Noncrystalline uric acid inhibits proteoglycan and glycosaminoglycan synthesis in distal tubular epithelial cells (MDCK)

Hyperuricemia is associated with renal stones, not only consisting of uric acid (UrAc) but also of calcium oxalate (CaOx). Glycosaminoglycans (GAGs) are well-known inhibitors of growth and aggregation of CaOx crystals. We analyzed the effect of noncrystalline UrAc on GAG synthesis in tubular distal cells. MDCK (Madin-Darby canine kidney) cells were exposed to noncrystalline UrAc (80 µg/mL) for 24 h. GAGs were labeled metabolically and characterized by agarose gel electrophoresis. The expression of proteoglycans and cyclooxygenase 2 (COX-2) was assessed by real-time PCR. Necrosis, apoptosis and prostaglandin E2 (PGE2) were determined by acridine orange, HOESCHT 33346, and ELISA, respectively. CaOx crystal endocytosis was evaluated by flow cytometry. Noncrystalline UrAc significantly decreased the synthesis and secretion of heparan sulfate into the culture medium (UrAc: 2127 ± 377; control: 4447 ± 730 cpm) and decreased the expression of perlecan core protein (UrAc: 0.61 ± 0.13; control: 1.07 ± 0.16 arbitrary units), but not versican. Noncrystalline UrAc did not induce necrosis or apoptosis, but significantly increased COX-2 and PGE2 production. The effects of noncrystalline UrAc on GAG synthesis could not be attributed to inflammatory actions because lipopolysaccharide, as the positive control, did not have the same effect. CaOx was significantly endocytosed by MDCK cells, but this endocytosis was inhibited by exposure to noncrystalline UrAc (control: 674.6 ± 4.6, CaOx: 724.2 ± 4.2, and UrAc + CaOx: 688.6 ± 5.4 geometric mean), perhaps allowing interaction with CaOx crystals. Our results indicate that UrAc decreases GAG synthesis in MDCK cells and this effect could be related to the formation of UrAc and CaOx stones.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FINEPFundacao Oswaldo Ramos (FOR)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo (UNIFESP) Departamento de Medicina Disciplina de NefrologiaUniversidade Federal de São Paulo (UNIFESP) Departamento de Bioquímica Disciplina de Biologia MolecularUNIFESP, Depto. de Medicina Disciplina de NefrologiaUNIFESP, Depto. de Bioquímica Disciplina de Biologia MolecularSciEL

Apolipoprotein E polymorphism modulation of asymmetric dimethylarginine in hypertensive patients is determined by renal function

Background: Endothelial dysfunction is considered an early step of atherosclerotic vascular disease. Asymmetric dimethylarginine (ADMA), the main endogenous inhibitor of nitric oxide synthase (NOS), plays a critical role in the process of atherosclerosis in a uremic environment. Increased plasma ADMA not only works as a cardiovascular morbidity biomarker but it is also involved in the genesis of atherosclerosis in renal disease. Considering the relationships of apolipoprotein E(ApoE) polymorphism with LDL cholesterol (LDL-C) levels and coronary risk, it is possible that it brings on susceptibility to endothelial dysfunction and atherogenesis seen on uremia. Methods: Six hundred twenty patients were stratified according to glomerular filtration rate (GFR) estimated by Chronic Kidney Disease Epidemiology Collaboration (CKDEPI) formula: group I > 60 mL/min, group II 15 mL/min, and group III <= 15 mL/min or in hemodialysis. Polymorphic ApoE analysis was performed by polymerase chain reaction amplification (PCR). Plasma ADMA levels were measured by high performance liquid chromatography (HPLC). Groups were compared on clinical and laboratory characteristics as well as allele and genotype distribution towards. Results: The epsilon 2 allele of ApoE was present in 62 (10.3 %) patients, epsilon 3 allele in 581 (96.2 %), and epsilon 4 allele in 114 (18.9 %). Their distribution among the 3 groups was uniform. Such uniformity was not observed when we considered endothelial function measured by asymmetric dimethylarginine. In group III, the frequency of epsilon 4 allele was significantly lower in the third tertile compared with the first tertile (14.7 versus 53.3 %, P = 0.000; Pearson chisquare). In groups I and II, there was no difference in allele frequency according to ADMA levels. This association remained significant even after confouding factors corrections (OR 0.329, 95 % CI 0.155 - 0.699, P = 0.004). Conclusions: The results of this study shows that the frequency of epsilon 4 allele of ApoE is significantly lower among hypertensive patients on hemodialysis with the highest levels of ADMA. Uremia is capable of determining lower plasma ADMA levels in hypertensive epsilon 4 allele carriers.Fundo de Amparo a Pesquisa do Estado de Sao Paulo/FAPESPUniv Fed Sao Paulo, Nephrol Div, R Pedro de Toledo 781 14 Andar, BR-04039032 Sao Paulo, BrazilTufts Univ, New England Med Ctr, Nephrol Div, Boston, MA 02111 USAHosp Israelita Albert Einstein, Res & Educ Inst, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Nephrol Div, R Pedro de Toledo 781 14 Andar, BR-04039032 Sao Paulo, BrazilWeb of Scienc

