The Rac1 And Mineralocorticoid Receptor MRNA Expression In Adrenal Tissues With Patients Of Primary Aldosteronism And Cushing Syndrome

Objectives:Primary aldosteronism (PA) is a group of disorders in which aldosterone production is inappropriately high, relatively autonomous from the renin-angiotensin system, and nonsuppressible by sodium loading. Studies on the genetic pathogenesis of primary aldosteronism is rare in foreign report. Rac1(Ras-related C3 botulinum toxinsubstrate 1) is a kind of oncogene, in recent years, some researches testified that overexpression of active Rac1 significantly potentiated aldosterone-induced mineralocorticoid receptor transcriptional activity. But, we didn't find any researches about primary aldosteronism with active Rac1.This study aims to analyze the mRNA expression level of Racl and mineralocorticoid recepto in adrenal tissues with patients of primary aldosteronism (including aldosterone-producing adenomas, idiopathic hyperaldosteronism) and cushing syndrome(including cortisol producing adenomas, ACTH-independent macronodular adrenal hyperplasia, cushing disease, and ectopic ACTH syndrome).Methods:1. Total RNA was extracted from 6 normal human adrenal cortex,19 primary aldosteronism include 15 adrenal aldoterone-producing adenomas(APA) and 4 idiopathic hyperaldosteronism(IHA),22 cushing syndrome include 10 cortisol-producing adenomas(CPA),7 ACTH-independent macronodular adrenal hyperplasia (AIMAH),2 cushing disease,3 ectopic ACTH syndrome and and then the total RNA was reverse transcripted to cDNA;2. RT-PCR was used to analyze the mRNA expression level of Rac1 and mineralocorticoid recepto in different tissues. The expression levels of the target genes were relatively quantified using (3-actin as internal control;3. The expression levels of the target genes were compared in different tissues. It was studied whether the mRNA expression of Racl and mineralocorticoid receptor correlated with clinical data in different adrenocortical disease.Results:1. Racl mRNA expression in adrenal tumors or adrenal tissues of primary aldosteronism and cushing syndrome patients:1) Expression in primary aldosteronism and cushing syndrome were significant higher than that in normal adrenal cortex(all, p<0.01). There was no statistical significance between primary aldosteronism and cushing syndrome(p>0.05);2) Expression in aldosterone-producing adenoma was significant higher than that in idiopathic hyperaldosteronism (p<0.01);3) Expression in cortisol-producing adenoma and adrenal hyperplasia were significant higher than that in normal adrenal cortex(all, p<0.01). There was no statistical significance between cortisol-producing adenoma and adrenal hyperplasia(p>0.05);4) Expression in aldosterone-producing adenoma was higher than that in adrenal hyperplasia caused by cushing disease and ectopic ACTH syndrome (p<0.05);5) Expressed in lung carcinoid tumor tissue of 2 ectopic ACTH syndrome cases (0.9 and 0.93, respectively).2. Mineralocorticoid receptor mRNA expression in adrenal tumors or adrenal tissues of primary aldosteronism and cushing syndrome patients:1)Expression in primary aldosteronism and cushing syndrome were significant higher than that in normal adrenal cortex(all, p<0.01), expression in cushing syndrome was significance higher than that in primary aldosteronism (p<0.01);2)Expression in idiopathic hyperaldosteronism tended to be higher than that in normal adrenal cortex, but the difference showed no statistical significance(p>0.05). There was no statistical significance between idiopathic hyperaldosteronism and aldosterone-producing adenoma(p>0.05);3)Expression in cortisol-producing adenoma and adrenal hyperplasia were significant higher than that in normal adrenal cortex(all, p<0.01), expression in adrenal hyperplasia was statistically significant higher than that in cortisol-producing adenoma(p<0.01).4) Expressed in lung carcinoid tumor tissue of 2 ectopic ACTH syndrome cases (0.85 and 0.37, respectively).3. The correlation between Racl, mineralocorticoid receptor mRNA expression and the clinical manifestations of primary aldosteronism and cushing syndrome patients:1) Primary aldosteronism Racl and mineralocorticoid receptor mRNA expression were not related to clinical manifestations(all, p>0.05);2) Cushing syndromea)Racl mRNA expression was positively related to HOMA-IR (r=0.588, p =0.034), and positively related to area under glucose curve (r=0.547, p =0.035);b) Mineralocorticoid receptor mRNA expression