Publications by authors named "Daisuke Mikami"

45 Publications

Fanconi syndrome in an elderly patient with membranous nephropathy during treatment with the immunosuppressant mizoribine.

CEN Case Rep 2022 Jun 24. Epub 2022 Jun 24.

Department of Nephrology, Faculty of Medical Sciences, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Eiheiji-cho, Yoshida-gun, Fukui, 910-1193, Japan.

We report on an 80-year-old man diagnosed with Fanconi syndrome induced by mizoribine after 4 weeks of administration to treat membranous nephropathy. Mizoribine is an oral immunosuppressant that inhibits inosine monophosphate dehydrogenase and is widely used in Japan for the treatment of autoimmune diseases and nephrotic syndrome, as well as after renal transplantation. Acquired Fanconi syndrome is often caused by drugs (antibacterial, antiviral, anticancer, and anticonvulsant drugs) and is sometimes caused by autoimmune diseases, monoclonal light chain-associated diseases, or heavy metal poisoning. In our patient, hypokalemia, hypophosphatemia, glucosuria, hypouricemia, and severe proteinuria resolved gradually after discontinuation of mizoribine administration, despite oral administration of prednisolone followed by a single intravenous injection of rituximab. The patient was ultimately diagnosed with Fanconi syndrome induced by mizoribine based on his clinical course and his typical laboratory data with the absence of proximal tubular acidosis. To our knowledge, this is the first report of Fanconi syndrome possibly induced by mizoribine. Although the precise mechanism by which mizoribine induces proximal tubular dysfunction is unknown, we suggest that nephrologists should be aware of the onset of Fanconi syndrome, a rare complication during mizoribine treatment.
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http://dx.doi.org/10.1007/s13730-022-00715-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9243880PMC
June 2022

Penta-deuterium-labeled 4E, 8Z-sphingadienine for rapid analysis in sphingolipidomics study.

Chem Phys Lipids 2022 07 22;245:105202. Epub 2022 Mar 22.

Graduate School of Life Science, Kita 21 Nishi 11, Sapporo 001-0021, Japan; Faculty of Advanced Life Science, Hokkaido University, Kita 21 Nishi 11, Sapporo 001-0021, Japan. Electronic address:

The use of deuterium-incorporated bioactive compounds is an efficient method for tracing their metabolic fate and for quantitative analysis by mass spectrometry without complicated HPLC separation even if their amounts are extremely small. Plant sphingolipids and their metabolites, which have C4, 8-olefins on a common backbone as a sphingoid base, show unique and fascinating bioactivities compared to those of sphingolipids in mammals. However, the functional and metabolic mechanisms of exogenous plant sphingolipids have not been elucidated due to the difficulty in distinguishing exogenous sphingolipids from endogenous sphingolipids having the same polarity and same molecular weight by mass spectrometric analysis. Their roles might be elucidated by the use of deuterated probes with original biological and physicochemical properties. In this study, we designed (2S,3R,4E,8Z)-2-aminooctadeca-4,8-diene-17,17,18,18,18-d-1,3-diol (penta-deuterium-labeled 4E, 8Z-sphingadienine) as a tracer for exogenous metabolic studies. In addition, the sphingadienine was confirmed to be metabolized in HEK293 cells and showed distinct peaks in mass spectrometric analysis.
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http://dx.doi.org/10.1016/j.chemphyslip.2022.105202DOI Listing
July 2022

Fenofibrate reduces cisplatin-induced apoptosis by inhibiting the p53/Puma/Caspase-9 pathway and the MAPK/Caspase-8 pathway rather than by promoting autophagy in murine renal proximal tubular cells.

Biochem Biophys Rep 2022 Jul 28;30:101237. Epub 2022 Feb 28.

Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

The main lesion of cisplatin nephrotoxicity is damage to proximal tubular cells due to increased apoptosis via the mitochondrial and death receptor pathways, which may be alleviated by appropriate promotion of autophagy. Fenofibrate, a peroxisome proliferator-activated receptor-alpha (PPAR-α) activator, is recently reported to promote autophagy as well as protect against cisplatin nephrotoxicity, although the mechanisms were only partially analyzed. Here, the detailed mechanisms of these putative protective effects were investigated in a murine renal proximal tubular (mProx) cell line. Fenofibrate attenuated cisplatin-induced apoptosis of mProx cells based on flow cytometry. As for the mitochondrial apoptotic pathway, the reagent reduced cisplatin-stimulated caspase-3 activation by decreasing the phosphorylation of p53, JNK, and 14-3-3, cytosolic and mitochondrial Puma accumulation, cytochrome C release to the cytosol, and resulting cytosolic caspase-9 activation. Fenofibrate also decreased cisplatin-stimulated activation of caspases-8 by suppressing MAPK and NFkB pathways and reducing the gene expression of TNF-α, TL1A, and Fas, main mediators of the death receptor apoptotic pathway. Autophagy defined by p62 reduction and an increase in LC3 II/I was promoted by fenofibrate in mProx cells under starvation. Autophagy inhibition using 3-MA further increased basal and cisplatin-induced caspase-3 and -8 activation, but had no influence on the inhibitory effects of fenofibrate on caspase activation. In conclusion, our study suggests fenofibrate to be a candidate agent to mitigate cisplatin nephrotoxicity by inhibiting the mitochondrial and death apoptotic pathways rather than by promoting autophagy.
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http://dx.doi.org/10.1016/j.bbrep.2022.101237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8889369PMC
July 2022

Konjac Ceramide (kCer)-Mediated Signal Transduction of the Sema3A Pathway Promotes HaCaT Keratinocyte Differentiation.

Biology (Basel) 2022 Jan 12;11(1). Epub 2022 Jan 12.

Lipid Biofunction Section, Frontier Research Center for Advanced Material and Life Science, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.

