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.

Association of urinary non-albumin protein with the different urinary marker for glomerular and tubular damage in patients with type 2 diabetes.

BACKGROUND/AIM: In recent years, the diagnostic utility of urinary protein levels has been demonstrated for the early detection and progression of kidney disease. This study aimed to evaluate the associations of the non-albumin protein (NAP) with different urinary marker for tubular and glomerular damage in patients with type 2 diabetes (T2D). METHODS: In this observational cross-sectional study, 424 patients with T2D duration > 10 years were classified into two groups according to estimated glomerular filtration rate (eGFR). The ratios of different urinary markers (albumin, NAP, total protein, transferrin, retinol-binding protein (RBP), and neutrophil gelatinase-associated lipocalin (NGAL) to creatinine were analyzed. RESULTS: The levels of urinary biomarkers increased significantly with decrease in eGFR levels. In the group with moderately decreased eGFR, the albumin to-creatinine ratio (ACR), non-albumin protein-to-creatinine ratio (NAPCR), and total protein-to-creatinine ratio (PCR) were independently associated with all urinary markers after being adjusted for risk factors. The area under the receiver operating characteristics (ROC) curve for ACR and PCR had a better diagnostic value than other urinary biomarkers. Comparing ROC curve of NAPCR with other urinary biomarkers, it was significantly better than NGAL/Cr (p = 0.033). CONCLUSIONS: The findings of the present study confirm that ACR and PCR are diagnostic biomarkers in T2D patients with decreased eGFR. NAPCR in these patients diagnostically only outperformed NGAL/Cr.

Urinary transferrin pre-emptively identifies the risk of renal damage posed by subclinical tubular alterations.

Nephrotoxicity is an important limitation to the clinical use of many drugs and contrast media. Drug nephrotoxicity occurs in acute, subacute and chronic manifestations ranging from glomerular, tubular, vascular and immunological phenotypes to acute kidney injury. Pre-emptive risk assessment of drug nephrotoxicity poses an urgent need of precision medicine to optimize pharmacological therapies and interventional procedures involving nephrotoxic products in a preventive and personalized manner. Biomarkers of risk have been identified in animal models, and risk scores have been proposed, whose clinical use is abated by their reduced applicability to specific etiological models or clinical circumstances. However, our present data suggest that the urinary level of transferrin may be indicative of risk of renal damage, where risk is induced by subclinical tubular alterations regardless of etiology. In fact, urinary transferrin pre-emptively correlates with the subsequent renal damage in animal models in which risk has been induced by drugs and toxins affecting the renal tubules (i.e. cisplatin, gentamicin and uranyl nitrate); whereas transferrin shows no relation with the risk posed by a drug affecting renal hemodynamics (i.e. cyclosporine A). Our experiments also show that transferrin increases in the urine in the risk state (i.e. prior to the damage) precisely as a consequence of reduced tubular reabsorption. Finally, urinary transferrin pre-emptively identifies subpopulations of oncological and cardiac patients at risk of nephrotoxicity. In perspective, urinary transferrin might be further explored as a wider biomarker of an important mechanism of predisposition to renal damage induced by insults causing subclinical tubular alterations.

Utility of urinary transferrin and ceruloplasmin in patients with systemic lupus erythematosus for differentiating patients with lupus nephritis. / Transferrina y ceruloplasmina en orina de pacientes con lupus eritematoso sistémico. ¿Son útiles para diferenciar pacientes con nefritis lúpica?