Diversity of Apolipoprotein E genetic polymorphism significance on cardiovascular risk is determined by the presence of Metabolic Syndrome among hypertensive patients

Background: Hypertension has a significant relevance as a cardiovascular risk factor. A consistent increase on world's Metabolic Syndrome (MetS) incidence has been associated with an epidemic cardiovascular risk in different populations. Dislipidemia plays a major role determining the epidemic CV burden attributed to MetS. Apolipoprotein E (ApoE) is involved on cholesterol and triglycerides metabolism regulation. Once ApoE polymorphism may influence lipid metabolism, it is possible that it brings on individual susceptibility consequences for the development of MetS and cardiovascular risk. the objective of the study is to measure the discriminatory power of ApoE polymorphism in determining cardiovascular risk stratification based on the presence MetS in a cohort of hypertensive patients.Methods: It was enrolled 383 patients, divided in two groups, classified by MetS presence (IDF criteria): Group 1: 266 patients with MetS (MetS +) and Group 2: 117 patients without Mets (MetS -). Patient's data were collected by clinical evaluation, physical exam, file reviews and laboratory testing. Polymorphic ApoE analysis was performed by PCR amplification. Groups were compared on clinical and laboratory characteristics as well as allele and genotype distribution towards ApoE polymorphism. Mets CVD prevalence was analysed according to E4 allele prevalence.Results: the results evidenced 184 men (48%), 63,7% whites, 45,1% diabetics and 11,7% of patients were smokers. Mean age was 64,0 +/- 12,0 years. When genotypic distribution was analyzed, E3/3 genotype and E3 allele frequencies were more prevalent. Among patients with MetS, we observed an independent association between CVD prevalence and E4 allele frequency (OR 2.42 (1.17-5.0, p < 0,05)). On the opposite direction, in those without MetS, there was lesser CVD burden in E4 allele carriers (OR 0,14 (0,02-0,75)). These associations remained significant even after confounding factor corrections.Conclusions: the results presented demonstrate that the association between ApoE gene and CVD may be modulated by the presence of MetS, with an increased CV burden observed among E4 allele carriers with the syndrome. On the opposite way, E4 allele carriers without visceral obesity had lesser prevalence of CVD.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Div Nephrol, BR-04039032 São Paulo, BrazilTufts Univ, New England Med Ctr, Div Nephrol, Boston, MA 02111 USAHosp Israelita Albert Einstein, Res & Educ Inst, São Paulo, BrazilUniversidade Federal de São Paulo, Div Nephrol, BR-04039032 São Paulo, BrazilWeb of Scienc

