Association of Serum Zinc Level with severity of chronic kidney disease in diabetic patients: a cross-sectional study.

BACKGROUND: In recent years, it has been reported that diabetic patients tend to have a lower zinc intake due to unbalanced diet accompanying changes in lifestyle habits. We investigated serum zinc concentration in diabetic patients according to the stage of nephropathy. METHODS: We enrolled 227 diabetic patients (119 men, 108 women, average age 65.7 ± 14.7 [mean ± standard deviation]) who were hospitalized for diabetes treatment due to poor blood glucose control. We investigated the relationship between fasting serum zinc concentration and estimated glomerular filtration rate (eGFR) and albuminuria (urinary albumin-to-creatinine ratio, UACR), as well as serum zinc concentration by stage of diabetic kidney disease and chronic kidney disease. RESULTS: The mean HbA1c value was 10.5 ± 2.1%. Serum zinc concentration was 75.5 ± 16.0 µg/dL in males and 75.7 ± 12.2 µg/dL in females, showing no gender difference and no significant relationship with diabetes type. The serum zinc concentration was negatively correlated with age (r = - 0.309, P < 0.001) and positively correlated with eGFR (r = 0.144, P = 0.030). A tendency was observed of serum zinc concentration to decrease after overt nephropathy, with values of 76.4 ± 14.1 µg/dL in pre-nephropathy (stage 1, n = 131), 78.5 ± 13.2 µg/dL in incipient nephropathy (stage 2, n = 65), 66.4 ± 14.3 µg/dL in overt nephropathy (stage 3, n = 25), and 65.7 ± 11.9 µg/dL in kidney failure (stage 4, n = 6). Serum zinc showed a negative trend with estimated GFR (P = 0.004) and significant reduction in albuminuria, with stage A3 (n = 29, 65.7 ± 13.9 µg/dL) having lower levels than A1 (n = 131, 76.4 ± 14.1 µg/dL, P = 0.001) and A2 (n = 67, 78.4 ± 13.1 µg/dL, P < 0.001). CONCLUSIONS: In diabetic patients, serum zinc concentration tended to decrease as age increased and also as renal function deteriorated. This study suggests that consideration of zinc deficiency is necessary in patients with overt albuminuria.

ATP1A1 Mutant in Aldosterone-Producing Adenoma Leads to Cell Proliferation.

The molecular mechanisms by which ATP1A1 mutation-mediated cell proliferation or tumorigenesis in aldosterone-producing adenomas (APAs) have not been elucidated. First, we investigated whether the APA-associated ATP1A1 L104R mutation stimulated cell proliferation. Second, we aimed to clarify the molecular mechanisms by which the ATP1A1 mutation-mediated cell proliferated. We performed transcriptome analysis in APAs with ATP1A1 mutation. ATP1A1 L104R mutation were modulated in human adrenocortical carcinoma (HAC15) cells (ATP1A1-mutant cells), and we evaluated cell proliferation and molecular signaling events. Transcriptome and immunohistochemical analysis showed that Na/K-ATPase (NKA) expressions in ATP1A1 mutated APA were more abundant than those in non-functioning adrenocortical adenoma or KCNJ5 mutated APAs. The significant increase of number of cells, amount of DNA and S-phase population were shown in ATP1A1-mutant cells. Fluo-4 in ATP1A1-mutant cells were significantly increased. Low concentration of ouabain stimulated cell proliferation in ATP1A1-mutant cells. ATP1A1-mutant cells induced Src phosphorylation, and low concentration of ouabain supplementation showed further Src phosphorylation. We demonstrated that NKAs were highly expressed in ATP1A1 mutant APA, and the mutant stimulated cell proliferation and Src phosphorylation in ATP1A1-mutant cells. NKA stimulations would be a risk factor for the progression and development to an ATP1A1 mutant APA.

Antithyroid arthritis syndrome caused by methimazole in a patient with Graves' disease.

