Urinary serpin-A3 is an early predictor of clinical response to therapy in patients with proliferative lupus nephritis.

We have previously reported that urinary excretion of serpin-A3 (uSerpA3) is significantly elevated in patients with active lupus nephritis (LN). Here, we evaluated the course of uSerpA3 during the first year of treatment and its association with response to therapy in patients with proliferative LN. The observational longitudinal study included 60 Mexican adults with proliferative LN followed during the first year after LN flare. uSerpA3 was detected by Western blot analysis at flare and after 3, 6, and 12 mo. The response to therapy was determined 1 yr after the LN flare. We evaluated the correlation between uSerpA3 and histological parameters at LN flare. The temporal association between uSerpA3 and response to therapy was analyzed with linear mixed models. uSerpA3 prognostic performance for response was evaluated with receiver-operating characteristic curves. Among the 60 patients studied, 21 patients (35%) were class III and 39 patients (65%) were class IV. uSerpA3 was higher in class IV than in class III LN (6.98 vs. 2.89 dots per in./mg creatinine, P = 0.01). Furthermore, uSerpA3 correlated with the histological activity index (r = 0.29, P = 0.02). There was a significant association between the temporal course of uSerpA3 and response to therapy. Responders showed a significant drop in uSerpA3 at 6 mo compared with LN flare (P < 0.001), whereas nonresponders persisted with elevated uSerpA3. Moreover, uSerpA3 was significantly lower at flare in responders compared with nonresponders (2.69 vs. 6.98 dots per in./mg creatinine, P < 0.05). Furthermore, uSerpA3 was able to identify nonresponders since 3 mo after LN flare (area under the curve: 0.77). In conclusion, uSerpA3 is an early indicator of kidney inflammation and predictor of the clinical response to therapy in patients with proliferative LN.NEW & NOTEWORTHY LN requires aggressive immunosuppression to improve long-term outcomes. Current indicators of remission take several months to normalize, prolonging treatment regiments in some cases. Serpin-A3 is present in urine of patients with proliferative LN. We evaluated the excretion of serpin-A3 in serial samples of patients with proliferative LN during the first year after flare. We found that uSerpA3 correlates with kidney inflammation and its decline at early points predicts the response to therapy 1 yr after flare.

Shotgun Proteomics of Isolated Urinary Extracellular Vesicles for Investigating Respiratory Impedance in Healthy Preschoolers.

Urine proteomic applications in children suggested their potential in discriminating between healthy subjects from those with respiratory diseases. The aim of the current study was to combine protein fractionation, by urinary extracellular vesicle isolation, and proteomics analysis in order to establish whether different patterns of respiratory impedance in healthy preschoolers can be characterized from a protein fingerprint. Twenty-one 3-5-yr-old healthy children, representative of 66 recruited subjects, were selected: 12 late preterm (LP) and 9 full-term (T) born. Children underwent measurement of respiratory impedance through Forced Oscillation Technique (FOT) and no significant differences between LP and T were found. Unbiased clustering, based on proteomic signatures, stratified three groups of children (A, B, C) with significantly different patterns of respiratory impedance, which was slightly worse in group A than in groups B and C. Six proteins (Tripeptidyl peptidase I (TPP1), Cubilin (CUBN), SerpinA4, SerpinF1, Thy-1 membrane glycoprotein (THY1) and Angiopoietin-related protein 2 (ANGPTL2)) were identified in order to type the membership of subjects to the three groups. The differential levels of the six proteins in groups A, B and C suggest that proteomic-based profiles of urinary fractionated exosomes could represent a link between respiratory impedance and underlying biological profiles in healthy preschool children.

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.

Vaspin in Serum and Urine of Post-Partum Women with Excessive Gestational Weight Gain.

