SERPINA1 Peptides in Urine as A Potential Marker of Preeclampsia Severity.

Preeclampsia (PE) is a multisystem disorder associated with pregnancy and its frequency varies from 5 to 20 percent of pregnancies. Although a number of preeclampsia studies have been carried out, there is no consensus about disease etiology and pathogenesis so far. Peptides of SERPINA1 (α1-antitrypsin) in urine remain one of the most promising peptide markers of PE. In this study the diagnostic potential of urinary α1-antitrypsin peptides in PE was evaluated. The urinary peptidome composition of 79 pregnant women with preeclampsia (PE), chronic arterial hypertension (CAH), and a control group was investigated. Mann-Whitney U-test (p < 0.05) revealed seven PE specific SERPINA1 peptides demonstrating 52% sensitivity and 100% specificity. SERPINA1 in urine has been associated with the most severe forms of preeclampsia (p = 0.014), in terms of systolic hypertension (p = 0.01) and proteinuria (p = 0.006). According to Spearman correlation analysis, the normalized intensity of SERPINA1 urinary peptides has a similar diagnostic pattern with known diagnostic PE markers, such as sFLT/PLGF. SERPINA1 peptides were not urinary excreted in superimposed PE (PE with CAH), which is a milder form of PE. An increase in expression of SERPINA1 in the structural elements of the placenta during preeclampsia reflects a protective mechanism against hypoxia. Increased synthesis of SERPINA1 in the trophoblast leads to protein accumulation in fibrinoid deposits. It may block syncytial knots and placenta villi, decreasing trophoblast invasion. Excretion of PE specific SERPINA1 peptides is associated with syncytiotrophoblast membrane destruction degradation and increased SERPINA1 staining. It confirms that the placenta could be the origin of SERPINA1 peptides in urine. Significant correlation (p < 0.05) of SERPINA1 expression in syncytiotrophoblast membrane and cytoplasm with the main clinical parameters of severe PE proves the role of SERPINA1 in PE pathogenesis. Estimation of SERPINA1 peptides in urine can be used as a diagnostic test of the severity of the condition to determine further treatment, particularly the need for urgent surgical delivery.

SerpinA3 in the Early Recognition of Acute Kidney Injury to Chronic Kidney Disease (CKD) transition in the rat and its Potentiality in the Recognition of Patients with CKD.

Recognizing patients at early phases of chronic kidney disease (CKD) is difficult, and it is even more challenging to predict acute kidney injury (AKI) and its transition to CKD. The gold standard to timely identify renal fibrosis is the kidney biopsy, an invasive procedure not usually performed for this purpose in clinical practice. SerpinA3 was identified by high-resolution-mass-spectrometry in urines from animals with CKD. An early and progressive elevation of urinary SerpinA3 (uSerpinA3) was observed during the AKI to CKD transition together with SerpinA3 relocation from the cytoplasm to the apical tubular membrane in the rat kidney. uSerpinA3/alpha-1-antichymotrypsin was significantly increased in patients with CKD secondary to focal and segmental glomerulosclerosis (FSGS), ANCA associated vasculitis (AAV) and proliferative class III and IV lupus nephritis (LN). uSerpinA3 levels were independently and positively associated with renal fibrosis. In patients with class V LN, uSerpinA3 levels were not different from healthy volunteers. uSerpinA3 was not found in patients with systemic inflammatory diseases without renal dysfunction. Our observations suggest that uSerpinA3 can detect renal fibrosis and inflammation, with a particular potential for the early detection of AKI to CKD transition and for the differentiation among lupus nephritis classes III/IV and V.

Discovery and Qualification of Candidate Urinary Biomarkers of Disease Activity in Lupus Nephritis.

Lupus nephritis (LN) is a severe clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Assessment of severity and activity of renal involvement in SLE requires a kidney biopsy, an invasive procedure with limited prognostic value. Noninvasive biomarkers are needed to inform treatment decisions and to monitor disease activity. Proteinuria is associated with disease progression in LN; however, the composition of the LN urinary proteome remains incompletely characterized. To address this, we profiled LN urine samples using complementary mass spectrometry-based methods:  protein gel fractionation, chemical labeling using tandem mass tags, and data-independent acquisition. Combining results from these approaches yielded quantitative information on 2573 unique proteins in urine from LN patients. A multiple-reaction monitoring (MRM) method was established to confirm eight proteins in an independent cohort of LN patients, and seven proteins (transferrin, α-2-macroglobulin, haptoglobin, afamin, α-1-antitrypsin, vimentin, and ceruloplasmin) were confirmed to be elevated in LN urine compared to healthy controls. In this study, we demonstrate that deep mass spectrometry profiling of a small number of patient samples can identify high-quality biomarkers that replicate in an independent LN disease cohort. These biomarkers are being used to inform clinical biomarker strategies to support longitudinal and interventional studies focused on evaluating disease progression and treatment efficacy of novel LN therapeutics.

