The Influence Of High Glucose On Human Mesangial Cells To Synthesize Aldosterone And Spironolactone On The Harmful Effects Of High Sugar Intervention Evaluation,

Background:As the incidence of diabetes mellitus rises,the prevalence of diabetic nephropathy(DN) is increased.The mortality associated with DN is remarkably higher than that with non-diabetic nephropathy.Diabetic nephropathy has become the leading cause of end stage renal disease(ESRD) in the United States,Europe and Japan,and the third leading cause of ESRD in China.Blockade of the renin-angiotensin-aldosterone systeme(RAAS) plays an important role in the treatment of diabetic nephropathy.However,the efficacy of angiotensin-converting enzyme inhibitors(ACEI) and angiotensinⅡreceptor blockers(ARB) has not been always optimal in reducing proteinuria as expected.Recently,it is thought that aldosterone escape or breakthrough might be part of the reasons,since add of aldosterone antognist has further decreased proteinuria.Aldosterone can induce myocardial hypertrophy and fibrosis.Cellular hypertrophy and extracellular matrix accumulation are also important pathologies in diabetic nephropathy.Studies have showed that aldosterone exerts similar effects on the kidney.Moreover,given the universal existence of mineralocorticoid receptor in tissues and local aldosterone secretion in the kidney,the local aldosterone might be involved in the progression of DN.Mesangial cells play an important role in the pathogenesis of DN.It is unknown yet whether human mesangial cells(HMCs) can produce aldosterone under high glucose and whether an aldosterone antagonist(spironolactone,SPI) would show protective effect agnaist high glucose.Objects:The aims of the study were 1) to investigate the capability of HMCs to express aldosterone synthase(CYP11B2) and to produce aldosterone in the supernatants under high glucose;2) to observe the effect of spironolactone on the activation of extracellular-signal regulated kinase(ERK) induced by high glucose; and 3) to find out whether spironolactone can influence the levels of TGF-β,PAI-1 and MCP-1 in supernatants produced by HMC under high glucose.Methods:HMCs were divided into four groups,which were cultured under DMEM/L, DMEM/H,DMEDM/L+SPI and DMEM/H+SPI,respectively.These four groups were cultured for 24hours,and then the RNA were extracted using Trizol reagent. Reverse transcription PCR and Real-time PCR were performed to detect the expression of CYP11B2.HMCs were cultured under DMEM/L and DMEM/H for 24h, 48h and 72h,respectively.The supernatants were collected and the level of aldosterone in each sample was determined by radioimmunoassay(RIA).All the four groups were cultured for 1h,after which the protein was extracted using RIPA reagent, and then Western Blot were applied to detect the activation of ERK.After being cultured for 48h,the supernatants of the four groups were collected and submitted to determination of the level of TGF-β,PAI-1 and MCP-1 by ELISA.Results:After 24 hours of high glucose stimulation,the expression of CYP11B2 by HMC was increased by about 6-fold compared with that in DMEM/L group;whereas after treatment with SPI,the expression of CYP11B2 in the DMEMH+SPI group were significantly decreased compared with DMEM/H group.There is also the expression of mineralocorticoid receptor(MR) and 11β-hydroxysteroid dehydrogenase2 (11β-HSD2),the gene protecting the specific ligand for MR.The expression of these two genes was not affected by high glucose or SPI treatment.After treatment with high glucose for 24h,48h and 72h,the level of aldosterone in the supernatants were around the lower limit of the kit,and no valid statistics could be established.After 1 hour treated with high glucose,the p44 component of ERK was significantly activated compared with low glucose,while after co-cultured with SPI,the p44 components in the DMEM/H+SPI group was significantly less activated compared with that in DMEM/H group.After stimulation by high glucose,the p42 component showed a tendency of activation,which approached the statistical significance.After treatment with SPI,the level of p42 component activation in the DMEM/H+SPI group was decreased.The levels of TGF-β,PAI-1 and MCP-1 in the DMEM/H group were significantly elevated compared with that in DMEM/L group.After treatment with SPI,the levels of all the three cytokines were significantly decreased in DMEM/H+SPI group compared with DMEM/H.Conclusions:Under the experiment condition,HMCs showed increased expression of CYP11B2 after treatment with high glucose for 24h.However,after 24h,48h and 72h treatment with high glucose,the levels of aldosterone in the supernatants were around the lower limit of the kit,and no valid statistics could be established.High glucose could activate ERK in HMCs while spironolactone of 100nM could significantly blunt the activation of ERK induced by high glucose.High glucose could also increase the levels of TGF-β,PAI-1 and MCP-1 produced by HMCs and the effect of high glucose on those cytokines were blocked by spironolactone of 100nM.Thus,we inferred that HMCs probably produced aldosterone under high glucose,and the local aldosterone secretion might be involved in the progression of diabetic nephropathy.