The Effect Of Calcium Channel Blockers On The Aldosterone Secretion Function Of Aldosteronoma

Part One.The primary culture nd identification the cells of human aldosterone-producing adenomaObjectivePrimary aldosteronism is the first reason of secondary hypertension.Aldosterone-producing adenoma is the second subtype of primary aldosteronism.Somatic mutation of KCNJ5/CACNA1D/ATP2B3 and ATP1A1 are found in aldosteronism-producing adenoma.Researchers usually transfect these genes into HEK293 cell lines or human adrenal cortical cancer cell lines to study the function of these genes.Studies about primary culture of aldosteronism-producing adenoma are rare.The aim of this study is to establishing primary culture of aldosterone-producing adenoma cells.This method may contribute to the functional study of aldosteronism-producing adenoma.MethodsThe fresh tumor tissue was cut into pieces and digested by collagen type I.After removal of red blood cells,digested tumor cells were cultured in complete DMEM/F12 medium.We continuously observed the morphology of tumor cells.Aldosterone concentration in culture medium was detected by radioimmunoassay and it was compared with the concentration in medium of adrenal non-functional tumor and cortisol-producing adenoma.The expression of aldosterone synthase in the cultureed cells was detected by immunofluorescence.ResultsAldosterone-producing adenoma cells grew adherently in a round or approximately round shape.The aldosterone concentration in the culture medium in the 5th culture day was 1084.0 ± 323.4 ng/dl which was much higher than the aldosterone concentration of adrenal non-functional tumor and cortisol-producing adenoma.During the primary culture,aldosterone secretion was the strongest in the first day,and decreased afterwards.It kept stable in day 3 to 7.ConclusionWe successfully established the primary culture of aldosterone-producing adenoma cells for the first time in China,which would be important for the future studying on the mechanism and function of aldosterone-producing adenoma.Part Two.The effect of calcium channel blocker and the common aldosterone stimulus on the function of aldosterone-producing adenomaObjectiveThe main pathogenic genes of aldosterone-producing adenoma(APA)include KCNJ5?ATP2B3?ATP1A1 and CACNA1D.All the four mutated genes will open voltage-gated calcium channel and increase the calcium concentration in cytosol.Upregulated calcium signal will stimulate the production of aldosterone.Calcium channel blocker could inhibit aldosterone secretion in mutated KCNJ5 gene transfected cell lines.However,the inhibitory effect of calcium channel blocker(CCB)on APA remains unkown.The stimulatory effect of aldosterone stimulus on aldosterone-producing adenoma is also unclear.The purpose of this part is to investigate the inhibitory effect of different calcium channel blocker on APA in vitro and the stimulatory effect of potassium,angiotensin ?,ACTH and sodium on APA in vitro.MethodsTwenty-three APAs were successfully cultured in this part.Sixteen patients' primary tumor cells were treated with benidipine,mibefradil and nifedipine with a final concentration of 10?M.Forty-eight hours later,the medium was harvested for the detection of aldosterone.Total RN A was harvested for the detection of C YP11B2 mRNA expression.Four patients' primary tumor cells were treated with potassium(15mM),ATII(100nM),ACTH(100nM)and different sodium concentrations.Twenty-four hours later,the medium was harvested for the detection of aldosterone.New medium with CCK-8 was added to detect cell proliferation.All the hot mutation sites of KCNJ5 were sequenced in these 23 tumors.ResultsBenidipine,mibefradil and nifedipine could significantly inhibit aldosterone secretion and CYP11B2 mRNA expression.ACTH could significantly stimulate the aldosterone secretion of APA while ATII had a moderate effect and potassium had no effect.ACTH could also promote cell proliferation while ATII and potassium had no effect.The aldosterone concentration increased along with the sodium concentration rise.Different sodium concentration had no effect on cell proliferation.KCNJ5 mutation rate was 91.3%in these 23 tumors.ConclusionThe three CCBs could significantly inhibit aldosterone secretion and CYP11B2 mRNA expression.ACTH was a potent stimulus on the secretary fuction of APA.ATII could partly stimulate the secretary function of APA.On the contrary to normal zona glomerulosar,high sodium concentration other than high potassium concentration could stimulate the aldosterone concentration of APA cells.Besides,ACTH increased the proliferation of APA cells.KCNJ5 mutation was quite common in APA patients with hypokalemia.Twenty-one tumors had KCNJ5 mutation in all the twenty-three tumors.Part Three.The effect of nifedipine on the transcriptome of APA cellsObjectiveIt is recongnized