A panel of urinary proteins predicts active lupus nephritis and response to rituximab treatment.

OBJECTIVES: ∼30% of patients with SLE develop LN. Presence and/or severity of LN are currently assessed by renal biopsy, but biomarkers in serum or urine samples may provide an avenue for non-invasive routine testing. We aimed to validate a urinary protein panel for its ability to predict active renal involvement in SLE. METHODS: A total of 197 SLE patients and 48 healthy controls were recruited, and urine samples collected. Seventy-five of the SLE patients had active LN and 104 had no or inactive renal disease. Concentrations of lipocalin-like prostaglandin D synthase (LPGDS), transferrin, alpha-1-acid glycoprotein (AGP-1), ceruloplasmin, monocyte chemoattractant protein 1 (MCP-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were quantified by MILLIPLEX® Assays using the MAGPIX Luminex platform. Binary logistic regression was conducted to examine whether proteins levels associate with active renal involvement and/or response to rituximab treatment. RESULTS: Urine levels of transferrin (P <0.005), AGP-1 (P <0.0001), MCP-1 (P <0.001) and sVCAM-1 (P <0.005) were significantly higher in SLE patients when compared with healthy controls. Furthermore, levels of transferrin, AGP-1, ceruloplasmin, MCP-1 and sVCAM-1 (all P <0.0001) were higher in SLE patients with active LN when compared with patients without active LN. A combination of five urine proteins, namely LPGDS, transferrin, ceruloplasmin, MCP-1 and sVCAM-1 was a good predictor of active LN (AUC 0.898). A combined model of LPGDS, transferrin, AGP-1, ceruloplasmin, MCP-1 and sVCAM-1 predicted response to rituximab treatment at 12 months (AUC 0.818). CONCLUSIONS: Findings support the use of a urinary protein panel to identify active LN and potentially predict response to treatment with rituximab in adult SLE patients. Prospective studies are required to confirm findings.

Elevated Urine Levels of Macrophage Migration Inhibitory Factor in Inflammatory Bladder Conditions: A Potential Biomarker for a Subgroup of Interstitial Cystitis/Bladder Pain Syndrome Patients.

OBJECTIVE: To investigate whether urinary levels of macrophage migration inhibitory factor (MIF) are elevated in interstitial cystitis/bladder pain syndrome (IC/BPS) patients with Hunner lesions and also whether urine MIF is elevated in other forms of inflammatory cystitis. METHODS: Urine samples were assayed for MIF by enzyme-linked immunosorbent assay. Urine samples from 3 female groups were examined: IC/BPS patients without (N = 55) and with Hunner lesions (N = 43), and non-IC/BPS patients (N = 100; control group; no history of IC/BPS; cancer or recent bacterial cystitis). Urine samples from 3 male groups were examined: patients with bacterial cystitis (N = 50), radiation cystitis (N = 18) and noncystitis patients (N = 119; control group; negative for bacterial cystitis). RESULTS: Urine MIF (mean MIF pg/mL ± standard error of the mean) was increased in female IC/BPS patients with Hunner lesions (2159 ± 435.3) compared with IC/BPS patients without Hunner lesions (460 ± 114.5) or non-IC/BPS patients (414 ± 47.6). Receiver operating curve analyses showed that urine MIF levels discriminated between the 2 IC groups (area under the curve = 72%; confidence interval 61%-82%). Male patients with bacterial and radiation cystitis had elevated urine MIF levels (2839 ± 757.1 and 4404 ± 1548.1, respectively) compared with noncystitis patients (681 ± 75.2). CONCLUSION: Urine MIF is elevated in IC/BPS patients with Hunner lesions and also in patients with other bladder inflammatory and painful conditions. MIF may also serve as a noninvasive biomarker to select IC/BPS patients more accurately for endoscopic evaluation and possible anti-inflammatory treatment.