Association of Vitamin D Levels With Kidney Volume in Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Vitamin D possesses renoprotective effects beyond mineral metabolism, potentially reducing arterial blood pressure and inflammation and vitamin D enzymes (CYP24A1 and CYP27B1) as well as vitamin D receptor (VDR) contribute to its homeostasis. In the present study, we aimed to determine vitamin D association with kidney volume, blood pressure parameters and inflammatory markers in ADPKD. This cross-sectional study, conducted from August 2011 through May 2016, evaluated 25(OH)D, 1,25(OH)2D and other hormonal/biochemical serum and urinary parameters, inflammatory markers and monocyte expression of VDR, CYP24A1, CYP27B1 in 74 ADPKD patients. The height-adjusted total kidney volume (htTKV) was determined by MRI and blood pressure (BP) measured through 24-h ambulatory BP monitoring (ABPM).Vitamin D insufficiency was present in 62% of patients and CYP24A1 was overexpressed in this group, raising a hypothesis of 25(OH)D increased catabolism. Serum 25(OH)D levels and VDR expression were negatively correlated with htTKV as was VDR with IL-6, IL-10, CRP, and NFκB. A multiple linear regression analysis with htTKV as dependent variable, including hypertension, CRP, eGFR, age, time since diagnosis, VDR, and 25(OH)D adjusted for season of the year showed that only the first three parameters were independent predictors of the former. There has been no association of serum 25(OH)D and VDR expression with ABPM parameters. Present findings suggested that low levels of serum 25(OH)D and VDR expression are associated with a higher kidney volume in ADPKD patients, but do not represent independent risk factors for htTKV

Acute aerobic session increased anti-inflammatory cytokine (IL-10) in CKD patients / A sessão aeróbica aguda aumentou a citocina anti-inflamatória (IL-10) em pacientes com CKD

In patients with chronic kidney disease (CKD), the presence of inflammation is a predictor of mortality. The aerobic exercise has been described to modulate the inflammatory response in general population. However, CKD patients have some limitations to performed aerobic exercise frequently. Thus, the present study evaluated the effect of an acute aerobic exercise session on markers of inflammation in non-dialytic CKD patients. MATERIALS AND METHODS: 10 stage 3 CKD patients and 10 subjects with normal kidney function performed experimental sessions of aerobic exercise on a cycle ergometer for 45 min, 50% at VO2peak. Before and after the session, blood samples were collected for the analysis of IL-6, TNF-?, IL-10, CRP, NGAL and Cystatin C.  The general linear model (GLM) test were used to evaluated the differences and interaction between group and time and Pearson's correlations were used to evaluate the association between the variables. RESULTS: Interestingly, after an aerobic exercise session, IL-10 levels increased in both group and more prominent in CKD group (p &lt;0.0001). As expected, CKD patients were more inflamed compared to the control group and IL-6 and TNF-? had a positive correlation with Cystatin-C and NGAL, markers of renal failure (p &lt; 0.001). CONCLUSION: In this study, an acute aerobic exercise session was able to increase IL-10 levels in CKD patients, suggesting that acute aerobic exercise may contribute to an anti-inflammatory response. However, it is necessary future studies to evaluate the impact of increased IL-10 and acute aerobic exercise on outcomes in patients with CKD

Effects of simvastatin on cytokines secretion from mononuclear cells from critically ill patients with acute kidney injury

Purpose: To assess the in vitro effects of simvastatin on IL-10 and TNF-alpha secretion from peripheral blood mononuclear cells (PBMC) of critically ill patients with and without acute kidney injury (AKI).Methods: PBMC were collected from 63 patients admitted to the intensive care unit (ICU) and from 20 healthy controls. Patients were divided in 3 subgroups: with AKI, with sepsis and without AKI and with AKI and sepsis. After isolation by ficoll-gradient centrifugation cells were incubated in vitro with LPS 1 ng/mL, simvastatin (10(-8)M) and with LPS plus simvastatin for 24 h. TNF-alpha and IL-10 concentrations on cells surnatant were determined by ELISA.Results: Cells isolated from critically ill patients showed a decreased spontaneous production of TNF-alpha and IL-10 compared to healthy controls (6.7(0.2-12) vs 103(64-257) pg/mL and (20 (13-58) vs 315(105-510) pg/mL, respectively, p < 0.05). Under LPS-stimulus, IL-10 production remains lower in patients compared to healthy control (451 (176-850) vs 1150(874-1521) pg/mL,p < 0.05) but TNF-alpha production was higher (641 (609-841) vs 406 (201-841) pg/mL, p < 0.05). the simultaneous incubation with LPS and simvastatin caused decreased IL-10 production in cells from patients compared to control (337 (135-626) vs 540 (345-871) pg/mL, p < 0.05) and increased TNF-alpha release (711 (619-832) vs 324 (155-355) pg/mL, p < 0.05). Comparison between subgroups showed that the results observed in TNF-alpha and IL-10 production by PBMC from critically ill patients was independent of AKI occurrence.Conclusions: the PBMC treatment with simvastatin resulted in attenuation on pro-inflammatory cytokine spontaneous production that was no longer observed when these cells were submitted to a second inflammatory stimulus. Our study shows an imbalance between pro and anti-inflammatory cytokine production in PBMC from critically ill patients regardless the presence of AKI. (C) 2011 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Instituto de Ensino e Pesquisa do Hospital Israelita Albert EinsteinUniversidade Federal de São Paulo, Div Nephrol, Dept Med, São Paulo, BrazilIAEH IEP Hosp Israelita Albert Einstein Inst Ensi, São Paulo, BrazilUniversidade Federal de São Paulo, Div Nephrol, Dept Med, São Paulo, BrazilWeb of Scienc

