Targeting Kidney Inflammation After Brain Death and Cold Storage: Investigating the Potential of an NLRP3 Inflammasome Inhibitor (MCC950) for Preconditioning Donor Kidneys.

BACKGROUND: Brain death (BD) and cold storage (CS) are critical factors that induce inflammation in donor kidneys, compromising organ quality. We investigated whether treating kidneys from BD rats with an inflammasome Nod-like receptor family pyrin domain containing 3 (NLRP3) inhibitor (MCC950) followed by CS could reduce kidney inflammation. METHODS: BD rats were assigned to MCC950-treated or nontreated (NT) groups. Kidneys were evaluated immediately before CS (T0) and after 12 h (T12) and 24 h (T24) of CS. Mean arterial pressure, serum creatinine, gene/protein expression, and histology were evaluated. RESULTS: At T0, MCC950 treatment did not affect mean arterial pressure but tended to reduce serum creatinine and ameliorated the histological score of acute tubular necrosis. However, MCC950 reduced NLRP3, caspase-1, interleukin (IL)-1ß, IL-6, Kim-1, nuclear factor kappa B, tumor necrosis factor alpha, and caspase-3 gene expression while increasing IL-10 cytokine gene expression. After 12 h of CS, only the expression of the NLRP3 and caspase-1 genes decreased, and after 24 h of CS, no further changes in the gene expression profile were observed. The levels of the inflammasome proteins NLRP3, caspase-1, and IL-1ß consistently decreased across all time points (T0, T12, and T24). CONCLUSIONS: These findings suggest that MCC950 treatment holds promise for mitigating the proinflammatory state observed in kidneys after BD and CS.

Different Patterns of Foxp3 Gene Expression in Pre-and Post-Transplantation Kidney Biopsies and the Effect of Use Mammalian Target of Rapamycin Inhibitors.

BACKGROUND: Trafficking of regulatory T cells (Tregs) modulates the inflammatory response after kidney transplantation (KTx). There is scarce information on whether circulating and intragraft Tregs are similarly affected by immunosuppressive drugs and the type of deceased kidney donor. METHODS: FOXP3 gene expression was measured in the pretransplant kidney biopsies (PIBx) from donors who met extended (ECD) and standard (SCD) criteria donors. In the third month after KTx, the patients were divided according to tacrolimus (Tac) or everolimus (Eve) and the type of kidney they had received. FOXP3 gene expression in the peripheral blood (PB) and kidney biopsies (Bx) was analyzed using real-time polymerase chain reaction. RESULTS: FOXP3 gene expression in the PIBx was higher in ECD kidneys. FOXP3 gene expression in the PB and Bx was greater in Eve- than in Tac-treated patients. However, SCD recipients treated with Eve (SCD/Eve) had higher FOXP3 expression than ECD/Eve. CONCLUSION: Pretransplant kidney biopsies from ECD kidneys had higher FOXP3 gene expression than SCD, and the use of Eve may affect the expression of the FOXP3 gene only in SCD kidneys.

Clinical outcomes of COVID-19 in patients undergoing chronic hemodialysis and peritoneal dialysis / Desfechos clínicos da COVID-19 em pacientes submetidos à hemodiálise crônica e diálise peritoneal

Abstract Background: The reported incidence and fatality rate of the severe acute respiratory syndrome coronavirus 2 in patients receiving chronic dialysis are higher than in the general population. We sought to study the outcomes following coronavirus disease 2019 (COVID-19) diagnosis in patients undergoing chronic hemodialysis (HD) or peritoneal dialysis (PD) in a single center in Brazil. Methods: Of the 522 patients on dialysis evaluated between March 1, 2020, and October 1, 2021, those presenting symptoms or with a history of close contact with COVID-19 patients were tested with reverse-transcription polymerase chain reaction of samples from nasopharyngeal swabs. Results: Of the 522 patients, 120 were positive for COVID-19 infection, of which 86% were on HD and 14% in the PD program. The incidence per 10,000 inhabitants was higher in the HD group than in the PD group (2,423.5 vs. 1,752.5). The mortality per 10,000 inhabitants (470.5 vs. 927.8) and the fatality rate (19.4 vs. 52.9%, p = 0.005) were higher in the PD group. The PD group also had a higher need for hospitalization, intensive care, and mechanical ventilation. Conclusions: We advise caution when considering strategies to transfer patients from HD to the PD program to minimize the risk of COVID-19 for patients on HD.

