News | 2012

Diabetes. 2012 Dec;61(12):3304-13.
Urinary Proteomics for Early Diagnosis in Diabetic Nephropathy.
Zürbig P, Jerums G, Hovind P, MacIsaac R, Mischak H, Nielsen SE, Panagiotopoulos S, Persson F, Rossing P.

Abstract

Diabetic nephropathy (DN) is a progressive kidney disease, a well-known complication of long-standing diabetes. DN is the most frequent reason for dialysis in many Western countries. Early detection may enable development of specific drugs and early initiation of therapy, thereby postponing/preventing the need for renal replacement therapy. We evaluated urinary proteome analysis as a tool for prediction of DN. Capillary electrophoresis-coupled mass spectrometry was used to profile the low-molecular weight proteome in urine. We examined urine samples from a longitudinal cohort of type 1 and 2 diabetic patients (n = 35) using a previously generated chronic kidney disease (CKD) biomarker classifier to assess peptides of collected urines for signs of DN. The application of this classifier to samples of normoalbuminuric subjects up to 5 years prior to development of macroalbuminuria enabled early detection of subsequent progression to macroalbuminuria (area under the curve [AUC] 0.93) compared with urinary albumin routinely used to determine the diagnosis (AUC 0.67). Statistical analysis of each urinary CKD biomarker depicted its regulation with respect to diagnosis of DN over time. Collagen fragments were prominent biomarkers 3-5 years before onset of macroalbuminuria. Before albumin excretion starts to increase, there is a decrease in collagen fragments. Urinary proteomics enables noninvasive assessment of DN risk at an early stage via determination of specific collagen fragments..

 

 

Eur Heart J. 2012 Sep;33(18):2342-50.
Urinary proteome analysis in hypertensive patients with left ventricular diastolic dysfunction.
Kuznetsova T, Mischak H, Mullen W, Staessen JA

Abstract

Aims:
Despite the significant heart failure (HF) burden on society, easily applicable screening techniques particularly for the early detection of asymptomatic left ventricular (LV) dysfunction are lacking. The present study aimed to identify and test a set of urinary polypeptides that may indicate early LV diastolic dysfunction as defined by echocardiography in hypertensive patients in a cross-sectional case-control study nested within the FLEMish study on ENvironment, Genes and Health Outcome (FLEMENGHO).

Methods
To identify potentially discriminating urinary biomarkers for LV diastolic dysfunction, we applied capillary electrophoresis coupled to mass spectrometry. In the discovery set, we compared 19 hypertensive patients with asymptomatic LV diastolic dysfunction with 19 healthy controls. In the absence of adjustment for multiple testing, 85 urinary peptides were different between cases and controls at a P-value of < 0.033. With adjustment for multiple testing, 3 potential biomarkers remained significantly different between cases and controls (P0.02). We combined the 85 potential biomarkers in a high-dimensional model (classifier), which we applied in a blinded manner to an independent test-set of 16 hypertensive patients with symptomatic HF and 16 healthy controls. Upon unblinding, the AUC of the HF classification was 0.84 (95CI%: 0.70 – 0.98, P=0.001)

Conclusion
In asymptomatic hypertensive patients with LV diastolic dysfunction, we identified a set of urinary polypeptides specific for essential hypertension with LV diastolic dysfunction that subsequently distinguished hypertensive patients with overt HF from healthy controls.

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PLoS One. 2012;7(5):e35879. Epub 2012 May 16.
Urinary proteomics to support diagnosis of stroke.
Dawson J, Walters M, Delles C, Mischak H, Mullen W.

Abstract
Accurate diagnosis in suspected ischaemic stroke can be difficult. We explored the urinary proteome in patients with stroke (n = 69), compared to controls (n = 33), and developed a biomarker model for the diagnosis of stroke. We performed capillary electrophoresis online coupled to micro-time-of-flight mass spectrometry. Potentially disease-specific peptides were identified and a classifier based on these was generated using support vector machine-based software. Candidate biomarkers were sequenced by liquid chromatography-tandem mass spectrometry. We developed two biomarker-based classifiers, employing 14 biomarkers (nominal p-value ,0.004) or 35 biomarkers (nominal p-value ,0.01). When tested on a blinded test set of 47 independent samples, the classification factor was significantly different between groups; for the 35 biomarker model, median value of the classifier was 0.49 (20.30 to 1.25) in cases compared to 21.04 (IQR 21.86 to 20.09) in controls, p,0.001. The 35 biomarker classifier gave sensitivity of 56%, specificity was 93% and the AUC on ROC analysis was 0.86. This study supports the potential for urinary proteomic biomarker models to assist with the diagnosis of acute stroke in those with mild symptoms. We now plan to refine further and explore the clinical utility of such a test in large prospective clinical trials.