Nutrient intake and environmental enteric dysfunction among Nepalese children 9-24 months old-the MAL-ED birth cohort study.

BACKGROUND: Nutrient deficiencies limit the growth and turnover of intestinal mucosa, but studies assessing whether specific nutrients protect against or improve environmental enteric dysfunction (EED) are scarce. We aimed to investigate associations between nutrient intake and EED assessed by lactulose:mannitol (L:M) ratio, anti-1-antitrypsin, myeloperoxidase (MPO), and neopterin (NEO) among children 9-24 months in Bhaktapur, Nepal. METHODS: Among 231 included children, nutrient intake was assessed monthly by 24 h recalls, and 3-month usual intake was estimated using Multiple Source Method. Associations between nutrient intake and L:M ratio (measured at 15 months) were assessed using multiple linear regression, while associations between nutrient intake and fecal markers (measured quarterly) were assessed using Generalized Estimating Equations (GEE) models. RESULTS: We found that associations between nutrient intake from complementary food and L:M ratio, alpha-1-antitrypsin (AAT), MPO and NEO were generally negative but weak. The only significant associations between nutrient intake (potassium, magnesium, phosphorous, folate, and vitamin C) and markers for intestinal inflammation were found for MPO. CONCLUSION: Negative but weak associations between nutrient intake and markers of intestinal inflammation were found. Significant associations between several nutrients and MPO might merit further investigation.

Beta-2-glycoprotein-1 and alpha-1-antitrypsin as urinary markers of renal cancer in von Hippel-Lindau patients.

CONTEXT: Von Hippel-Lindau disease (VHLD) is a rare inherited neoplastic syndrome. Among all the VHLD-associated tumors, clear cell renal cell carcinoma (ccRCC) is the major cause of death. OBJECTIVE: The aim of this paper is the discovery of new non-invasive biomarker for the monitoring of VHLD patients. MATERIALS AND METHODS: We compared the urinary proteome of VHLD patients, ccRCC patients and healthy volunteers. RESULTS: Among all differentially expressed proteins, alpha-1-antitrypsin (A1AT) and APOH (beta-2-glycoprotein-1) are strongly over-abundant only in the urine of VHLD patients with a history of ccRCC. DISCUSSION AND CONCLUSION: A1AT and APOH could be promising non-invasive biomarkers.

Proteomic approach for identification of IgA nephropathy-related biomarkers in urine.

Proteinuria is often used as a surrogate marker in monitoring and predicting outcome in patients with chronic kidney diseases, but it is non-specific. IgAN belongs to the most common primary glomerulonephritis worldwide with serious prognosis. The main aim of this work was to assess differences in urine proteins in patients with IgA nephropathy and to identify abnormal proteins as potential biomarkers of IgA nephropathy or the renal disease. In our pilot project, we selected 20 patients and compared them with 20 healthy volunteers. Protein quantification was performed using iTRAQ (isobaric tag for relative and absolute quantitation) labeling method. The peptides were separated by the isoelectric focusing method (IEF) and nano-LC with C18 column and identified by mass spectrometry using MALDI-TOF/TOF MS. Proteins´ lists obtained from IEF-LC-MS-MS/MS analysis were combined and contained 201 proteins. It was found out that 113 proteins were common in both experiments. 30 urinary proteins were significantly up- or down-regulated in patients with IgA nephropathy. We characterized potential biomarkers such as alpha-1-antitrypsin, apolipoprotein A-I, CD44 antigen or kininogen. Potential biomarkers of IgAN should be validated in further studies.

Comparative differential proteomic analysis of minimal change disease and focal segmental glomerulosclerosis.

