Plasma donor-derived cell-free DNA levels correlate strongly with AMR activity, acute kidney injury, and in a complex way with TCMR activity. Parenchymal injury PF-543 Citrate correlates with both reduced glomerular filtration rate and increased risk of graft loss. In kidneys with rejection, injury features, not rejection activity, are the strongest predictors of graft survival. Both TCMR and AMR produce injury, but TCMR induces immediate nephron injury and accelerates atrophy-fibrosis, whereas AMR induces microcirculation and glomerular damage that slowly leads to nephron failure and atrophy-fibrosis. Plasma donor-derived cell-free DNA levels correlate strongly with AMR activity, acute kidney injury, and in a complex way with TCMR activity. Thus, the MMDx project has documented the molecular processes that underlie the clinical and histologic states in kidney transplants, and provides a diagnostic tool that can be used to calibrate biomarkers, optimize histology interpretation, and guide clinical trials. == INTRODUCTION == From the first success in identical twins in 1954, clinical kidney transplantation approaches its 70th anniversary as a work in progress.1,2Long-term outcomes continue to fall short of ideal. New technologies such as donor-specific antibody (DSA)3testing using Luminex beads, virus detection and management, blood or urine gene expression profiling, and donor-derived cell-free DNA (dd-cfDNA)46have offered incremental utility, but the field needs new understanding and ultimately PF-543 Citrate new interventions to prevent and solve problems. The molecular processes at work can offer insights in this regard. This review outlines the stepwise exploration of the genome-wide change in gene expression in kidney transplant biopsies in the Molecular Microscope Diagnostic System (MMDx) project, which aims to discover the molecular basis of the rejection and parenchymal injury states in kidney transplants. (The term parenchyma here refers to the elements of the tissue responsible for organ function, plus their supporting matrix and microvasculature, as distinct from infiltrating cells.) MMDx also aims to define the relationship between the genome-wide molecular phenotype of the transplant and other diagnostic systems such as histology, DSA, and dd-cfDNA. Some aspects have previously been described, 711and the technology and workflow are outlined in a recent MMDx-Heart review.12 The assignment of the probability PF-543 Citrate that a disease is present is typically a 2-step approach: step 1 1, measure designated Rabbit Polyclonal to CBR1 features; and step 2 2, interpret feature measurements using predefined algorithms. In MMDx step 1 1, microarrays assess messenger RNA (mRNA) expression in the biopsy, currently measuring expression of 49 495 probe sets reflecting the expression of 19 462 unique genes. MMDx step 2 2 interprets these measurement using ensembles of predefined machine learningderived algorithms and compares the biopsy to a reference set of previously characterized biopsies. MMDx assesses rejection statesT cellmediated rejection (TCMR) and antibody-mediated rejection (AMR)and parenchymal injury states, including recent injury (acute kidney injury [AKI]), irreversible atrophy-fibrosis, and probability PF-543 Citrate of survival. The extent of injury correlates with impaired function and is critical PF-543 Citrate to understanding the risk of graft loss. The timeline of MMDx development is shown in Figure1. The reference set grows over time as new biopsies are acquired, and the diagnostic algorithms are periodically rederived. The use of a stable, fixed platform is essential to permit new biopsies to be added indefinitely to the reference set. == FIGURE 1. == Timeline of work done over the course of the MMDx-Kidney project. AMR, antibody-mediated rejection; dd-cfDNA, donor-derived cell-free DNA; DSA, donor-specific antibody; IRRAT, injury- and rejection-associated transcript; MMDx, Molecular Microscope Diagnostic System; NK, natural killer; TCMR, T cellmediated rejection. Table S1(SDC,http://links.lww.com/TP/C772) is a list of abbreviations.Table S2(SDC,http://links.lww.com/TP/C772) lists the collaborators in the project. == BACKGROUND FOR THE MMDx PROJECT == The MMDx project arose from earlier studies of the mechanisms operating in organ transplants, particularly the role of antibodies and the regulation of gene expression. An early focus was on interactions of Fc receptors with antibodies against MHC antigens,13,14a theme that surprisingly re-emerged decades later with the discovery of the association of natural killer (NK) cells with AMR. Another early theme was IFNG induction of MHC and other genes in donor tissue,1518acting through the donor IFNG receptor19and transcription factor IRF1.20Increased expression of IFNG-inducible genes continues to be a robust feature of all rejection. Understanding behavior of specific genes must acknowledge complexity. For example, IFNG is highly expressed in TCMR and therefore might be considered harmful. However, in the early stages of TCMR, IFNG acting on the donor tissue protects it from early necrosis,1921temporarily stabilizing the microcirculation as the inflammatory infiltrate develops. This.