Urinary beta-trace protein gene expression analysis in type 2 diabetes mellitus patients / Análise da expressão do gene da proteína beta-traço na urina de pacientes com diabetes mellitus tipo 2

ABSTRACT Objective: To evaluate the gene expression of beta-trace protein in urine of diabetic patients, with no reduction in glomerular filtration rate, which was defined as below 60mL/min/1.73m2. Methods: Type 2 diabetes mellitus patients were recruited, and a group of non-diabetic individuals served as control. Beta-trace protein gene expression was analyzed by quantitative PCR. Blood samples were collected to establish glucose levels and baseline kidney function. Accuracy was analyzed using ROC curves. Results: Ninety type 2 diabetes mellitus patients and 20 non-diabetic individuals were recruited. The area under the curve was 0.601, sensitivity of 20% and specificity of 89.47%. Among diabetic participants, 18% showed an expression above the cutoff point. Conclusion: These results of accuracy of beta-trace protein gene expression in urine of diabetic patients are promising, although they did not achieve a higher area under the curve level.

RESUMO Objetivo: Avaliar a expressão do gene da proteína beta-traço na urina de pacientes diabéticos, sem redução na taxa de filtração glomerular, definida como abaixo de 60mL/min/1,73m2. Métodos: Foram recrutados pacientes com diabetes mellitus tipo 2, e um grupo de indivíduos não diabéticos serviu como controle. A expressão do gene da proteína beta-traço foi analisada por PCR quantitativa. Amostras de sangue foram coletadas para estabelecer níveis de glicemia e função renal inicial. A acurácia foi analisada utilizando curvas ROC. Resultados: Foram recrutados 90 pacientes com diabetes mellitus tipo 2 e 20 não diabéticos. A área sob a curva foi de 0,601, com sensibilidade de 20% e especificidade de 89,47%. Entre os diabéticos, 18% apresentaram expressão acima do ponto de corte. Conclusão: Estes resultados de acurácia da expressão do gene da proteína beta-traço na urina de pacientes diabéticos são promissores, apesar de não terem atingido um nível alto na área sob a curva.

Macrophage Migration Inhibitory Factor Predicts Outcome in Complex Aortic Surgery.

The perioperative inflammatory response is associated with outcome after complex aortic repair. Macrophage migration inhibitory factor (MIF) shows protective effects in ischemia-reperfusion (IR), but also adverse pro-inflammatory effects in acute inflammation, potentially leading to adverse outcome, which should be investigated in this trial. This prospective study enrolled 52 patients, of whom 29 (55.7%) underwent open repair (OR) and 23 (44.3%) underwent endovascular repair (ER) between 2014 and 2015. MIF serum levels were measured until 72 h post-operatively. We used linear mixed models and ROC analysis to analyze the MIF time-course and its diagnostic ability. Compared to ER, OR induced higher MIF release perioperatively; at 12 h after ICU admission, MIF levels were similar between groups. MIF course was significantly influenced by baseline MIF level (P = 0.0016) and acute physiology and chronic health evaluation (APACHE) II score (P = 0.0005). MIF level at 24 h after ICU admission showed good diagnostic value regarding patient survival [sensitivity, 80.0% (28.4-99.5%); specificity, 51.2% (35.1-67.1%); AUC, 0.688 (0.534-0.816)] and discharge modality [sensitivity, 87.5% (47.3-99.7%); specificity, 73.7% (56.9-86.6%), AUC, 0.789 (0.644-0.896)]. Increased perioperative MIF-levels are related to an increased risk of adverse outcome in complex aortic surgery and may represent a biomarker for risk stratification in complex aortic surgery.

Urine Biomarkers to Predict Response to Lupus Nephritis Therapy in Children and Young Adults.