Resumo Antecedentes: A incidência e a taxa de letalidade da síndrome respiratória aguda grave por coronavírus 2 relatadas em pacientes em diálise crônica são mais elevadas do que na população em geral. Procuramos estudar os desfechos após o diagnóstico da doença por coronavírus 2019 (COVID-19) em pacientes submetidos à hemodiálise crônica (HD) ou diálise peritoneal (DP) em um único centro no Brasil. Métodos: Dos 522 pacientes em diálise avaliados entre 1º de Março de 2020 e 1º de Outubro de 2021, aqueles que apresentaram sintomas ou tiveram histórico de contato próximo com pacientes com COVID-19 foram testados com reação em cadeia da polimerase de transcrição reversa por meio de amostras de esfregaços nasofaríngeos. Resultados: Dos 522 pacientes, 120 foram positivos para infecção por COVID-19, dos quais 86% estavam em HD e 14% no programa de DP. A incidência por 10.000 habitantes foi maior no grupo HD do que no grupo DP (2.423,5 vs. 1.752,5). A mortalidade por 10.000 habitantes (470,5 vs. 927,8) e a taxa de letalidade (19,4 vs. 52,9%, p = 0,005) foram mais elevadas no grupo DP. O grupo DP também apresentou uma maior necessidade de hospitalização, terapia intensiva e ventilação mecânica. Conclusões: Recomendamos cautela ao considerar estratégias de transferência de pacientes do programa de HD para o de DP a fim de minimizar o risco de COVID-19 para pacientes em HD.

Evaluation of doxorubicin in three-dimensional culture of breast cancer cells and the response in PI3K/AKT/PTEN signaling pathways: a pilot study.

Breast cancer (BC) has a high mortality rate, which is attributed to the absence of effective treatment markers. Doxorubicin (DOX) was evaluated by molecular docking in vitro in cultured BC spheroids and its association with genes involved in the PI3K/AKT/PTEN signaling pathway. Spheroids were obtained from a primary BC. The selected compound was used for molecular docking experiments. Spheroids were treated with DOX for 1 (D1) and 9 (D9) days. qPCR was used to evaluate PIK3CA, HIF-1α, VEGF-A, PTEN expression. Treatment with DOX (1 µM) significantly increased the number of spheroids (D1), whereas exposure to chemotherapy at 2 µM on D9 was more effective. DOX treatment resulted in significantly higher expression of VEGF-A, HIF-1α and PIK3CA by D1 and HIF-1α and PTEN were upregulated by D9. Compared to treatment on D1 with D9 (1 µM) had significantly higher PTEN and lower PIK3CA gene expression. The genes HIF-1α and PTEN were more expressed with 2 µM of DOX while VEGF-A was downregulated. D1 vs. D9 exhibited reduced VEGF-A, HIF-1α, and PIK3CA expression and upregulation of PTEN expression. DOX effects at the molecular mechanisms can be involved the modulation of genes related to angiogenesis cell proliferation and tumor growth in BC tissue spheroids.

Distinct global DNA methylation and NF-κB expression profile of preimplantation biopsies from ideal and non-ideal kidneys.

BACKGROUND: Epigenetic mechanisms may affect the ideal and non-ideal kidneys selected for transplantation and their inflammatory gene expression profile differently and may contribute to poor clinical outcomes. OBJECTIVE: Study the Global DNA methylation and the expression profiles of the DNA methyltransferases (DNMTs) and nuclear factor kappa B (NF-κB) in preimplantation kidney biopsies from ideal and non-ideal kidneys (expanded criteria donor (ECD) and with KDPI > 85%). METHODS: In a sample consisting of 45 consecutive pre-implantation biopsies, global DNA methylation levels were detected by LINE-1 repeated elements using bisulfite pyrosequencing. DNMT gene expression was assessed by real-time quantitative polymerase chain reaction, and NF-κB protein expression by immunofluorescence. RESULTS: ECD kidneys displayed increased methylation levels in LINE-1, and DNMT1 and DNMT3B expression was upregulated when comparing ECD to standard criteria donor kidneys. Similarly, kidneys with KDPI > 85% exhibited increased LINE-1 methylation and DNMT1 upregulation when compared to a KDPI ≤ 85%. NF-κB protein expression levels were greatly increased in both types of non-ideal kidneys compared to ideal kidneys. Moreover, hypermethylation of LINE-1 was associated with cold ischemia time > 20 h and ECD kidney classification. CONCLUSIONS: This study shows that global DNA hypermethylation and high expression of NF-κB occurred in both types of non-ideal kidneys and were associated with prolonged cold ischemia time. Global DNA methylation can be a useful tool to assess non-ideal kidneys and hence, could be used to expand the pool of kidneys donors.

