Luteolin Inhibits Indoxyl Sulfate-Induced ICAM-1 and MCP-1 Expression by Inducing HO-1 Expression in EA.hy926 Human Endothelial Cells.

In patients with chronic kidney disease, the uremic toxin indoxyl sulfate (IS) accelerates kidney damage and the progression of cardiovascular disease. IS may contribute to vascular diseases by inducing inflammation in endothelial cells. Luteolin has documented antioxidant and anti-inflammatory properties. This study aimed to investigate the effect of luteolin on IS-mediated reactive oxygen species (ROS) production and intercellular adhesion molecule (ICAM-1) and monocyte chemoattractant protein (MCP-1) expression in EA.hy926 cells and the possible mechanisms involved. IS significantly induced ROS production (by 6.03-fold, p < 0.05), ICAM-1 (by 2.19-fold, p < 0.05) and MCP-1 protein expression (by 2.18-fold, p < 0.05), and HL-60 cell adhesion (by 31%, p < 0.05), whereas, luteolin significantly decreased IS-induced ROS production, ICAM-1 and MCP-1 protein expression, and HL-60 cell adhesion. Moreover, luteolin attenuated IS-induced nuclear accumulation of p65 and c-jun. Luteolin dose-dependently increased heme oxygenase-1 (HO-1) expression and the maximum fold induction of HO-1 by luteolin was 3.68-fold (p < 0.05), whereas, HO-1 knockdown abolished the suppression of ICAM-1 and MCP-1 expression by luteolin. Luteolin may protect against IS-induced vessel damage by inducing HO-1 expression in vascular endothelial cells, which suppresses nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1) mediated ICAM-1 and MCP-1 expression.

Redrawing Urokinase Receptor (uPAR) Signaling with Cancer Driver Genes for Exploring Possible Anti-Cancer Targets and Drugs.

During tumorigenesis, urokinase (uPA) and uPA receptor (uPAR) play essential roles in mediating pathological progression in many cancers. To understand the crosstalk between the uPA/uPAR signaling and cancer, as well as to decipher their cellular pathways, we proposed to use cancer driver genes to map out the uPAR signaling. In the study, an integrated pharmaceutical bioinformatics approach that combined modulator identification, driver gene ontology networking, protein targets prediction and networking, pathway analysis and uPAR modulator screening platform construction was employed to uncover druggable targets in uPAR signaling for developing a novel anti-cancer modality. Through these works, we found that uPAR signaling interacted with 10 of 21 KEGG cancer pathways, indicating the important role of uPAR in mediating intracellular cancerous signaling. Furthermore, we verified that receptor tyrosine kinases (RTKs) and ribosomal S6 kinases (RSKs) could serve as signal hubs to relay uPAR-mediated cellular functions on cancer hallmarks such as angiogenesis, proliferation, migration and metastasis. Moreover, we established an in silico virtual screening platform and a uPAR-driver gene pair rule for identifying potential uPAR modulators to combat cancer. Altogether, our results not only elucidated the complex networking between uPAR modulation and cancer but also provided a paved way for developing new chemical entities and/or re-positioning clinically used drugs against cancer.

Urokinase Plasminogen Activator Deficiency Aggravates Cationic Bovine Serum Albumin-Induced Membranous Nephropathy Through T Helper Cell Type 2-Prone Immune Response in Mice.

Urokinase plasminogen activator (uPA) is a crucial activator of the fibrinolytic system that modulates tissue remodeling, cancer progression, and inflammation. However, its role in membranous nephropathy (MN) remains unclear. To clarify this issue, an established BALB/c mouse model mimicking human MN induced by cationic bovine serum albumin (cBSA), with a T helper cell type 2-prone genetic background, was used. To induce MN, cBSA was injected into Plau knockout (Plau-/-) and wild-type (WT) mice. The blood and urine samples were collected to measure biochemical parameters, such as serum concentrations of immunoglobulin (Ig)G1 and IgG2a, using enzyme-linked immunoassay. The kidneys were histologically examined for the presence of glomerular polyanions, reactive oxygen species (ROS), and apoptosis, and transmission electron microscopy was used to examine subepithelial deposits. Lymphocyte subsets were determined using flow cytometry. Four weeks post-cBSA administration, Plau-/- mice exhibited a significantly higher urine protein-to-creatine ratio, hypoalbuminemia, and hypercholesterolemia than WT mice. Histologically, compared to WT mice, Plau-/- mice showed more severe glomerular basement thickening, mesangial expansion, IgG granular deposition, intensified podocyte effacement, irregular thickening of glomerular basement membrane and subepithelial deposits, and abolishment of the glycocalyx. Moreover, increased renal ROS levels and apoptosis were observed in Plau-/- mice with MN. B-lymphocyte subsets and the IgG1-to-IgG2a ratio were significantly higher in Plau-/- mice after MN induction. Thus, uPA deficiency induces a T helper cell type 2-dominant immune response, leading to increased subepithelial deposits, ROS levels, and apoptosis in the kidneys, subsequently exacerbating MN progression in mice. This study provides a novel insight into the role of uPA in MN progression.

