Differential Urinary Proteome Analysis for Predicting Prognosis in Type 2 Diabetes Patients with and without Renal Dysfunction.

Renal dysfunction, a major complication of type 2 diabetes, can be predicted from estimated glomerular filtration rate (eGFR) and protein markers such as albumin concentration. Urinary protein biomarkers may be used to monitor or predict patient status. Urine samples were selected from patients enrolled in the retrospective diabetic kidney disease (DKD) study, including 35 with good and 19 with poor prognosis. After removal of albumin and immunoglobulin, the remaining proteins were reduced, alkylated, digested, and analyzed qualitatively and quantitatively with a nano LC-MS platform. Each protein was identified, and its concentration normalized to that of creatinine. A prognostic model of DKD was formulated based on the adjusted quantities of each protein in the two groups. Of 1296 proteins identified in the 54 urine samples, 66 were differentially abundant in the two groups (area under the curve (AUC): p-value < 0.05), but none showed significantly better performance than albumin. To improve the predictive power by multivariate analysis, five proteins (ACP2, CTSA, GM2A, MUC1, and SPARCL1) were selected as significant by an AUC-based random forest method. The application of two classifiers-support vector machine and random forest-showed that the multivariate model performed better than univariate analysis of mucin-1 (AUC: 0.935 vs. 0.791) and albumin (AUC: 1.0 vs. 0.722). The urinary proteome can reflect kidney function directly and can predict the prognosis of patients with chronic kidney dysfunction. Classification based on five urinary proteins may better predict the prognosis of DKD patients than urinary albumin concentration or eGFR.

Urinary proteomics links keratan sulfate degradation and lysosomal enzymes to early type 1 diabetes.

Diabetes is the leading cause of end-stage renal disease worldwide. Our understanding of the early kidney response to chronic hyperglycemia remains incomplete. To address this, we first investigated the urinary proteomes of otherwise healthy youths with and without type 1 diabetes and subsequently examined the enriched pathways that might be dysregulated in early disease using systems biology approaches. This cross-sectional study included two separate cohorts for the discovery (N = 30) and internal validation (N = 30) of differentially excreted proteins. Discovery proteomics was performed on a Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer. We then searched the pathDIP, KEGG, and Reactome databases to identify enriched pathways in early diabetes; the Integrated Interactions Database to retrieve protein-protein interaction data; and the PubMed database to compare fold changes of our signature proteins with those published in similarly designed studies. Proteins were selected for internal validation based on pathway enrichment and availability of commercial enzyme-linked immunosorbent assay kits. Of the 2451 proteins identified, 576 were quantified in all samples from the discovery cohort; 34 comprised the urinary signature for early diabetes after Benjamini-Hochberg adjustment (Q < 0.05). The top pathways associated with this signature included lysosome, glycosaminoglycan degradation, and innate immune system (Q < 0.01). Notably, all enzymes involved in keratan sulfate degradation were significantly elevated in urines from youths with diabetes (|fold change| > 1.6). Increased urinary excretion of monocyte differentiation antigen CD14, hexosaminidase A, and lumican was also observed in the validation cohort (P < 0.05). Twenty-one proteins from our signature have been reported elsewhere as potential mediators of early diabetes. In this study, we identified a urinary proteomic signature for early type 1 diabetes, of which lysosomal enzymes were major constituents. Our findings highlight novel pathways such as keratan sulfate degradation in the early kidney response to hyperglycemia.

TGFBI Protein Is Increased in the Urine of Patients with High-Grade Urothelial Carcinomas, and Promotes Cell Proliferation and Migration.

Here, we discovered TGFBI as a new urinary biomarker for muscle invasive and high-grade urothelial carcinoma (UC). After biomarker identification using antibody arrays, results were verified in urine samples from a study population consisting of 303 patients with UC, and 128 urological and 58 population controls. The analyses of possible modifying factors (age, sex, smoking status, urinary leukocytes and erythrocytes, and history of UC) were calculated by multiple logistic regression. Additionally, we performed knockdown experiments with TGFBI siRNA in bladder cancer cells and investigated the effects on proliferation and migration by wound closure assays and BrdU cell cycle analysis. TGFBI concentrations in urine are generally increased in patients with UC when compared to urological and population controls (1321.0 versus 701.3 and 475.6 pg/mg creatinine, respectively). However, significantly increased TGFBI was predominantly found in muscle invasive (14,411.7 pg/mg creatinine), high-grade (8190.7 pg/mg) and de novo UC (1856.7 pg/mg; all p < 0.0001). Knockdown experiments in vitro led to a significant decline of cell proliferation and migration. In summary, our results suggest a critical role of TGFBI in UC tumorigenesis and particularly in high-risk UC patients with poor prognosis and an elevated risk of progression on the molecular level.

