Rationale and design of the Japanese Biomarkers in Nephrotic Syndrome (J-MARINE) study.

INTRODUCTION: Disease subtyping and monitoring are essential for the management of nephrotic syndrome (NS). Although various biomarkers for NS have been reported, their clinical efficacy has not been comprehensively validated in adult Japanese patients. METHODS: The Japanese Biomarkers in Nephrotic Syndrome (J-MARINE) study is a nationwide, multicenter, and prospective cohort study in Japan, enrolling adult (≥18 years) patients with minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), membranoproliferative glomerulonephritis (MPGN), C3 glomerulopathy (C3G), and lupus nephritis (LN). Baseline clinical information and plasma and urine samples will be collected at the time of immunosuppressive therapy initiation or biopsy. Follow-up data and plasma and urine samples will be collected longitudinally based on the designated protocols. Candidate biomarkers will be measured: CD80, cytotoxic T-lymphocyte antigen 4, and soluble urokinase plasminogen activator receptor for MCD and FSGS; anti-phospholipase A2 receptor and thrombospondin type-1 domain-containing protein 7A antibodies for MN; fragment Ba, C3a, factor I, and properdin for MPGN/C3G; and CD11b, CD16b, and CD163 for LN. Outcomes include complete and partial remission, relapse of proteinuria, a 30% reduction in estimated glomerular filtration rate (eGFR), eGFR decline, and initiation of renal replacement therapy. The diagnostic accuracy and predictive ability for clinical outcomes will be assessed for each biomarker. RESULTS: From April 2019 to April 2023, 365 patients were enrolled: 145, 21, 138, 10, and 51 cases of MCD, FSGS, MN, MPGN/C3G, and LN, respectively. CONCLUSION: This study will provide valuable insights into biomarkers for NS and serve as a biorepository for future studies.

Extracellular status of thrombospondin-2 in type 2 diabetes mellitus and utility as a biomarker in the determination of early diabetic kidney disease.

OBJECTIVE: Thrombospondin-2 (TSP-2) is a multifunctional matricellular glycoprotein correlated with glucose homeostasis, insulin sensitivity, and estimated glomerular filtration rate. Investigation of the association of TSP-2 with type 2 diabetes mellitus (T2DM) and the potential diagnostic value of serum TSP-2 for detecting early diabetic kidney disease (DKD) is needed. RESEARCH DESIGN AND METHODS: An enzyme-linked immunosorbent assay was used for detection serum TSP-2 levels in 494 Chinese T2DM subjects. The protein expression of TSP-2 in the kidney and other tissues were tested by western blotting. RESULTS: Serum TSP-2 levels in T2DM subjects were significantly higher than in healthy individuals. Serum TSP-2 correlated positively with triglycerides, serum uric acid, creatinine, platelets, and urinary albumin-to-creatinine ratio (UACR), but negatively with estimated glomerular filtration rate, after adjusting for age, sex, and T2DM duration. Logistic regression analysis demonstrated an independent association between serum TSP-2 and early DKD. Furthermore, the high UACR identified at risk of early DKD increased significantly from 0.78 (95%CI 0.73-0.83) to 0.82 (95%CI 0.77-0.86, p < 0.001) when added to a clinical model consisting of TSP-2 and age. In db/db mice, serum TSP-2 levels were elevated. TSP-2 expression was markedly increased in the kidney tissue compared with that in db/m and m/m mice. Furthermore, serum TSP-2 expression correlated well with UACR in mice. CONCLUSIONS: TSP-2 is a novel glycoprotein associated with early DKD in patients with T2DM. The paradoxical increase of serum TSP-2 in T2DM individuals may be due to a compensatory response to chronic inflammatory and renal vascular endothelial growth, warranting further investigation.

Serum levels of the novel adipokine isthmin-1 are associated with obesity in pubertal boys.

