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.     

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.     

Human kidney glomerulus proteome and biomarker discovery of kidney diseases

The kidney glomerulus is the site of plasma filtration and production of primary urine in the kidney. The structure not only plays a pivotal role in ultrafiltration of plasma into urine but also is the locus of kidney diseases progressing to chronic renal failure. Patients afflicted with these glomerular diseases frequently progress to irreversible loss of renal function and inevitably require replacement therapies. The diagnosis and treatment of glomerular diseases are now based on clinical manifestations, urinary protein excretion level, and renal pathology of needle biopsy specimens. The molecular mechanisms underlying the progression of glomerular diseases are still obscure despite a great number of clinical and experimental studies. Proteomics is a particularly promising approach for the discovery of proteins relevant to physiological and pathophysiological processes, and has been recently employed in nephrology. Although until now most efforts of proteomic analysis have been conducted with urine, the biological fluid that is easily collected without invasive procedures, proteomic analysis of the glomerulus, the tissue most proximal to the disease loci, is the most straightforward approach. In this review, we attempt to outline the current status of clinical proteomics of the glomerulus and provide a perspective of protein biomarker discovery of glomerular diseases.     

Identification of melanoma‐specific serological markers using proteomic analyses

The discovery of novel melanoma markers for not only early detection but also monitoring disease status is promising to improve the clinical outcome of patients. In the present study, we performed proteomic comparative analysis of plasma proteins between healthy volunteers and melanoma patients using NanoLC and MALDI‐TOF‐MS. As a result, we were successful in identifying nine proteins that were specifically expressed in melanoma plasma compared with healthy plasma, most of which had not previously been identified as plasma markers of melanoma. The mRNA expression levels of four proteins [pro‐platelet basic protein precursor (PPBP), serum amyloid A2 (SAA2), complement factor H‐related protein 1 precursor (FHR1), inter‐alpha‐trypsin inhibitor heavy chain H4 precursor (IAIH4)] were prominently up‐regulated in several melanoma cell lines compared with melanocytes. Moreover, two proteins (PPBP, SAA) were shown to be expressed in tumor specimens from melanoma patients. In the survival time analysis regarding melanoma patients, the semi‐quantified plasma PPBP levels were statistically negatively correlated with the survival time. Most interestingly, the significant survival benefit was seen in low PBPP level group (< index 20) versus high level (≥ index 20) group. The results suggest that PPBP might be a novel promising serological marker and a prognostic factor specific to melanomas.     

A proteomic approach to immune-mediated epithelial-mesenchymal transition

Increasing evidence suggests epithelial-mesenchymal transition (EMT) plays an important role in renal fibrosis. Initial renal injury enables the infiltration of mononuclear cells into the interstitium, and the resulting generation of inflammatory mediators that favour EMT may have a direct role in the development of renal fibrosis. The aim of this study was to investigate the proteome of renal tubular epithelial cells undergoing EMT in vitro. The human proximal tubular cell line (HK-2), exposed to conditioned medium from activated peripheral blood mononuclear cells (PBMC-CM), undergo phenotypic change, from an epithelial towards a fibroblastic phenotype, as evidenced by decreased E-cadherin and increased fibronectin protein expression. Further proteomic analysis, using 2-DE and Progenesis software, revealed the down-regulation of 4 proteins and up-regulation of 23 proteins. MS analysis allowed the positive identification of 15 differentially expressed proteins, including annexin A2, adipocyte plasma membrane-associated protein, T-complex protein 1, reticulocalbin-1 precursor and moesin among others. Western blotting and quantitative real-time PCR confirmed the increase in annexin A2 at the protein and gene level, respectively. Since annexin A2 and S100A6 exist as complexes in B-1 cells, we investigated the S100A6 gene expression further and show an increased expression in HK-2 cells following exposure to activated PBMC-CM. Therefore, we have identified several potential proteins that could play key roles in immune-mediated EMT.     

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.     

Analysis of protein profiling studies of β‐thalassemia/Hb E disease

A number of studies have used global protein profiling technologies on a range of patient samples to detect proteins that are differentially expressed in β‐thalassemia/Hb E as an aid for understanding the physiopathology of this disease. Seven studies have identified a total of 111 unique, differentially expressed proteins. Seven proteins (prothrombin, alpha‐1‐antichymotrypsin, fibrinogen beta chain, hemoglobin beta, selenium‐binding protein, microtubule‐actin cross‐linking factor and adenomatous polyposis coli protein 2) have been identified in two independent studies, whereas two proteins (carbonic anhydrase 1 and peroxiredoxin‐2) have been identified in three independent studies. Both of these latter two proteins were consistently upregulated in the studies that identified them. Ontological analysis of all differentially regulated proteins identified “response to inorganic substances” as the most significant functional annotation cluster, which is consistent with iron overload being a major pathological consequence of this disease. Despite the range of samples investigated and the relatively small number of studies undertaken, a coherent picture of the mediators of the pathological consequences of β‐thalassemia/Hb E disease is starting to emerge.     