Prediction of pneumoconiosis by serum and urinary biomarkers in workers exposed to asbestos-contaminated minerals.

Workers processing nephrite, antigorite, or talc may be exposed to paragenetic asbestos minerals. An effective screening method for pneumoconiosis in workers exposed to asbestos-contaminated minerals is still lacking. The objective of this study was to assess the diagnostic accuracy of serum and urinary biomarkers for pneumoconiosis in workers exposed to asbestos-contaminated minerals. We conducted a case-control study in a cohort of stone craft workers in Hualien, where asbestos, nephrite, antigorite, and talc are produced. A total of 140 subjects were screened between March 2013 and July 2014. All subjects received a questionnaire survey and a health examination that included a physical examination; chest X-ray; and tests for standard pulmonary function, fractional exhaled nitric oxide, serum soluble mesothelin-related peptide (SMRP), fibulin-3, carcinoembryonic antigen (CEA), and urinary 8-Oxo-2'-deoxyguanosine (8-OHdG)/creatinine. After excluding subjects with uraemia and chronic obstructive pulmonary disease (COPD), we included 48 subjects with pneumoconiosis and 90 control subjects without pneumoconiosis for analysis. In terms of occupational history, 43/48 (90%) case subjects and 68% (61/90) of the control subjects had processed asbestos-contaminated minerals, including nephrite, antigorite, and talc. The case group had decreased pulmonary function in forced vital capacity (FVC), forced expiratory volume in one second, and forced expiratory flow between 25% and 75% of the FVC. The levels of SMRP, fibulin-3, urinary 8-OHdG/creatinine, and CEA were higher in the case group than in the control group. Subjects exposed to nephrite had significantly higher SMRP levels (0.84 ± 0.52 nM) than subjects exposed to other types of minerals (0.60 ± 0.30 nM). A dose-response relationship was observed between the SMRP level and the severity of pneumoconiosis. Machine learning algorithms, including variables of sex, age, SMRP, fibulin-3, CEA, and 8-OHdG/creatinine, can predict pneumoconiosis with high accuracy. The areas under the receiver operating characteristic curves ranged from 0.7 to 1.0. We suggest that SMRP and fibulin-3 could be used as biomarkers of pneumoconiosis in workers exposed to asbestos-contaminated minerals.

Fibroblast Growth Factor-23 and Matrix Extracellular Phosphoglycoprotein Levels in Healthy Children and, Pregnant and Puerperal Women.

INTRODUCTION AND OBJECTIVE: Fibroblast growth factor (FGF-23) and matrix extracellular phosphoglycoprotein (MEPE) are bone-related factors and their role in physiologic conditions and in different life stages are unknown. We aimed to evaluate age- and pregnancy-related changes in MEPE and FGF-23 levels and their correlations with calcium (Ca)-phosphate (PO4) metabolism. METHODS: The study population included 96 healthy children (50 females) and 31 women (11 healthy, 10 pregnant, and 10 lactating). Intact FGF-23 (iFGF-23), MEPE, ferritin, parathyroid hormone (PTH), 25-OH vitamin D, alkaline phosphatase (ALP), IGF-I, IGFBP-3 and, Ca, PO4 and creatine (Cre) in serum (S) and urine (U) samples were determined. The renal phosphate threshold (TmPO4/GFR) and z-scores for the parameters that show age-related changes were calculated. RESULTS: Serum iFGF-23 concentrations showed nonsignificant changes with age; however, MEPE decreased with age, reaching the lowest levels after 7 years. Additionally, higher serum MEPE concentrations were observed during pregnancy. Other than ALP, all other examined parameters demonstrated age-related changes. ALP, BUN, S-Cre, and U-Ca/Cre showed puerperal and pregnancy related changes together with MEPE. iFGF-23 was positively correlated with S-PO4 and TmPO4/GFR. MEPE was positively correlated with S-Ca, S-PO4 and TmPO4/GFR and negatively correlated with PTH, IGF-1, and IGFBP-3. CONCLUSION: Not iFGF-23 but MEPE showed age-dependent changes and was affected by pregnancy. Although, MEPE and iFGF-23 did not correlate with each other, they seem to affect serum and urinary phosphate in the same direction. Additionally, we found evidence that ferritin and growth factors might have a role in serum calcium and phosphate regulation.