OBJECTIVES: To evaluate whether there is an association between the serum levels of the novel insulin-like adipokine isthmin-1 (ISM1) and obesity-related phenotypes in a population of Spanish children and to investigate the plausible molecular alterations behind the alteration of the serum levels of this protein in children with obesity. METHODS: The study population is a sub-cohort of the PUBMEP research project, consisting of a cross-sectional population of 119 pubertal children with overweight (17 boys, 19 girls), obesity (20 boys, 25 girls), and normal weight (17 boys, 21 girls). All subjects were classified into experimental groups according to their sex, obesity, and insulin resistance (IR) status. They were counted anthropometry, glucose and lipid metabolism, inflammation and cardiovascular biomarkers as well as isthmin-1 (ISM1) serum levels. This population was intended as a discovery population to elucidate the relationship between obesity and ISM1 levels in children. Furthermore, the study population had blood whole-genome DNA methylation examined, allowing deepening into the obesity-ISM1 molecular relationship. RESULTS: Higher serum ISM1 levels were observed in boys with obesity than in normal weight (P = 0.004) and overweight (P = 0.007) boys. ISM1 serum levels were positively associated with body mass index (BMI) Z-score (P = 0.005) and fat mass (P = 0.058) and negatively associated with myeloperoxidase (MPO) (P = 0.043) in boys. Although we did not find associations between ISM1 serum levels and metabolic outcomes in girls, which may indicate a putative sexual dimorphism, fat mass was positively associated in all children, including boys and girls (P = 0.011). DNA methylation levels in two-enhancer-related CpG sites of ISM1 (cg03304641 and cg14269097) were associated with serum levels of ISM1 in children. CONCLUSIONS: ISM1 is associated with obesity in boys at the pubertal stage, elucidating how this protein might be of special relevance as a new biomarker of obesity in children. Further studies including a longitudinal design during puberty are needed.

Serum isthmin-1 levels are positively and independently correlated with albuminuria in patients with type 2 diabetes mellitus.

INTRODUCTION: Isthmin-1 (Ism-1), as a novel adipokine, plays a role in glucose homeostasis and lipid metabolism. However, the relationship between Ism-1 and type 2 diabetes mellitus (T2DM) remains unclear. This study aims to investigate the association of serum Ism-1 levels with albuminuria and insulin resistance in patients with T2DM and preserved renal function. RESEARCH DESIGN AND METHODS: A total of 150 patients with T2DM were recruited. The presence of albuminuria was evaluated by urinary albumin:creatinine ratio (UACR) in first morning urine sample. Serum Ism-1 levels were tested by ELISA. Homeostasis model assessments were used to evaluate insulin resistance. Binary logistic regression and multivariable linear regression analyses were used to assess the association of serum Ism-1 levels with albuminuria. Multivariable linear regression analyses were performed to explore the correlation of serum Ism-1 levels with insulin resistance. RESULTS: Compared with the normal-albuminuria and microalbuminuria groups, serum Ism-1 levels were significantly higher in the macroalbuminuria group (p<0.01). Binary logistic regression analyses showed that serum Ism-1 was positively associated with odds of albuminuria even after multiple adjustments (OR=4.766, p=0.013). Serum Ism-1 was positively associated with log10-transformed UACR (ß=0.625, p<0.001). However, the associations between serum Ism-1 levels and insulin resistance were not observed in patients with T2DM. CONCLUSIONS: Serum Ism-1 levels were positively and independently correlated with the severity of albuminuria in patients with T2DM but not with insulin resistance.

Serum Thrombospondin-2 Levels Are Closely Associated With the Severity of Metabolic Syndrome and Metabolic Associated Fatty Liver Disease.

