Polyunsaturated fatty acids-induced ferroptosis suppresses pancreatic cancer growth.

Despite recent advances in science and medical technology, pancreatic cancer remains associated with high mortality rates due to aggressive growth and no early clinical sign as well as the unique resistance to anti-cancer chemotherapy. Current numerous investigations have suggested that ferroptosis, which is a programed cell death driven by lipid oxidation, is an attractive therapeutic in different tumor types including pancreatic cancer. Here, we first demonstrated that linoleic acid (LA) and α-linolenic acid (αLA) induced cell death with necroptotic morphological change in MIA-Paca2 and Suit 2 cell lines. LA and αLA increased lipid peroxidation and phosphorylation of RIP3 and MLKL in pancreatic cancers, which were negated by ferroptosis inhibitor, ferrostatin-1, restoring back to BSA control levels. Similarly, intraperitoneal administration of LA and αLA suppresses the growth of subcutaneously transplanted Suit-2 cells and ameliorated the decreased survival rate of tumor bearing mice, while co-administration of ferrostatin-1 with LA and αLA negated the anti-cancer effect. We also demonstrated that LA and αLA partially showed ferroptotic effects on the gemcitabine-resistant-PK cells, although its effect was exerted late compared to treatment on normal-PK cells. In addition, the trial to validate the importance of double bonds in PUFAs in ferroptosis revealed that AA and EPA had a marked effect of ferroptosis on pancreatic cancer cells, but DHA showed mild suppression of cancer proliferation. Furthermore, treatment in other tumor cell lines revealed different sensitivity of PUFA-induced ferroptosis; e.g., EPA induced a ferroptotic effect on colorectal adenocarcinoma, but LA or αLA did not. Collectively, these data suggest that PUFAs can have a potential to exert an anti-cancer effect via ferroptosis in both normal and gemcitabine-resistant pancreatic cancer.

Urinary growth differentiation factor 15 predicts renal function decline in diabetic kidney disease.

Sensitive biomarkers can enhance the diagnosis, prognosis, and surveillance of chronic kidney disease (CKD), such as diabetic kidney disease (DKD). Plasma growth differentiation factor 15 (GDF15) levels are a novel biomarker for mitochondria-associated diseases; however, it may not be a useful indicator for CKD as its levels increase with declining renal function. This study explores urinary GDF15's potential as a marker for CKD. The plasma and urinary GDF15 as well as 15 uremic toxins were measured in 103 patients with CKD. The relationship between the urinary GDF15-creatinine ratio and the uremic toxins and other clinical characteristics was investigated. Urinary GDF15-creatinine ratios were less related to renal function and uremic toxin levels compared to plasma GDF15. Additionally, the ratios were significantly higher in patients with CKD patients with diabetes (p = 0.0012) and reduced with statin treatment. In a different retrospective DKD cohort study (U-CARE, n = 342), multiple and logistic regression analyses revealed that the baseline urinary GDF15-creatinine ratios predicted a decline in estimated glomerular filtration rate (eGFR) over 2 years. Compared to the plasma GDF15 level, the urinary GDF15-creatinine ratio is less dependent on renal function and sensitively fluctuates with diabetes and statin treatment. It may serve as a good prognostic marker for renal function decline in patients with DKD similar to the urine albumin-creatinine ratio.

Effects of Ndufs4 Deletion on Hearing after Various Acoustic Exposures.

