RESUMO
This study aimed to investigate the role of hypoxia-inducible factor-2α (HIF-2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia conditions. The expression level of HIF-2α and tight junction proteins (occludin and ZO-1) in rGENCs were examined following 5% oxygen density exposure at different treatment times. HIF-2α lentivirus transfection was used to knockdown HIF-2α expression. Cells were divided into four groups: 1) control group (rGENCs were cultured under normal oxygen conditions), 2) hypoxia group (rGENCs were cultured under hypoxic conditions), 3) negative control group (rGENCs were infected with HIF-2α lentivirus negative control vectors and cultured under hypoxic conditions), and 4) Len group (rGENCs were transfected with HIF-2α lentivirus and cultured under hypoxic conditions). The hypoxia, negative control, and Len groups were kept in a hypoxic chamber (5% O2, 5% CO2, and 90% N2) for 24 h and the total content of occludin and ZO-1, and the permeability of rGENCs were assessed. With increasing hypoxia time, the expression of HIF-2α gradually increased, while the expression of occludin decreased, with a significant difference between groups. ZO-1 expression gradually decreased under hypoxia conditions, but the difference between the 24 and 48 h groups was not significant. The permeability of cells increased following 24-h exposure to hypoxia compared to the control group (P<0.01). The knockdown of HIF-2α expression significantly increased occludin and ZO-1 content compared with hypoxia and negative control groups (P<0.01), while permeability was reduced (P<0.01). Hypoxia increased HIF-2α content, inducing permeability of rGENCs through the reduced expression of occludin and ZO-1.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Endoteliais/química , Glomérulos Renais/citologia , Ocludina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Animais , Hipóxia Celular/fisiologia , Proliferação de Células , Células Endoteliais/metabolismo , Permeabilidade , Ratos , Fatores de TempoRESUMO
This study aimed to investigate the role of hypoxia-inducible factor-2α (HIF-2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia conditions. The expression level of HIF-2α and tight junction proteins (occludin and ZO-1) in rGENCs were examined following 5% oxygen density exposure at different treatment times. HIF-2α lentivirus transfection was used to knockdown HIF-2α expression. Cells were divided into four groups: 1) control group (rGENCs were cultured under normal oxygen conditions), 2) hypoxia group (rGENCs were cultured under hypoxic conditions), 3) negative control group (rGENCs were infected with HIF-2α lentivirus negative control vectors and cultured under hypoxic conditions), and 4) Len group (rGENCs were transfected with HIF-2α lentivirus and cultured under hypoxic conditions). The hypoxia, negative control, and Len groups were kept in a hypoxic chamber (5% O2, 5% CO2, and 90% N2) for 24 h and the total content of occludin and ZO-1, and the permeability of rGENCs were assessed. With increasing hypoxia time, the expression of HIF-2α gradually increased, while the expression of occludin decreased, with a significant difference between groups. ZO-1 expression gradually decreased under hypoxia conditions, but the difference between the 24 and 48 h groups was not significant. The permeability of cells increased following 24-h exposure to hypoxia compared to the control group (P<0.01). The knockdown of HIF-2α expression significantly increased occludin and ZO-1 content compared with hypoxia and negative control groups (P<0.01), while permeability was reduced (P<0.01). Hypoxia increased HIF-2α content, inducing permeability of rGENCs through the reduced expression of occludin and ZO-1.
Assuntos
Animais , Ratos , Células Endoteliais/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ocludina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Glomérulos Renais/citologia , Permeabilidade , Fatores de Tempo , Hipóxia Celular/fisiologia , Células Endoteliais/metabolismo , Proliferação de CélulasRESUMO
Activation of the intrarenal reninangiotensin system (RAS), which has been identified in podocytes and mesangial cells, is a novel mechanism in the progression of diabetic kidney disease (DKD). The present study aimed to identify the local RAS in glomerular endothelial cells (GEnCs). Rat GEnCs were stimulated by culture medium containing 30 mmol/l glucose for 12, 24, 48 and 72 h. Angiotensin II (Ang II) concentrations in cell lysates and culture media were examined by ELISA and mRNA levels of angiotensinogen and renin in cell lysates were analyzed by quantitative polymerase chain reaction. Ang II type 1 receptor (AT1R), Ang II type 2 receptor (AT2R), renin and angiotensinogen levels in cell lysates were determined by western blot analysis. Localization of intracellular AT1R, AT2R, angiotensinogen and renin was identified by confocal immunofluorescence microscopy. Consequently, high glucose (HG) increased intracellular and extracellular Ang II levels. Captopril and chymostatin (inhibitor of chymase, an enzyme that converts Ang I to Ang II) were able to antagonize HGinduced Ang II generation. Moreover, HG increased angiotensinogen production in GEnCs and reduced renin mRNA expression without altering renin protein production. However, HG decreased AT1R levels and resulted in AT2R shifting from the nuclear to perinuclear region in GEnCs. In conclusion, HG activated the intracellular RAS in rat GEnCs and the underlying mechanism may involve angiotensinconverting enzyme (ACE) and nonACE pathways. The effects of HG on GEnCs may also involve the substrate and receptors of Ang II.
Assuntos
Nefropatias Diabéticas/metabolismo , Glucose/administração & dosagem , Sistema Renina-Angiotensina/efeitos dos fármacos , Renina/biossíntese , Angiotensina II/biossíntese , Angiotensinogênio/biossíntese , Angiotensinogênio/metabolismo , Animais , Células Cultivadas , Nefropatias Diabéticas/patologia , Células Endoteliais/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor Tipo 2 de Angiotensina/biossíntese , Renina/metabolismo , Sistema Renina-Angiotensina/genéticaRESUMO
OBJECTIVE: To investigate the relationship between the length of telomere DNA and age at different altitude areas. METHODS: All 172 peripheral blood samples were randomly selected from healthy individuals of different ages from 25 to 65 years old. High altitude group (47 males, 48 females) living at an altitude of 4380 m (HA group), sea level group (39 males, 38 females) living at an altitude of 43 m (SL group). The terminal restriction fragment (TRF) length of telomere DNA was measured by Southern blotting analysis. The plasma levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were assayed. RESULTS: Average TRF lengths of males and females in HA groups were 10.45 +/- 1.35 and 10.50 +/- 1.45. Average TRF lengths of males and females in SL groups were 11.29 +/- 1.10 and 11.31 +/- 1.13. A negative correlation was shown between the average TRF length and age of males in two groups (P < 0.01). This was also the case for females. ANOVA test was used to analyze the difference between TRF length and gender at different ages (P < 0.001). It was shown that there was significant difference in TRF length between the male (25 years old and 55 years old) and female (25 years old and 55 years old) in two groups at different ages (P < 0.05). The plasma levels of SOD and MDA were significant different between HA groups and SL groups (25-44 years old groups/45-65 years old groups) (P < 0.05). CONCLUSION: Obviously shortening of telomere was observed by increasing of ages in high altitude groups. There was a negative correlation between the length of telomere DNA and ages. Telomere shortening became more obviously in high altitude group than in sea level group in keeping with the age increases.