Modifications by Olmesartan medoxomil treatment of the platelet protein profile of moderate hypertensive patients

Olmesartan medoxomil is a new angiotensin II receptor blockers (ARB) which exhibits pleiotropic effects that are not fully understood. Our aims were: i) to determine the effect of Olmesartan medoxomil on blood pressure, lipid profile and renal functionality in moderately hypertensive patients with non‐controlled blood pressure, ii) to determine if Olmesartan medoxomil may exert anti‐inflammatory effects and modify the expression profile of platelet proteins. Thirteen moderate hypertensive patients with non‐controlled systolic blood pressure (SBP) and renal function classified as Kidney Disease Outcome Quality Initiative stage 2–3 were included. Patients were treated with Olmesartan medoxomil (20 mg/day) for 6 months. SBP, proteinuria and the plasma levels of cholesterol and low density lipoprotein (LDL)‐cholesterol were reduced after the treatment. Olmesartan medoxomil did not modify the circulating plasma levels of a number of proteins associated with inflammation, but reduced the expression level of different platelet proteins including tropomyosin‐β chain isotypes 3 and 4, serotransferrin isotypes 1 to 5, the leukocyte elastase inhibitor and the chloride intracellular channel‐protein isotype 1. The expression of the gelsolin precursor isotype 4 was increased in the platelets after the treatment. In summary, Olmesartan medoxomil reduced SBP, total and LDL‐cholesterol plasma levels and urinary protein excretion and induced changes in the expression of platelet proteins which may be related to some action of the drug at the megakaryocyte level.     

Proteomic profile of differentially expressed plasma proteins from dystrophic mice and following suberoylanilide hydroxamic acid treatment

Purpose: Histone Deacetylase Inhibitors (DI) ameliorates dystrophic muscle regeneration restoring muscular strength in the mdx mouse model of Duchenne muscular dystrophy (DMD). The further development of these compounds as drugs for DMD treatment is currently hampered by the lack of knowledge about DIs effect in large dystrophic animal models and that of suitable biomarkers to monitor their efficacy. Experimental design: In this study we applied proteomic analysis to identify differentially expressed proteins present in plasma samples from mdx mice treated with the Suberoylanilide hydroxamic acid (SAHA) and relative normal controls (WT). Results: Several differentially expressed proteins were identified between untreated wild type and mdx mice. Among these, fibrinogen, epidermal growth factor 2 receptor, major urinary protein and glutathione peroxidase 3 (GPX3) were constitutively up‐regulated in mdx, while complement C3, complement C6, gelsolin, leukaemia inhibitory factor receptor (LIFr), and alpha 2 macroglobulin were down‐regulated compared to WT mice. SAHA determined the normalization of LIFr and GPX3 protein level while apoliprotein E was de novo up‐regulated in comparison to vehicle‐treated mdx mice. Conclusions and clinical relevance: Collectively, these data unravel potential serological disease biomarkers of mdx that could be useful to monitor muscular dystrophy response to DI treatment.     

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.     

Proteins and protein fragments in nephrotic syndrome: Clusters, specificity and mechanisms

Proteinuria is the hallmark of renal diseases and the characterization of the urinary protein composition may become an important source of information for diagnosis and research. So far, protein analysis in urine has been utilized for a generic individuation of site-specific defects (glomerular vs. tubular) but there is a need for an extension of proteomics to specific urinary biomarkers in selected clinical conditions. The identification of fragments of proteins in plasma and urine may increase the spectrum of urinary biomarkers. The unique speculative application so far proposed for protein fragments is nephrotic syndrome, and specifically focal segmental glomerulosclerosis, in which case they reflect intrinsic proteolysis occurring in plasma and represent surrogate biomarkers of the disease activity. Albumin is probably the most studied protein. Several of the albumin fragments present a peculiar distribution of the fingerprint peptide pattern containing both the N-terminal region and the C-terminal domain with a complete lack of any MS signals for the internal sequence region. Their characterization utilizing new strategies based on 2-D nondenaturing electrophoresis is now in progress. Studies on a direct characterization of proteases in plasma and urine will also define the participation of proteases to the genesis of renal diseases.     

