| Background:Diabetic nephropathy (DN) shows higher incidence year after year, and has already become an important cause of end-stage renal disease. DN manifests principally as glomerular sclerosis and renal interstitial fibrosis. The pathogenesis of DN is complex. Disorders of glucose metabolism and mechanical disturbances of blood flow constitute the basis of kidney disease; A large number of growth factors and activated cytokines are directly involved in the formation of DN. In recent years, the role of aldosterone in kidney disease, an important component of the RAAS system, is receiving more and more attention from scholars. Aldosterone is implicated in 5/6 of cases based on the nephrectomy model of chronic renal failure, and development of spontaneous hypertension, obstructive nephropathy, diabetic nephropathy and other kidney diseases. Aldosterone, in combination with the mineralocorticoid receptor, can regulate transcription of multiple target genes leading to abnormal sodium and water retention, induction of oxidative stress, promotion of inflammation, and expression of a variety of profibrotic factors, which promotes the progress of diabetic nephropathy.Recently, it was confirmed that aldosterone induced ENaC gene expression by histone methyltransferase Dot1. Dot1, a non-SET domain lysine histone methyltransferase, could build and maintain silent chromatin by catalyze H3K79 methylation to participate in a variety of diseases. Zhang et al. have found that Dot1 binds with AF9 transcription factors, and forms complexes with the promoter of ENaC gene, to regulate the expression of ENaCa. Aldosterone can down-regulate Dotl and AF9. Dot1/AF9 mediated the expression of ENaC induced by aldosterone. The mechanism that aldosterone regulates ENaC expression by Dot1/AF9 maybe present in other genes regulated by aldosterone.Studies have shown that endothelin 1(ET1) is one of the target genes transcriptionally regulated by aldosterone. ET1 is the strongest vasoconstrictor in vivo, has many biological effects, such as effecting renal blood vessels, reducing renal blood flow, increasing in insulin resistance, recruiting inflammatory cells and promoting extracellular matrix synthesis, and is very important in the development of DN. There is as yet no research which proves that aldosterone-regulated expression of ET1 by Dotl/AF9 in diabetic nephropathy during the last ten years.Objective:To observe the effect of aldosterone on ET-1 expression in NRK-52E cells cultured in high glucose environment; To explore the effect of Dot 1/AF9 on aldosterone inducing ET-1 expression; To study the effect of aldosterone on the expression of Dot 1, AF9, ET-1 in a rat model of diabetic nephropathy and interventional effect of spironolactone.Methods1. Rat renal tubular epithelial cells (normal rat kidney cells, NRK-52E) cultured in low-glucose DMEM (including glucose 1000mg/L), high-glucose DMEM (with glucose 4500mg/L), and high glucose DMEM containing different concentrations of aldosterone (10nM,25nM,50nM, 100nM). RT-PCR, Western blotting, immunofluorescence testing were used to detect the expression of Dot 1, AF9, ET-1 mRNA and protein in each group;2. Using low-glucose DMEM, high-glucose DMEM, high-glucose DMEM containing aldosterone (50nM) or spironolactone (100nM) to culture NRK-52E cells, RT-PCR, Western blotting, immunofluorescence testing were used to detect Dotl, AF9, ET-1 mRNA and protein expression in each group;3. We established the stable cell line which was transfected with Dotl or AF9 plasmid. Using low-glucose DMEM, high-glucose DMEM, high-glucose DMEM containing aldosterone (50nM) to culture NRK-52E cells and the cell line which stably transfected with Dot1 or AF9 plasmid, RT-PCR, Western blot, immunofluorescence testing were used to detect Dot1, AF9, ET-1 mRNA and protein expression in each group.4. To establish a DN rat model by STZ. The rats were divided into 3 groups-the normal group (N group), diabetic nephropathy (DN group), and the spironolactone intervention group (SP group, administer spironolactone (40mg*kg-1*d-1) to DN rats). Urine protein, serum creatinine, aldosterone of serum and kidney tissue were determined; RT-PCR, immunohistochemistry, Western blotting were used to detect Dot1, AF9, ET-1 expression.Results:1. High glucose decreased Dot1 and AF9 expression, and increased ET-1 mRNA and protein expression in NRK-52E cells. After treated with aldosterone, Dot1 and AF9mRNA and protein exp ression declined, however, ET-1 expression was higher.2. Spironolactone increased Dotl and AF9mRNA and protein expression, reduced ET-1 mRNA and protein expression at the same time.3. After treated with aldosterone, it showed a high level of expression of Dotl/AF9 in Dotl/AF9 plasmid transfected NRK-52E cells, and ET-1 expression was significantly lower than that in NRK-52E cells which didn't transfected Dotl/AF9 plasmids and transfected with empty plasmids.4. Blood sugar, blood pressure, urine protein excretion, serum creatinine increased and renal pathology showed pathological changes in early stages of diabetic nephropathy in the DN group. Compared with the N group, there was no significant change in serum aldosterone level, but local aldosterone level in kidney tissue had significantly elevated in the DN group.5. While local aldosterone level in kidney tissue had significantly elevated, Dotl, AF9mRNA and protein expression declined, and ET-1 mRNA and protein expression increased significantly in DN rats.6. Treated DN rats with spironolactone, compared to the DN group, it showed no significant change in blood pressure or blood sugar in the SP group, but urinary protein and serum creatinine were decreased and renal pathological changes alleviated, Dot1mRNA, AF9mRNA and protein expression of kidney tissue increased in the SP group, while ET-1 mRNA and protein expression decreased.Conclusion:1. Aldosterone decreased Dotl and AF9 expression, and increased ET-1 expression in NRK-52E cells cultured in high-glucose DMEM. After treated with spironolactone and transfected with Dotl/AF9 plasmid, Dot land AF9mRNA and protein expression increased, however, ET-1 expression was decreased. It confirmed that aldosterone induced ET-1 expression mediated by Dot1 and AF9.2. With the renal interstitial injury, elevated level of aldosterone in kidney tissue down-regulated of Dotl and AF9 and increased ET-1 expression in DN rats. It meaned that the local aldosterone level in kidney tissue maybe take part in the pathogenesis of DN, and related with the role of aldosterone-induced ET-1 expression mediated by Dot1 and AF9.3. Spironolactone reduced renal interstitial injury in diabetic nephropathy and increased Dotl and AF9 expression, thus reduced renal ET-1 gene expression in kidney tissue. It was showed that Spironolactone played a renoprotective role in kidney disease, maybe by blockade the role of aldosterone-induced ET-1 mediated by Dot1 and AF9.We comfirmed that aldosterone induced endothelin-1 expression mediated by DOT1/AF9 in NRK-52E is very important in diabetic nephropathy. |
