Continuous renal replacement therapy with vitamin E-coated polysulfone hemofilter reduces inflammatory responses in a porcine lipopolysaccharide-treated model.

INTRODUCTION: Biological invasions may promote the onset of systemic inflammatory response syndrome in patients eligible for continuous renal replacement therapy (CRRT), leading to poor prognosis. Hence, we aimed to examine the inflammatory reactions in circulation using vitamin E-coated polysulfone hollow fiber membrane (ViLIFE). METHODS: Lipopolysaccharides were intravenously administered to pigs (2 µg/kg/30 min) to establish an acute inflammation model. Extracorporeal circulation was performed for 6 h in continuous venovenous hemodiafiltration mode using a hemofilter for CRRT filled with a polysulfone hollow fiber membrane or ViLIFE, and the differences in inflammatory reactions were evaluated. RESULTS: The ViLIFE group exhibited low platelet and cytokine levels (p < 0.05 vs. sham-CRRT group). Additionally, the ViLIFE group had lower lactate and high mobility group box 1 levels than the other groups. CONCLUSION: ViLIFE represents a promising CRRT modality that can inhibit the inflammatory response in circulation and inhibit further biological invasions.

Constructing a continuous hemodiafiltration-type circulatory model of acute kidney injury in pigs.

INTRODUCTION: Animal-model experimental systems capable of reflecting the effects of devices for continuous renal replacement therapy (CRRT) on living organisms are limited; thus, aimed to construct an animal model of AKI-CRRT using pigs. METHODS: Pigs were subjected to renal artery ischemia-reperfusion injury (IRI) and then to a maximum of 24 h of continuous hemodiafiltration (CHDF)-type CRRT. RESULTS: Post-IRI, pigs' creatinine levels rose threefold, and they exhibited 24 h of anuria and clear aggravation of oxidative stress, demonstrating successful induction of AKI for CRRT. Post-CRRT, no significant changes in their vital signs or hematological parameters were observed. Creatinine and blood urea nitrogen clearance, as well as suppression of increases in oxidative stress, were also confirmed. CONCLUSION: We believe that the use of our model can enable the preclinical evaluation of the effects of under-development CRRT devices on living organisms under conditions similar to those encountered in an actual clinical setting.

Use of 13C-NMR Chemical Shifts; Application of Principal Component Analysis for Categorizing Structurally Similar Methoxyflavones and Correlation Analysis between Chemical Shifts and Cytotoxicity.

The 13C-NMR spectral data for the 15-carbon flavonoid skeleton in eleven methoxyflavones isolated from Kaempferia parviflora (Zingiberaceae) were processed by principal component analysis (PCA). Based on the PCA score plots, the methoxyflavones were categorized into three groups according to their structural features. The cytotoxicities of the methoxyflavones toward 3T3-L1 murine preadipocyte cells were evaluated by 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and found to differ according to structure. The relationship between the 13C-NMR chemical shifts of the methoxyflavones and their cytotoxicities was investigated using Pearson's correlation analysis. The 13C-NMR signal at C-10, a quaternary carbon, was correlated with cytotoxicity. Based on these results, a structural design which lowers the 13C-NMR chemical shift at C-10 would be important for the development of cytotoxic compounds. Although quantitative structure-activity and structure-property relationships are well established paradigms for predicting trends among a series of compounds, quantitative property-activity relationships have been relatively unstudied. This approach offers a new strategy for directing structure-activity relationship research.

First report of pear chlorotic leaf spot-associated virus on Japanese and European pears in Japan and its detection from an eriophyid mite.