Histamines suppress epidermal keratinocyte differentiation. Previously, we reported that konjac ceramide (kCer) suppresses histamine-stimulated cell migration of HaCaT keratinocytes. kCer specifically binds to Nrp1 and does not interact with histamine receptors. The signaling mechanism of kCer in HaCaT cells is also controlled by an intracellular signaling cascade activated by the Sema3A-Nrp1 pathway. In the present study, we demonstrated that kCer treatment induced HaCaT keratinocyte differentiation after migration of immature cells. kCer-induced HaCaT cell differentiation was accompanied by some features of keratinocyte differentiation markers. kCer induced activating phosphorylation of p38MAPK and c-Fos, which increased the protein levels of involucrin that was the latter differentiation marker. In addition, we demonstrated that the effects of both kCer and histamines are regulated by an intracellular mechanism of Rac1 activation/RhoA inhibition downstream of the Sema3A/Nrp1 receptor and histamine/GPCR pathways. In summary, the effects of kCer on cell migration and cell differentiation are regulated by cascade crosstalk between downstream Nrp1 and histamine-GPCR pathways in HaCaT cells.
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http://dx.doi.org/10.3390/biology11010121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8772740PMC
January 2022

Profiles of 5α-Reduced Androgens in Humans and Eels: 5α-Dihydrotestosterone and 11-Ketodihydrotestosterone Are Active Androgens Produced in Eel Gonads.

Front Endocrinol (Lausanne) 2021 23;12:657360. Epub 2021 Mar 23.

Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan.

Although 11-ketotestosterone (11KT) and testosterone (T) are major androgens in both teleosts and humans, their 5α-reduced derivatives produced by steroid 5α-reductase (SRD5A/srd5a), i.e., 11-ketodihydrotestosterone (11KDHT) and 5α-dihydrotestosterone (DHT), remains poorly characterized, especially in teleosts. In this study, we compared the presence and production of DHT and 11KDHT in Japanese eels and humans. Plasma 11KT concentrations were similar in both male and female eels, whereas T levels were much higher in females. In accordance with the levels of their precursors, 11KDHT levels did not show sexual dimorphism, whereas DHT levels were much higher in females. It is noteworthy that plasma DHT levels in female eels were higher than those in men. In addition, plasma 11KDHT was undetectable in both sexes in humans, despite the presence of 11KT. Three srd5a genes (, and ) were cloned from eel gonads. All three genes were expressed in the ovary, whereas only both srd5a2 genes were expressed in the testis. Human was expressed in testis, ovary and adrenal, whereas was expressed only in testis. Human SRD5A1, SRD5A2 and both eel srd5a2 isoforms catalyzed the conversion of T and 11KT into DHT and 11KDHT, respectively, whereas only eel srd5a1 converted T into DHT. DHT and 11KDHT activated eel androgen receptor (ar)α-mediated transactivation as similar fashion to T and 11KT. In contrast, human AR and eel arβ were activated by DHT and11KDHT more strongly than T and 11KT. These results indicate that in teleosts, DHT and 11KDHT may be important 5α-reduced androgens produced in the gonads. In contrast, DHT is the only major 5α-reduced androgens in healthy humans.
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http://dx.doi.org/10.3389/fendo.2021.657360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021924PMC
December 2021

Structure-dependent absorption of atypical sphingoid long-chain bases from digestive tract into lymph.

Lipids Health Dis 2021 Mar 1;20(1):24. Epub 2021 Mar 1.

Laboratory of Biomembrane and Biofunctional Chemistry, Graduate School of Advanced Life Science, and Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Kita-21 Nishi-11, Kita-ku, Sapporo, 001-0021, Japan.

Background: Dietary sphingolipids have various biofunctions, including skin barrier improvement and anti-inflammatory and anti-carcinoma properties. Long-chain bases (LCBs), the essential backbones of sphingolipids, are expected to be important for these bioactivities, and they vary structurally between species. Given these findings, however, the absorption dynamics of each LCB remain unclear.

Methods: In this study, five structurally different LCBs were prepared from glucosylceramides (GlcCers) with LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH moieties derived from konjac tuber (Amorphophallus konjac), from GlcCers with an LCB 18(9Me):2(4E,8E);2OH moiety derived from Tamogi mushroom (Pleurotus cornucopiae var. citrinopileatus), and from ceramide 2-aminoethyphosphonate with LCB 18:3(4E,8E,10E);2OH moiety and LCB 18(9Me):3(4E,8E,10E);2OH moiety derived from giant scallop (Mizuhopecten yessoensis), and their absorption percentages and metabolite levels were analyzed using a lymph-duct-cannulated rat model via liquid chromatography tandem mass spectrometry (LC/MS/MS) with a multistage fragmentation method.

Results: The five orally administered LCBs were absorbed and detected in chyle (lipid-containing lymph) as LCBs and several metabolites including ceramides, hexosylceramides, and sphingomyelins. The absorption percentages of LCBs were 0.10-1.17%, depending on their structure. The absorption percentage of LCB 18:2(4E,8Z);2OH was the highest (1.17%), whereas that of LCB 18:3(4E,8E,10E);2OH was the lowest (0.10%). The amount of sphingomyelin with an LCB 18:2(4E,8Z);2OH moiety in chyle was particularly higher than sphingomyelins with other LCB moieties.

Conclusions: Structural differences among LCBs, particularly geometric isomerism at the C8-C9 position, significantly affected the absorption percentages and ratio of metabolites. This is the first report to elucidate that the absorption and metabolism of sphingolipids are dependent on their LCB structure. These results could be used to develop functional foods that are more readily absorbed.
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http://dx.doi.org/10.1186/s12944-021-01448-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919070PMC
March 2021

PPAR-δ activation reduces cisplatin-induced apoptosis via inhibiting p53/Bax/caspase-3 pathway without modulating autophagy in murine renal proximal tubular cells.

Clin Exp Nephrol 2021 Jun 1;25(6):598-607. Epub 2021 Mar 1.

Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

Background: Cisplatin-induced injury of renal proximal tubular cells results basically from increased apoptosis via mitochondrial damage, and is mitigated by appropriate enhancement of autophagy. Peroxisome proliferator-activated receptor-delta (PPAR-δ) reportedly protects against not only mitochondrial damages but also enhances autophagy. Thus, PPAR-δ may protect against cisplatin-induced kidney injury.

Methods: We examined the protective effects of PPAR-δ activation on cisplatin-induced cellular injury and their detailed mechanisms in a murine renal proximal tubular (mProx) cell line using GW0742, an authentic PPAR-δ activator. Cisplatin-induced cell damages were evaluated by TUNEL assay and immunoblot analyses for p53, 14-3-3, Bax, Bcl2, cytochrome C, and activated caspases. Autophagy status was examined by immunoblot analyses for p62 and LC3.

