Ursolic acid inhibits epithelial-mesenchymal transition in vitro and in vivo.

CONTEXT: Ursolic acid (UA; 3ß-hydroxy-urs-12-en-28-oic acid), one of the pentacyclic triterpenoids found in various plants and herbs, possesses some beneficial effects under pathological conditions, including combating hepatic fibrosis. OBJECTIVE: This study investigates the effects of UA on renal tubulointerstitial fibrosis in vivo and in vitro. MATERIALS AND METHODS: In vivo, 24 male C57BL6 mice were divided into four groups. Eighteen mice were subjected to unilateral ureteral obstruction (UUO) and the remaining six sham-operated mice served as control. UUO mice received either vehicle or UA (50 or 100 mg/kg) by gastric gavage for 6 days. In vitro, HK-2 cells were treated with 10 or 50 µM UA and 10 ng/mL recombinant human transforming growth factor-ß1 (TGF-ß1). The molecular mechanisms of fibrosis were investigated. RESULTS: UUO induced marked interstitial collagen I and fibronectin deposition and epithelial-mesenchymal transition (EMT), as evidenced by increased α-smooth muscle actin (α-SMA) and decreased E-cadherin. However, UA treatment significantly reduced collagen I and fibronectin accumulation in the fibrotic kidney. UA treatment also decreased α-SMA and preserved E-cadherin in vivo. In vitro, TGF-ß1-treated HK-2 cells demonstrated elevated α-SMA, snail1, slug, TGF-ß1, and p-smad3, as well as diminished E-cadherin. UA pretreatment prevented E-cadherin loss and diminished α-SMA expression in HK-2 cells. UA downregulated mRNA expression of snail1 and slug. UA also lowered TGF-ß1 protein expression and p-Smad3 in HK-2 cells. CONCLUSIONS: UA attenuated renal tubulointerstitial fibrosis by inhibiting EMT, and such inhibition may be achieved by decreasing profibrotic factors. UA may be a novel therapeutic agent for renal fibrosis.

Baicalein ameliorates renal interstitial fibrosis by inducing myofibroblast apoptosis in vivo and in vitro.

OBJECTIVE: To investigate the anti-fibrotic effects of baicalein and its influence on myofibroblasts in vivo and in vitro. MATERIALS AND METHODS: An in vivo unilateral ureteric obstruction (UUO) mouse model and an in vitro transforming growth factor ß1 (TGF-ß1) activated normal rat kidney (NRK)-49F cell model were established. Baicalein treatment was then investigated in these models to assess its anti-fibrotic effects and potential mechanisms of action. RESULTS: Baicalein attenuated renal fibrosis by ameliorating kidney injury, reducing deposition of fibronectin and collagen type 1, and inducing apoptosis in myofibroblasts in the UUO mouse model. Baicalein also induced apoptosis of TGF-ß1-activated myofibroblasts in vitro in a dose-dependent manner. Furthermore, baicalein triggered a cascade of mitochondrion-associated apoptosis by upregulating cleaved-caspase-3, Bcl2-associated X protein (Bax), and cleaved-caspase-9 while downregulating the protein expression of B-cell lymphoma 2 (Bcl-2). Additionally, down-regulation of phosphorylated protein kinase B (pAkt) was found in the baicalein-induced pro-apoptotic components. CONCLUSIONS: The present findings show that baicalein can ameliorate tubulointerstitial fibrosis by inducing myofibroblast apoptosis through the mitochondrion-associated intrinsic pathway, which may be mediated by the inhibition of the phosphoinositide-3-kinase/Akt (PI3k/Akt) pathway.

In vitro dissolution of calcium oxalate stones with ethylenediaminetetraacetic acid and snake venom thrombin-like enzyme.

OBJECTIVE: The aim of this study was to determine the feasibility of using snake venom thrombin-like enzyme (SVTLE) and/or ethylenediaminetetraacetic acid (EDTA) to dissolve calcium oxalate stones in vitro. METHODS: Seven calcium oxalate stones were incubated with various chemolytic agents [EDTA, Tris-HCl/EDTA (TE) buffer or SVTLE diluted in TE buffer]. The pH, calcium concentration, stone weight and stone surface integrity were recorded, as well as related pathological changes to bladder mucosae. RESULTS: Compared to all other solutions, those containing SVTLE and buffered EDTA had higher concentrations of mobilized calcium and caused significantly more stone weight loss, stone fragility and gaps in the calcium crystals. Also, there were no adverse pathological effects on rabbit bladder mucosae from any of the solutions. CONCLUSIONS: The data indicate that buffered EDTA and SVTLE can be used to dissolve calcium oxalate stones and, at the concentrations used here, do not damage tissue.

