Published papers

Objective: Diabetic kidney disease (DKD) has been the most common cause of end-stage renal disease (ESRD) which leads to hemodialysis for more than 20 years in Japan. Various reports have been published on the mechanisms underlying the progression of DKD, however, many details remain unclear. We investigated the mechanisms underlying the progression of renal fibrosis and kidney injury in hyperglycemia from the viewpoint of metabolic alteration in the kidney by using streptozotocin-treated mice (STZ mice). Design/Methods: 8 weeks old ICR mice were treated with streptozotocin (200 mg/kg intra-peritoneally), to produce type 1 diabetes mellitus model mice. The kidneys of those mice were collected 4 weeks after STZ treatment and examined for metabolites that were altered in the kidneys of STZ mice by metabolome analysis. Amino acids that were increasing in the kidneys of STZ mice were administered to L929 murine fibroblast cells, and expression of pro-fibrotic genes were examined. Results: STZ-treatment caused progression of renal fibrosis in mice under both low- and high-protein diets. Metabolome analysis of the kidneys in STZ mice showed marked alterations of amino acids in the kidneys. Increased levels of branched chain amino acids (BCAAs) such as valine and leucine were observed. Among these amino acids, leucine administration to L929 cells resulted in increased expression of genes promoting renal fibrosis. Conclusions: Alterations of BCAAs in the kidneys due to hyperglycemia in STZ mice may affect progression of renal fibrosis and kidney injury. We are now investigating the mechanism by which BCAAs contribute to renal fibrosis.

Obesity is associated with dysregulation in blood pressure control and whole-body glucose metabolism, but the precise underlying mechanism(s) remain unclear. Here, we generated and characterized mice with vascular endothelial-cell specific deletion of nicotinamide phosphoribosyltransferase (NAMPT), which mediates the biosynthesis of nicotinamide adenine dinucleotide (NAD+), a key regulator of cellular energy metabolism. We found that vascular endothelial cell-specific Nampt knockout (VeNKO) mice exhibited 1) higher blood pressure (systolic blood pressure; flox/flox mice: 111 ± 2.6 mmHg, VeNKO mice: 134 ± 4.0 mmHg, p < 0.05) with cardiac hypertrophy (flox/flox mice: 5.0 mg/g-body weight, VeNKO mice: 6.1 mg/g-body weight, p < 0.05) and 2) insulin resistance with abnormal fat distribution, under high-fat diet feeding. These deleterious alterations were partially rescued by administering nicotinamide mononucleotide (NMN), a key NAD+ intermediate. Taken together, our findings demonstrate that vascular endothelial NAMPT-mediated NAD+ biosynthesis is involved in the regulation of blood pressure and whole-body glucose metabolism. Our study provides mechanistic and therapeutic insights into vascular endothelia NAD+ biology related to obesity-associated hypertension and insulin resistance.

Objectives: Total adrenalectomy of an affected adrenal gland is the standard therapy of aldosterone-producing adenoma (APA), a curable subtype of primary aldosteronism. However, the APA is occasionally inoperable in cases with bilateral adenomas or high-risk patients under general anesthesia. Computed tomography (CT)-guided radiofrequency ablation (RFA) of the adrenal gland is a less invasive, cortical-sparing therapy, and may become a treatment of choice for such cases. A multicenter clinical trial of RFA was conducted in patients with APA in Japan from 2015 to 2016. To establish its long-term efficacy and safety, the study subjects enrolled in Keio University Hospital have been annually followed up. Following the progress report at ISH 2018, here we update clinical and biochemical outcomes of this cohort of patients 6 years after the RFA treatment. Methods: Follow-up medical interviews, blood pressure measurements, blood tests, and urinalysis were done annually. In addition, at least one CT scan was performed since 2018. Data on antihypertensive medication, blood pressure, hormone and electrolyte levels, urinary albumin, and tumor size were collected and analyzed. Results: 10 cases of APA were treated with RFA between June 2015 and March 2016. As of April 2022, 6 cases were successfully followed up. Of these patients, the aldosterone-to-renin ratio (ARR) remained normalized in 5 cases (83%), albeit above the upper limit in 1 case (17%). In this particular patient, serum potassium level remained normalized and antihypertensive medication was not needed. Antihypertensive medication was needed in 1 case (17%), while urinary albumin levels were improved in all cases. Of note, follow-up CT scan showed that the tumor was undetectable in 1 case (17%), reduced in size in 3 cases (50%) and unchanged in 2 cases (33%). None of the cases have developed adrenal insufficiency since the RFA. Conclusion: The follow-up study on APA patients 6-year after the RFA revealed no cases of recurrence. RFA is likely to be effective and safe in patients with APA.