BACKGROUND AND OBJECTIVE: Diagnosis of lupus nephritis (LN) is usually based on renal biopsy, which is an invasive technique that involves multiple risks. Therefore, different biomarkers have emerged as alternatives for the diagnosis of LN. Nonetheless, studies regarding urinary biomarkers in Latin American patients are limited. The objective of this study was to assess the diagnostic value of urinary transferrin and ceruloplasmin to differentiate patients who have renal involvement from those who do not. MATERIALS AND METHODS: Systemic lupus erythematosus (SLE) patients that met the revised American College of Rheumatology (ACR) classification criteria were recruited. Patients with another autoimmune disease, active infection (urinary tract or systemic infection), renal replacement therapy, human immunodeficiency virus infection or pregnancy were excluded. A urine sample was collected from each patient. LN was diagnosed according to ACR criteria. The activity and chronicity of LN were measured using the Austin indices. Urinary transferrin and ceruloplasmin levels were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits. Mann-Whitney U test and Student's t-test were used to compare data. Spearman's rank correlation was used to determine associations. Lastly, receiver operating characteristic (ROC) curves were created. RESULTS: The study involved 120 SLE patients. In all, 85% were female, 76% mestizo, the mean age was 32.8±12.1years and mean systemic lupus erythematosus disease activity index (SLEDAI) was 8.4±8.9; 64% had renal involvement. Urinary levels of the two biomarkers were significantly higher in patients with LN compared to those without LN. Similarly, urinary levels of both biomarkers were significantly higher in patients with active LN compared to those with inactive LN. Furthermore, urinary transferrin levels were significantly higher in Afro-Latin American patients. On the other hand, urinary transferrin levels correlated with SLEDAI and proteinuria, and transferrin and ceruloplasmin levels correlated with each other. The diagnostic value of ROC curves for these urinary biomarkers for LN were good. CONCLUSIONS: In our cohort of SLE patients, we found that transferrin and ceruloplasmin were potential biomarkers for LN, and can even differentiate active LN.

Differential urinary proteomics analysis of myocardial infarction using iTRAQ quantification.

Myocardial infarction (MI) is a disease characterized by high morbidity and mortality rates. MI biomarkers are frequently used in clinical diagnosis; however, their specificity and sensitivity remain unsatisfactory. Urinary proteome is an easy, efficient and noninvasive source to examine biomarkers associated with various diseases. The present study, to the best of the authors' knowledge, is the first to examine the urinary proteome using the isobaric tags for relative and absolute quantitation (iTRAQ) technology to identify potential diagnostic biomarkers of MI. The urinary proteome was analyzed within 12 h following the first symptoms of early­onset MI and at day 7 following percutaneous coronary intervention via iTRAQ labeling and two­dimensional liquid chromatography­tandem mass spectrometry. Candidate biomarkers were validated by multiple reaction monitoring (MRM) analysis. A total of 233 urinary proteins were differentially expressed. Gene enrichment analysis identified that the urinary proteome in patients with MI was associated with atherosclerosis, abnormal coagulation and abnormal cell metabolism. In total, 12 differentially expressed urinary proteins were validated by MRM analysis, five of which were associated with MI for the first time in the present study. Binary logistic regression analysis suggested that the combination of five urinary proteins (antithrombin­III, complement C3, α­1­acid glycoprotein 1, serotransferrin and cathepsin Z) may be used to diagnose MI with 94% sensitivity and 93% specificity. In addition, the protein expression levels of three proteins were significantly restored to normal levels following surgical treatment. The verified candidate biomarkers may be used for the diagnosis of MI, and for monitoring the disease status and the effects of treatments for MI. The present results may facilitate future clinical applications of the urinary proteome to diagnose MI.

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.

Correlation between endocapillary proliferative and nephrotic-range proteinuria in children with Henoch-Schönlein purpura nephritis.

BACKGROUND: The endocapillary proliferative (EP) lesion is not included in the International Study of Kidney Disease in Children (ISKDC) pathological classification of Henoch-Schönlein purpura nephritis (HSPN). The main objective of the study was to determine the pathological importance of EP in the development of proteinuria in children with Henoch-Schönlein purpura nephritis (HSPN). METHODS: The pathological features of 148 HSPN children with nephrotic-range proteinuria were investigated retrospectively. Urinary IgG, transferrin, and albumin levels were measured by immunonephelometry. The correlations between EP lesion and 24-h proteinuria, urinary IgG, urinary transferrin, and urinary albumin were analyzed. Renal biopsy specimens were immunohistochemically stained for nephrin and podocalyxin. RESULTS: Of the total 581 cases of children with HSPN who underwent renal biopsy, 148 cases (25.5%) presented with nephrotic-range proteinuria. The pathological types of HSPN with nephrotic-range proteinuria were categorized as IIb, IIIa, IIIb, IIIb with diffuse EP, IVb, pure focal EP type, and pure diffuse EP type. Among these types, pure diffuse EP type accounted for 7.4%. The levels of 24-h proteinuria and urinary albumin were the highest in pure diffuse EP type among all pathological types, and the percentage of EP correlated with 24-h proteinuria and urinary albumin levels. 24-h proteinuria was significantly higher in pure diffuse EP type relative to HSPN IIb type, and significantly higher in IIIb with EP, compared with HSPN IIIb. Nephrin, but not podocalyxin, was downregulated in EP segment. CONCLUSIONS: EP is an independent pathogenic factor in HSPN with nephrotic-range proteinuria. Downregulation of nephrin in EP segment is a potential molecular mechanism of nephrotic-range proteinuria. Albumin is the major urinary protein component in HSPN with EP.

