Infrared vibrational spectroscopy: a rapid and novel diagnostic and monitoring tool for cystinuria.

Cystinuria is the commonest inherited cause of nephrolithiasis (~1% in adults; ~6% in children) and is the result of impaired cystine reabsorption in the renal proximal tubule. Cystine is poorly soluble in urine with a solubility of ~1 mM and can readily form microcrystals that lead to cystine stone formation, especially at low urine pH. Diagnosis of cystinuria is made typically by ion-exchange chromatography (IEC) detection and quantitation, which is slow, laboursome and costly. More rapid and frequent monitoring of urinary cystine concentration would significantly improve the diagnosis and clinical management of cystinuria. We used attenuated total reflection - Fourier transform infrared spectroscopy (ATR-FTIR) to detect and quantitate insoluble cystine in 22 cystinuric and 5 healthy control urine samples. Creatinine concentration was also determined by ATR-FTIR to adjust for urinary concentration/dilution. Urine was centrifuged, the insoluble fraction re-suspended in 5 µL water and dried on the ATR prism. Cystine was quantitated using its 1296 cm-1 absorption band and levels matched with parallel measurements made using IEC. ATR-FTIR afforded a rapid and inexpensive method of detecting and quantitating insoluble urinary cystine. This proof-of-concept study provides a basis for developing a high-throughput, cost-effective diagnostic method for cystinuria, and for point-of-care clinical monitoring.

Mass spectrometry of extracellular vesicles.

The review briefly summaries main features of extracellular vesicles, a joint terminology for exosomes, microvesicles, and apoptotic vesicles. These vesicles are in the center of interest in biology and medical sciences, and form a very active field of research. Mass spectrometry (MS), with its specificity and sensitivity, has the potential to identify and characterize molecular composition of these vesicles; but as yet there are only a limited, but fast-growing, number of publications that use MS workflows in this field. MS is the major tool to assess protein composition of extracellular vesicles: qualitative and quantitative proteomics approaches are both reviewed. Beside proteins, lipid and metabolite composition of vesicles might also be best assessed by MS techniques; however there are few applications as yet in this respect. The role of alternative analytical approaches, like gel-based proteomics and antibody-based immunoassays, are also mentioned. The objective of the review is to give an overview of this fast-growing field to help orient MS-based research on extracellular vesicles.

Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease.

PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EXPERIMENTAL DESIGN: EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. RESULTS: Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology.

The urinary proteome and metabonome differ from normal in adults with mitochondrial disease.

We studied the extent and nature of renal involvement in a cohort of 117 adult patients with mitochondrial disease, by measuring urinary retinol-binding protein (RBP) and albumin; established markers of tubular and glomerular dysfunction, respectively. Seventy-five patients had the m.3243A>G mutation and the most frequent phenotypes within the entire cohort were 14 with MELAS, 33 with MIDD, and 17 with MERRF. Urinary RBP was increased in 29 of 75 of m.3243A>G patients, whereas albumin was increased in 23 of the 75. The corresponding numbers were 16 and 14, respectively, in the 42 non-m.3243A>G patients. RBP and albumin were higher in diabetic m.3243A>G patients than in nondiabetics, but there were no significant differences across the three major clinical phenotypes. The urine proteome (mass spectrometry) and metabonome (nuclear magnetic resonance) in a subset of the m.3243A>G patients were markedly different from controls, with the most significant alterations occurring in lysosomal proteins, calcium-binding proteins, and antioxidant defenses. Differences were also found between asymptomatic m.3243A>G carriers and controls. No patients had an elevated serum creatinine level, but 14% had hyponatremia, 10% had hypophosphatemia, and 14% had hypomagnesemia. Thus, abnormalities in kidney function are common in adults with mitochondrial disease, exist in the absence of elevated serum creatinine, and are not solely explained by diabetes.

Human Hsp60 with its mitochondrial import signal occurs in solution as heptamers and tetradecamers remarkably stable over a wide range of concentrations.

