Yasuyuki Shinozaki

Fibrocytes are involved in the pathogenesis of human chronic kidney disease

The presence of chronic kidney disease in humans is associated with a risk of kidney function loss as well as the development of cardiovascular disease. Fibrocytes have been shown to contribute to organ fibrosis. In this study, the presence of fibrocytes was investigated immunohistochemically in kidney biopsy specimens from 100 patients with chronic kidney disease. In addition, 6 patients with thin basement membrane disease were studied as a disease control. In patients with chronic kidney disease, the infiltration of fibrocytes was observed mainly in the interstitium. The number of interstitial fibrocytes in patients with chronic kidney disease was higher than that in patients with thin basement membrane disease. The number of infiltrated fibrocytes in the interstitium correlated well with the severity of tubulointerstitial lesions, such as interstitial fibrosis, in patients with chronic kidney disease. In addition, there were significant correlations between the number of interstitial fibrocytes and the number of CD68-positive macrophages in the interstitium as well as urinary monocyte chemoattractant protein-1/CCL2 levels. In particular, there was an inverse correlation between the number of interstitial fibrocytes and kidney function at the time of biopsy. Finally, the numbers of interstitial fibrocytes and macrophages as well as urinary CCL2 levels were significantly decreased during convalescence induced by glucocorticoid therapy. These results suggest that fibrocytes may be involved in the pathogenesis of chronic kidney disease through the interaction with macrophages as well as CCL2.

Dendritic Cells Contribute to Autoimmune Kidney Injury in MRL-Faslpr Mice

Objective. Dendritic cells (DC) contribute to autoimmune disease progression and pathogenesis. Mature DC have been reported to secrete high mobility group box protein (HMGB-1), a novel inflammatory cytokine, via p38 mitogen-activated protein kinase (MAPK) activation. We investigated whether DC are involved in progression of autoimmune diseases followed by secretion of HMGB-1 via p38 MAPK activation in a lupus-prone mouse model. Methods. FR167653, a specific inhibitor of p38 MAPK, was given orally from 3 months of age in MRL-Faslpr mice. Cultured DC, treated with or without FR167653, were stimulated with tumor necrosis factor-α. Results. Inhibition of p38 MAPK led to a reduction in the number of CD11c-positive cells, including those with the mature phenotype, in the diseased kidney and spleen, which resulted in improvement of kidney pathology in MRL-Faslpr mice. The number of CD11c-positive cells in circulation was also reduced. HMGB-1 protein and transcripts detected in the diseased kidney, and the number of cells dual-positive for HMGB-1 and CD11c, were reduced by inhibition of p38 MAPK. Maturation of cultured DC and increased cytokines, including HMGB-1, in the supernatant were inhibited by FR167653 treatment. These results suggest that DC are involved in the progression of autoimmune kidney diseases in MRL-Faslpr mice followed by HMGB-1 secretion via p38 MAPK activation. Conclusion. Our results indicated that DC secrete HMGB-1 via p38 MAPK activation to participate in autoimmunity in MRL-Faslpr mice.

Inhibition of CTGF ameliorates peritoneal fibrosis through suppression of fibroblast and myofibroblast accumulation and angiogenesis

Peritoneal fibrosis (PF) is a serious complication in various clinical settings, but the mechanisms driving it remain to be fully determined. Connective tissue growth factor (CTGF) is known to regulate fibroblast activities. We therefore examined if CTGF inhibition has anti-fibrotic effects in PF. PF was induced by repetitive intraperitoneal injections of chlorhexidine gluconate (CG) in mice with type I pro-collagen promoter-driven green fluorescent protein (GFP) expression to identify fibroblasts. FG-3019, an anti-CTGF monoclonal antibody, was used to inhibit CTGF. CG-induced PF was significantly attenuated in FG-3019-treated mice. CG challenges induced marked accumulations of proliferating fibroblasts and of myofibroblasts, which were both reduced by FG-3019. Levels of peritoneal CTGF expression were increased by CG challenges, and suppressed in FG-3019-treated mice. FG-3019 treatment also reduced the number of CD31+ vessels and VEGF-A-positive cells in fibrotic peritoneum. In vitro studies using NIH 3T3 fibroblasts and peritoneal mesothelial cells (PMCs) showed that CTGF blockade suppressed TGF-β1-induced fibroblast proliferation and myofibroblast differentiation, PMC mesothelial-to-mesenchymal transition, and VEGF-A production. These findings suggest that the inhibition of CTGF by FG-3019 might be a novel treatment for PF through the regulation of fibroblast and myofibroblast accumulation and angiogenesis.

