Urinary proteomic analysis of chronic allograft nephropathy

The pathogenesis of progressive renal allograft injury, which is termed chronic allograft nephropathy (CAN), remains obscure and is currently defined by histology. Prospective protocol-biopsy trials have demonstrated that clinical and standard laboratory tests are insufficiently sensitive indicators of the development and progression of CAN. The study aim was to determine if CAN could be characterized by urinary proteomic data and identify the proteins associated with disease. The urinary proteome of 75 renal transplant recipients and 20 healthy volunteers was analyzed using surface enhanced laser desorption and ionization MS. Patients could be classified into subgroups with normal histology and Banff CAN grades 2-3 with a sensitivity of 86% and a specificity of 92% by applying the classification algorithm Adaboost to urinary proteomic data. Several urinary proteins associated with advanced CAN were identified including α1-microglobulin, β2-microglobulin, prealbumin, and endorepellin, the antiangiogenic C-terminal fragment of perlecan. Increased urinary endorepellin was confirmed by ELISA and increased tissue expression of the endorepellin/perlecan ratio by immunofluoresence analysis of renal biopsies. In conclusion, analysis of urinary proteomic data has further characterized the more severe CAN grades and identified urinary endorepellin, as a potential biomarker of advanced CAN.     

Detection of urinary biomarkers for early diagnosis of acute renal allograft rejection by proteomic analysis

Acute allograft rejection has been recognized as a major impediment to improved success in renal transplantation. Timely detection and control of rejection are very important for the improvement in long-term renal allograft survival. Thus, biomarkers for early diagnosis of acute rejection are required urgently to clinical medication. This study seeks to search for such biomarker candidates by comparing patients' pre-treatment urinary protein profiling with their post-treatment urinary protein profiling. A total of 15 significantly and consistently down-regulated protein candidates were identified. Among them, alpha-1-antichymotrypsin precursor (AACT), tumor rejection antigen gp96 (GP96) and Zn-Alpha-2-Glycoprotein (ZAG) were selected for further analysis. The results indicated that Western Blot assay of AACT, GP96 and ZAG had advanced the diagnosis time of acute renal rejection by 3 days, compared with current standard clinical observation and laboratory examination. Furthermore, the double-blind detection revealed that the accuracy, sensitivity and specificity of the diagnosis of acute renal rejection of AACT, GP96 and ZAG were 66.67%/100%/60%, 83.33%/100%/80% and 66.67%/100%/60%, respectively, and 100%/100%/100% in combination. In conclusion, urinary protein AACT, GP96 and ZAG could be a set of potential biomarkers for early non-invasive diagnosis of the acute rejection after renal transplantation.     

The discovery of biomarkers for type 2 diabetic nephropathy by serum proteome analysis

Diabetic nephropathy (DN) is a serious kidney complication of diabetes, and constitutes the leading cause of end-stage renal disease. The earliest clinical evidence of DN is microalbuminuria, a term which refers to the appearance of small but abnormal amounts of albumin in the urine. However, screening methods for DN, such as biomarker assays, are yet to be developed for type 2 DN. In the present study, in an attempt to identify the biomarkers for initial diagnoses of type 2 DN, the protein profiles of human sera collected from 30 microalbuminuric type 2 diabetic patients were compared with those collected from 30 normoalbuminuric type 2 diabetic patients, via 2-DE. As a result, a total of 18 spots were determined to have different protein levels in the microalbuminuric patients. Twelve spots had lower protein levels of approximately 50%, and the other six had higher levels of approximately 100-300% as compared to the spots of normoalbuminuric patients. These spots were identified with ESI-Q-TOF (ESI-quadrupole-TOF) MS. Among the identified proteins, vitamin D-binding protein (DBP) and pigment epithelium-derived factor (PEDF) were verified by Western blotting. The results of this study indicate that the DBP may be employed as diagnostic and monitoring biomarkers of type 2 DN, contingent on further study into the matter.     