BACKGROUND: Minimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS) are glomerular diseases characterized by nephrotic syndrome. Their diagnosis requires a renal biopsy, but it is an invasive procedure with potential complications. In a small biopsy sample, where only normal glomeruli are observed, FSGS cannot be differentiated from MCD. The correct diagnosis is crucial to an effective treatment, as MCD is normally responsive to steroid therapy, whereas FSGS is usually resistant. The purpose of our study was to discover and validate novel early urinary biomarkers capable to differentiate between MCD and FSGS. METHODS: Forty-nine patients biopsy-diagnosed of MCD and primary FSGS were randomly subdivided into a training set (10 MCD, 11 FSGS) and a validation set (14 MCD, 14 FSGS). The urinary proteome of the training set was analyzed by two-dimensional differential gel electrophoresis coupled with mass spectrometry. The proteins identified were quantified by enzyme-linked immunosorbent assay in urine samples from the validation set. RESULTS: Urinary concentration of alpha-1 antitrypsin, transferrin, histatin-3 and 39S ribosomal protein L17 was decreased and calretinin was increased in FSGS compared to MCD. These proteins were used to build a decision tree capable to predict patient's pathology. CONCLUSIONS: This preliminary study suggests a group of urinary proteins as possible non-invasive biomarkers with potential value in the differential diagnosis of MCD and FSGS. These biomarkers would reduce the number of misdiagnoses, avoiding unnecessary or inadequate treatments.

Toward Noninvasive Diagnosis of IgA Nephropathy: A Pilot Urinary Metabolomic and Proteomic Study.

IgA nephropathy is diagnosed by renal biopsy, an invasive procedure with a risk of significant complications. Noninvasive approaches are needed for possible diagnostic purposes and especially for monitoring disease activity or responses to treatment. In this pilot project, we assessed the utility of urine samples as source of biomarkers of IgA nephropathy. We used spot urine specimens from 19 healthy controls, 11 patients with IgA nephropathy, and 8 renal-disease controls collected on day of renal biopsy. Urine samples were analyzed using untargeted metabolomic and targeted proteomic analyses by several experimental techniques: liquid chromatography coupled with mass spectrometry, immunomagnetic isolation of target proteins coupled with quantitation by mass spectrometry, and protein arrays. No single individual biomarker completely differentiated the three groups. Therefore, we tested the utility of several markers combined in a panel. Discriminant analysis revealed that combination of seven markers, three metabolites (dodecanal, 8-hydroxyguanosine, and leukotriene C4), three proteins (α1-antitrypsin, IgA-uromodulin complex, and galactose-deficient IgA1), and heparan sulfate, differentiated patients with IgA nephropathy from patients with other renal diseases and healthy controls. Future studies are needed to validate these preliminary findings and to determine the power of these urinary markers for assessment of responses to therapy.

Urinary alpha-1 antitrypsin and CD59 glycoprotein predict albuminuria development in hypertensive patients under chronic renin-angiotensin system suppression.

BACKGROUND: Hypertension is a multi-factorial disease of increasing prevalence and a major risk factor for cardiovascular mortality even in the presence of adequate treatment. Progression of cardiovascular disease (CVD) occurs frequently during chronic renin-angiotensin-system (RAS) suppression, and albuminuria is a marker of CV risk. High prevalence of albuminuria in treated hypertensive patients has been demonstrated, but there are no available markers able to predict evolution. The aim of this study was the identification of novel indicators of albuminuria progression measurable in urine of diabetic and non-diabetic patients. METHODS: 1143 hypertensive patients under chronic treatment were followed for a minimum period of 3 years. Among them, 105 diabetic and non-diabetic patients were selected and classified in three groups according to albuminuria development during follow-up: (a) patients with persistent normoalbuminuria; (b) patients developing de novo albuminuria; (c) patients with maintained albuminuria. Differential urine analysis was performed by 2D gel electrophoresis (2D-DIGE) and further confirmed by liquid chromatography-mass spectrometry. Non-parametric statistical tests were applied. RESULTS: CD59 glycoprotein and alpha-1 antitrypsin (AAT) resulted already altered in patients developing albuminuria de novo, with a similar response in those with maintained albuminuria. A prospective study in a sub-group of normoalbuminuric patients who were clinically followed up for at least 1 year from urine sampling, revealed CD59 and AAT proteins significantly varied in the urine collected from normoalbuminurics who will negatively progress, serving as predictors of future albuminuria development. CONCLUSIONS: CD59 and AAT proteins are significantly altered in hypertensive patients developing albuminuria. Interestingly, CD59 and AAT are able to predict, in normoalbuminuric individuals, who will develop albuminuria in the future, being potential predictors of vascular damage and CV risk. These findings contribute to early identify patients at risk of developing albuminuria even when this classical predictor is still in the normal range, constituting a novel strategy towards a prompt and more efficient therapeutic intervention with better outcome.

