The early identification of a subclinical rejection (SCR) can improve the long-term outcome of the transplanted kidney through intensified immunosuppression. However, the only approved diagnostic method is the protocol biopsy, which remains an invasive method and not without minor and/or major complications. The protocol biopsy is defined as the sampling of allograft tissue at pre-established times even in the absence of an impaired renal function; however, it does not avoid histological damage. Therefore, the discovery of new possible biomarkers useful in the prevention of SCR has gained great interest. Among all the possible candidates, there are microRNAs (miRNAs), which are short, noncoding RNA sequences, that are involved in mediating numerous post-transcriptional pathways. They can be found not only in tissues, but also in different biological fluids, both as free particles and contained in extracellular vesicles (EVs) released by different cell types. In this study, we firstly performed a retrospective miRNA screening analysis on biopsies and serum EV samples of 20 pediatric transplanted patients, followed by a second screening on another 10 pediatric transplanted patients’ urine samples at one year post-transplant. In both cohorts, we divided the patients into two groups: patients with histological SCR and patients without histological SCR at one year post-transplantation. The isolated miRNAs were analyzed in an NGS platform to identify different expressions in the two allograft states. Although no statistical data were found in sera, in the tissue and urinary EVs, we highlighted signatures of miRNAs associated with the histological SCR state.

Viral infections can lead to transplant dysfunction, and their possible role in rejection is described. In total, 218 protocol biopsies performed in 106 children at 6, 12 and 24 months after transplantation were analyzed according to Banff ’15. RT-PCR for cytomegalovirus, Epstein-Barr virus, BK virus and Parvovirus B19 was performed on blood and bioptic samples at the time of transplant and each protocol biopsy. The prevalence of intrarenal viral infection increases between 6 and 12 months after transplantation (24% vs. 44%, p = 0.007). Intrarenal Parvovirus B19 infection is also associated with antibody-mediated rejection (ABMR) (50% ABMR vs. 19% T-cell-mediated rejection, p = 0.04). Moreover, Parvovirus infection is higher at 12 months of follow-up and it decreases at 48 months (40.4% vs. 14%, p = 0.02), while in 24% of grafts, Parvovirus is already detectable at the moment of transplantation. Intrarenal Parvovirus B19 infection seems to be related to ABMR in pediatric kidney recipients. The graft itself may be the way of transmission for Parvovirus, so performance of a PCR test for Parvovirus B19 should be considered to identify high-risk patients. Intrarenal Parvovirus infection presents mainly during the first-year post-transplantation; thus, we recommend an active surveillance of donor-specific antibodies (DSA) in patients with intrarenal Parvovirus B19 infection during this period. Indeed, it should be considered a treatment with intravenous immunoglobulins in patients with intrarenal Parvovirus B19 infection and DSA positivity, even in the absence of ABMR criteria for kidney biopsy.

Antibody-mediated rejection (AMR) is the leading cause of premature kidney transplant failure. The role of alloantibodies against Human Leukocyte Antigens (HLA) has been a primary focus in AMR. More recently autoantibodies and alloantibodies against the angiotensin II receptor type 1 (AT1R) and the endothelin A receptor (ETAR) have been linked to poor allograft outcomes in kidney transplantation. Nevertheless, evidence supporting routine testing remains insufficient. ELISA testing for anti-AT1R and anti-ETAR antibodies was performed in a pediatric renal transplant cohort. We selected 12 pediatric recipients who had undergone protocol biopsies and antibody measurements at 6 and 24 months post-transplant. Immunohistochemistry was performed on biopsies for AT1R and ETAR as well as the adhesion molecules ICAM-1 and VCAM-1. The analysis showed that ICAM-1 and VCAM-1 expression was significantly increased, along with the presence of circulating antibodies, in patients at 24 months post-transplant compared to patients without circulating antibodies. The presence of anti-AT1R and anti-ETAR antibodies does not seem to influence the expression of their receptors in the transplanted organ. Instead, the increase in adhesion molecules may precede the development of histological damage. Therefore, enlarging the cohort and extending long-term observation would help to understand the impact of anti-AT1R and anti-ETAR antibodies after transplantation.