A 89-year-old woman was admitted to our hospital for syncope in 20XX-13. During the hospitalization, hypertension was pointed out. Despite 4 cm left adrenal mass suggestive of myelolipoma in contrast-enhanced magnetic resonance imaging (MRI), the serum and urine hormone levels in relation to secondary hypertension was within normal limits. Diagnosis of essential hypertension was made and treatment with olmesartan and azelnidipine was begun. In 20XX-5, her blood pressure was well controlled, and serum aldosterone level and active renin concentration (ARC) were 167 pg/ml and 6.8 pg/ml, respectively. Annual computed tomography (CT) scans showed virtually the same size of the adrenal tumor over 5 years. The trend toward increase in her blood pressure prompted us to repeat a blood test in 20XX, which turned out that serum aldosterone concentration was extremely high (1144 pg/ml), while ARC was suppressed to 1.7 pg/ml. In light of positive captopril challenge test, primary aldosteronism was suspected, and she was admitted to our hospital. However, systolic blood pressure was only mildly elevated (130 mmHg), though blood pressure medication had not been changed for 5 years. In addition, serum potassium value was 4.7 mEq/L and 24-hr urinary aldosterone was merely 3.4 μg/day under the salt-loaded state, which was inconsistent with exceedingly high serum aldosterone concentration and argued against the diagnosis of primary aldosteronism. Hence, validity of the method to quantitate serum aldosterone levels was questioned. To determine serum aldosterone concentration, radioimmunoassay (RIA) was applied in 20XX-5, yet chemiluminescent enzyme immunoassay (CLEIA) has been introduced since 20XX. Strikingly, reexamination of the same serum sample discovered that serum aldosterone value of 770 pg/ml in CLEIA was equivalent to 59 pg/ml by RIA method. As such, marked dissociation seems present between the two types of assays in this particular patient. Although tendency of correlation with regard to serum aldosterone levels between the two methods have been clarified, attention needs to be paid for the exception as in this case. Thus, it is fundamental to thoroughly evaluate patients with suspected primary aldosteronism as to whether the clinical course and serum aldosterone levels are compatible, and if not, it may be necessary to change the measurement method.

Case report: A 43-year-old man had been followed up for hypertension and hypokalemia, but self-interrupted his hospital visits for the last four years. Based on the serum potassium level of 2.7 mEq/L, plasma aldosterone concentration of 202 pg/mL, and plasma renin activity of 1.1 ng/mL/hr, he was suspected of primary aldosteronism and was admitted to the previous hospital. A saline suppression test and a rapid ACTH stimulation test were both positive, and 24-hr urinary aldosterone level was 19 μg/day under sodium loading of 170 mEq/day, leading to the diagnosis of primary aldosteronism. Since contrast-enhanced CT showed an 8 mm-sized adenoma in the lower portion of the left adrenal gland, an aldosterone-producing adenoma was suspected and a non-segmental adrenal venous sampling (AVS) was performed. However, it yielded apparent bilateral aldosterone suppression in the main trunk of the bilateral adrenal veins, where the aldosterone-to-cortisol (A/C) ratio was lower than that in the inferior vena cava even after ACTH loading. The patient was referred to our hospital for a segmental AVS. Arteriography was first performed from the left inferior adrenal artery to confirm the outflow vessels from the adenoma. Strikingly, there was an aberrant vein which drained into the proximal segment of the left adrenal vein. CT imaging at the time of arteriography revealed, in addition to the known adenoma, a 6 mm-sized micronodule in the lateral region of the left adrenal gland, and the aberrant vein appeared to be the main outflow vessel from the micronodule. A segmental AVS was performed, and blood samples were taken from the bilateral adrenal veins including the aberrant vein and the main trunk of the left adrenal vein proximal to its confluence. The A/C ratio was highest in the aberrant vein, followed by in the main trunk of the proximal left adrenal vein, while it was markedly suppressed in other branches and the main trunk of the left adrenal vein distal to the confluence of the aberrant vein. Based on these results, the presence of aldosterone-producing adenoma in the left adrenal gland was suggested. Laparoscopic partial left adrenalectomy was performed without postoperative complications. Hypertension and hypokalemia were fully resolved afterwards. Conclusions: Adrenal arteriography and segmental AVS should be considered in such cases that apparent bilateral aldosterone suppression is noted in conventional AVS in patients with clinically suspected aldosterone-producing adenoma.