Elevated urinary CRELD2 is associated with endoplasmic reticulum stress-mediated kidney disease.

ER stress has emerged as a signaling platform underlying the pathogenesis of various kidney diseases. Thus, there is an urgent need to develop ER stress biomarkers in the incipient stages of ER stress-mediated kidney disease, when a kidney biopsy is not yet clinically indicated, for early therapeutic intervention. Cysteine-rich with EGF-like domains 2 (CRELD2) is a newly identified protein that is induced and secreted under ER stress. For the first time to our knowledge, we demonstrate that CRELD2 can serve as a sensitive urinary biomarker for detecting ER stress in podocytes or renal tubular cells in murine models of podocyte ER stress-induced nephrotic syndrome and tunicamycin- or ischemia-reperfusion-induced acute kidney injury (AKI), respectively. Most importantly, urinary CRELD2 elevation occurs in patients with autosomal dominant tubulointerstitial kidney disease caused by UMOD mutations, a prototypical tubular ER stress disease. In addition, in pediatric patients undergoing cardiac surgery, detectable urine levels of CRELD2 within postoperative 6 hours strongly associate with severe AKI after surgery. In conclusion, our study has identified CRELD2 as a potentially novel urinary ER stress biomarker with potential utility in early diagnosis, risk stratification, treatment response monitoring, and directing of ER-targeted therapies in selected patient subgroups in the emerging era of precision nephrology.

Increased RHAMM expression relates to ovarian cancer progression.

BACKGROUND: Elevated hyaluronan-mediated motility receptor (RHAMM) has been reported to contribute to disease progression, aggressive phenotype and poor prognosis in multiple cancer types, however, RHAMM's role in ovarian cancer (OC) has not been elucidated. Therefore, we sought to evaluate the role for RHAMM in epithelial OC. RESULTS: Despite little to no expression in normal ovarian surface epithelium, western immunoblotting, immunohistochemical staining and enzyme linked immunosorbent assay showed elevated RHAMM levels in clinical tissue sections, omental metastasis and urine specimens of serous OC patients, as well as in cell lysates. We also found that RHAMM levels increase with increasing grade and stage in serous OC tissues and that RHAMM localizes to the apical cell surface and inclusion cysts. Apical localization of RHAMM suggested protein secretion which was validated by detection of significantly elevated urinary RHAMM levels (p < 0.0001) in OC patients (116.66 pg/mL) compared with normal controls (8.16 pg/mL). Likewise, urinary RHAMM levels decreased following cytoreductive surgery in OC patients suggesting the source of urinary RHAMM from tumor tissue. Lastly, we validated RHAMM levels in OC cell lysate and found at least 12× greater levels compared to normal ovarian surface epithelial cells. CONCLUSION: This pilot study shows, for the first time, that RHAMM may contribute to OC disease and could potentially be used as a prognostic marker.

Epidermal Growth Factor-Containing Fibulin-Like Extracellular Matrix Protein 1 (EFEMP1) Acts as a Potential Diagnostic Biomarker for Prostate Cancer.

