Combined evaluation of glomerular phospholipase A2 receptor and immunoglobulin G subclass in membranous nephropathy.

Background: Phospholipase A2 receptor (PLA2R) is a major target antigen in idiopathic membranous nephropathy (MN). Anti-PLA2R antibodies are mainly of the immunoglobulin G (IgG) subclass IgG4, although other IgG subclass depositions in glomeruli may also be detected. However, the importance of the subclass of the IgG deposit has not been proven. Thus we investigated clinical findings from patients with idiopathic MN in relation to glomerular PLA2R deposition and IgG subclass. Methods: We enrolled 132 Japanese patients with biopsy-proven idiopathic MN in a multicentre retrospective observational study. We investigated the complete remission rate as the primary outcome and the development of end-stage kidney disease (ESKD) as the secondary outcome in relation to glomerular PLA2R deposition. Moreover, we evaluated prognostic factors, including glomerular IgG subclass, in the PLA2R-positive group. Results: The percentage of cases with glomerular PLA2R deposition was 76.5% (n = 101). The first complete remission rate of the PLA2R-positive group was worse than that of the PLA2R-negative group (logrank test P < .001). ESKD incidence did not significantly differ between the glomerular PLA2R-negative and PLA2R-positive MN groups (logrank test P = .608). In the PLA2R-positive group, higher PLA2R intensities and IgG2 staining were associated with a poorer first complete remission rate (logrank test P < .001 and P = .032, respectively). Cox proportional hazards analysis also showed that strong PLA2R deposition and positive IgG2 staining were significantly associated with a failure to reach complete remission [hazard ratio 2.09 (P = .004) and 1.78 (P = .030), respectively]. Conclusions: Our results suggest that intense glomerular PLA2R and IgG2 positivity predict a poor proteinuria remission rate in idiopathic MN.

Association between Hemoglobin A1c and Renal Arteriolar Sclerosis in Subjects Presenting without any Apparent Kidney Dysfunction.

AIMS: Diabetic kidney disease is a major vascular complication in patients with diabetes mellitus (DM). However, the association between the hemoglobin (Hb)A1c levels, notably the prediabetic levels, and renal pathological changes remains unclear. We investigated the association between the HbA1c levels and renal arteriolar lesions in subjects without any apparent kidney dysfunction using a living kidney donor cohort. METHODS: Between January 2006 and May 2016, 393 living kidney donors underwent a "zero-time" biopsy at Kyushu University Hospital. The patients were divided into four groups (HbA1c levels ＜5.6%, 5.6%-5.7%, 5.8%-6.4%, and ≥ 6.5%, or diagnosed with DM [DM group]). Renal arteriolar hyalinization and wall thickening were assessed using semi-quantitative grading. We then investigated the association between the HbA1c levels and renal pathological changes. RESULTS: 158 (40.2%) patients had arteriolar hyalinization and 148 (37.6%) showed wall thickening. A significant correlation was observed between the HbA1c levels and wall thickening (p for trend ＜0.001). An elevated HbA1c level was significantly associated with wall thickening according to a multivariable logistic analysis in subjects with HbA1c levels of 5.6%-5.7% and 5.8%-6.4%, and the DM group, compared with those with HbA1c levels of ＜5.6% (odds ratio [OR], 1.91; 95% confidence interval [CI]: [1.03-3.54] for 5.6%-5.7%, OR, 1.96; 95% CI: [1.09-3.53] for 5.8%-6.4%, and OR, 2.86; 95% CI: [0.91-9.01] for the DM group), whereas arteriolar hyalinization did not increase within the nondiabetic HbA1c levels. CONCLUSIONS: Elevated high-normal HbA1c levels are considered to be independent risk factors for arteriolar wall thickening. Subclinical renal arteriolar sclerosis may develop in patients with prediabetic HbA1c levels.

S-Palmitoylation of the serotonin transporter promotes its cell surface expression and serotonin uptake.

