Hiroshi Kajiyama

Relative transcriptional activities of SAA1 promoters polymorphic at position −13(T/C): Potential association between increased transcription and amyloidosis

The risk associated with the serum amyloid A (SAA) 1 gene and developing AA-amyloidosis is still controversial. In familial Mediterranean fever or Caucasoid rheumatoid arthritis (RA), the SAA1.1 allele is a risk factor for the development of AA-amyloidosis. However, individuals with the SAA1.3 allele are susceptible to AA-amyloidosis in the Japanese RA population, but those with the SAA1.1 are not. Previous reports have indicated that the − 13T/C single nucleotide polymorphism (SNP) at the 5’-flanking region of SAA1 appears to be a better marker of AA-amyloidosis than the exon-3 based haplotype, i.e., SAA1.1 or SAA1.3, in both Japanese and American Caucasian populations. So far, it is unknown why the − 13T SNP increases the amyloidogenicity of the patients. In the present study, a luciferase reporter gene assay showed that the transcriptional activity of the SAA1 having the − 13T-containing promoter was significantly higher than activities of those with − 13C-containing promoters (Fishers protected least significance difference test). We suggest that having the − 13T SNP in the SAA1 promoter correlates with the amyloidogenicity in part as a result of this increased transcriptional activity.

Original Research: Potential of urinary nephrin as a biomarker reflecting podocyte dysfunction in various kidney disease models.

Urinary nephrin is a potential non-invasive biomarker of disease. To date, however, most studies of urinary nephrin have been conducted in animal models of diabetic nephropathy, and correlations between urinary nephrin-to-creatinine ratio and other parameters have yet to be evaluated in animal models or patients of kidney disease with podocyte dysfunction. We hypothesized that urinary nephrin-to-creatinine ratio can be up-regulated and is negatively correlated with renal nephrin mRNA levels in animal models of kidney disease, and that increased urinary nephrin-to-creatinine ratio levels are attenuated following administration of glucocorticoids. In the present study, renal nephrin mRNA, urinary nephrin-to-creatinine ratio, urinary protein-to-creatinine ratio, and creatinine clearance ratio were measured in animal models of adriamycin nephropathy, puromycin aminonucleoside nephropathy, anti-glomerular basement membrane glomerulonephritis, and 5/6 nephrectomy. The effects of prednisolone on urinary nephrin-to-creatinine ratio and other parameters in puromycin aminonucleoside (single injection) nephropathy rats were also investigated. In all models tested, urinary nephrin-to-creatinine ratio and urinary protein-to-creatinine ratio increased, while renal nephrin mRNA and creatinine clearance ratio decreased. Urinary nephrin-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA in almost all models, as well as a significant positive correlation with urinary protein-to-creatinine ratio and a significant negative correlation with creatinine clearance ratio. Urinary protein-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA. Following the administration of prednisolone to puromycin aminonucleoside (single injection) nephropathy rats, urinary nephrin-to-creatinine ratio was significantly suppressed and exhibited a significant positive correlation with urinary protein-to-creatinine ratio. In addition, the decrease in number of glomerular Wilms tumor antigen-1-positive cells was attenuated, and urinary nephrin-to-creatinine ratio exhibited a significant negative correlation in these cells. In conclusion, these results suggest that urinary nephrin-to-creatinine ratio level is a useful and reliable biomarker for predicting the amelioration of podocyte dysfunction by candidate drugs in various kidney disease models with podocyte dysfunction. This suggestion will also be validated in a clinical setting in future studies.

Splicing variant of WDFY4 augments MDA5 signalling and the risk of clinically amyopathic dermatomyositis

Objectives Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of rare autoimmune diseases in which both genetic and environmental factors play important roles. To identify genetic factors of IIM including polymyositis, dermatomyositis (DM) and clinically amyopathic DM (CADM), we performed the first genome-wide association study for IIM in an Asian population. Methods We genotyped and tested 496 819 single nucleotide polymorphism for association using 576 patients with IIM and 6270 control subjects. We also examined the causal mechanism of disease-associated variants by in silico analyses using publicly available data sets as well as by in in vitro analyses using reporter assays and apoptosis assays. Results We identified a variant in WDFY4 that was significantly associated with CADM (rs7919656; OR=3.87; P=1.5×10−8). This variant had a cis-splicing quantitative trait locus (QTL) effect for a truncated WDFY4isoform (tr-WDFY4), with higher expression in the risk allele. Transexpression QTL analysis of this variant showed a positive correlation with the expression of NF-κB associated genes. Furthermore, we demonstrated that both WDFY4 and tr-WDFY4 interacted with pattern recognition receptors such as TLR3, TLR4, TLR9 and MDA5 and augmented the NF-κB activation by these receptors. WDFY4 isoforms also enhanced MDA5-induced apoptosis to a greater extent in the tr-WDFY4-transfected cells. Conclusions As CADM is characterised by the appearance of anti-MDA5 autoantibodies and severe lung inflammation, the WDFY4 variant may play a critical role in the pathogenesis of CADM.