It has been established that Hsp60 can accumulate in the cytosol in various pathological conditions, including cancer and chronic inflammatory diseases. Part or all of the cytosolic Hsp60 could be naïve, namely, bear the mitochondrial import signal (MIS), but neither the structure nor the in solution oligomeric organization of this cytosolic molecule has still been elucidated. Here we present a detailed study of the structure and self-organization of naïve cytosolic Hsp60 in solution. Results were obtained by different biophysical methods (light and X ray scattering, single molecule spectroscopy and hydrodynamics) that all together allowed us to assay a wide range of concentrations of Hsp60. We found that Naïve Hsp60 in aqueous solution is assembled in very stable heptamers and tetradecamers at all concentrations assayed, without any trace of monomer presence.

Enrichment specificity of micro and nano-sized titanium and zirconium dioxides particles in phosphopeptide mapping.

Owning to their anion-exchange properties, titanium and zirconium dioxides are widely used in phosphopeptide enrichment and purification protocols. The physical and chemical characteristics of the particles can significantly influence the loading capacity, the capture efficiency and phosphopeptide specificity and thus the outcome of the analyses. Although there are a number of protocols and commercial kits available for phosphopeptide purification, little data are found in the literature on the choice of the enrichment media. Here, we studied the influence of particle size on the affinity capture of phosphopeptides by TiO2 and ZrO2. Bovine milk casein derived phosphopeptides were enriched by micro and nanoparticles using a single-tube in-solution protocol at different peptide-to-beads ratio ranging from 1 : 1 to 1 : 200. Unsupervised hierarchical cluster analysis based on the whole set of Matrix Assisted Laser Desorption/Ionization time-of-flight mass spectra of the phosphopeptide enriched samples revealed 62 clustered peptide peaks and shows that nanoparticles have considerably higher enrichment capacity than bulk microparticles. Moreover, ZrO2 particles have higher enrichment capacity than TiO2. The selectivity and specificity of the enrichment was studied by monitoring the ion abundances of monophosphorylated, multiphosphorylated and non-phosphorylated casein-derived peptide peaks at different peptide-to-beads ratios. Comparison of the resulting plots enabled the determination of the optimal peptide-to-beads ratios for the different beads studied and showed that nano-TiO2 have higher selectivity for phosphopeptides than nano-ZrO2 particles.

Differential in gel electrophoresis (DIGE) comparative proteomic analysis of macrophages cell cultures in response to perthamide C treatment.

Secondary metabolites contained in marine organisms disclose diverse pharmacological activities, due to their intrinsic ability to recognize bio-macromolecules, which alter their expression and modulate their function. Thus, the identification of the cellular pathways affected by marine natural products is crucial to provide important functional information concerning their mechanism of action at the molecular level. Perthamide C, a marine sponge metabolite isolated from the polar extracts of Theonella swinhoei and endowed with a broad and interesting anti-inflammatory profile, was found in a previous study to specifically interact with heat shock protein-90 and glucose regulated protein-94, also disclosing the ability to reduce cisplatin-mediated apoptosis. In this paper, we evaluated the effect of this compound on the whole proteome of murine macrophages cells by two-dimensional DIGE proteomics. Thirty-three spots were found to be altered in expression by at least 1.6-fold and 29 proteins were identified by LC ESI-Q/TOF-MS. These proteins are involved in different processes, such as metabolism, structural stability, protein folding assistance and gene expression. Among them, perthamide C modulates the expression of several chaperones implicated in the folding of proteins correlated to apoptosis, such as Hsp90 and T-complexes, and in this context our data shed more light on the cellular effects and pathways altered by this marine cyclo-peptide.

Unraveling amyloid toxicity pathway in NIH3T3 cells by a combined proteomic and 1 H-NMR metabonomic approach.