Results: GW0742 suppressed cisplatin-induced apoptosis of mProx cells by reducing the activation of caspase-3 via attenuating the phosphorylation of p53 and 14-3-3, mitochondrial Bax accumulation, cytochrome C release from mitochondria to the cytosol and ensuing cytosolic caspase-9 activation. In contrast, GW0742 did not diminish cisplatin-enhanced activation of caspases-8 or -12 as extrinsic or endothelium reticulum apoptotic pathways, respectively. The inhibitory effect of GW0742 on cisplatin-induced caspase-3 activation was significantly diminished by silencing of the PPAR-δ gene expression. GW0742 itself had no influence on starvation-stimulated or cisplatin-induced autophagy in mProx cells, suggesting that the protective effects were not mediated by autophagy modification.

Conclusion: Our results indicate that GW0742 may serve as a candidate agent to mitigate cisplatin nephrotoxicity via inhibiting the mitochondrial apoptotic pathway considerably depending on PPAR-δ, without modulating autophagy.
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http://dx.doi.org/10.1007/s10157-021-02039-2DOI Listing
June 2021

11-Ketotestosterone is a major androgen produced in porcine adrenal glands and testes.

J Steroid Biochem Mol Biol 2021 06 17;210:105847. Epub 2021 Feb 17.

Center for Animal Disease Control, University of Miyazaki, Miyazaki 889-1692, Japan.

Porcine steroid hormone profiles have some unique characteristics. We previously studied human and murine steroidogenesis using steroidogenic cells-derived from mesenchymal stem cells (MSCs). To investigate porcine steroidogenesis, we induced steroidogenic cells from porcine subcutaneous preadipocytes (PSPA cells), which originate from MSCs. Using cAMP, adenovirus-mediated introduction of steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP) induced the differentiation of PSPA cells into sex steroid-producing cells. Introducing SF-1/Ad4BP also induced the aldo-keto reductase 1C1 (AKR1C1) gene. Porcine AKR1C1 had 17β-hydroxysteroid dehydrogenase activity, which converts androstenedione and 11-ketoandrostenedione into testosterone (T) and 11-ketotestosteorne (11KT). Furthermore, differentiated cells expressed hydroxysteroid 11β-dehydrogenase 2 (HSD11B2) and produced 11KT. HSD11B2 was expressed in testicular Leydig cells and the adrenal cortex. 11KT was present in the plasma of both immature male and female pigs, with slightly higher levels in the male pigs. T levels were much higher in the male pigs. It is noteworthy that in the female pigs, the 11KT levels were >10-fold higher than the T levels. However, castration altered the 11KT and T plasma profiles in the male pigs to near those of the females. 11KT induced endothelial nitric oxide synthase (eNOS) in porcine vascular endothelial cells. These results indicate that 11KT is produced in porcine adrenal glands and testes, and may regulate cardiovascular functions through eNOS expression.
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http://dx.doi.org/10.1016/j.jsbmb.2021.105847DOI Listing
June 2021

Mevalonate Pathway-mediated ER Homeostasis Is Required for Haploid Stability in Human Somatic Cells.

Cell Struct Funct 2021 Feb 22;46(1):1-9. Epub 2020 Dec 22.

Graduate School of Life Science, Hokkaido University.

The somatic haploidy is unstable in diplontic animals, but cellular processes determining haploid stability remain elusive. Here, we found that inhibition of mevalonate pathway by pitavastatin, a widely used cholesterol-lowering drug, drastically destabilized the haploid state in HAP1 cells. Interestingly, cholesterol supplementation did not restore haploid stability in pitavastatin-treated cells, and cholesterol inhibitor U18666A did not phenocopy haploid destabilization. These results ruled out the involvement of cholesterol in haploid stability. Besides cholesterol perturbation, pitavastatin induced endoplasmic reticulum (ER) stress, the suppression of which by a chemical chaperon significantly restored haploid stability in pitavastatin-treated cells. Our data demonstrate the involvement of the mevalonate pathway in the stability of the haploid state in human somatic cells through managing ER stress, highlighting a novel link between ploidy and ER homeostatic control.Key words: haploid, ER stress, Mevalonate pathway.
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http://dx.doi.org/10.1247/csf.20055DOI Listing
February 2021

Blood-brain barrier permeability analysis of plant ceramides.

PLoS One 2020 2;15(11):e0241640. Epub 2020 Nov 2.

Lipid Biofunction Section, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Hokkaido, Japan.

Ceramides, a type of sphingolipid, are cell membrane components and lipid mediators that modulate a variety of cell functions. In plants, ceramides are mostly present in a glucosylated glucosylceramide (GlcCer) form. We previously showed that oral administration of konjac-derived GlcCer to a mouse model of Alzheimer's disease reduced brain amyloid-β and amyloid plaques. Dietary plant GlcCer compounds are absorbed as ceramides, but it is unclear whether they can cross the blood-brain barrier (BBB). Herein, we evaluated the BBB permeability of synthetic plant-type ceramides (4, 8-sphingadienine, d18:2) using mouse and BBB cell culture models, and found that they could permeate the BBB both in vivo and in vitro. In addition, administrated ceramides were partially metabolized to other sphingolipid species, namely sphingomyelin (SM) and GlcCer, while crossing the BBB. Thus, plant ceramides can cross the BBB, suggesting that ceramides and their metabolites might affect brain functions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241640PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605672PMC
December 2020

PNU-120596, a positive allosteric modulator of α7 nicotinic acetylcholine receptor, directly inhibits p38 MAPK.

Biochem Pharmacol 2020 12 22;182:114297. Epub 2020 Oct 22.

Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical University, Asahikawa 078-8510, Japan.

PNU-120596 is a classical positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (α7 nAChR) and widely used to investigate the effect of α7 nAChR activation on several inflammation-associated diseases including rheumatoid arthritis, inflammatory bowel disease and cerebral ischemia. In this study, we report that PNU-120596 directly inhibits p38 mitogen-activated protein kinase (MAPK) activity. In 293A cells, p38 MAPK phosphorylation by several factors (oxidative stress, osmotic stress, TNF-α, or muscarinic stimulation) was inhibited by PNU-120596 as well as p38 MAPK inhibitor BIRB-796. Inhibition of p38 MAPK phosphorylation by PNU-120596 was not affected by α7 nAChR antagonist, methyllycaconitine (MLA). In vitro kinase assay revealed that PNU-120596 directly inhibits p38α MAPK-induced activating transcription factor 2 (ATF2) phosphorylation. MKK6-induced phosphorylation of p38α MAPK was also inhibited by PNU-120596. Real-time monitoring of binding to p38α MAPK using fluoroprobe SKF-86002 showed quite rapid binding of PNU-120596 compared to BIRB-796 which is known as a slow binder. Finally, we showed that PNU-120596 suppressed LPS-induced phosphorylation of p38 MAPK and expression of inflammatory factors including TNF-α, IL-6 and COX-2, independent on α7 nAChR activity in microglial cell BV-2. Thus, PNU-120596 might exert an anti-inflammatory effect through not only α7 nAChR potentiation but also direct inhibition of p38 MAPK.
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http://dx.doi.org/10.1016/j.bcp.2020.114297DOI Listing
December 2020

Lysosomal-associated transmembrane protein 4B regulates ceramide-induced exosome release.