A substructure-based screening approach to uncover N-nitrosamines in drug substances.

Drug substances are at risk of contamination with N-nitrosamines (NAs), well-known carcinogenic agents, during synthesis processes and/or long-term storage. Therefore, in this study, we developed an efficient data-based screening approach to systemically assess marketed products and investigated its scalability for benefiting both regulatory agencies and pharmaceutical industries. A substructure-based screening method employing DataWarrior, an open-source software, was established to evaluate the risks of NA impurities in drug substances. Eight NA substructures containing susceptible amino sources for N-nitrosation have been identified as screening targets: dimethylamine (DMA), diethylamine, isopropylethylamine, diisopropylamine, N-methyl-2-pyrrolidone, dibutylamine, methylphenylamine, and tetrazoles. Our method detected 192 drug substances with a theoretical possibility of NA impurity, 141 of which had not been reported previously. In addition, the DMA moiety was significantly dominant among the eight NA substructures. The results were validated using data from the literature, and a high detection sensitivity of 0.944 was demonstrated. Furthermore, our approach has the advantage of scalability, owing to which 31 additional drugs with suspected NA-contaminated substructures were identified using the substructures of 1-methyl-4-piperazine in rifampin and 1-cyclopentyl-4-piperazine in rifapentine. In conclusion, the reported substructure-based approach provides an effective and scalable method for the screening and investigation of NA impurities in various pharmaceuticals and might be used as an ancillary technique in the field of pharmaceutical quality control for risk assessments of potential NA impurities.

Reduced Expression of Urokinase Plasminogen Activator in Brown Adipose Tissue of Obese Mouse Models.

In recent decades, the obesity epidemic has resulted in morbidity and mortality rates increasing globally. In this study, using obese mouse models, we investigated the relationship among urokinase plasminogen activator (uPA), metabolic disorders, glomerular filtration rate, and adipose tissues. Two groups, each comprised of C57BL/6J and BALB/c male mice, were fed a chow diet (CD) and a high fat diet (HFD), respectively. Within the two HFD groups, half of each group were euthanized at 8 weeks (W8) or 16 weeks (W16). Blood, urine and adipose tissues were collected and harvested for evaluation of the effects of obesity. In both mouse models, triglyceride with insulin resistance and body weight increased with duration when fed a HFD in comparison to those in the groups on a CD. In both C57BL/6J and BALB/c HFD mice, levels of serum uPA initially increased significantly in the W8 group, and then the increment decreased in the W16 group. The glomerular filtration rate declined in both HFD groups. The expression of uPA significantly decreased in brown adipose tissue (BAT), but not in white adipose tissue, when compared with that in the CD group. The results suggest a decline in the expression of uPA in BAT in obese m models as the serum uPA increases. There is possibly an association with BAT fibrosis and dysfunction, which may need further study.

The relationship of irisin with metabolic syndrome components and insulin secretion and resistance in schoolchildren.

ABSTRACT: Irisin, a novel myokine, is believed to be the crucial factor in converting white adipose tissue to beige adipose tissue. For this paper, we studied the relationship among irisin and components of metabolic syndrome (MetS), and insulin secretion and resistance in schoolchildren of Taiwan.Subjects receiving routine annual health examination at elementary school were enrolled. Demographic data, anthropometry, MetS components, irisin, and insulin secretion and resistance were collected. Subjects were divided into normal, overweight, and obese groups for evaluation of irisin in obesity. Finally, the relationship between irisin and MetS was analyzed.There were 376 children (179 boys and 197 girls), aged 10.3 ±â€Š1.5 years, were enrolled. In boys, irisin levels were not associated with body mass index percentile, body fat, blood pressure, lipid profiles, insulin secretion or resistance. After adjusting for age, the irisin level in boys was negatively related to fasting plasma glucose (FPG) (r = -0.21, P = .006). In girls, after adjusting for age, the irisin levels were positively related only to FPG (r = 1.49, P = .038). In both genders, irisin levels were similar among normal, overweight, and obese groups, and between subjects with and without MetS.The irisin levels were not associated with MetS in either boys or girls. In girls, circulating irisin levels have a nonsignificant declining trend in overweight and obese girls. However, irisin levels were negatively related to FPG in boys and positively related to FPG in girls. The contrary relationship between irisin and FPG in boys and girls needs further exploration.