BACKGROUND The aim of this study was to detect the expression of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) and estimate its diagnostic value in prostate cancer (PCa). MATERIAL AND METHODS EFEMP1 expression in serum and urine of patients with PCa, benign controls and healthy controls at mRNA and protein level were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) analysis, respectively. The chi-square test was used to analyze the relationship between EFEMP1 expression and clinical factors of patients with PCa. A receiver operating characteristic (ROC) curve was established to evaluate the potential values of EFEMP1 for the diagnosis of PCa. RESULTS The relative expression of EFEMP1 was significantly decreased in patients with PCa compared with that in the benign controls and healthy individuals, both at mRNA and protein levels (P<0.05). In the postoperative serum, the EFEMP1 expression was significantly higher than that in preoperative serum at 2 levels. Urine EFEMP1 expression was also down-regulated in patients with PCa compared to that in the other 2 control groups. The low expression of EFEMP1 was obviously affected by Gleason's score, serum PSA, pathological stage, and lymph node metastasis. Moreover, there was a significant inverse correlation between EFEMP1 expression and PSA levels. The ROC curve revealed that EFEMP1 distinguished PCa patients from healthy controls, with a high AUC of 0.908, corresponding with high sensitivity and specificity, which was significantly higher than the PSA value. CONCLUSIONS Serum EFEMP1 is down-regulated and involved in the progression of PCa. It may serve as a useful diagnostic biomarker, with better diagnostic accuracy than PSA in PCa.

The value of cartilage biomarkers in progressive knee osteoarthritis: cross-sectional and 6-year follow-up study in middle-aged subjects.

To determine the possible diagnostic and prognostic value of cartilage biomarkers in early-stage progressive and nonprogressive knee osteoarthritis (OA) in a population-based cohort of middle-aged subjects with chronic knee pain. Design tibiofemoral (TF) and patellofemoral (PF) radiographs were graded in 128 subjects (mean age at baseline, 45 ± 6.2 years) in 2002, 2005, and 2008. Cartilage degradation was assessed by urinary C-telopeptide fragments of type II collagen (uCTx-II), synthesis by serum type II A procollagen N-terminal propeptide (sPIIANP), and articular tissue turnover in general by cartilage oligomeric matrix protein (sCOMP). Several diagnostic associations were found between all studied biomarkers and progressive osteophytosis. COMP and CTx-II had a predictive value for subsequent progressive osteophytosis in multiple knee compartments and in case of CTx-II-also for progressive JSN. Over the first 3 years (2002-2005), significant associations were observed between COMP and progressive osteophytosis, whereas 3 years later (2005-2008) between CTx-II and progressive JSN. Thus, the associations between cartilage markers (COMP, CTx-II) and progression of radiographic OA features--osteophytes and JSN--were different between 2002-2005 and 2005-2008. Logistic regression revealed that for every unit increase in COMP level, there was 33 % higher risk for TF osteophyte progression. During early-stage OA, the presence and progression of osteophytosis is accompanied by increased level of cartilage biomarkers. This is the first study to demonstrate biochemical differences over the course of knee OA, illustrating a phasic nonpersistent character of OA with periods of progression and stabilization.

Expression of CD147, BIGH3 and Stathmin and their potential role as diagnostic marker in patients with urothelial carcinoma of the bladder.

BACKGROUND: Urothelial carcinoma of the bladder is characterised by very high recurrence rate, followed up by cystoscopy which being invasive technique makes the need for non-invasive markers important for Transitional Cell Carcinoma (TCC) detection. CD147 is a transmembrane protein highly expressed in tumour cells which aids in tumour invasion and growth. BIGH3, an Extracellular matrix protein (ECM) which interacts with various ECM component in different tissue system and Stathmin(STMN1) is cytosolic microtubule destabilising protein also called as Oncoprotein18 due to its role in tumour promotion. So far the expression of BIGH3 and STMN1 remains undetermined in cancer subjects including TCC. We therefore studied the levels and molecular expression of these molecules in TCC patients, to evaluate their usefulness as diagnostic markers. METHODS: Thirty consecutive TCC patients and two sets of control- 15 Benign prostatic hyperplasia (BPH) patient and 15 healthy were taken. Serum and urine levels of these molecules were estimated by ELISA and relative mRNA expression by Q-PCR from tumour and normal urothelium. Post-Hoc analysis and ROC curve were determined to evaluate the significance and sensitivity and specificity. RESULTS: The mean concentrations of these molecules were found to be significantly increased (p<0.001) in the serum and urine of TCC patients, with varying significance in each grade for different molecules. The urinary levels of CD147 (67 pg/ml) and serum STMN1 concentration (1.38 ng/ml) showed a specific increase as compared to the controls, while BIGH3 was elevated in both serum and urine samples. Molecular (mRNA) expression was elevated in the high grade (Muscle Invasive) stage of the disease for all the molecules, with a significant 3-fold increase that correlated with disease severity being observed for STMN1. ROC analysis gave optimal combination of sensitivity and specificity for diagnosis of the disease in urine and serum sample for STMN1. CONCLUSION: Of CD147, BIGH3 and STMN1, significant results were obtained for STMN1 and it could serve as the best possible diagnostic marker for TCC detection in future.

