Comparison of Cardiovas cular Complications in Patients with and without KCNJ5 Gene Mutations Harboring Aldosterone-producing Adenomas

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1 Original Article 191 Comparison of Cardiovas cular Complications in Patients with and without KCNJ5 Gene Mutations Harboring Aldosterone-producing Adenomas Takumi Kitamoto, Sachiko Suematsu, Yoko Matsuzawa, Jun Saito, Masao Omura and Tetsuo Nishikawa Endocrinology and Diabetes Center, Yokohama Rosai Hospital, Yokohama, Japan Aim: Our objective was to evaluate the incidence of cardiovascular complications before and after unilateral adrenalectomy in patients with and without KCNJ5 gene mutations harboring aldosterone-producing adenoma (APA). Methods: A total of 108 APA patients were evaluated in the present study. We compared the clinical characteristics and laboratory findings according to the cardiovascular complications in the patients with or without KCNJ5 gene mutations harboring APA after excluding five APA patients with ATPase or CACNA1D gene mutations. Results: There were 75 and 28 APA patients with somatic mutations of KCNJ5 (p.g151r, p.l168r, p.e145q, p.t158a or 157del) and no mutations, respectively. There were no double mutations in any of the subjects. The KCNJ5-mutated and wild type groups demonstrated similar advances in left ventricular hypertrophy prior to, although the mutated group was significantly younger, with higher plasma and urine aldosterone levels, than the wild type group (48.2 vs (years old); p 0.001, vs. 247 (pg/ml); p 0.001, 22.2 vs ( g/day); p 0.008). Both groups displayed postoperative improvements in hyperaldosteronism and hypertension. Moreover, the LV mass index (LVMI) significantly improved after in the mutated group (p 0.001), but not in the wild type group (p 0.256). A multiple linear regression analysis showed that an improvement in the LVMI was independently associated with KCNJ5 mutations and the plasma aldosterone level in that order (p 0.034, 0.050, respectively). Conclusion: The present findings clearly demonstrated that KCNJ5 mutations are common among Japanese APA patients (frequency: 69.4%). In this study, the KCNJ5-mutated group demonstrated significant postoperative improvements in LVMI, possibly due to strong autonomous aldosterone production. Hence, it is necessary to precisely diagnose younger APA patients possessing a strong capacity for aldosterone production due to KCNJ5 gene mutations, as such cases may be easily complicated by cardiovascular events. J Atheroscler Thromb, 2015; 22: Key words: Aldosterone-producing adenoma, KCNJ5, Left ventricular hypertrophy Introduction Recent research on the pathogenesis of primary Address for correspondence: Tetsuo Nishikawa, Endocrinology and Diabetes Center, Yokohama Rosai Hospital, 3211 Kozukue-cho, Kohoku-ku, Yokohama City, Kanagawa , Japan tetsuon@yokohamah.rofuku.go.jp Received: February 19, 2014 Accepted for publication: August 5, 2014 hyperaldosteronism has made great progress, such as the identification of mutations in the KCNJ5 gene affecting the selectivity filter of the encoded GIRK4 potassium (K ) channel 1), mutations in the ATPase gene increasing the intracellular calcium ion concentration 2) and mutations in the CACNA1D gene increasing calcium ion influx 3), thereby inducing the autonomous production of aldosterone in patients with aldosterone-producing adenoma (APA). We previously demonstrated that somatic mutations of the

2 192 KCNJ5 gene occur only in APA tissue, while the aldosterone production pathway becomes dominant and the cortisol production pathway is downregulated in KCNJ5-mutated APA tissue, resulting in highly autonomous aldosterone production 4). Moreover, it was recently reported that 15 of 23 (65.2%) Japanese patients with APA possessed two somatic mutations in the KCNJ5 gene 5), suggesting that mutations of the KCNJ5 gene are closely related to the pathogenesis of APA in Japanese patients 5). It is well known that aldosterone plays a crucial role on the onset of cardiovascular complications, such as myocardial fibrosis and proteinuria 6-10). The prognostic impact of the abovementioned cardiovascular complications in APA patients is reported to be significantly improved by unilateral