Mitochondrial dysfunction can cause cochlear dysfunction and accelerate noise-induced hearing loss (NIHL). NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) is one of the subunits of mitochondrial complex I and has a role in the assembly and stabilization of complex I. However, the involvement of Ndufs4 in the pathogenesis of NIHL has not been reported. The aim of this study was to evaluate whether Ndufs4 deletion causes vulnerability to noise exposures. The wild-type (WT) and Ndufs4 knockout (KO) mice with C57BL/6J genetic background were used. Cochlear histology and hearing thresholds were assessed after noise exposure at 100 or 86 dB sound pressure level (SPL). Immunostaining showed the widespread expression of Ndufs4 in the cochlea. After noise exposure at 100 dB SPL, auditory brainstem response (ABR) threshold shifts at 4 kHz in Ndufs4 KO mice were significantly higher than that in WT mice. After noise exposure at 86 dB SPL, ABR threshold shifts, wave 1 amplitudes, and the number of synapses in the inner hair cells were not significantly different. RNA sequencing revealed the decreased expression of energy generation-related genes inNdufs4 KO mice. Ndufs4 deficiency accelerates permanent low-frequency threshold shifts after moderate noise exposure.

Mitochonic Acid 5 Improves Duchenne Muscular Dystrophy and Parkinson's Disease Model of Caenorhabditis elegans.

Mitochonic Acid 5 (MA-5) enhances mitochondrial ATP production, restores fibroblasts from mitochondrial disease patients and extends the lifespan of the disease model "Mitomouse". Additionally, MA-5 interacts with mitofilin and modulates the mitochondrial inner membrane organizing system (MINOS) in mammalian cultured cells. Here, we used the nematode Caenorhabditis elegans to investigate whether MA-5 improves the Duchenne muscular dystrophy (DMD) model. Firstly, we confirmed the efficient penetration of MA-5 in the mitochondria of C. elegans. MA-5 also alleviated symptoms such as movement decline, muscular tone, mitochondrial fragmentation and Ca2+ accumulation of the DMD model. To assess the effect of MA-5 on mitochondria perturbation, we employed a low concentration of rotenone with or without MA-5. MA-5 significantly suppressed rotenone-induced mitochondria reactive oxygen species (ROS) increase, mitochondrial network fragmentation and nuclear destruction in body wall muscles as well as endogenous ATP levels decline. In addition, MA-5 suppressed rotenone-induced degeneration of dopaminergic cephalic (CEP) neurons seen in the Parkinson's disease (PD) model. Furthermore, the application of MA-5 reduced mitochondrial swelling due to the immt-1 null mutation. These results indicate that MA-5 has broad mitochondrial homing and MINOS stabilizing activity in metazoans and may be a therapeutic agent for these by ameliorating mitochondrial dysfunction in DMD and PD.

Mitochonic acid-5 ameliorates chlorhexidine gluconate-induced peritoneal fibrosis in mice.

Peritoneal fibrosis is a serious complication of long-term peritoneal dialysis, attributable to inflammation and mitochondrial dysfunction. Mitochonic acid-5 (MA-5), an indole-3-acetic acid derivative, improves mitochondrial dysfunction and has therapeutic potential against various diseases including kidney diseases. However, whether MA-5 is effective against peritoneal fibrosis remains unclear. Therefore, we investigated the effect of MA-5 using a peritoneal fibrosis mouse model. Peritoneal fibrosis was induced in C57BL/6 mice via intraperitoneal injection of chlorhexidine gluconate (CG) every other day for 3 weeks. MA-5 was administered daily by oral gavage. The mice were divided into control, MA-5, CG, and CG + MA-5 groups. Following treatment, immunohistochemical analyses were performed. Fibrotic thickening of the parietal peritoneum induced by CG was substantially attenuated by MA-5. The number of α-smooth muscle actin-positive myofibroblasts, transforming growth factor ß-positive cells, F4/80-positive macrophages, monocyte chemotactic protein 1-positive cells, and 4-hydroxy-2-nonenal-positive cells was considerably decreased. In addition, reduced ATP5a1-positive and uncoupling protein 2-positive cells in the CG group were notably increased by MA-5. MA-5 may ameliorate peritoneal fibrosis by suppressing macrophage infiltration and oxidative stress, thus restoring mitochondrial function. Overall, MA-5 has therapeutic potential against peritoneal fibrosis.

