LOXL2 identifies inflammation-associated myofibroblasts predicting kidney allograft dysfunction and fibrosis

Chronic allograft nephropathy (CAN) is a major obstacle in kidney transplantation, often leading to long-term graft failure. Early identification of patients at high risk for CAN could be instrumental to improve outcomes. Here, we investigate Lysyl oxidase ligand 2 (LOXL2) as a predictive biomarker for CAN. Using single cell RNA sequencing of transplant biopsies, immunohistochemistry and immunofluorescence microscopy, we find that LOXL2 labels a distinct intertubular myofibroblast subtype with a smooth muscle actin (SMA)-, CXCL12- and PDGFRb-negative and CD68-positive phenotype. These cells are present in non-fibrotic and fibrotic regions utilizing collagen 3 as scaffold, and are associated with a high expression of extracellular matrix (ECM) proteins and related pathways. Native kidneys also harbor LOXL2+ myofibroblasts, albeit at much lower levels, and their presence correlates with the incidence of chronic kidney disease. Following transplant surgery, LOXL2+ cells can rapidly emerge within days, particularly during episodes of rejection, where they associate with leukocyte aggregates. Notably, elevated cell numbers can be normalized as shown in follow up biopsies. A retrospective analysis of 118 biopsies revealed a significant association between LOXL2+ cell abundance and transplant fibrosis, inflammation, and kidney function. However, no correlation was found with transplant age, rejection type and other Banff parameters. Non-rejecting allografts displayed high variability in the number of LOXL2+ cells, with high abundance serving as a long-term predictor of fibrosis and reduced allograft function. Collectively, our findings suggest that LOXL2+ intertubular cells are a subset of inflammation-associated SMA- myofibroblasts and may be useful as a risk biomarker for early fibrogenesis in non-rejecting allografts. Overall design: Renal graft biopsies of KTx patients were analysed in this study. The indication for graft biopsy was unexplained graft injury as assessed by creatinine or proteinuria. Per patient, 1 biopsy was analysed. In total, 18 patients were included in this study.