Comparative membrane proteomics: a technical advancement in the search of renal cell carcinoma biomarkers.

Renal Cell Carcinoma (RCC) is the most common kidney cancer, accounting for 3% of adult malignancies, with high metastatic potential and radio-/chemo-resistance. To investigate the protein profile of membrane microdomains (MD), plasma membrane supramolecular structures involved in cell signaling, transport, and neoplastic transformation, we set up a proteomic bottom-up approach as a starting point for the identification of potential RCC biomarkers. We purified MD from RCC and adjacent normal kidney (ANK) tissues, through their resistance to non-ionic detergents followed by ultracentrifugation in sucrose density gradient. MD from 5 RCC/ANK tissues were then pooled and analysed by LC-ESI-MS/MS. In order to identify the highest number of proteins and increase the amount of membrane and hydrophobic ones, we first optimized an enzymatic digestion protocol based on Filter Aided Sample Preparation (FASP), coupled to MD delipidation. The MS analysis led to the identification of 742 ANK MD and 721 RCC MD proteins, of which, respectively, 53.1% and 52.6% were membrane- bound. Additionally, we evaluated RCC MD differential proteome by label-free quantification; 170 and 126 proteins were found to be, respectively, up-regulated and down-regulated in RCC MD. Some differential proteins, namely CA2, CD13, and ANXA2, were subjected to validation by immunodecoration. These results show the importance of setting up different protocols for the proteomic analysis of membrane proteins, specific to the different molecular features of the samples. Furthermore, the subcellular proteomic approach provided a list of differentially expressed proteins among which RCC biomarkers may be looked for.

Preparation of urinary exosomes: methodological issues for clinical proteomics.

Urinary exosomes are small (<100 nm) vesicles secreted into urine from renal epithelial cells. They are coated with lipid bilayer, they contain an array of membrane and cytosolic proteins, and selected RNA species, reflecting the molecular composition of their cell of origin. Thus, urinary exosomes have received considerable attention as potential biomarker source, as their proteomic analysis could lead to the discovery of new non-invasive site-specific biomarkers for renal diseases. Here, we describe a robust method for urinary exosome preparation, additional protocols for their biochemical characterization and for the quantitation of different preparations, to be used for comparative proteomic studies.

Urinary exosomes in the diagnosis of Gitelman and Bartter syndromes.

BACKGROUND: Gitelman syndrome (GS) and Bartter syndrome (BS) are hereditary salt-losing tubulopathies (SLTs) resulting from defects of renal proteins involved in electrolyte reabsorption, as for sodium-chloride cotransporter (NCC) and furosemide-sensitive sodium-potassium-chloride cotransporter (NKCC2) cotransporters, affected in GS and BS Type 1 patients, respectively. Currently, definitive diagnosis is obtained through expensive and time-consuming genetic testing. Urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, represent an ideal source of markers for renal dysfunction and injury, because UE molecular composition stands for the cell of origin. On these assumptions, the aim of this work is to evaluate the relevance of UE for the diagnosis of SLTs. METHODS: UE were purified from second morning urines collected from 32 patients with genetically proven SLTs (GS, BS1, BS2 and BS3 patients), 4 with unclassified SLTs and 22 control subjects (age and sex matched). The levels of NCC and NKCC2 were evaluated in UE by SDS-PAGE/western blotting with specific antibodies. RESULTS: Due to their location on the luminal side of tubular cells, NCC and NKCC2 are well represented in UE proteome. The NCC signal is significantly decreased/absent in UE of Gitelman patients compared with control subjects (Mann-Whitney t-test, P < 0.001) and, similarly, the NKCC2 in those of Bartter type 1 (P < 0.001). The difference in the levels of the two proteins allows recognition of Gitelman and Bartter type 1 patients from controls and, combined with clinical data, from other Bartter patients. Moreover, the receiver operating characteristic curve analysis using UE NCC densitometric values showed a good discriminating power of the test comparing GS patients versus controls and BS patients (area under the curve value = 0.92; sensitivity 84.2% and specificity 88.6%). CONCLUSIONS: UE phenotyping may be useful in the diagnosis of GS and BS, thus providing an alternative/complementary, urine-based diagnostic tool for SLT patient recognition and a diagnostic guidance in complex cases.

The urinary proteome and peptidome of renal cell carcinoma patients: a comparison of different techniques.

Renal cell carcinomas, originating from the renal cortex, account for about 80% of kidney primary malignancies. Small localized tumors rarely produce symptoms and diagnosis is often delayed until the disease is advanced. In contrast to other urological cancers, renal cell carcinomas are associated with a high degree of metastases and a low 5-year survival rate. The identification of diagnostic and prognostic markers, especially in the urine, remains an area of intense investigation. Different proteomic strategies have been applied so far to biomarker discovery in urine at the proteome or the peptidome level. Gel-based and gel-free strategies combined with mass spectrometry are the most-used strategies, have different success rates, and will be depicted here. We also prefigure a scenario in which the limitations of a single approach are overcome by applying new and complementary research strategies, relying on the excellent availability coupled to the intrinsic richness typical of urine samples.