SGLT-1-specific inhibition ameliorates renal failure and alters the gut microbial community in mice with adenine-induced renal failure.

Sodium-dependent glucose cotransporters (SGLTs) have attracted considerable attention as new targets for type 2 diabetes mellitus. In the kidney, SGLT2 is the major glucose uptake transporter in the proximal tubules, and inhibition of SGLT2 in the proximal tubules shows renoprotective effects. On the other hand, SGLT1 plays a role in glucose absorption from the gastrointestinal tract, and the relationship between SGLT1 inhibition in the gut and renal function remains unclear. Here, we examined the effect of SGL5213, a novel and potent intestinal SGLT1 inhibitor, in a renal failure (RF) model. SGL5213 improved renal function and reduced gut-derived uremic toxins (phenyl sulfate and trimethylamine-N-oxide) in an adenine-induced RF model. Histological analysis revealed that SGL5213 ameliorated renal fibrosis and inflammation. SGL5213 also reduced gut inflammation and fibrosis in the ileum, which is a primary target of SGL5213. Examination of the gut microbiota community revealed that the Firmicutes/Bacteroidetes ratio, which suggests gut dysbiosis, was increased in RF and SGL5213 rebalanced the ratio by increasing Bacteroidetes and reducing Firmicutes. At the genus level, Allobaculum (a major component of Erysipelotrichaceae) was significantly increased in the RF group, and this increase was canceled by SGL5213. We also measured the effect of SGL5213 on bacterial phenol-producing enzymes that catalyze tyrosine into phenol, following the reduction of phenyl sulfate, which is a novel marker and a therapeutic target for diabetic kidney disease DKD. We found that the enzyme inhibition was less potent, suggesting that the change in the microbial community and the reduction of uremic toxins may be related to the renoprotective effect of SGL5213. Because SGL5213 is a low-absorbable SGLT1 inhibitor, these data suggest that the gastrointestinal inhibition of SGLT1 is also a target for chronic kidney diseases.

Ndufs4 ablation decreases synaptophysin expression in hippocampus.

Altered function of mitochondrial respiratory chain in brain cells is related to many neurodegenerative diseases. NADH Dehydrogenase (Ubiquinone) Fe-S protein 4 (Ndufs4) is one of the subunits of mitochondrial complex I and its mutation in human is associated with Leigh syndrome. However, the molecular biological role of Ndufs4 in neuronal function is poorly understood. In this study, upon Ndufs4 expression confirmation in NeuN-positive neurons, and GFAP-positive astrocytes in WT mouse hippocampus, we found significant decrease of mitochondrial respiration in Ndufs4-KO mouse hippocampus. Although there was no change in the number of NeuN positive neurons in Ndufs4-KO hippocampus, the expression of synaptophysin, a presynaptic protein, was significantly decreased. To investigate the detailed mechanism, we silenced Ndufs4 in Neuro-2a cells and we observed shorter neurite lengths with decreased expression of synaptophysin. Furthermore, western blot analysis for phosphorylated extracellular regulated kinase (pERK) revealed that Ndufs4 silencing decreases the activity of ERK signalling. These results suggest that Ndufs4-modulated mitochondrial activity may be involved in neuroplasticity via regulating synaptophysin expression.

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.

Prediction of Functional Outcome in Patients with Acute Stroke by Measuring tRNA Derivatives.