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PLoS ONE. 2012;7(4): e34606
Proteomic Candidate Biomarkers of Drug-Induced Nephrotoxicity in the Rat
Rouse R, Siwy J, Mullen W, Mischak H, Metzger J, Hanig J

Abstract
Improved biomarkers of acute nephrotoxicity are coveted by the drug development industry, regulatory agencies, and clinicians. In an effort to identify such biomarkers, urinary peptide profiles of rats treated with two different nephrotoxins were investigated. 493 marker candidates were defined that showed a significant response to cis-platin comparing a cisplatin treated cohort to controls. Next, urine samples from rats that received three consecutive daily doses of 150 or
300 mg/kg gentamicin were examined. 557 potential biomarkers were initially identified; 108 of these gentamicinresponse markers showed a clear temporal response to treatment. 39 of the cisplatin-response markers also displayed a clear response to gentamicin. Of the combined 147 peptides, 101 were similarly regulated by gentamicin or cis-platin and 54 could be identified by tandem mass spectrometry. Most were collagen type I and type III fragments up-regulated in
response to gentamicin treatment. Based on these peptides, classification models were generated and validated in a longitudinal study. In agreement with histopathology, the observed changes in classification scores were transient, initiated after the first dose, and generally persistent over a period of 10–20 days before returning to control levels. The data support the hypothesis that gentamicin-induced renal toxicity up-regulates protease activity, resulting in an increase in several specific urinary collagen fragments. Urinary proteomic biomarkers identified here, especially those common to both nephrotoxins, may serve as a valuable tool to investigate potential new drug candidates for the risk of nephrotoxicity.

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Mol Cell Proteomics. 2012 Jul
Urine proteome analysis reflects atherosclerotic disease in an ApoE-/- mouse model and allows the discovery of new biomarkers in mouse and human atherosclerosis
von Zur Muhlen C, Schiffer E, Sackmann C, Zürbig P, Neudorfer I, Zirlik A, Htun N, Iphöfer A, Jänsch L, Mischak H, Bode C, Chen YC, Peter K.

Summary
Non-invasive diagnosis of atherosclerosis via single biomarkers has been attempted but remains elusive. However, a previous polymarker or pattern approach of urine polypeptides in humans reflected coronary artery disease with high accuracy. The aim of the current study is to use urine proteomics in ApoE-/- mice to discover proteins with pathophysiological roles in atherogenesis and to identify urinary polypeptide patterns reflecting early stages of atherosclerosis. Urine of ApoE-/- mice either on high fat diet (HFD) or chow diet (CD) was collected over 12 weeks; urine of wild type mice on HFD was used to exclude diet-related proteome changes. Capillary electrophoresis coupled to mass spectrometry (CE-MS) of samples identified 16 polypeptides specific for ApoE-/- mice on HFD. In a blinded test set, these polypeptides allowed identification of atherosclerosis at a sensitivity of 90% and specificity of 100%, as well as monitoring of disease progression. Sequencing of the discovered polypeptides identified fragments of α1-antitrypsin, EGF, kidney androgen regulated protein (KAP) and collagen. Using immunohistochemistry, α1-antitrypsin, EGF and collagen type I were shown to be highly expressed in atherosclerotic plaques of ApoE-/- mice on HFD. Urinary excretion levels of collagen and α1-antitrypsin fragments also significantly correlated with intraplaque collagen and α1-antitrypsin content, mirroring plaque protein expression in the urine proteome. To provide further confirmation that the newly identified proteins are relevant in humans, presence of collagen type I, α1-antitrypsin, and EGF was also confirmed in human atherosclerotic disease. Urine proteome analysis in mice exemplifies the potential of a novel multimarker approach for the non-invasive detection of atherosclerosis and monitoring of disease progression. Furthermore, this approach represents a novel discovery tool for the identification of proteins relevant in murine and human atherosclerosis and thus also defines potential novel therapeutic targets.

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Pediatrics. 2012 Feb
Urinary Proteome Analysis to Exclude Severe Vesicoureteral Reflux
Drube J, Schiffer E, Lau E, Petersen C, Kirschstein M, Kemper M, Lichtinghagen R, Ure B, Mischak H, Pape L, Ehrich JH

Objectives:
High-grade vesicoureteral reflux (VUR, grade IV or V) is a risk factor for renal scarring, impaired renal function, and arterial hypertension. Voiding cystourethrography is the gold standard for detecting the severity of VUR. High-grade VUR is present in the minority of children with urinary tract infection (UTI), thus exposing the majority to invasive diagnostics that have no surgical consequence. We therefore aimed at establishing a noninvasive test to identify children with highgrade VUR.
Methods:
In a case-control study, a specific urinary proteome pattern was established by capillary electrophoresis coupled to mass spectrometry in 18 patients with primary VUR grade IV or V, distinguishing these from 19 patients without VUR after UTI. This proteome pattern was independently validated in a blinded cohort of 17 patients with VUR grade IV or V and 19 patients without VUR.
Results:
Sensitivity in detecting VUR grade IV or V in the blinded study was 88%, specificity was 79%. The test’s accuracy was independent of age, gender, and grade of VUR in the contralateral kidney. The odds ratio of suffering from VUR grade IV or V when tested positive was 28 (95% confidence interval: 4.5 to 176.0).
CONCLUSIONS: This noninvasive test is ready for prospective validation in large cohorts with the aim of identifying those children with UTI and hydronephrosis in need of further invasive diagnostics, such as voiding cystourethrography, thus sparing most children without pathologic urinary proteome patterns from additional diagnostics.

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