Urinary Proteomics – a Potential Method for Characterization of Acute Rejection Biomarkers

Håvard Loftheim¹, Karsten Midtvedt², Pål Falck¹, Anders Hartmann², Léon Reubsaet¹, Anders Åsberg¹.

¹School of Pharmacy, University of Oslo; ²Laboratory for Renal Physiology, Oslo University Hosptial, Rikshospitalet, Oslo, Norway.

Proteomics for determination of clinically relevant biomarkers is interesting for potential preemptive approaches to prevent acute rejections.

Twenty renal transplant recipients (average age 54 years) on cyclosporine, mycophenolate and steroids were followed prospectively in the early posttransplant phase. Urinary samples were collected three times weekly the first two weeks, twice weekly the next four weeks followed by 1-2 samples per week for the rest of the 3 months follow-up. Midstream urine were collected, left at 4 °C for up to one hour, centrifuged at 800g for 10 minutes and stored at -70 °C. Clinical information was collected during the whole period for the definition of acute rejection episodes. Proteomic analyses of the urine samples were performed with a recently published method using (16)O/(18)O-labeling for quantification. Definition of a regulated protein was based on relevant differences in protein levels between samples.

Seven of the 20 patients developed biopsy proven acute rejections, after an average of 44 ±32 days posttransplant. One of these rejections developed eight days posttransplant and was hence not analyzed. Two main patterns were shown in 18 urinary proteins that were more than two-fold up-regulated in at least three of the six remaining patients. Eight proteins showed an early rise, before clinical signs of the acute rejection, and stayed high up to the rejection episode. Four proteins showed less regulation early but a significant elevation at the time of acute rejection.

The present study show that the proteomics methodology used in this study is applicable for analysis of clinical urinary samples from renal transplant recipients. This method can hence be used in the search for relevant protein markers for acute rejection. Further investigations are needed to characterize the regulated proteins found in the present study. Especially the early rising proteins are interesting candidates as biomarkers for acute rejection.