Increased urinary excretion of nephrin, podocalyxin, and ßig-h3 in women with preeclampsia.

Emerging evidence has shown that podocyte injury and reduced specific podocyte protein expressions contribute to proteinuria in preeclampsia. We collected urine specimens from women with preeclampsia to study whether podocyte-specific protein shedding is associated with renal barrier dysfunction. Urine specimens from women with normal pregnancies and from pregnant women complicated by chronic hypertension were used for comparison. We determined soluble podocyte slit protein nephrin levels in the urine specimens. Podocalyxin, ßig-h3, and VEGF concentrations were also measured. We found that nephrin and podocalyxin were barely detectable in the urine specimens from normal pregnant women and from women with chronic hypertension. In preeclampsia, urinary nephrin and podocalyxin concentrations were significantly increased and highly correlated to each other, r(2) = 0.595. Nephrin and podocalyxin were also correlated with urine protein concentrations. ßig-h3 was detected in the urine specimens from women with preeclampsia, and it is highly correlated with nephrin and podocalyxin concentrations in preeclampsia. ßig-h3 was undetectable in normal pregnancy and pregnancy complicated by chronic hypertension. Elevated VEGF levels were also found in women with preeclampsia compared with those of normal pregnancy and pregnancy complicated by chronic hypertension. These results provide strong evidence that podocyte protein shedding occurs in preeclampsia, and their levels are associated with proteinuria. The finding of urinary ßig-h3 excretion in preeclampsia suggests that increased transforming growth factor activity might also be involved in the kidney lesion in this pregnancy disorder.

Fibulin 3 peptides Fib3-1 and Fib3-2 are potential biomarkers of osteoarthritis.

OBJECTIVE: This study was undertaken to identify new biomarkers of osteoarthritis (OA) by proteomics analysis and to develop specific immunoassays to detect and quantify them. METHODS: Proteomics analysis was performed in urine samples from 10 women (mean±SD age 76.0±5.0 years) undergoing knee replacement surgery due to severe OA and 5 healthy women (mean±SD age 25.6±2.6 years). Protein content was analyzed by 2-dimensional differential gel electrophoresis. Protein spots that exhibited an OA:control abundance ratio of ≥1.5 were identified by mass spectrometry. Specific enzyme-linked immunosorbent assays were developed and validated in serum obtained from 236 healthy subjects ages 20-64 years and from 76 patients with severe radiologic knee OA (mean±SD age 68.8±11.9 years). Immunohistochemical analysis was performed on articular cartilage from tibial plateaus. RESULTS: Thirteen proteins within spots that were significantly modified between groups were identified. Two peptides of fibulin 3, named Fib3-1 and Fib3-2, were of particular interest. Two antisera directed against these peptides were used to develop immunoassays. Compared with age-matched healthy subjects, median levels of serum Fib3-1 and Fib3-2 were elevated in OA patients (54.6 pM versus 85.1 pM [P<0.0001] and 144.4 pM versus 191.4 pM [P<0.0001], respectively). Using area under the receiver operating characteristic curve analysis, we demonstrated that Fib3-1 and Fib3-2 levels discriminate between OA and normal populations. Immunostaining revealed the presence of Fib3-1 and Fib3-2 in chondrocytes and in the extracellular matrix of the superficial layer of the fibrillated cartilage. CONCLUSION: Our findings indicate that Fib3-1 and Fib3-2 are potential biochemical markers for the diagnosis of OA.

Mindin: a novel marker for podocyte injury in diabetic nephropathy.