BACKGROUND AND PURPOSE: Transfer RNA (tRNA) is a noncoding RNA that delivers amino acids to ribosomes for protein synthesis. tRNA is also involved in cell stress response programs. Oxidative stress induces direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation. Using an antibody against tRNA-specific modified nucleoside 1-methyladenosine (m1A), we can detect tRNA derivatives such as conformationally changed tRNA, tRNA-derived fragments, and mononucleotide-free m1A. Based on these findings, tRNA derivatives may have potential as an early tissue damage marker. The purpose of this study was to investigate the plasma tRNA derivatives in stroke patients to clarify whether tRNA derivatives in the acute phase can detect early brain damage and then predict the functional outcome. METHODS: Patients (75 patients with ischemic and 66 with hemorrhagic stroke) and 22 healthy volunteers were prospectively enrolled for this study between November 2016 and February 2019. Plasma samples were collected within 24 h and at 1 day, 7 days, and 30 days from the onset. Plasma tRNA derivative concentrations were measured by ELISA kit using the anti-m1A antibody. RESULTS: The plasma tRNA derivative level on admission was significantly increased in both ischemic (mean ± standard error, 232.2 ± 33.1 ng/mL) and hemorrhagic stroke patients (212 ± 23.4 ng/mL) compared to the healthy volunteers (86.0 ± 7.9 ng/mL) (p = 0.00042 and p = 0.00018, respectively). The infarction size (r = 0.445, p = 0.00018) and hematoma volumes (r = 0.33, p = 0.0072) were also significantly correlated with tRNA derivatives. The concentrations of tRNA derivatives were associated with poor functional outcome (Modified Rankin Scale score 3-6 at 30 days from the onset) in patients with ischemic stroke at 7 days after onset (p = 0.020). CONCLUSIONS: Stress-induced tRNA derivatives can detect brain tissue damage, predicting functional outcome in patients with ischemic stroke.

Mitochondrial dysfunction in GnRH neurons impaired GnRH production.

The onset establishment and maintenance of gonadotropin-releasing hormone (GnRH) secretion is an important phenomenon regulating pubertal development and reproduction. GnRH neurons as well as other neurons in the hypothalamus have high-energy demands and require a constant energy supply from their mitochondria machinery to maintain active functioning. However, the involvement of mitochondrial function in GnRH neurons is still unclear. In this study, we examined the role of NADH Dehydrogenase (Ubiquinone) Fe-S protein 4 (Ndufs4), a member of the mitochondrial complex 1, on GnRH neurons using Ndufs4-KO mice and Ndufs4-KO GT1-7 cells. Ndufs4 was highly expressed in GnRH neurons in the medial preoptic area (MPOA) and NPY/AgRP and POMC neurons in the arcuate (ARC) nucleus in WT mice. Conversely, there was a significant decrease in GnRH expression in MPOA and median eminence of Ndufs4-KO mice, followed by impaired peripheral endocrine system. In Ndufs4-KO GT1-7 cells, Gnrh1 expression was significantly decreased with or without stimulation with either kisspeptin or NGF, whereas, stimulation significantly increased Gnrh1 expression in control cells. In contrast, there was no difference in cell signaling activity including ERK and CREB as well as the expression of GPR54, TrkA and p75NTR, suggesting that Ndufs4 is involved in the transcriptional regulation system for GnRH production. These findings may be useful in understanding the mitochondrial function in GnRH neuron.

Germ-Free Conditions Modulate Host Purine Metabolism, Exacerbating Adenine-Induced Kidney Damage.

Alterations in microbiota are known to affect kidney disease conditions. We have previously shown that germ-free conditions exacerbated adenine-induced kidney damage in mice; however, the mechanism by which this occurs has not been elucidated. To explore this mechanism, we examined the influence of germ-free conditions on purine metabolism and renal immune responses involved in the kidney damage. Germ-free mice showed higher expression levels of purine-metabolizing enzymes such as xanthine dehydrogenase, which converts adenine to a nephrotoxic byproduct 2,8-dihydroxyadenine (2,8-DHA). The germ-free mice also showed increased urinary excretion of allantoin, indicating enhanced purine metabolism. Metabolome analysis demonstrated marked differences in the purine metabolite levels in the feces of germ-free mice and mice with microbiota. Furthermore, unlike the germ-free condition, antibiotic treatment did not increase the expression of purine-metabolizing enzymes or exacerbate adenine-induced kidney damage. Considering renal immune responses, the germ-free mice displayed an absence of renal IL-17A expression. However, the adenine-induced kidney damage in wild-type mice was comparable to that in IL-17A-deficient mice, suggesting that IL-17A does not play a major role in the disease condition. Our results suggest that the enhanced host purine metabolism in the germ-free mice potentially promotes the conversion of the administered adenine into 2,8-DHA, resulting in exacerbated kidney damage. This further suggests a role of the microbiota in regulating host purine metabolism.