Growing international evidence for urinary biomarker panels identifying lupus nephritis in children - verification within the South African Paediatric Lupus Cohort.

BACKGROUND: A urinary biomarker panel including alpha-1-acid-glycoprotein (AGP), lipocalin-like-prostaglandin-D-synthase (LPGDS), transferrin and ceruloplasmin demonstrates an 'excellent' ability for identifying active lupus nephritis in UK/US children. This study aimed to assess whether this panel identifies active lupus nephritis within the South African Paediatric Lupus Cohort. METHODS: Juvenile-onset-systemic lupus erythematosus (JSLE) patients aged < 19 years at diagnosis and healthy controls were recruited. Patients were categorized as having active lupus nephritis (renal BILAG score; A/B and previous histological confirmation) or inactive lupus nephritis (renal BILAG score: D/E). Urinary biomarkers were quantified by ELISA. Mann-Whitney U-test compared biomarker levels between groups. Binary logistic regression and receiver operating curve analysis assessed biomarker combinations. RESULTS: Twenty-three juvenile-onset-systemic lupus erythematosus patients were recruited with a median age of 13.5 years (interquartile range (IQR) 12.7-14.9) and disease duration of 2.6 years (IQR 1.8-4.0). Eighteen healthy controls had a median age of 11.0 years (IQR 10.0-12.0). AGP, LPGDS, transferrin, ceruloplasmin and VCAM-1 were significantly higher in active than in inactive lupus nephritis patients (corrected p-values, all pc < 0.05), with no difference between inactive lupus nephritis patients and healthy controls (all pc = 1.0). The optimal biomarker combination included AGP, ceruloplasmin, LPGDS and transferrin (area under the curve = 1.0). CONCLUSIONS: A urinary biomarker panel comprising AGP, ceruloplasmin, LPGDS and transferrin previously validated within UK/US cohorts also performed excellently within a racially distinct South African cohort which displayed more severe lupus nephritis.

Study on the Correlation Between Urinary Transferrin and Nonalcoholic Fatty Liver Disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) can cause renal injury, and urinary transferrin (UTRF) is extremely sensitive to renal injury. We aimed to investigate the correlation between UTRF and NAFLD and to observe the distribution of NAFLD at different levels of UTRF. METHODS: A total of 711 subjects fulfilled the diagnostic criteria of NAFLD and 1,396 healthy control participants were enrolled in this study. UTRF levels and other clinical and laboratory parameters were measured. RESULTS: The UTRF level was higher in NAFLD than in non-NAFLD patients. Unit and multiple regression analysis showed that UTRF was an independent risk factor for NAFLD, with OR values of 1.474 (1.328 - 1.635, p < 0.001) and 1.435 (1.267 - 1.625, p < 0.001), respectively. The prevalence of UTRF (groups 1, 2, 3, 4) was 25.61%, 26.56%, 38.14%, and 44.59%, respectively (p < 0.001), and the prevalence of NAFLD in the high UTRF group was significantly higher than in the low UTRF group. CONCLUSIONS: UTRF is an independent risk factor for NAFLD.

Markers of Glomerular and Tubular Damage in the Early Stage of Kidney Disease in Type 2 Diabetic Patients.

