Browsing by Author "Reis, F."
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- Aging is associated with impaired renal function, INF-gamma induced inflammation and with alterations in iron regulatory proteins gene expressionPublication . Costa, E.; Fernandes, J.; Ribeiro, S.; Garrido, P.; Rocha-Pereira, P.; Coimbra, S.; Catarino, C.; Reis, F.; Belo, L.; Bronze-da-Rocha, E.; Vala, Helena; Alves, R.; Santos-Silva, A.Our aim was to contribute to a better understanding of the pathophysiology of anemia in elderly, by studying how aging affects renal function, ironmetabolism, erythropoiesis and the inflammatory response, using an experimental animal model. The study was performed in male Wistar, a group of young rats with 2 months age and an old one with 18 months age. Old rats presented a significant higher urea, creatinine, interferon (INF)-gamma, ferritin and soluble transferrin receptor serum levels, as well as increased counts of reticulocytes and RDW. In addition, these rats showed significant lower erythropoietin (EPO) and iron serum levels. Concerning gene expression of iron regulatory proteins, old rats presented significantly higher mRNA levels of hepcidin (Hamp), transferrin (TF), transferrin receptor 2 (TfR2) and hemojuvelin (HJV); divalent metal transporter 1 (DMT1) mRNA levels were significantly higher in duodenal tissue; EPO gene expression was significantly higher in liver and lower in kidney, and the expression of the EPOR was significantly higher in both liver and kidney. Our results showed that aging is associated with impaired renal function, which could be in turn related with the inflammatory process and with a decline in EPO renal production. Moreover, we also propose that aging may be associatedwith INF-gamma-induced inflammation and with alterations upon iron regulatory proteins gene expression.
- Biochemical and histological effects of sitagliptin on Zucker Diabetic Fatty rat pancreasPublication . Mega, C.; Vala, Helena; Teixeira de Lemos, E.; Fernandes, R.; Oliveira, J.; Ferreira, L.; Teixeira, F.; Reis, F.Background: Inhibition of dipeptidyl peptidase-4 (DPP-4) activity by sitagliptin has been shown to improve glycemic control in patients with type 2 diabetes Mellitus (T2DM) by prolonging the actions of incretin hormones, but the really impact of low-dose sitagliptin treatment on pancreas lesions is almost unknown. This study aimed to evaluate the effects of sitagliptin on biochemical and histological (pancreas) parameters of Zucker Diabetic Fatty (ZDF, fa/fa) rats, an animal model of T2DM. Methods: Diabetic (fa/fa) ZDF male rats were treated with vehicle or sitagliptin (10 mg/kg BW/day) during 6 weeks (n=8 each). The following parameters were assessed: serum glycaemia, HbA1c, insulin and lipid profile; serum and pancreas oxidative stress (MDA) and endocrine and exocrine pancreas histology, estimating and rating inflammatory infiltrate, fibrosis, vacuolization and congestion in a semiquantitative score ranging from 0 (minimal) to 3 (severe and extensive damage). Results: Sitagliptin in diabetic ZDF rats promoted beneficial effects on dysglycaemia, dyslipidaemia, inflammatory profile and pancreatic oxidative stress. Endocrine and exocrine pancreas presented a reduction/amelioration of fibrosis severity, inflammatory infiltrate, intra-islet vacuolation, and congestion vs the vehicle-treated diabetic rats. Conclusions: The favourable biochemical profile promoted by sitagliptin in the diabetic rats, together with a protection against endocrine and exocrine pancreas lesions, might represent a further advantage of low doses of sitagliptin in the management of T2DM.
