Following transplantation, kidney transplant recipients are routinely screened for signs of an acute rejection (AR). Previous studies have indicated that patients with AR have an increased risk of developing chronic allograft nephropathy and reduced long-term graft survival.1,2 Elevated creatinine levels, along with histology of graft biopsies, can indicate AR is occuring;3 however, creatinine levels can vary in the rate at which they increase and can be elevated by other causes.
Biopsies are also not an ideal method for diagnostics, as the procedure carries risks and is inconvenient and invasive at best. Concerned with the flaws of this diagnostic approach, the Reubsaet and Asberg groups sought to investigate potential urinary biomarkers of AR using a shotgun proteomic approach. Previous studies have sought to identify urinary biomarkers of AR in the urinary proteome, including one study employing shotgun proteomics to characterize protein found in pooled urine from pediatric kidney transplants with AR.4
In their small principle study,5 the Reubsaet and Asberg groups obtained urine samples from six renal transplant patients with acute AR, as well stable grafts from matched controls.
Urine samples were collected from 4.7±2.7 days following transplantation, and patients were followed for 8-10 weeks. Protein concentrations were measured using Bradford’s method.6 Samples were depleted of albumin and digested with trypsin. 2D LC-MS/MS analysis was performed on an LTQ Orbitrab Mass Spectrometer (Thermo Scientific).
MS data was analysed using Proteome Discoverer 1.2 (Thermo Scientific). A strict target false discovery rate (FDR) of 0.01 and a relaxed FDR of 0.05 were obtained through searching for the reverse sequence of proteins. Only proteins with an FDR of .05 were accepted.
In all, 11 proteins were significantly upregulated, with 10 of these proteins functioning in growth regulation and immune response. In the AR group, six proteins functioning in growth regulation were upregulated, and in five out of six of the patients these included IGFBP7, Vasorin, EGF, and Galectin-3-binding protein. Four out of six of the patients in the AR group also had upregulated proteins involved in immune response MASP2, C3, CD59, Ceruloplasmin, PiGR and CD74. Another protein, MEP1A, was also upregulated in the AR group and not present in the healthy controls. This protein is not involved in immune response or growth, and further studies may show it is indeed a biomarker for AR.
Interestingly, the increased levels of proteins were detected in the AR group days before creatinine levels increased. While these results look promising, further proteomic studies are needed with larger population sizes before a diagnostic biomarker for AR can be validated.
1. Nankivell B.J., Chapman J.R. (2006) ‘Chronic allograft nephropathy: current concepts and future directions‘, Transplantation, 8 (5) (pp. 643-654)
2. Kaplan, B. (2006) ‘Overcoming barriers to long-term graft survival‘, American Journal of Kidney Diseases, 47 (4) (pp. 52-S64)
3. Racusen LC, et al. (1999) ‘The Banff 97 working classification of renal allograft pathology‘, Kidney International, 55 (2) (pp. 713-723)
4. Sigdel, T.K., et al. (2010) ‘Shotgun proteomics identifies proteins specific for acute renal transplant rejection‘, Proteomics Clinical Applications, 4 (1), (pp. 32-47)
5. Loftheim, H., et al. (2012) ‘Urinary proteomic shotgun approach for identification of potential acute rejection biomarkers in renal transplant recipients‘, Transplantation Research, 1 (1) 9. doi: 10.1186/2047-1440-1-9
6. Bradford, M.M. (1976) ‘Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing Principle of Protein-Dye Binding‘, Analytical Biochemistry, May 7 (72) (pp. 248-254)