Extracellular vesicles as biomarkers for kidney allograft rejection

Kidney transplantation is the preferred treatment for patients with end-stage kidney disease. However, immune rejection remains the main reason for premature allograft loss and poor long-term survival. The incidence of acute rejection in the first year after kidney transplantation from a deceased donor in Slovenia is 13% (comparable to worldwide data), and the 10-year allograft failure rate is 30%. Survival and function of a transplanted kidney largely depend on early detection of (subclinical) rejection and rejection phenotype, followed by appropriate therapeutic interventions. There are two main phenotypes of allograft rejection, T-cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR), which underlie different immune processes and therefore require different treatment approaches. All non-invasive measures currently used for routine allograft monitoring (i.e., serum creatinine, estimated glomerular filtration rate, and proteinuria) cannot detect subclinical rejection or reliably distinguish between TCMR and ABMR, whereas invasive kidney biopsy is not compatible with routine monitoring of allograft function. Therefore, novel non-invasive biomarkers that allow frequent monitoring and detection of subclinical kidney allograft rejection are needed to improve transplant outcomes. Extracellular vesicles (EVs) are emerging as a promising new analyte of liquid biopsies. They are a heterogeneous group of phospholipid bilayer-enclosed nanosized particles that are secreted by all cells and accumulate in body fluids. Their molecular composition, concentration and size reflect their cell of origin and disease state. Urine and blood EVs are a novel, understudied source of non-invasive biomarkers for kidney allograft rejection, and our preliminary data support EV-bound DNA as a promising biomarker candidate. The aim of our project is to identify non-invasive EV or EV-bound DNA (evDNA)-based biomarkers of kidney allograft rejection that will allow early detection of rejection-related injury and identify the TCMR and ABMR phenotype. To this end, we will address the following five specific objectives in specific work packages: (i) establish a repository of time-matched biological samples and clinical data from kidney transplant recipients (WP1), (ii) demonstrate that urine and plasma EVs or evDNA reflect the histological diagnosis of kidney allograft rejection (WP2), (iii) demonstrate, that urine and plasma EVs or evDNA are early messengers of subclinical kidney allograft rejection (WP3), (iv) provide evidence that immune-related protein cargo of urine and plasma EVs reflects TCMR and ABMR phenotypes (WP4), and (v) disseminate and implement project findings (WP5). We hypothesize that evDNA in urine and plasma is a complementary biomarker for kidney allograft rejection that, together with analysis of the immune-related protein EV cargo, will allow detection of TCMR and ABMR phenotypes, thus enabling personalized therapeutic interventions for prolonged kidney allograft survival. The project will be implemented in collaboration between researchers from leading Slovenian institutions in the field of medical research, who have complementary expertise in kidney transplantation medicine, EV biomarkers and DNA quantification. They have already successfully collaborated on several research projects, research papers, PhD theses and outreach activities. The proposal is the first study to systematically investigate EV and evDNA from paired urine and blood samples as a source of non-invasive biomarkers to detect and monitor kidney allograft rejection and rejection phenotype. To this end, we will use state-of-the-art EV and DNA quantification techniques. Project results may lead to an innovative approach for non-invasive kidney allograft monitoring and personalized treatment interventions in the future, with important implications for allograft longevity, patient quality of life, and cost reduction.