SARS-CoV-2 assessment in an outpatient dialysis facility of a single center in Brazil

ABSTRACT Background: The reported incidence and fatality rates of SARS-CoV-2 infection in patients receiving maintenance dialysis are higher than those of the general population. Objective: This study sought to characterize the clinical characteristics and outcomes following COVID-19 infection in this population in a single center in Brazil. Methods: Out of 497 dialysis patients evaluated between March 1st, 2020 and February 1st, 2021, those presenting symptoms or history of close contact with COVID-19 patients were tested. Disease severity was categorized as mild, moderate, or severe. Results: Out of the 497 patients, 8.8% tested positive for COVID-19. These patients were predominantly male (59%), mean age 57.5 ± 17. Hospitalization was required for 45.4% of patients and 15.9% received mechanical ventilation. Symptoms such as fever, cough, dyspnea and asthenia were more frequent in the severe group. Neutrophil to lymphocyte ratio, C- reactive protein, glutamic oxalacetic transaminase and lactic dehydrogenase were significantly higher in the severe group, while hemoglobin and lymphocyte counts were significantly lower. Chest CT >50% of ground glass lesions was the risk factor associated with severe disease and need for hospitalization. The incidence of a thromboembolic event was of 22.7% in this population. The incidence, mortality, and case fatality rates were 954.4/10,000 patients, 151.8/10,000 patients, and 15.9%, respectively. Conclusions: The incidence, mortality and case fatality rates in our cohort were significantly higher than those reported for the general population. To institute appropriate control measures and early vaccination in dialysis facilities is imperative to prevent the spread of COVID-19 infection.

Therapeutic potential of human induced pluripotent stem cells and renal progenitor cells in experimental chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is a global public health problem. Cell therapy using pluripotent stem cells represents an attractive therapeutic approach for the treatment of CKD. METHODS: We transplanted mitomycin C (MMC)-treated human induced pluripotent stem cells (hiPSCs) and renal progenitor cells (RPCs) into a CKD rat model system. The RPC and hiPSC cells were characterized by immunofluorescence and qRT-PCR. Untreated 5/6 nephrectomized rats were compared to CKD animals receiving the same amount of MMC-treated hiPSCs or RPCs. Renal function, histology, and immunohistochemistry were evaluated 45 days post-surgery. RESULTS: We successfully generated hiPSCs from peripheral blood and differentiated them into RPCs expressing renal progenitor genes (PAX2, WT1, SIX2, and SALL1) and podocyte-related genes (SYNPO, NPHS1). RPCs also exhibited reduced OCT4 expression, confirming the loss of pluripotency. After cell transplantation into CKD rats, the body weight change was significantly increased in both hiPSC and RPC groups, in comparison with the control group. Creatinine clearance (CCr) was preserved only in the hiPSC group. Similarly, the number of macrophages in the kidneys of the hiPSC group reached a statistically significant reduction, when compared to control rats. Both treatments reduced positive staining for the marker α-smooth muscle actin. Histological features showed decreased tubulointerstitial damage (interstitial fibrosis and tubular atrophy) as well as a reduction in glomerulosclerosis in both iPSC and RPC groups. CONCLUSIONS: In conclusion, we describe that both MMC-treated hiPSCs and RPCs exert beneficial effects in attenuating CKD progression. Both cell types were equally efficient to reduce histological damage and weight loss caused by CKD. hiPSCs seem to be more efficient than RPCs, possibly due to a paracrine effect triggered by hiPSCs. These results demonstrate that the use of MMC-treated hiPSCs and RPCs improves clinical and histological CKD parameters, avoided tumor formation, and therefore may be a promising cell therapy strategy for CKD.

Does protein supplementation and exercise interfere with renal function and structure? / A suplementação de proteína e o exercício interferem na função e estrutura renal? / ¿la suplementación de proteínas y el ejercicio interfieren en la función y estructura renal?