Clinical practice guideline for drug-induced kidney injury in Japan 2016: digest version

The definition of DKI is a new onset of kidney injury or the worsening of an existing kidney injury due to drug administration. DKI can be classified based on the mechanism of pathogenesis, as well as on the damaged segment of the kidney. The classification based on the mechanism of pathogenesis is as follows: (1) toxic kidney injury (direct toxicity); (2) acute interstitial nephritis (AIN) due to allergic mechanism (hypersensitivity and direct toxicity); (3) indirect toxicity, such as electrolyte abnormalities and decrease of renal blood flow; and (4) obstruction of urinary tract. The classification based on the damaged segment of kidney is as follows: (1) glomerular injury; (2) tubular injury; (3) interstitial injury; and (4) vascular injury. The criteria for diagnosis are as follows: (1) new onset of kidney injury after the start of the administration of the candidate agent and (2) improvement or stoppage of the progression of the kidney injury after the cessation of the candidate agent, and all other causes can be ruled out. The cornerstone of treatment is the identification and cessation of the candidate agent as soon as possible.

FRI0042 Altered Profiles of Histone Lysine Methylation Affect Mmp Gene Transcription in Rheumatoid Arthritis Synovial Fibroblasts

Background Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes progressive joint destruction. A line of evidence suggests that synovial fibroblasts (SFs) play an important role in the pathogenesis of RA. RASFs produce matrix metalloproteinases (MMPs) that degrade articular cartilage. Although interleukin-6 (IL-6) is an inflammatory cytokine and is proved to be involved in the pathogenesis of RA, it is unknown whether IL-6 affects MMPs gene transcription in RASFs. Furthermore, recent advances have revealed that epigenetic mechanisms, including histone modifications, are considered to be important regulators in gene transcription. We hypothesized that epigenetic dysregulation might induce RASF activation. Objectives The aim of this study is to examine whether IL-6 regulates MMPs expression in RASFs and whether histone modifications are associated with the MMPs gene activation in RASFs. Methods We compared MMPs gene expression by quantitative RT-PCR and histone methylation in the MMP promoters by chromatin immunoprecipitation (ChIP) assay after stimulation with IL-6 and/or soluble IL-6 receptor α (sIL-6Rα) in RASFs and control, osteoarthritis (OA) SFs. Chromatin structures in the MMP promoters were evaluated with micrococcal nuclease (MNase) assay in RASFs and OASFs. We investigated the change in the MMPs gene expression after silencing of WDR5 that is required for generating H3K4me3, an active histone marker. IL-6 signal induces Signal Transducer and Activator of Transcription 3 (STAT3) activation. To elucidate the mechanisms of IL-6-induced MMPs gene activation in RASFs, we investigated cell surface expression of the IL-6 receptor (gp130 and membrane-bound IL-6Rα) by flow cytometry, phospho-STAT3 (p-STAT3) expression by immunoblotting, and binding of STAT3 to the MMPs 1, 3, 9, and 13 promoters after IL-6 stimulation by ChIP assay in RASFs and OASFs. Results MMPs 1, 3, 9 and 13 genes were actively transcribed in RASFs. The histone methylation profiles (H3K4me3 and H3K27me3) and the result of MNase assay indicated that chromatin structures were open in the MMPs 1, 3, 9 and 13 promoters in RASFs. The depletion of WDR5 reduced the levels of H3K4me3 as well as the MMPs 1, 3, 9 and 13 gene expression. Interestingly, IL-6 and sIL-6Rα significantly increased the expression of MMPs 1, 3 and 13, but not MMP-9, in RASFs. Although the expression levels of gp130 as well as membrane-bound IL-6Rα were comparable and STAT3 was similarly phosphorylated after IL-6 stimulation in RASFs and OASFs, STAT3 bound to the MMPs 1, 3 and 13 promoters, but not the MMP-9 promoter, after stimulation with IL-6 and sIL-6Rα only in RASFs. It was suggested that binding of STAT3 to the promoters resulted in MMPs 1, 3 and 13 gene activation after IL-6 stimulation in RASFs. Conclusions Our data indicate that altered profiles of histone methylation and binding of STAT3 to the promoters regulate constitutive and IL-6-induced MMPs gene activation in RASFs and possibly arthritogenic properties of RASFs. References Araki Y et al. Arthritis Rheum. Epub. 2015 Dec 29. Disclosure of Interest None declared