Background and objectives: Data concerning vaspin in obstetric aspects are limited and conflicting. The aim of the study was to evaluate vaspin concentrations in the serum and urine of women with excessive gestational weight gain (EGWG) in the early post-partum period (i.e., 48 h after delivery), when placental function no longer influences the results. Materials and Methods: The study subjects were divided into two groups of 28 healthy controls and 38 mothers with EGWG. Maternal body composition and hydration status were evaluated by the bioelectrical impedance analysis (BIA) method. Concentrations of vaspin, fatty acid-binding protein 4 (FABP4), leptin, and ghrelin were determined via enzyme-linked immunosorbent assay (ELISA). Results: Serum vaspin levels were lower in the EGWG group, whereas no significant differences were noted between the groups, with regard to the urine vaspin concentrations. In both studied groups, the serum vaspin concentrations correlated positively with the urine FABP4 levels and negatively with gestational weight gain, body mass index gain in the period from pre-pregnancy to 48 h after delivery (ΔBMI), and fat tissue index (FTI). In the multiple linear regression models, the serum vaspin concentrations were positively dependent on the serum FABP4 levels, as well as negatively dependent on triglycerides, FTI, and ΔBMI. Conclusions: Our study revealed that the EGWG mothers were characterized by significantly lower serum vaspin concentrations in the early post-partum period compared with the subjects that had appropriate gestational weight gain. Our observation supports previous hypotheses that vaspin might be used as a marker of lipid metabolism in pregnancy and maternal adipose tissue. Considering the fact that FABP4 is widely referred to as a pro-inflammatory adipokine, further research on the protective role of vaspin seems crucial, especially in the context of its relationship to FABP4.

Discovery of SERPINA3 as a candidate urinary biomarker of lupus nephritis activity.

Objectives: We used an unbiased proteomics approach to identify candidate urine biomarkers (CUBMs) predictive of LN chronicity and pursued their validation in a larger cohort. Methods: In this cross-sectional pilot study, we selected urine collected at kidney biopsy from 20 children with varying levels of LN damage (discovery cohort) and performed proteomic analysis using isobaric tags for relative and absolute quantification (iTRAQ). We identified differentially excreted proteins based on degree of LN chronicity and sought to distinguish markers exhibiting different relative expression patterns using hierarchically clustered log10-normalized relative abundance data with linked and distinct functions by biological network analyses. For each CUBM, we performed specific ELISAs on urine from a validation cohort (n = 41) and analysis of variance to detect differences between LN chronicity, with LN activity adjustment. We evaluated for CUBM expression in LN biopsies with immunohistochemistry. Results: iTRAQ detected 112 proteins in urine from the discovery cohort, 51 quantifiable in all replicates. Simple analysis of variance revealed four differentially expressed, chronicity-correlated proteins (P-values < 0.05). Further correlation and network analyses led to selection of seven CUBMs for LN chronicity. In the validation cohort, none of the CUBMs distinguished LN chronicity degree; however, urine SERPINA3 demonstrated a moderate positive correlation with LN histological activity. Immunohistochemistry further demonstrated SERPINA3 staining in proximal tubular epithelial and endothelial cells. Conclusion: We identified SERPINA3, a known inhibitor of neutrophil cathepsin G and angiotensin II production, as a potential urine biomarker to help quantify LN activity.

Hormone-dependent changes in female urinary proteome.

Human urine represents a good source for proteomic research for clinically related studies as it can be collected and processed easily and can give information about kidney-related mechanisms. Little is known about the urinary proteomic changes resulting from physiological (normal), pathological, or environmental variations, and there are few reports on hormone-related modifications of urine proteome. In our study, we highlighted the variations of urinary proteins associated with menstrual cycle or estro-progestin pill in females. We also described an association between some urinary proteins and the renin-angiotensin-aldosterone system, which might help to improve the understanding of physiological and pathological processes when a gender-specific pattern such as the menopause-related hypertension or eclampsia is evident. We therefore support the usefulness of urinary proteomics as a valuable tool for clinically related study as it can provide information on candidate biomarkers which, in turn, need to be confirmed by multiple approaches before the use in a clinical setting.

Urinary protease inhibitor Serpin B3 is higher in women and is further increased in female patients affected by aldosterone producing adenoma.