CONTEXT: Metabolic associated fatty liver disease (MAFLD) is the hepatic manifestation of obesity-related metabolic syndrome (MetS). Noninvasive biomarkers for monitoring the progression and severity of these metabolic comorbidities are needed. OBJECTIVES: To investigate the associations of serum thrombospondin-2 (TSP2) with MetS and MAFLD severity, and the potential diagnostic value of serum TSP2 for identifying at-risk metabolic associated steatohepatitis (MASH). METHODS: Blood samples, clinical data, and liver biopsies were collected from consecutively recruited 252 individuals with morbid obesity receiving bariatric surgery. Histopathology samples of liver biopsies were examined in a blinded fashion by 3 independent pathologists. Serum TSP2 levels were measured by enzyme-linked immunosorbent assay. RESULTS: Serum TSP2 levels were significantly elevated in MetS (1.58 [1.07-2.20] ng/mL) compared with non-MetS (1.28 [0.84-1.73] ng/mL; P = .006) in obese patients and positively correlated with increasing number of the MetS components, fasting glucose, glycated hemoglobin, fasting insulin, C-peptide, and homeostatic model assessment of insulin resistance after adjustment of conventional confounders. Serum TSP2 levels differentiated MASH (1.74 [1.32-3.09] ng/mL) from the other non-MASH less severe groups: normal liver (1.41 [1.04-1.63] ng/mL), simple steatosis (1.45 [0.89-1.92] ng/mL), and borderline MASH (1.30 [0.99-2.17] ng/mL) (P < .05). Elevated serum TSP2 was positively associated with the severity of hepatic steatosis, inflammation, fibrosis, and abnormal liver function independent of age, sex and adiposity. Furthermore, high serum TSP2 identified at-risk MASH with area under the operating curve of 0.84 (95% CI 0.70-0.98). CONCLUSION: Serum TSP2 is closely associated with severity and progression of MetS and MAFLD, and is a promising noninvasive biomarker for differentiating MASH from benign steatosis and identifying at-risk MASH patients among individuals with obesity.

Thrombospondin-2 as a diagnostic biomarker for distal cholangiocarcinoma and pancreatic ductal adenocarcinoma.

PURPOSE: Distal cholangiocarcinoma and pancreatic ductal adenocarcinoma are malignancies with poor prognoses that can be difficult to distinguish preoperatively. Thrombospondin-2 has been proposed as a novel diagnostic biomarker for early pancreatic ductal adenocarcinoma. The aim of the present study was to evaluate thrombospondin-2 as a diagnostic and prognostic biomarker in combination with current biomarker CA 19-9 for distal cholangiocarcinoma and pancreatic ductal adenocarcinoma. METHODS: Thrombospondin-2 was measured in prospectively collected serum samples from patients who underwent surgery with a histopathological diagnosis of distal cholangiocarcinoma (N = 51), pancreatic ductal adenocarcinoma (N = 52) and benign pancreatic diseases (N = 27) as well as healthy blood donors (N = 52) using an enzyme-linked immunosorbent assay. RESULTS: Thrombospondin-2 levels (ng/ml) were similar in distal cholangiocarcinoma 55 (41-77) and pancreatic ductal adenocarcinoma 48 (35-80) (P = 0.221). Thrombospondin-2 + CA 19-9 had an area under the curve of 0.92 (95% CI 0.88-0.97) in differentiating distal cholangiocarcinoma and pancreatic ductal adenocarcinoma from healthy donors which was superior to CA 19-9 alone (P < 0.001). The diagnostic value of adding thrombospondin-2 to CA 19-9 was larger in early disease stages. Thrombospondin-2 did not provide additional value to CA 19-9 in differentiating the benign disease group; however, heterogeneity was notable in the benign cohort. Three of five patients with autoimmune pancreatitis patients had greatly elevated thrombospondin-2 levels. Thrombospondin-2 levels had no correlation with prognoses. CONCLUSIONS: Serum thrombospondin-2 in combination with CA 19-9 has potential as a biomarker for distal cholangiocarcinoma and pancreatic cancer.

Wnt/ß-catenin pathway proteins in end-stage renal disease.

Aim: To delineate the association of end-stage renal disease (ESRD) and Wnt-proteins including the agonist R-spondin-1, the transducer ß-catenin and the antagonists DKK1 and sclerostin. Materials & methods: Serum Wnt-pathway proteins levels were measured by ELISA in 60 ESRD patients and 30 normal controls. Results: DKK1 and sclerostin were significantly higher in ESRD than in controls, and ß-catenin and the catenin + R-spondin-1/DKK1 + sclerostin ratio, reflecting the ratio of agonist and transducer on antagonists (AT/ANTA), were significantly lower in ESRD. Estimated glomerular filtration rate was significantly associated with DKK1 and sclerostin (inversely), ß-catenin (positively) and the AT/ANTA ratio (r = 0.468, p < 0.001). Conclusion: Wnt/ß-catenin pathway proteins show significant alterations in ESRD, indicating significantly increased levels of antagonists.