OBJECTIVE: To delineate urine biomarkers that forecast response to therapy of lupus nephritis (LN). METHODS: Starting from the time of kidney biopsy, patients with childhood-onset systemic lupus erythematosus who were diagnosed with LN were studied serially. Levels of 15 biomarkers were measured in random spot urine samples, including adiponectin, α-1-acid glycoprotein (AGP), ceruloplasmin, hemopexin, hepcidin, kidney injury molecule 1, monocyte chemotactic protein-1, lipocalin-like prostaglandin D synthase (LPGDS), transforming growth factor-ß (TGF-ß), transferrin, and vitamin D binding protein (VDBP). RESULTS: Among 87 patients (mean age 15.6 yrs) with LN, there were 37 treatment responders and 50 nonresponders based on the American College of Rheumatology criteria. At the time of kidney biopsy, levels of TGF-ß (p < 0.0001) and ceruloplasmin (p = 0.006) were significantly lower among responders than nonresponders; less pronounced differences were present for AGP, hepcidin, LPGDS, transferrin, and VDBP (all p < 0.05). By Month 3, responders experienced marked decreases of adiponectin, AGP, transferrin, and VDBP (all p < 0.01) and mean levels of these biomarkers were all outstanding (area under the receiver-operating characteristic curve ≥ 0.9) for discriminating responders from nonresponders. Patient demographics and extrarenal disease did not influence differences in biomarker levels between response groups. CONCLUSION: Low urine levels of TGF-ß and ceruloplasmin at baseline and marked reduction of AGP, LPGDS, transferrin, or VDBP and combinations of other select biomarkers by Month 3 are outstanding predictors for achieving remission of LN. If confirmed, these results can be used to help personalize LN therapy.

International validation of a urinary biomarker panel for identification of active lupus nephritis in children.

BACKGROUND: Conventional markers of juvenile-onset systemic lupus erythematosus (JSLE) disease activity fail to adequately identify lupus nephritis (LN). While individual novel urine biomarkers are good at detecting LN flares, biomarker panels may improve diagnostic accuracy. The aim of this study was to assess the performance of a biomarker panel to identify active LN in two international JSLE cohorts. METHODS: Novel urinary biomarkers, namely vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein 1 (MCP-1), lipocalin-like prostaglandin D synthase (LPGDS), transferrin (TF), ceruloplasmin, alpha-1-acid glycoprotein (AGP) and neutrophil gelatinase-associated lipocalin (NGAL), were quantified in a cross-sectional study that included participants of the UK JSLE Cohort Study (Cohort 1) and validated within the Einstein Lupus Cohort (Cohort 2). Binary logistic regression modelling and receiver operating characteristic curve analysis [area under the curve (AUC)] were used to identify and assess combinations of biomarkers for diagnostic accuracy. RESULTS: A total of 91 JSLE patients were recruited across both cohorts, of whom 31 (34 %) had active LN and 60 (66 %) had no LN. Urinary AGP, ceruloplasmin, VCAM-1, MCP-1 and LPGDS levels were significantly higher in those patients with active LN than in non-LN patients [all corrected p values (p c) < 0.05] across both cohorts. Urinary TF also differed between patient groups in Cohort 2 (p c = 0.001). Within Cohort 1, the optimal biomarker panel included AGP, ceruloplasmin, LPGDS and TF (AUC 0.920 for active LN identification). These results were validated in Cohort 2, with the same markers resulting in the optimal urine biomarker panel (AUC 0.991). CONCLUSION: In two international JSLE cohorts, urinary AGP, ceruloplasmin, LPGDS and TF demonstrate an 'excellent' ability for accurately identifying active LN in children.

Macrophage migration inhibitory factor urinary excretion revisited ­ MIF a potent predictor of the immunosuppressive treatment outcomes in patients with proliferative primary glomerulonephritis.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a cytokine that shares many activities with other pro-inflammatory cytokines in primary glomerulonephritis (GN). This study assesses the influence of immunosuppressive treatment on serum and urine MIF in patients with proliferative (PGN) and non-proliferative (NPGN) glomerulonephritis, and evaluates the potential of MIF in predicting outcomes. METHODS: Eighty-four patients (45 males and 39 females) with primary GN were included. Urinary excretion of MIF (ng/mg of urinary creatinine) was measured both pre- and post-treatment with combined steroids and cyclophosphamide. After a 12-month follow-up, the patients were retrospectively divided into four subgroups: responders of proliferative GN (R-PGN), non-responders of proliferative GN (NR-PGN), responders of non-proliferative GN (R-NPGN) and non-responders of non-proliferative GN (NR-NPGN). RESULTS: The median pre-treatment urinary MIF values were higher in PGN than in NPGN (3.6 versus 2.2; ANOVA P = 0.039). The highest pre-treatment urinary excretion of MIF was observed in NR-PGN (median 6.1), which was significantly higher than other subgroups (ANOVA P < 0.05). The treatment significantly reduced MIF urinary excretion only in R-PGN (P < 0.01). In NR-PGN, pre- (5.9 ± 2.9 pg/mgCr) and post-treatment mean MIF excretion (4.9 ± 2.3 pg/mgCr) exceeded the calculated cut off value (3.3 pg/mgCr). CONCLUSION: MIF urinary excretion appears to be a prognostic marker of therapy outcomes only in proliferative glomerulonephritis, in which lower urinary MIF may be linked with good prognosis, whereas a higher MIF urinary excretion value was a marker of unfavorable therapy outcomes. In Non-Responders, urinary MIF measurements may help to reconsider the choice of the immunosuppressive regimen at early stages of the treatment and act as an impulse to search for new therapeutic strategies.