The neurotransmitter serotonin (5-HT) is transported back into serotonergic neurons by the serotonin transporter (SERT). SERT is a main target of antidepressants, and much effort has therefore focused on finding relationships between SERT and depression. However, it is not fully understood how SERT is regulated at the cellular level. Here, we report post-translational regulation of SERT by S-palmitoylation, in which palmitate is covalently attached to cysteine residues of proteins. Using AD293 cells (a human embryonic kidney 293-derived cell line with improved cell adherence) transiently transfected with FLAG-tagged human SERT, we observed S-palmitoylation of immature SERT containing high-mannose type N-glycans or no N-glycan, which is presumed to be localized in the early secretory pathway, such as the endoplasmic reticulum. Mutational analysis by alanine substitutions shows that S-palmitoylation of immature SERT occurs at least at Cys-147 and Cys-155, juxtamembrane cysteine residues within the first intracellular loop. Furthermore, mutation of Cys-147 reduced cellular uptake of a fluorescent SERT substrate that mimics 5-HT without decreasing SERT on the cell surface. On the other hand, combined mutation of Cys-147 and Cys-155 inhibited SERT surface expression and reduced the uptake of the 5-HT mimic. Thus, S-palmitoylation of Cys-147 and Cys-155 is important for both the cell surface expression and 5-HT uptake capacity of SERT. Given the importance of S-palmitoylation in brain homeostasis, further investigation of SERT S-palmitoylation could provide new insights into the treatment of depression.

Epithelial cell-derived cytokine TSLP activates regulatory T cells by enhancing fatty acid uptake.

Epithelial cells control a variety of immune cells by secreting cytokines to maintain tissue homeostasis on mucosal surfaces. Regulatory T (Treg) cells are essential for immune homeostasis and for preventing tissue inflammation; however, the precise molecular mechanisms by which epithelial cell-derived cytokines function on Treg cells in the epithelial tissues are not well understood. Here, we show that peripheral Treg cells preferentially respond to thymic stromal lymphoprotein (TSLP). Although TSLP does not affect thymic Treg differentiation, TSLP receptor-deficient induced Treg cells derived from naïve CD4+ T cells are less activated in an adoptive transfer model of colitis. Mechanistically, TSLP activates induced Treg cells partially through mTORC1 activation and fatty acid uptake. Thus, TSLP modulates the activation status of induced Treg through the enhanced uptake of fatty acids to maintain homeostasis in the large intestine.

The effect of the Kasuya CKD network on prevention of the progression of chronic kidney disease: successful collaboration of a public health service, primary care physicians and nephrologists-community based cohort study.

BACKGROUND: In 2012, we established a CKD network in collaboration with the public health service, primary care physicians, and nephrologists in the Kasuya area. The aim of this study was to clarify if our CKD network was effective in preventing CKD progression. METHODS: 1591 subjects, who had CKD in health checks in 2012 were included in this study. The slope of estimated glomerular filtration rate (eGFR) was compared before and after 2012. Parameters at the first health check visit before 2012, visit in 2012, and the last visit after 2012, were compared. Paired t test, analysis of variance for repeated measurements, and the Friedman test were used for the analysis. RESULTS: Mean age was 65 years. There were 781 men and 810 women. Mean eGFR was 59 ml/min/1.73 m2. The mean slope of eGFR before 2012 was -1.833 ml/min/1.73 m2/year and significantly reduced to - 0.297 after 2012. Low-density lipoprotein cholesterol showed a significant serial lowering. Uric acid was significantly elevated in 2012 compared to the first visit and had decreased by the last. The dipstick urinary protein significantly increased in 2012 compared to the first visit and decreased by the last. The number of current smokers showed a significant reduction over time. On the other hand, systolic blood pressure (SBP) and HbA1c significantly elevated at the last visit. CONCLUSION: The Kasuya CKD network may be effective in preventing CKD progression.

Current antihypertensive treatment and treatment-resistant hypertension in Japanese patients with chronic kidney disease.

