Detection Of Urinary Biomarkers Research Articles

Identification of Low-Abundance Urinary Biomarkers in Lupus Nephritis Using Electrochemiluminescence Immunoassays.

To investigate the utility of a sensitive platform using electrochemiluminescence (ECL) for the identification of low-abundance urinary protein biomarkers in lupus nephritis (LN). Forty-eight urine samples were obtained from subjects in 2 independent cohorts, each consisting of 3 groups (matched for age, sex, and race) of 8 patients with active LN (renal Systemic Lupus Erythematosus Disease Activity Index [SLEDAI] >0), 8 patients with inactive SLE (renal SLEDAI 0), and 8 healthy controls. Samples were tested using a preexisting 40-plex ECL panel. A custom 5-plex ECL panel was then developed for further validation studies and used to test 140 urine samples (from 44 patients with active LN, 41 patients with inactive SLE, 28 healthy controls, and 27 patients with other kidney diseases). Levels of 17 urinary proteins were elevated (P < 0.05 by 2-tailed Mann-Whitney U test) in samples from patients with active LN compared to samples from patients with inactive SLE and healthy controls in cohort 1, while 9 were similarly elevated in cohort 2. Of these, interleukin-7 (IL-7), IL-12p40, IL-15, interferon-γ-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC) were chosen for further validation. These 5 proteins were undetectable by enzyme-linked immunosorbent assay (ELISA). Hence, a custom 5-plex ECL panel was developed and used to validate the results from the initial 40-plex screening panel. Urinary IL-7, IL-12p40, IL-15, IP-10, and TARC levels were again significantly elevated in patients with active LN compared to those with inactive SLE and healthy controls, and correlated well with the renal SLEDAI and physician's global assessment of disease activity (R > 0.67, P < 0.05). All 5 urinary proteins were more frequently elevated in LN compared to controls with other chronic kidney diseases, although overall group differences attained significance only for urinary IL-7 and IL-15. Urinary levels of IL-7, IL-12p40, IL-15, IP-10, and TARC are potentially useful diagnostic tools in LN. The use of ECL assays may allow detection of urinary biomarkers that are below ELISA detection limits.

Detection of urinary biomarkers in reservoir hosts of leptospirosis by capillary electrophoresis-mass spectrometry.

Pathogenic leptospires colonize the renal tubules of reservoir hosts of infection and are excreted via urine into the environment. Asymptomatic reservoir hosts include a wide range of domestic and wild animal species and include cattle, dogs, and rats that can persistently excrete large numbers of pathogenic leptospires over many months. A similar presentation has been observed in humans categorized as "long-term asymptomatic individuals" as they excreted leptospires in the absence of any clinical symptoms or positive serology. In the current study, the urine of experimentally infected rats, which showed no clinical signs or positive serology, was analyzed by CE-MS to identify urinary biomarkers of chronic infection. A discriminating peptide pattern of 43 polypeptides provided a sensitivity of 93%, a specificity of 83%, and an accuracy of 90% for the identification of urine from chronically infected rats (p < 0.05, AUC > 90%). The majority of discriminating peptides were decreased in abundance in urine of chronically infected rats, including a peptide derived from neprilysin, a membrane metalloendopeptidase, the expression of which has previously been shown to be diminished in infected urine. Results highlight the diagnostic capabilities of urinary biomarkers to identify reservoir hosts of leptospirosis using CE coupled to MS.

Clinical implication of urinary protein markers in diabetic nephropathy and interventional effects of Chinese herbal medicine

In clinic, some urinary protein makers can dynamically and noninvasively reflect the degree of renal tubular injury in patients with diabetic nephropathy (DN). These urinary biomarkers of tubular damage are broadly divided into two categories. One is newfound, including kidney injury molecule-1 (Kim-1), neutrophil getatinase-associated lipocalin (NGAL), liver-type fatty acid-binding protein (L-FABP) and cystatin C (CysC); the other one is classical, including beta2 microglobulin (beta2-MG), retinal binding protein (RBP) and N-acetyl-beta-D-glucosaminidase (NAG). It is reported that, the increases in urinary protein markers are not only closely related to the damage of tubular epithelial cells in DN patients, but also can be ameliorated by the treatment with Chinese herbal compound preparations or Chinese herbal medicine. Recently, although urinary proteomics are used in the protein separation and identification, the traditional associated detection of urinary protein markers is more practical in clinic. At present, it is possible that the associated detection of urinary biomarkers of glomerular and tubular damages may be a feasible measure to reveal the clinical significance of urinary protein markers in DN patients and the interventional effects of Chinese herbal medicine.