Background: Renovascular hypertension (RVH) is one of the most common forms of secondary hypertension, and accounts for nearly 1% of all hypertension cases. It is characterized by compromised renal perfusion pressure, resultant renin secretion, and hypokalemia due to secondary hyperaldosteronism. Arteriosclerosis is a common underlying pathogenesis in middle-aged and older adults, whilst fibromuscular dysplasia is occasionally seen in younger patients with RVH. Since cardiovascular complications are of grave concern in RVH, early detection and appropriate therapeutic intervention are particularly important. Here, we report a case of renovascular hypertension from fibromuscular dysplasia which was incidentally unveiled during close examination of an adrenal tumor. Case report: A 55-year-old woman was being treated for hypertension by her primary care physician. She was referred to our hospital for a thorough examination of her right ureteral stone, which necessitated a CT scan incidentally revealing an left adrenal nodule measuring 8 mm in size. Primary aldosteronism was unlikely based on the lack of renin suppression (active renin concentration, 36.5 pg/mL; plasma aldosterone levels, 263 pg/mL and aldosterone-renin ratio < 40 under treatment with nifedipine 40 mg/day. Ultrasound of the kidney demonstrated a notable difference in size between the right and left kidneys of 87 mm and 106 mm, respectively, albeit Renal Artery Doppler failed to suggest the renal artery stenosis. CT scan with contrast material revealed the remarkable irregularity and “string on beads” sign on the distal main trunk of the right renal artery. Renovascular hypertension from fibromyalgia dysplasia was suspected, and percutaneous renal artery angioplasty was performed. After the pressure gradient across the stenotic lesion (105/99 mmHg on the distal side and 125/67 mmHg on the proximal side of the renal artery) was verified, balloon dilation was applied. The procedure was successful as the pressure gradient virtually resolved (105/95 mmHg on the distal side and 109/101 mmHg on the proximal side of the renal artery). Digital subtraction angiography indeed reaffirmed improvement in blood flow. Conclusion: Renovascular hypertension can go unnoticed solely based on renal Doppler Ultrasound. This was a case in which renovascular hypertension from fibromuscular dysplasia was incidentally identified in CT scan designated as a work-up for an adrenal tumor.

Objective: In the part of the Japan Primary Aldosteronism Study (JPAS), the prediction algorithm for the subtype diagnosis of primary aldosteronism (PA) was advocated and published in 2018 (Umakoshi.H et.al, J Clin Endocrinol Metab, 2018, 103(3):900–908). In the algorithm, patients with PA with bilateral normal results on CT and normokalemia likely have a low prior probability of a lateral form of PA in AVS (6.2%) and could be treated medically, whereas those with unilateral visible mass on CT and presence of hypokalemia have a high probability of a lateralized form of PA in AVS (70.6%). To validate the efficacy of the algorithm, we applied the algorithm for recent PA cases in our institute, Keio University hospital. Patients: A total of 77 patients with PA who underwent adrenal venous sampling (AVS) between November 2018 and October 2019 were enrolled in this study. They were classified into four groups according to CT findings and potassium status. Subtype diagnosis of PA was determined by AVS. Results: In our case, the prevalence for unilateral hyperaldosteronism on AVS was as follows: CT-negative with normokalemia, 7.9%; CT-positive with normokalemia, 15.4%; CT-negative with hypokalemia 22.2%, and CT-positive, 71.4%. The results were quite similar to the results in the previous report. In the algorithm, it was stated that AVS could be spared in patients with unilateral lesion on CT and hypokalemia if they were younger than 35 years, however, in our cases, there was one patient whose age was 30 with unilateral lesion on CT and hypokalemia and the AVS result of whom indicated bilateral PA. In the algorithm, it was also stated that AVS could be spared in patients with bilateral lesions on CT and normokalemia if plasma aldosterone concentration was less than 8 ng/dl and it was consistent in our cases. Conclusion: Overall, the prediction algorithm for the subtype diagnosis of PA developed in JPAS could be applied well in our recent PA cases.

