Urinary Proteome Profile Research Articles

Rx Report

Rx Report

Bacterial Overgrowth Affects Urinary Proteome Analysis: Recommendation for Centrifugation, Temperature, Duration, and the Use of Preservatives during Sample Collection

Bacterial overgrowth is one of the major concerns in collection and storage of biofluids, particularly 24-h urine. However, there is no previous systematic analysis of effects of bacterial overgrowth on urinary proteome analysis, and necessity, type, and appropriate concentration of preservatives to prevent bacterial overgrowth in the urine remain unclear. We, therefore, performed such systematic evaluation. Pooled normal urine was either centrifuged at 1500 g (to remove cell debris) or uncentrifuged. The samples were then added with either sodium azide (NaN3) or boric acid with various concentrations, and kept at room temperature (RT) or at 4 degrees C. Bacterial overgrowth was determined by UV-visible spectrophotometry (lambda620 nm) and Gram staining. At both temperatures, centrifugation to remove cell debris could effectively delay the bacterial overgrowth. At RT, both centrifuged and uncentrifuged samples without any preservative had the detectable overgrowth of Gram-positive and Gram-negative cocci and bacilli as early as 12 and 8 h, respectively, whereas 0.1-1 mM NaN3 and 2-20 mM boric acid could delay bacterial overgrowth, which started at 16-20 h in the centrifuged urine and 12-16 h in the uncentrifuged urine. Greater delay (for at least 48 h) was achieved with 10 mM NaN3 and 200 mM boric acid. At 4 degrees C, no bacterial overgrowth was detected in all centrifuged samples. However, it was observed at 20 h in the uncentrifuged urine without preservative, and at 48 h for the uncentrifuged urine with 0.1 mM NaN3 or 2 mM boric acid. There was no bacterial overgrowth detectable in the uncentrifuged urine preserved with higher concentrations of NaN3 or boric acid. 2-DE showed obvious changes in the urinary proteome profile of the sample with bacterial contamination, and the bacterial proteins could be identified by MALDI-TOF MS. Our data suggest that the urine should be centrifuged to remove cell debris and kept at 4 degrees C, rather than at RT, during the collection interval prior to long-term storage in the freezer. Moreover, the addition of 200 mM boric acid or 10 mM NaN3 is highly recommended for the prevention of bacterial overgrowth in the urine.

Identification of a urinary proteomic signature for lupus nephritis in children

The quest for reliable biomarkers of systemic lupus erythematosus (SLE) nephritis is an area of intense contemporary research. In this study, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology was used for urinary proteomic profiling of patients with SLE nephritis. Clinical, laboratory, and kidney biopsy data from pediatric patients with SLE (n = 32) were analyzed. Children with juvenile idiopathic arthritis (n = 11) served as controls. SELDI-TOF-MS was performed using ProteinChips with different chromatographic surfaces. The resulting spectra were analyzed with Bio-Rad Biomarker Wizard software. A consistent urinary proteomic signature for SLE nephritis was found, comprising eight biomarker proteins with peaks at m/z of 2.7, 22, 23, 44, 56, 79, 100, and 133 kDa. The peak intensities of these biomarkers were significantly greater in patients with SLE nephritis compared with controls and SLE patients without nephritis. These biomarkers were strongly correlated with renal disease activity and moderately with renal damage. For the diagnosis of active nephritis, the area under the receiver operating characteristic curve was > or =0.90 for 22, 23, 44, 79, and 100 kDa biomarkers. Thus, SELDI-TOF-MS has identified a urine proteomic signature strongly associated with SLE renal involvement and active SLE nephritis.

Markedly Increased Urinary Preprohaptoglobin and Haptoglobin in Passive Heymann Nephritis: A Differential Proteomics Approach

Membranous nephropathy (MN), a common cause of idiopathic nephrotic syndrome in adults, remains a potentially devastating problem worldwide. At present, there is no reliable noninvasive method for predicting and/or monitoring this glomerular disease, and its pathophysiology remains poorly understood. In the present study, the urinary proteome profile of rats after 10 days of an induction of passive Heymann nephritis (PHN), which resembles human MN, was compared to that of the baseline (control) urine prior to the induction of PHN by anti-Fx1A injection. Each pool of PHN and control urine samples (n = 10 each) was labeled with different fluorescent dyes (Cy3 or Cy5), and equal amounts of the labeled proteins of both pools were resolved in the same 2D gel, together with an internal standard labeled with Cy2. Two-dimensional difference gel electrophoresis revealed a number of protein spots whose expression levels were altered during PHN. Eighteen protein spots with >1.5-fold changes and p < 0.05 were selected for subsequent identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. They were successfully identified as serum albumin precursor, alpha-1-antitrypsin, preprohaptoglobin, liver-regeneration-related protein, and transthyretin (which increased during PHN) and E-cadherin, MPP7, tropomyosin beta, kallikrein, and alpha-2u globulin (which decreased in the PHN urine). Among these proteins, the increase in urinary preprohaptoglobin has particularly drawn our attention because of its byproduct, haptoglobin (Hp), which is involved in the protection of tissue damage from hemoglobin-induced oxidative stress. Western blotting and enzyme-linked immunosorbent assay clearly showed a markedly increased level of Hp in the urine, but not in the serum, of the PHN animals. Our findings may lead to a significant advance in the attempt to define a new therapeutic target and/or novel biomarker for human MN.