adrenalectomy 6, 9-12). Therefore, in the present study, we attempted to characterize the pre- and postoperative clinical characteristics and cardiovascular complications observed in APA patients with KCNJ5 gene mutations compared with that noted in APA patients without KCNJ5 gene mutations after excluding subjects with ATPase and CACNA1D gene mutations. Patients and Methods The clinical records of 108 patients with APA were retrospectively analyzed. Blood pressure (BP) measurements were obtained from the right arm by the attending physician after the subject had been seated for five minutes. We evaluated the BP at the first visit to the outpatients unit and at six months to one year after surgical treatment. ABPM was performed in 84 APA patients to detect non-dipper hypertension 13). We confirmed the presence of diabetes and dyslipidemia according to the guidelines developed by the Japan Diabetes Society (JDS) 14) and Japan Atherosclerosis Society (JAS) 15), respectively. The subjects were definitively diagnosed with APA during screening of hypertensive patients who visited our general outpatient clinics between 2007 and 2013, according to the guidelines developed by the Japan Endocrine Society (JES) 16). The diagnosis was confirmed on endocrinological examinations, such as the furosemide plus upright test, saline-loading test, captopril-loading test 16-18) and rapid adrenocorticotropic hormone (ACTH) test 17-20). CT imaging and ACTHloading adrenal venous sampling (ACTH-AVS) were performed to determine the laterality of hyperaldosteronism, as previously reported 17-19). We diagnosed the laterality of aldosterone hypersecretion when the aldosterone concentration in the adrenal venous effluent was greater than 2,500 pg/ml before and 14,000 pg/ ml 30 minutes after ACTH stimulation, respectively. We also calculated the lateralized ratio (cutoff value: 2.6) according to the guidelines of the JES 12). All patients included in this study exhibited obvious unilateral hyperaldosteronism and underwent surgical resection of the affected adrenal gland. In addition, all patients displayed postoperative improvements in their endocrinological abnormalities, such as hyporeninemic hyperaldosteronemia and BP. In addition, all subjects understood the objectives of the study and provided their written informed consent. The study protocol was approved by the research ethics committee of Yokohama Rosai Hospital. Measurements of the Plasma Aldosterone Concentrations, Plasma Renin Activity and Serum Cortisol Concentrations Some of the patients took budralazine, -blockers and/or calcium channel blockers for several weeks prior to undergoing the blood sampling procedure. No other antihypertensive drugs, including diuretics, mineralocorticoid receptor antagonists or -blockers, were administered according to the guidelines developed by the JES 16). Blood samples were collected in the morning after the patient had rested in the supine position for 30 minutes. The plasma aldosterone concentration (PAC), serum cortisol concentration and plasma renin activity (PRA) were measured with specific radioimmunoassays (SPAC-S Aldosterone Kit (TFB, Inc.) and Beads solid phase RIA (YAMASA, Co.)), as previously reported 17-19). Patients who exhibited a PRA of 1.0 ng/ml/hr and PAC of 100 pg/ ml were diagnosed with hyporeninemic hyperaldosteronemia 17-19), and the PAC/PRA ratio (ARR) was calculated. The criterion of an ARR of 200 was used for PA screening. PRA was undetectable (less than 0.1 ng/ml/hr) in some cases, and we assigned such patients a PRA of 0.05 ng/ml/hr for the purpose of the subsequent statistical analyses. Evaluation of Cardiovascular Complications and Unilateral Adrenalectomy We evaluated the incidence of cardiovascular complications, such as left ventricular hypertrophy (LVH) and atherosclerosis, before and after adrenalectomy. The above-mentioned complications were assessed based on the left ventricular mass index (LVMI) 21, 22), brachial-ankle pulse wave velocity (bapwv) 23) and maximum carotid intima-media thickness (max IMT) 24-26). The LVM was calculated using the method of Devereux corrected with the appropriate regression equation 21, 22) and normalized for the body surface area to obtain the LVMI (model.