Shotgun proteomics identifies proteins specific for acute renal transplant rejection

Purpose: Acute rejection (AR) remains the primary risk factor for renal transplant outcome; development of non‐invasive diagnostic biomarkers for AR is an unmet need. Experimental design: We used shotgun proteomics applying LC‐MS/MS and ELISA to analyze a set of 92 urine samples, from patients with AR, stable grafts (STA), proteinuria (NS), and healthy controls. Results: A total of 1446 urinary proteins (UP) were identified along with a number of non‐specific proteinuria‐specific, renal transplantation specific and AR‐specific proteins. Relative abundance of identified UP was measured by protein‐level spectral counts adopting a weighted fold‐change statistic, assigning increased weight for more frequently observed proteins. We have identified alterations in a number of specific UP in AR, primarily relating to MHC antigens, the complement cascade and extra‐cellular matrix proteins. A subset of proteins (uromodulin, SERPINF1 and CD44), have been further cross‐validated by ELISA in an independent set of urine samples, for significant differences in the abundance of these UP in AR. Conclusions and clinical relevance: This label‐free, semi‐quantitative approach for sampling the urinary proteome in normal and disease states provides a robust and sensitive method for detection of UP for serial, non‐invasive clinical monitoring for graft rejection after kidney transplantation.     

Whole cell profiling and identification of galectin-1 as a potential marker of renal cell carcinoma

For most cancers, the patient's prognosis improves dramatically if the disease is detected at an early stage. Although advancements in imaging technology have improved early detection, many cancers remain undetected until it is too late for curative intervention. We have established, for the first time, expression difference mapping analysis of whole cell proteins from renal cell carcinoma (RCC) cell lines using ProteinChip technology. A total of 20 different RCC cell lines were cultured in vitro directly on ProteinChip arrays for 24 h. Direct MS analysis of proteins from the attached cells showed identical protein profiles by all analysed RCC lines. Comparative on‐chip analysis of isolated malignant cells from native tumour specimens revealed protein patterns highly similar to those from the continuous RCC lines. However, cultured primary cortex cells showed specific protein differences. Differential protein profiling of isolated cytosolic and enriched membrane fractions from the RCC lines revealed that the protein pattern of the membrane proteins included or were identical to those of the entire cells. Proteomics analysis of the chip‐binding membrane fractions allowed the identification of three forms of galectin‐1 as potential RCC marker. ProteinChip analysis with a bound‐specific antibody certified that galectin‐1 could be an RCC marker. Immunostaining methods confirmed the overexpression of galectin‐1 in renal carcinoma in comparison to healthy tissue.     

Identification of cancer-associated proteins by proteomics and downregulation of β-tropomyosin expression in colorectal adenoma and cancer

Elucidating the molecular mechanism underlying the development of adenoma, the major precursor lesion of colorectal cancer (CRC), would provide a basis for early detection, prevention as well as treatment of CRC. Using the highly sensitive 2-D DIGE method coupled with MS, we identified 24 differentially expressed proteins in adenoma tissues compared with matched normal colonic mucosa and CRC tissues. Fifteen proteins were downregulated and three proteins were upregulated in adenoma tissues when compared with individual-matched normal colonic mucosa. Five proteins were downregulated, while one protein was upregulated in adenoma tissues when compared with matched CRC tissues. A protein, β-tropomyosin (TM-β), recently suggested to be a biomarker of esophageal squamous carcinoma, was downregulated in both adenoma and CRC tissues. Additionally, the reduction in the level of TM-β in adenoma and CRC tissues was further validated by Western blotting (p<0.05) and RT-PCR (p<0.001). Our findings suggest that downregulation of TM-β is involved in the early development of CRC and that differentially expressed proteins might serve as potential biomarkers for detection of CRC.     

Intestinal proteomics in pig models of necrotising enterocolitis,short bowel syndrome and intrauterine growth restriction

Necrotising enterocolitis (NEC), short bowel syndrome (SBS) and intrauterine growth restriction (IUGR) are three conditions associated with intestinal dysfunction in newborn infants, particularly those born preterm. Piglet (Sus scrofa) models have recently been developed for NEC, SBS and IUGR, and tissue proteomic analyses have identified unknown pathways and new prognostic disease markers. Intestinal HSPs, iron metabolism proteins and proteins related to amino acid (e.g. arginine) and glucose metabolism are consistently affected by NEC progression and some of these proteins are also affected by SBS and IUGR. Parallel changes in some plasma and urinary proteins (e.g. haptoglobin, globulins, complement proteins, fatty acid binding proteins) may mirror the intestinal responses and pave the way to biomarker discovery. Explorative non-targeted proteomics provides ideas about the cellular pathways involved in intestinal adaptation during the critical neonatal period. Proteomics, combined with other -omic techniques, helps to get a more holistic picture of intestinal adaptation during NEC, SBS and IUGR. Explorative -omic research methods also have limitations and cannot replace, but only supplement, classical hypothesis-driven research that investigate disease mechanisms using a single or few endpoints.     