Bladder Cancer Associated Glycoprotein Signatures Revealed by Urinary Proteomic Profiling

Current methods in the noninvasive detection and surveillance of bladder cancer via urine analysis include voided urine cytology (VUC) and some diagnostic urinary protein biomarkers; however, due to the poor sensitivity of VUC and high false-positive rates of currently available protein assays, detection of bladder cancer via urinalysis remains a challenge. In the study presented here, a rapid, high-sensitivity technique was developed to profile the N-linked glycoprotein component in naturally micturated human urine specimens. Concanavalin A (Con A) affinity chromatography coupled to nanoflow liquid chromatography was utilized to separate the complex peptide mixture prior to a linear ion trap MS analysis. Of 186 proteins identified with high confidence by multiple analyses, 40% were secreted proteins, 18% membrane proteins, and 14% extracellular proteins. In this study, the presence of several proteins appeared to be associated with the presence of bladder cancer, including alpha-1B-glycoprotein that was detected in all tumor-bearing patient samples but in none of the samples obtained from non-tumor-bearing individuals. The combination of Con A affinity chromatography and nano-LC/MS/MS provides an initial investigation of N-glycoproteins in complex biological samples and facilitates the identification of potential biomarkers of bladder cancer in noninvasively obtained human urine.

Urinary Proteome Profiling Using Microfluidic Technology on a Chip

Clinical diagnostics and biomarker discovery are the major focuses of current clinical proteomics. In the present study, we applied microfluidic technology on a chip for proteome profiling of human urine from 31 normal healthy individuals (15 males and 16 females), 6 patients with diabetic nephropathy (DN), and 4 patients with IgA nephropathy (IgAN). Using only 4 microL of untreated urine, automated separation of proteins/peptides was achieved, and 1-7 (3.8 +/- 0.3) spectra/bands of urinary proteins/peptides were observed in the normal urine, whereas 8-16 (11.3 +/- 1.2) and 9-14 (10.8 +/- 1.2) spectra were observed in urine samples of DN and IgAN, respectively. Coefficient of variations of amplitudes of lower marker (1.2 kDa), system spectra (6-8 kDa), and upper marker (260.0 kDa) were 22.84, 24.92, and 32.65%, respectively. ANOVA with Tukey post-hoc multiple comparisons revealed 9 spectra of which amplitudes significantly differed between normal and DN urine (DN/normal amplitude ratios ranged from 2.9 to 3102.7). Moreover, the results also showed that 3 spectra (with molecular masses of 12-15, 27-28, and 34-35 kDa) were significantly different between DN and IgAN urine (DN/IgAN amplitude ratios ranged from 3.9 to 7.4). In addition to the spectral amplitudes, frequencies of some spectra could differentiate the normal from the diseased urine but could not distinguish between DN and IgAN. There was no significant difference, regarding the spectral amplitude or frequency, observed between males and females. These data indicate that the microfluidic chip technology is applicable for urinary proteome profiling with potential uses in clinical diagnostics and biomarker discovery.

IgA nephropathy associated with Hodgkin’s disease in children: a case report, literature review and urinary proteome analysis

We report herein a rare case of IgAN associated with Hodgkin's disease in a 14-year-old boy. Clinical manifestations and laboratory parameters were improved after treatment with CHOP chemotherapy and enalapril. Urinary proteins were isolated and examined using state-of-the-art proteomic technology, before and during the treatment course. Two-dimensional gel electrophoresis showed obvious alterations in the urinary proteome profile in response to such therapy. Quantitative intensity analysis and gel mapping revealed 14 altered proteins with reduced excretion levels during the treatment course, including albumin, albumin complexed with decanoic acid, alpha-1 antitrypsin, cadherin-11 precursor, collagen alpha 1 (VI) chain precursor, complement C1q tumor necrosis factor-related protein, Ig heavy chain, Ig light chain, kininogen, inter-alpha-trypsin inhibitor (alpha-1 microglobulin), inter-alpha-trypsin inhibitor heavy chain, leucine-rich alpha-2 glycoprotein, beta-2 microglobulin, and transferrin precursor. Their potential roles in the pathogenesis and pathophysiology of IgAN are discussed.