3 193 SIMENS SEQUOIA512, TOSHIBA Aplio80 SSH- 770A or TOSHIBA Aplio Artida SSH-880CV). These examinations were performed only by authorized experts of echocardiographic assessments. We again determined the plasma and urinary aldosterone concentrations, BP, need for antihypertensive drugs, serum potassium levels and cardiovascular complications six to 12 months after unilateral adrenalectomy. RNA Extraction and Detection of Mutations in the KCNJ5, ATPase and CACNA1D Genes on PCR and Direct Sequencing The APA tissue specimens were frozen in liquid nitrogen immediately after excision and maintained at 80 until use. Total RNA was prepared from each tissue sample using the TriPure isolation reagent and High Pure RNA isolation kit (Roche Diagnostics), as previously reported 27, 28). Then cdna was reverse transcribed from total RNA using a high-capacity cdna reverse transcription kit (Applied Biosystems). In order to sequence KCNJ5, ATPase and CACNA1D cdna, 1.0 L of cdna was subjected to PCR using Tks Gflex TM DNA polymerase (Takara-bio). For KCNJ5 PCR, the forward primer was 5 -ACCTGGA- CCATGTTGGCGACC-3, and the reverse primer was 5 -TCCCGCATGGAGATGACTGCG-3 (PCR products: 286 bp; positions 664~949; NM_ ) 29). We subsequently analyzed gene mutations in ATP1A1, ATP2B3 and CACNA1D according to the methods reported by other investigators 2, 3). The PCR products were purified using electrophoresis (2% agarose gel) and a NucleoSpin Gel and PCR clean-up kit (MACH- EREY-NAGEL GmbH &Co.KG) before being directly sequenced by a customized service (Premix sequence, Takara-bio). Statistical Analysis All data are expressed as the mean SD for parameters with a normal distribution or the median (25-75th percentile) for parameters with a non-normal distribution. The LVMI, max IMT and bapwv values were logarithmically transformed (base 10) for the parametric statistical analysis. The data were compared between groups using Student s t -test or the Wilcoxon rank-sum test. The relative proportions of categorical variables were assessed using Yates chisquare test or Fisher s exact test. The paired t -test, Wilcoxon signed-rank test or McNemar s test were used to compare differences in the mean values obtained before and after. A multiple linear regression analysis was performed to evaluate the strength of the correlation between each variable. We used dummy variables for categorical variables and employed a stepwise regression analysis to select the best cutoff points. A p value of 0.05 was considered to indicate statistical significance. All analyses were performed using a commercially available statistical software package (SAS version 9.3; SAS Institute, Cary, North Carolina). Results Preoperative Clinical Characteristics of the APA Patients with or without KCNJ5 Mutations Of the 108 APA patients, 75 had KCNJ5 mutations (KCNJ5-mutated group), three possessed ATP1A1 or ATP2B3 mutations, two possessed CACNA1D mutations and 28 did not display KCNJ5, ATPase or CACNA1D mutations (wild type group). Concomitant KCNJ5 and ATP1A1 or ATP2B3 and CACNA1D mutations were not observed in any tumors. We subsequently excluded patients possessing ATP1A1 or ATP2B3 and CACNA1D mutations from the present analysis. The present data showed that the KCNJ5- mutated group accounted for 69.4% of the APA patients (75 of 108 APA patients). Of the 75 KCNJ5- mutated APA patients, 46 displayed a glycine-to-arginine substitution at codon 151 (p.g151r), seven of which involved mutations from G to C at 451 bp from the translation start site of the KCNJ5 gene (c.451g C) and 39 involved G to A mutations (c.451g A). Another 24 cases exhibited a leucine-toarginine mutation at codon 168 (p.l168r); all of these changes involved T to G mutations at 503 bp (c.503t G). Two patients exhibited a glutamine acid-to-glutamine mutation at codon 145 (p.e145q: c.433g C), one patient showed a threonine-to-alanine at codon 158 (p.t158a: c.472a G) and two patients displayed a deletion at codon 157 (157 del: c.dell ; c.469a T, dell ). Hence, in the KCNJ5-mutated group, p.g151r mutations were much more frequent than p.l168r mutations (61.3% vs. 32%). A comparison of the preoperative clinical characteristics of the 75 patients in the KCNJ5-mutated group and the 28 patients in the wild type group is summarized in Table 1. The subjects in the KCNJ5- mutated group were significantly younger than those in the wild type group. In addition, the plasma and urinary aldosterone concentrations were significantly higher in the KCNJ5-mutated group than in the wild type group, whereas the serum potassium levels were significantly lower in the KCNJ5-mutated group than in the wild type group. Moreover, the number of patients being treated with potassium replacement

4 194 Table 1. Comparison of the preoperative clinical features of the KCNJ5 mutated and wild type APA patients KCNJ5 mutated group (n 75) Wild type group (n 28) p value Frequency of genetic type (%) KCNJ5 mutations (G151R/L168R/E145Q/T158A/157de1) Gender (male/female) Age (yr) Duration of HT (yr) DM (patients) DL (patients) Numbers of smoker BMI (kg/m 2 ) Numbers of antihypertensive drugs used SBP (mmhg) DBP (mmhg) PR (beats/min) Plasma aldosterone (pg/ml) Plasma renin activity (ng/ml/hr) Urinary aldosterone ( g/day) Urinary cortisol ( g/day) Serum potassium (meq/l) Cases being treated with potassium replacement Serum creatinine (mg/dl) egfr (ml/min/1.73 m 2 ) Proteinuria ( ) Type of hypertension (dipper/non-dipper) DST ( ) Tumor size (mm) Laterality (right/left) LV mass index (g/m 2 ) Max IMT (mm) bapwv (cm/s) /24/2/1/2 25/ (2-11) (12%) (1-2) ( ) 0.2 ( ) 22.2 ( ) 46.2 ( ) (81.3%) / /35 a) ( ) 0.8 ( ) ( ) 25.9 ( ) 12/ (2-14) (29%) (1-3) ( ) 0.2 ( ) 12.6 ( ) 39.4 ( ) (57.1%) / / ( ) 1.1 ( ) ( ) p 0.05 vs. the wild group, p 0.01 vs. the wild type group The data are expressed as the mean SD, except for the duration of hypertension, number of antihypertensive drugs, plasma and urinary aldosterone levels, serum cortisol levels, plasma renin activity, LV mass index, max IMT and bapwv, which are shown as the median (range). HT: hypertension, SBP: systolic blood pressure, DBP: diastolic blood pressure, PR: pulse rate, DM: diabetes mellitus, DL: dyslipidemia, DST( ): cortisol 3.0 g/dl after overnight suppression with 1 mg of dexamethasone. We used the highest value of PAC at the time of blood sampling for the statistical calculations in this table in order to examine the basal levels before each confirmatory test. The number of patients evaluated for each cardiovascular complication was as follows (mutated group vs. wild type group): LVMI (n 71 vs. n 24), max IMT (n 71 vs. n 27), bapwv (n 57 vs. n 18). a) One patient had bilateral adenoma. We treated this patient with left total adrenalectomy and right partial adrenalectomy and evaluated both sides of the adenoma. therapy was significantly higher in the KCNJ5- mutated group than in the wild type group. The KCNJ5-mutated group exhibited higher egfr levels than the wild type group. In addition, the KCNJ5- mutated group contained 1.2 times more women than men (50 of 67 women and 25 of 41 men). The tumor size was significantly larger in the KCNJ5-mutated group than in the wild type group. Other findings, such as the duration of hypertension, body mass index (BMI), BP and proportion of smokers, non-dipper hypertensives and subjects with diabetes or dyslipidemia, did not differ between the two groups. Postoperative Clinical Characteristics of the APA Patients with or without KCNJ5 Mutations We were able to reassess 86 patients whose hyperaldosteronemia had completely normalized at six months to one year after, including 65 patients

