Resveratrol inhibits the intracellular calcium increase and angiotensin/endothelin system activation induced by soluble uric acid in mesangial cells

Resveratrol (Resv) is natural polyphenol found in grapes. This study evaluated the protective effect of Resv against the effects of uric acid (UA) in immortalized human mesangial cells (ihMCs). ihMCs were preincubated with Resv (12.5 µM) for 1 h and treated with UA (10 mg/dL) for 6 or 12 h. The intracellular calcium concentration [Ca2+]i was quantified by fluorescence using flow cytometry. Angiotensinogen (AGT) and pre-pro endothelin-1 (ppET-1) mRNA were assayed by quantitative real-time RT-PCR. Angiotensin II (AII) and endothelin-1 (ET-1) were assayed by ELISA. UA significantly increased [Ca2+]i. Pre-incubation with Resv significantly reduced the change in [Ca2+]i induced by UA. Incubation with UA for 6 or 12 h also increased AGT mRNA expression and AII protein synthesis. Resv blunted these increases in AGT mRNA expression and AII protein. Incubation with UA in the ihMCs increased ppET-1 expression and ET-1 protein synthesis at 6 and 12 h. When ihMCs were pre-incubated with Resv, UA had a significantly diminished effect on ppET-1 mRNA expression and ET-1 protein synthesis at 6 and 12 h, respectively. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. The renin-angiotensin system may contribute to the pathogenesis of renal function and chronic kidney disease. Resv can minimize the impact of UA on AII, ET-1 and the increase of [Ca2+]i in mesangial cells, suggesting that, at least in part, Resv can prevent the effects of soluble UA in mesangial cells.Universidade Federal de São Paulo (UNIFESP) Departamento de Medicina Divisão de NefrologiaUNIFESP, Depto. de Medicina Divisão de NefrologiaSciEL

Extracellular vesicles: structure, function, and potential clinical uses in renal diseases

Interest in the role of extracellular vesicles in various diseases including cancer has been increasing. Extracellular vesicles include microvesicles, exosomes, apoptotic bodies, and argosomes, and are classified by size, content, synthesis, and function. Currently, the best characterized are exosomes and microvesicles. Exosomes are small vesicles (40- 100 nm) involved in intercellular communication regardless of the distance between them. They are found in various biological fluids such as plasma, serum, and breast milk, and are formed from multivesicular bodies through the inward budding of the endosome membrane. Microvesicles are 100-1000 nm vesicles released from the cell by the outward budding of the plasma membrane. the therapeutic potential of extracellular vesicles is very broad, with applications including a route of drug delivery and as biomarkers for diagnosis. Extracellular vesicles extracted from stem cells may be used for treatment of many diseases including kidney diseases. This review highlights mechanisms of synthesis and function, and the potential uses of wellcharacterized extracellular vesicles, mainly exosomes, with a special focus on renal functions and diseases.Univ Fed Paulista, Dept Med, Div Nefrol, Escola Paulista Med, São Paulo, BrazilUniv Cruzeiro Sul, Programa Posgrad Ciencias Saude Ciencias Biol & S, São Paulo, BrazilUniv Fed Paulista, Dept Med, Div Nefrol, Escola Paulista Med, São Paulo, BrazilWeb of Scienc

Phyllanthus niruri as a promising alternative treatment for nephrolithiasis

In spite of considerable efforts to identify effective treatments for urolithiasis, this is a goal yet to be achieved. This review summarizes experimental and clinical data evaluating the effect of the plant Phyllanthus niruri, a plant with worldwide distribution, as a potential agent to prevent and/or to treat urolithiasis The review is based on data from the literature and on the results obtained by our group from either in vivo/in vitro experiments or clinical studies. Phyllanthus niruri has been shown to interfere with many stages of stone formation, reducing crystals aggregation, modifying their structure and composition as well as altering the interaction of the crystals with tubular cells leading to reduced subsequent endocytosis. The clinical beneficial effects of Phyllanthus niruri may be related to ureteral relaxation, helping to eliminate calculi or to clear fragments following lithotripsy, or also to a putative reduction of the excretion of urinary crystallization promoters such as calcium. No adverse renal, cardiovascular, neurological or toxic effects have been detected in either of these studies. Altogether, these studies suggest a preventive effect of Phyllanthus niruri in stone formation or elimination, but still longer-term randomized clinical trials are necessary to confirm its therapeutic properties.Federal University of São Paulo Renal DivisionUNIFESP, Renal DivisionSciEL