Evaluation of biomarkers of cell cycle arrest and inflammation in prediction of dialysis or recovery after kidney transplantation.

Early prediction of delayed graft function (DGF) after kidney transplantation would facilitate patient management. Cell cycle arrest and inflammation are implicated in the pathogenesis of DGF. We assessed the utility of two novel acute kidney injury (AKI) biomarkers, urinary tissue inhibitor of metalloproteinases-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7), and five inflammatory markers to predict DGF following deceased-donor kidney transplantation. Serial urine concentrations of TIMP-2 and IGFBP7 were measured immediately after transplantation in 56 recipients along with vascular endothelial growth factor-A (VEGF-A), macrophage migration inhibitory factor (MIF), monocyte chemotactic protein-1 (MCP-1), trefoil factor 3 (TFF3) and chemokine (C-X-C) ligand 16 (CXCL16). Delayed graft function (DGF) was defined as requirement for dialysis within 7 days. Integrated discrimination improvement analysis was used to evaluate whether these biomarkers enhanced prediction of DGF independently of a validated clinical risk prediction model. DGF occurred in 22 patients (39%). At 4 h after kidney reperfusion, the area under the receiver operator characteristic curve (AUC) for VEGF-A was good (AUC > 0.80); for TIMP-2, IGFBP7 and [TIMP-2] × [IGFBP7] fair (AUCs 0.70-0.79); and for MIF, MCP-1, TFF3 and CXCL16 poor (AUC < 0.70). Only TIMP-2 and VEGF significantly enhanced the DGF prediction at 4 and 12 h. The cell cycle arrest marker urinary TIMP-2 and the inflammatory biomarker VEGF-A are potentially useful adjuncts to clinical data for early prediction of DGF.

Urinary proteomics revealed prostaglandin H(2)D-isomerase, not Zn-α2-glycoprotein, as a biomarker for active lupus nephritis.

Although renal histopathology is the gold standard for the diagnosis and prognosis of lupus nephritis (LN), the invasiveness of renal biopsy warrants the discovery of novel non-invasive diagnostic and prognostic biomarkers. In the present study, urine samples from 10 LN patients (5 active and 5 inactive) were analyzed by two-dimensional gel electrophoresis (2-DE) to screen for potential biomarkers of active LN. Quantitative analysis and statistics revealed 16 protein spots whose levels significantly differed between groups. These proteins were successfully identified by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS). Among these potential candidates, differential levels of urinary Zn-α2-glycoprotein (ZA2G) and prostaglandin H(2)D-isomerase (PGDS) were further validated by enzyme-linked immunosorbent assay (ELISA) in another independent group of 78 subjects, including 30 active LN, 26 inactive LN, 14 non-LN glomerular diseases, and 8 healthy normal individuals. Whereas ZA2G levels were elevated in urine of patients with active LN and non-LN glomerular diseases, PGDS was elevated only in the urine of the active LN group. Urinary PGDS, not ZA2G, may serve as a biomarker for active LN and upon validation in larger studies, may become the non-invasive test to evaluate the disease activity in future management of LN.

Evaluation of renal damage by urinary beta-trace protein in patients with chronic kidney disease.