5 195 Table 2. Comparison of the clinical characteristics of the patients in the KCNJ5 mutated and wild type groups at 6 to 12 postoperative months KCNJ5 mutated group (n 65) p value 1) Wild type group (n 21) p value 2) p value 3) Gender (male/female) Age (yr) Duration of HT (yr) Fast plasma glucose (mg/dl) Trigiyceride (mg/dl) HDL-Cholesterol (mg/dl) LDL-Cholesterol (mg/dl) BMI (kg/m 2 ) Numbers of antihypertensive drugs used SBP (mmhg) DBP (mmhg) PR (beats/min) Plasma aldosterone (pg/ml) Plasma renin activity (ng/ml/hr) Urinary aldosterone ( g/day) Serum potassium (meq/l) Senum creatinine (mg/dl) egfr (ml/min/1.73 m 2 ) Proteinuria ( ) LV mass index (g/m 2 ) Max IMT (mm) bapwv (cm/s) 23/ (3-12) (0-1) (61-104) 0.8 ( ) 2.6 ( ) ( ) 0.9 ( ) ( ) / (2-14) (0-1) (61-91) 0.9 ( ) 2.8 ( ) ( ) 1.0 ( ) ( ) p 0.05 vs. the wild group, p 0.01 vs. the wild type group The presentation of the data and abbreviations are the same as in Table 1. 1) and 2) paired t-test between before and after. 3) Student s t-test between the KCNJ5-mutated and wild type groups after. The number of patients in the mutated and wild type groups able to be evaluated for each cardiovascular complication was as follows: LVMI (n 49 vs. n 13), max IMT (n 48 vs. n 14) and bapwv (n 40 vs. n 8). in the KCNJ5-mutated group and 21 patients in the wild type group. Table 2 shows the postoperative findings of the 86 APA patients in the KCNJ5-mutated and wild type groups. The plasma and urinary aldosterone concentrations in the KCNJ5-mutated group decreased to the same levels as those seen in the wild type group after. None of the patients in either group required postoperative potassium replacement therapy, as all subjects exhibited normal potassium levels. In addition, the systolic blood pressure values significantly improved in the KCNJ5-mutated group compared to that noted in the wild type group, and the number of patients using antihypertensive drugs tended to be smaller in the KCNJ5-mutated group (p 0.064). The egfr values in the KCNJ5-mutated group significantly decreased after, while those in the wild type group did not. The number of patients with proteinuria decreased equally in both groups after. Cardiovascular Complications and Unilateral Adrenalectomy We evaluated each cardiovascular parameter before and after. Consequently, the LVMI was assessed preoperatively, postoperatively or both in 95, 62 and 58 patients, respectively, the max IMT was measured in 98, 62 and 59 patients, respectively, and the bapwv was determined in 75, 48 and 38 patients, respectively. The pre- and postoperative LVMI, max IMT and bapwv data for the KCNJ5-mutated and wild type groups are summarized in Tables 1 and 2 and Fig. 1. As shown in Table 1, the groups displayed similar LVMI and max IMT values before, whereas the individuals in the KCNJ5-mutated group were significantly younger than those in the wild type group before treatment. Meanwhile, the bapwv values were significantly lower in the KCNJ5-mutated group than in the wild type group; this result may be due to the younger ages of the KCNJ5-mutated APA

6 196 Fig. 1a: LVMI Fig. 1b: Max IMT Fig. 1c: bapwv KCNJ5 mutated group (n=47) Wild type group (n=11) KCNJ5 mutated group (n=46) Wild type group (n=13) KCNJ5 mutated group (n=33) Wild type group (n=5) 200 P<0.001 n.s P=0.052 n.s P=0.001 n.s ,400 2,400 LV mass index (g/m 2 ) LV mass index (g/m 2 ) Max IMT (mm) Max IMT (mm) bapwv (cm/s) 1,800 1, bapwv (cm/s) 1,800 1, Fig.1. Postoperative changes in various cardiovascular complications in the KCNJ5 mutated and wild type groups. We evaluated the postoperative improvements in each cardiovascular parameter compared with that observed before. Consequently, the LVMI and bapwv values improved significantly, while the max IMT tended to decrease, in the KCNJ5-mutated group (p values for the comparisons between the KCNJ5-mutated group and wild type group: p 0.001, p for LVMI; p 0.052, p for max IMT; and p 0.001, p for bapwv). Table 3. Multi regression analysis of the factors associated with improvement of LV mass index around Variables Standard error t P t KCNJ5 mutation Age (yr) BMI (kg/m 2 ) PAC (pg/ml) R ; adjusted R , F value 3.28 (p 0.050) Abbreviations: BMI, body mass index; PAC, plasma aldosterone concentration We performed a multiple linear regression analysis to determine which factors were significantly associated with an improvement in