Yoshitaka Ando

Associations between circulating microRNAs (miR-21, miR-34a, miR-122 and miR-451) and non-alcoholic fatty liver

BACKGROUND In many industrialized countries, non-alcoholic fatty liver disease (NAFLD) is recognized as an important disease that increases the risk of cardiovascular disease, type-2 diabetes, and metabolic syndrome. Most people with NAFLD are asymptomatic, and the disease is discovered incidentally during clinical laboratory tests. Present screening methods for NAFLD use ultrasound, and CT scans that are time-consuming, and a simple screening method for NAFLD is needed. In this study, we investigated whether serum miRNAs are associated with NAFLD and assessed the potential of serum miRNAs as a biomarker for NAFLD. METHODS We assessed intrahepatic fat by ultrasound scan, and the serum levels of five miRNAs (miR-21, miR-34a, miR-122, miR-145, and miR-451), which help regulate cholesterol and fatty acid homeostasis in liver tissue, by real-time PCR in a cross-sectional sample of 403 participants who attended health examinations. RESULTS Serum levels of miRNAs, miR-21, miR-34a, miR-122, and miR-451 were higher in participants with NAFLD. The serum level of miR-122 was correlated with the severity of liver steatosis. CONCLUSION Serum levels of miRNAs, particularly miR-122, may be a useful biomarker for NAFLD.

Simple evaluation of aortic arch calcification by chest radiography in hemodialysis patients

Vascular calcification is associated with a poor prognosis in dialysis patients. It can be assessed with computed tomography but simple inoffice techniques may provide useful information. We compared the results obtained with a simple noninvasive technique with those obtained using multidetector computed tomography for aortic arch calcification volume (AoACV) in chronic hemodialysis (HD) patients. The enrolled study subjects were 63 (32 men and 31 women) maintenance HD patients. Calcification of the aortic arch was semiquantitatively estimated with a AoAC score (AoACS) on plain chest radiology. The AoACV was increased, with a mean value of 6.6 ranging from 0% to 36.5%. The coefficient of intraobserver variation was less than 2.5%. Aortic arch calcification score was highly correlated with AoACV (r=0.635, P<0.001). Multiple regression analysis showed age (F value=12.62, P<0.001) and pulse pressure (F value=4.54, P=0.037) to be significant independent determinants of AoACS. In conclusion, a simple measurement of AoACS may be useful for inoffice imaging to choose a therapeutic regimen in HD patients.

Relation of oral 1α-hydroxy vitamin D3 to the progression of aortic arch calcification in hemodialysis patients

The role of decreased active vitamin D levels on vascular calcification has not been elucidated in hemodialysis (HD) patients. The aim of the present study was to evaluate the relationship between progression of aortic arch calcification (AoAC) and prescribed dose of 1α-hydroxy vitamin D. The enrolled study subjects were 65 (40 men and 25 women) HD patients. Calcification of the aortic arch was semiquantitatively estimated with a score (AoACS) on plain chest radiology. Change in AoACS (ΔAoACS) was obtained by subtracting the baseline AoACS value from the follow-up AoACS value. The second assessment was performed from 2 years after the first determination. The nonprogressors (63.2 ± 14.5 years) were significantly younger than the progressors (68.2 ± 10.8 years) (P = 0.0419). In addition, prescribed dose of 1α-hydroxy vitamin D3 was significantly higher in the nonprogressors (125.5 ± 109.1 μg) than progressors (84.8 ± 81.1 μg) (P = 0.0371). Multiple regression analysis revealed prescribed dose of 1α-hydroxy vitamin D3 (β value = −0.324, P = 0.0051) as well as DBP (β value = −0.418, P = 0.007), serum levels of P (β value = 0.333, P = 0.006) and C-reactive protein (β value = 0.237, P = 0.0048) to be significant independent determinants of ΔAoACS. In conclusion, the evaluation of AoACS on chest radiography is a very simple tool in HD patients. Active vitamin D therapy seems to protect patients from developing vascular calcification.

