ABSTRACT: T cell-mediated rejection (TCMR) remains a challenge in kidney transplantation. Based on a histopathological biopsy examination, patients can be classified into groups defining their kidney’s status, i.e. no rejection (NR), borderline rejection (BR) and acute rejection (AR). Yet, this classification is not perfect, since for the borderline cases, a number of patients may or may not require further clinical intervention. Although in other studies, detection of early signs of rejection in the urine proteome was already attempted, these results failed to be replicated. Thus, a robust method of classification by biopsy proteome profiling may provide a possible solution. Unlike for urine or small extracellular vesicle’s analysis, biopsy proteome directly reflects the subclinical changes leading to allograft loss. Kidney tissue biopsies from patients classified into NR, BR and AR groups based on the current Banff guidelines were subjected to a mass spectrometry based proteomic profiling. Using qualitative, quantitative, profile and ROC analysis, a set of four proteins (i.e., GNB4, PDK1, AGXT and CD73) was chosen for further validation using immunohistochemical (IHC) methods. Proteomic analysis identified 2547 proteins across all groups with their abundance profiles showing high degree of similarity between the BR and AR datasets. In a quantitative comparison, GNB4 and AGXT emerged as the proteins significantly differentiating the groups with the highest fold change. Moreover, AGXT was indicated as a potential biomarker candidate following a ROC analysis. Morover, PDK1 and CD73 were found to best classify the samples in a binary fashion. These candidate biomarkers were validated using an IHC approach, where only upregulation of GNB4 in immune cells and PDK1 in macrophages could be confirmed, with no significant changes in the tubular epithelium. Based on these results, GNB4 and PDK1 presence in the immune cells and macrophages has been identified as an interesting target for further extensive studies. If their relevance was to be confirmed in a large patient cohort, their analysis could potentially serve an extension of the already established histopathological classification in the context of kidney transplant rejection. This retrospective study was approved by the Bioethics Committee of the Medical University of Gdańsk no. NKBBN/201/2021.
Daniel Fochtman 