Characterization of THSD7A-antibodies not binding to glomerular THSD7A in a patient with diabetes mellitus but no membranous nephropathy.

Membranous nephropathy (MN) is an autoimmune disease caused by autoantibodies against the podocyte antigens phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type 1 domain containing protein 7A (THSD7A) in 80% and 2-3% of patients, respectively. THSD7A antibodies are considered to be pathogenic and highly specific for MN patients. Using an indirect immunofluorescence test (IIFT) we detected THSD7A-antibodies (titre 1:10) in the serum of a patient with high proteinuria who, however, in the kidney biopsy was diagnosed with diabetic nephropathy and MN was excluded as a possible cause of proteinuria. Different immunofluorescence assays and Western blot techniques using recombinant THSD7A (rTHSD7A) or THSD7A from different human tissues revealed that the circulating THSD7A-autoantibodies were only of the IgG3 subclass. The patient serum reacted exclusively with rTHSD7A and only when the antigen was present in reducing Western blot conditions, or on formaldehyde-fixed cells for the IIFT. Our findings show for the first time the existence of circulating THSD7A-antibodies recognizing denatured/reduced rTHSD7A, which do not react with glomerular THSD7A in vivo and are thus presumptively non-pathogenic. As a consequence, kidney biopsy or Western blot analyses of THSD7A under non-reducing conditions should be performed to confirm the diagnosis of THSD7A-associated MN, especially in cases with low THSD7A-antibody levels in the IIFT.

Transcriptomics Identify Thrombospondin-2 as a Biomarker for NASH and Advanced Liver Fibrosis.

BACKGROUND AND AIMS: NAFLD is the most common liver disease worldwide. NASH, the progressive form of NAFLD, and advanced fibrosis are associated with poor outcomes. We searched for their noninvasive biomarkers. APPROACH AND RESULTS: Global RNA sequencing of liver tissue from 98 patients with biopsy-proven NAFLD was performed. Unsupervised hierarchical clustering well distinguished NASH from nonalcoholic fatty liver (NAFL), and patients with NASH exhibited molecular abnormalities reflecting their pathological features. Transcriptomic analysis identified proteins up-regulated in NASH and/or advanced fibrosis (stage F3-F4), including matricellular glycoprotein thrombospondin-2 (TSP-2), encoded by the thrombospondin 2 (THBS2) gene. The intrahepatic THBS2 expression level showed the highest areas under the receiver operating characteristic curves (AUROCs) of 0.915 and 0.957 for diagnosing NASH and advanced fibrosis, respectively. THBS2 positively correlated with inflammation and ballooning according to NAFLD activity score, serum aspartate aminotransferase and hyaluronic acid (HA) levels, and NAFLD Fibrosis Score (NFS). THBS2 was associated with extracellular matrix and collagen biosynthesis, platelet activation, caspase-mediated cleavage of cytoskeletal proteins, and immune cell infiltration. Serum TSP-2 expression was measured in 213 patients with biopsy-proven NAFLD, was significantly higher in NASH than in NAFL, and increased parallel to fibrosis stage. The AUROCs for predicting NASH and advanced fibrosis were 0.776 and 0.856, respectively, which were comparable to Fibrosis-4 index, serum HA level, and NFS in advanced fibrosis diagnosis. Serum TSP-2 level and platelet count were independent predictors of NASH and advanced fibrosis. Serum TSP-2 levels could stratify patients with NAFLD according to the risk of hepatic complications, including liver cancer and decompensated cirrhotic events. CONCLUSIONS: TSP-2 may be a useful biomarker for NASH and advanced fibrosis diagnosis in patients with NAFLD.

Distribution of serum Thrombospondin-2, a novel tumor marker, in general population and cancer patients in China.