FASEB J 2020 12 8;34(12):16022-16033. Epub 2020 Oct 8.

Lipid Biofunction Section, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.

Exosomes are extracellular vesicles that mediate the transport of intracellular molecules, including neurodegenerative agents. Exogenously administrated ceramides have been implicated in the acceleration of exosome production by neurons; however, the molecular machinery involved in this process is unknown. Here, we found that ceramides, especially those consisting of long fatty acids, were internalized into the endocytic pathway in neuroblastoma SH-SY5Y cells to induce exosome secretion through lysosome-associated protein transmembrane 4B (LAPTM4B). Knockdown of LAPTM4B inhibited the ceramide-mediated increase in exosome release completely. Fluorescence microscopy observations indicated that exogenous ceramides promote the transport of multivesicular bodies to the plasma membranes in a LAPTM4B-dependent manner. Similarly, inhibition of acid ceramidase, which tends to induce intracellular ceramide accumulation, increased exosome production by SH-SY5Y cells in a LAPTM4B-dependent manner. Furthermore, the level of amyloid-ß protein (Aß) was decreased in neuronal cells following treatment with exogenous ceramide or inhibition of acid ceramidase, and this effect was attributed to the LAPTM4B-dependent efflux of Aß-containing exosomes. Overall, these findings reveal the novel machinery involved in exosome secretion regulated by ceramides and LAPTM4B, and may contribute to efforts to ameliorate the cellular accumulation of neurodegenerative agents such as Aß.
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http://dx.doi.org/10.1096/fj.202001599RDOI Listing
December 2020

AR420626, a selective agonist of GPR41/FFA3, suppresses growth of hepatocellular carcinoma cells by inducing apoptosis HDAC inhibition.

Ther Adv Med Oncol 2020 20;12:1758835920913432. Epub 2020 Mar 20.

Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

Background: Hepatocellular carcinoma (HCC) is a major cause of cancer death worldwide and establishment of new chemotherapies for HCC is urgently needed. GPR41 [free fatty acid receptor 3 (FFA3)] is a G protein-coupled receptor for short chain fatty acids, including acetate, propionate, and butyrate. In our previous study, we showed that propionate enhances the cytotoxic effect of cisplatin in HCC cells and that this mechanism is dependent on inhibition of histone deacetylases (HDACs) GPR41/FFA3. However, the antitumor action of GPR41/FFA3 has not been elucidated.

Methods: In this study, we examined AR420626 as a GPR41-selective agonist in HepG2 and HLE cells. Nude mice were used for HepG2 xenograft studies. The apoptotic effect of AR420626 was evaluated using flow cytometry analysis. Expression of apoptosis-related proteins and HDACs was evaluated by Western immunoblot. Gene silencing of HDAC 3/5/7 and GPR41 was performed using small interfering RNA. Expression of TNF-α mRNA was evaluated by TaqMan real-time polymerase chain reaction.

Results: We found that AR420626, a selective GPR41/FFA3 agonist, suppressed growth of HepG2 xenografts and inhibited proliferation of HCC cells by inducing apoptosis. AR420626 induced proteasome activation through mTOR phosphorylation, which reduced HDAC proteins, and then increased expression of TNF-α.

Conclusion: AR420626, a selective GPR41/FFA3 agonist, may be a candidate as a therapeutic agent for HCC.
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http://dx.doi.org/10.1177/1758835920913432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7517987PMC
March 2020

Preemptive HLA Antibody Screening Prior to Episodic Transplant Renal Biopsy Enables Early Diagnosis and Therapeutic Response in Asymptomatic Chronically Active Antibody-Related Rejection: A Case Report.

Transplant Proc 2020 Nov 18;52(9):2750-2753. Epub 2020 Sep 18.

Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

Common management of renal transplant recipients includes episodic renal biopsy based on clinical findings such as an increase in proteinuria or serum creatinine. When antibody-related rejection is suspected from the renal biopsy, subsequent testing for donor-specific antibodies (DSAs) is performed. We instead performed preemptive screening of asymptomatic post-renal transplant recipients for DSAs prior to renal biopsy. In this case, a 30-year-old woman with a secondary transplant was positive for 61 anti-HLA antibodies of class I and class II, among which DQ2 was a DSA with a mean fluorescence index of 2039. The patient had a living kidney transplant 9 years earlier. She had never been diagnosed with rejection, her serum creatinine was around 1.0 mg/dL, and her proteinuria was negative. Following the positive DSA result, a renal biopsy was performed, and she was diagnosed as C4d-negative chronic-active antibody-mediated rejection (CAABMR) with a Banff score of cg1b, (g + ptc) ≥ 2, and C4d 0. Intravenous steroid pulse, deoxyspagarin, antithymocyte globulin, rituximab, and oral everolimus were administrated. The treatment resulted in a gradual decrease in the DSA, which became negative 1 year later. The patient's serum creatinine remains around 1.0 mg/dL, and proteinuria remains negative. Treatments for advanced CAABMR are often expensive and ineffective. Our present case suggests that early detection and treatment through preemptive HLA antibody screening could improve the prognosis of renal transplants.
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http://dx.doi.org/10.1016/j.transproceed.2020.08.034DOI Listing
November 2020

Chronic hypoxia exacerbates diabetic glomerulosclerosis through mesangiolysis and podocyte injury in db/db mice.

Nephrol Dial Transplant 2020 10;35(10):1678-1688

Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

Background: Chronic hypoxia may play a pivotal role in the development of diabetic nephropathy (DN). However, the precise mechanisms underlying progressive hypoxia-induced glomerular injury remain unclear.