A correct diagnosis of primary aldosteronism (PA) requires adrenal venous sampling (AVS) for the classification of subtypes (bilateral hyperplasia, BAH, or adenoma, APA). Since such testing is not easily practicable, appropriate markers for the definition of subtypes are desirable. We hypothesized that an aldosterone excess was associated with abnormalities in urinary proteome, specific for PA subtypes. The project work was divided into 3 phases: (1) screening/identification by proteomic analysis and further characterization by RT-PCR and immunohistochemistry of the candidate protein; (2) clinical validation by quantitative ELISA assay of 57 (33 M, 24 F) PA patients and 50 normotensive controls (21 M, 29 F); (3) analysis of adrenal tissue of 8 individuals who had undergone adrenalectomy for APA or other adrenal tumors. The proteomic analysis showed a different expression of Serpin B3 Inhibitor-SCCA1 (SB3) in APA and BAH patients. Urine SB3 concentrations in normotensive controls, quantified by ELISA assay and normalized by urinary creatinine, resulted much lower in males (6.72 ng SB3 per mg creatinine, C.I. 4.43-10.19) than in females (20.56 ng SB3 per mg creatinine, C.I. 12.43-33.99, p < 0.00001). SB3 concentrations were not significantly different in males affected by different PA subtypes (BAH, n = 19 and APA, n = 14) compared with normotensive subjects (n = 21). In contrast, in PA females, SB3 was significantly higher in APA (n = 13) than in BAH patients (n = 11) or in normotensive controls (n = 29) (P < 0.01 and <0.05, respectively). Neither messenger RNA nor SB3 protein were identified in tissue obtained from adrenal tumors and from the surrounding normal gland. In conclusion urine SB3 concentrations are physiologically much lower in males than in females. Hypertensive women, affected by APA, present urinary SB3 concentrations significantly higher than women affected by BAH.

Urine protein biomarkers for the diagnosis and prognosis of necrotizing enterocolitis in infants.

OBJECTIVES: To test the hypothesis that an exploratory proteomics analysis of urine proteins with subsequent development of validated urine biomarker panels would produce molecular classifiers for both the diagnosis and prognosis of infants with necrotizing enterocolitis (NEC). STUDY DESIGN: Urine samples were collected from 119 premature infants (85 NEC, 17 sepsis, 17 control) at the time of initial clinical concern for disease. The urine from 59 infants was used for candidate biomarker discovery by liquid chromatography/mass spectrometry. The remaining 60 samples were subject to enzyme-linked immunosorbent assay for quantitative biomarker validation. RESULTS: A panel of 7 biomarkers (alpha-2-macroglobulin-like protein 1, cluster of differentiation protein 14, cystatin 3, fibrinogen alpha chain, pigment epithelium-derived factor, retinol binding protein 4, and vasolin) was identified by liquid chromatography/mass spectrometry and subsequently validated by enzyme-linked immunosorbent assay. These proteins were consistently found to be either up- or down-regulated depending on the presence, absence, or severity of disease. Biomarker panel validation resulted in a receiver-operator characteristic area under the curve of 98.2% for NEC vs sepsis and an area under the curve of 98.4% for medical NEC vs surgical NEC. CONCLUSIONS: We identified 7 urine proteins capable of providing highly accurate diagnostic and prognostic information for infants with suspected NEC. This work represents a novel approach to improving the efficiency with which we diagnose early NEC and identify those at risk for developing severe, or surgical, disease.

Urinary pigment epithelium-derived factor as a marker of diabetic nephropathy.

BACKGROUND: Pigment epithelium-derived factor (PEDF), a serine protease inhibitor, regulates extracellular matrix production in the kidney. We sought the association between urinary PEDF (uPEDF) and development of nephropathy among patients with type 2 diabetes (T2DM). METHODS: Two human studies were performed in which uPEDF was determined by ELISA. These studies included (1) a cross-sectional study of T2DM (n = 228) and healthy controls (n = 46) [corrected] and (2) a longitudinal study of hypertensive T2DM with microalbuminuria (MA; n = 42) treated with irbesartan for 6 months. An animal study was performed in which PEDF was measured in the kidney and urine samples of control rats, rats rendered diabetic with streptozotocin that were also fed a high-fat diet, and diabetic rats treated with irbesartan for 3 months. RESULTS: Cross-sectional study: compared to controls, uPEDF was significantly higher in patients with diabetic nephropathy. uPEDF independently correlated with MA. In the MA group, uPEDF in patients with diabetic retinopathy was significantly higher than that in patients without diabetic retinopathy. Longitudinal study: irbesartan significantly decreased uPEDF in T2DM with MA. Animal study: in diabetic rats, increased PEDF was observed in both the urine and kidney samples. uPEDF showed a significant correlation with the expression of PEDF in the kidney. Irbesartan could significantly decrease the PEDF expression in the kidneys of diabetic rats as well as uPEDF. CONCLUSION: uPEDF may serve as a novel marker for screening for nephropathy among patients with T2DM and monitoring the response to therapy.