1,25(OH)2 D3 attenuates indoxyl sulfate-induced epithelial-to-mesenchymal cell transition via inactivation of PI3K/Akt/ß-catenin signaling in renal tubular epithelial cells.

OBJECTIVES: Indoxyl sulfate (IS), a uremic toxin, has been shown to promote the epithelial-to-mesenchymal transition (EMT) of human proximal tubular cells and to accelerate the progression of chronic kidney disease (CKD). Despite the well-known protective role of 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] in EMT, the effect of 1,25(OH)2 D3 on IS-induced EMT in human proximal tubular epithelial cells and the underlying mechanism remain unclear. The aim of this study was to determine whether IS (0-1 mM) dose-dependently inhibited the protein expression of E-cadherin and increased the protein expression of alpha-smooth muscle actin, N-cadherin, and fibronectin. METHODS: This study investigated the molecular mechanism by which 1,25(OH)2 D3 attenuates IS-induced EMT. HK-2 human renal tubular epithelial cells was used as the study model, and the MTT assay, Western Blotting, siRNA knockdown technique were used to explore the effects of 1,25(OH)2 D3 on EMT in the presence of IS. RESULTS: Pretreatment with 1,25(OH)2 D3 inhibited the IS-induced EMT-associated protein expression in HK-2 cells. IS induced the phosphorylation of Akt (S473) and ß-catenin (S552) and subsequently increased the nuclear accumulation of ß-catenin. Pretreatment with 1,25(OH)2 D3 and LY294002 (phosphoinositide 3-kinase [PIK3] inhibitor) significantly inhibited the IS-induced phosphorylation of Akt and ß-catenin, nuclear ß-catenin accumulation, and EMT-associated protein expression. CONCLUSIONS: Results from the present study revealed that the anti-EMT effect of 1,25(OH)2 D3 is likely through inhibition of the PI3K/Akt/ß-catenin pathway, which leads to down-regulation of IS-driven EMT-associated protein expression in HK-2 human renal tubular epithelial cells.

Deficiency of Urokinase Plasminogen Activator May Impair ß Cells Regeneration and Insulin Secretion in Type 2 Diabetes Mellitus.

: Background: The relationship between urokinase-type plasminogen activator (uPA) and the development of type 2 diabetes mellitus (T2DM) was investigated in the study by using mice and cell models, as well as patients with T2DM. METHODS: In mice models, wild-type and uPA knockout (uPA-/-) BALB/c mice were used for induction of T2DM. In cell models, insulin secretion rate and ß cell proliferation were assessed in normal and high glucose after treating uPA siRNA, uPA, or anti-uPA antibody. In our clinical study, patients with T2DM received an oral glucose-tolerance test, and the relationship between uPA and insulin secretion was assessed. RESULTS: Insulin particles and insulin secretion were mildly restored one month after induction in wild-type mice, but not in uPA-/- mice. In cell models, insulin secretion rate and cell proliferation declined in high glucose after uPA silencing either by siRNA or by anti-uPA antibody. After treatment with uPA, ß cell proliferation increased in normal glucose. In clinical study, patients with T2DM and higher uPA levels had better ability of insulin secretion than those with lower uPA levels. CONCLUSION: uPA may play a substantial role in insulin secretion, ß cell regeneration, and progressive development of T2DM. Supplementation of uPA might be a novel approach for prevention and treatment of T2DM in the future.

Atorvastatin from target screening attenuates endothelial cell tube formation and migration by regulating urokinase receptor-related signaling pathway and F/G actin.