ABSTRACT Introduction: During physical activity, the body diverts blood to essential areas such as skeletal muscle, reducing the supply to non-essential areas such as the kidney. Whey protein is one of the most widely used supplements in gyms. Objectives: To evaluate renal function and renal structure in rats submitted to physical exercise with and without the use of protein supplementation. Methods: The protein used was Whey Hydro PRO 2 - Probiotica®. It was administered orally (by gavage), diluted in mineral water (1.8 g/kg of body weight, shortly after swimming training). The rats were divided into four groups: rats with exercise (Exc), rats without exercise (ñExc), rats with exercise and with protein supplementation (Prot/Exc) and rats without exercise and with protein supplementation (Prot/ñExc). The training consisted of swimming for 30 minutes, using load equivalent to 2% of body weight, five times a week for a total of 10 weeks. The protein was administered by gavage, once daily, immediately after the training. Results: A reduced glomerular filtration rate was observed in the animals of the Exc group compared to those of the Prot/Exc group. Plasma creatinine values were similar between the groups submitted to exercise and those not submitted to exercise. Plasma sodium and the sodium excretion fraction were lower in the Prot/Exc group compared to the Exc group. Urinary excretion was similar between groups. Histological analysis: Significant hydropic degeneration was observed in the animals that received protein supplementation and submitted to exercise. Conclusion: These results show that exercise associated with protein supplementation (2g/day/kg) leads to changes in the tubular mechanisms of sodium adjustments and structural changes in the renal parenchyma. Level of evidence II; Therapeutic studies - Investigation the results of treatment.

RESUMO Introdução: Durante a atividade física o corpo faz remanejamento sanguíneo para áreas essenciais como a musculatura esquelética, reduzindo o suprimento em áreas não essenciais como o rim. O whey protein (proteína do soro do leite) é um dos suplementos mais usados nas academias. Objetivos: Avaliar a função e a estrutura renal em ratos submetidos ao exercício físico sem e com o uso da suplementação de proteína. Métodos: A proteína usada foi Whey Hydro PRO 2 - Probiótica®, sendo administrada por via oral (gavagem), diluída em água mineral (1,8 g/kg de peso corporal logo após o treino de natação). Os ratos foram divididos em quatro grupos: ratos com exercício (Exc), ratos sem exercício (ñExc), ratos com exercício e com suplementação alimentar de proteína (Prot/Exc) e ratos sem exercício e com suplementação alimentar de proteína (Prot/ñExc). O treinamento consistia em natação por 30 minutos, com utilização de carga, equivalente a 2% do peso corporal, 5 vezes por semana em um total de 10 semanas. A proteína foi administrada por gavagem, uma vez ao dia e logo depois do treino. Resultados: Observou-se queda da filtração glomerular renal nos animais do grupo Exc vs. Prot/Exc. Os valores de creatinina plasmática foram semelhantes entre os grupos que praticaram o exercício vs. os que não praticaram. Para o sódio plasmático e a fração de excreção de sódio, os valores foram menores no grupo Prot/Exc quando comparados com o grupo Exc. A excreção urinária de ureia foi semelhante entre os grupos. Análise histológica: Observou-se degeneração hidrópica significativa nos animais que receberam a suplementação de proteína e realizaram o exercício. Conclusão: Esses resultados mostram que o exercício em conjunto com a suplementação de proteína (2 g/dia/Kg), determina alterações nos mecanismos tubulares de ajustes do sódio e alterações estruturais no parênquima renal. Nível de evidência II; Estudos terapêuticos - Investigação dos resultados do tratamento.

RESUMEN Introducción: Durante la actividad física el cuerpo hace reubicación sanguínea hacia áreas esenciales como la musculatura esquelética, reduciendo el suministro en áreas no esenciales como el riñón. La whey protein (proteína del suero de la leche) es uno de los suplementos más usados en los gimnasios. Objetivos: Evaluar la función y la estructura renal en ratones sometidos al ejercicio físico sin y con el uso de la suplementación de proteína. Métodos: La proteína utilizada fue Whey Hydro PRO 2 - Probiótica® siendo administrada vía oral (gavaje), diluida en agua mineral (1,8 g/kg de peso corporal luego después del entrenamiento de natación). Los ratones fueron divididos en cuatro grupos: ratones con ejercicio (Exc), ratones sin ejercicio (ñExc), ratones con ejercicio y con suplementación alimentaria de proteína (Prot/Exc) y ratones sin ejercicio y con suplementación alimenticia de proteína (Prot/ñExc). El entrenamiento consistía en natación por 30 minutos, con uso de carga, equivalente al 2% del peso corporal, 5 veces por semana en un total de 10 semanas. La proteína fue administrada por gavaje, una vez al día y luego después del entrenamiento. Resultados: Se observó caída de la filtración glomerular renal en los animales del grupo Exc vs Prot/Exc. Los valores de creatinina plasmática fueron semejantes entre los grupos que practicaron el ejercicio vs los no practicaron. Para el sodio plasmático y la fracción de excreción de sodio, los valores fueron menores en el grupo Prot/Exc cuando comparados con el Exc. La excreción urinaria de urea fue semejante entre los grupos. Análisis histológico: Se observó degeneración hidrópica significativa en los animales que recibieron la suplementación de proteínas y no realizaron el ejercicio. Conclusión: Estos resultados muestran que el ejercicio en conjunto con la suplementación de proteína (2 g/día/Kg), determina alteraciones en los mecanismos tubulares de ajustes del sodio y alteraciones estructurales en el parénquima renal. Nivel de evidencia II; Estudios terapéuticos - Investigación de los resultados del tratamiento.

