Chronopharmacology of diuresis via metabolic profiling and key biomarker discovery of the traditional Chinese prescription Ji-Ming-San using tandem mass spectrometry in rat models.

BACKGROUND: Ji-Ming-Shan (JMS) is a traditional prescription used for patients with rheumatism, tendons swelling, relief of foot pain, athlete's foot, diuresis, gout. Although many studies have investigated the active compounds in each herb, the functional mechanism behind its therapeutic effect remains unclear. STUDY DESIGN: Metabolic cages for sample collection. The serum components obtained from the experimental animals were analyzed using LC-MS/MS. Furthermore, cross-analysis using the software MetaboAnalyst and Venn diagrams were used to investigate chronopharmacology of JMS in the animal models. PURPOSE: The aim of this study is to analyze the diuretic effects of JMS and to explore their chronopharmacology involved in organ regulation through four-quarter periods from serum samples of rat models. METHODS: Metabolic cages were used for collecting the urine samples and PocketChem UA PU-4010, Fuji DRI-CHEM 800 were used to examine the urine biochemical parameters. The serum components were identified through ultra-performance liquid chromatography-quadrupole time-of-flight (UPLC-Q-TOF) with a new developed method. Cross analysis, Venn diagram, MetaboAnalyst were used to investigate the key biomarker and major metabolism route with the oral administration of the drug. RESULT: JMS significantly changed the 6 h urine volume with no observed kidney toxicity. Urine pH value ranges from 7.0 to 7.5. The chronopharmacology of JMS diuresis activity were 0-6 and 6-12 groups. UPLC-Q-TOF analyses identified 243 metabolites which were determined in positive mode and negative mode respectively. With cross analysis in the Venn diagram, one key biomarker naringenin-7-O-glucoside has been identified. Major metabolic pathways such as 1: Glycerophospholipid metabolism, 2: Primary bile acid biosynthesis, 3: Sphingolipid metabolism, 4: Riboflavin metabolism, 5: Linoleic acid metabolism, 6: Butanoate metabolism. CONCLUSION: JMS significantly changed the urine output of animals in the 0-6 and 6-12 groups. No change in urine pH was observed and also kidney toxicity. A new UPLC-Q-TOF method was developed for the detection of the metabolites of JMS after oral administration. The cross analysis with Venn diagram and identified the key biomarker of JMS namely naringenin-7-O-glucoside. The results showed that six major pathways are involved in the gastrointestinal system and the liver. This study demonstrated the capability of JMS prescription in the regulation of diuresis and identified a key biomarker that is responsible for its therapeutic effect.

Oxidative Stress, Transfer RNA Metabolism, and Protein Synthesis.

Significance: Oxidative stress refers to excessive intracellular levels of reactive oxygen species (ROS) due to an imbalance between ROS production and the antioxidant defense system. Under oxidative stress conditions, cells trigger various stress response pathways to protect themselves, among which repression of messenger RNA (mRNA) translation is one of the key hallmarks promoting cell survival. This regulation process minimizes cellular energy consumption, enabling cells to survive in adverse conditions and to promote recovery from stress-induced damage. Recent Advances: Recent studies suggest that transfer RNAs (tRNAs) play important roles in regulating translation as a part of stress response under adverse conditions. In particular, research relying on high-throughput techniques such as next-generation sequencing and mass spectrometry approaches has given us detailed information on mechanisms such as individual tRNA dynamics and crosstalk among post-transcriptional modifications. Critical Issues: Oxidative stress leads to dynamic tRNA changes, including their localization, cleavage, and alteration of expression profiles and modification patterns. Growing evidence suggests that these changes not only are tightly regulated by stress response mechanisms, but also can directly fine-tune the translation efficiency, which contributes to cell- or tissue-specific response to oxidative stress. Future Directions: In this review, we describe recent advances in the understanding of the dynamic changes of tRNAs caused by oxidative stress. We also highlight the emerging roles of tRNAs in translation regulation under the condition of oxidative stress. In addition, we discuss future perspectives in this research field. Antioxid. Redox Signal. 40, 715-735.

tRNA-derived RNAs: Biogenesis and roles in translational control.