BACKGROUND: GeneChip Expression Analysis was employed to survey the glomerular gene expression profile in a type 2 diabetes (T2D) model of KK/Ta mice fed with a high-calorie diet (HC), and we focused on the role of mindin (also called spondin 2), whose expression is upregulated by HC. METHODS: Isolated glomeruli from three 20-week-old KK/Ta mice fed with HC or a standard diet (SD) were dissected. Total RNA was extracted and labelled for hybridization using the Affymetrix GeneChip Mouse Genome 430 2.0 Array. The gene expression profile was compared between the HC and SD groups using GeneSpring 7.3.1 software. Mindin expression was examined using real-time PCR, western blot analysis and immunohistochemical staining in the glomeruli, cultured podocytes and urine samples of both mice and humans. RESULTS: Podocyte foot process effacement was observed in mice fed with HC. The mindin protein expression levels in mice were localized in the podocytes, and their levels in the glomeruli were increased in the HC group compared with the SD group. The levels of urinary mindin in the HC group at 16 weeks of age were also significantly higher than those in the SD group although albumin/creatinine ratio (ACR) did not differ between the groups. Furthermore, the levels in patients with T2D were higher than those in healthy individuals and increased gradually with increases in ACR. CONCLUSIONS: Mindin could be related to podocyte injury and appears to be an early biomarker of the progression of diabetic nephropathy.

Role of mindin in diabetic nephropathy.

A number of studies have shown that proinflammatory cytokines have important roles in determining the development of microvascular diabetic complications, including nephropathy. Inflammatory biomarkers should be useful for diagnosis or monitoring of diabetic nephropathy. Mindin (spondin 2) is a member of the mindin-/F-spondin family of secreted extracellular matrix (ECM) proteins. Recent studies showed that mindin is essential for initiation of innate immune response and represents a unique pattern-recognition molecule in the ECM. Previously, we demonstrated that the levels of urinary mindin in patients with type 2 diabetes were higher than those in healthy individuals. We propose that urinary mindin is a potent biomarker for the development of diabetic nephropathy.

Detection of cartilage oligomeric matrix protein using a quartz crystal microbalance.

Current methods for diagnosing early stage osteoarthritis (OA) based on the magnetic resonance imaging and enzyme-linked immunosorbent assay methods are specific, but require specialized laboratory facilities and highly trained personal to obtain a definitive result. In this work, a user friendly and non-invasive quartz crystal microbalance (QCM) immunosensor method has been developed to detect Cartilage Oligomeric Matrix Protein (COMP) for early stage OA diagnosis. This QCM immunosensor was fabricated to immobilize COMP antibodies utilizing the self-assembled monolayer technique. The surface properties of the immunosensor were characterized by its FTIR and electrochemical impedance spectra (EIS). The feasibility study was based on urine samples obtained from 41 volunteers. Experiments were carried out in a flow system and the reproducibility of the electrodes was evaluated by the impedance measured by EIS. Its potential dynamically monitored the immunoreaction processes and could increase the efficiency and sensitivity of COMP detection in laboratory-cultured preparations and clinical samples. The frequency responses of the QCM immunosensor changed from 6 kHz when testing 50 ng/mL COMP concentration. The linear regression equation of frequency shift and COMP concentration was determined as: y=0.0872 x+1.2138 (R2=0.9957). The COMP in urine was also determined by both QCM and EIS for comparison. A highly sensitive, user friendly and cost effective analytical method for the early stage OA diagnosis has thus been successfully developed.

Effect of eplerenone, enalapril and their combination treatment on diabetic nephropathy in type II diabetic rats.