Serum soluble-Fas is a predictor of red blood cell transfusion in critically ill patients

OBJECTIVE: To investigate the relation between the need for red blood cell transfusion and serum levels of soluble-Fas, erythropoietin and inflammatory cytokines in critically ill patients with and without acute kidney injury. METHODS: We studied critically ill patients with acute kidney injury (n=30) and without acute kidney injury (n=13), end-stage renal disease patients on hemodialysis (n=25) and healthy subjects (n=21). Serum levels of soluble-Fas, erythropoietin, interleukin 6, interleukin 10, iron status, hemoglobin and hematocrit concentration were analyzed in all groups. The association between these variables in critically ill patients was investigated. RESULTS: Critically ill patients (acute kidney injury and non-acute kidney injury patients) had higher serum levels of erythropoietin than the other groups. Hemoglobin concentration was lower in the acute kidney injury patients than in other groups. Serum soluble-Fas levels were higher in acute kidney injury and end-stage renal disease patients. Critically ill patients requiring red blood cell transfusions had higher serum levels of soluble-Fas (5,906±2,047 and 1,920±1,060; p<0.001), interleukin 6 (518±537 and 255+502; p=0.02) and interleukin 10 (35.8±30.7 and 18.5±10.9; p=0.02), better iron status and higher mortality rates in the first 28 days in intensive care unit. Serum soluble-Fas levels were independently associated with the number of red blood cell units transfused (p=0.02). Serum soluble-Fas behaved as an independent predictor of the need for red blood cell transfusion in critically ill patients (p=0.01). CONCLUSIONS: Serum soluble-Fas level is an independent predictor of the need for red blood cell transfusion in critically ill patients with or without acute kidney injury. Further studies are warranted to reconfirm this finding.OBJETIVO: Investigar a relação entre a transfusão de hemácias e os níveis séricos de Fas solúvel, eritropoietina e citocinas inflamatórias em pacientes gravemente enfermos, com e sem insuficiência renal aguda. MÉTODOS: Os seguintes grupos foram estudados: pacientes gravemente enfermos com insuficiência renal aguda (n=30) e sem insuficiência renal aguda (n=13), pacientes portadores de doença renal crônica terminal em hemodiálise (n=25) e indivíduos saudáveis (n=21). Os níveis séricos de Fas solúvel, eritropoietina, interleucina 6, interleucina 10 e ferro, além da concentração de hemoglobina e de hematócrito, foram analisados em todos os grupos. A associação entre tais variáveis foram estudadas nos pacientes gravemente enfermos. RESULTADOS: Os níveis séricos de eritropoietina mostraram-se mais elevados nos pacientes gravemente enfermos do que nos dos demais grupos. Concentrações mais baixas de hemoglobina foram documentadas nos pacientes com insuficiência renal aguda em relação aos demais. Níveis séricos mais elevados de Fas solúvel foram observados nos pacientes com insuficiência renal aguda e doença renal crônica terminal. Pacientes gravemente enfermos transfundidos apresentaram níveis séricos mais elevados de Fas solúvel (5.906±2.047 e 1.920±1.060; p<0,001), interleucina 6 (518±537 e 255±502; p=0,02), interleucina 10 (35,8±30,7 e 18,5±10,9; p=0,02) e ferro, além de maior mortalidade em 28 dias. Os níveis séricos de Fas solúvel mostraram-se independentemente associados ao número de transfusões (p=0,02). O nível sérico de Fas solúvel foi um preditor independente da necessidade de transfusão de hemácias em pacientes gravemente enfermos (p=0,01). CONCLUSÃO: O nível sérico de Fas solúvel é um preditor independente da necessidade de transfusão de hemácias em pacientes gravemente enfermos, com ou sem insuficiência renal aguda. Mais estudos clínicos e laboratoriais são necessários para confirmar tal resultado.Universidade Federal de São Paulo (UNIFESP)Hospital Israelita Albert EinsteinUNIFESPSciEL

