Studi di proteomica subcellulare nelle patologie renali: carcinoma renale e nefropatia diabetica

ABSTRACT Renal cell carcinoma (RCC) is among the 10 leading causes of cancer-related deaths worldwide and its incidence is increasing steadily. At the time of diagnosis, 30% of patients have metastases and another third will develop metastatic disease within 10 years. Moreover metastatic RCC is radio- and chemotherapy resistant. Diabetic nephropathy (DN) is a common diabetic complication that causes a renal massive functional deficit making dialysis or transplantation essential for the survival of patients and it is associated with alterations in the expression of several renal proteins. Therefore, it is important to identify differentially expressed proteins to be used as potential diagnostic and/or prognostic markers in RCC and DN. A promising strategy is comparative subcellular proteomics of urinary exosomes, membranous vesicles released from cells and membrane microdomains, lipid arfts and caveolae. About 30 ml of urines were collected from 29 clear cell RCC patients and 28 healthy matched controls and stored at -80°C, after antiproteases addition. Diabetes was induced in 10 Sprague Dawley rats by streptozotocin injection. Control rats (n=4) underwent only buffer injection. Out of 10 diabetic rats, 4 were chronically treated with insulin. After 3 months, 24 hours urines were collected. In both cases (RCC patients and diabetic rats with their healthy controls respectively) exosomes were then isolated from total urines by ultracentrifugation after cells and debris clearing. As regards RCC esults show that the protein profile of urinary exosomes is peculiar: the most abundant urinary proteins of plasmatic origin (i.e. albumin) are markedly reduced, while the Tamm Horsfall protein (THP) is highly enriched and affected by a significant biological variability. Moreover, protein profiles of exosomal fractions isolated from urine of ccRCC and matched controls show some difference. Mass spectrometry analysis of two pools of exosomes isolated from CTRL and RCC patients urines permits the identification of 261 proteins in CTRL and 187 proteins in RCC. Some of these protein Aquaporin-1, Matrix Metallo-protease 9 and Carbonic Anhydrase 9, display differential amount in ccRCC patient urine exosomes by EF/WB. Moreover subcellular fractions were prepared by differential centrifugation from surgical samples of RCC and adjacent normal kidney (ANK). MD were isolated from plasma-membrane-enriched fractions after treatment with Triton X-100 and sucrose density gradient ultracentrifugation. MD derived from RCC and ANK tissues of 7 patients were pooled, and proteins separated by 4-12% and 12% gel electrophoresis. After Coomassie Blue staining, bands were excised and analyzed by LC-MS/MS after trypsin digestion. We identified 83 proteins from microdomains isolated from RCC tissue, and 95 proteins from ANK. About 60% of the identified proteins are membrane-associated and about half of these resulted microdomain-associated. GRAVY scores assignment shows that most identified proteins (about 70%) are in the hydrophobic range. From a functional point of view, we found proteins involved in signal transduction (Ras related proteins), channels (Aquaporin-1), carriers (P-glycoprotein) and cytoskeleton structural constituents (Spectrin). We chose some promising proteins such as Renal dipeptidase (identified only in ANK MD) or Emmprin (overexpressed in RCC tissue) and investigated their differential expression of by WB About DN study, protein profiles of exosomes obtained from the three groups of rat (healthy controls CTRL, diabetic D and diabetic treated with insulin DI) show differences, mainly at low molecular weights. Some differential bands were excised and analyzed by LC-MS/MS after digestion by tripsin. In particular we identified the Major Urinary Proteins, known as MUPs from CTRL and DI gels, while these proteins are not present in the same bands excised from D gels. MUPs regulate glucose and lipid metabolism suggesting an involvement in hyperglycemia, insulin resistance and/or glucose intolerance in diabetes.