Oral administration of Bifidobacterium longum in a gastro-resistant seamless capsule decreases serum phosphate levels in patients receiving haemodialysis

When kidney function decreases, phosphate (P) per se can initiate and promote hyperparathyroidism and vascular calcification. Therefore, hyperphosphataemia is a widely recognized risk factor for mortality and cardiovascular disease in patients with chronic kidney disease (CKD) [1]. Despite the introduction in the market of new P-binders, such as sevelamer hydrochloride and lanthanum carbonate, the control of hyperphosphataemia in haemodialysis (HD) patients remains difficult because of adverse effects such as constipation and faecal impaction, drug compliance and metal accumulation. We here report an interesting observation that serum P levels can be reduced by modifying the intestinal flora with probiotic administration. This may suggest a new potential approach to better control hyperphosphataemia in HD patients. In patients receiving HD, intestinal aerobic bacteria, such as enterobacteria and enterococci, occur in numbers ∼100 times higher than those in healthy subjects. These aerobes produce putrefactive substances [2]. Oral administration of antibiotic-resistant lactic acid bacteria for 4 weeks can restore the composition of disturbed microflora characterized by overgrowth of aerobes and decreased levels of serum uraemic toxins, including indicant, in HD patients [2]. When administered orally, Bifidobacteria cannot survive exposure to gastric juice before they reach the intestine. In order to enable Bifidobacteria to reach the intestine, a colony-forming unit of Bifidobacterium longum JBL01, a strain of human Bifidobacteria, measuring 2.0 × 109, combined with 0.11 g of oligosaccharides (lactulose and raffinose) as a stimulant to bacteria proliferation, were encapsulated in a gastro-resistant seamless capsule (B capsule, MorishitaJintan Co., Ltd, Osaka, Japan). Bifidobacteria can survive in this gastro-resistant capsule even in a solution of 1.2 pH at 37°C for 120 min. In contrast, Bifidobacteria without the capsule (a powder formulation) cannot be detected immediately after mixing with the solution [3]. Moreover, significant decreases in serum levels of indoxyl sulphate and homocysteine were observed by oral administration of B. longum in the gastro-resistant capsule, but not by the administration of B. longum without the capsule in HD patients [4]. Thus, to improve the composition of disturbed microflora and alleviate faecal impaction, B capsules containing B. longum JBL01 were administered orally once daily for 4 weeks in 15 patients receiving HD [age: 62.2 ± 9.8 years; 10 males, 5 females; 5 diabetes mellitus (DM), 10 non-DM; HD duration: 9.3 ± 7.0 years; serum albumin levels: 3.9 ± 0.3 g/dL; serum corrected calcium (Ca) levels: 9.1 ± 0.8 mg/dL; serum P levels: 6.7 ± 0.6 mg/dL; serum intact parathyroid hormone (PTH) levels: 363 ± 221 pg/mL]. Results were compared with those of 16 HD patients who did not receive B capsules as a control group (age:58.1 ± 15.6 years; 10 males, 6 females; 3 DM, 13 non-DM; HD duration: 9.1 ± 6.8 years; serum albumin levels: 3.8 ± 0.3 g/dL; serum-corrected Ca levels: 9.0 ± 0.9 mg/dL; serum P levels: 7.0 ± 0.8 mg/dL; serum intact PTH levels: 219 ± 124 pg/mL). No patient had undergone treatment with B. longum preparations before participating in this study. The dose of drugs affecting P metabolism were fixed through the study period in the control group (Ca carbonate: 2.79 ± 1.44 g, n= 14; sevelamer hydrochloride: 3.20 ± 2.94 g, n= 5; calcitriol injection: 1.67 ± 0.24 μg/week, n= 3; maxacalcitol: 15.0 ± 4.1 μg/week, n= 3; oral alfacalcidol: 0.5 μg, n= 2, oral calcitriol: 0.38 μg, n= 2) and in the B capsule-treated group (Ca carbonate: 2.50 ± 0.71 g, n= 12; sevelamer hydrochloride: 3.75 ± 0.53 g, n= 4; calcitriol injection: 2.75 μg/week, n= 2; maxacalcitol: 12.5 ± 8.5 μg/week, n= 4; oral alfacalcidol: 0.25 ± 0 μg, n= 3, oral calcitriol: 0.25 μg, n= 1). No patients received lanthanum carbonate or cinacalcet