BACKGROUND: Hypertension is an important prognostic predictor in patients with chronic kidney disease (CKD), and the recommended target blood pressure has been continuously revised. This study aimed to reveal the current antihypertensive practices in Japanese patients with CKD. METHODS: In the Fukuoka Kidney disease Registry, we extracted 3664 non-dialysis-dependent patients with CKD. Apparent treatment-resistant hypertension (aTRH) was defined as a failure of blood-pressure control treated with three antihypertensive medication classes or a treatment with ≥ 4 classes regardless of blood pressure. The blood-pressure control complied with the target blood pressure recommended by the KDIGO 2012 guideline. RESULTS: The median age of the patients was 67 years, body mass index (BMI) was 23 kg/m2, and estimated glomerular filtration rate (eGFR) was 40 mL/min/1.73 m2. The number of patients with unachieved blood-pressure control was 1933, of whom 26% received ≥ 3 classes of antihypertensive medications. The first choice of medication was renin-angiotensin system inhibitors, followed by calcium-channel blockers. The rate of thiazide use was low in all CKD stages (3-11%). The prevalence of aTRH was 16%, which was significantly associated with BMI (odds ratio [95% confidence interval] per 1-standard deviation change, 1.38 [1.25-1.53]), decreased eGFR (1.87 [1.57-2.23]), as well as age, diabetes mellitus, and chronic heart disease. CONCLUSIONS: Renal dysfunction and obesity are important risk factors of aTRH. Even under nephrologist care, most patients were treated with insufficient antihypertensive medications. It is important to prescribe sufficient classes of antihypertensive medications, including diuretics, and to improve patients' lifestyle habits.

Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium.

OBJECTIVES: Methotrexate (MTX) is an anchor drug for the treatment of rheumatoid arthritis (RA). However, the precise mechanisms by which MTX stalls RA progression and alleviates the ensuing disease effects remain unknown. The aim of the present study was to identify novel therapeutic target molecules, the expression patterns of which are affected by MTX in patients with RA. METHODS: CD4+ T cells from 28 treatment-naïve patients with RA before and 3 months after the initiation of MTX treatment were subjected to DNA microarray analyses. The expression levels of semaphorin 3G, a differentially expressed gene, and its receptor, neuropilin-2, were evaluated in the RA synovium and collagen-induced arthritis synovium. Collagen-induced arthritis and collagen antibody-induced arthritis were induced in semaphorin3G-deficient mice and control mice, and the clinical score, histological score, and serum cytokines were assessed. The migration and proliferation of semaphorin 3G-stimulated bone marrow-derived macrophages were analyzed in vitro. The effect of local semaphorin 3G administration on the clinical score and number of infiltrating macrophages during collagen antibody-induced arthritis was evaluated. RESULTS: Semaphorin 3G expression in CD4+ T cells was downregulated by MTX treatment in RA patients. It was determined that semaphorin 3G is expressed in RA but not in the osteoarthritis synovium; its receptor neuropilin-2 is primarily expressed on activated macrophages. Semaphorin3G deficiency ameliorated collagen-induced arthritis and collagen antibody-induced arthritis. Semaphorin 3G stimulation enhanced the migration and proliferation of bone marrow-derived macrophages. Local administration of semaphorin 3G deteriorated collagen antibody-induced arthritis and increased the number of infiltrating macrophages. CONCLUSIONS: Upregulation of semaphorin 3G in the RA synovium is a novel mechanism that exacerbates joint inflammation, leading to further deterioration, through macrophage accumulation.

Prevalence, treatment status, and predictors of anemia and erythropoietin hyporesponsiveness in Japanese patients with non-dialysis-dependent chronic kidney disease: a cross-sectional study.