long time ago that calcium signal is important for aldosterone synthesis in physiological condition.In KCNJ5.ATP2B3.ATP1A1 and CACNA1D gene mutated APA,calcium signal is also crucial for aldosterone production.However,the downstream signals after calcium in either physiological condition or pathological condition are not clear yet.Transcriptome is a method studying the specific subset of transcripts present in a particular condition.It may make contributrion in the finding of new transcrits.The study aims to finding out the influence of nifedipine on the transcripts of APA cells.MethodsThe cultured APA cells were treat with nifedipine with a final concentration of 50?M or as vehicle.Forty-eight hours later,the medium was harvested to detect aldosterone concentration and the cells were harvested to extrat total RNA.The next procedure of RNA sequencing was done by a company in Shanghai.Several differential genes were selected and validated through real-time PCR.ResultsWe select two APAs in this study of transcriptome.The two APAs had the same protein change of KCNJ5 gene.Fourty highly expressed genes were differently expressed between the control and nifedipine group.The downregulated genes were mainly asscociated with cholesterol and terpenoids synthesis such as HMGCR,HMGCSl,NSDHL,ACAT2,SDLE.The decreased expression of HMGCS1 were confirmed by real-time PCR.ConclusionFourty highly expressed genes were differently expressed between the two groups.It was discoveried the first time that nifedipine inhibited HMGCS1 mRNA expression and that APA cells expressed many genes involved in cholesterol synthesis.Part Four.The effect of benidipine and nifedipine on aldosterone concentration in patients with primary alsosteronismObjectiveCCB inhibited the aldosterone secretion in primary cultured APA cells.Benidipine was inclined to have a stronger effect than nifedipine.Few reseach studied the effect of CCB on aldosterone concentration in patients with primary alsosteronism.The purpose of this part is to evaluate the effect of L/T type CCB and L type CCB on the aldosterone concentration in patients with primary alsosteronism.MethodsTwo idiopathic hyperaldosteronism patients,eleven APA patients and one familial hyperaldosteronism patient were enrolled in this study.All anti-hypertensive drugs except prazosin were quitted.The study was done in three days respectively.No drugs were taken in the morning of the first day.Ten milligrams of nifedipine were taken in the second day and 8 milligrams of benidipine were taken in the third day.Plasma renin activity(PRA),plasma aldosterone concentration(PAC),cortisol,serum potassium and serum sodium concentration were detected at eight o'clock and ten o'clock or before medicine and two hours after medicine in a seated position.Blood pressure were measured every fifteen minutes.Hotspot mutation of KCNJ5,CACNA1D,ATP2B3 and ATP1A1 were sequenced in eight available tumor speciments.ResultsPAC after medicine was decrease significantly in contrast to PAC before medicine in the day of benidipine.PAC after medicine didn't change in the day of nifedipine.Benidipine and nifedipine could significantly decrease blood pressure in PA patients.The tumor tissues were available in eight patients and all of them were identified with KCNJ5 gene mutation.ConclusionThis is the first study to evaluate the inhibitory effect of benidipine in PA patients.Aldosterone in benidipine group was significantly decreased.Part Five.Clinical and genetic analysis of 64 patients with Gitelman syndromeObjectiveTo study the clinical and genetic profile of the patients with Gitelman syndrome(GS).MethodsWe retrospectively analyzed the genotype and phenotype of 64 GS patients diagnosed in Peking Union Medical Hospital from 2012 to 2016.ResultsThe age at diagnosis of these 64 patients(39 male,25 female)were 35± 14 years.At admission,the serum potassium level of the patients was 2.866±0.44mmol/L,serum magnesium level was 0.62±0.14mmol/L,24-hour urine potassium was 82.27 ±39.73mmol/d,24-hour urine calcium was 0.94±0.83mmol/d and their average blood pressure was 110/69mmHg.The genotype was divided into four groups including homozygous(N=5),compound heterozygous(N=40),multiple mutations(N=9)and single heterozygous mutation(N=10)group.The compound heterozygous group had higher serum potassium concentration(P<0.05)and the homozygous group had a relatively higher serum magnesium concentration but without significance.A total of 74 distinctly different mutation alleles were identified,of which 24 were new mutation alleles.p.Asp486Asn was a hotspot in our series which was found in 16 patients(25.0%).ConclusionsThere exists great heterogeneity of genotype and phenotype in Gitelman syndrome.Patients with compound heterozygous have a relatively milder phenotype.p.Asp486Asn mutation is a hotspot in Chinese patients.