Insights into the gut-kidney axis and implications for chronic kidney disease management in cats and dogs.

Chronic kidney disease (CKD) in cats and dogs presents significant clinical challenges, with emerging research highlighting the pivotal role of the gut-kidney axis in its pathogenesis and management. Gut dysbiosis, characterized by alterations in the gut microbiome composition and function, contributes to microbial dysmetabolism of key nutrients causing uremic toxin accumulation and disruptions in amino acid, bile acid and fatty acid profiles. These disturbances in turn exacerbate renal dysfunction and systemic inflammation. Recent research in veterinary medicine, particularly in cats, supports the gut microbiome and microbial-derived metabolites as novel therapeutic targets. Potential therapeutic strategies targeting the gut microbiome and microbial dysmetabolism, including dietary management, probiotics, adsorbents, and addressing constipation, offer promising avenues for intervention to restore metabolic balance and preserve renal function. This review highlights the microbial influence on renal health and focuses on potential therapeutic strategies available to veterinarians to optimize the management of CKD in cats and dogs.

Microbe-derived uremic solutes enhance thrombosis potential in the host.

p-Cresol sulfate (pCS) and indoxyl sulfate (IS), gut microbiome-derived metabolites, are traditionally associated with cardiovascular disease (CVD) risks in the setting of impaired kidney function. While pharmacologic provision of pCS or IS can promote pro-thrombotic phenotypes, neither the microbial enzymes involved nor direct gut microbial production have been linked to CVD. Untargeted metabolomics was performed on a discovery cohort (n = 1,149) with relatively preserved kidney function, followed by stable isotope-dilution mass spectrometry quantification of pCS and IS in an independent validation cohort (n = 3,954). Genetic engineering of human commensals to produce p-cresol and indole gain-of-function and loss-of-function mutants, followed by colonization of germ-free mice, and studies on host thrombosis were performed. Systemic pCS and IS levels were independently associated with all-cause mortality. Both in vitro and within colonized germ-free mice p-cresol productions were recapitulated by collaboration of two organisms: a Bacteroides strain that converts tyrosine to 4-hydroxyphenylacetate, and a Clostridium strain that decarboxylates 4-hydroxyphenylacetate to p-cresol. We then engineered a single organism, Bacteroides thetaiotaomicron, to produce p-cresol, indole, or both metabolites. Colonizing germ-free mice with engineered strains, we show the gut microbial genes for p-cresol (hpdBCA) and indole (tryptophanase) are sufficient to confer a pro-thrombotic phenotype in vivo. Moreover, human fecal metagenomics analyses show that abundances of hpdBCA and tryptophanase are associated with CVD. These studies show that pCS and IS, two abundant microbiome-derived metabolites, play a broader potential role in CVD than was previously known. They also suggest that therapeutic targeting of gut microbial p-cresol- and indole-producing pathways represent rational targets for CVD.IMPORTANCEAlterations in gut microbial composition and function have been linked to numerous diseases. Identifying microbial pathways responsible for producing molecules that adversely impact the host is an important first step in the development of therapeutic interventions. Here, we first use large-scale clinical observations to link blood levels of defined microbial products to cardiovascular disease risks. Notably, the previously identified uremic toxins p-cresol sulfate and indoxyl sulfate were shown to predict 5-year mortality risks. After identifying the microbes and microbial enzymes involved in the generation of these uremic toxins, we used bioengineering technologies coupled with colonization of germ-free mice to show that the gut microbial genes that generate p-cresol and indole are sufficient to confer p-cresol sulfate and indoxyl sulfate formation, and a pro-thrombotic phenotype in vivo. The findings and tools developed serve as a critical step in both the study and targeting of these gut microbial pathways in vivo.

p-Cresol Sulfate Is a Sensitive Urinary Marker of Fecal Microbiota Transplantation and Antibiotics Treatments in Human Patients and Mouse Models.