Nephrolithiasis in patients with inflammatory bowel disease in the community

Background: Inflammatory bowel disease (IBD) has been associated with renal stone formation. The objective of this study was to determine prospectively the prevalence of nephrolithiasis in a community-based population of patients with IBD and to analyze factors associated with renal calculus formation. Methods: Screening renal ultrasound was performed in a well characterized cohort of patients seen between 2009 and 2012 at an IBD clinic. We enrolled 168 patients, including 93 with Crohn’s disease and 75 with ulcerative colitis. Clinical and phenotypic variables associated with asymptomatic nephrolithiasis were determined. Results: Nephrolithiasis was detected in 36 patients with Crohn’s disease and in 28 patients with ulcerative colitis (38% for both). Although none of the patients had been previously hospitalized for symptomatic nephrolithiasis, nine with Crohn’s disease and five with ulcerative colitis had recurrent urinary tract infections or hydronephrosis. In patients with Crohn’s disease, ileocolonic (L3) disease was associated with a greater risk of nephrolithiasis than was ileal (L1) or colonic (L2) disease (odds ratio [OR] 2.3, 95% confidence interval [CI] 1.8–7). Active ulcerative colitis (regardless of severity) represented a significant risk factor for formation of renal calculi (OR 4.2, 95% CI 1.1–15, P = 0.02). Conclusion: In surgery-naïve patients with IBD in the community, asymptomatic nephrolithiasis is common and should be considered when renal dysfunction or infection is detected

The effect of an HIV-1 viral protease inhibitor on staurosporine-induced apoptosis in immortalized mesangial cells

CONTEXT: Progressive glomerular sclerosis is a condition characterized by the accumulation of glomerular extracellular matrix and a decrease in the number of glomerular cells. The mechanisms involved in the progressive loss of glomerular cells are not well understood but may involve the process of apoptosis. The principal mediators for the apoptotic pathway are a class of protease enzymes called caspases. It is not known how other therapeutic protease inhibitors affect the caspase cascade and therefore whether they would be effective in preventing excessive apoptosis in the late stages of progressive glomerular sclerosis. OBJECTIVE: To evaluate whether an inhibitor of the HIV-1 viral protease Ac-Leu-Val-phenylalanine (PI) could inhibit apoptosis in immortalized mesangial cells. DESIGN: Experimental. SETTING: Nephrology Division, Universidade Federal de São Paulo (UNIFESP)/Escola Paulista de Medicina. PARTICIPANTS: Immortalized mesangial cells. PROCEDURES: Cell culture. MAIN MEASUREMENTS: Viability and rate of apoptosis. RESULTS: Immortalized mesangial cells were treated with staurosporine (at concentrations of 10-100 nM for 8-28 hours) to induce apoptosis. Staurosporine at 10 nM for 8 hours had no effect on viability, but did cause a significant increase in the rate of apoptosis (p = 0.0411, n = 6). Increasing the incubation time elicited a greater increase in the rate of apoptosis (p = 0.0001, n = 6), although there was also a significant decrease in viability (p=0.0002). Increasing the concentration of staurosporine to 100 nM resulted in a marked increase in apoptosis (p <0.0001) but resulted in unacceptable viability (<40%, p <0.0001, n = 6). CONCLUSIONS: Incubation of immortalized mesangial cells with PI (900 nM) alone for 2-24 hours had no effect on cell viability or the rate of apoptosis when compared with vehicle (methanol) controls. Co-incubation of the cells with staurosporine (10 nM) and PI for 24 hours had no significant effect on the rate of apoptosis. Therefore, in immortalized mesangial cells, staurosporine-induced apoptosis was not significantly affected by the HIV-1 viral protease inhibitor Ac-Leu-Val-phenylalanine.CONTEXTO: A glomeruloesclerose progressiva (GEP) é uma situação caracterizada pela acumulação de matriz extracelular glomerular e pela diminuição no número de células no glomérulo. Os mecanismos envolvidos na perda progressiva do número glomerular da célula não são bem compreendidos, mas podem envolver o processo da apoptose. Os principais mediadores na evolução da apoptose são uma classe de enzimas de protease chamadas caspases. Não se sabe como outros inibidores de proteases com potencial uso terapêuticos efetuam a cascata dos caspases e, conseqüentemente, se podem ser eficazes em impedir a apoptose nos estágios anteriores da glomeruloesclerose progressiva. OBJETIVO: Avaliar se um inibidor da protease HIV-1 viral (Ac-Leu-Val-phenylalanine; o PI) poderia prevenir a apoptose em células mesangiais glomerulares imortalizadas. TIPO DE ESTUDO: Estudo experimental LOCAL: Disciplina de Nefrologia da Universidade Federal de São Paulo (UNIFESP)/Escola Paulista de Medicina, São Paulo, Brasil. PARTICIPANTES: Células Mesangiais Imortalizadas. PROCEDIMENTOS: Cultura de Célula. VARIÁVEIS ESTUDADAS: Viabilidade e taxa de apoptose. RESULTADOS: As células mesangiais imortali zadas foram tratadas com a estaurosporina (10-100 nM por 8 a 28h) para induzir a apoptose. A estaurosporina (10 nM; 8 h) não teve nenhum efeito viável, mas causou um significativo aumento na taxa de apoptose (p = 0,0411, n = 6). O aumento do tempo de incubação proporcionou um grande aumento da taxa de apoptose (p = 0,0001, n = 6), entretanto, há também uma significativa diminuição da viabilidade (p = 0,0002). O aumento da concentração de estaurosporina 100 nM resultou em aumento da apoptose (p <0,0001), porém com níveis inaceitáveis de viabilidade (< 40%, p <0,0001, n = 6). CONCLUSÕES: A incubação de células mesangiais imortalizadas com PI (900 nM) isolado por 2-24 h não teve efeito na viabilidade celular nem na taxa de apoptose comparada aos meios de controle (metanol). Co-incubação das células com estaurosporina (10 nM) e PI por 24 horas não adicionou efeito significativo na taxa de apoptose. Conseqüentemente, nas células mesangiais imortalizadas a apoptose induzida por estaurosporina não foi afetada significativamente pelo inibidor viral da protease HIV-1 Ac-Leu-Val-phenylalanine.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Department of MedicineUNIFESP, EPM, Department of MedicineSciEL