LVMI ( LVMI). We selected the reduction of LVMI from before to after ( LVMI) as the dependent variable and KCNJ5 mutations and age as the explanatory variables. Other effective factors were selected according to a stepwise analysis, in which only BMI and PAC remained explanatory variables. KCNJ5 mutations and PAC were found to be significantly associated with the LVMI, in that order. : standard partial regression coefficient We also attempted to determine which parameters (among KCNJ5 mutations, sex, age, BMI, systolic BP, diastolic BP, BMI, smoking, non-dipper hypertension and PAC) were associated most significantly with an improvement in LVMI. In this analysis, the existence of KCNJ5 mutations and differences in age and time course should be taken into consideration. Therefore, we used the degree of change in LVMI from before to after ( LVMI) as the dependent variable and KCNJ5 mutations and age as the explanatory variables for each adjustment. As to other variables, we employed a stepwise regression model to select the best cutoff points. Table 3 shows the results of the multiple linear regression analysis of the relationship between each parameter and the LVMI. This analysis demonstrated that the LVMI was indepatients. The cardiovascular complications after are described in Table 2. The LVMI, max IMT and bapwv values each improved after. Moreover, the postoperative LVMI values were lower in the KCNJ5-mutated group than in the wild type group, although the difference was not statistically significant, whereas the two groups exhibited almost equal preoperative values. We also examined the postoperative improvements in cardiovascular complications in each group. As shown in Fig. 1, the patients in the KCNJ5-mutated group exhibited a significant improvement in LVMI, bapwv after with a tendency toward improvement in the max IMT values, whereas these parameters did not demonstrate any such improvements in the wild type group.

7 197 pendently associated with KCNJ5 mutations itself as well as PAC, in that order. Discussion In the present study, of the 108 APA patients, 75 (69.4%) possessed somatic mutations of the KCNJ5 gene, and the frequency did not differ between men and women. However, it should be noted that this study was performed retrospectively. Furthermore, the high frequency of KCNJ5 gene mutations is not consistent with that reported in Western countries (from 34% to 45%), although it is similar to the results of Taguchi et al., who found mutations in 65.2% of APA patients 1, 5, 30-32). Therefore, KCNJ5 mutations may play an important role in the development of APA in Japanese patients due to the higher prevalence of APA compared with bilateral adrenal hyperplasia among the various types of PA in Japan 33). The present findings clearly demonstrated that the plasma and urinary aldosterone concentrations were significantly higher in the KCNJ5-mutated group than in the wild type group. The clinical characteristics of the KCNJ5-mutated group, such as the young age of onset, high levels of aldosterone secretion and severe hypokalemia, were similar to the typical characteristics of APA 34). Therefore, it is possible that the KCNJ5-mutated group suffered from an early onset form of APA that induces excess aldosterone production, thus resulting in the typical and classical features of APA, with a high risk of various cardiovascular conditions. We do not know the exact reason why the incidence of KCNJ5 mutation-carrying APA is so high in Japan; however, clarifying the mechanisms underlying the strong autonomous aldosterone production induced by KCNJ5 mutation-carrying APA, which is closely related to the risk of cardiovascular complications, is very important for determining the most appropriate treatment for Japanese APA patients. In the present analysis, cardiovascular parameters, including the LVMI and max IMT, did not differ significantly between the KCNJ5-mutated group and the wild type group, although the patients in the KCNJ5-mutated group were