BACKGROUND: Beta-trace protein (BTP) was found to be increased in the serum and urine of patients with renal diseases. The aim of this study was to compare the urinary levels of beta-trace protein with levels of other urinary proteins: albumin, beta2-microglobulin (B2M), alpha1-microglobulin (A1M), and cystatin C and to determine its clinical usefulness for detection of renal dysfunction in chronic kidney disease (CKD). METHODS: These markers were measured in 24-hour urine samples from 134 patients with CKD. RESULTS: BTP correlated significantly with A1M (r = 0.871), cystatin C (r = 0.759), total protein (r = 0.684), B2M (r = 0.497), and albumin (r = 0.448) in 24-hour urine samples (P < 0.05). Urinary BTP concentrations in patients with albuminuria below 30 mg/day were significantly lower than in patients with albuminuria above 30 mg/day (P < 0.0001). ROC analysis showed high diagnostic accuracy of BTP for detection of > 30 mg/day albuminuria (AUC 0.908). Urinary BTP was also in significant correlation with the estimated glomerular filtration rate (r = -0.580). CONCLUSIONS: The results of our study suggest that BTP may be a useful and reliable urinary marker of renal dysfunction and may have a place in addition to urinary alpha1-microglobulin and albumin as an alternative marker for tubular damage and the magnitude of renal impairment in patients with chronic kidney disease.

Study of urinary proteomes in Anderson-Fabry disease.

BACKGROUND: Anderson-Fabry disease (AFD) is an X-linked genetic disorder with deficient α-galactosidase A activity. The main aim of this work was to investigate possible differences in urine proteins between healthy controls and AFD patients and to identify abnormal proteins as potential biomarkers of disease. MATERIAL AND METHODS: We studied 2D electrophoresis images of urine samples collected from AFD patients and healthy subjects. The proteins were separated using isoelectric focusing method followed by SDS-PAGE. The proteins were then visualized by silver staining and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: We found out that the urinary spectra of all the Fabry disease samples included identical proteins with molecular weight around 20-40 kDa. The concentration of some proteins was more than three times higher in the AFD samples, compared to the controls. The abundant proteins were identified by MALDI-TOF MS and included the following: alpha-1-antitrypsin, alpha-1-microglobulin, prostaglandin H2 d-isomerase, complement-c1q tumor necrosis factor-related protein, and Ig kappa chain V-III. Possible glycosylation at Asn51 and Asn78 sites of the prostaglandin H2 d-isomerase was detected. CONCLUSIONS: AFD urinary proteomics revealed increased secretion of several proteins. We postulate that the observed difference in the amount of prostaglandin H2 d-isomerase and its position on two-dimensional gels might be related to different glycosylation in AFD subjects.

Serum and urinary markers of early impairment of GFR in chronic kidney disease patients: diagnostic accuracy of urinary ß-trace protein.

The screening for chronic kidney diseases (CKD) patients with impaired GFR needs the measurement of serum creatinine (SCr) or cystatin C (SCys). GFR can also be predicted from SCr or SCys with different formulas. The aim of this study, performed in a group of CKD patients with different levels of GFR, was to evaluate the possibility to select the patients with a GFR <90 ml·min(-1)·1.73 m(-2) by means of serum levels and urinary excretion of different low-molecular-weight proteins (LMWP), cystatin C (Cys), ß2-microglobulin (ß2M), retinol-binding protein (RBP), ß-trace protein (BTP), and derived prediction equations for GFR. In the 295 CKD patients (137 women), at all stages of GFR impairment a very high correlation was found between GFR ((99m)Tc-DTPA) and serum Cr, Cys, ß2M, and BTP. All these serum markers showed a similar accuracy as indicators of different GFR impairments. RBP had the lowest correlation with GFR and was also significantly less accurate. The different prediction formulas derived from gender, anthropometric data and SCr or S-LMWP had a diagnostic accuracy similar to that of serum Cr, Cys, ß2M, and BTP. Urinary albumin was inadequate as an indicator of any level of GFR impairment. Urinary excretion of Cys and ß2M increased significantly only in patients with a GFR <30 ml·min(-1)·1.73 m(-2), while urinary BTP increased already at GFR <90 ml·min(-1)·1.73 m(-2). In this selected group of CKD patients, the positive predictive value of urinary BTP for a GFR <90 ml·min(-1)·1.73 m(-2) was 85%, indicating that, in CKD patients, a urine-based test can predict a slight GFR impairment.