Soluble Fas(aFas): a new inflammation marker in ESRD patients

A Doenca Cardiovascular (DCV) tem sido reconhecida como uma doenca inflamatoria. DCV e frequente em pacientes com InsufiCiência renal Cronica (IRC) e contribui para elevada taxa de mortalidade nessa populacao. Estudos recentes mostram que niveis sericos de Fas soluvel (sFas), uma molecula anti-apoptotica e pro-inflamatoria, estao elevados em pacientes com DCV. Nos investigarmos os niveis sericos de sFas em pacientes uremicos e sua correlacao com marcadores de inflamacao e DCV. Foram estudados 25 pacientes com insufiCiência renal cronica (14 em hemodialise (HD) e 11 em Dialise Peritoneal Ambulatorial Continua (DPAC), 27 pacientes pre-dialise com clearance de creatinina < 50 ml/min/1,73 m2) e 14 individuos saudaveis. Foram quantificados os niveis sericos de sFas e marcadores inflamatorios (.orno: Proteina C-Reativa (PCR) e Albumina. Alem disso, investigamos a associacao de niveis sericos de sFas com a presenca de DCV e com dose de Eritropoietina ; EPO). Os niveis de sFas estavam elevados em pacientes uremicos comparados ao grupo controle (p < 0,05). Os niveis de sFas correlacionaram-se negativamente com clearance de creatinina. Observamos ainda, que os niveis de sFas foram maiores nos pacientes com Doenca Cardiovascular (p = 0,02). Houve correlacao dos niveis de sFas com niveis sericos de PCR (r= 0,33; p = 0,009) e albumina (r= - 0,35; p = 0,02). Tambem houve correlacao entre sFas e dose de EPO (U/semana) no grupo de HD (r = 0,72; p = 0,004) e DPAC (r = 0,62; p = 0,04). Estes resultados sugerem que o sFas pode ser um marcador de inflamacao na IRBV UNIFESP: Teses e dissertaçõe

Pro-inflammatory and oxidative effects of noncrystalline uric acid in human mesangial cells: contribution to hyperuricemic glomerular damage

Hyperuricemia is associated with cardiovascular and renal diseases, as glomerulosclerosis. Noncrystalline uric acid induces deleterious effects on endothelial and vascular smooth muscle cells. in the present study, we analyzed the damage induced by UA on human mesangial cells (HMC), the potential mechanism involved in this injury, and its consequences during infection. HMC were exposed to noncrystalline UA (8 mg/dl) and/or lipopolysaccharide (LPS, 100 mu g/ml) for 24 h. in the experiments of cellular viability, HMC were exposed to 8-50 mg/dl of UA. Necrosis was assessed by acridine orange and ethidium bromide. Reactive oxygen species (ROS) were analyzed by 2',7'-dichlorofluorescein. Prostaglandin E2 (PGE2) was evaluated by ELISA. Cyclooxygenase 2 (COX-2) expression was assessed by real-time PCR. UA induced necrosis only at supraphysiological concentrations. Nevertheless, it significantly increased ROS production at 8 mg/dl. LPS increased necrosis and ROS production. Interestingly, the association between UA and LPS decreased ROS and necrosis. UA associated or not with LPS induced COX-2 expression and PGE2 increases in HMC. Results suggest that UA has pro- and anti-oxidant effects in HMC. During infections, it acts like scavenger increasing cellular viability, but alone it can induce ROS production and cellular death in higher concentrations. Additionally, UA has direct pro-inflammatory effects inducing COX-2 expression and PGE2 synthesis. It is concluded that elevated concentrations of uric acid potentially contributes to glomerular damage.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Financiadora de Estudos e Projetos (FINEP)Fundacao Oswaldo Ramos (FOR)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fed Univ São Paulo UNIFESP, Dept Med, Div Nephrol, BR-04023900 São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Med, Div Nephrol, BR-04023900 São Paulo, BrazilWeb of Scienc