Prognostic significance of proteomics and multi-omics studies in renal carcinoma.

INTRODUCTION: Renal carcinoma, and in particular its most common variant, the clear cell subtype, is often diagnosed incidentally through abdominal imaging and frequently, the tumor is discovered at an early stage. However, 20% to 40% of patients undergoing nephrectomy for clinically localized renal cancer, even after accurate histological and clinical classification, will develop metastasis or recurrence, justifying the associated mortality rate. Therefore, even if renal carcinoma is not among the most frequent nor deadly cancers, a better prognostication is needed. AREAS COVERED: Recently proteomics or other omics combinations have been applied to both cancer tissues, on the neoplasia itself and surrounding microenvironment, cultured cells, and biological fluids (so-called liquid biopsy) generating a list of prognostic molecular tools that will be reviewed in the present paper. EXPERT OPINION: Although promising, none of the approaches listed above has been yet translated in clinics. This is likely due to the peculiar genetic and phenotypic heterogeneity of this cancer, which makes nearly each tumor different from all the others. Attempts to overcome this issue will be also revised. In particular, we will discuss how the application of omics-integrated approaches could provide the determinants of response to the different targeted drugs.

Effects of Hematuria on the Proteomic Profile of Urinary Extracellular Vesicles: Technical Challenges.

Hematuria is a common sign of many renal and urologic pathologic conditions and it may affect the proteomic analysis of urinary extracellular vesicles (UEv), nanovesicles released from all cells in contact with the urinary space. This condition hinders UEv based proteomic studies aiming to discover biomarkers. Therefore, we studied the effects of hematuria on the proteome of UEv and introduced a possible solution to reduce its misleading impact. We mimicked hematuria by adding increasing amount of blood to nonaffected urine and investigated its effects on UEv isolation, purity, and proteomic composition. We proposed a trypsin treatment able to reduce the impact of hematuria on UEv. The effects of the treatment were investigated by evaluating the UEv proteomic profile, the enrichment of known UEv markers, and by assessing differential protein content by MS-based label-free quantification. Results showed that as the blood contamination increased, it affected both the proteome profile and the yield of UEv isolated from urine. Our treatment with trypsin was able to counteract completely these effects for low/medium levels of hematuria, which are most commonly encountered. This promising finding could lead to the reliable use of hematuria samples for UEv proteomic investigation.

Urinary proteomics for the study of genetic kidney diseases.

Despite their low prevalence, genetic kidney diseases (GKD) still represent a serious health problem. They often lead to kidney failure and to the consequent need of dialysis or kidney transplant. To date, reliable diagnosis requires laborious genetic tests and/or a renal biopsy. Moreover, only scant and non-specific markers exist for prognostic purposes. Biomarkers assayed in an easily available and low-cost sample, such as urine, would be highly valuable. Urinary proteomics can provide clues related to their development through the identification of differentially expressed proteins codified by the affected genes, or other dis-regulated species, in total or fractionated urine, providing novel mechanistic insights. In this review, the authors summarize and discuss the results of the main proteomic investigations on GKD urine samples and in urinary extracellular vesicles.

The proteomic landscape of renal tumors.

INTRODUCTION: Renal cell carcinoma (RCC) is the most fatal of the common urologic cancers, with approximately 35% of patients dying within 5 years following diagnosis. Therefore, there is a need for non-invasive markers that are capable of detecting and determining the severity of small renal masses at an early stage in order to tailor treatment and follow-up. Proteomic studies have proved to be very useful in the study of tumors. Areas covered: In this review, we will detail the current knowledge obtained by the different proteomic approaches, focusing on MS-based strategies, used to investigate RCC biology in order to identify diagnostic, prognostic and predictive biomarkers on tissue, cultured cells and biological fluids. Expert commentary: Currently, no reliable biomarkers or targets for RCC have been translated into the clinical setting. Moreover, despite the efforts of proteomics and other -omics disciplines, only a small number of them have been observed as shared targets between the different analytical platforms and biological specimens. The difficulty to define a specific molecular pattern for RCC and its subtypes highlights a peculiar profile and a heterogeneity that must be taken into account in future 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.

A hyphenated microLC-Q-TOF-MS platform for exosomal lipidomics investigations: application to RCC urinary exosomes.