Association of PAX2 and Other Gene Mutations with the Clinical Manifestations of Renal Coloboma Syndrome

Background Renal coloboma syndrome (RCS) is characterized by renal anomalies and optic nerve colobomas. PAX2 mutations contribute to RCS. However, approximately half of the patients with RCS have no mutation in PAX2 gene. Methods To investigate the incidence and effects of mutations of PAX2 and 25 candidate genes, patient genes were screened using next-generation sequence analysis, and candidate mutations were confirmed using Sanger sequencing. The correlation between mutations and clinical manifestation was evaluated. Result Thirty patients, including 26 patients (two families of five and two, 19 sporadic cases) with RCS, and 4 optic nerve coloboma only control cases were evaluated in the present study. Six PAX2 mutations in 21 probands [28%; two in family cohorts (n = 5 and n = 2) and in 4 out of 19 patients with sporadic disease] including four novel mutations were confirmed using Sanger sequencing. Moreover, four other sequence variants (CHD7, SALL4, KIF26B, and SIX4) were also confirmed, including a potentially pathogenic novel KIF26B mutation. Kidney function and proteinuria were more severe in patients with PAX2 mutations than in those without the mutation. Moreover, the coloboma score was significantly higher in patients with PAX2 gene mutations. Three out of five patients with PAX2 mutations had focal segmental glomerulosclerosis (FSGS) diagnosed from kidney biopsies. Conclusion The results of this study identify several new mutations of PAX2, and sequence variants in four additional genes, including a novel potentially pathogenic mutation in KIF26B, which may play a role in the pathogenesis of RCS.

Impairment of the carnitine/organic cation transporter 1–ergothioneine axis is mediated by intestinal transporter dysfunction in chronic kidney disease

Carnitine/organic cation transporter 1 (OCTN1) is a specific transporter of the food-derived antioxidant ergothioneine. Ergothioneine is absorbed by intestinal OCTN1, distributed through the bloodstream, and incorporated into each organ by OCTN1. OCTN1 expression is upregulated in injured tissues, and promotes ergothioneine uptake to reduce further damage caused by oxidative stress. However, the role of the OCTN1-ergothioneine axis in kidney-intestine cross-talk and chronic kidney disease (CKD) progression remains unclear. Here we assessed ergothioneine uptake via intestinal OCTN1 and confirmed the expression of OCTN1. The ability of OCTN1 to absorb ergothioneine was diminished in mice with CKD. In combination with OCTN1 dysfunction, OCTN1 localization on the intestinal apical cellular membrane was disturbed in mice with CKD. Proteomic analysis, RT-PCR, Western blotting, and immunohistochemistry revealed that PDZ (PSD95, Dlg, and ZO1), a PDZK1 domain-containing protein that regulates the localization of transporters, was decreased in mice with CKD. Decreased intestinal ergothioneine uptake from food decreased ergothioneine levels in the blood of mice with CKD. Despite increased OCTN1 expression and ergothioneine uptake into the kidneys of mice with CKD, ergothioneine levels did not increase. To identify the role of the OCTN1-ergothioneine axis in CKD, we evaluated kidney damage and oxidative stress in OCTN1-knockout mice with CKD and found that kidney fibrosis worsened. Oxidative stress indicators were increased in OCTN1-knockout mice. Moreover, ergothioneine levels in the blood of patients with CKD decreased, which were restored after kidney transplantation. Thus, a novel inter-organ interaction mediated by transporters is associated with CKD progression.

Messenger RNA expression profile of sleep-related genes in peripheral blood cells in patients with chronic kidney disease

BackgroundVarious sleep abnormalities, such as delayed sleep onset, frequent awakening and daytime sleepiness, deteriorate the quality of life in patients with chronic kidney disease (CKD), including those on haemodialysis (HD). Although there are some candidate causative molecules in the central nervous system, the contribution of peripheral blood cells (PBCs) remains unclear. In this study, we performed polysomnographic analysis in CKD patients and used PBCs to examine the expression of genes related to sleep and wakefulness states.MethodsPolysomnographic analysis was performed in 9 CKD patients and 6 controls. Genes related to sleep and wakefulness were evaluated by RNA microarray in 19 subjects, including CKD patients and control subjects.ResultsPolysomnographic analysis revealed that the duration of the rapid eye movement (REM)/non-REM phases during total sleep time was different between CKD patients and healthy controls. In mRNA microarray evaluation, hierarchical clustering analysis showed different patterns of sleep-related gene expression in HD patients. mRNA expression levels of GABA receptor (GABBR2), noradrenaline receptor (ADRA1A), dopamine receptor (DRD1) and histamine receptor (HRH1) showed an inverse correlation with renal function. Moreover, the mRNA expression of orexin and its receptor (HCRTR1 and HCRTR2) was also inversely correlated with renal function.ConclusionThese data indicate that the expression of sleep-related genes in PBCs of CKD patients may be associated with sleep abnormalities.