Summary: This is a report on antithyroid arthritis syndrome (AAS) which is a rare adverse effect of antithyroid agents. AAS presents with severe symptoms including myalgia, arthralgia, arthritis, fever, and skin eruption due to the use of antithyroid agents. We encountered a 55-year-old woman with severe pain in the hand and forearm and arthralgia in multiple joints, including the knee, ankle, hand, and wrist on day 23 after initiation of methimazole (MMI) for Graves' disease. Blood tests revealed elevated inflammation markers such as C-reactive protein and interleukin-6, and magnetic resonance imaging of the hands confirmed inflammation findings. After withdrawing MMI on day 25, symptoms showed a tendency toward improvement. Afterwards, inflammation markers also dropped to an almost normal range. In addition to the above findings, the absence of anti-neutrophil cytoplasmic antibodies and most vasculitis symptoms such as nephritis, skin, or pulmonary lesions led to the diagnosis of AAS. A resolution of symptoms, except for mild arthralgia in the second to fourth fingers of the right hand, was observed 61 days after discontinuation of MMI. Although the pathogenesis is unclear, the positive drug lymphocyte stimulation test for MMI and the several weeks before the onset of AAS suggested involvement of a type IV allergic reaction. Based on a discussion of definitive treatment for Graves' disease, radioactive iodine ablation with 131I, which was selected by the patient, was performed and improved her thyroid function. Our case demonstrates the importance of awareness regarding AAS, which is a rare and under-recognized, but life-threatening adverse effect of antithyroid agents. Learning points: Clinicians should be aware of the possibility of developing antithyroid arthritis syndrome (AAS) in patients treated with antithyroid medications, which can lead to severe migratory polyarthritis. Prompt cessation of the antithyroid agent is essential for the resolution of AAS. Anti-neutrophil cytoplasmic antibody (ANCA) negativity is needed to differentiate from antithyroid agent-induced ANCA-associated vasculitis, which shows arthritis similar to AAS.

Hypomethylation associated vitamin D receptor expression in ATP1A1 mutant aldosterone-producing adenoma.

DNA methylation alteration is tissue-specific and play a pivotal role in regulating gene transcription during cell proliferation and survival. We aimed to detect genes regulated by DNA methylation, and then investigated whether the gene influenced cell proliferation or survival in adrenal cells. DNA methylation and qPCR analyses were performed in nonfunctioning adrenocortical adenoma (NFA, n = 12) and aldosterone-producing adenoma (APA, n = 35) samples. The VDR gene promoter was markedly hypomethylated in APA with ATP1A1 mutation, and the promoter methylation levels showed a significant inverse association with the transcripts in APA. ATP1A1 mutation led to VDR transcription in HAC15 cells, and VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. We demonstrated that APA with ATP1A1 mutation showed entire hypomethylation in the VDR promoter and abundant VDR mRNA and protein expression. VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. Abundant VDR expression would be essential for ATP1A1 mutation-mediated cell proliferation.

Endoplasmic Reticulum Chaperone Calmegin Is Upregulated in Aldosterone-Producing Adenoma and Associates With Aldosterone Production.

The endoplasmic reticulum (ER) plays a pivotal role in syntheses of proteins and steroid hormones and regulation of intracellular Ca2+ level. We aimed to investigate ER-associated genes in aldosterone-producing adenomas (APAs) and clarify their effect on aldosterone production. Microarray analysis targeting 288 ER-associated genes was conducted using nonfunctioning adrenocortical adenomas (n=5) and APAs (n=19). Immunohistochemistry and quantitative polymerase chain reaction analyses were performed with 13 nonfunctioning adrenocortical adenoma and 48 APA samples. Functional studies were performed with human adrenocortical carcinoma (HAC15) cells, some of which were genetically modified using lentiviruses. The ER chaperone calmegin (CLGN) was the most highly expressed ER-associated gene in APAs relative to nonfunctioning adrenocortical adenomas. Analysis with quantitative polymerase chain reaction revealed CLGN to be 9.5-fold upregulated in APAs relative to nonfunctioning adrenocortical adenomas. There were no differences among different APA genotypes affecting aldosterone production. Immunohistochemistry analysis revealed that CLGN was strongly expressed in APAs and aldosterone-producing cell clusters. Angiotensin II stimulation or KCNJ5 T158A overexpression in HAC15 cells did not affect CLGN mRNA levels. CLGN overexpression in HAC15 cells increased aldosterone levels but did not stimulate CYP11B2 mRNA levels. Pathway and gene ontology analyses using RNA sequencing results showed that tRNA aminoacyl metabolism was the most enriched pathway in CLGN-overexpressing cells. CYP11B2 (aldosterone synthase) and HSD3B2 (3 beta-hydroxysteroid dehydrogenase/delta 5->4-isomerase type 2) protein expression were more abundant in CLGN-overexpressing cells. CLGN knockdown using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) method in HAC15 cells that carry the KCNJ5 mutation did not affect aldosterone production. To summarize, CLGN was upregulated and associated with aldosterone production via translational regulation of CYP11B2 in APAs.

