Differential expressed proteins in urine samples from Von Hippel-Lindau disease and renal cell carcinoma patients versus healthy people.

Background: Von Hippel-Lindau (VHL) disease is an autosomal dominant, inherited syndrome occurring in 1 out of 35,000 births. VHL is characterized by the development of retinal and CNS haemangioblastomas, phaeochromocytomas, pancreatic neuroendocrine tumours, clear-cell renal cancers (RCC) and renal cysts. In particular, RCC occurs in about 40% of patients affected by VHL disease and is often bilateral and multifocal. Actually the only method to identify abdominal lesion is the yearly radiological imaging. There are no reliable methods and markers to classify the VHL patients based on the risk of developing renal cancer (RCC). In order to identify differentially expressed proteins, that could be useful as predictors of the VHL related RCC, we performed 2DE analysis on urine samples from healthy subjects, patients with sporadic RCC and VHL syndrome patients. The latter were collected during the annual follow-up in our clinical care VHL-centre. Materials and Methods: Urine samples were collected from 9 healthy subjects, 10 patients affected by VHL syndrome and 9 patients with RCC. Proteins were obtained through Acetone Precipitation and solubilised in Lysis Buffer (9M Urea, 4% CHAPS, 1mM Na 3 VO 4 , 80 mM DTT, protease inhibitors). Following protein quantification, 200 μg of each sample were loaded on IPG strip gels (7 cm IPG strips, pH 3-10 NL) after dialysis. For second dimension 10% poly-acrylamide gels were run. Colloidal Comassie-stained gels were analysed by PD Quest 2D analysis software and statistical analysis was performed (T test). The study was approved by the internal institution ethical committee. Results: From January 2012 to January 2013 we collected urine samples (100 ml) from 10 VHL patients, 9 sporadic RCC and 9 healthy people and we compared the protein expression profile among them. Mean age of the VHL group was 34.33 years (range 24-58), 6 male and 3 female, 3 patients had positive history of renal cancer. Mean age of sporadic RCC patients was 65 years (range 43-78), 4 male and 5 female, all with histological diagnosis of clear cell RCC. The healthy urine samples were collected from 9 blood donors, with mean age of 42 years (range 25-58) 4 male and 5 female. Image analysis of the 2DE maps showed 35 statistically significant (p<0.05) differentially expressed spots among the three groups. Conclusion: Through PD Quest 2D analysis software of the 2DE urine maps we demonstrated the presence of 35 statistically significant (p<0.05) differentially expressed proteins in VHL patients versus RCC and healthy people. These preliminary evidence could suggest the possibility to develop a risk assessment tool for RCC in VHL patients.