hydrochloride in either group. All data are expressed as mean ± SD or without SD in n= 1 or 2. Oral administration of B capsules for 4 weeks had no significant effect on defecation frequency or faecal hardness determined by the Bristol stool form scale (data not shown). The explanation for this result may be related to the low incidence of obstinate constipation from the beginning in this study (mean defecation frequency: 0.9/day). Serum P levels unexpectedly decreased significantly in the second and fourth weeks due to oral administration of the B capsule (Figure ​(Figure1).1). Serum P levels remained unchanged in the control group. Subsequently, serum P levels returned to original levels 2 weeks after the completion of oral treatment. No significant changes were found in other serum parameters in the B capsule-treated group compared with baseline data and with the control group (data not shown). Fig. 1. Effect of once-daily oral administration of encapsulated B. longum JBL01 with oligosaccharides (B capsule) for 4 weeks on serum P levels in patients on haemodialysis. Open circle: untreated control group (n= 16); filled circle: B capsule-treated group ... The main mechanism for a decrease in serum P levels during B capsule treatment is its ability to lower intestinal pH levels. In the faeces of HD patients, pH levels and ammonia concentrations are elevated due to bacteria-mediated hydrolysis of urea [4]. High levels of ammonia are responsible for elevated pH in faeces and lead to overgrowth of aerobic and putrefactive bacteria. Bifidobacteria can ferment carbohydrates to produce acetic acid and lactic acid, resulting in acidification of the intestinal lumen [4]. Low pH levels in the intestinal milieu inhibit the growth of aerobic and putrefactive bacteria, thus normalizing the intestinal flora. In another study, the administration of B. longum decreased faecal pH levels, and pretreatment levels were restored 1 week after the completion of treatment [5]. Decreased intestinal pH levels due to B capsule treatment may increase the ionization of intestinal Ca. Ca2+ binds with intestinal P ions as an intrinsic P binder. In this study, increased intestinal Ca2+ did not lead to elevated serum Ca levels. Ca2+ may be immediately used in the formation of calcium phosphate in the intestine, and thus not absorbed. Further studies measuring faecal pH, P and Ca excretions will examine this as a possible mechanism in B capsule-treated patients. In this study, although the number of faecal bacteria was not determined during B capsule treatment, the decrease in serum P levels cannot be due to increased uptake of P into the intestinal B. longum for their proliferation. Because feeding of B. longum increases their number, but does not affect the total number of bacteria through a reduction in aerobes in the faeces of healthy subjects [5]. In addition, increased faecal volume and water content are not responsible for low serum P levels, as evidenced by unchanged defecation frequency and faecal hardness among the patients in the present study. It is increasingly recognized that uraemic toxins originating from intestinal microbial metabolism may contribute to the deterioration of renal function, cardiovascular calcification, metabolic bone disease and mortality in CKD/HD patients [6, 7]. The administration of Bifidobacteria can decrease the serum concentrations of indoxyl sulphate and P-cresol in HD patients [4, 7]. In addition, Bifidobacteria produce vitamin B12 and folate, which can normalize serum homocysteine levels in HD patients [3]. This is an important property since most HD patients show hyperhomocysteinaemia, which is a risk factor for cardiovascular disease. Although further investigation is clearly required to clarify the mechanism for the P-reducing effect of the B capsule, Bifidobacteria preparation is a simple and safe treatment for persistent hyperphosphataemia in patients receiving HD. Further studies may identify additional potential benefits of probiotic treatments in CKD/HD patients [6, 7].