A 41-year-old woman was admitted to the obstetrics department because of epigastric pain, nausea, and fatigue at 31 weeks of gestation. She was suspected of subclinical Cushing's syndrome based on high plasma cortisol and low ACTH levels along with an incidentally discovered tumor in the left adrenal gland 4 years prior to conception. 3 months before admission, 75 g oral glucose tolerance test yielded one point positive (81–169–174 mg/dL) and diagnosis of gestational diabetes mellitus was made. Diet and exercise therapy was started on an outpatient basis. While blood glucose control had been favorable, she discontinued visits to outpatient clinics due to difficulty in physical activity. At the time of admission, blood glucose level was running more than 200 mg/dl, and self-monitoring of blood glucose (SMBG) and insulin self-injection therapy were proposed for immediate blood glucose control accordingly. However, the patient had psychiatric symptoms that were presumably associated with delusional disorder and subclinical Cushing's syndrome. As such, she refused to implement both procedures on account of a strong fear against puncture, which caused blood glucose management difficult. Despite understanding the necessity of SMBG and potential risks to the fetus, the patient frequently expressed anxiety and could not perform SMBG. Given the difficult circumstance, intermittently scanned continuous glucose monitoring was installed to reduce invasive procedures. As a result, blood glucose levels were under good control with 0.43 U/kg of insulin per day until the delivery by cesarean section at 34 weeks of gestation. A healthy infant weighing 2442 g was delivered without complications. Here we highlight utility and efficacy of intermittently scanned continuous glucose monitoring in a difficult patient with gestational diabetes mellitus and subclinical Cushing's syndrome.

Surveillance of metastatic lesions and biochemical control of catecholamines in patients with malignant pheochromocytoma remain a fundamental problem to be solved. Here we present a 60-year-old male who visited a local clinic because of headache, sweating, malaise, and loss of body weight. Hypertension, orthostatic hypotension, along with remarkable increase in serum and urine noradrenaline, and urine metanephrine and vanillylmandelic acid were noted, and pheochromocytoma was suspected accordingly. Magnetic resonance imaging failed to reveal adrenal gland or sympathetic plexuses, yet it uncovered metastatic lesions in the liver and spine. Functional imaging study using 131I-Metaiodobenzylguanidine (MIBG) scan detected notable uptake in the spinal lesion, albeit not in the hepatic lesion. On the other hand, positron emission tomography with fluorodeoxyglucose (FDG-PET) disclosed FDG uptake both in the hepatic and spinal masses. Biopsy of the hepatic tumors revealed chromogranin A-positive clusters of cells, a finding consistent with metastatic pheochromocytoma. Transarterial embolization (TAE) of the metastatic tumors in the liver was performed for the purpose of tumor mass reduction. Additionally, TAE followed by resection of the spinal lesion was conducted. Pathological examination of the spinal tumor also showed chromogranin A-positive cells, leading to diagnosis of malignant pheochromocytoma. Crucially, TAE of the liver and spinal tumors resulted in dramatic reduction of catecholamine production, likely constituting an effective therapeutic strategy against metastatic pheochromocytoma (Homma, et al. Hypertens Res 2006). Afterwards, the patient became unable to walk because of weakness in the left leg at the age of 64. It seemed attributed to spinal cord compression from the tumor at the level of thoracic vertebrae 7. The patient underwent posterior decompression and fusion, and his muscle strength of the left leg was recovered. At the age of 65, the patient developed paralytic ileus following a surgery against metastatic tumors in the skull. In this case report, we discuss early detection of metastatic lesions and biochemical control of catecholamines in patients with malignant pheochromocytoma.