Abstract LB-84: Multiplex detection of urinary biomarkers for rapid bladder cancer diagnosis using an automated cartridge-based platform.

Abstract Introduction and Objectives Bladder cancer, a common cancer with high recurrence rate, currently depends on invasive endoscopy for diagnosis and surveillance. A non-invasive, rapid, and accurate urine test will significantly improve management. We have developed a near-patient, fully automated bladder cancer test using the GeneXpert® platform (GX, Cepheid, Sunnyvale CA) for multiplex detection of urinary biomarkers. This test, requires simply mixing voided urine with a liquid preservative followed by loading into the GX cartridge. Materials and Methods Thirty candidate mRNA markers from published studies were selected for evaluation. These markers were screened in urine using RT-qPCR targeting intron spanning amplicons for each candidate mRNA. Urinary expression was measured relative to ABL1 reference gene. Four markers, IGF2, KRT20, ANXA10 and CRH, were chosen based on sensitivity and specificity with analysis of &amp;gt;200 patient cases and control urines. A urine preservative was developed to preserve urothelial cells and their RNA and to lyse red blood cells and white blood cells. Four mL preserved urine was added to an integrated cartridge and inserted into a GX module where the on-board sample preparation steps and RT-qPCR took place. The test results, cancer detected or not, are returned within 90 minutes. With local IRB approval, clinical urine samples were collected from research subjects prior to undergoing cystoscopy for cancer screening or resection. The Xpert® Bladder assay results were compared to urine cytology, tissue histopathology and in selected samples, UroVysion, a commercially available bladder cancer test based on fluorescent in-situ hybridization. Results In our pilot validation study, the Xpert® Bladder assay demonstrated 89.7% sensitivity (61/68) for the detection of high grade bladder cancer; 54.1% sensitivity (33/61) for the detection of low grade bladder cancer; 77.5% specificity (248/320) for cystoscopy negative patients; and 92.7% specificity (51/55) for the healthy control group. In the split sample study the Xpert® Bladder assay demonstrated equal performance compared to UroVysion for patients with high grade bladder cancer and performed better than UroVysion for patients with low grade bladder cancer. Analysis of tumor biopsy tissue from patients whose urine samples were falsely negative showed that all tumors tested had up-regulation of at least one of the four markers, suggesting the failure to detect these markers in the urine may be due to insufficient quantity of urinary tumor cells. Conclusion We have developed a rapid, on-demand urine test for bladder cancer diagnosis based on multiplex detection of a 4-marker panel using a well-established molecular diagnostic platform. Pending further validation, the Xpert® Bladder assay may be integrated as an important part of bladder cancer management. Citation Format: Ellen Wallace, Edwin W. Lai, Kathleen E. Mach, Neda Haque, Leena McCann, Shelly Hsiao, Daniel Bui, Ruchika Mohan, Malini Satya, Edith Wong, Julia A. Bridge, David Persing, Russell Higuchi, Joseph C. Liao. Multiplex detection of urinary biomarkers for rapid bladder cancer diagnosis using an automated cartridge-based platform. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-84. doi:10.1158/1538-7445.AM2013-LB-84

Urinary proteomics analysis for sepsis biomarkers with iTRAQ labeling and two-dimensional liquid chromatography–tandem mass spectrometry

Proteomics has only recently been applied to the field of critical care research. Sepsis is a major factor contributing to intensive care unit admissions and deaths. The purpose of this study was to screen potential urinary biomarkers for sepsis using A proteomics approach. Fifteen sepsis and 15 systemic inflammatory response syndrome patients were involved in this study. Urinary proteins were identified by isobaric tag for relative and absolute quantitation coupled with liquid chromatography-tandem mass spectrometry. Mass spectroscopy analysis was performed with the Mascot software and the International Protein Index. Bioinformatics analyses were performed using the hierarchy cluster analysis, the STRING software, the Gene Ontology, and the Kyoto Encyclopedia of Genes and Genome database. One hundred thirty proteins were identified, and 34 differentially expressed proteins were selected (fold change, >1.5). On the basis of the Gene Ontology and the Kyoto Encyclopedia of Genes and Genome database, these 34 proteins were identified to be involved in inflammation, immunity, and structural or cytoskeletal processes. Five proteins were selected by a protein-protein interaction network for sepsis differentiation: cadherin 1, haptoglobin, complement 3, alpha-1-antitrypsin, and ceruloplasmin. Urinary proteomics may represent a suitable approach for sepsis-related research. The detection of urinary biomarkers is expected to become a noninvasive and acceptable method, which facilitates the close surveillance of diseases and reduces medical costs. Diagnostic study, level IV.

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.