PURPOSE: Distribution of serum thrombospondin-2 in general population and cancer patients in China have not been reported. METHODS: This study evaluated the expression level of serum thrombospondin-2 in general population and various cancer patients, the 95% confidence interval was used for the derivation of reference range. The comparison of the expression levels in controls for age and gender was performed. The associations between candidate biomarkers (thrombospondin-2 [THBS2]) expression and tumor metastasis status were also explored. RESULTS: 125 healthy controls and 193 various cancer patients were enrolled. The mean ± SD in serum THBS2 levels in general population was 42.37 ± 12.24 ng/ml, there was no significant sex and age difference, the reference range is 18.37-66.36 ng/ml. Most cancer patients present a decreased serum THBS2 level except hepatoma and lymphoma which most patients showed a relatively high level of THBS2. There was no statistical difference of serum THBS2 level between metastasis and non-metastasis group in breast, lung, cervical, colorectal cancer, nasopharyngeal carcinoma and hepatoma (P > 0.05) while a significant negative correlation was observed in ovarian cancer (P = 0.0209). CONCLUSIONS: The distribution of serum THBS2 displayed an obvious heterogeneity among various cancers comparing to health controls, ovarian cancer patients detected with low THBS2 expression may be more prone to develop metastasis in China.

Circulating Levels of Thrombospondin-1 and Thrombospondin-2 in Patients with Common Brain Tumors.

AIM: To measure serum levels of thrombospondin-1 (TSP-1) and thrombospondin-2 (TSP-2) in patients with common brain tumors, namely high-grade glioma (HGG), low-grade glioma (LGG), and meningioma. MATERIAL AND METHODS: For this prospective study, a total of 56 patients were operated on for supratentorial gliomas and meningiomas, and 18 healthy subjects were evaluated. Serum levels of angiostatic molecules were measured with enzyme-linked immunosorbent assay. The results of patients were compared with those of healthy subjects. RESULTS: High serum levels of TSP-1 were seen in HGG, followed by LGG, meningioma groups, and controls. The only significant difference was found between HGGs and controls (p=0.004). There was a trend to decrease from HGG to controls. High serum levels of TSP-2 were seen in controls, followed by meningioma, LGG, and HGG. None of the patient groups showed significant differences compared with controls. Among the patient groups, TSP-2 was significantly higher in the meningioma group than the HGG group (p=0.01). No correlation was found with any of the molecules and the clinical parameters, including the presence of peritumoral edema or seizure, the anterior-posterior diameter of the tumor, and, more importantly, the grade of glioma. CONCLUSION: Our results indicate that TSP-2 might be more important than TSP-1 in preventing angiogenesis and a major angiostatic factor in glioma cells.

Serum thrombospondin 2 is a novel predictor for the severity in the patients with NAFLD.

AIM: Thrombospondins are a family of multidomain and secretory glycoproteins. Among them, thrombospondin 2 (TSP2) encoded by TSP2 gene has been reported to be involved in various functions such as collagen/fibrin formation, maintenance of normal blood vessel density and cell adhesion properties. Microarray analyses ranked TSP2 as one of the most highly up-regulated genes in the fibrotic liver in patients with non-alcoholic fatty liver disease (NAFLD). Since TSP2 possesses unique properties as a secretory protein, we hypothesized that hepatic TSP2 gene expression levels would be reflected in serum TSP2 levels. In this study, we examined the relationship between serum TSP2 concentrations and clinicopathological findings in NAFLD patients. METHODS: One hundred and thirty NAFLD patients who had undergone liver biopsy between 2009 and 2015 were retrospectively enrolled. Serum samples were collected at the time of biopsy, and TSP2 was measured by enzyme immunoassays. RESULTS: Serum TSP2 levels moderately correlated with ballooning (r = 0.56, P < .001) and fibrosis stage (r = 0.53, P < .001). The AUC values of TSP2 for predicting mild fibrosis (≧F1), moderate fibrosis (≧F2) and severe fibrosis (≧F3) were 0.73, 0.76 and 0.82 respectively. Additionally, NAFLD activity score (NAS) correlated best with TSP2 (r = 0.52, P < .001) compared to conventional NAFLD-related biomarkers, such as cytokeratin 18 M30, hyaluronic acid, type IV collagen 7S, APRI and FIB-4 index. CONCLUSION: Serum TSP2 levels reflected hepatocyte ballooning, fibrosis and NAS in NAFLD patients. For clinical application of serum TSP2 as a predictor of NAFLD histological activity, additional validation and mechanistic investigations are required.