Induced Pluripotent Stem Cells Reduce Progression of Experimental Chronic Kidney Disease but Develop Wilms' Tumors.

The therapeutic effect of induced pluripotent stem cells (iPSs) on the progression of chronic kidney disease (CKD) has not yet been demonstrated. In this study, we sought to assess whether treatment with iPSs retards progression of CKD when compared with bone marrow mesenchymal stem cells (BMSCs). Untreated 5/6 nephrectomized rats were compared with CKD animals receiving BMSCs or iPSs. Renal function, histology, immunohistochemistry, and gene expression were studied. Implanted iPSs were tracked by the SRY gene expression analysis. Both treatments minimized elevation in serum creatinine, significantly improved clearance, and slowed down progression of disease. The proteinuria was reduced only in the iPS group. Both treatments reduced glomerulosclerosis, iPSs decreased macrophage infiltration, and TGF-ß was reduced in kidneys from the BMSC group. Both types of treatments increased VEGF gene expression, TGF-ß was upregulated only in the iPS group, and IL-10 had low expression in both groups. The SRY gene was found in 5/8 rats treated with iPSs. These 5 animals presented tumors with histology and cells highly staining positive for PCNA and Wilms' tumor protein antibody characteristics of Wilms' tumor. These results suggest that iPSs may be efficient to retard progression of CKD but carry the risk of Wilms' tumor development.

Validation of an Experimental Model to Study Less Severe Chronic Renal Failure.

PURPOSE: The 5/6 nephrectomy, mimics the stages of human chronic renal failure (CRF), but the procedure causes severe renal functional and morphological damage that could interfere with the evaluation of therapies for slowing the progression of the disease. This study summarizes the results of renal function, histology, and immunohistochemical findings in rats undergoing a 2/3 nephrectomy. METHODS: The rats were distributed in groups according to the type of nephrectomy: CRF5/6: induced by a 5/6 renal mass reduction and CRF2/3: less severe CRF. The body weight and blood pressure were monitored, and the serum creatinine (SCr), creatinine clearance (CCr), urine osmolality, and 24-h proteinuria (PT24h) were measured. CRF progression was evaluated by the rate of decline of CCr (RCCr). Histology and immunohistochemistry were performed in the remnant kidneys. Statistical analysis was done by unpaired t-test, and a P-value < 0.05 was taken as a statistical significance. RESULTS: Compared to the CRF5/6 group, the CRF2/3 model had a lower SCr, PT24h, CCr, and variations of the SCr from baseline. The disease progression was also significantly slower. The renal histopathological findings revealed fewer chronic lesions in rats with CRF2/3. Similarly, we observed less macrophage accumulation as well as lower proliferative activity and expression of fibronectin and a-smooth muscle-actin in the CRF2/3 model. CONCLUSIONS: The CRF2/3 model presented with a pattern of less severe CRF, functionally and morphologically, compared to the classical CRF5/6 model, and the CRF2/3 model may be useful for evaluating therapeutic interventions that target the early stages of CRF.

Comparative effects of mesenchymal stem cell therapy in distinct stages of chronic renal failure.