Bronchoalveolar lavage fluid proteomic patterns of sulfur mustard-exposed patients

Sulfur mustard is an alkylating agent that reacts with ocular, respiratory, cutaneous, and bone marrow tissues. Main late respiratory complications are chronic obstructive pulmonary disease, bronchiectasis, asthma, and bronchiolitis obliterans. The aim of the present study was to identify differentially expressed proteins in bronchoalveolar lavage (BAL) fluid of control healthy and sulfur mustard-exposed lung disease patients. The BAL protein profile of ten healthy and 30 exposed patients with mild, moderate, and severe conditions (ten males in each group) were separated with 2-D SDS-PAGE and differentially expressed protein spots were successfully identified with MALDI TOF TOF MS. Among the differentially expressed proteins we observed a significant increase in vitamin D binding protein isoforms, haptoglobin isoforms, and fibrinogen especially in exposed moderate and severe lung diseases patients (p<0.01). Moreover, compared with healthy controls, significant decreases was noted in calcyphosine, surfactant protein A, and transthyretin in these patients (p<0.01). Apolipoprotein A1 was detected in all patients' BAL fluid but none of the healthy controls. Furthermore, S100 calcium-binding protein A8 was only detected in BAL fluid of moderate and severe groups. These findings will be useful to improve current methods of monitoring and helps to identify new therapeutic targets for treatment of this complicated illness.     

Profiling of rat urinary proteomic patterns associated with drug-induced nephrotoxicity using CE coupled with MS as a potential model for detection of drug-induced adverse effects

We have investigated urine obtained from Sprague Dawley rats before and after administration of cis-Platin, aiming at the definition of biomarkers for drug-induced cytotoxicity. Rats were treated with 3 or 6 mg/kg cis-Platin (i.p., single injection) and urine samples were collected before and after drug or saline treatment. Analysis of the low molecular weight proteome (<20 kDa) using capillary-electrophoresis coupled mass spectrometry allowed us to tentatively identify 34 urinary peptides that show significant differences between control and treated animals, and hence may serve as a potential biomarker for cis-Platin-induced nephrotoxicity. These biomarkers were confirmed in a blinded assessment of additional samples. The blinded data also revealed time-dependency of induced changes. Some of the potential biomarkers could be sequenced. This information revealed great similarity between cis-Platin-induced changes and significant changes in the urinary proteome of patients suffering from tubular injury (Fanconi syndrome). Our study strongly suggests that (drug-induced) nephrotoxicity can be detected with high accuracy in laboratory rodents using urinary proteome analysis. The effects observed are very similar to those seen in corresponding human diseases and similar approaches may be very helpful in evaluating drug-induced organ damage in preclinical animal models. This study aiming at the definition of biomarkers for drug-induced cytotoxicity may serve as a proof-of-principle for the use of urinary proteomics in assessment of drug-induced nephrotoxicity.     

Contrast-enhanced ultrasound in optimization of treatment plans for diabetic nephropathy patients based on deep learning

The Journal of Supercomputing - To evaluate the efficacy of atorvastatin in the treatment of diabetic nephropathy, contrast-enhanced ultrasound (CEUS) based on Poisson three-dimensional (3D)...     

Fine-needle aspiration for proteomic study of tumour tissues

Fine-needle aspiration (FNA) is a technique largely applied in the diagnosis of tumours. FNA is a safe diagnostic procedure that is widely employed in the examination of masses at relatively low cost and minimal risk to the patient. In this review, we report on the state-of-the-art and the potential role of FNA to search for protein biomarkers by the proteomic approach.     

A simple method for improving local binary patterns by considering non-uniform patterns

The basic idea behind LBP is that an image is composed of micropatterns. A histogram of these micropatterns contains information about the local features in an image. These micropatterns can be divided into two types: uniform and non-uniform. In standard applications using LBP, only the uniform patterns are used. The non-uniform patterns are considered in only a single bin of the histogram that is used to extract features in the classification stage. Non-uniform patterns have undesirable characteristics: they are of a high dimension, partially correlated, and introduce unwanted noise. To offset these disadvantages, we explore using random subspace, well-known to work well with noise and correlated features, to train features based also on non-uniform patterns. We find that a stand-alone support vector machine performs best with the uniform patterns and random subspace with histograms of 50 bins performs best with the non-uniform patterns. Superior results are obtained when the two are combined. Based on extensive experiments conducted in several domains using several benchmark databases, it is our conclusion that non-uniform patterns improve classifier performance.     