significantly younger than those in the wild type group. It has been reported that the influence of a high BP on vascular mortality is much greater in younger patients than in elderly patients with hypertension 35). Moreover, PA patients easily develop severe cardiovascular disorders, such as coronary heart disease, stroke and sustained arrhythmia, which may occur independently from BP 8, 36-38). Furthermore, our findings suggest that the patients in the KCNJ5-mutated group were more susceptible to the early onset of hypertension, with stronger autonomous aldosterone production, than those in the wild type group. Therefore, the subjects in the KCNJ5- mutated group may exhibit progressively advancing vascular damage compared to that seen in the wild type group. Next, we evaluated the cardiovascular parameters at six to 12 months after unilateral adrenalectomy. In this analysis, we were able to postoperatively analyze 86 patients among all subjects. Consequently, the KCNJ5-mutated group showed greater improvements in aldosterone secretion, including significant improvements in renal hyperfiltration after and significantly lower SBP values than that noted in the wild type group. Such changes resulting from normalized aldosterone secretion may affect the prognosis of cardiovascular complications. The present data also demonstrated that the LVMI, max IMT and bapwv values were significantly decreased at 6~12 months after in the KCNJ5-mutated group, whereas no such changes were observed in the wild type group. Our findings clearly show that surgical resection of APA has a significantly greater beneficial effect in inhibiting cardiac enlargement and atherosclerotic changes in the KCNJ5-mutated group than in the wild type group. In essential hypertensives, the LVH is well known to be affected by age, height, systolic BP and BMI 37, 39). Moreover, the LVMI is influenced by the aldosterone and fibrinogen levels, independent of the above factors 40, 41), and is improved by mineralocorticoid receptor blockade 40, 42). As expected from these reports, PA patients display greater LV mass values than subjects with essential hypertension 6, 36, 43-46). However, in patients with APA, the LVMI has been demonstrated to decrease markedly within one year after adrenalectomy, but not after medical therapy. Variables most strongly associated with improvements in this parameter include the preoperative PAC and SBP values 6, 11, 41). These results suggest that aldosterone, the level of which is independent of the hypertension-related hemodynamic load, plays an important role in promoting LVH. In the present study, the multiple linear regression analysis clearly demonstrated that KCNJ5 mutations and PAC have strong independent relationships with improvements in the LVMI. Furthermore, the LVMI values exhibited a significant postoperative improvement in the KCNJ5-mutated group, whose higher blood and urine aldosterone levels decreased to the same levels as those seen in the wild type group after. It has been reported that the initiation of LVH may be a compensatory response to mechanical stress,

8 198 resulting in progressive and continuous adverse remodeling, which soon improves following normalization of pressure overload 47, 48). Excess aldosterone secretion induces both pressure overload and hypervolemia via sodium retention and vasoconstriction and directly triggers the onset of cardiac fibrosis, possibly induced by various inflammatory reactions 7, 49, 50). In the present study, the patients in the wild type group did not display any changes in the LVMI values at one year postoperatively. However, in a previous study, adrenalectomy was demonstrated to have an effect on LVH at three years postoperatively 12). Hence, such patients should be followed up for more than one year in order to confirm the effects of. The pathogenesis of arteriosclerosis is very complex and multifactorial. In the current study, the patients with APA harboring KCNJ5 mutations showed significant improvements in