Independent and incremental prognostic value of novel cardiac biomarkers in chronic hemodialysis patients

UNLABELLED End-stage renal disease is a major clinical and public health problem, and cardiovascular disease accounts for half of the mortality in hemodialysis patients. An existing mortality risk score (AROii score) or N-terminal pro-brain natriuretic peptide (NT-proBNP) level have modest predictive power, but there is room for improvement. There are emerging cardiac biomarkers (soluble isoforms of ST2 [sST2], galectin-3 [Gal-3]), and uremic toxicity (indoxyl sulfate). We sought to determine whether these biomarkers predict cardiovascular outcomes in hemodialysis patients and have incremental prognostic value over the clinical score and NT-proBNP level. METHODS A total of 423 hemodialysis patients were prospectively followed up for primary (all-cause death) and secondary end points (a composite of all-cause death or cerebrocardiovascular events). RESULTS During a mean follow-up of 2.1 ± 0.4 years, there were 48 all-cause deaths and 78 composite outcomes. Soluble isoforms of ST2, Gal-3, and NT-proBNP were associated with all-cause deaths but indoxyl sulfate was not in both log-rank test and receiver operating characteristic analysis. Both sST2 and Gal-3 had independent and incremental prognostic value for both outcomes over the AROii score and NT-proBNP. Although adding sST2 did not reclassify over the model-based AROii score and NT-proBNP for all-cause death, further addition of Gal-3 did. Subgroup analyses of patients with left ventricular ejection fraction measurement (n = 301) corroborated these results, where the 2 biomarkers remained independent and incremental for both all-cause death and composite outcome after adjusting for the risk score and the ejection fraction. CONCLUSIONS Both sST2 and Gal-3 had independent and incremental prognostic values over NT-proBNP and an established risk score in patients with hemodialysis. Assessment of sST2 and Gal-3 further enhances risk stratification.

Measurement of serum remnant-like lipoprotein particle-triglyceride (RLP-TG) and RLP-TG/total TG ratio using highly sensitive triglyceride assay reagent.

BACKGROUND Serum concentration of remnant-like lipoprotein particles (RLP) have been measured by cholesterol as RLP-C for clinical diagnostic purpose. However, the measurement of TG in RLP and the ratio of RLP-TG/total TG has not been well established. METHOD Highly sensitive triglyceride assay reagent (TG-EX) was used for RLP-TG assay and compared with the previously used TG reagent (Determiner LTGII). Sera in health check-up populations, cardiovascular disease, diabetes and oral fat load cases were used for the evaluation of the new RLP-TG assay. Serum TC, TG, HDL-C, LDL-C and RLP-C concentrations were also determined in above cases. RESULTS The detection limit of new RLP-TG using TG-EX was 2.0mg/dl. The within-run imprecision (n=10) was CV=3.0% (RLP-TG: 4.1 mg ± 0.7 mg/dl), CV = 1.4% (RLP-TG: 42.0 ± 0.6 mg/dl) and CV=0.5% (RLP-TG: 100.6 ± 0.6 mg/dl). Cut-off value (75 percentile) of RLP-TG determined in the fasting Japanese population was 13.1mg/dl in men and 9.9 mg/dl in women. In patients with metabolic syndrome, cardiovascular disease and diabetes, RLP-TG levels were significantly higher than those in normal control subjects. RLP-TG levels increased significantly after an oral fat load and the ratio of RLP-TG/total TG increased > 3-fold compared to the ratio in the fasting state. Approximately 80% of TG increased after an oral fat load was TG derived from remnant lipoproteins. CONCLUSION Normal range of plasma RLP-TG in the fasting Japanese population was first determined using a highly sensitive TG assay reagent. RLP-TG was shown to be higher in cases with metabolic syndrome, cardiovascular disease, etc and a better marker than RLP-C for the measurement of postprandial remnant lipoproteins, together with total TG for RLP-TG/total TG ratio.

