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.

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.

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.

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.

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.

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.

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.

Mitochonic Acid 5 Binds Mitochondria and Ameliorates Renal Tubular and Cardiac Myocyte Damage.

Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroid-resistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.

Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD.

The accumulation of uremic toxins is involved in the progression of CKD. Various uremic toxins are derived from gut microbiota, and an imbalance of gut microbiota or dysbiosis is related to renal failure. However, the pathophysiologic mechanisms underlying the relationship between the gut microbiota and renal failure are still obscure. Using an adenine-induced renal failure mouse model, we evaluated the effects of the ClC-2 chloride channel activator lubiprostone (commonly used for the treatment of constipation) on CKD. Oral administration of lubiprostone (500 µg/kg per day) changed the fecal and intestinal properties in mice with renal failure. Additionally, lubiprostone treatment reduced the elevated BUN and protected against tubulointerstitial damage, renal fibrosis, and inflammation. Gut microbiome analysis of 16S rRNA genes in the renal failure mice showed that lubiprostone treatment altered their microbial composition, especially the recovery of the levels of the Lactobacillaceae family and Prevotella genus, which were significantly reduced in the renal failure mice. Furthermore, capillary electrophoresis-mass spectrometry-based metabolome analysis showed that lubiprostone treatment decreased the plasma level of uremic toxins, such as indoxyl sulfate and hippurate, which are derived from gut microbiota, and a more recently discovered uremic toxin, trans-aconitate. These results suggest that lubiprostone ameliorates the progression of CKD and the accumulation of uremic toxins by improving the gut microbiota and intestinal environment.

Exonic mutations in the SLC12A3 gene cause exon skipping and premature termination in Gitelman syndrome.

A variety of genetic backgrounds cause the loss of function of thiazide-sensitive sodium chloride cotransporter, encoded by SLC12A3, responsible for the phenotypes in Gitelman syndrome. Recently, the phenomenon of exon skipping, in which exonic mutations result in abnormal splicing, has been associated with various diseases. Specifically, mutations in exonic splicing enhancer (ESE) sequences can promote exon skipping. Here, we used a bioinformatics program to analyze 88 missense mutations in the SLC12A3 gene and identify candidate mutations that may induce exon skipping. The three candidate mutations that reduced ESE scores the most were further investigated by minigene assay, and two (p.A356V and p.M672I) caused abnormal splicing in vitro. Furthermore, we identified the p.M672I (c.2016G>A) mutation in a patient with Gitelman syndrome and found that this single nucleotide mutation causes exclusion of exon 16 in the SLC12A3 mRNA transcript. Functional analyses revealed that the protein encoded by the aberrant SLC12A3 transcript does not transport sodium. These results suggest that aberrant exon skipping is one previously unrecognized mechanism by which missense mutations in SLC12A3 can lead to Gitelman syndrome.

Mitochonic Acid 5 (MA-5), a Derivative of the Plant Hormone Indole-3-Acetic Acid, Improves Survival of Fibroblasts from Patients with Mitochondrial Diseases.

Mitochondria are key organelles implicated in a variety of processes related to energy and free radical generation, the regulation of apoptosis, and various signaling pathways. Mitochondrial dysfunction increases cellular oxidative stress and depletes ATP in a variety of inherited mitochondrial diseases and also in many other metabolic and neurodegenerative diseases. Mitochondrial diseases are characterized by the dysfunction of the mitochondrial respiratory chain, caused by mutations in the genes encoded by either nuclear DNA or mitochondrial DNA. We have hypothesized that chemicals that increase the cellular ATP levels may ameliorate the mitochondrial dysfunction seen in mitochondrial diseases. To search for the potential drugs for mitochondrial diseases, we screened an in-house chemical library of indole-3-acetic-acid analogs by measuring the cellular ATP levels in Hep3B human hepatocellular carcinoma cells. We have thus identified mitochonic acid 5 (MA-5), 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid, as a potential drug for enhancing ATP production. MA-5 is a newly synthesized derivative of the plant hormone, indole-3-acetic acid. Importantly, MA-5 improved the survival of fibroblasts established from patients with mitochondrial diseases under the stress-induced condition, including Leigh syndrome, MELAS (myopathy encephalopathy lactic acidosis and stroke-like episodes), Leber's hereditary optic neuropathy, and Kearns-Sayre syndrome. The improved survival was associated with the increased cellular ATP levels. Moreover, MA-5 increased the survival of mitochondrial disease fibroblasts even under the inhibition of the oxidative phosphorylation or the electron transport chain. These data suggest that MA-5 could be a therapeutic drug for mitochondrial diseases that exerts its effect in a manner different from anti-oxidant therapy.

Conformational change in transfer RNA is an early indicator of acute cellular damage.

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.

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.

Metabolomic profiling of the autosomal dominant polycystic kidney disease rat model.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is an inherited systemic disease characterized by renal cyst expansion, resulting in renal failure. With the progression of renal damage, the accumulation of uremic compounds is recently reported to subsequently cause further renal damage and hypertension. Finding uremic toxins and sensitive markers for detecting the early stage of ADPKD is necessary to clarify its pathophysiological process and to prevent its progression. The aim of this study was to analyze the profile of uremic retention solutes of ADPKD by capillary electrophoresis-mass spectrometry (CE-MS) using the Han:SPRD rat model. METHODS: Two hundred and ninety-seven cations and 190 anions were comprehensively analyzed by CE-MS in Han:SPRD rats and control rats. RESULTS: We found 21 cations and 19 anions that accumulated significantly in the heterozygous (Cy/+) ADPKD rat model compared with control rats. Among the compounds, increases in 5-methyl-2'-deoxycytidine, glucosamine, ectoine, allantoate, α-hydroxybenzoate, phenaceturate and 3-phenylpropionate and decreases in 2-deoxycytidine, decanoate and 10-hydroxydecanoate were newly identified in the ADPKD Cy/+ rats. CONCLUSION: We identified uremic retention solutes in ADPKD Cy/+ rats. Compounds related to ADPKD could be useful markers for detecting the early stage of ADPKD.

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.

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.

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.