Diabetic kidney disease develops in half of genetically predisposed patients with type 2 diabetes (T2DM). Early diagnosis of kidney damage and nephroprotective treatment are the ways of preventing the disease progression. Our aim was to evaluate selected laboratory markers of glomerular and tubular damage in T2DM patients with early stages of chronic kidney disease (G1/G2, A1/A2) for their associations with A2 albuminuria and early decline in the estimated glomerular filtration rate (eGFR). Among 80 T2DM patients with median eGFR of 92.4 ml/min/1.73 m2 and median urinary albumin to creatinine ratio (uACR) of 4.69 mg/g, 19 had uACR > 30 mg/g (A2). Higher serum cystatin C, serum and urine neutrophil gelatinase associated lipocalin (NGAL), urine kidney injury molecule 1 (KIM-1), detectable urine transferrin and IgG, and lower serum uromodulin significantly predicted A2 albuminuria, urine KIM-1/creatinine ratio, and IgG being the best predictors. Albuminuria, urine NGAL/creatinine, and IgG correlated with diabetes duration. Albuminuria, urine NGAL, transferrin, IgG, and uromodulin correlated with diabetes control. In a subgroup of 29 patients, retrospective data were available on changes in eGFR and uACR over one year. Decline in eGFR was observed in 17 patients and increase in uACR in 10 patients. Serum and urine NGAL correlated with eGFR changes. Higher urine NGAL, KIM-1/creatinine ratio, and detectable IgG were significantly associated with the increase in uACR. Widely available markers, serum cystatin C, urine IgG, transferrin, and NGAL, may help in early assessment of kidney disease in T2DM patients; however, large prospective studies are needed to confirm the conclusion.

[Relevance of urinary specific protein assay in the diagnosis of kidney diseases]. / Intérêt du dosage des protéines spécifiques urinaires dans le diagnostic des pathologies rénales.

The analysis of urinary protein composition is an important step in the evaluation and monitoring of kidney diseases. Among the various approaches, the determination of urinary-specific proteins makes it possible to non-invasively detect a preferentially tubular or glomerular injury, to orientate towards a pathological process, to guide the indication of a kidney biopsy, and to follow the evolution of the disease and the effectiveness of a therapy. No study systematically evaluated the performance of urinary-specific proteins for the diagnosis of a renal disease. We conducted this retrospective study to perform an exhaustive analysis of the correlations that may exist between histologically proven kidney disease and the corresponding specific urinary protein composition it in order to evaluate the diagnostic value of each of its components. Urinary concentrations of total protein, albumin, transferrin, alpha1microglobulin, beta2microglobulin, retinol binding protein, and immunoglobulin G were analyzed in more than 500 patients who underwent renal biopsy and concomitant urine specific protein analysis. Our analysis shows that these markers have a limited positive predictive value in this cohort of complex and unselected kidney diseases. In particular, low molecular weight proteins, and especially alpha1microglobulin, are frequently associated with glomerular diseases. We identified transferrin as an independent predictor of minimal changes disese and renal amyloidosis, and beta2microglobulin as an independent predictor of acute tubulointerstitial nephropathy and myelomatous tubulopathy. Finally, we defined the thresholds at which these parameters had excellent negative predictive values.

Lime powder treatment reduces urinary excretion of total protein and transferrin but increases uromodulin excretion in patients with urolithiasis.

Our previous study has shown that lime powder (LP) had an inhibitory effect against calcium oxalate stone formation. However, the precise mechanisms underlying such beneficial effect remained unclear. Our present study thus aimed to address the effect of LP on excretory level and compositions of urinary proteins using a proteomics approach. From a total of 80 calcium oxalate stone formers recruited into our 2-year randomized clinical trial of LP effect, 10 patients with comparable age and clinical parameters were selected for this proteomic study. 24-h urine specimens were collected from all subjects, at baseline (before) and after LP treatment for 6 months, and then subjected to quantitative proteomics analysis and subsequent validation by ELISA. Total urinary protein excretion was significantly decreased by LP treatment, but unaffected by placebo. Nanoflow liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS) followed by quantitative analysis revealed 17 proteins whose levels were significantly altered (16 decreased and 1 increased) exclusively by LP treatment. Among these, the decrease of transferrin and increase of uromodulin were validated by ELISA. Moreover, there was a significant correlation between microalbuminuria and urinary transferrin level by Pearson's correlation test. In summary, LP treatment caused significant reduction in total urinary protein excretion and changes in urinary protein compositions that could be linked to stone inhibitory effects and might be relevant mechanisms responsible for the beneficial effects of LP to prevent kidney stone formation and recurrence.

Renal iron accumulation occurs in lupus nephritis and iron chelation delays the onset of albuminuria.