Affinity-based microarrays for proteomic analysis of cancer tissues

Based on about a decade of technical developments in analysing the human proteome with antibody microarrays and experience in performing such analyses, now there are the means at hand for detailed and simultaneously global investigations of this kind. Many technical aspects have been dealt with of both the microarray format itself – such as overcoming kinetic and mass transport limitations and thus achieving accurate measurements – and ancillary processes – such as extraction procedures that provide good protein solubilisation, produce reproducible yields and preserve the native protein conformation as much as possible. The overall analysis process is robust and reproducible, highly sensitive down to the level of single-molecule detection and permits an analysis of several parameters on many molecules at a time. While the study of body liquids is widely applied, analyses of tissue proteomes are still scarce. However, conditions do exist to perform the latter at a quality level that meets the standards for clinical applications. This review highlights methodological aspects relevant for a biomedically useful analysis of cellular samples and discusses the potential of such studies, in particular, in view of personalised medicine approaches.     

Sources of urinary proteins and their analysis by urinary proteomics for the detection of biomarkers of disease

Renal disorders account for a substantial fraction of the budget for health care in many countries. Proteinuria is a frequent manifestation in afflicted patients, but the origin of the proteins varies based on the nature of the disorder. The emerging field of urinary proteomics has the potential to replace kidney biopsy as the diagnostic procedure of choice for patients with some glomerular forms of renal disease. To fully realize this potential, it is vital to understand the basis for the urinary excretion of protein in physiological and pathological conditions. In this review, we discuss the structure of the nephron, the functional unit of the kidney, and the process by which proteins/peptides enter the urine. We discuss several aspects of proteinuria that impact the proteomic analysis of urine of patients with renal diseases.     

Proteomic analysis of early urinary biomarkers of renal changes in type 2 diabetic patients

Diabetic nephropathy (DN) is a complication associated with diabetes, leading to end-stage renal disease (ESRD). Despite significant progress in understanding DN, the cellular mechanisms leading to the renal damage are incompletely defined. In this study, with the aim to identify urine biomarkers for the early renal alterations in type 2 diabetes mellitus (T2D), we performed urinary proteomic analysis of 10 normoalbuminuric patients with T2D, 12 patients with type 2 DN (T2DN), and 12 healthy subjects. Proteins were separated by 2-DE and identified with ESI-Q-TOF MS/MS. Comparing the patients proteomic profiles with those of normal subjects, we identified 11 gradually differently changed proteins. The decreased proteins were the prostatic acid phosphatase precursor, the ribonuclease and the kallikrein-3. Eight proteins were progressively increased in both patients groups: transthyretin precursor, Ig κ chain C region, Ig κ chain V-II region Cum, Ig κ-chain V-III region SIE, carbonic anhydrase 1, plasma retinol-binding protein, β-2-microglobulin precursor, β-2-glycoprotein 1. The proteomic analysis allowed us to identify several increased urinary proteins, not only in T2DN but also in T2D patients in which the microalbuminuria was in the normal range. These patterns of urinary proteins might represent a potential tool for a better understanding of diabetic renal damage.     