BACKGROUND: Hyporesponsiveness to erythropoiesis-stimulating agents (ESAs) has been highlighted as a potential risk factor for cardiovascular disease in patients with chronic kidney disease (CKD). METHODS: We assessed cross-sectionally the prevalence, associated factors, and treatment status of anemia and ESA hyporesponsiveness in 4460 non-dialysis-dependent CKD patients enrolled in a multicenter cohort in Japan. Anemia was defined as a hemoglobin (Hb) level of less than 11 g/dL or receiving ESA therapy. ESA hyporesponsiveness was defined by the erythropoietin-resistance index (ERI), which was the erythropoietin dose per week divided by body weight and Hb level (U/kg/week/g/dl). RESULTS: Of the 4460 patients, 1050 (23.5%) had anemia. ESAs were administered to 626 patients, reaching a percentage of 57.5% of patients with stage G5 CKD. However, the ESA treatment rate was only 49.0% in patients with a hemoglobin level of < 11 g/dL. The proportion of patients receiving iron supplementation was lower than that of patients receiving ESAs regardless of CKD stage or hemoglobin level, and a significant proportion of patients did not receive iron supplementation, even those with iron deficiency. The ERI increased with CKD stage progression, and the multiple regression analysis showed that age, female sex, body mass index, cholesterol, glomerular filtration rate, and intact parathyroid hormone level were independent contributors. CONCLUSIONS: Our findings demonstrate that many Japanese patients with non-dialysis-dependent CKD receiving ESAs fail to maintain adequate hemoglobin levels. These results suggest the need for interventions for ESA hyporesponsiveness factors in addition to iron supplementation.

Potential role of inducible GPR3 expression under stimulated T cell conditions.

G protein-coupled receptor 3 (GPR3) constitutively activates Gαs proteins without any ligands and is predominantly expressed in neurons. Since the expression and physiological role of GPR3 in immune cells is still unknown, we examined the possible role of GPR3 in T lymphocytes. The expression of GPR3 was upregulated 2 h after phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation and was sustained in Jurkat cells, a human T lymphocyte cell line. In addition, the expression of nuclear receptor 4 group A member 2 (NR4A2) was highly modulated by GPR3 expression. Additionally, GPR3 expression was linked with the transcriptional promoter activity of NR4A in Jurkat cells. In mouse CD4+ T cells, transient GPR3 expression was induced immediately after the antigen receptor stimulation. However, the expression of NR4A2 was not modulated in CD4+ T cells from GPR3-knockout mice after stimulation, and the population of Treg cells in thymocytes and splenocytes was not affected by GPR3 knockout. By contrast, spontaneous effector activation in both CD4+ T cells and CD8+ T cells was observed in GPR3-knockout mice. In summary, GPR3 is immediately induced by T cell stimulation and play an important role in the suppression of effector T cell activation.

GPR3 accelerates neurite outgrowth and neuronal polarity formation via PI3 kinase-mediating signaling pathway in cultured primary neurons.

During neuronal development, immature neurons extend neurites and subsequently polarize to form an axon and dendrites. We have previously reported that G protein-coupled receptor 3 (GPR3) levels increase during neuronal development, and that GPR3 has functions in neurite outgrowth and neuronal differentiation in cerebellar granular neurons. Moreover, GPR3 is transported and concentrated at the tips of neurite, thereby contributing to the local activation of protein kinase A (PKA). However, the signaling pathways for GPR3-mediated neurite outgrowth and its subsequent effects on neuronal polarization have not yet been elucidated. We therefore analyzed the signaling pathways related to GPR3-mediated neurite outgrowth, and also focused on the possible roles of GPR3 in axon polarization. We demonstrated that, in cerebellar granular neurons, GPR3-mediated neurite outgrowth was mediated by multiple signaling pathways, including those of PKA, extracellular signal-regulated kinases (ERKs), and most strongly phosphatidylinositol 3-kinase (PI3K). In addition, the GPR3-mediated activation of neurite outgrowth was associated with G protein-coupled receptor kinase 2 (GRK2)-mediated signaling and phosphorylation of the C-terminus serine/threonine residues of GPR3, which affected downstream protein kinase B (Akt) signaling. We further demonstrated that GPR3 was transiently increased early in the development of rodent hippocampal neurons. It was subsequently concentrated at the tip of the longest neurite, and was thus associated with accelerated polarity formation in a PI3K-dependent manner in rat hippocampal neurons. In addition, GPR3 knockout in mouse hippocampal neurons led to delayed neuronal polarity formation, thereby affecting the dephosphorylation of collapsing response mediator protein 2 (CRMP2), which is downstream of the PI3K signaling pathway. Taken together, these findings suggest that the intrinsic expression of GPR3 in differentiated neurons constitutively activates PI3K-mediated signaling pathway predominantly, thus accelerating neurite outgrowth and further augmenting polarity formation in primary cultured neurons.