- Cyclosporine A-induced nephrotoxicity is ameliorated by dose reduction and conversion to Sirolimus in the ratPublication . Sereno, J.; Vala, Helena; Nunes, S.; Rocha-Pereira, P.; Carvalho, E.; Alves, R.; Teixeira, F.; Reis, F.Side-effect minimization strategies to avoid serious side-effects of cyclosporine A (CsA), such as nephrotoxicity, have been mainly based on dose reduction and conversion to other putatively less nephrotoxic drugs, such as sirolimus (SRL), an inhibitor of the mammalian target of rapamycin. This study intended to evaluate the impact of protocols based on CsA dose reduction and further conversion to SRL on kidney function and lesions, based on serum, urine and renal tissue markers. The following 3 groups (n=6) were tested during a 9-week protocol: control (vehicle); CsA (5 mg/kg/day) and Red + Conv (CsA 30 mg/kg/day during 3 weeks + 3 weeks with CsA 5 mg/kg/day + SRL 1 mg/kg/day during the last 3 weeks). The following parameters were analysed: blood pressure, heart rate and biochemical data; serum and urine contents and clearances of creatinine, urea and neutrophil gelatinase-associated lipocalin, as well as, glomerular filtration rate; kidney lipid peroxidation and clearance; kidney lesions were evaluated and protein expression was performed by immunohistochemistry. After the first 3 weeks of CsA (30 mg/kg/day) treatment animals showed body weight loss, hypertension, tachycardia, as well as, increased serum levels of non-HDL cholesterol, glucose, triglycerides, creatinine and urea, accompanied by decreased GFR and insulin levels. In addition, a significant increase in the expression of connective tissue growth factor, Kim-1, mammalian target of rapamycin, nuclear factor-κβ1 and transforming growth factor-β was found in the kidney, accompanied by extensive renal damage. The following 3 weeks with CsA dose reduction revealed amelioration of vascular and glomerular lesions, but without significant tubular improvement. The last 3 weeks with the conversion to sirolimus revealed high serum and urine NGAL contents but the CsA-evoked renal damage was substantially ameliorated, by reduced of connective tissue growth factor, mammalian target of rapamycin, nuclear factor-κβ1 protein expression. In conclusion, CsA nephrotoxicity is dose dependent and moderate dysfunction could be ameliorated/prevented by SRL conversion, which could be pivotal for the preservation of kidney function and structure.
- Diabetic nephropathy amelioration by a low-dose sitagliptin in an animal model of type 2 diabetes (Zucker Diabetic Fatty rat)Publication . Mega, C.; Teixeira de Lemos, E.; Vala, Helena; Fernandes, R.; Oliveira, J.; Mascarenhas-Melo, F.; Teixeira, F.; Reis, F.This study was performed to assess the effect of chronic low-dose sitagliptin, a dipeptidyl peptidase 4 inhibitor, on metabolic profile and on renal lesions aggravation in a rat model of type-2 diabetic nephropathy, the Zucker Diabetic Fatty (ZDF) rat. Diabetic and obese ZDF (fa/fa) rats and their controls ZDF (+/+) were treated for 6 weeks with vehicle (control) or sitagliptin (10 mg/kg/bw). Blood/serum glucose, HbA1c, insulin, Total-c, TGs, urea and creatinine were assessed, as well as kidney glomerular and tubulointerstitial lesions (interstitial fibrosis/tubular atrophy), using a semi-quantitative rating from 0 (absent/normal) to 3 (severe and extensive damage). Vascular lesions were scored from 0-2. Sitagliptin in the diabetic rats promoted an amelioration of glycemia, HbA1c, Total-c and TGs, accompanied by a partial prevention of insulinopenia. Furthermore, together with urea increment prevention, renal lesions were ameliorated in the diabetic rats, including glomerular, tubulointerstitial and vascular lesions, accompanied by reduced lipid peroxidation. In conclusion, chronic low-dose sitagliptin treatment was able to ameliorate diabetic nephropathy, which might represent a key step forward in the management of T2DM and this serious complication.
- Exercise training decreases proinflammatory profile in Zucker diabetic (type 2) fatty ratsPublication . Teixeira-Lemos, Edite; Reis, F.; Baptista, S.; Pinto, R.; Sepodes, B.; Vala, Helena; Rocha-Pereira, P.; et al.Objective In the present study we evaluated the effect of exercise on the plasma levels of proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), and the anti-inflammatory molecule uric acid in the Zucker diabetic fatty (ZDF) rats that are more prone to develop type 2 diabetes mellitus. Methods Sixteen obese ZDF (Gmi fa/fa) rats (8 wk old, 228.40 ± 4.05 g) were randomly assigned to one of two groups (n = 8 each): an exercise-trained group and a sedentary one. In addition, 16 lean ZDF (Gmi +/+) rats (8 wk old, 199.00 ± 3.50 g) were subjected to identical sedentary and exercise conditioning (n = 8 each). Initially, rats swam 15 min/d (5 d/wk) in a 36°C bath. The exercise protocol was gradually increased by 15 min/d until a swimming period of 1 h/d (1 wk) was attained. Thereafter, rats swam 1 h/d, 3 d/wk, for an additional period of 11 wk. Rats were sacrificed 48 h after the last training period and the blood and pancreas were collected. Circulating levels of glucose, glycosylated hemoglobin, total cholesterol, triglycerides, insulin, uric acid, IL-6, and TNF-α were assessed. The concentrations of proinflammatory cytokines in the pancreas were also evaluated. Results In the diabetic ZDF (fa/fa) rats, exercise decreased hyperuricemia (−37.3%) and IL-6 and TNF-α levels (−16.9% and −12.7% respectively) and maintained the weight of the pancreas at near normal. Immunohistochemistry revealed a marked decrease in the expression of TNF-α and IL-6 in the pancreatic islet cells of ZDF (fa/fa) rats. Conclusion These results indicate that aerobic exercise is anti-inflammatory in nature.