BACKGROUND: Recent data suggest that aldosterone antagonists have beneficial effects on diabetic nephropathy. In this study, we investigated the dose-dependent effect of eplerenone and a combined treatment with eplerenone and enalapril compared with each drug alone on renal function in type II diabetic rats. To further explore the molecular mechanism of action of combination therapy, we also performed in vitro study. METHODS: The animals were divided into six groups as follows: normal control Long-Evans Tokushima Otsuka (LETO) rats, Otsuka Long-Evans Tokushima Fatty (OLETF) rats, OLETF rats treated with low dose of eplerenone (50 mg/kg/day), OLETF rats treated with high dose of eplerenone (200 mg/kg/day), OLETF rats treated with enalapril (10 mg/kg/day) and OLETF rats treated with a combination of both drugs (eplerenone 200 mg/kg/day and enalapril 10 mg/kg/day) for 6 months. RESULTS: Treatment of OLETF rats had no significant effect on body weight, kidney weight and blood glucose levels. However, urinary albumin excretion, glomerular filtration rate and glomerulosclerosis were significantly improved in the enalapril group and improvement was observed in a dose-dependent manner in the eplerenone groups; the most dramatic decreases were observed in the combination group. In accordance with these findings, renal expressions of TGF-beta1, type IV collagen and PAI-1 were also markedly decreased in the treatment groups, with the combined treatment providing the most significant level of improvement. In cultured mesangial cells, combined treatment resulted in an additive decrease in TGF-beta1, PAI-1 and collagen gene expressions and protein production induced by high glucose and aldosterone stimulation. CONCLUSIONS: Aldosterone receptor antagonism provided additional benefits beyond blockade of the renin-angiotensin system in type II diabetic nephropathy.

Analysis of cartilage oligomeric matrix protein (COMP) in synovial fluid, serum and urine from 51 racehorses with carpal bone fracture.

We investigated the relationship between cartilage oligomeric matrix protein (COMP) levels in synovial fluid (SF), serum and urine and the development of osteochondral damage and osteophyte (OP) formation following intraarticular fractures of the carpus in racehorses in order to assess the clinical usefulness of COMP as a diagnostic biomarker of developmental osteoarthritis (OA). Two monoclonal antibodies (mAb clones 2A11 and 3C8) raised against equine COMP were shown to be capable of detecting the molecule in serum and urine as well as SF. Fifty-one samples were obtained from 26 OP-positive (OP(+)) and 25 OP-negative (OP(-)) racehorses with carpal bone fracture, in whom OP was ascertained arthroscopically and radiographically. The COMP measurements obtained using the two mAbs were highly correlated with each other in SF, serum, or urine. Horses with OP(+) showed a significantly higher [urinary COMP (microg)]/[urinary creatinine (mg)] ratio (4.94 +/- 5.10 and 1.46 +/- 1.19, using mAbs 2A11 and 3C8, respectively) than OP(-) horses (2.80 +/- 1.72 and 0.93 +/- 0.49, respectively). The relationship between serum and urine COMP levels and the period from injury to surgery were extrapolated using a polynomial expression. Measurement of COMP, especially in urine, has potential as a predictive marker of advanced OA following carpal bone fractures in racehorses.

Effects of ACE inhibition and angiotensin II receptor blockade on glomerular basement membrane protein excretion and charge selectivity in type 2 diabetic patients.

Angiotensin-converting enzyme (ACE) inhibitors may reduce urinary albumin excretion (UAE) by decreasing glomerular pressure and increasing glomerular charge selectivity through preservation of glycosaminoglycans. The effect of Angiotensin II antagonism on glomerular charge selectivity remains to be determined. The aim of this study was to compare the effects of an AT1 blocker losartan and an ACE inhibitor (ACE-I) enalapril on UAE, extracellular matrix proteins, glycosaminoglycan excretion (UGAG) and red blood cell anionic charge (RBCCh) which are the indirect markers of glomerular basement membrane anionic content in hypertensive Type 2 diabetic patients. Twenty-four patients were randomised into two groups and received either enalapril (520 mg/d) or losartan (50100 mg/d). All parameters were measured at baseline and after six months of treatment. At the end of six months, systolic and diastolic blood pressures (BP), UAE rates, UGAG excretion and RBCCh were significantly and equally reduced in both treatment groups compared with baseline. RBCCh was negatively correlated with UAE (r=-0.57, p<0.0001) and UGAG excretion (r=-0.57, p<0.0001); UAE was correlated with UGAG excretion (r=0.58, p<0.0001). In conclusion, enalapril and losartan treatment were equally effective in reducing BP, UAE as well as UGAG excretion and preserving RBCCh in hypertensive Type 2 diabetic patients. ACE inhibition and AT1-receptor blockade may have favourable effects on preserving glomerular anionic content in hypertensive diabetic patients.