Association between Multimorbidity and Kidney Function among Patients with Non-Dialysis-Dependent CKD: The Fukuoka Kidney Disease Registry Study.

AIM: Individuals with chronic kidney disease (CKD) have a high prevalence of comorbidities, including cardiovascular disease (CVD) and its risk factors. However, epidemiological results to assess the association between multimorbidity and kidney function among the CKD population remains limited. METHODS: We performed a cross-sectional analysis of the association between 23 comorbid conditions and reduced kidney function in 4,476 patients with non-dialysis-dependent CKD enrolled in a multicenter cohort in Japan. Reduced kidney function was defined as an estimated glomerular filtration rate of ≤ 60 mL/min/1.73 m2. RESULTS: The mean age of patients was 67 years (male, 56.0%). The prevalence of hypertension, diabetes mellitus, dyslipidemia, prior CVD, cancer, and bone fracture, which are the major comorbidities, was 83.3%, 28.7%, 45.9%, 23.3%, 12.7%, and 6.3%, respectively. Multivariable-adjusted analyses revealed that age, male sex, hypertension, dyslipidemia, prior CVD, body mass index, urinary protein excretion, and underlying kidney disease were independent factors associated with reduced kidney function. Importantly, the odds ratios (ORs) for reduced kidney function increased linearly as the number of major comorbid conditions increased (OR for 1-2 conditions: 2.22, 95% confidence interval [CI]: 1.65-2.97; OR for 3-4 conditions: 3.04, 95% CI: 2.12-4.37; OR for ≥ 5 conditions: 4.37, 95% CI: 1.75-10.9). The upward trend in OR was more pronounced with cardiovascular comorbidities but not significant with non-cardiovascular comorbidities. CONCLUSIONS: In conclusion, we observed an independent association between cardiovascular comorbidity and its risk factors and reduced kidney function. The results of this study highlight the importance of managing multimorbidity among patients with CKD.

Inhibition of Interleukin-21 prolongs the survival through the promotion of wound healing after myocardial infarction.

Ly6Clow macrophages promote scar formation and prevent early infarct expansion after myocardial infarction (MI). Although CD4+ T cells influence the regulation of Ly6Clow macrophages after MI, the mechanism remains largely unknown. Based on the hypothesis that some molecule(s) secreted by CD4+ T cells act on Ly6Clow macrophages, we searched for candidate molecules by focusing on cytokine receptors expressed on Ly6Clow macrophages. Comparing the transcriptome between Ly6Chigh macrophages and Ly6Clow macrophages harvested from the infarcted heart, we found that Ly6Clow macrophages highly expressed the receptor for interleukin (IL)-21, a pleiotropic cytokine which is produced by several types of CD4+ T cells, compared with Ly6Chigh macrophages. Indeed, CD4+ T cells harvested from the infarcted heart produce IL-21 upon stimulation. Importantly, the survival rate and cardiac function after MI were significantly improved in IL-21-deficient (il21-/-) mice compared with those in wild-type (WT) mice. Transcriptome analysis of infarcted heart tissue from WT mice and il21-/- mice at 5 days after MI demonstrated that inflammation is persistent in WT mice compared with il21-/- mice. Consistent with the transcriptome analysis, the number of neutrophils and matrix metalloproteinase (MMP)-9 expression were significantly decreased, whereas the number of Ly6Clow macrophages and MMP-12 expression were significantly increased in il21-/- mice. In addition, collagen deposition and the number of myofibroblasts in the infarcted area were significantly increased in il21-/- mice. Consistently, IL-21 enhanced the apoptosis of Ly6Clow macrophages. Finally, administration of neutralizing IL-21 receptor Fc protein increased the number of Ly6Clow macrophages in the infarcted heart and improved the survival and cardiac function after MI. Thus, IL-21 decreases the survival after MI, possibly through the delay of wound healing by inducing the apoptosis of Ly6Clow macrophages.