Fulminant Type 1 Diabetes Mellitus Presenting 15 Days after Delivery Diagnosed in Cooperation with Obstetricians.

The patient was a 32-year-old Japanese woman who was given a 75-g oral glucose tolerance test at the 35th week of pregnancy and was normoglycemic. She had excessive thirst and polyuria from 15 days after delivery. When she visited for the 1-month postpartum checkup, her plasma glucose level was 479 mg/dL, HbA1c was 7.4%, and urinary C-peptide was 1.1 µg/mL; she was therefore diagnosed with fulminant type 1 diabetes mellitus associated with pregnancy. All physicians should be aware of this disease so as to provide a prompt diagnosis and emergency treatment and consequently improve the maternal prognosis.

Aberrant G protein-receptor expression is associated with DNA methylation in aldosterone-producing adenoma.

This study aimed to evaluate the methylation levels of G protein-coupled receptor (GPCR) related genes and the effects of methylation on mRNA expression levels in aldosterone-producing adenoma (APA). DNA methylation array and transcriptome analysis were applied in non-functioning adrenocortical adenoma (NFA) and APA. We investigated 192 GPCR-related genes and found hypo-methylation in the promoter region of 66 of these genes in APA. An integration study between microarray and methylation analysis revealed that HTR4, MC2R, TACR1, GRM3, and PTGER1 showed hypo-methylation and up-regulation of mRNA in APA. qPCR analysis showed that HTR4 and PTGER1 expression was 9.3-fold and 6.6-fold higher in APAs than in NFAs, respectively, whereas expression of the other genes was not different between the groups. Methylation of HTR4 and PTGER1 at positions -229 and -666 from the transcription start site, respectively, showed a significant inverse correlation with their mRNA levels. Methylation levels were not associated with KCNJ5 or ATP1A1 mutations in human adrenal samples. We demonstrated an increased incidence of CpG island demethylation of GPCR-related gene in APA. The expression of two receptors, HTR4 and PTGER1, showed a strong association with DNA methylation.

Purkinje Cell Protein 4 Expression Is Associated With DNA Methylation Status in Aldosterone-Producing Adenoma.

Context: Aldosterone production is stimulated by activation of calcium signaling in aldosterone-producing adenomas (APAs), and epigenetic factors such as DNA methylation may be associated with the expression of genes involved in aldosterone regulation. Objective: Our aim was to investigate the DNA methylation of genes related to calcium signaling cascades in APAs and the association of mutations in genes linked to APAs with DNA methylation levels. Methods: Nonfunctioning adrenocortical adenoma (n = 12) and APA (n = 35) samples were analyzed. The KCNJ5 T158A mutation was introduced into human adrenocortical cell lines (HAC15 cells) using lentiviral delivery. DNA methylation array analysis was conducted using adrenal tumor samples and HAC15 cells. Results: The Purkinje cell protein 4 (PCP4) gene was one of the most hypomethylated in APAs. DNA methylation levels in two sites of PCP4 showed a significant inverse correlation with messenger RNA expression in adrenal tumors. Bioinformatics and multiple regression analysis revealed that CCAAT/enhancer binding protein alpha (CEBPA) may bind to the methylation site of the PCP4 promoter. According to chromatin immunoprecipitation assay, CEBPA was bound to the PCP4 hypomethylated region by chromatin immunoprecipitation assay. There were no significant differences in PCP4 methylation levels among APA genotypes. Moreover, KCNJ5 T158A did not influence PCP4 methylation levels in HAC15 cells. Conclusions: We showed that the PCP4 promoter was one of the most hypomethylated in APAs and that PCP4 transcription may be associated with demethylation as well as with CEBPA in APAs. KCNJ5 mutations known to result in aldosterone overproduction were not related to PCP4 methylation in either clinical or in vitro studies.

Calneuron 1 Increased Ca2+ in the Endoplasmic Reticulum and Aldosterone Production in Aldosterone-Producing Adenoma.