Down-regulation of the two-component system and cell-wall biosynthesis-related genes was associated with the reversion to daptomycin susceptibility in daptomycin non-susceptible methicillin-resistant Staphylococcus aureus

Daptomycin (DAP) is widely used in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection. The emergence of DAP non-susceptible MRSA strains during therapy is a major concern in clinical settings. Recent studies revealed that MRSA spontaneously reverts to a subsequent methicillin-susceptible S. aureus (MSSA) strain. However, it is not clear whether DAP non-susceptible MRSA has the ability to revert to a susceptible strain. We obtained an MRSA strain pair, DAP non-susceptible strain and subsequent DAP susceptible strain, from a patient. To understand the underlying mechanism by which DAP non-susceptible MRSA reverts to a susceptible strain, we performed genetic and phenotypic analysis in the strain pair. Although whole-genome analysis revealed four missense mutations, including L826F in mprF, in both strains, the net cell-surface charge was similar between the DAP non-susceptible and susceptible strains. However, the thickness of the cell wall was higher in the DAP non-susceptible strain, which was decreased to the same level as the control after reversion to the DAP susceptible strain. Moreover, the non-susceptible strain showed higher mRNA expression of the two-component system (TCS), such as VraSR, yycG and GraS, with the up-regulated transcription levels of cell-wall biosynthesis-related genes. The expression levels of those genes were decreased after reversion to the susceptible strain. These results indicated that DAP non-susceptibility due to up-regulation of the TCS and cell-wall biosynthesis-related genes may be reversible by the discontinuation of DAP, leading to reversion to the DAP susceptible phenotype.

Pro-inflammatory/Th1 gene expression shift in high glucose stimulated mesangial cells and tubular epithelial cells

Diabetic nephropathy (DN) is a major cause of end stage kidney disease and a strong risk factor for cardiovascular diseases. Growing data show chronic inflammation plays an important role for the progression of DN. As for the immune cells, anti-inflammatory leukocytes as well as pro-inflammatory leukocytes play an important role in DN. In addition to leukocytes, renal resident cells contribute to the inflammatory process of DN. However, precise functions, phenotypes and immune balance of renal resident cells remain to be explored. Therefore, we hypothesized that the aberrant immune balance of renal resident cells contributes to the pathogenesis of DN. To explore this possibility, we performed genome-wide transcriptome profiling in mesangial cells and tubular epithelial cells (TECs), which were stimulated by high glucose (HG) and detected the expression of inflammation associated genes. HG increased the mRNA expression of oxidative stress, inflammasome and mammalian target of rapamycin (mTOR) related genes in mesangial cells. Pro-inflammatory/Th1 gene expression was upregulated, but Th2 related gene expression was downregulated in mesangial cells. In TECs, HG stimulation increased pro-inflammatory/Th1/Th2 gene expression. Phosphorylation of signaling proteins shifted towards pro-inflammatory phenotype with suppressed phosphorylation of Th2 related signaling in TECs. The data taken together indicate that HG shifts the immune balance toward pro-inflammatory/Th1 phenotype in mesangial cells and TECs, which might initiate and/or prolong inflammation, thereby resulting in diabetic nephropathy.

Involvement of p38MAPK in Impaired Neutrophil Bactericidal Activity of Hemodialysis Patients: Role of p38MAPK in Impaired Neutrophil Function

Mortality from infections has been reported to be higher in hemodialysis (HD) patients. Although dysfunction of neutrophils against bacterial infection was reported in HD patients, the precise mechanism remains to be clarified. We therefore examined the impacts of neutrophil inflammatory signaling on bactericidal activity in HD patients. Comprehensive analyses of intracellular signalings were performed in whole blood of HD patients and control using a microarray system. To confirm the contribution of the signaling to bactericidal activity in neutrophils, we examined the phosphorylation, bacterial killing function, reactive oxygen species (ROS) production, and myeloperoxidase (MPO) release in neutrophils against Staphylococcus aureus. RNA microarray analysis showed the suppression of p38 mitogen activated protein kinase (MAPK) signaling in HD patients. Neutrophils in HD patients showed the impairment of bactericidal activity against S. aureus compared to healthy subjects. Phosphorylation rate of p38MAPK of neutrophils in response to S. aureus was lower in HD patients than healthy subjects. The levels of ROS produced by neutrophils after co‐culture with S. aureus were lower in HD patients, on the other hand, there was no difference of MPO release between HD patients and healthy subjects. A selective pharmacological inhibitor of p38MAPK suppressed bacterial killing function as well as ROS production in neutrophils of healthy subjects. Impairment of p38MAPK signaling pathway might contribute to the suppression of neutrophil bactericidal activity in HD patients through less production of ROS.

Occult Hepatitis B Virus Infection With Increased Virus DNA Levels in a Chronic Hemodialysis Patient: Occult HBV Infection in a Hemodialysis Patient

Dear Editor, Controlling hepatitis B virus (HBV) infection is a serious issue in dialysis hospitals. Occult HBV infection (OBI) is defined as the presence of HBV genome in the serum or liver tissue without detectable hepatitis B surface antigen (HBsAg). A previous study reported OBI prevalence in Japanese chronic hemodialysis (HD) patients to be 0.3% (1). The patients in that study tested negative for anti‐ hepatitis B surface antibody (HBsAb) and positive for anti‐hepatitis B core antibody (anti‐HBc), and HBV DNA was detected in their sera with a quantitative sensitivity of 2.1 log copies/mL (20 IU/mL). Herein, we report an OBI patient with increased serum HBV DNA levels who was undergoing HD.