Non-invasive fibrosis assessments of non-alcoholic fatty liver disease associated with low estimated glomerular filtration rate among CKD patients: the Fukuoka Kidney disease Registry Study.

BACKGROUND: A growing body of evidence has shown that non-alcoholic fatty liver disease (NAFLD) is associated with chronic kidney disease (CKD). Non-invasive fibrosis assessments of NAFLD such as Fibrosis-4 (FIB-4) index and NAFLD fibrosis score (NFS) have been developed to substitute liver biopsy. Little is known about the association between FIB-4 index or NFS and the components of CKD. METHODS: In the present cross-sectional study, we assessed of 3640 Japanese CKD patients. We examined the association between FIB-4index or NFS and the odds of having low estimated glomerular filtration rate (eGFR) defined as eGFR < 60 mL/min/1.73 m2 or albuminuria defined as urinary albumin-to-creatinine ratio (UACR) ≥ 30 mg/g. Patients were divided into quartiles according to their baseline FIB-4 index and NFS levels. Linear and logistic regression analysis were conducted, with adjustment for potential confounding factors. RESULTS: FIB-4 index and NFS were negatively associated with eGFR, but not UACR, after adjustment for potential confounding factors. Both FIB-4 index and NFS were significantly associated with low eGFR after adjustment for potential confounding factors. Meanwhile, in the multivariable-adjusted model, no associations were found between FIB-4 index or NFS and albuminuria. The addition of FIB-4 index or NFS to the established clinical CKD risk factors improved diagnostic accuracy of prevalence of low eGFR. We also found that there was a significant trend of higher FIB-4 index and NFS with more advanced renal fibrosis using the kidney biopsy data. CONCLUSIONS: Higher non-invasive fibrosis assessments of NAFLD were associated with higher odds of decreased eGFR.

Associations Between Surrogates of Skeletal Muscle Mass and History of Bone Fracture in Patients with Chronic Kidney Disease: The Fukuoka Kidney disease Registry (FKR) Study.

Patients with chronic kidney disease (CKD) are at increased risks of both sarcopenia and fragility fractures. However, information on the association between skeletal muscle mass (SMM) and the risk of bone fractures in patients with CKD is lacking. We performed a cross-sectional analysis of 4146 patients with CKD using the baseline dataset of the Fukuoka Kidney disease Registry Study, as a multicenter, prospective cohort study of pre-dialysis CKD patients. The main measure was estimated SMM (eSMM) calculated using an equation validated by bioelectrical impedance analysis with two independent datasets of 100 and 81 CKD patients. The main outcome was historical bone fractures. The associations between sex-specific quartiles (Q1-Q4) of eSMM and fracture history were assessed by logistic regression analyses. The prevalence of a history of fractures increased and eSMM decreased with progressive CKD stages. Among the 4146 patients, 249 had prior bone fractures, including 111 patients in Q1 (lowest quartile), 65 in Q2, 46 in Q3, and 27 in Q4 (highest quartile). A multivariable-adjusted model revealed that patients in Q1 had a significantly higher odds ratio (95% confidence interval) for bone fracture history than those in Q4 (reference): Q1, 2.77 (1.32-5.80); Q2, 1.95 (1.05-3.65); and Q3, 1.57 (0.90-2.75) (P-value for trend < 0.001). Similar associations were obtained when other skeletal muscle surrogates were applied: serum creatinine to serum cystatin C and daily urinary creatinine excretion. These results suggest that a lower eSMM is associated with an increased prevalence of historical bone fractures in pre-dialysis CKD patients.

Positive outcome of first-line therapy for a SMARCA4-deficient thoracic sarcomatoid tumor.

SMARCA4-deficient thoracic sarcomatoid tumor is a rare malignancy indicating some characteristics of a smoking-related disease. The purpose of this report is to describe a case of aggressive thoracic tumor with loss of immunochemical SMARCA4 expression and detail the results of our treatment regimen. The patient was a 58-year-old male and clinicopathologically diagnosed with a SMARCA4-deficient thoracic sarcomatoid tumor. Pembrolizumab plus carboplatin and pemetrexed resulted in significant response. This combination therapy showed potential for first-line systemic treatment of SMARCA4-deficient thoracic sarcomatoid tumors.

Stronger Effect of Azilsartan on Reduction of Proteinuria Compared to Candesartan in Patients with CKD: A Randomized Crossover Trial.

INTRODUCTION: Angiotensin receptor blockers (ARBs) are preferably used in hypertensive patients with CKD. Azilsartan is a strong antihypertensive ARB, but its antiproteinuric effects are not well understood. We compared the antiproteinuric effect of azilsartan and candesartan in CKD patients in an open-label, randomized, crossover trial. METHODS: A total of 111 patients were treated with 20 mg of azilsartan daily for 2 months as a run-in period. After the run-in period, patients were randomized into 2 arms and received either 20 mg of azilsartan or 8 mg of candesartan daily for 3 months in a crossover trial. The primary outcome was the percent change in urinary protein-to-Cr ratio (UPCR). RESULTS: Ninety-five patients completed the trial. The mean age was 64.3 years. The estimated glomerular filtration rate (eGFR) and UPCR were 41.5 mL/min/1.73 m2 and 1.8 g/gCr, respectively. The baseline systolic and diastolic blood pressures were 131.4 and 71.0 mm Hg, respectively. The mean percent change in the UPCR was -3.8% in the azilsartan group and 30.8% in the candesartan group at the 1st endpoint (p = 0.0004), and 6.1% in the azilsartan group and 25.8% in the candesartan group at the 2nd (final) endpoint (p = 0.029). The incidence of adverse events, including eGFR levels and serum potassium levels, was not significantly different between the groups. CONCLUSION: A 20 mg azilsartan dose had potent antiproteinuric effects compared with an 8 mg candesartan dose, without an increase in adverse events. Azilsartan may provide renal protection in addition to antihypertensive effects in CKD patients.

Risk of bladder cancer in male Japanese workers exposed to ortho-toluidine and other aromatic amines.

PURPOSE: Nine bladder cancer (BCa) cases were reported among aromatic amine-exposed male workers at a factory manufacturing organic dye/pigment intermediates in Japan. We aimed to evaluate the characteristics of aromatic amine-exposed workers by cross-sectional observation, and the risk of BCa by assessing the standardized incidence ratio (SIR). METHODS: In the cross-sectional study, our subjects were: 9 BCa patients, 36 aromatic amine-exposed non-patients, and 79 non-exposed workers from 3 factories. We evaluated the subjects' medical history, urinalysis, qualitative determination of nuclear matrix protein 22, and urinary cytology. For SIR assessment, 98 aromatic amine-exposed workers from 1 factory were included, and the Japanese general male population was used as a referent population. Since no direct aromatic amine-exposure data were available, we calculated surrogate exposure levels using information on job sites, exposure potency, and duration. RESULTS: Coexistent aromatic amines were ortho-toluidine (OT), aniline, para-toluidine, ortho-anisidine, 2,4-xylidine, and ortho-chloroaniline. The prevalence rates of cystitis and bladder lesion-related symptoms in both BCa patients and aromatic amine-exposed non-patient workers were significantly higher than those of non-exposed workers. Overall, the SIR for BCa in OT-exposed workers was 56.8 (95% CI 27.7-104.3) and apparent dose-response relationships were revealed between the SIR and the surrogate exposure level in the 0-10-year lagged analyses. Overall, SIRs in other aromatic amine-exposed workers were also significantly high but no or unclear dose-response relationships were observed. CONCLUSIONS: We conclude that OT may be responsible for the increased risk of BCa. Regular monitoring of bladder lesion-related symptoms is essential for the early identification of BCa.