A range of debilitating human diseases is known to be associated with the formation of stable highly organized protein aggregates known as amyloid fibrils. The early prefibrillar aggregates behave as cytotoxic agents and their toxicity appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increase in free Ca(2+) that lead to apoptotic or necrotic cell death. However, specific signaling pathways that underlie amyloid pathogenicity remain still unclear. This work aimed to clarify cell impairment induced by amyloid aggregated. To this end, we used a combined proteomic and one-dimensional (1) H-NMR approach on NIH-3T3 cells exposed to prefibrillar aggregates from the amyloidogenic apomyoglobin mutant W7FW14F. The results indicated that cell exposure to prefibrillar aggregates induces changes of the expression level of proteins and metabolites involved in stress response. The majority of the proteins and metabolites detected are reported to be related to oxidative stress, perturbation of calcium homeostasis, apoptotic and survival pathways, and membrane damage. In conclusion, the combined proteomic and (1) H-NMR metabonomic approach, described in this study, contributes to unveil novel proteins and metabolites that could take part to the general framework of the toxicity induced by amyloid aggregates. These findings offer new insights in therapeutic and diagnostic opportunities.

Genomic and proteomic approaches to renal cell carcinoma.

Renal cell carcinoma (RCC) is a frequent adult malignancy comprising different subtypes, with clear cell renal cell carcinoma (CCRCC) the most common. CCRCC is associated with the loss of function of the von Hippel-Lindau (VHL) gene. pVHL, the product of the VHL gene, is a component of an E3 ubiquitin ligase complex which targets proteins for degradation. In the absence of functional pVHL, a series of proteins accumulate in the cells, including hypoxic-inducible factor-1a (HIF-1a) and the products of the target genes of pVHL, such as vascular endothelial growth factor (VEGF). The activation of this pathway explains the use of both VEGF and VEGF-receptor inhibitors (bevacizumab, sunitinib and sorafenib) in the therapy of advanced CCRCC. In addition, mammalian target of rapamycin (mTOR), an intracellular serine/threonine kinase, is also implicated in HIF-1a regulation, thus reinforcing the rationale for using mTOR inhibitors (rapamycin) in CCRCC. Proteomic technologies have been applied to human renal cancer to detect biomarkers able to guide physicians for diagnostic and prognostic purposes. Among others, vimentin, heat shock protein 27 (Hsp27), annexin IV and serum amyloid alpha-1 (SAA-1) have been identified as reliable markers of RCC that are potentially useful in the clinical setting.

Proteomic analysis of peritoneal fluid of patients treated by peritoneal dialysis: effect of glucose concentration.

BACKGROUND: Depending on both membrane composition and solute transport rate across the membrane, protein composition of the dialysate of patients receiving peritoneal dialysis (PD) has recently become of great interest. Unfortunately, thus far few studies have focused on dialysate characterization, and further investigations are required to better understand the biological mechanisms influencing PD efficiency. METHODS: Different classical proteomic approaches were combined with advanced mass spectrometric (MS) techniques to analyse peritoneal fluid (PF) protein composition of adult patients receiving PD. Characterization was performed by using 1D gel electrophoresis combined with nano-RP-HPLC-ESI-MS/MS and shotgun proteomics, while comparative analyses were performed coupling 2D gel electrophoresis with MALDI-TOF MS. RESULTS: The study allowed the identification of 151 different proteins from PF, which are mainly of plasmatic origin. Comparison of PD effluents characterized by different glucose concentrations demonstrated four proteins (apolipoprotein A-IV, fibrinogen beta chain, transthyretin and alpha-1-antitrypsin) to be under-expressed in the highest osmolar solution having 4.25% compared to others having 1.5% and 2.5% glucose. All of them were found to be involved in the inflammatory processes. CONCLUSIONS: This study provides a possible platform for future diagnostic and therapeutic applications in the field of PD and allowed the identification of potential targets to be used in preventing inflammatory processes induced by the exposure to dialysis solutions.

Proteomics and tubulopathies.