In May 2018, three leaf samples were collected from Japanese pear trees cv. "Hosui" that exhibited typical chlorotic spot symptoms (Supplementary Figure S1) in a germplasm nursery in Tsukuba, Ibaraki. Total RNA was prepared using the rapid CTAB method (Gambino et al. 2008) for high-throughput sequencing, as described by Kubota et al. (2020). In brief, after removing ribosomal RNAs, a library was constructed by fragmenting RNA, synthesizing cDNA, and polymerase chain reaction (PCR) amplification. Sequencing was performed using NovaSeq 6000 sequencer (Illumina, San Diego, CA, U.S.A.) with paired-end 150 nt reads. De novo assembly was performed using CLC Genomics Workbench 11.0 Software (Qiagen, Hilden, Germany), with a minimum length of 500 bp. A total of 36,017 contigs derived from 33,565,182 reads were obtained and subjected to BLASTX search against the GenBank sequence database as of January 2019. Viruses commonly found in stone fruits, i.e., apple stem pitting virus, apple green crinkle-associated virus, apricot latent virus (foveaviruses), and apple stem grooving virus (a capillovirus), were detected. In addition, five contigs with amino acid sequence homologies to P1-P4 of known emaraviruses and the P7 of High Plains wheat mosaic virus (Tatineni et al. 2014) were detected and designated as PEV-Jp. The complete nucleotide (nt) sequences of the five segments of PEV-Jp were determined by Sanger sequencing of cloned reverse transcription (RT)-PCR amplification products using the primers shown in Supplementary Table S1; the 5'- and 3'-terminal sequences were RACE verified (Takara Bio, Shiga, Japan). In pairwise comparisons, the complete RNA1 to RNA5 of PEV-Jp (LC554756-760) shared 90.7% to 98.7% nt identities with those of PCLSaV-CG1 (MK602177-181), indicating that PEV-Jp is an isolate of PCLSaV. Using newly designed segment-specific primers (Supplementary Table S1), 12 symptomatic Japanese pear trees cv. "Kosui" sampled in 2020 from the same nursery tested positive for PCLSaV by RT-PCR while 12 symptomless trees were negative for the virus. Similar chlorotic spots, sometimes accompany necrotic spots, were observed on European pear (Pyrus communis) cv. "Le Lectier." (Fig. S1F) in Niigata in 2019; PCLSaV was detected by RT-PCR in leaf tissue samples from symptomatic trees (n = 3/3) but not in symptomless trees (n = 0/2). No vector for PCLSaV has been identified (Liu et al. 2020) but acaricide sprays in the early spring are effective for preventing occurrence of chlorotic spots in pear orchards (Nakai et al. 2018). Since the infestations of Eriophyes chibaensis Kadono, an eriophyid mite often observed on the Japanese pear (Fig. S1G to S1I) (Kadono, 1981), has been associated with occurrences of the chlorotic spots (Shimizu et al. 2019), samples of E. chibaensis individuals were collected from PCLSaV-positive Japanese pear cvs. "Akizuki" and "Kosui"and P. communis cv. "Le Lectier." for total nucleic acid isolations via phenol-chloroform extraction, followed by quantitative RT-PCR (Supplementary Table S1). The expected RNA1 and RNA5 specific 150 bp products were detected from mite samples collected from Akizuki (n = 6/12), Kosui (n = 13/18), and Le Lectier (n = 6/8). The results indicate that E. chibaensis can ingest PCLSaV and may be a potential vector for the virus, although additional experiments are needed to demonstrate its vector competency. To our knowledge, this is the first report of PCLSaV in Japan and the first report of its detection in E. chibaensis.

Impact of altered dietary calcium-phosphorus ratio caused by high-phosphorus diets in a rat chronic kidney disease (CKD) model created by partial ligation of the renal arteries.

This study aimed to establish a rat chronic kidney disease (CKD) model by studying the effects of a high-phosphorus diet in rats that had undergone partial ligation of the renal arteries (RL). Separate groups of 10-week-old male Slc:Sprague-Dawley rats underwent RL and were fed diets with varying phosphorous levels for a period of 48 days. A marked suppression of body weight gain necessitating humane euthanization occurred on day 28 in rats that had undergone RL and were given high-phosphorus feed. By contrast, the group of intact animals on a high-phosphorus feed exhibited a slightly decreased body weight gain from day 21 and survived until scheduled euthanization. In rats with RL, hematological, blood biochemical, and histopathological analyses demonstrated the presence of CKD-like conditions, particularly in the group that were fed a high-phosphorus diet. Hyperphosphatemia and hypocalcemia were induced by a high-phosphorus diet in both the RL and intact groups, both of which had high levels of FGF23 and parathyroid hormone in the blood. Rats with RL on a high-phosphorus diet showed decreased hematopoiesis by the hematopoietic cell area being narrower in the medullary cavity, proliferation of mesenchymal cells and osteoblasts/osteoclasts, and expansion of the osteoid area, a furthermore generalized vascular lesions, such as calcification, were observed. These findings demonstrate that the partial ligation of the renal arteries combined with a calcium-phosphorus imbalance induced by a high-phosphorus diet serves as an animal model for CKD-like conditions accompanied by bone lesions, helping to elucidate this clinical condition and its underlying molecular mechanisms.

Suppression of adipocyte hypertrophy by polymethoxyflavonoids isolated from Kaempferia parviflora.