Circulating Levels of Thrombospondin-1 and Thrombospondin-2 in Patients with Temporal Lobe Epilepsy Before and After Surgery.

AIM: To measure the serum levels of strong angiostatic and synaptogenetic molecules thrombospondin-1 (TSP-1) and thrombospondin-2 (TSP-2) in patients with temporal lobe epilepsy (TLE) before and after surgery. MATERIAL AND METHODS: In this prospective study, 20 patients operated for TLE and 20 healthy subjects were included. Serum levels of TSP-1 and TSP-2 were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: Our findings showed that both groups had higher serum levels of both molecules "before" surgery than 10 days ?after? SURGERY: However, a significant difference was noted between ?before? and "after" surgery regarding TSP-1 (p=0.00001). Although a marked decrease was found "after" surgery with respect to TSP-2, the difference did not reach statistical significance (p=0.22). In patients with TLE, serum levels of both molecules ?before? surgery were found to be significantly higher than in healthy controls (TSP-1, p=0.00001; TSP-2, p=0.007). CONCLUSION: Serum levels of TSP-1 and TSP-2 are determined to be higher in patients with TLE than in healthy subjects, and the resection of epileptogenic tissues decreases the serum levels of these molecules. Future studies should involve a higher number of patients with serial serum levels of TSP-1 and TSP-2 at the long-term follow-up to correlate with seizure outcome.

THBS2/CA19-9 Detecting Pancreatic Ductal Adenocarcinoma at Diagnosis Underperforms in Prediagnostic Detection: Implications for Biomarker Advancement.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed too late for effective therapy. The classic strategy for early detection biomarker advancement consists of initial retrospective phases of discovery and validation with tissue samples taken from individuals diagnosed with disease, compared with controls. Using this approach, we previously reported the discovery of a blood biomarker panel consisting of thrombospondin-2 (THBS2) and CA19-9 that together could discriminate resectable stage I and IIa PDAC as well as stages III and IV PDAC, with c-statistic values in the range of 0.96 to 0.97 in two phase II studies. We now report that in two studies of blood samples prospectively collected from 1 to 15 years prior to a PDAC diagnosis (Mayo Clinic and PLCO cohorts), THBS2 and/or CA19-9 failed to discriminate cases from healthy controls at the AUC = 0.8 needed. We conclude that PDAC progression may be heterogeneous and for some individuals can be more rapid than generally appreciated. It is important that PDAC early-detection studies incorporate high-risk, prospective prediagnostic cohorts into discovery and validation studies.Prevention Relevance: A blood biomarker panel of THBS2 and CA19-9 detects early stages of pancreatic ductal adenocarcinoma at diagnosis, but not when tested across a population up to 1 year earlier. Our findings suggest serial sampling over time, using prospectively collected samples for biomarker discovery, and more frequent screening of high-risk individuals.

Plasma proteomic signature of the risk of developing mobility disability: A 9-year follow-up.