The guanylate cyclase C agonist linaclotide ameliorates the gut-cardio-renal axis in an adenine-induced mouse model of chronic kidney disease.

BACKGROUND: Cardiorenal syndrome is a major cause of mortality in patients with chronic kidney disease (CKD). However, the involvement of detrimental humoral mediators in the pathogenesis of cardiorenal syndrome is still controversial. Trimethylamine-N-oxide (TMAO), a hepatic metabolic product of trimethylamine generated from dietary phosphatidylcholine or carnitine derived by the gut microbiota, has been linked directly with progression of cardiovascular disease and renal dysfunction. Thus, targeting TMAO may be a novel strategy for the prevention of cardiovascular disease and chronic kidney disease. METHODS: Linaclotide, a guanylate cyclase C agonist, was administered to adenine-induced renal failure (RF) mice and changes in renal function and levels of gut-derived uremic toxins, as well as the gut microbiota community, were analyzed using metabolomic and metagenomic methods to reveal its cardiorenal effect. RESULTS: Linaclotide decreased the plasma levels of TMAO at a clinically used low dose of 10 µg/kg in the adenine-induced RF mouse model. At a high concentration of 100 µg/kg, linaclotide clearly improved renal function and reduced the levels of various uremic toxins. A reduction in TMAO levels following linaclotide treatment was also observed in a choline-fed pro-atherosclerotic model. Linaclotide ameliorated renal inflammation and fibrosis and cardiac fibrosis, as well as decreased the expression of collagen I, transforming growth factor-ß, galectin-3 (Gal-3) and ST2 genes. Plasma levels of Gal-3 and ST2 were also reduced. Because exposure of cardiomyocytes to TMAO increased fibronectin expression, these data suggest that linaclotide reduced the levels of TMAO and various uremic toxins and may result in not only renal, but also cardiac, fibrosis. F4/80-positive macrophages were abundant in small intestinal crypts in RF mice, and this increased expression was decreased by linaclotide. Reduced colonic claudin-1 levels were also restored by linaclotide, suggesting that linaclotide ameliorated the 'leaky gut' in RF mice. Metagenomic analysis revealed that the microbial order Clostridiales could be responsible for the change in TMAO levels. CONCLUSION: Linaclotide reduced TMAO and uremic toxin levels and could be a powerful tool for the prevention and control of the cardiorenal syndrome by modification of the gut-cardio-renal axis.

Drugs Repurposed as Antiferroptosis Agents Suppress Organ Damage, Including AKI, by Functioning as Lipid Peroxyl Radical Scavengers.