- Main difficulties encountered in implementation of techniques for apoptosis detection in animal tissuesPublication . Garcia, C.; Cruz, R.; Mega, A.; Ortiz, A. L.; Teixeira de Lemos, E.; Ferreira, D.; Reis, F.; Vala, HelenaIntroduction: The implementation of methodologies capable of identifying apoptotic cells is a valuable tool in many biomedical studies. Much of the knowledge acquired about the mechanisms regulating apoptosis comes from observations in isolated cells; however, the properties shown by these assays do not always apply to the study of tissue samples. For detection of apoptosis in tissues, many investigators recommend the TUNEL method, nonetheless, other histochemical methodologies are available, and their choice must be carefully evaluated, taking into account the morphologic alterations which they intend to detect. Objective: To share encountered difficulties in the implementation of three different methods for the detection of apoptotic cells in animal tissue. Methods: Animal tissues, fixed in 10% neutral-buffered formalin and embedded in paraffin were submitted to immunohistochemical techniques using the TUNEL method (In situ Cell Death Kit, Roche) and polyclonal Bax and Bcl-2 antibodies (Santa Cruz Biotechnology). Results: After a long period of tests and costs, in which unspecific reactions, as well as several artifacts were obtained, the persistence to overcome these obstacles made all methods effective in the detection of apoptosis. Observed differences in immunolabeling were related to the type of tissues tested, which will be documented in the present study. Conclusions: Immunohistochemical methods are an approach to consider in the detection of apoptosis in animal tissues. Sharing the challenging difficulties found in our work, might obviate many tests and costs in its implementation in other laboratory facilities.
- Occurrence of Kimmelstiel-Wilson like lesions in the absence of diabetic nephropathy in dogs, may provide a natural model for further research in the corresponding human formPublication . Mega, C.; Mesquita, J.; Nóbrega, C.; Cruz, R.; Esteves, F.; Santos, C.; Lemos, C.; Reis, F.; Vala, HelenaBackground: Nodular glomerulosclerosis occurring in non-diabetic patients is an unexplained renal condition that resembles Kimmelstiel-Wilson lesions (K-W), associated almost exclusively with diabetic nephropathy. This study aimed to assess the presence of nodular glomerulosclerosis in dogs with non-diabetic chronic kidney disease (CKD) and evaluate similarities with human condition, and thus, establish the dog as a natural disease model for further studies. Methods: Kidneys from dogs (n=27) with non-diabetic CKD were evaluated for the presence of nodular glomerulosclerosis. Samples were stained with haematoxylin-eosin and periodic acid of Schiff. Lesions were classified as absent or present, the later, classified as non-convincing and convincing K-W like lesions. Results: Nodular glomerulosclerosis was present in 66.66% of evaluated cases (n=18), of which, 38.88% (n=7) were convincing K-W like lesions and the remaining 61.11% (n=11), although similar, didn’t exhibit typical microscopic characteristics and were classified as non-convincing. Fibrosis was positively associated to diagnosis of K-W lesions (OR=6; p<0.05). Conclusion: As in man, Kimmelstiel-Wilson like lesions may also occur in dogs in the absence of diabetic nephropathy and reveal similar microscopic aspects to the description related in humans and, that some factors, others than hyperglycaemia, may be responsible for the appearance of these lesions. Thus, dogs may provide a model of natural disease for further elucidation.