Transfer RNA (tRNA)-derived RNAs (tDRs) are a class of small non-coding RNAs that play important roles in different aspects of gene expression. These ubiquitous and heterogenous RNAs, which vary across different species and cell types, are proposed to regulate various biological processes. In this review, we will discuss aspects of their biogenesis, and specifically, their contribution into translational control. We will summarize diverse roles of tDRs and the molecular mechanisms underlying their functions in the regulation of protein synthesis and their impact on related events such as stress-induced translational reprogramming. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.

CD98 regulates the phosphorylation of HER2 and a bispecific anti-HER2/CD98 antibody inhibits the growth signal of human breast cancer cells.

Human epidermal growth factor receptor (HER) family proteins are currently major targets of therapeutic monoclonal antibodies against various epithelial cancers. However, the resistance of cancer cells to HER family-targeted therapies, which may be caused by cancer heterogeneity and persistent HER phosphorylation, often reduces overall therapeutic effects. We herein showed that a newly discovered molecular complex between CD98 and HER2 affected HER function and cancer cell growth. The immunoprecipitation of the HER2 or HER3 protein from lysates of SKBR3 breast cancer (BrCa) cells revealed the HER2-CD98 or HER3-CD98 complex. The knockdown of CD98 by small interfering RNAs inhibited the phosphorylation of HER2 in SKBR3 cells. A bispecific antibody (BsAb) that recognized the HER2 and CD98 proteins was constructed from a humanized anti-HER2 (SER4) IgG and an anti-CD98 (HBJ127) single chain variable fragment, and this BsAb significantly inhibited the cell growth of SKBR3 cells. Prior to the inhibition of AKT phosphorylation, BsAb inhibited the phosphorylation of HER2, however, significant inhibition of HER2 phosphorylation was not observed in anti-HER2 pertuzumab, trastuzumab, SER4 or anti-CD98 HBJ127 in SKBR3 cells. The dual targeting of HER2 and CD98 has potential as a new therapeutic strategy for BrCa.

Dual-targeting therapy against HER3/MET in human colorectal cancers.

BACKGROUND: Colorectal cancer (CRC) is the most common malignancy in the world, and novel molecular targeted therapies for CRC have been vigorously pursued. We searched for novel combination therapies based on the expression patterns of membrane proteins in CRC cell lines. RESULTS: A positive correlation was observed between the expression of human pidermal growth factor receptor (HER) 3 and mesenchymal-to-epithelial transition factor (MET) on the cell surface of CRC cell lines. The brief stimulation of HER3/MET-high SW1116 CRC cells with both neuregulin-1 (NRG1) and hepatocyte growth factor enhanced ERK phosphorylation and cell proliferation more than each stimulation alone. In addition, a prolonged NRG1 stimulation resulted in the tyrosine phosphorylation of MET. In this context, the Forkhead Box protein M1 (FOXM1)-regulated tyrosine phosphorylation of MET by NRG1 was demonstrated, suggesting the existence of a signaling pathway mediated by FOXM1 upon the NRG1 stimulation. Since the co-expression of HER3 and MET was also demonstrated in in vivo CRC tissues by immunohistochemistry, we investigated whether the co-inhibition of HER3 and MET could be an effective therapy for CRC. We established HER3-and/or MET-KO SW1116 cell lines, and HER3/MET-double KO resulted in the inhibition of in vitro cell proliferation and in vivo tumor growth in nude mice by SW1116 cells. Furthermore, the combination of patritumab, an anti-HER3 fully human mAb, and PHA665752, a MET inhibitor, markedly inhibited in vitro cell proliferation, 3D-colony formation, and in vivo tumor growth in nude mice by SW1116 cells CONCLUSION: The dual targeting of HER3/MET has potential as CRC therapy.

RTCB Complex Regulates Stress-Induced tRNA Cleavage.

Under stress conditions, transfer RNAs (tRNAs) are cleaved by stress-responsive RNases such as angiogenin, generating tRNA-derived RNAs called tiRNAs. As tiRNAs contribute to cytoprotection through inhibition of translation and prevention of apoptosis, the regulation of tiRNA production is critical for cellular stress response. Here, we show that RTCB ligase complex (RTCB-LC), an RNA ligase complex involved in endoplasmic reticulum (ER) stress response and precursor tRNA splicing, negatively regulates stress-induced tiRNA production. Knockdown of RTCB significantly increased stress-induced tiRNA production, suggesting that RTCB-LC negatively regulates tiRNA production. Gel-purified tiRNAs were repaired to full-length tRNAs by RtcB in vitro, suggesting that RTCB-LC can generate full length tRNAs from tiRNAs. As RTCB-LC is inhibited under oxidative stress, we further investigated whether tiRNA production is promoted through the inhibition of RTCB-LC under oxidative stress. Although hydrogen peroxide (H2O2) itself did not induce tiRNA production, it rapidly boosted tiRNA production under the condition where stress-responsive RNases are activated. We propose a model of stress-induced tiRNA production consisting of two factors, a trigger and booster. This RTCB-LC-mediated boosting mechanism may contribute to the effective stress response in the cell.