Comparison of different interdialytic intervals among hemodialysis patients on their echocardiogram-based cardiovascular parameters.

BACKGROUND Although cardiovascular events in hemodialysis (HD) patients are the most frequent on the day after a long (2 days) interdialytic interval (IDT), it has been uncertain whether accumulation or elimination of large extracellular fluid volume, electrolyte, and/or uremic substances is the culprit for this. We sought to test our hypothesis that the long IDT alters echocardiographic parameters at rest and during exercise in stable maintenance HD patients compared with other IDTs. METHODS We performed a cross-sectional comparison using 1-way repeated analysis of variance or Friedman test of echocardiograms at 3 different IDTs, just after HD, after short IDT (1 day), and after long IDT, among 80 stable Japanese outpatients (age 61 ± 9 years, 60 males) on thrice weekly maintenance. End-systolic elastance (Ees), arterial elastance (Ea), and pressure-volume area (PVA) were estimated using a noninvasive single-beat technique. Ventricular-arterial coupling was assessed by Ea/Ees ratio. Measurements were repeated after 2-minute handgrip stress to evaluate cardiac reserve. RESULTS Resting left ventricular end-diastolic volume index and stroke volume index were significantly larger after a 1-day IDT compared with just after HD and even more after a 2-day IDT. Although Ees, Ea, and Ea/Ees ratio at rest remained similar between short and long intervals, stroke work (SW) and PVA were higher after the long interval. During handgrip stress, a significant increase in Ea without corresponding rise in Ees was observed only after long IDT, resulting in decreased stroke volume index, SW, and SW/PVA efficiency. CONCLUSIONS In a selective Japanese outpatient population on maintenance HD, there were no differences in resting cardiovascular function measured by echocardiography at 3 different IDTs. However, exercise-induced afterload mismatch assessed by the changes in Ea, SV, SW, and SW/PVA efficiency was most pronounced in individuals after the long IDT compared with other IDTs. Our findings report potential pathophysiologic echocardiographic parameters that attempt to explain why cardiovascular events are highest on the day after the long IDT compared to other IDTs in dialysis patients.

Plasma adiponectin is a more specific marker of fatty liver than a marker of metabolic syndrome in Japanese men

Background The association of plasma cardiovascular risk markers and metabolic syndrome (MetS) with non-alcoholic fatty liver disease (NAFLD) has not been well defined. Methods Japanese men (n = 809) had standard anthropometric measurements done, and had their liver fat quantitated by ultrasound. Three groups were identified: (1) normal controls without significant disease, (2) preliminary-metabolic syndrome (pre-MetS) cases and (3) MetS cases. Plasma adiponectin, high sensitivity-C reactive protein (hs-CRP), HOMA-IR, lipids, lipoproteins and liver enzymes were evaluated among the three groups. Results The prevalence of fatty liver was 13% in controls, 39% in pre-MetS and 62% in MetS. Plasma adiponectin and high density lipoprotein cholesterol (HDL-C) were significantly decreased, and HOMA-IR, hs-CRP, TG, remnant lipoproteins (RLPs) and small dense-LDL-C (sd LDL-C) were significantly increased in subjects with fatty liver compared to those without fatty liver. Multivariate analyses of serum parameters associated with fatty liver revealed that adiponectin and hs-CRP were more strongly associated with the presence of fatty liver than waist circumference. However, HOMA-IR, HDL-C, TG, RLP-C, RLP-TG and sd LDL-C were more strongly associated with waist circumference than with fatty liver. Factor analysis revealed that adiponectin and HDL-C were linked to liver enzymes, lipoproteins and HOMA-IR associated with fatty liver, but not with waist circumference. Conclusions Adiponectin was found to be a more specific diagnostic marker for the presence of fatty liver regardless of MetS status, and was inversely correlated with liver enzyme concentrations. However, RLPs were found to be more specifically associated with the presence of MetS.