Proteins involved in iron homeostasis have been identified as biomarkers for lupus nephritis, a serious complication of systemic lupus erythematosus (SLE). We tested the hypothesis that renal iron accumulation occurs and contributes to renal injury in SLE. Renal non-heme iron levels were increased in the (New Zealand Black x New Zealand White) F1 (NZB/W) mouse model of lupus nephritis compared with healthy New Zealand White (NZW) mice in an age- and strain-dependent manner. Biodistribution studies revealed increased transferrin-bound iron accumulation in the kidneys of albuminuric NZB/W mice, but no difference in the accumulation of non-transferrin bound iron or ferritin. Transferrin excretion was significantly increased in albuminuric NZB/W mice, indicating enhanced tubular exposure and potential for enhanced tubular uptake following filtration. Expression of transferrin receptor and 24p3R were reduced in tubules from NZB/W compared to NZW mice, while ferroportin expression was unchanged and ferritin expression increased, consistent with increased iron accumulation and compensatory downregulation of uptake pathways. Treatment of NZB/W mice with the iron chelator deferiprone significantly delayed the onset of albuminuria and reduced blood urea nitrogen concentrations. Together, these findings suggest that pathological changes in renal iron homeostasis occurs in lupus nephritis, contributing to the development of kidney injury.

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.

Interest of the combined measurement of selected urinary proteins in the diagnosis approach in nephrology.

Determination of the protein composition of urine is a non-invasive method helping to diagnose renal lesions and evaluate therapeutic interventions. We present here five observations that highlight the performance and relevance of urine protein analysis combining selected glomerular and tubular protein measurements. Total urine protein level and measurements of urinary IgG, albumin, transferrin alpha-1 microglobulin and retinol binding protein were performed on a urine sample by immunonephelometry. The results were normalized for urine creatinine concentration and integrated in the MDI interpretation software that provides a "urine protein profile" (UPP). Sequential UPP were performed in two patients with drug-induced tubular toxicity. One resolved after drug withdrawal. The second concomitantly developped glomerular lesions and repeated UPP was warranted to follow evaluation of the distinct renal lesions. Two cases illustrate two distinct clinical situations in patients with multiple myeloma, respectively myeloma cast nephropathy and toxic acute tubular necrosis. Those differential diagnoses were early anticipated by UPP. In one case, UPP was in favour of the presence of large amount of urinary monoclonal light chain excretion before performing urine electrophoresis analysis and renal biopsy while this was not in the other case. In the last case, we compared sequential UPP, renal function and kidney biopsies in a patient with a diagnosis of membranous nephropathy, and demonstrated a good correlation between urine glomerular and tubular protein excretion and progression of the renal lesions. The use of the UPP in clinical practice, particularly through the accurate quantification of tubular markers, is a more efficient tool for the diagnosis and follow-up of renal diseases than the less sensitive semi-quantitative urine electrophoresis or combined assays of both total proteinuria and albuminuria.

Improved detection of alcohol consumption using the novel marker phosphatidylethanol in the transplant setting: results of a prospective study.

Phosphatidylethanol (PEth) is a new, highly specific alcohol marker. The aim of this study was to assess its diagnostic value in the liver transplant setting. In 51 pre- and 61 post-transplant patients with underlying alcoholic liver disease PEth, ethanol, methanol, carbohydrate-deficient transferrin (CDT), and ethyl glucuronide in urine (uEtG) and hair (hEtG) were tested and compared with patients' questionnaire reports. Twenty-eight (25%) patients tested positive for at least one alcohol marker. PEth alone revealed alcohol consumption in 18% of patients. With respect to detection of alcohol intake in the preceding week, PEth showed a 100% sensitivity. PEth testing was more sensitive than the determination of ethanol, methanol, CDT or uEtG alone [sensitivity 25% (confidence interval (CI) 95%, 7-52%), 25% (7-52%), 21% (6-45%) and 71% (41-91%), respectively], or ethanol, methanol and uEtG taken in combination with 73% (45-92%). Specificity of all markers was 92% or higher. Additional testing of hEtG revealed alcohol consumption in seven patients, not being positive for any other marker. Phosphatidylethanol was a highly specific and sensitive marker for detection of recent alcohol consumption in pre- and post-transplant patients. The additional determination of hEtG was useful in disclosing alcohol consumption 3-6 months retrospectively.

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.

Predicting decline of kidney function in lupus nephritis using urine biomarkers.