- Pancreatic lesions and metabolic aggravation are prevented by low doses of sitagliptin in a rat model of type 2 diabetesPublication . Mega, C.; Vala, Helena; Oliveira, J.; Fernandes, R.; Mascarenhas-Melo, F.; Parada, B.; Pinto, R.; Teixeira, F.; Teixeira de Lemos, E.; Reis, F.Introduction: The management of type 2 diabetes is designed to reduce disease-related complications and improve long-term outcomes. Inhibition of dipeptidyl peptidase-4 (DPP-4) activity by sitagliptin has been shown to improve glycaemic control in patients with type 2 diabetes Mellitus (T2DM) by prolonging the actions of incretin hormones, but the real impact of low-dose sitagliptin treatment on cardiometabolic risk factors and pancreatic lesions is almost unknown. This study aimed to evaluate the effects of low doses of sitagliptin on cardiovascular risk factors and histological pancreas parameters in Zucker Diabetic Fatty rats (ZDF (fa/fa)) an animal model of T2DM. Materials and Methods: Twenty weeks old diabetic obese (fa/fa) ZDF male rats were treated with vehicle or sitagliptin (10 mg/kg BW/day) during 6 weeks (n=8 each). The following parameters were assessed: glycaemia, HbA1c, insulin, lipidic profile; blood pressure. Specimens for pancreatic histopathology were stained with haematoxylin-eosin and periodic-acid-Shiff, examined by light microscopy. Endocrine and exocrine pancreas was evaluated semiquantitatively concerning inflammatory infiltrate, fibrosis, vacuolization and congestion, and scored from 0 (absent) to 3 (severe and extensive damage). Results: Sitagliptin in diabetic obese ZDF rats promoted a positive effect on dysglycaemia, dyslipidaemia and prevented the increase of blood pressure. Endocrine and exocrine pancreas presented a reduction/amelioration of fibrosis severity, inflammatory infiltrate, intra-islet vacuolation, and congestion vs the vehicle-treated diabetic rats. Conclusion: Simultaneous improvement of a sustainable glycaemic profile and of pancreatic histopathological lesions supports the favorable cardiovascular risk profile and may prove beneficial in decreasing long-term complications of T2DM.
- Pancreatic protection afforded by sitagliptin in a rat model of type 2 diabetes: putative mechanismsPublication . Mega, C.; Vala, Helena; Oliveira, J.; Teixeira, F.; Fernandes, R.; Teixeira de Lemos, E.; Reis, F.
- Presence of Kimmelstiel-Wilson like lesions in canine renal faillurePublication . Vala, Helena; Mega, A. C.; Mesquita, J.; Nóbrega, C.; Cruz, R.; Esteves, F.; Ortiz, A. L.; Santos, C.; Teixeira de Lemos, E.; Reis, F.Background: Nodular increases in mesangial matrix is the definition of Kimmelstiel-Wilson (K-W) lesions. In man, focal mesangiolyses and K-W nodules were positively associated with diabetes, proteinuria and hyalinization of afferent and efferent arterioles. Although, nodular sclerotic lesions may also occur in the absence of diabetic nephropathy and are clinically related to hypertension, smoking, hypercholesterolemia, among others. This study aimed to assess the presence of nodular glomerulosclerosis in dogs in non-diabetic chronic kidney disease. Materials and Methods A retrospective study including dogs with non-diabetic chronic kidney disease (n=27) was performed. Samples were stained with haematoxylin-eosin and periodic acid of Schiff and classified according to Maxie (2007). Lesions were classified as absent or present and the later, as non-convincing and convincing K-W like lesions. Univariate logistic regression models were performed to measure the association between a positive histological diagnosis of K-W lesions and the accessed histological features (variables membranes, inflammation, atrophy and fibrosis) by using statistical software R with Epicalc package. Results Nodular glomerulosclerosis was present in 66.66% of evaluated cases (n=18) of which, 38.88% (n=7) were classified as convincing and 61.11% (n=11) as non-convincing. The convincing K-W like lesions observed, revealed similar microscopic aspects to the descriptions in literature related to man. The presence of fibrosis was positively associated to the diagnosis of K-W lesions (OR=6; p<0.05). The remaining variables were not statistically associated (p>0.05). Conclusion Presence of K-W like lesions, in dogs with renal failure exposed a possible association with end-stage renal disease. Therefore, the diagnosis of K-W like lesions could be helpful to specify and define renal biopsies.
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