High Expression of SLC16A1 as a Biomarker to Predict Poor Prognosis of Urological Cancers.

OBJECTIVE: Tumor metabolism has always been the focus of cancer research. SLC16A1, as a key factor in catalysis of monocarboxylate transport across the plasma membrane, has been found to be associated with the occurrence and metastasis of a variety of cancers, but its prognostic significance and mechanism in different tumors are still unclear. METHODS: Based on the gene expression matrix and clinical information of human cancer tissues acquired from TCGA and GTEX databases, the differential expression of SLC16A1 in different tumors and normal tissues was analyzed. To confirm the association between its expression, the mutation of MMRS gene, and the expression level of DNMTs. Univariate Cox regression was applied to analyze the association between SLC16A1 expression and patient prognosis. The effect of SLC16A1 expression on patient survival was examined by Kaplan Meier analysis. GSEA was used to identify related signaling pathways. RESULTS: The expression of SLC16A1 was differentially expressed in most tumors, especially in the urinary tract where it is commonly highly expressed, and differential expression of SLC16A1 in different clinical stages. SLC16A1 expression was significantly positively correlated with MMRS gene mutation and DNMTS expression. Moreover, high SLC16A1 expression was associated with poorer overall survival (OS) and progression-free survival (PFS) in urological cancers. In particular, the results of the enrichment analysis showed that SLC16A1 was associated with processes such as cell adhesion and many signaling pathways affecting cell cycle were significantly enriched in the group with high-expressed SLC16A1. CONCLUSION: SLC16A1 expression was upregulated in urological cancer. SLC16A1 may promote tumor development by regulating the epigenetic process of urological cancer and demonstrated a great potential as a prognostic biomarker of urological cancer patients.

Dihydroquercetin protects against renal fibrosis by activating the Nrf2 pathway.

BACKGROUND: Dihydroquercetin (DHQ) is an antifibrotic agent. However, whether DHQ can prevent renal fibrosis remains unknown. PURPOSE: This study aimed to investigate the effects of DHQ on tubulointerstitial fibrosis and its underlying mechanisms in unilateral ureteral obstruction (UUO) mice in vivo and NRK-49F cells in vitro. METHODS: In vivo, UUO mice received vehicle or DHQ treatment. In vitro, NRK-49F cells were pretreated with DHQ and exposed to transforming growth factor-ß1 (TGF-ß1). Changes in fibroblast activation, collagen synthesis, oxidative stress, and related signaling pathways were assessed by immunohistochemical staining, Western blot analysis, real-time reverse transcription-PCR, and fluorescence microscopy. RESULTS: UUO induced tubular atrophy, inflammation, fibroblast differentiation into myofibroblast, and collagen deposition, whereas DHQ ameliorated these effects. UUO also resulted in decreased levels of nuclear factor-erythroid-2-related factor 2 (Nrf2), catalase, and heme oxygenase-1, but increased H2O2 and malondialdehyde levels. DHQ treatment corrected these changes. In vitro, the intracellular Nrf2 level of NRK-49F exposed to TGF-ß1 decreased. However, DHQ rescued intracellular Nrf2 level and promoted nuclear translocation of Nrf2. DHQ scavenged TGF-ß1-induced accumulation of reactive oxygen species, inhibited TGF-ß1-induced Smad3 phosphorylation, and prevented TGF-ß1-induced fibroblast activation and collagen synthesis in NRK-49F. Nrf2 knockdown could suppress the DHQ-mediated inhibitory effects on oxidative stress, Smad3 phosphorylation, fibroblast activation, and collagen deposition. Furthermore, DHQ ameliorated established renal fibrosis in UUO mice. CONCLUSIONS: DHQ posed remarkable preventive and therapeutic effects on UUO-induced renal fibrosis and suppressed fibroblast activation by reducing oxidative stress and Smad3 phosphorylation via Nrf2 signaling. This study provided a mechanistic basis for the clinical application of DHQ in renal fibrosis treatment.

Diagnostic and Prognostic Values of BMPER in Patients with Urosepsis following Ureteroscopic Lithotripsy.