BACKGROUND: Angiogenesis and cytoskeletal transformation are common denominators of many pathological developments. The relationship between angiogenesis, urokinase plasminogen activator receptor (uPAR) signaling pathway, and cytoskeletal transformation is still unknown. In this study, a pGL3-uPAR promoter reporter system combined with Bio-Plex mRNA analysis was established for discovering uPAR modulators to analyze this interconnection. METHODS: After screening a set of clinically used drugs, atorvastatin (Ator) was found to significantly affect uPAR expression and its ideal dose, 1 µM, was determined for cell study. Mouse endothelial cell (mEC) models, including tube formation for angiogenesis and wound healing assay for migration, were employed to test the effects on angiogenesis and cytoskeletal transformation with (Group Ator) and without (Group C) the treatment of Ator. RESULTS: The mEC tube formation and migration was significantly decreased in Group Ator. Regarding cytoskeleton changes, the ratio of F/G actin by Western blotting and the assembly of F-actin (lamellipodia) by immunofluorescence were attenuated. Furthermore, uPAR and all uPAR-related factors, including integrin α5ß3, phosphorylated-focal adhesion kinase, and Rac, revealed a significant reduction when compared with Group C. CONCLUSION: We conclude that close regulatory machinery spans angiogenesis, uPAR signaling, and cytoskeletal transformation, and that uPAR modulator Ator can decrease the reorganization of actin cytoskeleton, which may lead to a new approach in angiogenesis.

Significance of the urokinase-type plasminogen activator and its receptor in the progression of focal segmental glomerulosclerosis in clinical and mouse models.

BACKGROUND: suPAR biomarker generally considered a pathogenic factor in FSGS. However, studies have been published that dispute this conclusion. The current study was designed to investigate the roles of uPA and suPAR in FSGS in clinical and mouse models. METHODS: Clinical subjects including those with biopsy-proven FSGS and MCD were enrolled. To verify the role of uPA in FSGS, Adriamycin was used to induce FSGS in uPA knockout (uPA(-/-)) and BALB/c (WT) mice. Proteinuria and suPAR, the cleaved/intact forms of the circulating suPAR, and possible proteases involving cleavage of the suPAR were also studied. RESULTS: FSGS clinical cases presented significantly higher serum levels of suPAR and Cr and lower serum levels of uPA. In the mice model, the uPA(-/-) group exhibited faster disease progression and worsening proteinuria than the WT group. In addition, the uPA(-/-) group had higher plasma suPAR levels, glomerular cell apoptosis, and dysregulation of the Th1/Th2 balance. In an analysis of suPAR variants in FSGS, both the intact and cleaved forms of the suPAR were higher in clinical subjects and the mouse model. However, the process of suPAR cleavage was not mediated by enzymatic activities of the uPA, elastase, or cathepsin G. CONCLUSIONS: A deficiency of uPA accelerated the progression of Adriamycin-induced mouse FSGS model. Decrease of serum uPA levels may be an indicator of the progression of FSGS in clinical subjects and animal models.

Association among Fibrinolytic Proteins, Metabolic Syndrome Components, Insulin Secretion, and Resistance in Schoolchildren.

We investigated the role of urokinase plasminogen activator (uPA) and its soluble receptors (suPAR) and plasminogen activator inhibitor-1 (PAI-1) in metabolic syndrome (MetS) components, insulin secretion, and resistance in schoolchildren. We enrolled 387 children, aged 10.3 ± 1.5 years, in Taipei. Anthropometry, fibrinolytic proteins, MetS components, insulin secretion, and resistance were measured. Subjects were divided into normal, overweight, and obese groups. Finally, the relationship between fibrinolytic proteins and metabolic syndrome in boys and girls was analyzed. In boys, PAI-1 was positively associated with body mass index (BMI) percentile, hypertriglyceride, insulin secretion, and resistance. In girls, PAI-1 was positively associated with obesity, hypertriglyceridemia, and insulin secretion. In girls, uPA was positively associated with insulin secretion. suPAR was positively associated with high-sensitivity C-reactive protein in both boys and girls, and with BMI percentile and body fat in girls. The obese boys had higher suPAR and PAI-1 levels than the normal group. The obese girls had higher uPA, suPAR, and PAI-1 than the normal group. Boys and girls with MetS had higher PAI-1. Fibrinolytic proteins, especially PAI-1, are associated with MetS components and insulin secretion in children. Fibrinolytic proteins changes were more likely to occur in girls than in boys.

Antidepressant use and risk of recurrent stroke: a population-based nested case-control study.