BACKGROUND: The therapeutic potential of adult stem cells in the treatment of chronic diseases is becoming increasingly evident. In the present study, we sought to assess whether treatment with mesenchymal stem cells (MSCs) efficiently retards progression of chronic renal failure (CRF) when administered to experimental models of less severe CRF. METHODS: We used two renal mass reduction models to simulate different stages of CRF (5/6 or 2/3 mass renal reduction). Renal functional parameters measured were serum creatinine (SCr), creatinine clearance (CCr), rate of decline in CCr (RCCr), and 24-h proteinuria (PT24h). We also evaluated renal morphology by histology and immunohistochemistry. MSCs were obtained from bone marrow aspirates and injected into the renal parenchyma of the remnant kidneys of both groups of rats with CRF (MSC5/6 or MSC2/3). RESULTS: Animals from groups MSC5/6 and CRF2/3 seemed to benefit from MSC therapy because they showed significantly reduction in SCr and PT24h, increase in CCr and slowed the RCCr after 90 days. Treatment reduced glomerulosclerosis but significant improvement did occur in the tubulointerstitial compartment with much less fibrosis and atrophy. MSC therapy reduced inflammation by decreasing macrophage accumulation proliferative activity (PCNA-positive cells) and fibrosis (α-SM-actin). Comparisons of renal functional and morphological parameters responses between the two groups showed that rats MSC2/3 were more responsive to MSC therapy than MSC5/6. CONCLUSION: This study showed that MSC therapy is efficient to retard CRF progression and might be more effective when administered during less severe stages of CRF.

Supplementation with omega-3 polyunsaturated fatty acids and experimental tacrolimus-induced nephrotoxicity.

OBJECTIVES: Calcineurin inhibitors are effective immunosuppressive agents, but associated adverse effects such as nephrotoxicity may limit efficacy. Dietary fish oil may minimize nephrotoxicity caused by long-term use of calcineurin inhibitors. The purpose of the present study was to evaluate the effects of omega-3 fatty acids on calcineurin inhibitor nephrotoxicity in rats that had normal kidney function or chronic kidney failure. MATERIALS AND METHODS: Rats that had normal kidney function or chronic renal failure that was induced by mass reduction surgery were treated with tacrolimus without or with fish oil, fish oil alone, or olive oil. Kidney function and histology were evaluated after 14 days. RESULTS: Mean body weight loss, serum creatinine, change in serum creatinine, and rate of decrease in creatinine clearance were greater in normal rats that received than did not receive tacrolimus. Tacrolimus nephrotoxicity was greater in rats that had chronic renal failure than normal kidney function, but the mean change in serum creatinine was significantly lower in rats with chronic renal failure that were treated with tacrolimus and fish oil than tacrolimus alone. Fish oil supplementation was associated with fewer abnormal histopathologic lesions in the kidneys of tacrolimustreated rats that had normal kidney function or chronic renal failure (not signifant). CONCLUSIONS: Fish oil may be protective against the development of kidney dysfunction and histopathologic changes in rats treated with tacrolimus.

Gene expression profile of 5-fluorouracil metabolic enzymes in laryngeal cancer cell line: predictive parameters for response to 5-fluorouracil-based chemotherapy.

BACKGROUND: 5-fluorouracil (5-FU) is an antifolate chemotherapeutic that has become established in many therapeutic regimes, but sensitivity variations and development of resistance are common problems that limit the efficiency of the treatments. Inter-individual variations to 5-FU outcome have been attributed to different expression profiles of genes related to folate metabolism. METHODS: To elucidate the mechanisms of variations to 5-FU outcome, the authors investigated MTHFR, DHFR, TYMS and SLC19A1 folate genes expression for 5-FU response in laryngeal cancer cell line (Hep-2). Concentrations of 10, 50, and 100 ng/mL of 5-FU chemotherapeutic were added separately in Hep-2 cell line for 24 hours at 37 °C. Cell sensibility was evaluated with fluorescein isothiocyanate (FITC) label Bcl-2 by flow cytometry. The real-time quantitative PCR (qPCR) technique was performed for quantification of gene expression using TaqMan(®) Gene Expression Assay. ANOVA and Bonferroni's post hoc tests were utilized to statistical analysis. RESULTS: The numbers of viable Hep-2 cells with 10, 50, and 100 ng/mL concentrations of 5-FU chemotherapy were 15.87, 28.3 and 68.9%, respectively. Statistical analysis showed significant association between control group and increased expression for TYMS gene in cells treated with 100 ng/mL/5-FU chemotherapy (P<0.05). CONCLUSIONS: The authors found association between the highest 5-FU dose chemotherapy and increased expression levels for TYMS folate gene in laryngeal cancer cell line. Although these experiments were performed in vitro, the results suggest that genetic factors are thought to play an important role in drug metabolism and may be useful for predicting treatment outcomes.