The present study aims to investigate the risk factors for urosepsis and the diagnostic and prognostic values of the bone morphogenetic protein endothelial cell precursor-derived regulator (BMPER) in patients with urosepsis following ureteroscopic lithotripsy. A total of 305 patients with unilateral ureteral obstruction caused by calculi were included in the study. Patients were divided into three groups, namely, high, medium, and low perfusion pressure groups. The serum C-reactive protein, procalcitonin, lactate (LAC), and BMPER were measured after operation. A logistic regression model was used to assess the risk factors for postoperative urosepsis. The relationships of BMPER with laboratory parameters were explored with a multiple linear regression model. Receiver operating characteristic (ROC) curves were used to diagnosis urosepsis. The cumulative incidence of the adverse events after operation was calculated and compared by log-rank test. Forty-five patients (14.8%) had an episode of urosepsis after operation. Irrigation pressure was an independent risk factor for urosepsis. LAC and sequential organ failure assessment (SOFA) were associated with BMPER after operation. The area under curve value of BMPER for urosepsis was 0.829 (95% confidence interval [CI], 0.773 to 0.884). Uroseptic patients with higher BMPER concentration exhibited more adverse outcome. BMPER possesses valuable discriminative capacity for urosepsis and is a strong predictor of adverse outcome in patients with urosepsis.

Cryptotanshinone hinders renal fibrosis and epithelial transdifferentiation in obstructive nephropathy by inhibiting TGF-ß1/Smad3/integrin ß1 signal.

Recent studies have reported that CTS can alleviate cardiac fibrosis. However, the effects of CTS on kidney fibrosis and EMT are still unknown. This study explored whether CTS could attenuate tubulointerstitial fibrosis as well as EMT, and investigated the potential underlying mechanisms. In this study, an in vivo UUO mouse model and an in vitro TGF-ß1 stimulated normal renal tubular kidney epithelial cell model were established. In UUO model, administration of 50 mg kg-1 day-1 CTS markedly decreased the occurrence of kidney injury and the accumulation of fibronectin and collagen-1. In addition, CTS reduced the expression level of α-SMA but retained E-cadherin in obstructed kidneys. In vitro, CTS suppressed the expression of fibronectin, collagen-1 and α-SMA but retained that of E-cadherin. Furthermore, CTS selectively abolished the activation of Smad3 and suppressed the nuclear translocation of Smad2, Smad3 and Smad4. CTS could block the promoter activity of integrin ß1 induced by Smad3. Furthermore, CTS inhibited Smad3 binding to integrin ß1 promoter sequences. These data suggest that CTS can ameliorate kidney fibrosis and EMT, at least in part, by inhibiting the TGF-ß1/Smad3/integrin ß1 signaling pathway.

Histidine Metabolism and IGPD Play a Key Role in Cefquinome Inhibiting Biofilm Formation of Staphylococcus xylosus.

Staphylococcus xylosus (S. xylosus) is an AT-rich and coagulase-negative Staphylococcus (CNS). It is normally regarded as non-pathogenic, however, recent studies have demonstrated that it is related to human opportunistic infections and bovine mastitis. In addition, S. xylosus strains have the ability to form biofilm. Biofilms are also involved in chronic infections and antibiotic resistance, there are only a few reports about cefquinome inhibiting S. xylosus biofilm formation and the protein targets of cefquinome. In our study, we found that sub-MICs of cefquinome were sufficient to inhibit biofilm formation. To investigate the potential protein targets of cefquinome, we used iTRAQ for the analyses of cells at two different conditions: 1/2-MIC (0.125 µg/mL) cefquinome treatment and no treatment. Using iTRAQ technique and KEGG database analysis, we found that proteins differently expression in histidine metabolism pathway may play a role in the process by which 1/2-MIC (0.125 µg/mL) cefquinome inhibits S. xylosus biofilm formation. Interestingly, we found a sharply down-regulated enzyme [A0A068E9J3 imidazoleglycerol-phosphate dehydratase (IGPD)] involved in histidine metabolism pathway in cefquinome-treated cells. We demonstrated the important role of IGPD in sub-MICs cefquinome inhibiting biofilm formation of S. xylosus by gene (hisB) knockout, IGPD enzyme activity and histidine content assays. Thus, our data sheds light on important role of histidine metabolism in S. xylosus biofilm formation; especially, IGPD involved in histidine metabolism might play a crucial role in sub-MICs cefquinome inhibition of biofilm formation of S. xylosus, and we propose IGPD as an attractive protein target of cefquinome.

Inhibition of Streptococcus suis Adhesion and Biofilm Formation in Vitro by Water Extracts of Rhizoma Coptidis.