Urinary exosomes are released from every renal epithelial cell type facing the urinary space and therefore, they may carry molecular markers of renal dysfunction and structural injury. Here, we present a hyphenated microLC-Q-TOF-MS platform for lipidomics studies applied to investigate the urinary exosome lipid repertoire. Lipids were separated by reversed-phase chromatography using a linear gradient of formic acid 0.2% and tetrahydrofuran, in 40 min of analysis. Features (m/z with associated own retention time) were extracted by MarkerLynx(TM) (Waters) and processed, demonstrating good analytical performance in terms of repeatability and mass accuracy of the microLC Q-TOF MS platform. In particular, a stable retention time (RSD less than 4%) and relative intensity (RSD from 2.9% to 11%) were observed. Moreover, the method takes advantages by the use of a lock spray interface (Waters) that allows readjusting the m/z data after acquisition, obtaining inaccuracy below 6 ppm in measuring the m/z value of the reference compound during chromatographic run. The method was employed in a preliminary application to perform comparative analysis from healthy control subjects and renal cell carcinoma (RCC) patients, in order to possibly highlight differences in lipid composition to be exploited as potential tumor biomarker. Differential lipid composition in RCC urinary exosomes was achieved and tentatively identified by accurate mass, providing a preliminary indication of a relationship between lipid composition of urinary exosomes and RCC disease. Among the total features significantly different in RCC exosomes, the ion at m/z 502.3 was taken as an example for molecular confirmation by MS/MS fragmentation analysis.

Advances in membranous vesicle and exosome proteomics improving biological understanding and biomarker discovery.

Exosomes are membranous vesicles released by cells in extracellular fluids: they have been found and analyzed in blood, urine, amniotic fluid, breast milk, seminal fluid, saliva and malignant effusions, besides conditioned media from different cell lines. Several recent papers show that exosome proteomes of different origin include both a common set of membrane and cytosolic proteins, and specific subsets of proteins, likely correlated to cell-type associated functions. This is particularly interesting in relation to their possible involvement in human diseases. The knowledge of exosome proteomics can help not only in understanding their biological roles but also in supplying new biomarkers to be searched for in patients' fluids. This review offers an overview of technical and analytical issues in exosome proteomics, and it highlights the significance of proteomic studies in terms of biological and clinical usefulness.

Primary cell cultures from human renal cortex and renal-cell carcinoma evidence a differential expression of two spliced isoforms of Annexin A3.

Primary cell cultures from renal cell carcinoma (RCC) and normal renal cortex tissue of 60 patients have been established, with high efficiency (more than 70%) and reproducibility, and extensively characterized. These cultures composed of more than 90% of normal or tumor tubular cells have been instrumental for molecular characterization of Annexin A3 (AnxA3), never extensively studied before in RCC cells although AnxA3 has a prognostic relevance in some cancer and it has been suggested to be involved in the hypoxia-inducible factor-1 pathway. Western blot analysis of 20 matched cortex/RCC culture lysates showed two AnxA3 protein bands of 36 and 33 kDa, and two-dimensional Western blot evidenced several specific protein spots. In RCC cultures the 36-kDa isoform was significantly down-regulated and the 33-kDa isoform up-regulated. Furthermore, the inversion of the quantitative expression pattern of two AnxA3 isoforms in tumor cultures correlate with hypoxia-inducible factor-1alpha expression. The total AnxA3 protein is down-regulated in RCC cultures as confirmed also in tissues by tissue microarray. Two AnxA3 transcripts that differ for alternative splicing of exon III have been also detected. Real-time PCR quantification in 19 matched cortex/RCC cultures confirms the down-regulation of longer isoform in RCC cells. The characteristic expression pattern of AnxA3 in normal and tumor renal cells, documented in our primary cultures, may open new insight in RCC management.

Urinary Extracellular Vesicle Protein Profiles Discriminate Different Clinical Subgroups of Children with Idiopathic Nephrotic Syndrome.

Idiopathic nephrotic syndrome (INS) is the most frequent primary glomerular disease in children, displaying high grade proteinuria and oedema. The mainstay of therapy are steroids, and patients are usually classified according to the treatment response (sensitive vs. resistant). The mechanisms involved in INS pathogenesis and treatment responsiveness have not yet been identified. In this context, the analysis of urinary extracellular vesicles (UEv) is interesting, since they represent a molecular snapshot of the parental cells, offering a "fingerprint" for monitoring their status. Therefore, the aim of this study is to verify the feasibility of using UEv of INS patients as indicators of therapy response and its prediction. UEv were isolated from the urine of pediatric patients in remission after therapy; they showed characteristic electrophoresis profiles that matched specific patient subgroups. We then built a statistical model to interpret objectively each patient UEv protein profile: in particular, steroid-resistant patients cluster together with a very distinct pattern from other INS patients and controls. In conclusion, the evaluation of the UEv protein profile looks promising in the investigation of INS, showing a disease signature that might predict clinical evolution.