[The regulatory transport proteins in diagnostic and prognosis of preeclampsia].

The article considers the results of analysis of content of regulative transport proteins in blood serum and urine of pregnant women (term III) in case of uncomplicated pregnancy and pregnancy complicated by preeclampsia and eclampsia for elaborating their pathogenic role and evaluating prognostic significance. It is established that the more severe eclampsia is the higher is the level of a2-macroglobulin and the lower is the content of lactoferrin in blood. At that, excretion of a2-macroglobulin and albumin with urine increases with aggravation of severity of processes and in urine is detected a1-antitrypsin previously undetected. The excretion of lactoferrin reaches its peak values in case of preeclampsia and decreases in case of eclampsia. The alteration of levels of a2-macroglobulin and lactoferrin are uncharacteristic for classic inflammatory reaction and testify their active involvement into pathogenesis of eclampsia. The decreasing of in blood of levels of a1-antitrypsin lesser than 5 g/l and lactoferrin lesser than 0.8 mg/l at concentration of a2-macroglobulin higher than 3.5 g/l against the background of decreased levels of albumin and crude protein in blood and also increasing in urine of concentrations of a2-macroglobulin up to 0.0005 g/l and higher and occurrence of a1-antitrypsin and increasing of content of albumin up to 10 times can be recommended as criteria of high risk of development of eclampsia in regnant women with moderately expressed preeclampsia in term III.

[Value of determination of haptoglobin and α1-antitrypsin in predicting response to glucocorticoid therapy in children with primary nephrotic syndrome].

OBJECTIVE: To study the value of the determination of serum and urine haptoglobin (HP) and alpha 1-antitrypsin (AAT) in predicting the response to glucocorticoid therapy in children with primary nephrotic syndrome (PNS). METHODS: A total of 84 children with PNS were classified to steroid-sensitive nephrotic syndrome (SSNS) (n=58) and steroid-resistant nephrotic syndrome (SRNS) groups (n=26). Forty healthy children were randomly selected for the control group. HP and AAT levels in blood and urinary samples were determined using ELISA. The efficiency of HP and AAT in predicting the response to glucocorticoid treatment of PNS was evaluated by the receiver operating characteristic (ROC) curve. RESULTS: Compared with the control group, both the SSNS and SRNS groups had significantly higher serum HP concentrations and urine AAT/Cr ratio before treatment (P<0.05); compared with the SSNS group, the SRNS group had significantly higher serum HP concentrations and urine AAT/Cr ratio before treatment and after one week and four weeks of treatment (P<0.05). Serum HP had the highest efficiency in predicting the response to glucocorticoid treatment of PNS at the concentration of 37.935 mg/mL, with the sensitivity and specificity being 92.3% and 86.2% respectively. Urine AAT/Cr ratio had the highest prediction efficiency at 0.0696, with the sensitivity and specificity being 100% and 79.3% respectively. ROC curve analysis of serum HP combined with urine AAT/Cr ratio showed a better prediction efficiency, with the sensitivity and specificity being 92.3% and 96.6% respectively. CONCLUSIONS: The increase in serum HP level or urine AAT/Cr ratio may indicate glucocorticoid resistance in the early stage of PNS. A combination of the two can achieve better efficiency in the prediction of SRNS.

Investigation of CCL18 and A1AT as potential urinary biomarkers for bladder cancer detection.