Plasma proteome of buffaloes

The proteomic approach has aroused the interest of veterinary medicine researchers, especially regarding the production of biopharmaceuticals and diagnosis of diseases in farm animals. Water buffaloes have gained prominence in the world economy due to the quality of their milk, meat, and leather, in addition to being an important donor of blood components. This work aimed to identify and characterize the proteins present in the blood plasma of Murrah buffaloes (Bubalus bubalis) through 2D electrophoresis, in gel protein digestion followed by mass spectrometry technique and for albumin depletion, in solution protein digestion followed by shotgun analysis. Our results showed the identification of 112 protein spots and 35 individual proteins, respectively. The abundant proteins were represented by albumin, fibrinogen‐α, fibrinogen‐β, fibrinogen‐γ, immunoglobulins in general, α‐1‐antiproteinase, α‐1B‐glycoprotein, α‐2‐HS‐glycoprotein, α‐macroglobulin, apolipoprotein A1, antithrombin‐III, endopin 2B, fetuin‐B, retinol‐binding protein, serotransferrin, transthyretin and vitamin D‐binding protein. Most of these proteins are related to the signaling pathways of the complement system and coagulation cascade. The results allowed a better understanding of the protein composition of these blood components, thus promoting studies on animal health in the search for molecular markers of zoonotic diseases in buffaloes.     

Discovery of putative oocyte quality markers by comparative ExacTag proteomics

Purpose: Identification of the biomarkers of oocyte quality, and developmental and reprogramming potential is of importance to assisted reproductive technology in humans and animals. Experimental design: PerkinElmer ExacTag? Kit was used to label differentially proteins in pig oocyte extracts (oocyte proteome) and pig oocyte‐conditioned in vitro maturation media (oocyte secretome) obtained with high‐ and low‐quality oocytes. Results: We identified 16 major proteins in the oocyte proteome that were expressed differentially in high‐ versus low‐quality oocytes. More abundant proteins in the high‐quality oocyte proteome included kelch‐like ECH‐associated protein 1 (an adaptor for ubiquitin‐ligase CUL3), nuclear export factor CRM1 and ataxia‐telangiectasia mutated protein kinase. Dystrophin (DMD) was more abundant in low‐quality oocytes. In the secretome, we identified 110 proteins, including DMD and cystic fibrosis transmembrane conductance regulator, two proteins implicated in muscular dystrophy and cystic fibrosis, respectively. Monoubiquitin was identified in the low‐quality‐oocyte secretome. Conclusions and clinical implications: A direct, quantitative proteomic analysis of small oocyte protein samples can identify potential markers of oocyte quality without the need for a large amount of total protein. This approach will be applied to discovery of non‐invasive biomarkers of oocyte quality in assisted human reproduction and in large animal embryo transfer programs.     

Large-scale protein identification of human urine proteome by multi-dimensional LC and MS/MS

Urine is a human specimen that is easily obtained non-invasively for clinical diagnosis. We attempted to enhance the resolution of current human urine proteomes and construct a comprehensive reference database for advanced studies, such as the discovery of biomarkers for renal diseases. Multi-dimensional LC-MS/MS was coupled with de novo sequencing and database matching. The proposed approach improved the identification of not only the proteins, but also the post-translational sites of urinary proteins. We identified 165, 200 and 259 unique gene products in the urine proteomes from males, females and pregnant women, respectively. When all of the results were combined and the redundancies removed, a total of 1095 distinct peptides were identified. Of these, 1016 peptides were associated with 334 unique gene products. In this study, over 100 gene products, including some disease-related proteins, were detected in urine for the first time by proteomic approaches. Various proteins with novel post-translational hydroxylation were identified using the MASCOT program and de novo sequencing. All proteins with peptide information were summarized into a comprehensive urine protein database. We believe that this comprehensive urine proteome database will assist in the identification of urinary proteins/polypeptides whose spectra are difficult to interpret in the discovery of urinary biomarkers.     

Urinary extracellular vesicles for biomarker source to monitor polycystic kidney disease

Extracellular vesicles (EVs) are bilayered lipid vesicles, 50–1000 nm in diameter and secreted by most types of cells. They contain many proteins, mRNAs, miRNAs, and lipids that reflect the pathophysiological state of the cells they originate from, and are therefore considered to be a rich source of potential biomarkers. In this issue (Pocsfalvi, G. et al., Proteomics Clin. Appl. 2015, 9, 552–567), Pocsfalvi et al. conducted pioneering investigations to determine whether changes in the protein content of EVs occur during progression of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disorder that predominantly affects the kidneys. Most significantly, iTRAQ-based quantitative proteomics showed that cytoskeleton-regulating and Ca²⁺-binding proteins are differentially expressed in urinary EVs of ADPKD patients. Impressively, these proteins are involved in biological processes that are closely related to the pathogenic state of tubular epithelial cells in ADPKD, demonstrating the possibility to monitor the status of patients using urinary EVs.