the bapwv, an indicator of arterial stiffness, and early decreases in the max IMT, which reflects the arterial thickness, after normalization of the aldosterone levels and BP. In addition, major atherosclerotic risk factors, such as diabetes and dyslipidemia, were well controlled equally in each group before and after. Interestingly, Lin et al. found that the IMT and PWV improved within one year after APA resection 10). In that study, the APA patients were much younger and had higher aldosterone levels and BP values, with a much shorter duration of hypertension, than that observed in the patients in the mutated group in the present study. These findings suggest that surgical treatment have beneficial effects on arterial sclerosis within one year postoperatively in APA patients with high aldosterone levels. Limitations & Conclusion The present study was performed retrospectively in order to collect possible cases for the tissue gene analysis. We were able to conduct the statistical analysis even though the number of patients in the wild type group was not equal to that in the mutated group and the follow-up studies after surgical treatment were slightly limited. Therefore, it is necessary to investigate a large sample size in future reports, such as nationwide studies. The present data clearly demonstrated differences in the rate of improvement in cardiovascular complications between APA patients with and without KCNJ5 gene mutations undergoing unilateral adrenalectomy. Cases of APA associated with more severe phenotypes, such as APA with KCNJ5 gene mutations, should be diagnosed earlier in order to avoid rapid progression of fatal cardiovascular complications due to the early onset of hypertension and hyperaldosteronemia, although it is currently difficult to detect such mutations prior to. As the cardiovascular complications were more severe and surgical treatment was more effective in the KCNJ5-mutated group than in the wild type group, physicians should precisely diagnose cases of APA with KCNJ5 gene mutations as early as possible after carefully examining the clinical characteristics and laboratory findings of each APA patient. Acknowledgement This study was partly supported by a Grant for Research on Intractable Diseases provided by the Japanese Ministry of Health, Labour and Welfare. Conflicts of Interest The authors declare that they have no conflicts of interest. References 1) Choi, M., U.I. Scholl, P. Yue, P. Bjorklund, B. Zhao, C. Nelson-Williams, W. Ji, Y. Cho, A. Patel, C.J. Men, E. Lolis, M.V. Wisgerhof, D.S. Geller, S. Mane, P. Hellman, G. Westin, G. Akerstrom, W. Wang, T. Carling, and R.P. Lifton, K channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension. Science, (6018): p ) Beuschlein, F., S. Boulkroun, A. Osswald, T. Wieland, H.N. Nielsen, U.D. Lichtenauer, D. Penton, V.R. Schack, L. Amar, E. Fischer, A. Walther, P. Tauber, T. Schwarzmayr, S. Diener, E. Graf, B. Allolio, B. Samson-Couterie, A. Benecke, M. Quinkler, F. Fallo, P.F. Plouin, F. Mantero, T. Meitinger, P. Mulatero, X. Jeunemaitre, R. Warth, B. Vilsen, M.C. Zennaro, T.M. Strom, and M. Reincke, Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension. Nat Genet, (4): p , 444e1-2 3) Scholl, U.I., G. Goh, G. Stolting, R.C. de Oliveira, M. Choi, J.D. Overton, A.L. Fonseca, R. Korah, L.F. Starker, J.W. Kunstman, M.L. Prasad, E.A. Hartung, N. Mauras, M.R. Benson, T. Brady, J.R. Shapiro, E. Loring, C. Nelson-Williams, S.K. Libutti, S. Mane, P. Hellman, G. Westin, G. Akerstrom, P. Bjorklund, T. Carling, C. Fahlke, P. Hidalgo, and R.P. Lifton, Somatic and germline CAC- NA1D calcium channel mutations in aldosterone-producing adenomas and primary aldosteronism. Nat Genet, (9): p ) Matsuzawa Y., Kitamoto T, Suematsu S, Saito J, Omura M and Nishikawa T, Steroidogenic Activity of Aldosterone-Producing Adenoma with and without KCNJ5 Gene Mutations, Comparing with That in Each Adherent Normal Tissue. J Endocrinol Diab, (1): p. 1-6

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