In the last thirty decade, with the emergence of new trends in molecular biology and advances in high-throughput technologies, much progress has been made in basic renal physiology. Molecular genetics has allowed the identification and elucidation of the structure, function and effects of the mutations of several of the main transporters and ion channels involved in renal disorders. Some renal stone disorders, such as cystinuria and Dent's disease, have been found to be due to mutations in genes SLC3A1 (type I) (See the section "Molecular biology and genotype-phenotype correlation in tubular dysfunction") and SLC7A9 (type II and type III), (See the section "Molecular biology and genotype-phenotype correlation in tubular dysfunction") and in CLC5, respectively. Liddle syndrome, a rare cause of hypertension, is now known to be caused by a mutation in tubular transport, due to a mutation in the SCNN1B gene, encoding for a Na+ channel protein (ENaC). Nevertheless, numerous issues remain unsettled and warrant additional research. These important advances and discoveries are not without limitations and challenges as changes in individual gene expression do not always translate into changes in its protein or protein modification. This raises proteomics as the most logical next step in our understanding of biological processes, as proteins from these deregulated genes are the functional agents in the cells. Proteomics takes a global and comprehensive view of a system, involving in many cases some notion of high throughput; but in contrast to genomics, there is no single biochemical method that can be used for the analysis of all proteins. Genomics and proteomics can complement each other in clinical applications by balancing the strengths and weaknesses of each individual technology. Several proteomics approaches have been exploited to shed more light on the molecular pathophysiology of several hereditary tubular disorders, such as Fanconi and Gitelman syndromes, and have provided important insights into the defective molecular mechanisms underlying these tubulopathies. Here we summarize several of the most important discoveries arising from molecular genetic and proteomic studies on hereditary tubular dysfunctions and show how these results can complement each other to increase our comprehension of these disorders at the molecular level.

Application of proteomic techniques to the study of urine and renal tissue.

The increasing application of proteomic methods to biomedical research is providing us with important new information; it holds particular promise in advancing basic and clinical renal research, but whether proteomics can ever become a routine diagnostic tool in nephrology is still uncertain. Currently, proteomic techniques are used by many groups in the search for "biomarkers" of disease, especially kidney disease, because of the ready availability of urine as an "end-product" of renal function. However, the question as to whether any disease-specific biomarkers exist or can be identified by proteomics is also uncertain. A growing application of proteomics in biomedical research is to understand the mechanism(s) of disease. This brief review is selective; in it we consider examples of proteomic studies of human urine for biomarkers, others that have explored renal physiology, and still others that have begun to probe the proteome of organelles. No single approach is sufficiently comprehensive, and the pooled application of proteomics to renal research will undoubtedly improve our understanding of renal function and enable us to explore in more detail subcellular structures, and to characterize cellular processes at the molecular level. When combined with other techniques in renal research, proteomics, and related analytical methods could prove indispensable in modeling renal function, and perhaps also in diagnosis and management of renal disease.

Combined proteomic and metabonomic studies in three genetic forms of the renal Fanconi syndrome.

The renal Fanconi syndrome is a defect of proximal tubular function causing aminoaciduria and low-molecular-weight proteinuria. Dent's disease and Lowe syndrome are defined X-linked forms of Fanconi syndrome; there is also an autosomal dominant idiopathic form (ADIF), phenotypically similar to Dent's disease though its gene defect is still unknown. To assess whether their respective gene products are ultimately involved in a common reabsorptive pathway for proteins and low-molecular-mass endogenous metabolites, we compared renal Fanconi urinary proteomes and metabonomes with normal (control) urine using mass spectrometry and (1)H-NMR spectroscopy, respectively. Urine from patients with low-molecular-weight proteinuria secondary to ifosfamide treatment (tubular proteinuria; TP) was also analyzed for comparison. All four of the disorders studied had characteristic proteomic and metabonomic profiles. Uromodulin was the most abundant protein in normal urine, whereas Fanconi urine was dominated by albumin. (1)H-NMR spectroscopic data showed differences in the metabolic profiles of Fanconi urine vs. normal urine, due mainly to aminoaciduria. There were differences in the urinary metabolite and protein compositions between the three genetic forms of Fanconi syndrome: cluster analysis grouped the Lowe and Dent's urinary proteomes and metabonomes together, whereas ADIF and TP clustered together separately. Our findings demonstrate a distinctive "polypeptide and metabolite fingerprint" that can characterize the renal Fanconi syndrome; they also suggest that more subtle and cause-specific differences may exist between the different forms of Fanconi syndrome that might provide novel insights into the underlying mechanisms and cellular pathways affected.