Methods: We housed db/db mice in a hypoxia chamber (12% O2) for up to 16 weeks beginning at 8 weeks of age. Various urine, serum and kidney abnormalities and glomerular messenger RNA (mRNA) expression were compared with those in age-matched db/db mice housed under normoxia.

Results: Levels of urinary albumin and podocalyxin (PCX) were significantly higher in hypoxic mice early during hypoxia. Ultracentrifugation of urine samples revealed that podocytes in the hypoxic mice shed PCX-positive microparticles into the urine. After 16 weeks of hypoxia, the mice also had higher hematocrits with lower serum glucose and various degrees of mesangiolytic glomerulosclerosis with microaneurysms and the infrequent occurrence of nodular lesions. Immunohistologically, hypoxic mice showed significantly decreased endothelial cell densities early during hypoxia and decreased podocyte densities later. In both hypoxic and normoxic mice, glomerular macrophage and transforming growth factor-β1 (TGF-β1) staining significantly increased with aging, without changes in vascular endothelial growth factor or endothelial nitric oxide synthase (eNOS). Glomerular mRNA expression of monocyte chemoattractant protein-1, eNOS and TGF-β1 was significantly enhanced in the hypoxic mice.

Conclusions: These results indicate that chronic hypoxia induces advanced glomerulosclerosis with accelerated albuminuria triggered by mesangiolysis and podocyte injury in a murine model of DN.
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http://dx.doi.org/10.1093/ndt/gfaa074DOI Listing
October 2020

Analytical and clinical validation of rapid chemiluminescence enzyme immunoassay for urinary thioredoxin, an oxidative stress-dependent early biomarker of acute kidney injury.

Clin Chim Acta 2020 Aug 26;507:271-279. Epub 2020 Apr 26.

Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

Background: Oxidative stress is now recognized to be an important therapeutic target in kidney diseases. However, there are currently no biomarkers that can be used clinically to diagnose renal oxidative stress.

Methods: A rapid assay system for urinary thioredoxin 1, an oxidative stress-dependent biomarker of acute kidney injury (AKI), was developed as a chemiluminescence enzyme immunoassay and validated analytically and clinically.

Results: Analytic evaluation revealed that hemolytic hemoglobin caused measurements to be abnormally high, above the detectable range. However, urine sediment containing red blood cells did not affect the measurements. Assays using our proposed chemiluminescence enzyme immunoassay were completed within as little as 6 min, whereas a conventional ELISA > 4 h. Aciduria
Conclusions: These results suggest that the chemiluminescent enzyme immunoassay system for urinary thioredoxin 1 enables rapid and specific diagnosis of AKI associated with oxidative stress.
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http://dx.doi.org/10.1016/j.cca.2020.04.025DOI Listing
August 2020

Nrp1 is Activated by Konjac Ceramide Binding-Induced Structural Rigidification of the a1a2 Domain.

Cells 2020 02 24;9(2). Epub 2020 Feb 24.

Lipid Biofunction Section, Faculty of Advanced Life Science, Hokkaido University, 001-0021 Sapporo, Hokkaido, Japan.

Konjac ceramide (kCer) is a plant-type ceramide composed of various long-chain bases and a-hydroxyl fatty acids. The presence of d4t,8t-sphingadienine is essential for semaphorin 3A (Sema3A)-like activity. Herein, we examined the three neuropilin 1 (Nrp1) domains (a1a2, b1b2, or c), and found that a1a2 binds to d4t,8t-kCer and possesses Sema3A-like activity. kCer binds to Nrp1 with a weak affinity of mM dissociation constant (Kd). We wondered whether bovine serum albumin could influence the ligand-receptor interaction that a1a2 has with a single high affinity binding site for kCer (Kd in nM range). In the present study we demonstrated the influence of bovine serum albumin. Thermal denaturation indicates that the a1a2 domain may include intrinsically disordered region (IDR)-like flexibility. A potential interaction site on the a1 module was explored by molecular docking, which revealed a possible Nrp1 activation mechanism, in which kCer binds to Site A close to the Sema3A-binding region of the a1a2 domain. The a1 module then accesses a2 as the IDR-like flexibility becomes ordered via kCer-induced protein rigidity of a1a2. This induces intramolecular interaction between a1 and a2 through a slight change in protein secondary structure.
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http://dx.doi.org/10.3390/cells9020517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072815PMC
February 2020

Short-chain fatty acid mitigates adenine-induced chronic kidney disease via FFA2 and FFA3 pathways.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 06 13;1865(6):158666. Epub 2020 Feb 13.

Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

Short-chain fatty acids (SCFAs), including acetate, butyrate, and propionate, are produced when colonic bacteria in the human gastrointestinal tract ferment undigested fibers. Free fatty acid receptor 2 (FFA2) and FFA3 are G-protein-coupled receptors recently identified as SCFA receptors that may modulate inflammation. We previously showed through in vitro experiments that SCFAs activate FFA2 and FFA3, thereby mitigating inflammation in human renal cortical epithelial cells. This study used a murine model of adenine-induced renal failure to investigate whether or not SCFAs can prevent the progression of renal damage. We also examined whether or not these FFA2 and FFA3 proteins have some roles in this protective mechanism in vivo. Immunohistochemical analyses of mouse kidneys showed that FFA2 and FFA3 proteins were expressed mainly in the distal renal tubules and collecting tubules. First, we observed that the administration of propionate mitigated the renal dysfunction and pathological deterioration caused by adenine. Consistent with this, the expression of inflammatory cytokines and fibrosis-related genes was reduced. Furthermore, the mitigation of adenine-induced renal damage by the administration of propionate was significantly attenuated in FFA2 and FFA3 mice. Therefore, the administration of propionate significantly protects against adenine-induced renal failure, at least in part, via the FFA2 and FFA3 pathways. Our data suggest that FFA2 and FFA3 are potential new therapeutic targets for preventing or delaying the progression of chronic kidney disease.
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http://dx.doi.org/10.1016/j.bbalip.2020.158666DOI Listing
June 2020

β-Hydroxybutyrate enhances the cytotoxic effect of cisplatin via the inhibition of HDAC/survivin axis in human hepatocellular carcinoma cells.

J Pharmacol Sci 2020 Jan 5;142(1):1-8. Epub 2019 Nov 5.

Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

Ketone bodies, including acetoacetate and β-hydroxybutyrate (βOHB), are produced from acetyl coenzyme A in the liver and then secreted into the blood. These molecules are a source of energy for peripheral tissues during exercise or fasting. βOHB has been reported to inhibit histone deacetylases (HDACs) 1, 3, and 4 in human embryonic kidney 293 cells. Thus, βOHB may regulate epigenetics by modulating HDACs. There have been several reports that the administration of βOHB or induction of a physiological state of ketosis has an antitumor effect; however, the mechanism remains unclear. The aim of this study was to investigate whether βOHB enhances cisplatin-induced apoptosis in hepatocellular carcinoma (HCC) cells by modulating activity and/or expression of HDACs. We found that βOHB significantly enhanced cisplatin-induced apoptosis and cleavage of caspase-3 and -8 in HCC cells. Further, βOHB significantly decreased the expression of HDCA 3/5/6 and survivin in liver hepatocellular (HepG2) cells. In HDAC3/6 gene silencing, survivin expression was significantly decreased, and cisplatin-induced cleavage of caspase-3 was significantly enhanced compared with control in HepG2 cells. In conclusion, βOHB enhanced cisplatin-induced apoptosis via HDAC3/6 inhibition/survivin axis in HepG2 cells, which suggests that βOHB could be a new adjuvant agent for cisplatin chemotherapy.
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http://dx.doi.org/10.1016/j.jphs.2019.10.007DOI Listing
January 2020

Plant sphingolipids promote extracellular vesicle release and alleviate amyloid-β pathologies in a mouse model of Alzheimer's disease.

Sci Rep 2019 11 14;9(1):16827. Epub 2019 Nov 14.

Lipid Biofunction Section, Faculty of Advanced Life Science, Hokkaido University, Kita-21, Nishi-11, Kita-ku, Sapporo, 001-0021, Japan.

The accumulation of amyloid-β protein (Aβ) in brain is linked to the early pathogenesis of Alzheimer's disease (AD). We previously reported that neuron-derived exosomes promote Aβ clearance in the brains of amyloid precursor protein transgenic mice and that exosome production is modulated by ceramide metabolism. Here, we demonstrate that plant ceramides derived from Amorphophallus konjac, as well as animal-derived ceramides, enhanced production of extracellular vesicles (EVs) in neuronal cultures. Oral administration of plant glucosylceramide (GlcCer) to APP overexpressing mice markedly reduced Aβ levels and plaque burdens and improved cognition in a Y-maze learning task. Moreover, there were substantial increases in the neuronal marker NCAM-1, L1CAM, and Aβ in EVs isolated from serum and brain tissues of the GlcCer-treated AD model mice. Our data showing that plant ceramides prevent Aβ accumulation by promoting EVs-dependent Aβ clearance in vitro and in vivo provide evidence for a protective role of plant ceramides in AD. Plant ceramides might thus be used as functional food materials to ameliorate AD pathology.
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http://dx.doi.org/10.1038/s41598-019-53394-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856149PMC
November 2019

Evaluation of 17β-hydroxysteroid dehydrogenase activity using androgen receptor-mediated transactivation.

J Steroid Biochem Mol Biol 2020 02 12;196:105493. Epub 2019 Oct 12.

Department of Biochemistry, Asahikawa Medical University, Hokkaido 078-8510, Japan.

17β-Hydroxysteroid dehydrogenases (17β-HSDs) catalyze the reduction of 17-ketosteroids and the oxidation of 17β-hydroxysteroids to regulate the production of androgens and estrogens. Among them, 17β-HSD type 3 (HSD17B3) is expressed almost exclusively in testicular Leydig cells and contributes to development of male sexual characteristics by converting androstenedione (A4) to testosterone (T). Mutations of HSD17B3 genes cause a 46,XY disorder of sexual development (46,XY DSD) as a result of low T production. Therefore, the evaluation of 17β-HSD3 enzymatic activity is important for understanding and diagnosing this disorder. We adapted a method that easily evaluates enzymatic activity of 17β-HSD3 by quantifying the conversion from A4 to T using androgen receptor (AR)-mediated transactivation. HEK293 cells were transduced to express human HSD17B3, and incubated medium containing A4. Depending on the incubation time with HSD17B3-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the AR expression vector and androgen-responsive reporter. Culture medium from HSD17B1 and HSD17B5-expressing cells also increased the luciferase activities. This system is also applicable to detect the conversion of 11-ketoandrostenedione to 11-ketotestosterone by HSD17B3. Establishment of HEK293 cells expressing various missense mutations in the HSD17B3 gene associated with 46,XY DSD revealed that this system is effective to evaluate the enzymatic activities of mutant proteins.
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http://dx.doi.org/10.1016/j.jsbmb.2019.105493DOI Listing
February 2020

Malabaricone C as Natural Sphingomyelin Synthase Inhibitor against Diet-Induced Obesity and Its Lipid Metabolism in Mice.

ACS Med Chem Lett 2019 Aug 3;10(8):1154-1158. Epub 2019 Jul 3.

Faculty of Advanced Life Science, Hokkaido University, Kita 21 Nishi 11, Sapporo 001-0021, Japan.

The interaction between natural occurring inhibitors and targeted membrane proteins could be an alternative medicinal strategy for the treatment of metabolic syndrome, notably, obesity. In this study, we identified malabaricones A-C and E (-) isolated from the fruits of King as natural inhibitors for sphingomyelin synthase (SMS), a membrane protein responsible for sphingolipid biosynthesis. Having the most promising inhibition, oral administration of compound exhibited multiple efficacies in reducing weight gain, improving glucose tolerance, and reducing hepatic steatosis in high fat diet-induced obesity mice models. Liver lipid analysis revealed a crucial link between the SMS activities of compound and its lipid metabolism and . The nontoxic nature of compound makes it a suitable candidate in search of drugs which can be employed in the treatment and prevention of obesity.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691482PMC
August 2019

Store-operated calcium entry (SOCE) contributes to phosphorylation of p38 MAPK and suppression of TNF-α signalling in the intestinal epithelial cells.

Cell Signal 2019 11 8;63:109358. Epub 2019 Jul 8.

Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical University, Asahikawa 078-8510, Japan.

Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gα protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca-dependent early phase p38 MAPK phosphorylation and extracellular Ca-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.
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http://dx.doi.org/10.1016/j.cellsig.2019.109358DOI Listing
November 2019

Cyclooxygenase-2 is acutely induced by CCAAT/enhancer-binding protein β to produce prostaglandin E and F following gonadotropin stimulation in Leydig cells.