INTRODUCTION: Mobility disability is a powerful indicator of poor health in older adults. The biological and pathophysiological mechanism underlying the development of mobility disability remains unknown. This study conducted a data-driven discovery phase investigation to identify plasma proteins that predict the incidence of mobility disability in community-dwelling older adults without mobility disability at baseline. METHODS: We investigated 660 women and men, aged 71.9 ± 6.0 (60-94) years, who participated in the Invecchiare in Chianti, "Aging in the Chianti Area" study and completed the 400-m walk at fast pace (400-m walk) at enrollment. Median follow-up time was 8.57 [interquartile, 3.20-9.08] years. SOMAscan technology was used to measure 1,301 plasma proteins at enrollment. The incident of mobility disability was defined as inability to complete the 400-m walk. Protein-specific Cox proportional hazard model was adjusted for sex, age, and other important covariates. RESULTS: Plasma levels of 75 proteins predicted mobility disability (p < .05). Significant proteins were enriched for the KEGG "PI3K-Akt signaling," "phagosomes," and "cytokine-cytokine receptor interaction" pathways. After multiple comparison adjustment, plasma cathepsin S (CTSS; hazard ratio [HR] 1.33, 95% CI: 1.17, 1.51, q = 0.007), growth/differentiation factor 15 (GDF15; HR: 1.45, 95% CI: 1.23, 1.72, q = 0.007), and thrombospondin-2 (THBS2; HR: 1.44, 95% CI: 1.22, 1.69, q = 0.007) remained significantly associated with high risk of losing mobility. CONCLUSION: CTSS, GDF15, and THBS2 are novel blood biomarkers associated with new mobility disability in community-dwelling individuals. Overall, our analysis suggests that cellular senescence and inflammation should be targeted for prevention of mobility disability.

Role of Serum and Urine Biomarkers (PLA2R and THSD7A) in Diagnosis, Monitoring and Prognostication of Primary Membranous Glomerulonephritis.

Differentiating primary and secondary membranous glomerulonephritis (MGN) using biomarkers for MGN is essential in patients' diagnosis, treatment and follow-up. Although biopsy has been the primary tool in making the diagnosis, not all patients can withstand it due to its invasive nature, and it cannot be used to monitor treatment. Hence, there is the need for less invasive or even non-invasive biomarkers for effective diagnosis, treatment monitoring and prognostication. This study aimed at providing an alternative way of differentiating primary and secondary MGN using enzyme-linked immunosorbent assay (ELISA) technique for serum and urine biomarkers (M-type phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A)) for prompt diagnosis, treatment and prognosis. A total of 125 subjects, including 81 primary and 44 secondary MGN subjects, were diagnosed from January 2012 to October 2019 at Hospital Serdang and Hospital Kuala Lumpur from which 69 subjects consisting of 45 primary and 24 secondary MGN subjects participated in the study. Of these, 13 primary MGN subjects were positive for both serum and urine anti-PLA2R antibodies (Ab) whereas only one secondary MGN subject associated with hepatitis B virus was positive for both serum and urine anti-PLA2R Ab. At the same time, anti-THSD7A Ab was found positive in four primary MGN subjects and two secondary MGN subjects with malignancy.

Combined Plasmatic and Tissue Approach to Membranous Nephropathy-Proposal of a Diagnostic Algorithm Including Immunogold Labelling: Changing the Paradigm of a Serum-based Approach.

Membranous nephropathy represents the most frequent cause of nephrotic syndrome in the adult, leading to end-stage renal disease in one third of all the patients. In the last years, the discovery of circulating autoantibodies against phospholipase A2 receptor 1 (PLA2R) and thrombospondin type-1 containing 7A domain (THSD7A), shed light on the pathogenesis of idiopathic forms, being responsible for 70% and 3% of all the cases, respectively. These identifications allowed the development of serological and histologic tests to detect autoantibodies and relative targets for diagnostic and prognostic purposes. Rising evidences suggest that serum titer correlates with disease activity and response to therapy. For these reasons, for patients with nephrotic syndrome, a serum-based approach has been proposed, reserving renal biopsy only in cases with doubtful/negative serology. However, the recent introduction of useful criteria for the interpretation of PLA2R/THSD7A immunohistochemistry could lead to high values of sensitivity and specificity for the in situ detection of target antigens. The present multicentric study on a series of membranous nephropathy cases with available serum/histologic correlation will show the importance of the crosstalk among the different techniques, recovering the possible role of electron microscopy in challenging situations.

Thrombospondin-4 increases with the severity of peripheral arterial disease and is associated with diabetes.