was positively related to serum cortisol at 0 AM(r=0.641,p=0.025).Conclusion:1. Racl and mineralocorticoid receptor mRNA expressed in normal adrenal cortex, Racl and mineralocorticoid receptor mRNA expression level in primary aldosteronism and cushing syndrome were significant higher than that in normal adrenal cortex, which indicated that they might have tight correlation with adrenocortical disease(primary aldosteronism and cushing syndrome);2. Racl mRNA expression in cushing syndrome was positively related to HOMA-IR and area under glucose curve, which indicated that insulin resistance might improve Racl mRNA expression. Mineralocorticoid receptor mRNA expression in cushing syndrome was related to serum cortisol, which indicated that high serum cortisol improve mineralocorticoid receptor mRNA expression. Objectives:The purpose of this study is to detect the protein level of Rac1 and mineralocorticoid receptor in human different adrenocortical diseases, including primary aldosteronism, cushing syndrome and normal adrenal cortex, to investigate the relationship of Rac1 and mineralocorticoid receptor with adrenocortical diseases.Methods:1. Total protein was extracted from 6 normal human adrenal cortex,19 primary aldosteronism include 15 adrenal aldoterone-producing adenomas(APA) and 4 idiopathic hyperaldosteronism(IHA),22 cushing syndrome include 10 cortisol-producing adenomas(CPA),7 ACTH-independent macronodular adrenal hyperplasia (AIMAH),2 cushing disease,3 ectopic ACTH syndrome, and then, The concentration of protein was evaluated by the means of Bicinchoninic acid.2. Using western blot we analyzed the protein expression of Rac1,MR and P-actin. The protein expression of Racl and MR was semi-quantified by dividing the grayness value of Rac1/β-actin and MR/β-actin in the same sample and compared among the different tissues.3. It was studied whether the protein expression of Racl and MR correlated with clinical data in different adrenocortical diseases, and the difference between mRNA and protein expression of Rac1 and MR were also compared.Results:1. Racl protein expression in adrenal tumors or adrenal tissues of Primary aldosteronism and Cushing syndrome patients:1) Racl protein expression in primary aldosteronism and cushing syndrome werehigher than that in normal adrenal cortex (p<0.01, p<0.05), expression in primary aldosteronism was higher than that in cushing syndrome (p<0.05);2) Racl protein expression in aldosterone-producing adenoma and idiopathic hyperaldosteronism were significant higher than that in normaladrenal cortex(all, p<0.01). There was no statistical significance when compared with aldosterone-producing adenoma group and idiopathic hyperaldosteronism group(p>0.05);3) Racl protein expression in cortisol-producing adenoma was significant higher than that in normal adrenal cortex(p<0.01). There was no statistically significant difference between adrenal hyperplasia and normal adrenal cortex(p>0.05). There was no statistical significance between cortisol-producing adenoma and adrenal hyperplasia(p>0.05);4) Racl protein expressed in lung carcinoid tumor tissue of 2 ectopic ACTH syndrome cases (0.75 and 0.94 respectively).2. Mineralocorticoid receptor protein expression in adrenal tumors or adrenal tissues of primary aldosteronism and Cushing syndrome patients:1) Mineralocorticoid receptor protein expression in primary aldosteronism was significant higher than that in normal adrenal cortex(p<0.01). Mineralocorticoid receptor protein expression in primary aldosteronism was significant higher than that in cushing syndrome(p<0.01). There was no statistical significance when compared cushing syndrome with normal adrenal cortex(p>0.05);2) Mineralocorticoid receptor protein expression in aldosterone-producing adenoma and idiopathic hyperaldosteronism were significant higher than that in normal adrenal cortex (all, p<0.01), but was no statistical significance when compared aldosterone-producing adenoma with idiopathic hyperaldosteronism;3) Mineralocorticoid receptor protein expression in cortisol-producing adenoma was significant higher than that in normal adrenal cortex(p<0.01). Mineralocorticoid receptor protein expression in cortisol-producing adenoma was significant higher than that in adrenal hyperplasia(p<0.05). There was no statistically significant difference between adrenal hyperplasia and normal adrenal cortex(p>0.05); 4) MR protein expressed in lung carcinoid tumor tissue of 2 ectopic ACTH syndrome cases(1.22 