Establishment of a sandwich ELISA for human megsin, a kidney-specific serine protease inhibitor.

BACKGROUND: We previously identified a novel serine protease inhibitor (serpin), megsin, which is predominantly expressed in the kidney. Megsin expression is up-regulated in human and experimental renal diseases associated with mesangial proliferation and expansion, suggesting that urinary megsin may be a novel diagnostic marker for some renal diseases. METHODS: We established a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for megsin and measured urinary megsin of patients with various renal diseases. RESULTS: Megsin ELISA specifically detected megsin but not other serpins. The detection limit was 0.04 ng/ml, which allowed detection of urinary megsin in 3.6% of healthy individuals. The antigenic epitope in the urine detected by the ELISA was confirmed as megsin protein by time-of-flight mass spectrometry. Among patients with rapidly progressive glomerulonephritis (n = 18), 55.6% were urinary megsin-positive, while 24.1% in IgA nephropathy (n = 112) and 15.1% in chronic non-IgA glomerulonephritis (n = 245) were urinary megsin-positive, respectively. Among patients with chronic renal failure due to unknown causes (n = 74), 18.9% were positive for urinary megsin. In diabetic patients with or without nephropathy (n = 1073), 12.3% were urinary megsin-positive, while positivity of urinary megsin in patients with non-renal diseases (n = 768) was equivalent (3.3%) to that of healthy individuals. Of note, when urinary megsin-positive patients with diabetic nephropathy (n = 71) were classified into four stages by their proteinuria and estimated glomerular filtration rate, urinary megsin excretion increased as the stage progressed up to stage 3A, suggesting correlation of that with mesangial expansion level. Urinary megsin decreased in the advanced stage, probably reflecting development of glomerulosclerosis. CONCLUSION: We established a high-sensitive megsin ELISA, which detects urinary megsin in some patients with renal diseases and in only a few healthy subjects. Megsin ELISA may be a novel diagnostic tool for renal diseases.

Biodistribution and pharmacokinetics of 131I-labeled LEX 032, a recombinant hybrid of antichymotrypsin.

Pharmacokinetic and biodistribution studies were conducted in rats on a novel serine protease inhibitor, LEX 032, that was radiolabeled with 131I by the Bolton-Hunter reagent. LEX 032, a genetically engineered recombinant human nonglycosylated serpin, has been shown to have antiinflammatory properties in a number of animal models of inflammation and reperfusion injury. When 131I-LEX 032 was injected intravenously, a rapid whole body clearance of radioactivity was seen. Blood clearance followed a similar pattern. Forty-eight hours postinjection, 2.00 +/- 0.65 of the administered dose remained in the body. Greater than 59% of the radio-activity was excreted in the urine within the first 24 hr. Little radioactivity was found in the feces. With the exception of the thyroid, no significant organ-related uptake was noted. Radioactivity in the liver peaked at 20 min postinjection, with 1.00 +/- 0.13% administered dose/g and approximately 10% administered dose in the whole liver. At 1 hr, uptake in the kidney (9.30 +/- 1.52% administered dose/g) was the highest among all tissues, except for the thyroid. Gamma camera images were consistent with the biodistribution pattern. Pharmacokinetics and biodistribution were not affected by the dose of LEX 032 and were quite different from those of glycosylated wild type antichymotrypsin. These data indicate that LEX 032 exhibits the pharmacokinetics expected of a nonglycosylated 45 kDa protein.