Urinary Extracellular Vesicles and Salt-Losing Tubulopathies: A Proteomic Approach.

Renal tubular cells release urinary extracellular vesicles (uEV) that are considered a promising source of molecular markers for renal dysfunction and injury. We investigated uEV proteomes of patients with hereditary salt-losing tubulopathies (SLTs), focusing on those caused by Gitelman and Bartter (BS) syndromes, to provide potential markers for differential diagnosis. Second morning urine was collected from patients with genetically proven SLTs and uEV were isolated by the ultracentrifugation-based protocol. The uEV proteome was run through a diagonal bidimensional electrophoresis (16BAC/SDS-PAGE), to improve hydrophobic protein resolution. Sixteen differential spots from the proteome of two variants (BS2 and BS3) were analysed by nLC-ESI-MS/MS after in-gel tryptic digestion. A total of 167 protein species were identified from 7 BS2 spots and 9 BS3 spot. Most of these proteins were membrane-associated proteins, in particular transmembrane proteins, and were related to typical renal functions. The differential content of some uEV was then validated by immunoblotting. Our work suggests that uEV proteomics represents a promising strategy for the identification of differential SLT proteins. This could play a role in understanding the pathophysiological disease mechanisms and may support the recognition of different syndromes.

Development of an algorithm for ruling-out non-ST elevation myocardial infarction in the emergency department using high sensitivity troponin T assay.

INTRODUCTION: Chest pain and its clinical manifestations are the most common reasons for presentation to the emergency department (ED). Given that the prevalence of chest pain due to acute myocardial infarction (AMI) in the ED is modest, clinicians should use cardiac troponins to safely and rapidly rule out AMI, avoiding the delayed release of low risk patients. The study aims to develop and validate an algorithm to early rule-out of non-ST elevation myocardial infarction (NSTEMI) in subjects admitted to the ED with symptoms of myocardial infarction. METHODS: High sensitivity cardiac Troponin T (hs-cTnT) serial measurements (baseline, T0; after 1 h, T1; after 3 h, T3) were used to develop and validate the algorithm, respectively, in 6403 and 773 consecutive admissions suggestive of AMI. RESULTS: Patients were classified as having or not having NSTEMI according to clinical assessment, diagnostic imaging, and serial measurements ofhs-cTnT; ROC curve analysis allowed to find changes in consecutive hs-cTnT associated with diagnostic sensitivity close to 100%. Only patients with hs-cTnTat T0 lower than 14 ng/L resultedto be eligible for the safe rule-out of NSTEMI. CONCLUSIONS: Although some points remain to be improved, the results obtained indicate that algorithms for fast NSTEMI rule-out are feasible and safe.

Concentration and microsatellite status of plasma DNA for monitoring patients with renal carcinoma.

We verified the feasibility of plasma bound method for detecting renal cell carcinoma (RCC) combining the study of plasma DNA concentration and microsatellite alterations (LOH). Plasma DNA concentration was evaluated with real-time PCR in 54 patients with renal neoplasm before surgery and in 20 of these patients during a 26-64 month follow-up. Microsatellite study was performed on tumour tissue DNA of 33 RCC clear cell (RCCcc) and on plasma DNA of 14 RCCcc patients during preoperative and/or follow-up period. Patients had a significantly high (26.4+/-48.3 ng/ml versus controls 3.2+/-1.5 ng/ml; p=0.003) preoperative plasma DNA concentration that decreased after nephrectomy. During follow-up, plasma DNA increased in 12 patients without evidence of neoplasia; 3 patients successively relapsed. Tumour tissue DNA of 25 RCCcc patients (75.8%) displayed microsatellite LOH. Preoperative plasma DNA of 9 patients harboured LOH in 5 cases (55.6%). Augmented plasma DNA of 7 patients displayed LOH in 3 cases (42.9%) at follow-up, and in 1 case preceded the recurrence of disease. Plasma DNA concentration combined with microsatellite LOH in plasma DNA may predict disease recurrence in RCC patients.

AQP1 expression analysis in human diseases: implications for proteomic characterization.

