RESUMO
Objective: To investigate the effect of hyperglycaemic memory on the local aldosterone system, reactive oxygen species (ROS) and expression of oncofetal fibronectin (oncofetal FN) mRNA in human mesangial cells (HMCs),and to further understand the role of local aldosterone system in the process. Methods: In this study HMCs were divided into the following groups:normal glucose group (NG, 5 mmol/L D-glucose for 2 days),high glucose group (HG, 25 mmol/L D-glucose for 2 days), memory group (M, 25 mmol/L D-glucose for 2 days→5 mmol/L D-glucose for 4 days), memory + eplerenone group (MY,25 mmol/L D-glucose for 2 days→5 mmol/L D-glucose+10 μ mol/L eplerenone for 4 days), normal glucose + eplerenone group (NY, 5 mmol/L D-glucose for 2 days→5 mmol/L D-glucose+10 μmol/L eplerenone for 4 days), and persistent normal glucose group (SN, 5 mmol/L D-glucose for 6 days). ROS levels were tested by fluorescence microscope and fluorescence microplate reader. Aldosterone synthase (CYP11B2) protein expression was detected by Western blotting analysis. The mRNA expressions of llβ-hydroxysteroid dehydrogenase type 2, CYP11B2 and oncofetal FN were detected by RT-PCR. The expression and translocation of mineralocorticoid receptor (MR) was observed by laser scanning confocal microscope (LSCM). Aldosterone level in cell culture supernatant was detected by radioimmu noassay. Results: (DCYP11B2 mRNA and protein expression in group HG and in group M were all significantly increased, being 3. 45, 2. 09 and 3. 14, 2. 06 folds of those in group NG, respectively (all P<0. 05). The aldosterone levels in HMCs culture supernatant were significantly increased in group HG and group M, being 2. 01 and 1. 81 folds of that in group NG, respectively (P<0. 05). MR was activated and translocated from the cytosol to the nucleus in group HG and group M. Quantitative analysis showed that the ratios of cytosol/ nucleus fluorescence intensity in group HG and group M were decreased by 30% and 21% compared with that in group NG, respectively (all P<0. 05). (2)Oncofetal FN mRNA expression and ROS levels in group HG and group M were significantly increased,being2.23, 1. 99 and 2. 16, 1. 90 folds those of group NG, respectively (all P<0. 05). Oncofetal FN mRNA expression and ROS levels in group MY were significantly decreased,being 35% and 51% of those in group M (all P<0. 05). Conclusion: HMCs have hyperglycaemic memory effect, which might be mediated by the local aldosterone system.
RESUMO
Objective To investigate the role of local aldosterone system in oncofetal fibronectin ( oncofetal FN) mRNA expression and reactive oxygen species (ROS) production in human mesangial cells (HMCs) exposed to short-term intermittent high glucose and the effect of Fenofibrate.Methods The HMCs were divided into 8 groups:normal glucose(NG) ;osmotic fluctuation(OF) ;mean glucose load (MGL) ;stable high glucose (SHG),short-term intermittent high glucose (IHG) ; intermittent high glucose plus eplerenone (IHGE) ; intermittent high glucose plus fenofibrate(IHGF) ; and normal glucose plus fenofibrate (NGF) groups.The mRNA expression levels of Aldosterone synthase ( CYP11 B2 ),11 β-hydroxysteroid dehydrogenase type 2 ( 11βHSD2 ) and oncofetal FN were determined by RT-PCR.The expression of CYP11B2 protein was determined by western-blot.Aldosterone level in cell culture supernatant was detected by radioimmunoassay.The expression and translocation of mineralocorticoid receptor (MR)protein were assayed with confocal laser scanning microscopy. ROS levels were determined by Fluorescence microscopy and fluorescence microplate reader.Results ( 1 ) MGL,SHG,and IHG groups showed a 2.41,3.63,and 4.45 times increase in CYP11B2 mRNA expression,and a 1.83,2.15,and 2.78 times increase in CYP11B2 protein expression,respectively,compared with NG group (P ＜ 0.05 ).The aldosterone levels of HMCs culture supernatant in MGL,SHG,and IHG groups were also increased,being 1.49,2.04,and 2.54 times of that in NG group ( P＜0.05 ),and the degree of elevation in IHG group was more marked than that in SHG group( P＜0.05 ).MR was activated and translocated from cytosol to nucleus in MGL,SHG,and IHG groups.Quantitative analysis showed the ratioes of cytosol/nucleus fluorescence intensity in MGL,SHG,and IHG groups were 15％,38％,and 53％ decreased as compared with that in NG group,and the decrease was more marked in IHG group ( P＜0.05 ).(2) Oncofetal FN mRNA expression and ROS levels in MGL,SHG,and IHG groups were increased,being 1.54,2.31,3.65 and 1.26,1.91,2.48 times of those in NG group,respectively ( P＜0.05 ),and this increase was more marked in group IHG ( P＜O.05 ).Compared with IHG group,oncofetal FN mRNA expression and ROS levels in group IHGE were significantly decreased by 54％ and 53％,and in group IHGF by 45％ and 39％. ( 3 ) CYP11B2 mRNA,protein,and aldosterone levels in IHGF group were decreased by 74％,59％,and 50％,and the activation of MR in group IHGF was inhibited when the ratio of cytosol/nuclear fluorescence intensity was increased 1.88 fold as compared with that in group IHG ( P＜0.05 ).Conclusions Increased expressions of oncofetal FN and ROS by HMCs induced by short-term intermittent high glucose were nore marked than those induced by stable high glucose.The mechanism was associated with activation of local aldosterone system.Fenofibrate may inhibit the activation of local aldosterone system and alleviate the injury to HMCs induced by intermittent high glucose.