Microfiltration isolation of human urinary exosomes for characterization by MS

Purpose: The purpose of this study was to address the hypothesis that small vesicular urinary particles known as exosomes could be selectively microfiltered using low protein‐binding size exclusion filters, thereby simplifying their use in clinical biomarker discovery studies. Experimental design: We characterized a microfiltration approach using a low protein binding, hydrophilized polyvinylidene difluoride membrane to easily and efficiently isolate urinary exosomes from fresh, room temperature or 4°C urine, with a simultaneous depletion of abundant urinary proteins. Using LC‐MS, immunoblot analysis, and electron microscopy methods, we demonstrate this method to isolate intact exosomes and thereby enrich for a low abundant urinary proteome. Results: In comparison to other standard methods of exosome isolation including ultracentrifugation and nanofiltration, we demonstrate equivalent enrichment of the exosome proteome with reduced co‐purification of abundant urinary proteins. Conclusion and clinical relevance: In conclusion, we demonstrate a microfiltration isolation method that preserves the exosome structure, reduces contamination from higher abundant urinary proteins, and can be easily implemented into mass spectrometry analysis for biomarker discovery efforts or incorporation into routine clinical laboratory applications to yield higher sample throughput.     

Analysis of head gesture and prosody patterns for prosody-driven head-gesture animation

We propose a new two-stage framework for joint analysis of head gesture and speech prosody patterns of a speaker towards automatic realistic synthesis of head gestures from speech prosody. In the first stage analysis, we perform Hidden Markov Model (HMM) based unsupervised temporal segmentation of head gesture and speech prosody features separately to determine elementary head gesture and speech prosody patterns, respectively, for a particular speaker. In the second stage, joint analysis of correlations between these elementary head gesture and prosody patterns is performed using Multi-Stream HMMs to determine an audio-visual mapping model. The resulting audio-visual mapping model is then employed to synthesize natural head gestures from arbitrary input test speech given a head model for the speaker. In the synthesis stage, the audio-visual mapping model is used to predict a sequence of gesture patterns from the prosody pattern sequence computed for the input test speech. The Euler angles associated with each gesture pattern are then applied to animate the speaker head model. Objective and subjective evaluations indicate that the proposed synthesis by analysis scheme provides natural looking head gestures for the speaker with any input test speech, as well as in "prosody transplant" and gesture transplant" scenarios.     

Sample handling for mass spectrometric proteomic investigations of human urine

Because of its non-invasive sample collection method, human urine is an attractive biological material both for discovering biomarkers and for use in future screening trials for different diseases. Before urine can be used for these applications, standardized protocols for sample handling that optimize protein stability are required. In this explorative study, we examine the influence of different urine collection methods, storage temperatures, storage times, and repetitive freeze-thaw procedures on the protein profiles obtained by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Prospectively collected urine samples from 11 women were collected as either morning or midday specimens. The effects of storage temperature, time to freezing, and freeze-thaw cycles were assessed by calculating the number, intensity, and reproducibility of peaks visualized by SELDI-TOF-MS. On the CM10 array, 122 peaks were detected and 28 peaks were found to be significantly different between urine types, storage temperature and time to freezing. On the IMAC-Cu array, 65 peaks were detected and 1 peak was found to be significantly different according to time to freezing. No significant differences were demonstrated for freeze-thaw cycles. Optimal handling and storage conditions are necessary in clinical urine proteomic investigations. Collection of urine with a single and consistently performed protocol is needed to reduce analytical bias. Collecting only one urine type, which is stored for a limited period at 4°C until freezing at -80°C prior to analysis will provide the most stable profiles.     

Mass spectrometry imaging for the proteomic study of clinical tissue

Over the last decade, MALDI-MS imaging has been used by researchers to explore areas of proteomics, lipidomics and metabolomics in samples of clinical origin for both targeted and global biomarker analysis. Numerous technological advancements in MS and clinical tissue MS imaging have been accomplished; hence, in this article we aim to critically discuss whether MS imaging has now in fact become a true champion of the ‘Omics Era’. In order to assess the potential for it to be routinely used in the clinical setting, it is pertinent to discuss some of its limitations, and to examine how these have been addressed by researchers. The key limitations of the technique we will discuss in this viewpoint article are as follows: sample throughput; relevance to patients, the availability of validated/standardised techniques; and integration with conventional pathology and other medical imaging techniques. Good progress has been made over the last 5 years in overcoming these limitations that had previously restricted the use of this technology in the clinical setting.