Streptococcus suis is difficult to treat and responsible for various infections in humans and pigs. It can also form biofilms and induce persistent infections. Rhizoma Coptidis is a medicinal plant widely used in Traditional Chinese Medicine. Although the inhibitory effects of Rhizoma Coptidis on biofilm formation have been investigated in several studies, the ability of Rhizoma Coptidis to inhibit S. suis biofilm formation and the underlying mechanisms have not yet been reported. In this study, we showed that sub-minimal inhibitory concentrations (25 and 50 µg mL-1) of water extracts of Rhizoma Coptidis (Coptis deltoidea C.Y.Cheng & P.K.Hsiao, obtained from Sichuan Province) were sufficient to inhibit biofilm formation, as shown in the tissue culture plate (TCP) method and scanning electron microscopy. Real-time PCR and iTRAQ were used to measure gene and protein expression in S. suis. Sub-minimum inhibitory concentrations (25 and 50 µg mL-1) of Rhizoma Coptidis water extracts inhibited S. suis adhesion significantly in an anti-adherence assay. Some genes, such as gapdh, sly, and mrp, and proteins, such as antigen-like protein, CPS16V, and methyltransferase H, involved in adhesion were significantly modulated in cells treated with 50 µg mL-1 of Rhizoma Coptidis water extracts compared to untreated cells. The results from this study suggest that compounds in Rhizoma Coptidis water extracts play an important role in inhibiting adhesion of S. suis cells and, therefore, biofilm formation.

Homology Modeling and Virtual Screening to Discover Potent Inhibitors Targeting the Imidazole Glycerophosphate Dehydratase Protein in Staphylococcus xylosus.

The imidazole glycerophosphate dehydratase (IGPD) protein is a therapeutic target for herbicide discovery. It is also regarded as a possible target in Staphylococcus xylosus (S. xylosus) for solving mastitis in the dairy cow. The 3D structure of IGPD protein is essential for discovering novel inhibitors during high-throughput virtual screening. However, to date, the 3D structure of IGPD protein of S. xylosus has not been solved. In this study, a series of computational techniques including homology modeling, Ramachandran Plots, and Verify 3D were performed in order to construct an appropriate 3D model of IGPD protein of S. xylosus. Nine hits were identified from 2,500 compounds by docking studies. Then, these nine compounds were first tested in vitro in S. xylosus biofilm formation using crystal violet staining. One of the potential compounds, baicalin was shown to significantly inhibit S. xylosus biofilm formation. Finally, the baicalin was further evaluated, which showed better inhibition of biofilm formation capability in S. xylosus by scanning electron microscopy. Hence, we have predicted the structure of IGPD protein of S. xylosus using computational techniques. We further discovered the IGPD protein was targeted by baicalin compound which inhibited the biofilm formation in S. xylosus. Our findings here would provide implications for the further development of novel IGPD inhibitors for the treatment of dairy mastitis.

Comparative Proteomic Analysis Provides insight into the Key Proteins as Possible Targets Involved in Aspirin Inhibiting Biofilm Formation of Staphylococcus xylosus.

Staphylococcus xylosus is an opportunistic pathogen that causes infection in humans and cow mastitis. And S. xylosus possesses a strong ability to form biofilms in vitro. As biofilm formation facilitates resistance to antimicrobial agents, the discovery of new medicinal properties for classic drugs is highly desired. Aspirin, which is the most common active component of non-steroidal anti-inflammatory compounds, affects the biofilm-forming capacity of various bacterial species. We have found that aspirin effectively inhibits biofilm formation of S. xylosus by Crystal violet (CV) staining and scanning electron microscopy analyses. The present study sought to elucidate possible targets of aspirin in suppressing S. xylosus biofilm formation. Based on an isobaric tag for relative and absolute quantitation (iTRAQ) fold-change of >1.2 or <0.8 (P-value < 0.05), 178 differentially expressed proteins, 111 down-regulated and 67 up-regulated, were identified after application of aspirin to cells at a 1/2 minimal inhibitory concentration. Gene ontology analysis indicated enrichment in metabolic processes for the majority of the differentially expressed proteins. We then used the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database to analyze a large number of differentially expressed proteins and identified genes involved in biosynthesis of amino acids pathway, carbon metabolism (pentose phosphate and glycolytic pathways, tricarboxylic acid cycle) and nitrogen metabolism (histidine metabolism). These novel proteins represent candidate targets in aspirin-mediated inhibition of S. xylosus biofilm formation at sub-MIC levels. The findings lay the foundation for further studies to identify potential aspirin targets.

Baicalein attenuates renal fibrosis by inhibiting inflammation via down-regulating NF-κB and MAPK signal pathways.