Absence of peripheral blood mononuclear cells priming in hemodialysis patients

As a consequence of the proinflammatory environment occurring in dialytic patients, cytokine overproduction has been implicated in hemodialysis co-morbidity. However, there are discrepancies among the various studies that have analyzed TNF-alpha synthesis and the presence of peripheral blood mononuclear cell (PBMC) priming in this clinical setting. We measured bioactive cytokine by the L929 cell bioassay, and evaluated PBMC TNF-alpha production by 32 hemodialysis patients (HP) and 51 controls. No difference in TNF-alpha secretion was observed between controls and HP (859 ± 141 vs 697 ± 130 U/10(6) cells). Lipopolysaccharide (5 µg/ml) did not induce any further TNF-alpha release, showing no PBMC priming. Paraformaldehyde-fixed HP PBMC were not cytotoxic to L929 cells, suggesting the absence of membrane-anchored TNF-alpha. Cycloheximide inhibited PBMC cytotoxicity in HP and controls, indicating lack of a PBMC TNF-alpha pool, and dependence on de novo cytokine synthesis. Actinomycin D reduced TNF-alpha production in HP, but had no effect on controls. Therefore, our data imply that TNF-alpha production is an intrinsic activity of normal PBMC and is not altered in HP. Moreover, TNF-alpha is a product of de novo synthesis by PBMC and is not constitutively expressed on HP cell membranes. The effect of actinomycin D suggests a putative tighter control of TNF-alpha mRNA turnover in HP. This increased dependence on TNF-alpha RNA transcription in HP may reflect an adaptive response to hemodialysis stimuli.Universidade de São Paulo Instituto de Ciências Biomédicas Departamento de ImunologiaInstituto Butantan Laboratório de ImunogenéticaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MedicinaUNIFESP, EPM, Depto. de MedicinaSciEL

Mini review: Current molecular methods for the detection and quantification of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1

The detection of acute human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) infection is vital for controlling the spread of HIV, HBV, and HCV to uninfected individuals. Considering that these viruses have high replication rates and are undetectable by serological markers, early detection upon transmission is crucial. Various nucleic acid assays have been developed for diagnostics and therapeutic monitoring of infections. in the past decade, rapid and sensitive molecular techniques such as PCR have revolutionized the detection of a variety of infectious viruses, including HIV, HCV, and HBV. Here, we describe two of the most commonly used licensed methods for the detection and quantification of HIV, HCV, and HBV: the cobas TaqScreen MPX (PCR) test and the Tigris System. We used transcription-mediated amplification to review and compare the development and efficiency of these technologies. (C) 2014 the Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao Oswaldo Ramos (FOR)Fundo de Auxilio aos Docentes e Alunos (FADA)Fed Univ São Paulo UNIFESP, Dept Med, Div Nephrol, BR-04023900 São Paulo, BrazilColsan Assoc Beneficente Coleta Sangue, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Med, Div Nephrol, BR-04023900 São Paulo, BrazilWeb of Scienc

Heterogeneity of glomerular perfusion and filtration induced by epinephrine and norepinephrine

The role of catecholamines in the distribution of intrarenal blood flow and in single-nephron glomerular filtration rate (SNGFR) was evaluated in anesthetized Wistar rats by the Hanssen technique. Epinephrine (EPI) and norepinephrine (NOR) were infused to produce elevations of 20-30 mmHg in mean arterial pressure. Superficial and juxtamedullary nephron perfusion and filtration were determined by the presence of Prussian blue dye. In the control group, 100% of the nephrons presented a homogeneous pattern of perfusion and filtration. In contrast, a heterogeneous distribution of the dye was found even in the larger arteries (arciform and radial), indicating variable perfusion and filtration in both superficial and juxtamedullary nephrons. The effects of EPI and NOR were also evaluated in the superficial cortex by the micropuncture technique in two additional groups of Munich-Wistar rats. Mean SNGFR was 27% and 54% lower in the EPI- and NOR-treated groups, respectively. No change in mean intraglomerular hydraulic pressure was observed after EPI or NOR infusion in spite of a highly scattered pattern, indicating an important variability in perfusion along the superficial cortex, and/or different sensitivity of the pre- and post-glomerular arterioles. The present data suggest that EPI and NOR may affect intrarenal hemodynamics by modifying perfusion and filtration in both superficial and juxtamedullary glomeruli and not by shifting blood flow from superficial to juxtamedullary nephrons. The heterogeneous pattern of perfusion was a consequence of differential vasoconstriction along the intrarenal arteries, probably due to different density and/or sensitivity of the adrenergic receptor subtypes present in the intrarenal vascular tree.Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL