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Acute lung injury (ALI), which occurs in association with sepsis, trauma, and coronavirus disease 2019 (COVID‐19), is a serious clinical condition with high mortality. Excessive platelet‐leukocyte aggregate (PLA) formation promotes neutrophil extracellular trap (NET) release and thrombosis, which are involved in various diseases, including ALI. Macrophage‐1 antigen (Mac‐1, CD11b/CD18), which is expressed on the surface of leukocytes, is known to promote NET formation. This study aimed to elucidate the role of Mac‐1 in extracellular histone‐induced ALI. Exogenous histones were administered to Mac‐1‐deficient mice and wild‐type (WT) mice with or without neutrophil or platelet depletion, and several parameters were investigated 1 h after histone injection. Depletion of neutrophils or platelets improved survival time and macroscopic and microscopic properties of lung tissues, and decreased platelet‐leukocyte formation and plasma myeloperoxidase levels. These improvements were also observed in Mac‐1−/− mice. NET formation in Mac‐1−/− bone marrow neutrophils (BMNs) was significantly lower than that in WT BMNs. In conclusion, our findings suggest that Mac‐1 is associated with exacerbation of histone‐induced ALI and the promotion of NET formation in the presence of activated platelets.

Objectives: Cardiovascular and renal diseases are closely related. Brain natriuretic peptide (BNP) and urinary albumin are established predictors for cardiac and renal morbidities, respectively. To date, no reports have investigated the combined predictive value of BNP and urinary albumin for long-term cardiovascular–renal events in patients with chronic kidney disease (CKD). The aim of this study was to investigate this theme. Methods: Four hundred eighty-three patients with CKD were enrolled into this study and followed-up for 10 years. The endpoint was cardiovascular–renal events. Results: During the median follow-up period of 109 months, 221 patients developed cardiovascular–renal events. Log-transformed BNP and urinary albumin were identified as independent predictors for cardiovascular–renal events, with a hazard ratio of 2.59 (95% confidence interval [CI], 1.81–3.72) and 2.27 (95% CI, 1.82–2.84) for BNP and urinary albumin, respectively. For the combined variables, the group with high BNP and urinary albumin had a markedly higher risk (12.41-times; 95% CI 5.23–29.42) of cardiovascular–renal events compared with that of the group with low BNP and urinary albumin. Adding both variables to a predictive model with basic risk factors improved the C-index (0.767, 0.728 to 0.814, p=0.009), net reclassification improvement (0.497, p

Introduction: Immunoglobulin A1 (IgA1) with galactose-deficient O-glycans (Gd-IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). Mucosal-tissue infections increase IL-6 production and, in patients with IgAN, are often associated with macroscopic hematuria. IgA1-secreting cell lines derived from the circulation of patients with IgAN, compared to those of healthy controls (HCs), produce more IgA1 that has O-glycans with terminal or sialylated N-acetylgalactosamine (GalNAc). GalNAc residues are added to IgA1 hinge region by some of the 20 GalNAc transferases, the O-glycosylation-initiating enzymes. Expression of GALNT2, encoding GalNAc-T2, the main enzyme initiating IgA1 O-glycosylation, is similar in cells derived from patients with IgAN and HCs. In this report, we extend our observations of GALNT14 overexpression in IgA1-producing cell lines from patients with IgAN. Methods: GALNT14 expression was analyzed in peripheral blood mononuclear cells (PBMCs) from patients with IgAN and from HCs. Moreover, the effect of GALNT14 overexpression or knock-down on Gd-IgA1 production in Dakiki cells was assessed. Results: GALNT14 was overexpressed in PBMCs from patients with IgAN. IL-6 increased GALNT14 expression in PBMCs from patients with IgAN and HCs. We used IgA1-producing cell line Dakiki, a previously reported model of Gd-IgA1-producing cells, and showed that overexpression of GalNAc-T14 enhanced galactose deficiency of IgA1, whereas siRNA-mediated GalNAc-T14 knock-down reduced it. GalNAc-T14 was localized in trans-Golgi network, as expected. Conclusions: Overexpression of GALNT14 due to inflammatory signals during mucosal infections may contribute to overproduction of Gd-IgA1 in patients with IgAN.

A long-term high-fat diet (HFD) causes obesity and changes in renal lipid metabolism and lysosomal dysfunction in mice, causing renal damage. Sodium-glucose co-transporter inhibitors, including phlorizin, exert nephroprotective effects in patients with chronic kidney disease, but the underlying mechanism remains unclear. A HFD or standard diet was fed to adult C57BL/6J male mice, and phlorizin was administered. Lamellar body components of the proximal tubular epithelial cells (PTECs) were investigated. After phlorizin administration in HFD-fed mice, sphingomyelin and ceramide in urine and tissues were assessed and label-free quantitative proteomics was performed using kidney tissue samples. Mitochondrial elongation by fusion was effective in the PTECs of HFD-fed obese mice under phlorizin administration, and many lamellar bodies were found in the apical portion of the S2 segment of the proximal tubule. Phlorizin functioned as a diuretic, releasing lamellar bodies from the apical membrane of PTECs and clearing the obstruction in nephrons. The main component of the lamellar bodies was sphingomyelin. On the first day of phlorizin administration in HFD-fed obese mice, the diuretic effect was increased, and more sphingomyelin was excreted through urine than in vehicle-treated mice. The expressions of three peroxisomal β-oxidation proteins involved in fatty acid metabolism were downregulated after phlorizin administration in the kidneys of HFD-fed mice. Fatty acid elongation protein levels increased with phlorizin administration, indicating an increase in long-chain fatty acids. Lamellar bodies accumulated in the proximal renal tubule of the S2 segment of the HFD-fed mice, indicating that the urinary excretion of lamellar bodies has nephroprotective effects.

Objectives: Favipiravir is an antiviral that is being evaluated for the treatment of COVID-19. Use of favipiravir is associated with elevation of serum uric acid levels. Risk factors for the occurrence of hyperuricemia are unclear. Methods: Specimens from COVID-19 patients who received 10 days of favipiravir in a previous clinical trial (jRCTs041190120) were used. Serum favipiravir concentrations were measured by LC-MS. Factors associated with the development of hyperuricemia were investigated using logistic regression analysis. Optimal cut-off values for the baseline serum uric acid levels and steady-state serum favipiravir concentrations in predicting the occurrence of hyperuricemia were determined by ROC curve analysis. Results: Among the 66 COVID-19 patients who were treated with favipiravir for 10 days, the steady-state serum favipiravir concentrations were significantly correlated with serum uric acid levels. High baseline serum uric acid levels and steady-state serum favipiravir concentrations during therapy were factors associated with the development of hyperuricemia. The cut‑off baseline serum uric acid level and steady-state serum favipiravir concentration during favipiravir administration determined to predict hyperuricemia were 3.7 mg/dL and 46.14 μg/mL, respectively. Conclusions: Patients with high baseline serum uric acid levels or who achieved high steady-state serum favipiravir concentrations during therapy were susceptible to hyperuricemia.

Acute lung injury (ALI) occurs in patients with severe sepsis and has a mortality rate of 40%–60%. Severe sepsis promotes the release of histones from dying cells, which can induce platelet aggregation, activate coagulation and cause endothelial cell (EC) death. We previously reported that the expression of membrane complement receptor type 1‐related gene Y (Crry)/p65, which plays a principal role in defence against abnormal activation of complement in the blood, is reduced in response to peritoneal mesothelial cell injury, and we hence hypothesized that a similar mechanism occurs in pulmonary ECs. In this study, we examined the role of Crry/p65 in histone‐mediated ALI using an experimental animal model. In ALI model mice, exposure to extracellular histones induces lung injury and results in a decrease in Crry/p65 expression. The levels of lactic acid dehydrogenase (LDH), a marker of cell damage, were significantly increased in the serum of ALI model compared with vehicle mice. The significant inverse correlation between the expression of Crry/p65 and LDH levels in plasma revealed an association between Crry/p65 expression and cell damage. The levels of complement component 3a (C3a) were also significantly increased in the serum of the ALI model compared with vehicle mice. Notably, a C3a receptor antagonist ameliorated lung injury induced by histones. We hypothesize that extracellular histones induce complement activation via down‐regulation of Crry/p65 and that C3a might serve as a therapeutic target for the treatment of ALI.

Abstract The first line of treatment for pemphigus is systemic corticosteroids. When steroids alone do not result in remission, additional immunosuppressants are recommended. Twenty‐two patients with pemphigus vulugris were treated with steroids and other immunomodulators. However, they were refractory, and hence, methotrexate (MTX) was administered. The efficacy of MTX was assessed for 16 patients who were able to continue MTX therapy for 1 year. Steroid dose reduction was possible in 11 patients. One patient with severe infections had diabetes and was elderly. Our results suggested that MTX was useful as a steroid‐sparing agent in treating recalcitrant pemphigus, when an initial immunosuppressant treatment had failed. However, adverse effects should be closely monitored.

Abstract Dysfunction of immunoinhibitory signals and persistent T cell activation reportedly play important roles in the development of vasculitis. The skin is one of the most accessible organs, and it is suitable for the characterization of immune cell signatures. However, the inhibitory checkpoint molecules in the skin and their relevance to vasculitis have not been studied. Here, we investigated the profile of immune checkpoint molecules in the skin and peripheral blood of patients with vasculitis and healthy donors. We found that some of the inhibitory checkpoint molecules, including programmed cell death 1 receptor (PD-1), were elevated in T-cells in the blood of patients with systemic and cutaneous vasculitis. In addition, programmed death-ligand 1 (PD-L1) expression was elevated in the skin of patients with cutaneous vasculitis. Histologically, PD-L1 was highly expressed in the vessels in the skin along with CD4+ and CD8+ T-cell infiltration in patients with cutaneous vasculitis. Notably, plasma soluble PD-L1 levels were increased, and these correlated with C-reactive protein in patients with systemic vasculitis. Our findings suggest that inhibitory checkpoint molecules might be differentially modulated in the skin and peripheral blood of patients with vasculitis, and that the alteration of the PD-L1/PD-1 axis may be associated with the regulation of T-cell activation in vasculitis.

Summary: Galactose (Gal)-deficient IgA1 (Gd-IgA1) is involved in IgA nephropathy (IgAN) pathogenesis. To reflect racial differences in clinical characteristics, we assessed disease- and race-specific heterogeneity in the O-glycosylation of the IgA1 hinge region (HR). We determined serum Gd-IgA1 levels in Caucasians (healthy controls [HCs], n = 31; IgAN patients, n = 63) and Asians (HCs, n = 20; IgAN patients, n = 60) and analyzed profiles of serum IgA1 HR O-glycoforms. Elevated serum Gd-IgA1 levels and reduced number of Gal residues per HR were observed in Caucasians. Reduced number of N-acetylgalactosamine (GalNAc) residues per HR and elevated relative abundance of IgA1 with three HR O-glycans were common features in IgAN patients; these features were associated with elevated blood pressure and reduced renal function. We speculate that the mechanisms underlying the reduced GalNAc content in IgA1 HR may be relevant to IgAN pathogenesis.

Objectives: Vascular calcification is common in patients with advanced chronic kidney disease (CKD) and contributes to cardiovascular disease. Accumulating evidence indicates that CKD patients often acquire subclinical vitamin K deficiency, which is associated with vascular calcification. Methods: This prospective, randomized, parallel group, multicenter trial (UMINID000011490) will include 200 dialysis patients in an open-label, two-arm design. After baseline computed tomography of the abdominal aorta, patients will be randomized to two groups that will either (1) continue receiving standard care or (2) receive additional oral supplementation with menatetrenone (45 mg/day). The treatment duration will be 24 months, and the computed tomography scan will be repeated after 12 and 24 months. The primary endpoint is the progression of abdominal aortic calcification, which is calculated as absolute changes based on the Agatston score. The secondary endpoints are the decrease in bone mineral density (measured by dual-energy X-ray absorptiometry), the biomarkers associated with vitamin K, vitamin K intake (evaluated by the food frequency questionnaire), and the biomarkers associated with vascular calcification. Conclusions: This study aims to confirm whether vitamin K has inhibitory effects on calcification that can be clinically determined. Trial registration: UMINID000011490.

Background The benefits of vitamin D receptor activators (VDRAs) for patients with chronic kidney disease are well recognized. However, the optimal criteria for patient selection, dosage forms, and duration providing the highest benefit and the least potential risk remain to be confirmed. Materials and methods The study population was derived from the Aichi Cohort Study of Prognosis in Patients Newly Initiated into Dialysis, a multicenter prospective cohort study of 1520 incident dialysis patients. According to the VDRA usage status in March 2015 (interim report), the 967 patients surviving after March 2015 were classified into three groups: without VDRA (NV, n = 177), oral VDRA (OV, n = 447), and intravenous VDRA (IV, n = 343). Mortality rates were compared using the log-rank test, and factors contributing to all-cause mortality were examined using both univariate and multivariate Cox proportional hazard regression analyses. Results There were 104 deaths (NV, n = 27; OV, n = 53; IV, n = 24) during the follow-up period (1360 days, median), and significant differences in cumulative survival rates were observed between the three groups (p = 0.010). Moreover, lower all-cause mortality was associated with IV versus NV (hazard ratio, 0.46 [95% confidence interval 0.24–0.89]; p = 0.020). Conclusion This study demonstrated the impact of the VDRA dosage form on the short-term survival of incident hemodialysis patients during the introduction period. Our results suggest that relatively early initiation of intravenous VDRA in patients beginning hemodialysis may have some clinical potential.

Abstract Background The present study aimed to investigate associations between long-term renal function, whether IgG4-related tubulointerstitial nephritis (TIN) was diagnosed by renal biopsy at initial examination, chronic kidney disease (CKD) stage, and histological stage in patients with IgG4-related TIN. Methods This study used a retrospective cohort design including almost all patients who underwent renal biopsy at Fujita Health University Hospital and Nagoya University or its affiliated hospitals in Aichi between April 2003 and March 2015 (n = 6977 renal biopsies). The primary outcome was longitudinal changes in eGFR. Main exposures were whether IgG4-related TIN was diagnosed by renal biopsy at the initial examination, CKD stage, and its histological stage. Linear mixed models were performed to examine associations. Results Of the 6977 samples, there were 24 patients (with 201 records due to repeated measures) with IgG4-related TIN (20 men, mean age, 68.7 ± 9.7 years). They were followed up 6.6 ± 2.8 years after the renal biopsy and underwent glucocorticoid treatment. We found significant increase in eGFR from the baseline to 2 and 6 months after treatment initiation, which was maintained until 60 months. Patients initially diagnosed with IgG4-related TIN had higher eGFR from the baseline (at the start of treatment) to 60 months than those who were not. Compared with patients with CKD stage 3, patients with CKD stages 4 and 5 had lower eGFR at the baseline and other time points. Patients with histological stage B had comparatively lower eGFR at each point than stage A patients. Those mean differences of eGFR were stable from the baseline to 60 months. Conclusions After the treatment initiation, renal function rapidly improved and maintained for a long period, even with advanced CKD stage. We showed importance of early diagnosis of IgG4-related TIN in maintaining eGFR.

Abstract Background Although the 2018 revised International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification was proposed recently, until now, no reports have been made comparing the association of renal prognosis between the 2018 revised ISN/RPS classification and the 2003 ISN/RPS classification. The present study aimed to assess the usefulness, especially of activity and chronicity assessment, of the 2018 revised ISN/RPS classification for lupus nephritis (LN) in terms of renal prognosis compared to the classification in 2003. Methods We retrospectively collected medical records of 170 LN patients from the database of renal biopsy at Fujita Health University from January 2003 to April 2019. Each renal biopsy specimen was reevaluated according to both the 2003 ISN/RPS classification and the 2018 revised ISN/RPS classification. Renal endpoint was defined as a 30% decline of estimated glomerular filtration rate (eGFR). Results A total of 129 patients were class III/IV±V (class III, 44 patients; class IV, 35 patients; class III/IV+V, 50 patients). The mean age was 42 years, 88% were female, and the median observation period was 50.5 months. Renal prognosis was significantly different among the classes and significantly poor in the patients with higher modified National Institute of Health (mNIH) chronicity index (C index, ≥ 4) by a log-rank test (p = 0.05 and p = 0.02, respectively). By Cox proportional hazard models, only the C index was significantly associated with renal outcome (hazard ratio 1.32, 95% CI 1.11–1.56, p ≤ 0.01), while the classes, the 2003 activity and chronicity subdivision, and the mNIH activity index had no significant association with renal outcome. Each component of the C index was significantly associated with renal outcome in different models. Conclusion This study demonstrates that the 2018 revised ISN/RPS classification was more useful in terms of association with renal prognosis compared to the 2003 ISN/RPS classification.

Information on immune checkpoint inhibitor-induced vasculitides is limited, and predictors for this condition have not been identified. Therefore, we have examined the frequency of immune checkpoint inhibitor-induced vasculitides by analyzing the data recorded in the Japanese Adverse Drug Event Report database. Data from April 2004 to March 2020 were extracted, and vasculitides as an immune-related adverse event was defined according to the 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Adverse event signals were recognized as significant when the reporting odds ratio estimates and lower limits of the corresponding 95% confidence intervals exceeded 1. The use of nivolumab showed a significant signal for vasculitides. Furthermore, significant signals of polymyalgia rheumatica were found when the patients were treated with nivolumab, pembrolizumab, and ipilimumab. In addition, the frequencies of nivolumab- and pembrolizumab-induced polymyalgia rheumatica were higher in patients aged ≥70 years and female patients, respectively. Polymyalgia rheumatica was reported in 38 patients treated with nivolumab; 31 (82%) of these were either in recovery or in remission. Further, polymyalgia rheumatica was reported in 17 patients treated with pembrolizumab; 13 (76%) of these were in recovery or remission, while three (18%) were not. Polymyalgia rheumatica was reported in 12 patients treated with ipilimumab; seven (58%) of these were in recovery or remission. Our study highlights that careful monitoring for the symptom of PMR (e.g., bilateral pain in shoulder and pelvic girdles) is required when the patients are aged >70 years and have been treated with nivolumab and when the patients are women and have been treated with pembrolizumab.

BackgroundEarly detection and prediction of cisplatin-induced acute kidney injury (Cis-AKI) are essential for the management of patients on chemotherapy with cisplatin. This study aimed to evaluate the performance of a prediction model for Cis-AKI.MethodsJapanese patients, who received cisplatin as the first-line chemotherapy at Fujita Health University Hospital, were enrolled in the study. The main metrics for evaluating the machine learning model were the area under the curve (AUC), accuracy, precision, recall, and F-measure. In addition, the rank of contribution as a predictive factor of Cis-AKI was determined by machine learning.ResultsA total of 1,014 and 226 patients were assigned to the development and validation data groups, respectively. The current prediction model showed the highest performance in patients 65 years old and above (AUC: 0.78, accuracy: 0.77, precision: 0.38, recall: 0.70, F-measure: 0.49). The maximum daily cisplatin dose and serum albumin levels contributed the most to the prediction of Cis-AKI.ConclusionOur prediction model for Cis-AKI performed effectively in older patients.

Renal anemia in chronic kidney disease is treated with recombinant human erythropoietin (rhEPO). However, some patients with anemia do not respond well to rhEPO, emphasizing the need for a more biocompatible EPO. Differentiation protocols for hepatic lineages have been modified to enable production from human induced pluripotent stem cell (hiPSC)‐derived EPO‐producing cells (EPO cells). However, markers for hiPSC‐EPO cells are lacking, making it difficult to purify hiPSC‐EPO cells and therefore to optimize EPO production and cell counts for transplantation. To address these issues, we investigated whether CD140b and CD73 could be used as markers for hiPSC‐EPO cells. We measured the expression of EPO, CD140b, and CD73 in hiPSC‐EPO cells and the EPO concentration in the cell supernatant by immunohistochemistry and enzyme‐linked immunosorbent assays on culture day 13, revealing that expression levels of CD140b and CD73 are correlated with the level of EPO. In addition, rates of CD140b+ CD73+ cells were observed to be correlated with the concentration of EPO. Thus, our results suggest that CD140b and CD73 may be markers for hiPSC‐EPO cells.

Introduction There are few studies on the association between serum uric acid (UA) level and mortality in incident dialysis patients. We aimed to clarify whether the serum UA level at dialysis initiation is associated with mortality during maintenance dialysis. Methods We enrolled 1486 incident dialysis patients who participated in a previous multicenter prospective cohort study in Japan. We classified the patients into the following five groups according to their serum UA levels at dialysis initiation: G1 with a serum UA level

Monocarboxylates, such as lactate and pyruvate, are precursors for biosynthetic pathways, including those for glucose, lipids, and amino acids via the tricarboxylic acid (TCA) cycle and adjacent metabolic networks. The transportation of monocarboxylates across the cellular membrane is performed primarily by monocarboxylate transporters (MCTs), the membrane localization and stabilization of which are facilitated by the transmembrane protein basigin (BSG). Here, we demonstrate that the MCT/BSG axis sits at a crucial intersection of cellular metabolism. Abolishment of MCT1 in the plasma membrane was achieved by Bsg depletion, which led to gluconeogenesis impairment via preventing the influx of lactate and pyruvate into the cell, consequently suppressing the TCA cycle. This net anaplerosis suppression was compensated in part by the increased utilization of glycogenic amino acids (e.g., alanine and glutamine) into the TCA cycle and by activated ketogenesis through fatty acid β-oxidation. Complementary to these observations, hyperglycemia and hepatic steatosis induced by a high-fat diet were ameliorated in Bsg-deficient mice. Furthermore, Bsg deficiency significantly improved insulin resistance induced by a high-fat diet. Taken together, the plasma membrane–selective modulation of lactate and pyruvate transport through BSG inhibition could potentiate metabolic flexibility to treat metabolic diseases.

Introduction: As glomerular filtration rate (GFR) decreases, serum phosphate level increases. Previous reports indicated that serum phosphate level was associated with mortality in patients on dialysis. However, few reports have examined the association using dialysis initiation as the baseline period. Methods: This was a multicenter prospective cohort analysis including 1492 patients. Patients were classified into four quartiles based on the serum phosphate level at dialysis initiation, with Q1 being the lowest and Q4 the highest. All-cause mortality after dialysis initiation was compared using the log-rank test. The propensity score represented the probability of being assigned to group Q1 or Q2–4. All-cause mortality was compared in propensity score-matched patients by using the log-rank test for Kaplan–Meier curves. All-cause mortality of Q1 was compared with that for Q2–4 using multivariate Cox proportional hazard regression analysis. All-cause mortality was also determined among stratified groups with or without use of phosphate binders. Results: Significant differences in cumulative survival rates were observed between the four groups (p

IntroductionDegenerative aortic valve stenosis (AS) is a chronic progressive disease that resembles atherosclerosis development. Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is reportedly associated with accelerated atherosclerosis. This study aimed to examine the development of AS in patients with myeloperoxidase-AAV (MPO-AAV) with renal involvement at more than 1 year after the onset of vasculitis.MethodsWe performed a retrospective review of clinical records of MPO-AAV patients with renal involvement without AS at the onset of vasculitis who were treated in three hospitals and three dialysis clinics.ResultsThe study included 97 MPO-AAV patients with renal involvement and 230 control patients with chronic kidney disease (CKD). Among them, 64 patients had AS. The prevalence rates of AS were 28.9% and 15.7% in MPO-AAV and control patients, respectively (p = 0.006). The multivariable logistic regression analysis showed that MPO-AAV, dialysis dependence, and hypertension were independently associated factors for AS. In MPO-AAV patients, systolic blood pressure was positively significantly associated with AS, whereas glucocorticoid dose of induction therapy was negatively significantly associated. The use of cyclophosphamide tended to be negatively associated with AS. The survival rate was significantly lower for patients with AS than for those without AS.ConclusionsThe AS prevalence rate was significantly higher in MPO-AAV patients at more than 1 year after the onset of vasculitis than in control CKD patients. Therefore, regular monitoring of echocardiography during MPO-AAV treatment is suggested.

IgA1 with galactose (Gal)-deficient hinge-region (HR) O-glycans (Gd-IgA1) plays a key role in IgA nephropathy (IgAN), and the serum level of Gd-IgA1 is elevated in the majority of IgAN patients. To characterize the involvement of IgA1 in the development and progression of IgAN, O-glycan micro-heterogeneity and attachment sites need to be analyzed, as each HR has nine potential sites for O-glycosylation. We have developed an on-line liquid chromatography (LC) hybrid quadrupole mass filter/linear ion trap/orbitrap mass spectrometry (MS) protocol, which was used to analyze IgA1 from a patient with IgAN. LC-MS profiling provided the overall O-glycan micro-heterogeneity distribution of IgA1 HR O-glycoforms. The LC-extracted ion chromatogram (XIC) of HR O-glycoforms containing Gal-deficient O-glycans indicated that the Gal-deficient O-glycans attached at specific sites. Structural isomers based on changes in the amino acid position of the attached glycans were identified in relation to the IgA1 HR O-glycoforms containing Gal-deficient O-glycans. To identify the predominant O-glycoforms in the serum IgA1 from IgAN patients as candidate biomarkers, O-glycan micro-heterogeneity and attachment sites, including isomeric structures, need to be analyzed. Our MS-based approach is useful in this respect and should prove a valuable tool for the development of biomarkers for IgA1 HR O-glycosylation in IgAN.

Abstract Background Aortic stenosis (AS) is common in patients on dialysis as well as in the general population. AS leads to difficulty with dialysis therapy because of unstable conditions such as intradialytic hypotension due to low cardiac output. However, the precise morbidity rates and risk factors of AS in patients on dialysis are unknown. Moreover, there are no large-scale observational studies regarding the association between AS in patients on dialysis and mortality. Therefore, we will investigate whether morbidity of AS in patients on dialysis is associated with mortality. Methods This is a multicenter prospective cohort analysis in the Tokai region of Japan. The 75 participating centers in this study will enroll approximately 2400 patients during 12 months, with or without AS. We started enrollment in July 2017 and will follow patents until June 2023. Transthoracic echocardiography will be performed to evaluate aortic valve. Parameters used for evaluation of aortic valve are mean pressure gradient between left ventricle and ascending aorta, aortic valve area, and maximum aortic jet velocity. We will diagnose AS using the criteria based on the 2014 American Heart Association/ American College of Cardiology Guideline. We will also perform transthoracic echocardiography at 12, 24, 36, 48, and 60 months. Survival prognosis and CV events will be determined at the end of June 2019, 2020, 2021, 2022, and 2023. Development of AS will be also evaluated as new onset or annual change in AS parameters. We will classify patients based on the presence or absence of AS and the stages of AS and will compare outcomes. Study outcomes will include the following: 1) all-cause mortality rates; 2) incidence of cardiovascular (CV) events; 3) CV-related mortality rates; 4) infection-related mortality rates; 5) new onset or development of AS. Discussion We will consider the following hypotheses in this study, among others: The prevalence of AS is higher in dialysis patients; new onset and development of AS are associated with factors that are specific for dialysis, such as hyperphosphatemia, hyperparathyroidism, and medication; and outcomes in AS patients are poorer than in patients without AS at baseline. Trial registration UMIN000026756, Registered March 29 2017.

Background: Cell-free and concentrated ascites reinfusion therapy (CART) was approved by the National Insurance Scheme in 1981 in Japan and has since been used as a treatment modality for refractory ascites. Two filtration methods may be used for CART: the internal and external pressure filtration methods. However, the precise characteristics of each method are unknown. Methods: Ascitic fluid will be obtained by puncture from patients with refractory cancerous ascites. The quantity of fluid obtained from each patient will be divided in half, and each half will be processed using either the internal or external pressure filtration method. The primary endpoint will be the time required for the transmembrane pressure to reach 500 mmHg. The secondary endpoints will be serial changes in the weight of the ascitic and filtered fluid, serial changes in the pressure at the inlet and outlet of the filter, measurement of the components of the ascitic and filtered fluid, and observation of the filter by visual inspection and light and electron microscopy. Conclusion: This trial may clarify the characteristics of the two filtration methods. Trial registration: UMIN000025382.

IgA nephropathy (IgAN), the most common primary glomerular disease worldwide, is characterized by glomerular deposition of IgA1-containing immune complexes. The IgA1 hinge region (HR) has up to six clustered O-glycans consisting of Ser/Thr-linked N-acetylgalactosamine usually with β1,3-linked galactose and variable sialylation. Circulating levels of IgA1 with abnormally O-glycosylated HR, termed galactose-deficient IgA1 (Gd-IgA1), are increased in patients with IgAN. Current evidence suggests that IgAN is induced by multiple sequential pathogenic steps, and production of aberrantly glycosylated IgA1 is considered the initial step. Thus, the mechanisms of biosynthesis of aberrantly glycosylated IgA1 and the involvement of aberrant glycoforms of IgA1 in disease development have been studied. Furthermore, Gd-IgA1 represents an attractive biomarker for IgAN, and its clinical significance is still being evaluated. To elucidate the pathogenesis of IgAN, it is important to deconvolute the biosynthetic origins of Gd-IgA1 and characterize the pathogenic IgA1 HR O-glycoform(s), including the glycan structures and their sites of attachment. These efforts will likely lead to development of new biomarkers. Here, we review the IgA1 HR O-glycosylation in general and the role of aberrantly glycosylated IgA1 in the pathogenesis of IgAN in particular.

Some variables including age, comorbidity of diabetes, and so on at dialysis initiation are associated with patient prognosis. Cardiovascular (CV) events are a major cause of death, and adequate models that predict prognosis in dialysis patients are warranted. Therefore, we created models using some variables at dialysis initiation. We used a database of 1,520 consecutive dialysis patients (median age, 70 years; 492 women [32.4%]) from a multicenter prospective cohort study. We established the primary endpoint as a composite of the incidence of first CV events or all-cause death. A multivariable Cox proportional hazard regression model was used to construct a model. We considered a complex and a simple model. We used area under the receiver operating characteristic curve (AUROC) to assess and compare the predictive performances of the prediction models and evaluated the improvement in discrimination using the complex model versus the simple model using net reclassification improvement (NRI). We then assessed integrated discrimination improvement (IDI) to evaluate improvements in average sensitivity and specificity. Of 392 deaths, 152 were CV-related. Totally, 506 CV events occurred during the follow-up period (median 1,285 days). Finally, 692 patients reached the primary endpoint. Baseline data were set at dialysis initiation. AUROC for the primary endpoint was 0.737 (95% confidence interval [CI], 0.712-0.761) in the simple model and 0.765 (95% CI, 0.741-0.788) in the complex model. There were significant intergroup differences in NRI (0.44; 95% CI, 0.34-0.53; p < 0.001) and IDI (0.02; 95% CI, 0.02-0.03; p < 0.001). We prepared a Shiny R application for each model to automatically calculate the predicted occurrence probability (https://statacademy.shinyapps.io/App_inaguma_20190717/). The complex model made more accurate predictions than the simple model. However, the intergroup difference was not significant. Hence, the simple model was more useful than the complex model. The tool was useful in a real-world clinical setting because it required only routinely available variables. Moreover, we emphasized that the tool could predict the incidence of CV events or all-cause mortality for individual patients. In the future, we must confirm its external validity in other prospective cohorts.

We characterized 419 Mycoplasma pneumoniae isolates collected between 2011 and 2017 in Osaka prefecture of Japan. This analysis revealed high prevalence of macrolide-resistant M. pneumoniae (MRMP) in Osaka during 2011 and 2014 with annual detection rates of MRMP strains between 71.4% and 81.8%. However, in 2015 and after, the detection rate of MRMP decreased significantly and did not exceed 50%. Genotyping of the p1 gene of these isolates showed that most of MRMP strains harbored type 1 p1 gene. In contrast, strains expressing p1 gene type 2 or its variant were largely macrolide-susceptible M. pneumoniae (MSMP) strains. There was a strong correlation between p1 gene genotype and the presence of mutations conferring macrolide resistance in M. pneumoniae isolated in Osaka. These results indicate that lower incidence of MRMP strains in Osaka from 2015 was associated with the relative increase of p1 gene type 2 lineage strains. During these experiments, we also isolated three M. pneumoniae strains that showed irregular typing pattern in the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the p1 gene. Two of these strains harbored new variants of type 2 p1 gene and were designated as type 2f and 2g. The remaining strain with an irregular typing pattern had a large deletion in the p1 operon.

A major determinant in the change of the avian influenza virus host range to humans is the E627K substitution in the PB2 polymerase protein. However, the polymerase activity of avian influenza viruses with a single PB2-E627K mutation is still lower than that of seasonal human influenza viruses, implying that avian viruses require polymerase mutations in addition to PB2-627K for human adaptation. Here, we used a database search of H5N1 clade 2.2.1 virus sequences with the PB2-627K mutation to identify other polymerase adaptation mutations that have been selected in infected patients. Several of the mutations identified acted cooperatively with PB2-627K to increase viral growth in human airway epithelial cells and mouse lungs. These mutations were in multiple domains of the polymerase complex other than the PB2-627 domain, highlighting a complicated avian-to-human adaptation pathway of avian influenza viruses. Thus, H5N1 viruses could rapidly acquire multiple polymerase mutations that function cooperatively with PB2-627K in infected patients for optimal human adaptation.

Anti-endothelial cell antibodies (AECA) are frequently detected in patients with systemic lupus erythematosus (SLE), but their pathological role remains unclear. We recently developed a solubilized cell surface protein capture enzyme-linked immunosorbent assay (CSP-ELISA) to detect antibodies against membrane proteins involved in autoimmune reactions. In this study, sera from 51 patients with biopsy-proven lupus nephritis (LN), 25 with SLE without renal involvement (non-LN SLE), 42 disease control (DC) subjects, and 80 healthy control (HC) subjects were tested for IgG- and IgA-AECA for human umbilical vein endothelial cells (HUVEC) and human glomerular EC (HGEC) by using CSP-ELISA. IgG- and IgA-AECA titers were significantly higher in LN and non-LN SLE patients than in the DC or HC (P < 0.001) groups. IgG- and IgA-AECA titers for HUVEC corresponded well with those for HGEC. The IgA-AECA level correlated with the SLE disease activity index and with histological evidence of active lesions (cellular proliferations, hyaline thrombi and wire loops, leukocytic infiltration, and fibrinoid necrosis) in LN patients (P < 0.001). The sensitivity of IgA-AECA as a diagnostic test for histological evidence of active lesions in LN patients was 0.92, with a specificity of 0.70. The significant correlation of IgA-AECA with glomerular hypercellularity indicates that IgA-AECA are associated with endothelial damage in LN.

We investigated the associations between the self-evaluated pain status and two pain biomarker candidates, nerve growth factor and S100A8/A9, in exudate from venous leg ulcer to finally develop an objective pain evaluation method. Patients with venous leg ulcer participated in this cross-sectional observational study conducted between April and October 2014 at two medical facilities. During routine wound care, each participant self-evaluated their pain status at each examination using the 10-point numerical rating scale (present pain intensity) and the short-form McGill Pain Questionnaire 2 (continuous pain, intermittent pain, neuropathic pain, affective descriptors, and total score). Venous leg ulcer exudate sample was collected after wound cleansing. The nerve growth factor and S100A8/A9 concentrations in the venous leg ulcer exudate were measured by enzyme-linked immunosorbent assay and standardized according to the wound area. The association between each pain status and the two standardized protein concentrations was evaluated using Spearman's correlation coefficient. In 30 sample collected from 13 participants, the standardized nerve growth factor concentration was negatively correlated with continuous pain (ρ = -0.47, P = 0.01), intermittent pain (ρ = -0.48, P = 0.01), neuropathic pain (ρ = -0.51, P = 0.01), and total score (ρ = -0.46, P = 0.01). The standardized S100A8/A9 concentration was positively correlated with present pain intensity (ρ = 0.46, P = 0.03) and continuous pain (ρ = 0.48, P = 0.03). Thus, these two proteins may be useful for objective evaluation of wound pain in venous leg ulcer patients.

ABSTRACT A change in viral hemagglutinin (HA) receptor binding specificity from α2,3- to α2,6-linked sialic acid is necessary for highly pathogenic avian influenza (AI) virus subtype H5N1 to become pandemic. However, details of the human-adaptive change in the H5N1 virus remain unknown. Our database search of H5N1 clade 2.2.1 viruses circulating in Egypt identified multiple HA mutations that had been selected in infected patients. Using reverse genetics, we found that increases in both human receptor specificity and the HA pH threshold for membrane fusion were necessary to facilitate replication of the virus variants in human airway epithelia. Furthermore, variants with enhanced replication in human cells had decreased HA stability, apparently to compensate for the changes in viral receptor specificity and membrane fusion activity. Our findings showed that H5N1 viruses could rapidly adapt to growth in the human airway microenvironment by altering their HA properties in infected patients and provided new insights into the human-adaptive mechanisms of AI viruses. IMPORTANCE Circulation between bird and human hosts may allow H5N1 viruses to acquire amino acid changes that increase fitness for human infections. However, human-adaptive changes in H5N1 viruses have not been adequately investigated. In this study, we found that multiple HA mutations were actually selected in H5N1-infected patients and that H5N1 variants with some of these HA mutations had increased human-type receptor specificity and increased HA membrane fusion activity, both of which are advantageous for viral replication in human airway epithelia. Furthermore, HA mutants selected during viral replication in patients were likely to have less HA stability, apparently as a compensatory mechanism. These results begin to clarify the picture of the H5N1 human-adaptive mechanism.

Patients with IgA nephropathy (IgAN) have elevated circulating levels of IgA1 with some O-glycans consisting of galactose (Gal)-deficient N-acetylgalactosamine (GalNAc) with or without N-acetylneuraminic acid (NeuAc). We have analyzed O-glycosylation heterogeneity of naturally asialo-IgA1 (Ale) myeloma protein that mimics Gal-deficient IgA1 (Gd-IgA1) of patients with IgAN, except that IgA1 O-glycans of IgAN patients are frequently sialylated. Specifically, serum IgA1 of healthy controls has more α2,3-sialylated O-glycans (NeuAc attached to Gal) than α2,6-sialylated O-glycans (NeuAc attached to GalNAc). As IgA1-producing cells from IgAN patients have an increased activity of α2,6-sialyltransferase (ST6GalNAc), we hypothesize that such activity may promote premature sialylation of GalNAc and, thus, production of Gd-IgA1, as sialylation of GalNAc prevents subsequent Gal attachment. Distribution of NeuAc in IgA1 O-glycans may play an important role in the pathogenesis of IgAN. To better understand biological functions of NeuAc in IgA1, we established protocols for enzymatic sialylation leading to α2,3- or α2,6-sialylation of IgA1 O-glycans. Sialylation of Gal-deficient asialo-IgA1 (Ale) myeloma protein by an ST6GalNAc enzyme generated sialylated IgA1 that mimics the Gal-deficient IgA1 glycoforms in patients with IgAN, characterized by α2,6-sialylated Gal-deficient GalNAc. In contrast, sialylation of the same myeloma protein by an α2,3-sialyltransferase yielded IgA1 typical for healthy controls, characterized by α2,3-sialylated Gal. The GalNAc-specific lectin from Helix aspersa (HAA) is used to measure levels of Gd-IgA1. We assessed HAA binding to IgA1 sialylated at Gal or GalNAc. As expected, α2,6-sialylation of IgA1 markedly decreased reactivity with HAA. Notably, α2,3-sialylation also decreased reactivity with HAA. Neuraminidase treatment recovered the original HAA reactivity in both instances. These results suggest that binding of a GalNAc-specific lectin is modulated by sialylation of GalNAc as well as Gal in the clustered IgA1 O-glycans. Thus, enzymatic sialylation offers a useful model to test the role of NeuAc in reactivities of the clustered O-glycans with lectins.

BACKGROUND: In April 2009, a novel swine-derived influenza A virus (H1N1pdm) emerged and rapidly spread around the world, including Japan. It has been suggested that the virus can bind to both 2,3- and 2,6-linked sialic acid receptors in infected mammals, in contrast to contemporary seasonal H1N1 viruses, which have a predilection for 2,6-linked sialic acid. METHODS/RESULTS: To elucidate the existence and transmissibility of α2,3 sialic acid-specific viruses in H1N1pdm, amino acid substitutions within viral hemagglutinin molecules were investigated, especially D187E, D222G, and Q223R, which are related to a shift from human to avian receptor specificity. Samples from individuals infected during the first and second waves of the outbreak in Japan were examined using a high-throughput sequencing approach. In May 2009, three specimens from mild cases showed D222G and/or Q223R substitutions in a minor subpopulation of viruses infecting these individuals. However, the substitutions almost disappeared in the samples from five mild cases in December 2010. The D187E substitution was not widespread in specimens, even in May 2009. CONCLUSIONS: These results suggest that α2,3 sialic acid-specific viruses, including G222 and R223, existed in humans as a minor population in the early phase of the pandemic, and that D222 and Q223 became more dominant through human-to-human transmission during the first and second waves of the epidemic. These results are consistent with the low substitution rates identified in seasonal H1N1 viruses in 2008.

BackgroundPandemic influenza A(H1N1) virus infection quickly circulated worldwide in 2009. In Japan, the first case was reported in May 2009, one month after its outbreak in Mexico. Thereafter, A(H1N1) infection spread widely throughout the country. It is of great importance to profile and understand the situation regarding viral mutations and their circulation in Japan to accumulate a knowledge base and to prepare clinical response platforms before a second pandemic (pdm) wave emerges.MethodologyA total of 253 swab samples were collected from patients with influenza-like illness in the Osaka, Tokyo, and Chiba areas both in May 2009 and between October 2009 and January 2010. We analyzed partial sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 pdm influenza virus in the collected clinical samples. By phylogenetic analysis, we identified major variants of the 2009 pdm influenza virus and critical mutations associated with severe cases, including drug-resistance mutations.Results and conclusionsOur sequence analysis has revealed that both HA-S220T and NA-N248D are major non-synonymous mutations that clearly discriminate the 2009 pdm influenza viruses identified in the very early phase (May 2009) from those found in the peak phase (October 2009 to January 2010) in Japan. By phylogenetic analysis, we found 14 micro-clades within the viruses collected during the peak phase. Among them, 12 were new micro-clades, while two were previously reported. Oseltamivir resistance-related mutations, i.e., NA-H275Y and NA-N295S, were also detected in sporadic cases in Osaka and Tokyo.

With the severe acute respiratory syndrome epidemic of 2003 and renewed attention on avian influenza viral pandemics, new surveillance systems are needed for the earlier detection of emerging infectious diseases. We applied a "next-generation" parallel sequencing platform for viral detection in nasopharyngeal and fecal samples collected during seasonal influenza virus (Flu) infections and norovirus outbreaks from 2005 to 2007 in Osaka, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.1-0.25 ml of nasopharyngeal aspirates (N = 3) and fecal specimens (N = 5), and more than 10 microg of cDNA was synthesized. Unbiased high-throughput sequencing of these 8 samples yielded 15,298-32,335 (average 24,738) reads in a single 7.5 h run. In nasopharyngeal samples, although whole genome analysis was not available because the majority (>90%) of reads were host genome-derived, 20-460 Flu-reads were detected, which was sufficient for subtype identification. In fecal samples, bacteria and host cells were removed by centrifugation, resulting in gain of 484-15,260 reads of norovirus sequence (78-98% of the whole genome was covered), except for one specimen that was under-detectable by RT-PCR. These results suggest that our unbiased high-throughput sequencing approach is useful for directly detecting pathogenic viruses without advance genetic information. Although its cost and technological availability make it unlikely that this system will very soon be the diagnostic standard worldwide, this system could be useful for the earlier discovery of novel emerging viruses and bioterrorism, which are difficult to detect with conventional procedures.

Background/Aim: Sepsis is a life-threatening biological condition that induces systemic tissue and organ dysfunction and confers a high mortality risk. Although the use of hydrocortisone in combination with ascorbic acid and thiamine (HAT therapy) significantly reduced mortality from sepsis or septic shock in a previous study, it did not improve mortality in subsequent randomized controlled trials (RCTs). Therefore, no definitive conclusion has been established on the benefits of HAT therapy for sepsis or septic shock. We performed a meta-analysis to assess the treatment outcomes of HAT therapy in patients with sepsis or septic shock. Patients and Methods: We searched databases (PubMed/MEDLINE, Embase, Scopus and Cochrane Library) for RCTs using the terms "ascorbic acid", "thiamine", "sepsis", "septic shock", and "RCT". The primary outcome of this meta-analysis was the mortality rate, and the secondary outcomes were the incidence of new-onset acute renal injury (AKI), intensive care unit (ICU) length of stay (ICU-LOS), change in the Sequential Organ Failure Assessment (SOFA) score within 72 hours, and duration of vasopressor use. Results: Nine RCTs were identified and included in the outcome evaluation. HAT therapy did not improve the 28-day and ICU mortality, new-onset AKI, ICULOS, or SOFA scores. However, HAT therapy significantly shortened the duration of vasopressor use. Conclusion: HAT therapy did not improve mortality, the SOFA score, renal injury, or ICU-LOS. Further studies are needed to confirm whether it shortens the duration of vasopressor use. [ABSTRACT FROM AUTHOR]