Aldosterone production is initiated by angiotensin II stimulation and activation of intracellular Ca2+ signaling. In aldosterone-producing adenoma (APA) cells, the activation of intracellular Ca2+ signaling is independent of the renin-angiotensin-aldosterone systems. The purpose of our study was to clarify molecular mechanisms of aldosterone production related to Ca2+ signaling. Transcriptome analysis revealed that the CALN1 gene encoding calneuron 1 had the strongest correlation with CYP11B2 (aldosterone synthase) among genes encoding Ca2+-binding proteins in APA. CALN1 modulation and synthetic or fluorescent compounds were used for functional studies in human adrenocortical carcinoma (HAC15) cells. CALN1 expression was 4.4-fold higher in APAs than nonfunctioning adrenocortical adenomas. CALN1 expression colocalized with CYP11B2 expression as investigated using immunohistochemistry in APA and zona glomerulosa of male rats fed by a low-salt diet. CALN1 expression was detected in the endoplasmic reticulum (ER) by using GFP-fused CALN1, CellLight ER-RFP, and the corresponding antibodies. CALN1-overexpressing HAC15 cells showed increased Ca2+ in the ER and cytosol fluorescence-based studies. Aldosterone production was potentiated in HAC15 cells by CALN1 expression, and dose-responsive inhibition with TMB-8 showed that CALN1-mediated Ca2+ storage in ER involved sarcoendoplasmic reticulum calcium transport ATPase. The silencing of CALN1 decreased Ca2+ in ER, and abrogated angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells. Increased CALN1 expression in APA was associated with elevated Ca2+ storage in ER and aldosterone overproduction. Suppression of CALN1 expression prevented angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells, suggesting that CALN1 is a potential therapeutic target for excess aldosterone production.

Association between Serum Elaidic Acid Concentration and Insulin Resistance in Two Japanese Cohorts with Different Lifestyles.

AIM: Many cohort studies have shown that increased trans fatty acid (TFA) intake increases the risk of developing coronary heart disease. However, whether TFA intake is directly associated with the development of diabetes mellitus (DM) remains unknown. METHODS: We performed the 75-g oral glucose tolerance test in two Japanese cohorts: a cohort of 454 native Japanese living in Hiroshima, Japan, and a cohort of 426 Japanese-Americans living in Los Angeles, USA, who shared identical genetic predispositions but had different lifestyles. Serum elaidic acid concentration was measured and compared, and its association with insulin resistance was assessed. RESULTS: Serum elaidic acid concentrations were significantly higher in the Japanese-Americans (median, 18.2 µmol/L) than in the native Japanese (median, 11.0 µmol/L). The serum elaidic acid concentrations in the native Japanese DM group (16.0 µmol/L) were significantly higher compared with those in the normal glucose tolerance (10.8 µmol/L) and impaired glucose tolerance (11.7 µmol/L) groups. Multiple linear regression analyses showed that serum elaidic acid concentrations were significantly positively associated with homeostasis model assessment for insulin resistance (HOMA-IR) values after adjusting for various factors. CONCLUSIONS: These results suggest that excessive TFA intake worsens insulin resistance and increases the risk of developing DM even in the native Japanese, whose intakes of animal fat and simple carbohydrates were presumed to be lower than those of the Japanese-Americans.

Hypomethylation of CYP11B2 in Aldosterone-Producing Adenoma.

The purpose of this study was to evaluate the DNA methylation levels of steroidogenic enzyme genes in aldosterone-producing adenoma (APA) and the effects of gene mutations in APA on the DNA methylation levels. DNA methylation array analysis was conducted using nonfunctioning adrenocortical adenoma (n=12) and APA (n=35) samples, including some with a KCNJ5 mutation (n=21), an ATP1A1 mutation (n=5), and without the known mutations (n=9). The quantitative polymerase chain reaction assay was performed for the detection of CYP11B2 and CYP11B1 expression levels in nonfunctioning adrenocortical adenoma and APA. We introduced the KCNJ5 T158A mutation using lentivirus delivery in the human adrenocortical 15 cell line, and analyzed the effects of the mutation on DNA methylation levels. We analyzed the 83 presumed DNA methylation sites of steroidogenic enzymes. In APA, we found 7 hypomethylated sites in CYP11B2 and 1 hypomethylated and 6 hypermethylated sites in CYP11B1 There were no differences in the steroidogenic enzymes gene DNA methylation of peripheral leukocytes between nonfunctioning adrenocortical adenoma and APA. No CYP11B2 methylation level was associated with CYP11B2 transcription levels in APA. All methylation sites, except for a CYP11B2 region, showed no difference among APAs with or without gene mutations. Human adrenocortical 15 cells with the KCNJ5 mutation showed no changes in CYP11B2 or CYP11B1 methylation levels compared with control cells. We demonstrated that CYP11B2 in APA was extensively hypomethylated, and CYP11B2 methylation in the region with hypomethylation was not induced by KCNJ5 or ATP1A1 mutations that cause aldosterone overproduction in APA and a KCNJ5 mutation human adrenocortical 15 cells.