Thrombospondin-4 (TSP-4) is an extracellular matrix protein of the vessel wall. Despite bench evidence, its significance in the clinical setting of atherosclerosis is missing. TSP-4 (ng/ml) was measured in 365 PAD patientsusing a commercially available ELISA. PAD was diagnosed by the ankle-brachial index (ABI) and clinically graded using the Fontaine classification. TSP-4 levels were significantly higher in Fontaine II vs. Fontaine I (4.78 ± 0. 42, 4.69 ± 0.42, p = 0.043). TSP-4 significantly correlated with ABI (r = - 0.141, p = 0.023, n = 259) after the exclusion of mediasclerotic patients. Binary logistic regression analysis for Fontaine I vs. II showed an OR of 1.70 (1.02-2.82) in a multivariable model adjusted for traditional risk factors. Interestingly, TSP-4 levels were higher in patients with type 2 diabetes mellitus or prediabetes (DGT) compared with normal glucose tolerance (NGT) (4.76 ± 0.42 vs. 4.66 ± 0.41, p = 0.035). ANOVA for PAD and diabetes subgroups showed a linear increase with disease burden with the highest difference between Fontaine I-NGT and Fontaine II-DGT (4.59 ± 0.40, 4.79 ± 0.43, p = 0.015). TSP-4 levels increased with PAD severity and showed a former unknown association with diabetes. Thus, TSP-4 could be a novel marker of atherosclerotic activity, especially in the major subgroup of patients with concomitant diabetes.

Identification and Validation of Stage-Associated Serum Biomarkers in Colorectal Cancer Using MS-Based Procedures.

PURPOSE: Successful prevention of colorectal cancer (CRC) would benefit from a rapid serum screening for early detection. Here, a novel strategy for CRC biomarker discovery and validation exclusively based on MS procedures is reported. EXPERIMENTAL DESIGN: Identification of CRC serum biomarkers is initially made using label-free quantification on pooled serum samples from different CRC stages followed by two consecutive steps of targeted parallel reaction monitoring assays in different serum cohorts. Relevance of different protein depletion and peptide fractionation extent is investigated. Absolute quantification of a selected peptide is performed as a proof-of-concept. RESULTS: A total of 945 proteins showed differential abundance in the discovery phase. Based on their statistical significance and relative expression in disease stages, 123 potential biomarkers are selected for a training step. In the final validation step, five peptides belonging to four proteins are consistently quantified in individual CRC serum samples and controls. Different statistical analyses indicate that peptides GWVTDGFSSLK (APOC3) and LCNNPTPQFGGK (THBS1) are candidate biomarkers. Absolute quantification of LCNNPTPQFGGK shows statistical significance for the diagnosis of early respect to late CRC stages. CONCLUSIONS AND CLINICAL RELEVANCE: Two peptides from APOC3 and THBS1 are validated by PRM as potential biomarkers for non-invasive diagnosis of colorectal cancer.

Elevated Thrombospondin 2 Contributes to Delayed Wound Healing in Diabetes.

Impaired wound healing is a major complication of diabetes, and despite the associated risks, treatment strategies for diabetic wounds remain limited. This is due, in part, to an incomplete understanding of the underlying pathological mechanisms, including the effects of hyperglycemia on components of the extracellular matrix (ECM). In the current study, we explored whether the expression of thrombospondin 2 (TSP2), a matricellular protein with a demonstrated role in response to injury, was associated with delayed healing in diabetes. First, we found that TSP2 expression was elevated in diabetic mice and skin from patients with diabetes. Then, to determine the contribution of TSP2 to impaired healing in diabetes, we developed a novel diabetic TSP2-deficient model. Though the TSP2-deficient mice developed obesity and hyperglycemia comparable with diabetic control mice, they exhibited significantly improved healing, characterized by accelerated reepithelialization and increased granulation tissue formation, fibroblast migration, and blood vessel maturation. We further found that hyperglycemia increased TSP2 expression in fibroblasts, the major cellular source of TSP2 in wounds. Mechanistically, high glucose increased activation of the hexosamine pathway and nuclear factor-κB signaling to elevate TSP2 expression. Our studies demonstrate that hyperglycemia-induced TSP2 expression contributes to impaired healing in diabetes.