Aquaporin (AQP)1 belongs to a ubiquitous family of water channel proteins characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs existing in almost all organs and tissues. Currently, 13 human AQPs are known and they are divided into two subgroups according to their ability to transport only water molecules, such as AQP1, or also glycerol and other small solutes. The genomic, structural and functional aspects of AQP1 are briefly described. An in-depth discussion is devoted to proteomic approaches that are useful for identifying and characterizing AQP1, mainly through electrophoretic techniques combined with different extraction procedures followed by mass spectrometry analysis. Moreover, the relevance of AQP1 in human diseases is also explained. Its role in human tumors and, in particular, those of the kidney (e.g., clear cell renal carcinoma) is discussed.

Enhanced folate binding of cultured fibroblasts from Alzheimer's disease patients.

We compared the levels of serum folate from Alzheimer's disease (AD) patients and from age-matched healthy subjects and used primary cultures of fibroblasts, obtained from the two groups, to assess possible differences in their ability to bind folate. The results show that the levels of circulating folate are significantly (p<0.01; n=30) lower in AD patients than in controls (4.91+/-2.44 and 7.56+/-2.5 ng/mL, respectively). Moreover, the folate binding of AD fibroblasts is significantly (p<0.01; n=8) higher (2-4-fold) with respect to controls. RT-PCR experiments suggest that the higher folate binding could be due to an enhanced expression in AD fibroblasts of folate receptor alpha.

Enhanced GM1 ganglioside catabolism in cultured fibroblasts from Alzheimer patients.

The metabolic processing of GM1 ganglioside, exogenously administered to cultured skin fibroblasts, was investigated on cells obtained from patients affected with Alzheimer disease, in comparison with age-matched control subjects. Cultured fibroblasts were incubated with GM1 ganglioside, [(3)H]-radiolabelled at the sphingosine moiety. It was observed that the extent of tritiated GM2 and GM3 ganglioside formation was higher in AD fibroblasts than in control cells. The activity of acidic beta-D-galactosidase, responsible of GM1 hydrolysis to GM2 within lysosomes, assayed in vitro on cell lysates, was increased in AD fibroblasts in comparison with control cells. These data suggest that up-regulation of lysosomal enzymes could be responsible of the enhanced GM1 catabolism in AD fibroblasts. Finally, it was found that the extent of GM1 hydrolysis in AD fibroblasts was inversely correlated with the mini-mental score index of patients. The increased hydrolysis rate of sphingolipids could be taken as peripheral hallmark of Alzheimer's disease patients.

Proteomics of plasma membrane microdomains.

Plasma membrane microdomains represent subcompartments of the plasma membrane characterized by a specific lipid and protein composition. The recognition of microdomains in nearly all the eukaryotic membranes has accredited them with specialized functions in health and disease. Several proteomic studies have recently addressed the specific composition of plasma membrane microdomains, and will be reviewed in this paper. Peculiar information has been obtained, but a comprehensive view of the main protein classes required to define the microdomain proteome is still missing. The achievement of this information is slowed by the difficulties encountered in resolving and analyzing hydrophobic proteins, but it could help in understanding the overall function of plasma membrane microdomains and their involvement in human pathology.

Ghrelin regulates proliferation and differentiation of osteoblastic cells.

It has previously been reported that growth hormone secretagogues (GHS) may have a role in the regulation of bone metabolism in animals and humans. In this study we evaluated the effect of ghrelin, the endogenous ligand of GHS receptors, on the proliferation rate and on osteoblast activity in primary cultures of rat calvaria osteoblasts. In the same experiments, we compared the effects of ghrelin with those of hexarelin (HEXA) and EP-40737, two synthetic GHS with different characteristics. Both ghrelin and HEXA (10(-11)-10(-8) M) significantly stimulated osteoblast proliferation at low concentrations (10(-10) M). Surprisingly, EP-40737 demonstrated an antiproliferative effect at 10(-9)-10(-8) M, whereas lower concentrations had no effect on cell proliferation. Ghrelin and HEXA significantly increased alkaline phosphatase (ALP) and osteocalcin (OC) production. At variance with these peptides, EP-40737 did not significantly stimulate ALP and OC. The mRNA for GHS-R1a receptors and the corresponding protein were detected in calvarial osteoblasts by RT-PCR and Western blot respectively, indicating that ghrelin and GHS may bind and activate this specific receptor. We conclude that endogenous ghrelin and synthetic GHS modulate proliferation and differentiation of rat osteoblasts, probably by acting on their specific receptor.

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.

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.