Supplementary Material for: Salt Intake and All-Cause Mortality in Hemodialysis Patients

<b><i>Background:</i></b> Although some clinical practice guidelines regarding hemodialysis recommend salt restriction, few studies have examined the association between salt intake and clinical outcomes in hemodialysis patients. This study aimed to clarify the association between salt intake and mortality in hemodialysis patients. <b><i>Methods:</i></b> This retrospective cohort study was based on the Japanese Society for Dialysis Therapy renal data registry database (2008) and included 88,115 adult patients who had received hemodialysis for at least 2 years. Estimated salt intake was the main predictor and was calculated from intra-dialytic weight loss and pre- and post-dialysis serum sodium levels. Nonlinear logistic regression was used to determine the association between salt intake and mortality, adjusting for potential confounders. The outcomes considered were all-cause mortality and cardiovascular death at 1 year. <b><i>Results:</i></b> The median (25–75th percentile) salt intake at baseline was 6.4 (4.6–8.3) g/day. At 1 year, all-cause mortality occurred in 1,845 (2.1%) patients, including 807 cardiovascular deaths. The low salt intake group (< 6 g/day) demonstrated the highest all-cause mortality and cardiovascular deaths. No association was observed between high salt intake, all-cause mortality and cardiovascular deaths. The lowest risk for all-cause mortality and cardiovascular death occurred among patients with an estimated salt intake of 9 g/day. <b><i>Conclusion:</i></b> Low salt intake, but not high salt intake, was associated with all-cause and cardiovascular mortality in Japanese hemodialysis patients. Further studies to justify including a lower limit of salt intake for hemodialysis patients are suggested

Supplementary Material for: Dietary fat composition affects hepatic angiogenesis and lymphangiogenesis in HCV core gene transgenic mice

Introduction: Previous research has demonstrated that an isocaloric diet rich in trans fatty acid (TFA), saturated fatty acid (SFA), and cholesterol (Chol) promoted steatosis-derived hepatic tumorigenesis in hepatitis C virus core gene transgenic (HCVcpTg) mice in different manners. Growth factor signaling and ensuing angiogenesis/lymphangiogenesis are key factors in hepatic tumorigenesis that have become recent therapeutic targets for hepatocellular carcinoma. However, the influence of dietary fat composition on these factors remains unclear. This study investigated whether the type of dietary fat would have a specific impact on hepatic angiogenesis/lymphangiogenesis in HCVcpTg mice. Methods: Male HCVcpTg mice were treated with a control diet, an isocaloric diet containing 1.5% cholesterol (Chol diet), or a diet replacing soybean oil with hydrogenated coconut oil (SFA diet) for a period of 15 months, or with shortening (TFA diet) for 5 months. The degree of angiogenesis/lymphangiogenesis and the expression of growth factors, including fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF), were evaluated in non-tumorous liver tissues using quantitative mRNA measurement, immunoblot analysis, and immunohistochemistry. Results: Long-term feeding of SFA and TFA diets to HCVcpTg mice increased the expressions of vascular endothelial cell indicators, such as CD31 and TEK tyrosine kinase, in addition to lymphatic vessel endothelial hyaluronan receptor 1, indicating that angiogenesis/lymphangiogenesis were up-regulated only by these fatty acid-enriched diets. This promoting effect correlated with elevated VEGF-C and FGF receptor 2 and 3 levels in the liver. c-Jun N-terminal kinase (JNK) and hypoxia-inducible factor (HIF) 1α, both key regulators of VEGF-C expression, were enhanced in the SFA- and TFA-rich diet groups as well. The Chol diet significantly increased the expressions of such growth factors as FGF2 and PDGF subunit B (PDGF-B), without any detectable impact on angiogenesis/lymphangiogenesis. Conclusion: This study revealed that diets rich in SFA and TFA, but not Chol, might stimulate hepatic angiogenesis/lymphangiogenesis mainly through the JNK-HIF1α-VEGF-C axis. Our observations indicate an importance of dietary fat species for preventing hepatic tumorigenesis

Supplementary Material for: Reduced DEFA5 expression and STAT3 activation underlie the submucosal invasion of early gastric cancers