Selective Cleavage at CCA Ends and Anticodon Loops of tRNAs by Stress-Induced RNases.

Stress-induced tRNA cleavage has been implicated in various cellular processes, where tRNA fragments play diverse regulatory roles. Angiogenin (ANG), a member of the RNase A superfamily, induces cleavage of tRNAs resulting in the formation of tRNA-derived stress-induced RNAs (tiRNAs) that contribute to translational reprogramming aiming at cell survival. In addition to cleaving tRNA anticodon loops, ANG has been shown to cleave 3'-CCA termini of tRNAs in vitro, although it is not known whether this process occurs in cells. It has also been suggested that tiRNAs can be generated independently of ANG, although the role of other stress-induced RNases in tRNA cleavage is poorly understood. Using gene editing and biochemical approaches, we examined the involvement of ANG in stress-induced tRNA cleavage by focusing on its cleavage of CCA-termini as well as anticodon loops. We show that ANG is not responsible for CCA-deactivation under sodium arsenite (SA) treatment in cellulo, and although ANG treatment significantly increases 3'-tiRNA levels in cells, the majority of 3'-tiRNAs retain their 3'-CCA termini. Instead, other RNases can cleave CCA-termini in cells, although with low efficiency. Moreover, in the absence of ANG, other RNases are able to promote the production of tiRNAs in cells. Depletion of RNH1 (an endogenous inhibitor of RNase A superfamily) promotes constitutively-produced tiRNAs and CCA-deactivated tRNAs in cells. Interestingly, SA treatment in RNH1-depleted cells did not increase the amount of tiRNAs or CCA-deactivated tRNAs, suggesting that RNase A superfamily enzymes are largely responsible for SA-induced tRNA cleavage. We show that interplay between stress-induced RNases cause targeting tRNAs in a stress-specific manner in cellulo.

tiRNA signaling via stress-regulated vesicle transfer in the hematopoietic niche.

Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process in vivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulate protein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides a stress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.

In lysate RNA digestion provides insights into the angiogenin's specificity towards transfer RNAs.

Under adverse conditions, tRNAs are processed into fragments called tRNA-derived stress-induced RNAs (tiRNAs) by stress-responsive ribonucleases (RNases) such as angiogenin (ANG). Recent studies have reported several biological functions of synthetic tiRNAs lacking post-transcriptional modifications found on endogenous tiRNAs. Here we describe a simple and reproducible method to efficiently isolate ANG-cleaved tiRNAs from endogenous tRNAs. Using this in vitro method, more than 50% of mature tRNAs are cleaved into tiRNAs which can be enriched using complementary oligonucleotides. Using this method, the yield of isolated endogenous 5'-tiRNAGly-GCC was increased about fivefold compared to when tiRNAs were obtained by cellular treatment of ANG. Although the non-specific ribonuclease activity of ANG is much lower than that of RNase A, we show that ANG cleaves physiologically folded tRNAs as efficiently as bovine RNase A. These results suggest that ANG is highly specialized to cleave physiologically folded tRNAs. Our method will greatly facilitate the analysis of endogenous tiRNAs to elucidate the physiological functions of ANG.

CE-MS-Based Identification of Uremic Solutes Specific to Hemodialysis Patients.

Uremic toxins are suggested to be involved in the pathophysiology of hemodialysis (HD) patients. However, the profile of uremic solutes in HD patients has not been fully elucidated. In this study using capillary electrophoresis mass spectrometry (CE-MS), we comprehensively quantified the serum concentrations of 122 ionic solutes before and after HD in 11 patients. In addition, we compared the results with those in non-HD patients with chronic kidney disease (CKD) to identify HD patient-specific solutes. We identified 38 solutes whose concentrations were higher in pre-HD than in CKD stage G5. Ten solutes among them did not significantly accumulate in non-HD CKD patients, suggesting that these solutes accumulate specifically in HD patients. We also identified 23 solutes whose concentrations were lower in both pre- and post-HD than in CKD stage G5. The serum levels of 14 solutes among them were not affected by renal function in non-HD patients, suggesting that these solutes tend to be lost specifically in HD patients. Our data demonstrate that HD patients have a markedly different profile of serum uremic solute levels compared to that in non-HD CKD patients. The solutes identified in our study may contribute to the pathophysiology of HD patients.

Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5.

Sporadic inclusion body myositis (sIBM) is the most common idiopathic inflammatory myopathy, and several reports have suggested that mitochondrial abnormalities are involved in its etiology. We recruited 9 sIBM patients and found significant histological changes and an elevation of growth differential factor 15 (GDF15), a marker of mitochondrial disease, strongly suggesting the involvement of mitochondrial dysfunction. Bioenergetic analysis of sIBM patient myoblasts revealed impaired mitochondrial function. Decreased ATP production, reduced mitochondrial size and reduced mitochondrial dynamics were also observed in sIBM myoblasts. Cell vulnerability to oxidative stress also suggested the existence of mitochondrial dysfunction. Mitochonic acid-5 (MA-5) increased the cellular ATP level, reduced mitochondrial ROS, and provided protection against sIBM myoblast death. MA-5 also improved the survival of sIBM skin fibroblasts as well as mitochondrial morphology and dynamics in these cells. The reduction in the gene expression levels of Opa1 and Drp1 was also reversed by MA-5, suggesting the modification of the fusion/fission process. These data suggest that MA-5 may provide an alternative therapeutic strategy for treating not only mitochondrial diseases but also sIBM.

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome.

A major proportion of extracellular RNAs (exRNAs) do not copurify with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes are probably not silent from an immunological perspective and could possibly be decoded as damage-associated molecular patterns.

eIF4G has intrinsic G-quadruplex binding activity that is required for tiRNA function.

As cells encounter adverse environmental conditions, such as hypoxia, oxidative stress or nutrient deprivation, they trigger stress response pathways to protect themselves until transient stresses have passed. Inhibition of translation is a key component of such cellular stress responses and mounting evidence has revealed the importance of a class of tRNA-derived small RNAs called tiRNAs in this process. The most potent of these small RNAs are those with the capability of assembling into tetrameric G-quadruplex (G4) structures. However, the mechanism by which these small RNAs inhibit translation has yet to be elucidated. Here we show that eIF4G, the major scaffolding protein in the translation initiation complex, directly binds G4s and this activity is required for tiRNA-mediated translation repression. Targeting of eIF4G results in an impairment of 40S ribosome scanning on mRNAs leading to the formation of eIF2α-independent stress granules. Our data reveals the mechanism by which tiRNAs inhibit translation and demonstrates novel activity for eIF4G in the regulation of translation.

Isolation and initial structure-functional characterization of endogenous tRNA-derived stress-induced RNAs.

Recent transcriptome-wide studies have identified a diverse pool of transfer RNA (tRNA)-derived RNAs or tRNA-derived fragments (tRFs). Some of these RNAs have been demonstrated to be functional and involved in multiple biological processes ranging from the regulation of gene expression to transgenerational epigenetic inheritance. Post-transcriptional maturation of tRNAs includes various processing events including extensive decoration by various RNA modifications, which are required for correct tRNA folding and stability. Moreover, tRNA modifications determine the pattern and specificity of tRNA cleavage. The major drawbacks of many studies in the field of tRFs are that most of them used synthetic RNAs that closely mimic endogenous tRFs in their sequence, yet lack RNA modification that is found in vivo. Here, we developed a simple method to isolate tRNA-derived stress-induced RNAs (tiRNAs), a specific subset of tRFs. Our approach is scalable, cost-effective and relies on the purification of individual tiRNAs based on a sequence-specific RNA/DNA isolation technique using DNA probes. Our method facilitates functional studies of tiRNAs by addressing how physiological RNA modifications within tRNA fragments affect their biological activities. Here, we report pilot functional studies on selected endogenous tiRNAs, namely tiRNAAla and tiRNAGly. We show that natural 5'-tiRNAAla molecules assemble into G-quadruplex structures, and endogenous 5'-tiRNAGly possesses translation inhibition activity.