Prediction of Diabetic Nephropathy Using Urine Proteomic Profiling 10 Years Prior to Development of Nephropathy

We examined whether proteomic technologies identify novel urine proteins associated with subsequent development of diabetic nephropathy in subjects with type 2 diabetes before evidence of microalbuminuria. In a nested case-control study of Pima Indians with type 2 diabetes, baseline (serum creatinine <1.2 mg/dl and urine albumin excretion <30 mg/g) and 10-year urine samples were examined. Case subjects (n = 31) developed diabetic nephropathy (urinary albumin-to-creatinine ratio >300 mg/g) over 10 years. Control subjects (n = 31) were matched to case subjects (1:1) according to diabetes duration, age, sex, and BMI but remained normoalbuminuric (albumin-to-creatinine ratio <30 mg/g) over the same 10 years. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) was performed on baseline urine samples, and training (14 cases:14 controls) and validation (17:17) sets were tested. At baseline, A1C levels differed between case and control subjects. SELDI-TOF MS detected 714 unique urine protein peaks. Of these, a 12-peak proteomic signature correctly predicted 89% of cases of diabetic nephropathy (93% sensitivity, 86% specificity) in the training set. Applying this same signature to the independent validation set yielded an accuracy rate of 74% (71% sensitivity, 76% specificity). In multivariate analyses, the 12-peak signature was independently associated with subsequent diabetic nephropathy when applied to the validation set (odds ratio [OR] 7.9 [95% CI 1.5-43.5], P = 0.017) and the entire dataset (14.5 [3.7-55.6], P = 0.001), and A1C levels were no longer significant. Urine proteomic profiling identifies normoalbuminuric subjects with type 2 diabetes who subsequently develop diabetic nephropathy. Further studies are needed to characterize the specific proteins involved in this early prediction.

Serial Changes in Urinary Proteome Profile of Membranous Nephropathy: Implications for Pathophysiology and Biomarker Discovery

Membranous nephropathy is one of the most common causes of primary glomerular diseases worldwide. The present study adopted a gel-based proteomics approach to better understand the pathophysiology and define biomarker candidates of human membranous nephropathy using an animal model of passive Heymann nephritis (PHN). Clinical characteristics of Sprague-Dawley rats injected with rabbit anti-Fx1A antiserum mimicked those of human membranous nephropathy. Serial urine samples were collected at Days 0, 10, 20, 30, 40, and 50 after the injection with anti-Fx1A (number of rats = 6; total number of gels = 36). Urinary proteome profiles were examined using 2D-PAGE and SYPRO Ruby staining. Quantitative intensity analysis and ANOVA with Tukey post-hoc multiple comparisons revealed 37 differentially expressed proteins among 6 different time-points. These altered proteins were successfully identified by MALDI-TOF MS and classified into 6 categories: (i) proteins with decreased urinary excretion during PHN; (ii) proteins with increased urinary excretion during PHN; (iii) proteins with increased urinary excretion during PHN, but which finally returned to basal levels; (iv) proteins with increased urinary excretion during PHN, but which finally declined below basal levels; (v) proteins with undetectable levels in the urine during PHN; and (vi) proteins that were detectable in the urine only during PHN. Most of these altered proteins have functional significance in signaling pathways, glomerular trafficking, and controlling the glomerular permeability. The ones in categories (v) and (vi) may serve as biomarkers for detecting or monitoring membranous nephropathy. After normalization of the data with 24-h urine creatinine excretion, changes in 34 of initially 37 differentially expressed proteins remained statistically significant. These data underscore the significant impact of urinary proteomics in unraveling disease pathophysiology and biomarker discovery.

Urinary proteomic profiles distinguish between active and inactive lupus nephritis

Key aims of the treatment of lupus nephritis (LN) are to induce and maintain remission with minimal side effects. However, assessing ongoing renal inflammatory activity is poorly served by current diagnostic tests apart from renal biopsy, but frequent biopsies cannot be justified. Our long-term aim is to identify novel biomarkers from urinary protein profiles to improve diagnosis and monitoring of activity and response to therapy in LN. We used surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify biomarkers able to discriminate between urine samples from patients with inactive (n= 49) and active (n= 26) LN. Discriminant function analysis was used to define the minimum number of proteins whose levels best distinguished between the two patient groups. Serial urines of six biopsied patients were studied prospectively, and multiple regression (MR) scores calculated. Proteins with masses of 3340 and 3980 distinguished active from inactive LN with 92% sensitivity and specificity of 92% each. The prospective study of the biopsied patients demonstrated that MR scores could predict both relapse and remission earlier than traditional clinical markers. SELDI-TOF MS identified potential biomarker profiles strongly associated with activity in LN. Identification of these proteins will allow us to devise specific assays to routinely monitor disease progression, and alter immunosuppressive drug regimens accordingly. These proteins may also play a critical role in the pathogenesis of glomerulonephritis, and could therefore provide targets for therapeutic intervention.