OBJECTIVE: Antidepressants may carry an increased risk for incident stroke, but there is little safety evidence regarding poststroke antidepressant use. This study aimed to examine whether antidepressants are associated with an increased risk of stroke recurrence. METHOD: A population-based nested case-control study was conducted analyzing the Taiwan universal health care claims database from January 1, 2000, to December 31, 2008. We followed up 19,825 patients who survived a first admission for stroke at the age of ≥ 18 years, among which 3,536 hospitalized cases with stroke recurrence (ICD-9-CM codes 430.xx-437.xx) were identified and individually matched to 6,679 randomly-selected controls. Multivariate conditional logistic regression models were used to characterize the risk associated with antidepressant use. RESULTS: The study cohort had a mean age of 66 years and was followed up for a median of 2.9 person-years. Use of any tricyclic antidepressants (TCAs) was associated with a 1.41-fold (95% CI, 1.19-1.67) increased risk of stroke recurrence, whereas any use of selective serotonin reuptake inhibitors (SSRIs) or other antidepressants showed no association. Stopping TCAs for 1-30 days was associated with a 1.87-fold (95% CI, 1.22-2.86) increased risk of stroke recurrence, and the risk was attenuated for a longer discontinuation. The stroke risk associated with TCA use was not present in a dose-dependent or duration-dependent manner. CONCLUSIONS: Use of TCAs, but not SSRIs or other antidepressants, was associated with an increased risk of stroke recurrence. The risk is particularly elevated with abrupt cessation of TCA therapy. Health care professionals should be vigilant to that risk during TCA therapy in poststroke patients.

Urokinase plasminogen activator receptor and its soluble form in common biopsy-proven kidney diseases and in staging of diabetic nephropathy.

OBJECTIVES: Soluble urokinase plasminogen activator receptor (suPAR), derived from membrane bound uPAR, is associated with inflammatory diseases. In the present study, we explored the expression of uPAR/suPAR in common biopsy-proven kidney diseases and the relationship between suPAR and staging of type 2 diabetic nephropathy (DN). DESIGN AND METHODS: Serum samples for suPAR and renal tissues for uPAR staining were investigated in various common kidney diseases. The levels of serum suPAR were measured and adequate cut-off values of different stage of DN were calculated by receiver operating characteristic (ROC) curve. RESULTS: In our results, the expression of uPAR on renal tissues was pronounced in the majority of kidney diseases. Comparing of expression of uPAR among different kidney diseases, it was strongest in minimal change disease (MCD) and weakest in chronic interstitial nephritis. Serum suPAR in most kidney diseases, except of MCD, was significantly elevated and was highest in DN. As for DN and suPAR, we found that suPAR progressively increased with staging of DN. Moreover, suPAR was linearly and negatively related to estimated glomerular filtration rate and positively related to the amount of proteinuria. By ROC curve, the cut-off values of suPAR in DN for assessing development increased with the progression of the disease. CONCLUSIONS: We concluded that uPAR/suPAR is elevated in most kidney diseases and that suPAR is a useful biomarker for assessing stages of DN.

Chaperonin-containing t-complex protein-1 subunit ß as a possible biomarker for the phase of glomerular hyperfiltration of diabetic nephropathy.

In cell model, we discovered the association between chaperonin-containing t-complex polypeptide 1 subunit ß (TCP-1ß) and early diabetic nephropathy (DN). In this study, we further explored the relationships between TCP-1ß and type 2 diabetic mellitus (DM). To mimic the clinical hyperfiltration state, a type 2 DM mice model was established by feeding a high-fat diet in combination with treatment of streptozotocin and nicotinamide. Blood and urine were collected to determine creatinine clearance (C cr), and kidney tissues were harvested for evaluation of TCP-1ß expression by immunohistochemistry and Western blot. Meanwhile, clinical subjects of healthy controls and type 2 DM were recruited to strengthen the evidence with urine TCP-1ß. Results showed that C cr and the expression of TCP-1ß in kidney were significantly higher one week after hyperglycemia development, suggesting that the hyperfiltration state was successfully established in the mice model. TCP-1ß was expressed predominantly on renal tubules. By using the estimated glomerular filtration rate to index progression in clinical investigation, urine TCP-1ß level was associated with the hyperfiltration phase in type 2 DM patients. Conclusively, we confirmed that TCP-1ß is a possible biomarker for early nephropathy of type 2 DM, but further mechanistic study to elucidate its cause and pathway is needed.