Mol Reprod Dev 2019 07 13;86(7):786-797. Epub 2019 May 13.

Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan.

Cyclooxygenase 2 (COX-2) is an inducible rate-limiting enzyme for prostanoid production. Because COX-2 represents one of the inducible genes in mouse mesenchymal stem cells upon differentiation into Leydig cells, we investigated COX-2 expression and production of prostaglandin (PG) in Leydig cells. Although COX-2 was undetectable in mouse testis, it was transiently induced in Leydig cells by human chorionic gonadotropin (hCG) administration. Consistent with the finding that Leydig cells expressed aldo-keto reductase 1B7 (PGF synthase) and PGE synthase 2, induction of COX-2 by hCG caused a marked increase in testicular PGF and PGE levels. Using mouse Leydig cell tumor-derived MA-10 cells as a model, it was indicated by reporter assays and electron mobility shift assays that transcription of the COX-2 gene was activated by CCAAT/enhancer-binding protein β (C/EBPβ) with cAMP-stimulation. C/EBPβ expression was induced by cAMP-stimulation, whereas expression of C/EBP homolog protein (CHOP) was robustly downregulated. Transfection of CHOP expression plasmid inhibited cAMP-induced COX-2 promoter activity. In addition, CHOP reduced constitutive COX-2 expression in other mouse Leydig cell tumor-derived TM3 cells. These results indicate that COX-2 is induced in Leydig cells by activation of C/EBPβ via reduction of CHOP expression upon gonadotropin-stimulation to produce PGF and PGE .
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http://dx.doi.org/10.1002/mrd.23163DOI Listing
July 2019

Neurite Outgrowth and Morphological Changes Induced by 8-trans Unsaturation of Sphingadienine in kCer Molecular Species.

Int J Mol Sci 2019 Apr 29;20(9). Epub 2019 Apr 29.

Lipid Biofunction Section, Frontier Research Center for Advanced Material and Life Science, Faculty of Advanced Life Science, Hokkaido University, Sapporo 011-0021, Japan.

Konjac ceramide (kCer), which consists of plant-type molecular species of characteristic shingoid bases and fatty acids, is prepared from konjac glucosylceramide GlcCer by chemoenzymatical deglucosylation. kCer activates the semaphorin 3A (Sema3A) signaling pathway, inducing collapsin response mediator protein 2 (CRMP2) phosphorylation. This results in neurite outgrowth inhibition and morphological changes in remaining long neurites in PC12 cells. Whether a specific molecular species of kCer can bind to the Sema3A receptor (Neuropilin1, Nrp1) and activate the Sema3A signaling pathway remains unknown. Here, we prepared kCer molecular species using endoglycoceramidase I-mediated deglucosylation and examined neurite outgrowth and phosphorylation of collapsin response mediator protein 2 in nerve growth factor (NGF)-primed cells. The 8-trans unsaturation of sphingadienine of kCer was essential for Sema3A-like signaling pathway activation. Conversely, 8-cis unsaturation of kCer molecular species had no effect on Sema3A-like activation, and neurite outgrowth inhibition resulted in remaining short neurites. In addition, α-hydroxylation of fatty acids was not associated with the Sema3A-like activity of the kCer molecular species. These results suggest that 8-trans or 8-cis isomerization of sphingadienine determines the specific interactions at the ligand-binding site of Nrp1.
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http://dx.doi.org/10.3390/ijms20092116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540580PMC
April 2019

Isolation of Sphingoid Bases from Starfish Asterias amurensis Glucosylceramides and Their Effects on Sphingolipid Production in Cultured Keratinocytes.

J Oleo Sci 2019 May 9;68(5):427-441. Epub 2019 Apr 9.

Laboratory of Biomembrane and Biofunctional Chemistry, Frontier Research Center for Advanced Material and Life science, Hokkaido University.

Starfish Asterias amurensis produces sphingoid bases d18:3, 9-methyl-d18:3 (9Me-d18:3), and d22:2, which possess unique structural features. In this study, sphingoid bases prepared from A. amurensis glucosylceramides displayed unexpected elution behaviors from a general octadecyl silyl high-performance liquid chromatography (HPLC) column. For separation and isolation, sphingoid bases were fractionated by octadecyl silyl HPLC after N-acetylation, yielding d18:3, 9Me-d18:3, and two d22:2 isomers. To compare the biological activities of individual sphingoid bases, their effects on sphingolipid production in normal human keratinocytes were evaluated. Treatment with sphingoid bases increased the content of ceramides, glucosylceramides, and sphingomyelins in keratinocytes. Moreover, ceramides, which contain saturated ultra-long-chain fatty acids (C30-34), were significantly increased by treatment with d18:3, but not with other A. amurensis sphingoid bases. The mRNA level of the early differentiation marker keratin 10 was markedly decreased and sphingolipid synthesis-related genes were slightly increased in keratinocytes exposed to A. amurensis-derived d18:3, 9Me-d18:3, and d22:2 isomers. These results suggest that A. amurensis-derived sphingoid bases induce differentiation to varying degrees, sphingolipid production depends on their chemical structures, and d18:3 is the most promising functional sphingoid base.
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http://dx.doi.org/10.5650/jos.ess18256DOI Listing
May 2019

β-Hydroxybutyrate, a ketone body, reduces the cytotoxic effect of cisplatin via activation of HDAC5 in human renal cortical epithelial cells.

Life Sci 2019 Apr 6;222:125-132. Epub 2019 Mar 6.

Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

Aims: β-Hydroxybutyrate (βOHB) is a metabolic intermediate that constitutes about 70% of ketone bodies produced in liver from oxidation of fatty acids released from adipose tissue. A recent study showed that βOHB inhibits HDAC1, 3 and 4 (classes I and IIa) in human embryonic kidney 293 (HEK293) cells. Therefore, βOHB could regulate epigenetics via modulating HDACs. However, little is known about the protective effect of βOHB on renal cells through epigenetics. The aim of this study is to investigate whether βOHB reduces cisplatin-induced nephrotoxicity in human renal cortical epithelial (HRCE) cells by modulating HDACs.

Main Methods: In this study, we used human renal cortical epithelial (HRCE) cells. The anti-apoptotic effect of βOHB was evaluated using flow cytometry analysis. The expression of apoptosis-related proteins and HDACs was evaluated by western immunoblot.