Component of nicotine-induced intracellular calcium elevation mediated through α3- and α5-containing nicotinic acetylcholine receptors are regulated by cyclic AMP in SH-SY 5Y cells.

The pathway from the medial habenular nucleus to the interpeduncular nucleus, in which nicotinic acetylcholine receptor (nAChR) including the α3 and α5 subunits (α3 * and α5 * nAChRs) are expressed, is implicated in nicotine dependence. We investigated whether α3 * and α5 * nAChRs are regulated by cAMP using SH-SY5Y cells to clarify the significance of these receptors in nicotine dependence. We analyzed the nicotine-induced elevation of intracellular Ca2+ ([Ca2+]i). Nicotine induces a concentration-dependent increase in [Ca2+]i. The elimination of Ca2+ from extracellular fluid or intracellular stores demonstrated that the nicotine-induced [Ca2+]i elevation was due to extracellular influx and intracellular mobilization. The effects of tubocurarine on nicotine-induced [Ca2+]i elevation and current suggest that intracellular mobilization is caused by plasma membrane-permeating nicotine. The inhibition of α3 *, α5 *, α7 nAChR and voltage-gated Ca2+ channels by using siRNAs and selective antagonists revealed the involvement of these nAChR subunits and channels in nicotine-induced [Ca2+]i elevation. To distinguish and characterize the α3 * and α5 * nAChR-mediated Ca2+ influx, we measured the [Ca2+]i elevation induced by nonmembrane-permeating acetylcholine when muscarinic receptors, α7nAChR and Ca2+ channels were blocked. Under this condition, the [Ca2+]i elevation was significantly inhibited with a 48-h treatment of dibutyryl cAMP, which was accompanied by the downregulation of α3 and ß4 mRNA. These findings suggest that α3 * and α5 * nAChR-mediated Ca2+ influx is possibly regulated by cAMP at the transcriptional level.

RNA-Binding Protein ZFP36L2 Downregulates Helios Expression and Suppresses the Function of Regulatory T Cells.

The zinc finger protein 36-like 2, ZFP36L2, is a member of a small family of RNA-binding proteins composed by ZFP36 (also known as tristetraprolin, TTP), ZFP36L1 and ZFP36L2 in humans, with corresponding murine orthologs. These proteins bind to adenine uridine-rich element (ARE) in the 3'untranslated region of target messenger RNA and stimulate target degradation. ZFP36 functions as an anti-inflammatory modulator in murine models of inflammatory diseases by down-regulating the production of inflammatory cytokines such as tumor necrosis factor-α. However, how ZFP36L1 and ZFP36L2 alter the function of CD4+ T cells is not completely understood. We addressed this issue by searching for the target genes of ZFP36L2 by comprehensive transcriptome analysis. We observed that ZFP36L2 is highly expressed in naïve CD4+ T cells; however, when CD4+ T cells are stimulated through their T cell receptors, ZFP36L2 expression is rapidly reduced in both humans and mice. Among CD4+ T cell populations, the expression levels of ZFP36L2 in regulatory T cells (Tregs) were significantly lower than those in naïve or effector CD4+ T cells. RNA-sequence analysis revealed that the forced expression of ZFP36L2 decreased Ikzf2 (encoding Helios) expression in Foxp3+ Tregs and inhibited the ability of induced Tregs (iTregs). ZFP36L2 directly bound to and destabilized the 3'untranslated region of Ikzf2 mRNA, which contains AU-rich elements. These results indicate that ZFP36L2 reduces the expression of Ikzf2 and suppresses iTreg function, raising the interesting possibility that the inhibition of ZFP36L2 in iTregs could be a therapeutic strategy for autoimmune diseases.