BACKGROUND: In this study, we further investigated the association of two biomarkers, CCL18 and A1AT, with bladder cancer (BCa) and evaluated the influence of potentially confounding factors in an experimental model. METHODS: In a cohort of 308 subjects (102 with BCa), urinary concentrations of CCL18 and A1AT were assessed by enzyme-linked immunosorbent assay (ELISA). In an experimental model, benign or cancerous cells, in addition to blood, were added to urines from healthy controls and analyzed by ELISA. Lastly, immunohistochemical staining for CCL18 and A1AT in human bladder tumors was performed. RESULTS: Median urinary protein concentrations of CCL18 (52.84 pg/ml vs. 11.13 pg/ml, p < 0.0001) and A1AT (606.4 ng/ml vs. 120.0 ng/ml, p < 0.0001) were significantly elevated in BCa subjects compared to controls. Furthermore, the addition of whole blood to pooled normal urine resulted in a significant increase in both CCL18 and A1AT. IHC staining of bladder tumors revealed CCL18 immunoreactivity in inflammatory cells only, and there was no significant increase in these immunoreactive cells within benign and cancerous tissue and no association with BCa grade nor stage was noted. A1AT immunoreactivity was observed in the cytoplasm of epithelia cells and intensity of immunostaining increased with tumor grade, but not tumor stage. CONCLUSIONS: Further development of A1AT as a diagnostic biomarker for BCa is warranted.

[Molecular heterogeneity of alpha 1-antitrypsin in the urine of the urolithiasis].

Randall's plaque theory is regarded as the most plausible mechanism of urinary stone formation; however, we speculated that urine proteins are necessarily involved in the process of stone formation. We focused on alpha 1-antitrypsin (alpha1-AT), a protein verified to be present in urinary calculi, and which is considered as a protein of inflammation, comparing its presence in healthy subjects and patients with urolithiasis. Quantitative analysis of alpha1-AT was performed with ELISA, whereas qualitative analysis was performed with SDS PAGE, two-dimensional electrophoresis, and western blotting. The results revealed a molecular heterogeneity in alpha1-AT, which can be classified into four patterns, a concentration-independent difference in alpha1-AT molecules found in the urine of patients and healthy subjects. A wider distribution of protein isoelectric points was found in urolithiasis (3.0-8.0) than in healthy subjects (4.0-5.0). We suggest that this new finding with molecular heterogeneity was due to the urolithiasis.

Diagnostic potential of urinary α1-antitrypsin and apolipoprotein E in the detection of bladder cancer.

PURPOSE: The ability to reliably diagnose bladder cancer in voided urine samples would be a major advance. Using high throughput technologies, we identified a panel of bladder cancer associated biomarkers with potential clinical usefulness. In this study we tested 4 potential biomarkers for the noninvasive detection of bladder cancer. MATERIALS AND METHODS: We examined voided urine specimens from 124 patients, including 63 newly diagnosed with bladder cancer and 61 controls. Concentrations of proteins were assessed by enzyme-linked immunosorbent assay, including α1-antitrypsin, apolipoprotein E, osteopontin and pentraxin 3. Data were compared to the results of urinary cytology and the BTA Trak® enzyme-linked immunosorbent assay based bladder cancer detection assay. We used the AUC of ROC curves to compare the usefulness of each biomarker to detect bladder cancer. RESULTS: Urinary levels of α1-antitrypsin, apolipoprotein E and bladder tumor antigen were significantly increased in subjects with bladder cancer. α1-Antitrypsin (AUC 0.9087, 95% CI 0.8555-0.9619) and apolipoprotein E (AUC 0.8987, 95% CI 0.8449-0.9525) were the most accurate biomarkers. The combination of α1-antitrypsin and apolipoprotein E (AUC 0.9399) achieved 91% sensitivity, 89% specificity, and a positive and negative predictive value of 89% and 90%, respectively. Multivariate regression analysis highlighted only apolipoprotein E as an independent predictor of bladder cancer (OR 24.9, 95% CI 4.22-146.7, p = 0.0004). CONCLUSIONS: Alone or in combination, α1-antitrypsin and apolipoprotein E show promise for the noninvasive detection of bladder cancer (OR 24.9, 95% CI 4.22-146.7, p = 0.0004). Larger, prospective studies including more low grade, low stage tumors are needed to confirm these results.

Proteomic profiling of dog urine after European adder (Vipera berus berus) envenomation by two-dimensional difference gel electrophoresis.