Abstract Primary aldosteronism (PA) has two major subtypes: unilateral (uPA) and bilateral (bPA). Although several diagnostic models for subtype classification have been reported, the optimal combination of algorithms and clinical features remains unclear. This study aimed to identify machine learning models and clinical features that contribute to PA subtype prediction. A total of 274 PA patients who underwent successful adrenal venous sampling (AVS) at a single center were analyzed. Overall, 196 endocrine features were comprehensively collected and classified into four categories: A, PA-related features; B, challenge tests; C, general biochemistry; and D, urinary steroid profile. Five machine learning algorithms were applied; predictive performance of the models as well as predictive contribution of features and categories were evaluated. Among the models, the random forest model achieved the highest predictive accuracy (91.3%). The most contributing feature in the RF model was plasma aldosterone concentration after the captopril challenge test (CCT90-PAC). Category B showed the greatest contribution to RF, followed by Categories A, D, and C. Combining Categories A and B improved predictive performance. These findings indicate that machine learning models, particularly RF, are effective for PA subtype prediction, with challenge test–related features in Category B making a major contribution.

Primary aldosteronism (PA) has two major subtypes: unilateral (uPA) and bilateral (bPA). Although several diagnostic models for subtype classification have been reported, the optimal combination of algorithms and clinical features remains unclear. This study aimed to identify machine learning models and clinical features that contribute to PA subtype prediction. A total of 274 PA patients who underwent successful adrenal venous sampling (AVS) at a single center were analyzed. Overall, 196 endocrine features were comprehensively collected and classified into four categories: A, PA-related features; B, challenge tests; C, general biochemistry; and D, urinary steroid profile. Five machine learning algorithms were applied; predictive performance of the models as well as predictive contribution of features and categories were evaluated. Among the models, the random forest (RF) model achieved the highest predictive accuracy (91.3%). The most contributing feature in the RF model was plasma aldosterone concentration after the captopril challenge test (CCT90-PAC). Category B made the greatest contribution to RF, followed by Categories A, D, and C. Combining Categories A and B improved predictive performance. These findings indicate that machine learning models, particularly RF, are effective for PA subtype prediction, with challenge test-related features in Category B making a major contribution.

Objective Primary aldosteronism (PA) adversely affects cardiovascular and renal health and has been linked to increased urinary calcium excretion, elevated parathyroid hormone (PTH) levels, and reduced bone mineral density. Although adrenalectomy and mineralocorticoid receptor antagonists (MRAs) are established treatments, their differential effects on calcium dynamics across PA subtypes remain unclear. Design We conducted a retrospective observational study, analyzing a large, well-characterized cohort on calcium metabolism in PA. A total of 352 patients with PA and 57 controls were evaluated. Methods Clinical and biochemical parameters, including intact PTH (iPTH) and urinary electrolyte levels, were retrospectively assessed. Comparative analyses were performed before and after treatment in 43 patients with unilateral PA (UPA) and 131 patients with bilateral PA (BPA), with a direct comparison of adrenalectomy and MRAs in UPA. Results Patients with PA had higher urinary fractional excretion of calcium (FECa) (1.28% vs 1.04%, P <.001) and iPTH (60.0 vs 57.0 pg/mL, P =.047) than controls, with more pronounced abnormalities in UPA (FECa, 1.50%, iPTH 73.1 pg/mL). Both treatments reduced FECa and iPTH levels, but adrenalectomy resulted in greater improvement than MRAs in the UPA (FECa, 0.68% post-MRA vs 0.36% post-adrenalectomy). In the BPA group, MRAs decreased FECa, irrespective of disease severity. Conclusion Adrenalectomy was associated with greater improvements in calcium abnormalities than MRAs in patients with UPA, consistent with its cardiovascular benefits. These findings underscore the importance of subtype-specific strategies and suggest that adrenalectomy may offer additional benefits beyond cardiovascular outcomes.