Introduction Submucosal invasion is a core hallmark of early gastric cancer (EGC) with poor prognosis. However, the molecular mechanism of the progression from intramucosal gastric cancer (IMGC) to early submucosal-invasive gastric cancer (SMGC) is not fully understood. The objective of this study was to identify genes and pathways involved in the submucosal invasion in EGC using comprehensive gene expression analysis. Methods Gene expression profiling was performed for eight cases of IMGC and eight cases of early SMGC with submucosal invasion ≥ 500 μm. To validate the findings of gene expression analysis and to examine the gene expression pattern in tissues, immunohistochemical (IHC) staining was performed for 50 cases of IMGC and SMGC each. Results Gene expression analysis demonstrated that the expression levels of small intestine-specific genes were significantly decreased in SMGC. Among them, defensin alpha 5 (DEFA5) was the most downregulated gene in SMGC, which was further validated in SMGC tissues by IHC staining. Gene set enrichment analysis showed a strong association between SMGC, the JAK-STAT signaling pathway, and the upregulation of STAT3-activating cytokines. The expression of phosphorylated STAT3 was significant in the nucleus of tumor cells in SMGC tissues but not in areas expressing DEFA5. Conclusion The results of this study strongly suggest that the downregulation of DEFA5 and the activation of STAT3 play a significant role in the submucosal invasion of EGC

Supplementary Material for: Proton beam therapy versus radiofrequency ablation for patients with treatment-naïve single hepatocellular carcinoma: a propensity score analysis

Introduction: Proton beam therapy (PBT) is known to be an effective locoregional treatment for hepatocellular carcinoma (HCC). However, few comparative studies in treatment-naïve cases have been reported. The aim of this study was to compare the survival outcomes of PBT with those of radiofrequency ablation (RFA) in patients with treatment-naïve solitary HCC. Methods: Ninety-five consecutive patients with treatment-naïve HCC, a single nodule measuring ≤5 cm in diameter, and a Child-Pugh score of ≤8 who were treated with PBT at the University of Tsukuba Hospital between 2001 and 2013 were enrolled in the study. In addition, 836 patients with treatment-naïve HCC treated by RFA at the University of Tokyo Hospital during the same period were analyzed as controls. Recurrence-free survival (RFS) and overall survival (OS) were compared in 83 patient pairs after propensity score matching. Results: The 1-year, 3-year, and 5-year RFS rates were 86.6%, 49.5%, and 35.5%, respectively, in the PBT group and 59.5%, 34.0%, and 20.9% in the RFA group (p = 0.058); the respective OS rates were 97.6%, 77.8%, and 57.1% in the PBT group and 95.1%, 81.7%, and 67.7% in the RFA group (p = 0.16). Regarding adverse effects, no grade 3 or higher adverse events were noted in the PBT; however, two grade 3 adverse events occurred within 30 days of RFA in the RFA group, one hemoperitoneum and one hemothorax.. Discussion/Conclusion: After propensity score matching, PBT showed no significant difference in RFS and OS compared to RFA. PBT can be an alternative for patients with solitary treatment-naïve HCC

Supplementary Material for: Gastroesophageal Reflux Disease-Related Disorders of Systemic Sclerosis Based on the Analysis of 66 Patients

<b><i>Background/Aims:</i></b> Gastroesophageal reflux disease (GERD)-related disorders of systemic sclerosis (SSc) patients have not been adequately investigated. <b><i>Methods:</i></b> Sixty-six SSc patients (5 males and 61 females; 56.6 ± 14.6 years old) who underwent esophagogastroduodenoscopy were analyzed on the basis of 16 background factors. They were additionally compared with 116 matched non-SSc subjects controlling age, sex, and use of proton pump inhibitors (PPIs). <b><i>Results:</i></b> The mean disease duration of 66 patients was 5.1 ± 8.1 years, and their breakdown was as follows: 53 (80.3%) with GERD, 38 (57.6%) with GERD-related symptoms, and 20 (30.3%) with reflux esophagitis (RE; LA-A: 10, LA-B: 5, LA-C: 4, LA-D: 1). Use of PPI (<i>p</i> = 0.0455), complication of interstitial lung disease (<i>p</i> = 0.0242), and history of cyclophosphamide therapy (<i>p</i> = 0.0184) denoted significant association with GERD-related symptoms. Older age (<i>p</i> = 0.0211) was significantly associated with RE. None of GERD-related disorders showed any difference between 37 diffuse cutaneous SSc and 29 limited cutaneous SSc patients. The matched analysis indicated that SSc patients had higher prevalence of GERD (<i>p </i>< 0.0001), GERD-related symptoms (<i>p</i> = 0.0034), and RE (<i>p </i>= 0.0002). <b><i>Conclusion:</i></b> SSc patients tend to have worse GERD symptoms and severer RE. However, most SSc-associated factors did not show significant association with GERD-related disorders, indicating the difficulty in predicting GERD-related disorders among SSc patients