Urine proteomic profiling of pediatric nephrotic syndrome

The prognosis of pediatric nephrotic syndrome (NS) correlates with the responsiveness to glucocorticoid therapy. Steroid-resistant NS (SRNS) patients progress to end-stage renal disease, while steroid-sensitive NS (SSNS) and steroid-dependent (SDNS) patients do not. We have performed proteomic profiling of urine samples from a cross section of pediatric and adolescent subjects with SSNS, SRNS, and orthostatic proteinuria (OP) to identify urinary biomarkers of steroid resistance. We performed surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) on urine from 19 subjects with SSNS/SDNS in remission, 14 with SSNS/SDNS in relapse, 5 with SRNS in relapse, and 6 with OP. Genetic algorithm search of principal component space revealed a group of five peaks distinguishing SRNS subjects, with mass/charge (m/z) values of 3,917.07, 4,155.53, 6,329.68, 7,036.96, and 11,117.4. Our analyses identified the peak at m/z 11,117.4 with an accuracy of 95% for classifying SRNS. Multidimensional protein fractionation and mass spectrometric analysis of SRNS urine samples combined with immunodepletion identified the 11,117.4 protein as beta2-microglobulin (B2M). Using an unbiased protein profiling approach, we have validated previously reported findings of B2M as a biomarker associated with SRNS. Prospective studies are warranted to establish additional biomarkers that would be predictive of SRNS.

Proteomic Profiling of Urinary Protein Excretion in the Factor H-Deficient Mouse

Background: Since the 1970s a variety of experimental techniques have been employed in an attempt to identify urinary biomarkers of renal injury. While these approaches have met with some success, modern proteomic tools now permit broad based high-throughput analysis of the urinary proteome. Methods: Using the ICAT isotopic labeling based LC/MS/MS approach, comparative urinary protein profiling was performed in a murine model of membranoproliferative glomerulonephritis. Paired samples were analyzed mice with a targeted deletion of the complement regulatory protein factor H (FH^–/–) and control mice. Results: 25 distinct urinary proteins were identified of which 7 were differentially expressed in the FH^–/– mice. Two proteins were markedly altered in the urine of FH^–/– mice compared to controls: uromodulin (5.5-fold lower) and the MHC class II molecule H2^e (8.6-fold higher). Differential expression was confirmed by Western blot and RT-PCR. Immunofluorescent staining demonstrated a marked increased expression of H2^e and a reduction of uromodulin expression in the tubular epithelium of FH^–/– mice. Conclusions: These findings provide insight into early complement-dependent alterations in tubular protein expression which may play critical roles in the development of tubulointerstitial disease, and provide experimental support for the use of urinary proteomic profiling in murine models of renal injury.

Systematic Evaluation of Sample Preparation Methods for Gel-Based Human Urinary Proteomics: Quantity, Quality, and Variability

We performed systematic evaluation of 38 protocols to concentrate normal human urinary proteins prior to 2D-PAGE analysis. Recovery yield and pattern of resolved protein spots were compared among different methods and intra-/inter-individual variabilities were examined. Precipitation with 90% ethanol provided the greatest protein recovery yield (92.99%), whereas precipitation with 10% acetic acid had the least protein recovery (1.91%). In most of precipitation protocols, the higher percentage of applied organic compounds provided the greater recovery yield. With a fixed concentration at 75%, the urine precipitated with acetonitrile had the greatest number of protein spots visualized in 2D gel, whereas the acetic-precipitated sample had the smallest number of spots. For the intra-individual variability, the first morning urine had the greatest amount of total protein but provided the smallest number of protein spots visualized. Excessive water drinking, not caffeine ingestion, caused alterations in the urinary proteome profile with newly presenting spots and also proteins with decreased excretion levels. As expected, there was a considerable degree of inter-individual variability. Coefficients of variation for albumin and transferrin expression were greatest by inter-individual variables. Male urine had greater amount of total protein but provided smaller number of protein spots compared to female urine. These data offer a wealth of useful information for designing a high-quality, large-scale human urine proteome project.