Gut microbiome-derived phenyl sulfate contributes to albuminuria in diabetic kidney disease.

Diabetic kidney disease is a major cause of renal failure that urgently necessitates a breakthrough in disease management. Here we show using untargeted metabolomics that levels of phenyl sulfate, a gut microbiota-derived metabolite, increase with the progression of diabetes in rats overexpressing human uremic toxin transporter SLCO4C1 in the kidney, and are decreased in rats with limited proteinuria. In experimental models of diabetes, phenyl sulfate administration induces albuminuria and podocyte damage. In a diabetic patient cohort, phenyl sulfate levels significantly correlate with basal and predicted 2-year progression of albuminuria in patients with microalbuminuria. Inhibition of tyrosine phenol-lyase, a bacterial enzyme responsible for the synthesis of phenol from dietary tyrosine before it is metabolized into phenyl sulfate in the liver, reduces albuminuria in diabetic mice. Together, our results suggest that phenyl sulfate contributes to albuminuria and could be used as a disease marker and future therapeutic target in diabetic kidney disease.

Stress-induced tRNA cleavage and tiRNA generation in rat neuronal PC12 cells.

Transfer RNA (tRNA) plays a role in stress response programs involved in various pathological conditions including neurological diseases. Under cell stress conditions, intracellular tRNA is cleaved by a specific ribonuclease, angiogenin, generating tRNA-derived fragments or tRNA-derived stress-induced RNA (tiRNA). Generated tiRNA contributes to the cell stress response and has potential cell protective effects. However, tiRNA generation under stress conditions in neuronal cells has not been fully elucidated. To examine angiogenin-mediated tiRNA generation in neuronal cells, we used the rat neuronal cell line, PC12, in combination with analysis of SYBR staining and immuno-northern blotting using anti-1-methyladenosine antibody, which specifically and sensitively detects tiRNA. Oxidative stress induced by arsenite and hydrogen peroxide caused tRNA cleavage and tiRNA generation in PC12 cells. We also demonstrated that oxygen-glucose deprivation, which is an in vitro model of ischemic-reperfusion injury, induced tRNA cleavage and tiRNA generation. In these stress conditions, the amount of generated tiRNA was associated with the degree of morphological cell damage. Time course analysis indicated that generation of tiRNA was prior to severe cell damage and cell death. Angiogenin over-expression did not influence the amount of tiRNA in normal culture conditions; however, it significantly increased tiRNA generation induced by cell stress conditions. Our findings show that angiogenin-mediated tiRNA generation can be induced in neuronal cells by different cell stressors, including ischemia-reperfusion. Additionally, detection of tiRNA could be used as a potential cell damage marker in neuronal cells. Cover Image for this issue: doi: 10.1111/jnc.14191.

Mitochonic Acid 5 (MA-5) Facilitates ATP Synthase Oligomerization and Cell Survival in Various Mitochondrial Diseases.

Mitochondrial dysfunction increases oxidative stress and depletes ATP in a variety of disorders. Several antioxidant therapies and drugs affecting mitochondrial biogenesis are undergoing investigation, although not all of them have demonstrated favorable effects in the clinic. We recently reported a therapeutic mitochondrial drug mitochonic acid MA-5 (Tohoku J. Exp. Med., 2015). MA-5 increased ATP, rescued mitochondrial disease fibroblasts and prolonged the life span of the disease model "Mitomouse" (JASN, 2016). To investigate the potential of MA-5 on various mitochondrial diseases, we collected 25 cases of fibroblasts from various genetic mutations and cell protective effect of MA-5 and the ATP producing mechanism was examined. 24 out of the 25 patient fibroblasts (96%) were responded to MA-5. Under oxidative stress condition, the GDF-15 was increased and this increase was significantly abrogated by MA-5. The serum GDF-15 elevated in Mitomouse was likewise reduced by MA-5. MA-5 facilitates mitochondrial ATP production and reduces ROS independent of ETC by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin/Mic60. MA-5 reduced mitochondria fragmentation, restores crista shape and dynamics. MA-5 has potential as a drug for the treatment of various mitochondrial diseases. The diagnostic use of GDF-15 will be also useful in a forthcoming MA-5 clinical trial.

Selective embolization therapy for intrarenal artery stenosis causing renovascular hypertension: Efficacy and follow-up renal imaging.