OBJECTIVE: To evaluate candidate biomarkers to predict future renal function decline (RFD) in children and adults with lupus nephritis (LN). METHODS: At the time of enrollment into prospective observational LN cohort studies liver-type fatty acid binding protein (LFABP), albumin, monocyte chemoattractant protein-1 (MCP-1), uromodulin, transferrin, and hepcidin were measured in urine samples of two cohorts of patients with LN, one followed at a pediatric (cohort-1; n = 28) and one at an adult institution (cohort-2; n = 69). The primary outcome was RFD, defined in cohort-1 as a decrease in estimated glomerular filtration rate (eGFR) of ≥20% and in cohort-2 as a sustained increase of ≥25% in serum creatinine concentration (SCr), both from baseline. RESULTS: All patients (n = 97) had normal eGFR or SCr at the time of urine collection at baseline. RFD occurred in 29% (8/28) of patients in cohort-1 during a mean follow-up of 6.1 months, and in 30% (21/69) of those in cohort-2 during a mean follow-up of 60 months. Individually, in cohort-1, levels of MCP-1, transferrin, LFABP, and albumin were higher in the RFD group than those who maintained renal function, with statistical significance for LFABP and albumin. In cohort-2 the RFD group also had higher levels of urine MCP-1 and albumin than others. The combination of LFABP, MCP-1, albumin, and transferrin had good predictive accuracy for RFD in both cohorts (area under the ROC curve = 0.77-0.82). CONCLUSION: The combinatorial urine biomarker LFABP, MCP-1, albumin, and transferrin shows promise as a predictor of renal functional decline in LN, and warrants further investigation.

Urinary microprotein concentrations in the long-term follow-up of dilating vesicoureteral reflux patients who underwent medical or surgical treatment.

PURPOSE: This study examined the relationship between urinary microprotein concentrations and renal functional parameters in children with dilating (grade III-V) vesicoureteral reflux (VUR) who underwent either medical or surgical treatment. METHODS: All 44 dilating VUR patients who were followed for 4 years were screened for inclusion in this study. The patients' clinical features and clinical outcomes, as well as the urinary activities of albumin (ALB), transferrin (TRF), immunoglobulin G (IgG), alpha-1-microglobulin (α1-MG), and N-acetyl-ß-glucosaminidase (NAG), were retrospectively analyzed. RESULTS: High values of NAG, α1-MG, IgG, TRF, and ALB were noted in 73.33, 58.33, 43.33, 24.14, and 53.33 % of patients, respectively, at the first examination. Cystatin C, eGFR, and urinary microprotein levels were associated with a good prognosis after 4 years of follow-up. No differences in recurrent UTI, cystatin C concentration, most microprotein/creatinine (Cr) ratios, eGFR, or ΔGFR4 % were found between the groups. High levels of urinary proteins were found in 2.38-9.52 % of cases after 4 years of follow-up. ALB/Cr, IgG/Cr, and α1-MG/Cr levels were positively correlated with 99mTc-dimercaptosuccinic acid (DMSA) grade, and α1-MG excretion was inversely correlated with eGFR. CONCLUSIONS: The levels of microprotein were elevated at diagnosis in a higher proportion of patients than for the other markers examined. At long-term follow-up, the reflux level had decreased or completely resolved in all patients, and the proportions of microproteins that were elevated were significantly reduced. Renal impairment measured by eGFR and DMSA grade was related to increased urinary α1-MG levels.

Urinary albumin and transferrin as early diagnostic markers of chronic kidney disease.

Feline renal diseases are increasingly noted in veterinary practice. It is important to diagnose and identify the pathological basis of renal dysfunction accurately at an early stage, but there are only a few reports on this area in clinical veterinary medicine. We investigated the efficacy of measurement of urinary albumin (u-Alb) and urinary transferrin (u-Tf) for early diagnosis using 5-µl urine samples collected noninvasively by catheterization from normal (IRIS stage I) cats and cats with stage I chronic kidney disease (CKD). The u-Alb levels in normal and stage I CKD cats were 6.0 ± 4.5 and 11.2 ± 8.4 mg/dl, respectively, and the u-Tf levels were 0.09 ± 0.42 and 0.52 ± 0.79 mg/dl, respectively. Based on ROC curve analysis, the sensitivity and specificity of u-Alb and u-Tf were higher than those of the currently used biomarker, the plasma creatinine level. The sensitivity of u-Alb was higher than that of u-Tf, whereas the specificity of u-Tf was higher than that of u-Alb. The validity of the threshold albumin level (20 mg/dl) was confirmed by measurements using SDS-PAGE. Since leakage of u-Tf in urine precedes leakage of u-Alb, inclusion of u-Tf in biochemistry tests may be appropriate for IRIS staging as a diagnostic marker of early diagnosis of renal disorder in cats.