and 0.30 respectively).3. Rac1 protein expression in all adrenocortical diseases patients was positively related to mineralocorticoid receptor protein expression.4. The correlation between Rac1, mineralocorticoid receptor protein expression and the clinical manifestations of Primary aldosteronism and Cushing syndrome patients.1) Primary aldosteronisma)Racl protein expression was positively related to highest diastolic blood pressure in the course(r=0.543,p=0.016), positively related to recently systolic blood pressure (r=0.583,p=0.018), and positively related to the increase of plasma aldosterone level to upright stimulation(r=0.583,p=0.018);b) Mineralocorticoid receptor protein expression was negatively related to 24 hours urine sodium(r=-0.610, p=0.027);c)The protien levels of Rac1 and mineralocorticoid receptor in angiotensin-responsive primary aldosteronism were significant higher than that in angiotensin-unresponsive primary aldosteronism(all,p<0.01);d) The protien levels of mineralocorticoid receptor in primary aldosteronism with insulin resistance was higher than that in without insulin resistance(p<0.05);e) The protien levels of mineralocorticoid receptor in primary aldosteronism with high urine sodium was significant lower than that in mormal urine sodium (p <0.05); f) The protien levels of Rac1 in primary aldosteronism with bad control of blood pressure was significant higher than that in good control of blood pressure (p <0.05). 2) Cushing syndromea) Rac1 protein expression was negatively related to 24 hours urine sodium(r=-0.564, p=0.010);b) Mineralocorticoid receptor protein expression was negatively related to 24 hours urine sodium(r=-0.697, p=0.017), negatively related to tumor or adrenal weight(r=-0.652,p=0.002), negatively related to tumor or adrenal adrenal volume(r=-0.722,p=0.000);c) The protien levels of Racl and mineralocorticoid receptor protein expression were no statistical significance between with insulin resistance and without insulin resistance, high urine sodium and mormal urine sodium, bad control of blood pressure and good control of blood pressure(all,p>0.05).Concliusion:1. It is tested in protein level that Racl, mineralocorticoid receptor protein expression level in primary aldosteronism and cushing syndrome were significant higher than that in normal adrenal cortex, which indicated that they might have tight correlation with adrenocortical disease(primary aldosteronism and cushing syndrome);2. In primary aldosteronism patient, Rac1 protein expression was positively related to the increase of plasma aldosterone level to upright stimulation and blood pressure; The protien levels of Rac1 in angiotensin-responsive primary aldosteronism was higher than that in angiotensin-unresponsive primary aldosteronism, the protien levels of Rac1 in bad control of blood pressure was higher than that in good control of blood pressure, which indicated that the increase of plasma aldosterone level to upright stimulation and high blood pressure might improve Racl protein expression;3. In primary aldosteronism patient, mineralocorticoid receptor protein expression was negatively related to 24 hours urine sodium, the protien levels of mineralocorticoid receptor in high urine sodium was lower than that in mormal urine sodium, which indicated that the increase of urine sodium couldn't suppress mineralocorticoid receptor protein expression; the protien levels of mineralocorticoid receptor in angiotensin-responsive primary aldosteronism was higher than that in angiotensin-unresponsive primary aldosteronism, in with insulin resistance was higher than that in without insulin resistance, which indicated that the increase of plasma aldosterone level to upright stimulation and insulin resistance might improve mineralocorticoid receptor protein expression; 4. Racl protein expression was positively related to mineralocorticoid receptor protein expression, which indicated that activated Rac1 might improve mineralocorticoid receptor protein expression;5. In cushing syndrome, the mRNA levels of mineralocorticoid receptor was high in adrenal hyperplasia, but the protein levels of mineralocorticoid receptor wasn't high, which indicated that some other factors modify MR protein translation;6. In cushing syndrome, mineralocorticoid receptor protein expression was negatively related to tumor or adrenal weight and volume, which indicated that mineralocorticoid receptor protein expression might relate with the growth of adrenal cortex.