney transplant recipients with ABMR (n=14) or no rejection (NR; n=16) (cohort 1). Protein from these tissues was subjected to liquid chromatography mass spectrometry on Q-Exactive HFX and results were analyzed using MaxQuant and Perseus software. Significantly differentially expressed proteins (t-test, p<0.05) were mapped to signalling pathways via pathDIP and Reactome databases (FDR q value<0.05), and associated miRNAs were identified via the miRDIP database. miRNA was measured (HTG Molecular Diagnostics) in the sera of a second DSA+ patient cohort (n=40) and assessed for association with ABMR via LASSO and Essential Regression machine learning.
Results: We identified 184 proteins in glomeruli and 170 in the tubulointerstitium (FDR<1%) that were significantly differentially expressed between DSA+ ABMR and NR patients in cohort 1, with 61% and 73% upregulated in ABMR, respectively. pathDIP and Reactome analyses revealed that these proteins mapped significantly to pathways involved in the immune and cellular stimuli responses (Fig. 1A), particularly antigen processing (q= 6.49e-12) and oxidative stress (q= 1.75e-08). LASSO analysis of miRNA measured in the serum of a second cohort of DSA+ patients revealed 12 miRNA that significantly discriminated between patients with or without ABMR (Fig. 1B). Subsequent miRDIP analysis found that all 12 of the identified miRNAs target the genes encoding kidney proteins differentially expressed in cohort 1.

Conclusions: These preliminary results suggest that ABMR in DSA+ patients is strongly linked to dysregulated immune and cellular stress responses and that this dysregulation manifests in multiple, interconnected cellular products. These findings will ultimately help us identify targets for the development of novel therapeutics for kidney transplant recipients.