Key Findings: The results showed that βOHB significantly reduced cisplatin-induced apoptosis in HRCE cells. Furthermore, βOHB significantly reduced cisplatin-induced cleavage of caspase-3, acetylation of histone H3, and phosphorylation of AMP-activated kinase. This anti-apoptotic effect of βOHB was markedly attenuated by an inhibitor of HDAC4/5, and βOHB-mediated suppression of cleavage of caspase3 was significantly blocked by siRNA-induced gene silencing of HDAC5.

Significance: βOHB attenuates cisplatin-induced apoptosis by activation of HDAC5 in HRCE cells, suggesting that βOHB may be a new therapeutic agent for cisplatin nephropathy.
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http://dx.doi.org/10.1016/j.lfs.2019.03.008DOI Listing
April 2019

Elevated Levels of Urinary Extracellular Vesicle Fibroblast-Specific Protein 1 in Patients with Active Crescentic Glomerulonephritis.

Nephron 2019 12;141(3):177-187. Epub 2018 Dec 12.

Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

Background/aims: Extracellular vesicles (EVs), including exosomes, are present in various bodily fluids, including urine. We and others previously reported that cells expressing fibroblast-specific protein 1 (FSP1) accumulate within damaged glomeruli, and that urinary FSP1, as well as urinary soluble CD163, could potentially serve as a biomarker of ongoing glomerular injury.

Methods: To test that idea, we collected urine samples from 37 patients with glomerular disease; purified the urinary EVs; characterized them using Nanosight, western blotting, and immunoelectron microscopy; and determined FSP1 and soluble CD163 levels using enzyme-linked immunosorbent assays.

Results: Deemed to be mainly exosomes based on their size distribution, the EVs in urine contained FSP1, and a portion of the FSP1-positive vesicles was also positive for podocalyxin. FSP1 levels in urinary EVs were (1) positively correlated with rates of biopsy-proven cellular crescent formation (r = 0.562, p < 0.001) and total crescent formation (r = 0.448, p = 0.005) among total glomeruli; (2) significantly higher in patients with cellular crescents affecting 20% or more of their glomeruli than in those with fewer affected glomeruli (p = 0.003); and (3) significantly decreased after glucocorticoid and immunosuppressant therapy (p < 0.05). A positive correlation between FSP1 levels in urinary EVs and urinary soluble CD163 levels was confirmed (r = 0.367, p < 0.05).

Conclusion: These data suggest that a portion of urinary FSP1 is secreted as EVs originating from podocytes, and that FSP1 levels reflect active and ongoing glomerular injury and disease activity, such as cellular crescent formation.
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http://dx.doi.org/10.1159/000495217DOI Listing
December 2019

A short-chain fatty acid, propionate, enhances the cytotoxic effect of cisplatin by modulating GPR41 signaling pathways in HepG2 cells.

Oncotarget 2018 Jul 31;9(59):31342-31354. Epub 2018 Jul 31.

Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are generated by microbial fermentation of indigestible fiber by gut flora. SCFAs are ligands of two orphan G protein-coupled receptors, GPR41 and GPR43, that modulate cell proliferation and induce apoptosis. However, it is unclear if SCFAs enhance the effects of chemotherapy in a GPR41- or GPR43-dependent manner. The aim of this study was to investigate whether SCFAs, and particularly propionate, activate GPR41 or GPR43, and thereby enhance the antitumor effects of cisplatin in HepG2 human hepatocellular carcinoma (HCC) cells. The inhibitory effects of propionate and cisplatin on proliferation of HCC cells were determined by MTS assay. Changes in apoptosis rate were analyzed by flow cytometry. The effects of combined propionate and cisplatin on these properties in HCC cells were significantly higher than those of cisplatin alone. With combined treatment, the levels of cleaved caspase-3, active caspase-3 forms, and acetylated histone H3 were enhanced in a GPR41-dependent manner; expression of histone deacetylases (HDAC) 3, 4, 5, 6, 8 proteins was significantly reduced; and induction of TNF-α expression was significantly enhanced. These results suggest that propionate and cisplatin synergistically and significantly induce apoptosis of HepG2 cells by increasing expression of autocrine TNF-α via reduction of HDACs through GPR41 signaling. From clinical and translational perspectives, our data suggest that a combination of propionate with cisplatin may have better therapeutic effects on HCC compared with conventional treatment, and that a selective GPR41 agonist may be a candidate as an adjuvant therapeutic agent for HCC.
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http://dx.doi.org/10.18632/oncotarget.25809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101142PMC
July 2018

Tubulointerstitial Nephritis with IgM-Positive Plasma Cells.

J Am Soc Nephrol 2017 Dec 9;28(12):3688-3698. Epub 2017 Aug 9.

Departments of Nephrology.

Infiltration by IgG-positive plasma cells is a common finding in tubulointerstitial nephritis. Indeed, it has been thought that CD138-positive mature plasma cells secrete mainly IgG, and the occurrence of tubulointerstitial nephritis with CD138-positive plasma cells secreting IgM has rarely been reported. Routine immunofluorescence of fresh frozen sections is considered the gold standard for detection of immune deposits. However, the immunoenzyme method with formalin-fixed, paraffin-embedded sections is superior for detecting IgM- or IgG-positive cells within the renal interstitium, thus histologic variants may often go undetected. We recently discovered a case of tubulointerstitial nephritis showing IgM-positive plasma cell accumulation within the interstitium. To further explore the morphologic and clinical features of such cases, we performed a nationwide search for patients with biopsy-proven tubulointerstitial nephritis and high serum IgM levels. We identified 13 patients with tubulointerstitial nephritis and IgM-positive plasma cell infiltration confirmed with the immunoenzyme method. The clinical findings for these patients included a high prevalence of distal renal tubular acidosis (100%), Fanconi syndrome (92%), and anti-mitochondrial antibodies (82%). The pathologic findings were interstitial nephritis with diffusely distributed CD3-positive T lymphocytes and colocalized IgM-positive plasma cells, as well as tubulitis with CD3-positive T lymphocytes in the proximal tubules and collecting ducts. Additionally, levels of H-ATPase, H, K-ATPase, and the HCO-Cl anion exchanger were markedly decreased in the collecting ducts. We propose to designate this group of cases, which have a common histologic and clinical form, as IgM-positive plasma cell-tubulointerstitial nephritis.
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http://dx.doi.org/10.1681/ASN.2016101074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698061PMC
December 2017
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