We report the case of a young woman treated with selective renal embolization for renovascular hypertension caused by intrarenal artery stenosis and show follow-up imaging of the treated kidney. An 18-year-old woman had renin-dependent hypertension with intrarenal artery stenosis caused by fibromuscular dysplasia. A middle branch artery was nearly occluded, resulting in segmental renal ischemia with excessive renin secretion. Because our angioplasty attempt for revascularization failed as a result of technical difficulty, we performed selective embolization of the diseased vessel by anhydrous ethanol. The embolization promptly ameliorated hyperreninemia and resistant hypertension without deterioration of renal function. Findings from magnetic resonance imaging showed disappearance of the blood flow in the embolized area corresponding to the ischemic lesion that had been revealed by diffusion-weighted imaging. Thus, selective embolization can be effective in treating renovascular hypertension by intrarenal stenosis for which angioplasty is not feasible. Additionally, renal magnetic resonance imaging is useful for evaluating the causative ischemic lesion and embolized area.

A novel indole compound MA-35 attenuates renal fibrosis by inhibiting both TNF-α and TGF-ß1 pathways.

Renal fibrosis is closely related to chronic inflammation and is under the control of epigenetic regulations. Because the signaling of transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) play key roles in progression of renal fibrosis, dual blockade of TGF-ß1 and TNF-α is desired as its therapeutic approach. Here we screened small molecules showing anti-TNF-α activity in the compound library of indole derivatives. 11 out of 41 indole derivatives inhibited the TNF-α effect. Among them, Mitochonic Acid 35 (MA-35), 5-(3, 5-dimethoxybenzyloxy)-3-indoleacetic acid, showed the potent effect. The anti-TNF-α activity was mediated by inhibiting IκB kinase phosphorylation, which attenuated the LPS/GaIN-induced hepatic inflammation in the mice. Additionally, MA-35 concurrently showed an anti-TGF-ß1 effect by inhibiting Smad3 phosphorylation, resulting in the downregulation of TGF-ß1-induced fibrotic gene expression. In unilateral ureter obstructed mouse kidney, which is a renal fibrosis model, MA-35 attenuated renal inflammation and fibrosis with the downregulation of inflammatory cytokines and fibrotic gene expressions. Furthermore, MA-35 inhibited TGF-ß1-induced H3K4me1 histone modification of the fibrotic gene promoter, leading to a decrease in the fibrotic gene expression. MA-35 affects multiple signaling pathways involved in the fibrosis and may recover epigenetic modification; therefore, it could possibly be a novel therapeutic drug for fibrosis.

Evaluation of the impact of gut microbiota on uremic solute accumulation by a CE-TOFMS-based metabolomics approach.

Gut microbiota is involved in the metabolism of uremic solutes. However, the precise influence of microbiota to the retention of uremic solutes in CKD is obscure. To clarify this, we compared adenine-induced renal failure and control mice under germ-free or specific pathogen-free (SPF) conditions, examining the metabolite profiles of plasma, feces, and urine using a capillary electrophoresis time-of-flight mass spectrometry-based approach. Mice with renal failure under germ-free conditions demonstrated significant changes in plasma metabolites. Among 183 detected solutes, plasma levels of 11 solutes, including major uremic toxins, were significantly lower in germ-free mice than in SPF mice with renal failure. These 11 solutes were considered microbiota-derived uremic solutes and included indoxyl sulfate, p-cresyl sulfate, phenyl sulfate, cholate, hippurate, dimethylglycine, γ-guanidinobutyrate, glutarate, 2-hydroxypentanoate, trimethylamine N-oxide, and phenaceturate. Metabolome profiling showed that these solutes were classified into three groups depending on their origins: completely derived from microbiota (indoxyl sulfate, p-cresyl sulfate), derived from both host and microbiota (dimethylglycine), and derived from both microbiota and dietary components (trimethylamine N-oxide). Additionally, germ-free renal failure conditions resulted in the disappearance of colonic short-chain fatty acids, decreased utilization of intestinal amino acids, and more severe renal damage compared with SPF mice with renal failure. Microbiota-derived short-chain fatty acids and efficient amino acid utilization may have a renoprotective effect, and loss of these factors may exacerbate renal damage in germ-free mice with renal failure. Thus, microbiota contributes substantially to the production of harmful uremic solutes, but conversely, growth without microbiota has harmful effects on CKD progression.