Urinary proteomics in kidney transplant recipients and influence of demographics and treatments as potential confounders of graft lesions

In renal transplantation, there is a strong need for non-invasive biomarkers that can replace or complement biopsy to detect graft lesions. In this study, we employed a high-resolution mass spectrometry technique (micro-LC-TOF-MS) to analyze urinary proteomics from 311 kidney transplant recipients recruited across four European centers. Urine proteins were extracted using a Filter-Aided Sample Preparation (FASP) protocol, digested, and identified through Paragon® and Mascot® searches against the Swiss-Prot database. We detected 7260 peptides corresponding to 1777 proteins. In a subgroup of 183 patients with normal graft biopsies, we assessed the influence of donor and recipient age and sex, along with different immunosuppressive regimens, on the urinary proteome. Significant variations were observed in protein expression based on these variables, with recipient sex showing the most pronounced effect. STRING-based clustering highlighted key biological pathways affected by these variables, including immune modulation and metabolic regulation. Proteins such as MSMB and PPAP (males) and SPRR3 and S100A9 (females) were strongly discriminative, while treatments like tacrolimus and corticosteroids altered distinct protein networks related to coagulation and inflammation. These findings underscore the necessity of adjusting for demographic and treatment-related variables in biomarker discovery to avoid misleading conclusions.