Nicotinamide adenine dinucleotide (NAD) is a classical coenzyme regulating cellular energy metabolism. Emerging evidence demonstrates the causal relationship between defective NAD metabolism and various age-associated diseases. The major purpose of the present study was to investigate the role of adipocyte mitochondrial NAD biology in age-associated metabolic diseases. To this end, we focused on solute carrier family 25 member 51 (SLC25A51), a recently identified mitochondrial NAD transporter. We found that aging was associated with decreased adipose tissue SLC25A51 expression in both humans and mice. We next generated and analyzed novel knockout and overexpression models, which we have named adipocyte-specific Slc25a51 knockout (ASKO) and Slc25a51 overexpressing (ASLO) mice. ASKO mice had a marked decrease in adipose tissue mitochondrial NAD levels and exhibited age-associated systemic metabolic complications, such as obesity, glucose intolerance, insulin resistance, hyperinsulinemia, metabolic inflexibility, dyslipidemia, and hepatosteatosis. Mechanistically, loss of Slc25a51 reduced mitochondrial respiratory function, fatty acid oxidation capacity, and adiponectin production in adipose tissue, likely contributing to the development of systemic metabolic complications. Conversely, ASLO mice were protected from obesity and insulin resistance caused by aging. In conclusion, our results provide novel mechanistic and therapeutic insights into understanding the critical role of adipocyte mitochondrial NAD transporter SLC25A51 in the pathophysiology of age-associated metabolic diseases, particularly obesity and insulin resistance.

Nicotinamide adenine dinucleotide (NAD+) functions as an essential cofactor regulating a variety of biological processes. The purpose of the present study was to determine the role of nuclear NAD+ biosynthesis, mediated by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), in thermogenesis and whole-body energy metabolism. We first evaluated the relationship between NMNAT1 expression and thermogenic activity in brown adipose tissue (BAT), a key organ for non-shivering thermogenesis. We found that reduced BAT NMNAT1expression was associated with inactivation of thermogenic gene program induced by obesity and thermoneutrality. Next, we generated and characterized adiponectin-Cre-driven adipocyte-specific Nmnat1 knockout (ANMT1KO) mice. Loss of NMNAT1 markedly reduced nuclear NAD+ concentration by approximately 70% in BAT. Nonetheless, adipocyte-specific Nmnat1 deletion had no impact on thermogenic (rectal temperature, BAT temperature and whole-body oxygen consumption) responses to β-adrenergic ligand norepinephrine administration and acute cold exposure, adrenergic-mediated lipolytic activity, and metabolic responses to obesogenic high-fat diet feeding. In addition, loss of NMNAT1 did not affect nuclear lysine acetylation or thermogenic gene program in BAT. These results demonstrate that adipocyte NMNAT1 expression is required for maintaining nuclear NAD+ concentration, but not for regulating BAT thermogenesis or whole-body energy homeostasis.

Background/Aims: A significant role of (pro)renin receptor in the pathogenesis of end-organ damage has been suggested only in animal studies. This study was conducted to examine the mRNA expression of (pro)renin receptor in human artery. Methods: In 141 kidney failure patients, the mRNA was harvested from arterial fragments obtained during surgery constructing an arteriovenous access for hemodialysis therapy, and expression levels of (pro)renin receptor and other components of the renin-angiotensin system were determined. Results: Arterial (pro)renin receptor expression was similar in diabetic and non-diabetic patients, although plasma prorenin levels were significantly higher in the diabetic patients than in the non-diabetic patients. The arterial (pro)renin receptor mRNA levels of the hypertensive patients, who had not been treated with either angiotensin-converting enzyme (ACE) inhibitors or angiotensin II type 1 receptor blockers, were significantly lower than those of the patients who had been treated with either drug. Multiple regression analyses showed a significant association with a large coefficient between the arterial mRNA level of the (pro)renin receptor and the arterial mRNA level of ACE; this significant association disappeared in patients who had been treated with either drug. Conclusion: (Pro)renin receptor may contribute to the generation of arterial angiotensin II in kidney failure patients.

The (pro)renin receptor (P)RR is a receptor for renin and prorenin, not only allowing local production of angiotensin I from angiotensinogen, but also inducing intracellular signaling. Intriguingly, (P)RR is also called ATP6AP2 because a (P)RR fragment was demonstrated to be associated with vacuolar-type H+-ATPase (V-ATPase), which is of importance for the maintenance of intracellular pH. Recent studies implicate that deletion of (P)RR results in the dysfunction of V-ATPase, suggesting that the (P)RR is essential for its role as a proton pump. Furthermore, the novel function of (P)RR as an adaptor protein between the Wnt receptor complex and the V-ATPase was discovered. Thus, (P)RR is a multi-functional molecule with functions beyond renin and prorenin. This review focuses on the current knowledge and issues of (P)RR and V-ATPase.