Alterations in the expression pattern of MTHFR, DHFR, TYMS, and SLC19A1 genes after treatment of laryngeal cancer cells with high and low doses of methotrexate.

Inter-individual variations to methotrexate (MTX) outcome have been attributed to different expression profiles of genes related to folate metabolism. To elucidate the mechanisms of variations to MTX outcome, we investigated MTHFR, DHFR, TYMS, and SLC19A1 gene expression profiles by quantifying the mRNA level of the genes involved in folate metabolism to MTX response in laryngeal cancer cell line (HEP-2). For this, three different concentrations of MTX (0.25, 25, and 75 µmol) were added separately in HEP-2 cell line for 24 h at 37 °C. Apoptotis quantification was evaluated with fluorescein isothiocyanate-labeled Bcl-2 by flow cytometry. Real-time quantitative PCR technique was performed by quantification of gene expression with TaqMan® Gene Expression Assay. ANOVA and Bonferroni's post hoc tests were utilized for statistical analysis. The results showed that the numbers of apoptotic HEP-2 cells with 0.25, 25.0, and 75.0 µmol of MTX were 14.57, 77.54, and 91.58%, respectively. We found that the expression levels for MTHFR, DHFR, TYMS, and SLC19A1 genes were increased in cells with 75.0 µmol of MTX (p < 0.05). Moreover, SLC19A1 gene presented lower expression in cells treated with 0.25 µmol of MTX (p < 0.05). In conclusion, our data suggest that MTHFR, DHFR, TYMS, and SLC19A1 genes present increased expression after the highest application of MTX dose in laryngeal cancer cell line. Furthermore, SLC19A1 gene also presents decreased expression after the lowest application of MTX dose in laryngeal cancer cell line. Significant alterations of expression of these studied genes in cell culture model may give support for studies in clinical practice and predict interesting and often novel mechanisms of resistance of MTX chemotherapy.

Effects of polyunsaturated fatty acids (PUFAs) in the treatment of experimental chronic renal failure.

PURPOSE: A diet with polyunsaturated fatty acid (PUFA) supplementation has been reported to reduce renal and cardiac diseases. This study sought to elucidate whether PUFAs derived from plant or marine oils could have beneficial effects on the progression of experimental chronic renal failure (CRF). METHODS: Experimental CRF was achieved by a 5/6 nephrectomy model. Male Wistar rats were divided into groups and given daily supplements of fish oil (group FO), flaxseed oil (group FXO), or soybean oil (control-group SO) for 30 days. Serum creatinine (sCr), 24-h proteinuria, total cholesterol, triglycerides, and creatinine clearance (CLcr) were measured at day 0 and 30 days after surgery when the rats were euthanized for histological analysis of the remnant kidney. RESULTS: After 30 days, we observed lower levels of sCr in the groups supplemented with PUFA when compared with the control group (FO: 0.92 ± 0.13; FXO: 1.06 ± 0.28; SO: 1.32 ± 0.47 mg/dL) and significantly slower variations of sCr (ΔsCr) in the groups treated with PUFAs (FO = 0.35 ± 0.16; FXO = 0.47 ± 0.31; OS = 0.72 ± 0.43; mg/dL, P = 0.041). Similarly, the CLcr of both of the groups that received PUFAs was significantly slower than the rats in the control group (FO: 0.45 ± 0.15; FXO: 0.60 ± 0.09; SO: 0.28 ± 0.06 mL/min/day; P = 0.01). The rats that received PUFA supplements also presented significantly less histological lesions compared with the control group. CONCLUSIONS: These results suggest a beneficial effect of dietary supplementation with flaxseed or fish oil in rats with CRF.

[Blood mesenchymal stem cell culture from the umbilical cord with and without Ficoll-Paque density gradient method]. / Cultivo de células mesenquimais do sangue de cordão umbilical com e sem uso do gradiente de densidade Ficoll-Paque.