BACKGROUND: Ferroptosis, nonapoptotic cell death mediated by free radical reactions and driven by the oxidative degradation of lipids, is a therapeutic target because of its role in organ damage, including AKI. Ferroptosis-causing radicals that are targeted by ferroptosis suppressors have not been unequivocally identified. Because certain cytochrome P450 substrate drugs can prevent lipid peroxidation via obscure mechanisms, we evaluated their antiferroptotic potential and used them to identify ferroptosis-causing radicals. METHODS: Using a cell-based assay, we screened cytochrome P450 substrate compounds to identify drugs with antiferroptotic activity and investigated the underlying mechanism. To evaluate radical-scavenging activity, we used electron paramagnetic resonance-spin trapping methods and a fluorescence probe for lipid radicals, NBD-Pen, that we had developed. We then assessed the therapeutic potency of these drugs in mouse models of cisplatin-induced AKI and LPS/galactosamine-induced liver injury. RESULTS: We identified various US Food and Drug Administration-approved drugs and hormones that have antiferroptotic properties, including rifampicin, promethazine, omeprazole, indole-3-carbinol, carvedilol, propranolol, estradiol, and thyroid hormones. The antiferroptotic drug effects were closely associated with the scavenging of lipid peroxyl radicals but not significantly related to interactions with other radicals. The elevated lipid peroxyl radical levels were associated with ferroptosis onset, and known ferroptosis suppressors, such as ferrostatin-1, also functioned as lipid peroxyl radical scavengers. The drugs exerted antiferroptotic activities in various cell types, including tubules, podocytes, and renal fibroblasts. Moreover, in mice, the drugs ameliorated AKI and liver injury, with suppression of tissue lipid peroxidation and decreased cell death. CONCLUSIONS: Although elevated lipid peroxyl radical levels can trigger ferroptosis onset, some drugs that scavenge lipid peroxyl radicals can help control ferroptosis-related disorders, including AKI.

The indole compound MA-35 attenuates tumorigenesis in an inflammation-induced colon cancer model.

In inflammatory bowel disease, chronic inflammation results in the development of colon cancer known as colitis-associated cancer. This disease is associated with tumor necrosis factor-α (TNF-α) signaling. In addition, intestinal fibrosis is a common clinical complication that is promoted by transforming growth factor ß1 (TGF-ß1). In our previous study, MA-35 attenuated renal fibrosis by inhibiting both TNF-α and TGF-ß1 signaling. This study aimed to identify the possible antitumor effects and antifibrotic effects of MA-35 using an AOM/DSS mouse model. MA-35 was orally administered every day for 70 days in the AOM/DSS mouse model. There was no difference in weight loss between the AOM/DSS group and the AOMDSS + MA-35 group, but the disease activity index score and the survival rate were improved by MA-35. MA-35 blocked the anemia and shortening of the colon induced by AOM/DSS. MA-35 reduced the macroscopic formation of tumors in the colon. In the microscopic evaluation, MA-35 reduced inflammation and fibrosis in areas with dysplasia. Furthermore, the TNF-α mRNA level in the colon tended to be reduced, and the interleukin 6, TGF-ß1 and fibronectin 1 mRNA levels in the colon were significantly reduced by MA-35. These results suggested that MA-35 inhibited AOM/DSS-induced carcinogenesis by reducing inflammation and fibrosis.

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.

Canagliflozin reduces plasma uremic toxins and alters the intestinal microbiota composition in a chronic kidney disease mouse model.

Accumulation of uremic toxins, which exert deleterious effects in chronic kidney disease, is influenced by the intestinal environment; the microbiota contributes to the production of representative uremic toxins, including p-cresyl sulfate and indoxyl sulfate. Canagliflozin is a sodium-glucose cotransporter (SGLT) 2 inhibitor, and it also exerts a modest inhibitory effect on SGLT1. The inhibition of intestinal SGLT1 can influence the gastrointestinal environment. We examined the effect of canagliflozin on the accumulation of uremic toxins in chronic kidney disease using adenine-induced renal failure mice. Two-week canagliflozin (10 mg/kg po) treatment did not influence the impaired renal function; however, it significantly reduced the plasma levels of p-cresyl sulfate and indoxyl sulfate in renal failure mice (a 75% and 26% reduction, respectively, compared with the vehicle group). Additionally, canagliflozin significantly increased cecal short-chain fatty acids in the mice, suggesting the promotion of bacterial carbohydrate fermentation in the intestine. Analysis of the cecal microbiota showed that canagliflozin significantly altered microbiota composition in the renal failure mice. These results indicate that canagliflozin exerts intestinal effects that reduce the accumulation of uremic toxins including p-cresyl sulfate. Reduction of accumulated uremic toxins by canagliflozin could provide a potential therapeutic option in chronic kidney disease.

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.

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.