Between April and September every year, many dogs in Finland are bitten by Vipera berus berus, also known as the European adder, the only venomous snake in the area. Exposure to snake bite venom causes local and systemic symptoms and in severe cases can lead to death. Urine samples were collected from four dogs bitten by V. berus berus and treated in the intensive care unit of the Veterinary Teaching Hospital at the University of Helsinki. The inclusion criteria were a strong suspicion of an adder bite no more than two days before admission and clinical signs of an adder bite. Exclusion criteria were defined as ongoing treatment with glucocorticoids or a known history of liver or kidney diseases. Six privately owned, healthy dogs were obtained as controls. Samples were subjected to 2D-DIGE analysis. Image analysis was performed with DeCyder 7.0 2D software, and protein spots demonstrating a minimum 1.5-fold difference in average spot volume ratios between envenomed and control dogs with a Student's t-test p-value of less than 0.05 were picked and identified using LC-MS/MS. In 2D-DIGE analysis, seven proteins were significantly (p < 0.05) over-expressed in the urine of dogs bitten by V. berus berus compared to the control group. From these, five proteins were identified: beta-2-microglobulin (b2MG), alpha-1-antitrypsin (AAT), albumin, fetuin-B and superoxide dismutase (SOD1). Results indicate that envenomation by V. berus berus alter the urinary protein profile in dogs.

Uromodulin and α(1)-antitrypsin urinary peptide analysis to differentiate glomerular kidney diseases.

BACKGROUND/AIMS: Glomerular kidney disease (GKD) is suspected in patients based on proteinuria, but its diagnosis relies primarily on renal biopsy. We used urine peptide profiling as a noninvasive means to link GKD-associated changes to each glomerular entity. METHODS: Urinary peptide profiles of 60 biopsy-proven glomerular patients and 14 controls were analyzed by combining magnetic bead peptide enrichment, MALDI-TOF MS analysis, and ClinProTools v2.0 to select differential peptides. Tentative identification of the differential peptides was carried out by HPLC-MS/MS. RESULTS: The HPLC-MS/MS results suggest that uromodulin (UMOD; m/z: 1682, 1898 and 1913) and α(1)-antitrypsin (A1AT; m/z: 1945, 2392 and 2505) are differentially expressed urinary peptides that distinguish between GKD patients and healthy subjects. Low UMOD and high A1AT peptide abundance was observed in 80-92% of patients with GKD. Proliferative forms of GKD were distinguished from nonproliferative forms, based on a combination of UMOD and A1AT peptides. Nonproliferative forms correlated with higher A1AT peptide levels - focal segmental glomerulosclerosis was linked more closely to high levels of the m/z 1945 peptide than minimal change disease. CONCLUSION: We describe a workflow - urinary peptide profiling coupled with histological findings - that can be used to distinguish GKD accurately and noninvasively, particularly its nonproliferative forms.

Urine proteome analysis reflects atherosclerotic disease in an ApoE-/- mouse model and allows the discovery of new candidate biomarkers in mouse and human atherosclerosis.

Noninvasive 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 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 α(1)-antitrypsin, epidermal growth factor (EGF), kidney androgen-regulated protein, and collagen. Using immunohistochemistry, α(1)-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 α(1)-antitrypsin fragments also significantly correlated with intraplaque collagen and α(1)-antitrypsin content, mirroring plaque protein expression in the urine proteome. To provide further confirmation that the newly identified proteins are relevant in humans, the presence of collagen type I, α(1)-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 noninvasive 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.

Proteomic analysis of urinary biomarker candidates for nonmuscle invasive bladder cancer.

Nonmuscle invasive tumors of the bladder often recur and thereby bladder cancer patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method, e.g. a urine dipstick test, for monitoring recurrence would thus be advantageous. In this study, the complementary techniques mass spectrometry (MS) and Western blotting (WB)/dot blot (DB) were used to screen the urine samples from bladder cancer patients. High resolving MS was used to analyze and quantify the urinary proteome and 29 proteins had a significantly higher abundance (p<0.05) in bladder cancer samples compared with control urine samples. The increased abundance found in urine from bladder cancer patients compared with controls was confirmed with Western blot for four selected proteins; fibrinogen ß chain precursor, apolipoprotein E, α-1-antitrypsin, and leucine-rich α-2-glycoprotein 1. Dot blot analysis of an independent urine sample set pointed out fibrinogen ß chain and α-1-antitrypsin as most interesting biomarkers having sensitivity and specificity values in the range of 66-85%. Exploring the Human Protein Atlas (HPA) also revealed that bladder cancer tumors are the likely source of these proteins. They have the potential of being useful in diagnosis, monitoring of recurrence and thus may improve the treatment of bladder tumors, especially nonmuscle invasive tumors.