Baicalein is a natural flavonoid that possesses notable anti-inflammatory effects. In this study, we detected whether baicalein protects against inflammatory response in unilateral ureteral obstruction mice model to ameliorate tubulointerstitial fibrosis. Baicalein treatment significantly attenuated tubulointerstitial fibrosis by markedly reducing fibronectin and collagen-I. The downregulation of alpha-smooth muscle actin and upregulation of E-cadherin indicated that the epithelial-mesenchymal transition process was suppressed. Furthermore, baicalein administration blocked the infiltration of macrophages and lymphocytes, as evidenced by the significantly reduced CD68 and CD3 positive cells. Meanwhile, the mRNA expression of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1ß, and monocyte chemotactic protein in baicalein-treated groups was markedly reduced compared with the vehicle-treated group. More importantly, unilateral ureteral obstruction induced the activation of NF-κB and mitogen-activated protein kinase signal pathways to switch on inflammatory response to aggravate kidney fibrosis, but these effects were mitigated by baicalein. These data demonstrate that baicalein could inhibit inflammatory process via inactivation of NF-κB and MAPK signal pathways to execute its anti-fibrotic actions in obstructive kidney disease.

Plasma concentrations of adrenomedullin and natriuretic peptides in patients with essential hypertension.

This study was designed to assess any changes in the plasma concentrations of adrenomedullin (ADM) and atrial and brain natriuretic peptide (ANP and BNP, respectively), and to investigate their pathophysiological roles in patients with essential hypertension (EH). The plasma ADM, ANP and BNP concentrations were measured in 64 patients with untreated EH and 35 normotensive control subjects. After 4 weeks of effective antihypertensive therapy with oral drugs for the hypertensive patients, the plasma concentrations of ADM, ANP and BNP in the hypertensive patients were measured again. The plasma concentrations of ADM, ANP and BNP were significantly higher in the hypertensive patients than those in the control subjects, and the concentrations increased with the clinical stage. Furthermore, the hypertensive patients exhibited increased mean arterial pressure (MAP), blood urea nitrogen (BUN), serum creatinine (Scr) and decreased glomerular filtration rates (GFRs) compared with the control subjects. The plasma ADM concentration was not only correlated with BUN, Scr and the GFR, but was also associated with the MAP and the plasma levels of ANP and BNP. Following effective antihypertensive therapy with oral medication for 4 weeks, the plasma concentrations of ADM, ANP and BNP were significantly, but not sharply, decreased. In conclusion, ADM, along with ANP and BNP, may be involved in the mechanisms acting against a further increase in blood pressure and may be useful biomarkers for the diagnosis and treatment of hypertensive patients.

Pathophysiological functions of adrenomedullin and natriuretic peptides in patients with primary aldosteronism.

To measure the plasma concentrations of adrenomedullin (ADM),atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP), and investigate their pathophysiological functions in patients with primary aldosteronism (PA). Between June 2006 and December 2012, we recruited 25 patients with untreated PA, 30 patients with untreated low-renin essential hypertension (EH), and 35 healthy control subjects. The plasma concentrations of ADM, ANP, and BNP were measured in all the subjects. After 4 weeks of effective antihypertensive therapy with slow-release nifedipine, the three peptides were measured again in the PA and low-renin EH subjects. Unilateral laparoscopic adrenalectomy was performed in all the PA patients; 2 weeks after surgery, the three peptides were measured again. The PA patients had significantly higher plasma concentrations of ADM, ANP, and BNP than the low-renin EH and control subjects. The low-renin EH and control subjects significantly differed in the concentrations of the three peptides between low-renin EH and control subjects. ADM was the most important peptide associated with aldosterone or blood pressure in the PA patients. Plasma ADM concentration was not only correlated with plasma aldosterone concentrations, but also with systolic and diastolic blood pressures, and plasma ANP and BNP concentrations in the PA patients. By contrast, ADM concentration was not related to blood urea nitrogen levels, serum creatinine levels, and glomerular filtration rates. After antihypertensive treatment, the concentrations of the three peptides significantly decreased in the low-renin EH patients, but remained unchanged in the PA subjects. However, these concentrations significantly decreased 2 weeks after laparoscopic adrenalectomy in the PA subjects. ADM, ANP, and BNP possibly participate in the mechanisms counteracting further elevation of blood pressure or plasma volume expansion resulting from aldosterone hypersecretion in PA patients. An ADM/aldosterone local regulatory mechanism may be involved in regulating adrenal adenoma functions.