OBJECTIVES: Implantation of cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood with and without using the Ficoll-Paque gradient density method (d=1.077 g/ml). METHODS: Ten samples of the umbilical cord blood obtained from full-term deliveries were submitted to two different procedures of mesenchymal stem cell culture: a) Method without the Ficoll-Paque density gradient, which concentrates all nucleated cells; b) Method with the Ficoll-Paque density gradient, which selects only low-density mononuclear cells. Cells were initially plated into 25 cm(2) cultures flasks at a density of 1 x 10(7) nucleated cells/cm(2) and 1 x 10(6) mononuclear cells/cm(2). RESULTS: It was obtained 2-13 x 10(7) (median = 2.35 x 10(7)) nucleated cells/cm(2) by the method without the Ficoll-Paque gradient density, and 3.7-15.7 x 10(6) (median = 7.2 x 10(6)) mononuclear cells/cm(2) by the method with the Ficoll-Paque gradient density. In all cultures adherent cells were observed 24 hours after being cultured. Cells presented fibroblastoid and epithelioid morphology. In most of the cultures, cell proliferation occurred in the first week, but after the second week only some cultures - derived from the method without the Ficoll-Paque gradient density-maintained the growth rate reaching confluence. Those cultures were submitted to trypsinization with 0.25% trypsin/EDTA solution and cultured for two to three months. CONCLUSION: In the samples analyzed, cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood by the method without the Ficoll-Paque density gradient was more efficient than the method with the Ficoll-Paque density gradient.

Cultivo de células mesenquimais do sangue de cordão umbilical com e sem uso do gradiente de densidade Ficoll-Paque / Blood mesenchymal stem cell culture from the umbilical cord with and without Ficoll-Paque density gradient method

OBJETIVOS: Implantação de técnicas de isolamento e cultivo de células-tronco mesenquimais do sangue de cordão umbilical humano, com e sem uso de gradiente de densidade Ficoll-Paque (d=1,077g/ml). MÉTODOS: Dez amostras de sangue de cordão umbilical humano de gestação a termo foram submetidas a dois procedimentos de cultivo de células-tronco mesenquimais: sem gradiente de densidade Ficoll-Paque e com gradiente de densidade. As células foram semeadas em frascos de 25cm² a uma densidade de 1x10(7)células nucleadas/cm² (sem Ficoll) e 1,0x10(6) células mononucleares/cm² (com Ficoll). As células aderentes foram submetidas a marcação citoquímica com fosfatase ácida e reativo de Schiff. RESULTADOS: No procedimento sem gradiente de densidade Ficoll, foram obtidas 2,0-13,0x10(7) células nucleadas (mediana=2,35x10(7)) e, no procedimento com gradiente de densidade Ficoll, foram obtidas 3,7-15,7x10(6) células mononucleares (mediana=7,2x10(6)). Em todas as culturas foram observadas células aderentes 24 horas após o início de cultivo. As células apresentaram morfologias fibroblastóides ou epitelióides. Na maioria das culturas houve proliferação celular nas primeiras semanas de cultivo, mas após a segunda semana, somente três culturas provenientes do método sem gradiente de densidade Ficoll-Paque mantiveram crescimento celular, formando focos confluentes de células. Essas culturas foram submetidas a várias etapas de tripsinização para espalhamento ou subdivisão e permaneceram em cultivo por períodos que variaram de dois a três meses. CONCLUSÃO: Nas amostras estudadas, o isolamento e cultivo de células-tronco mesenquimais do sangue de cordão umbilical humano pelo método sem gradiente de densidade Ficoll-Paque foi mais eficiente do que o método com gradiente de densidade Ficoll-Paque.

OBJECTIVES: Implantation of cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood with and without using the Ficoll-Paque gradient density method (d=1.077g/ml). METHODS: Ten samples of the umbilical cord blood obtained from full-term deliveries were submitted to two different procedures of mesenchymal stem cell culture: a) Method without the Ficoll-Paque density gradient, which concentrates all nucleated cells; b) Method with the Ficoll-Paque density gradient, which selects only low-density mononuclear cells. Cells were initially plated into 25 cm² cultures flasks at a density of 1x10(7) nucleated cells/cm² and 1x10(6) mononuclear cells/cm². RESULTS: It was obtained 2-13x10(7) (median = 2.35x10(7)) nucleated cells/cm² by the method without the Ficoll-Paque gradient density, and 3.7-15.7x10(6) (median = 7.2x10(6)) mononuclear cells/cm² by the method with the Ficoll-Paque gradient density. In all cultures adherent cells were observed 24 hours after being cultured. Cells presented fibroblastoid and epithelioid morphology. In most of the cultures, cell proliferation occurred in the first week, but after the second week only some cultures - derived from the method without the Ficoll-Paque gradient density - maintained the growth rate reaching confluence. Those cultures were submitted to trypsinization with 0.25 percent trypsin/EDTA solution and cultured for two to three months. CONCLUSION: In the samples analyzed, cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood by the method without the Ficoll-Paque density gradient was more efficient than the method with the Ficoll-Paque density gradient.