We previously demonstrated that ethyl acetate extracts of Kaempferia parviflora Wall. Ex Baker (KPE) improve insulin resistance in TSOD mice and showed that its components induce differentiation and adipogenesis in 3T3-L1 preadipocytes. The present study was undertaken to examine whether KPE and its isolated twelve components suppress further lipid accumulation in 3T3-L1 mature adipocytes. KPE reduced intracellular triglycerides in mature adipocytes, as did two of its components, 3,5,7,3',4'-pentamethoxyflavone and 5,7,4'-trimethoxyflavone. Shrinkage of lipid droplets in mature adipocytes was observed, and mRNA expression levels of adipose tissue triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) were up-regulated by these two polymethoxyflavonoids (PMFs). Furthermore, the protein expression level of ATGL and the release level of glycerol into the cell culture medium increased. In contrast, the peroxisome proliferator-activated receptor γ (PPARγ) agonist, troglitazone, did not decrease intracellular triglycerides in mature adipocytes, and the mRNA expression level of PPARγ was not up-regulated in mature adipocytes treated with the two active PMFs. Therefore, suppression of lipid accumulation in mature adipocytes is unlikely to be enhanced by transcriptional activation of PPARγ. These results suggest that KPE and its active components enhance lipolysis in mature adipocytes by activation of ATGL and HSL independent of PPARγ transcription, thus preventing adipocyte hypertrophy. On the other hand, the full hydroxylated flavonoid quercetin did not show the suppressive effects of lipid accumulation in mature adipocyte in the same conditions. Consequently, methoxy groups in the flavones are important for the activity.

Influence of long-term enteral nutrition on pharmacokinetics of digoxin in rats.

This study was designed to clarify the influence of long-term enteral nutrition (EN) on the pharmacokinetics of digoxin. Rats were fed EN diets (semi-digested, digested, and elemental) for 4 weeks, then digoxin (0.05 mg/kg) was administered orally. The AUC(0-∞) and k(a) of digoxin were significantly reduced in the semi-digested diet group versus the control, while the AUC(0-∞) was significantly increased in the digested and elemental diet groups. The mRNA level of Slco1a4 was significantly reduced at the upper small intestine in all EN groups. Further, the expression levels of P-glycoprotein (P-gp) protein and Abcb1a mRNA were increased at the same site in all EN groups, and the increases were significant in the elemental diet group. Cyp3a2 protein and mRNA expressions were significantly reduced in the liver in the digested and elemental diet groups. Abcb1a mRNA was also significantly reduced in the kidney in these groups. These results indicate that the absorption kinetics at the small intestine is influenced by semi-digested diet, and the elimination kinetics in the liver and kidney are influenced by digested and elemental diet. Semi-digested diet also altered digoxin pharmacokinetics in humans. Thus, the effect of long-term EN on digoxin pharmacokinetics depended on the dietary components.

Polymethoxyflavonoids from Kaempferia parviflora induce adipogenesis on 3T3-L1 preadipocytes by regulating transcription factors at an early stage of differentiation.

We previously reported that Kaempferia parviflora WALL. ex BAKER (KP) and its ethyl acetate extract (KPE) improve various metabolic disorders in obesity-model mice. However the mechanism is not certain, and, in this study, in order to elucidate the mechanism of the suppressive effect of KP on fat accumulation, we focused on adipocytes, which are closely linked to metabolic diseases. The finding was that KPE and its components, 3,5,7,4'-tetramethoxyflavone and 3,5,7,3',4'-pentamethoxyflavone, strongly induced differentiation of 3T3-L1 preadipocytes to adipocytes. The above two polymethoxyflavonoids (PMFs) also induced adiponectin mRNA levels, and release of adiponectin into the medium. In addition, these PMFs enhanced the expression of peroxisome proliferator-activated receptor γ (PPARγ), but did not show PPARγ ligand activity. We then investigated the expression of the differentiation-regulator located upstream of PPARγ. Expression of CCAAT/enhancer-binding protein (C/EBP) ß and -δ mRNA, a transcriptional regulator of PPARγ, was induced, and expression of GATA-2 mRNA, a down-regulator of adipogenesis, was suppressed by these PMFs. These functions of the KP PMFs that enhance adipogenesis and secretion of adiponectin are, to some extent at least, involved in the mechanisms of anti-metabolic disorders effects.

Preventive effect of Kaempferia parviflora ethyl acetate extract and its major components polymethoxyflavonoid on metabolic diseases.

Previously, we reported that rhizome powder of Kaempferia parviflora Wall. Ex. Baker prevented obesity and a range of metabolic diseases. In this study, to clarify which molecular mechanisms and active ingredients of K. parviflora have an anti-obesity effect, we investigated the effect of ethyl acetate extract of K. parviflora (KPE) on TSOD mice, a spontaneously obese Type II diabetes model, and on pancreatic lipase. In the TSOD groups, KPE showed a suppressive effect on body weight increase and visceral fat accumulation and also showed preventive effects on symptoms related to insulin resistance, hypertension and fatty liver. In addition, KPE also suppressed body weight increase and food intake in TSNO mice groups, which served as reference animals, at an early stage of administration. Searching for the ingredients in KPE revealed that KPE contains at least 12 kinds of polymethoxyflavonoid (PMF). Furthermore, KPE and its component PMFs showed an inhibitory effect on pancreatic lipase. The above results suggest that KPE has a preventive effect on obesity and various metabolic diseases. The mechanisms of action probably involve inhibition of pancreatic lipase by the PMFs in KPE.