Incremental Prognostic Value of Ventricular-Arterial Coupling over Ejection Fraction in Patients with Maintenance Hemodialysis

Background: Left ventricular ejection fraction (LVEF) is a predictor of adverse outcomes in hemodialysis patients. LVEF is, however, an integral parameter determined by contractility, loading condition, and coupling. We sought to determine whether these components would better predict adverse outcomes and have incremental prognostic value over a validated clinical score and EF. Methods: Two hundred thirty‐four hemodialysis patients were prospectively followed up for primary composite endpoint: all‐cause death, nonfatal myocardial infarction, and hospitalization due to worsening heart failure (HF). Load‐independent contractility (end‐systolic elastance [Ees] and preload recruitable stroke work [PRSW]) and arterial afterload (arterial elastance [Ea]) were noninvasively estimated. Ventricular‐arterial coupling was assessed using the Ea/Ees ratio. LV global longitudinal strain (GLS) and mitral E‐wave over annular velocity E′ ratio (E/E′) were also measured. Results: During a median follow‐up of 776 days, 30 patients developed the primary endpoint. Ees, PRSW, GLS, S′, Ea/Ees, E/E′, and EF were independently associated with the outcome after adjusting for the clinical score and prior HF hospitalization, whereas end‐diastolic volume index or arterial afterload parameters were not. The nested Cox models indicated that Ea/Ees had independent and incremental predictive value over the model based on the score and either EF or E/E′. Furthermore, Ea/Ees continued to have predictive value after adjusting for GLS. The classification and regression analysis stratified event rates ranging from 4.2% to 68.8%. Conclusions: LV contractility and Ea/Ees were independently associated with adverse outcome in hemodialysis patients. Ea/Ees had an incremental prognostic value over the clinical score and EF.

Association Between Circulating Ketone Bodies and Worse Outcomes in Hemodialysis Patients

Background Cardiovascular disease is the leading cause of morbidity and mortality in patients receiving hemodialysis. Systemic metabolic perturbation is one of the hallmark abnormalities in patients at high cardiovascular risk. We sought to determine the relationship between circulating ketone body and clinical outcomes in patients with prevalent hemodialysis. Methods and Results We retrospectively assessed the relationship between serum β‐hydroxybutyrate (βOHB), the most abundant ketone body in the circulation, and prognosis in 405 stable hemodialysis patients. During a mean follow‐up of 3.2±0.9 years, there were 54 major adverse cardiovascular events (defined as cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, and hospitalization attributed to heart failure) and 67 all‐cause deaths. Major adverse cardiovascular events rates increased from 11.1 per 1000 person‐years in the lowest βOHB quintile (<89 μmol/L) to 80.1 per 1000 person‐years in the highest quintile (>409 μmol/L). After adjusting for demographic characteristics, coronary artery disease, and atrial fibrillation, the highest βOHB quintile was associated with increased risk of major adverse cardiovascular events compared with the lowest quintile (hazard ratio, 10.2; 95% confidence interval [3.35–44.0]; P<0.001). Increased quintiles of βOHB were independently and incrementally associated with major adverse cardiovascular events over the model based on an established risk score (the second Analyzing Data, Recognizing Excellence and Optimizing Outcomes cohort score) and N‐terminal pro‐B‐type natriuretic peptide (chi square 39.9 versus 21.7; P<0.001; c‐statistics, 0.713). Sensitivity analyses also confirmed the robustness of association between βOHB and all‐cause death. Conclusions Increased serum βOHB levels were independently associated with cardiovascular events and all‐cause death in patients receiving hemodialysis. These results highlight the need for future studies to understand the mechanisms underlying these observations.