Fecal microbiota transplantation (FMT) has emerged as a highly effective therapy for recurrent Clostridioides difficile infection (rCDI) and also a potential therapy for other diseases associated with dysbiotic gut microbiota. Monitoring metabolic changes in biofluids and excreta is a noninvasive approach to identify the biomarkers of microbial recolonization and to understand the metabolic influences of FMT on the host. In this study, the pre-FMT and post FMT urine samples from 11 rCDI patients were compared through metabolomic analyses for FMT-induced metabolic changes. The results showed that p-cresol sulfate in urine, a microbial metabolite of tyrosine, was rapidly elevated by FMT and much more responsive than other microbial metabolites of aromatic amino acids (AAAs). Because patients were treated with vancomycin prior to FMT, the influence of vancomycin on the microbial metabolism of AAAs was examined in a mouse feeding trial, in which the decreases in p-cresol sulfate, phenylacetylglycine, and indoxyl sulfate in urine were accompanied with significant increases in their AAA precursors in feces. The inhibitory effects of antibiotics and the recovering effects of FMT on the microbial metabolism of AAAs were further validated in a mouse model of FMT. Overall, urinary p-cresol sulfate may function as a sensitive and convenient therapeutic indicator on the effectiveness of antibiotics and FMT for the desired manipulation of gut microbiota in human patients.

SLC22A11 Inserts the Uremic Toxins Indoxyl Sulfate and P-Cresol Sulfate into the Plasma Membrane.

Chronic kidney disease (CKD) is a global health concern affecting millions worldwide. One of the critical challenges in CKD is the accumulation of uremic toxins such as p-cresol sulfate (pCS) and indoxyl sulfate (IS), which contribute to systemic damage and CKD progression. Understanding the transport mechanisms of these prominent toxins is essential for developing effective treatments. Here, we investigated whether pCS and IS are routed to the plasma membrane or to the cytosol by two key transporters, SLC22A11 and OAT1. To distinguish between cytosolic transport and plasma membrane insertion, we used a hyperosmolarity assay in which the accumulation of substrates into HEK-293 cells in isotonic and hypertonic buffers was measured in parallel using LC-MS/MS. Judging from the efficiency of transport (TE), pCS is a relevant substrate of SLC22A11 at 7.8 ± 1.4 µL min-1 mg protein-1 but not as good as estrone-3-sulfate; OAT1 translocates pCS less efficiently. The TE of SLC22A11 for IS was similar to pCS. For OAT1, however, IS is an excellent substrate. With OAT1 and p-aminohippuric acid, our study revealed an influence of transporter abundance on the outcomes of the hyperosmolarity assay; very high transport activity confounded results. SLC22A11 was found to insert both pCS and IS into the plasma membrane, whereas OAT1 conveys these toxins to the cytosol. These disparate transport mechanisms bear profound ramifications for toxicity. Membrane insertion might promote membrane damage and microvesicle release. Our results underscore the imperative for detailed structural inquiries into the translocation of small molecules.

Total analysis system for the determination of uremic toxins in human plasma based on bead injection solid phase extraction hyphenated to mass spectrometry.

Indoxyl sulfate (INDS) and p-cresol sulfate (pCS) are two of the most relevant uremic toxins that are recognized to have an essential role in chronic kidney disease (CKD) progression and associated cardiovascular risk. Thus, it is crucial to accurately assess their circulating levels in the body. Aiming at establishing an analytical strategy for quantification of INDS and pCS in human plasma, an automatic on-line micro-solid-phase extraction (µSPE) procedure hyphenated to tandem mass spectrometry (MS/MS) detection without previous chromatographic separation was herein developed. The bead injection (BI) concept was used to implement the µSPE procedure in the lab-on-valve (LOV) format. After studying the extraction conditions, the anion-exchange OASIS WAX sorbent beads (10 mg) and 99% ACN-H2O (15:85, v/v)-1% (v/v) NH4OH were chosen as sorbent and eluent, respectively, as they provided the highest analyte recoveries. Subsequently, the µSPE-BI-LOV system was hyphenated on-line to a MS/MS detector and the full analytical cycle, comprising sample preparation and analytes detection, was completed in <20 min. The developed µSPE-BI-LOV-MS methodology presented good linearity (r2 > 0.999) for quantification of the target analytes at concentrations ranging from 18 to 360 µg mL-1 in plasma. LOQ values were 2 µg mL-1 for INDS and 7 µg mL-1 for pCS in plasma. Human plasma samples from healthy subjects and individuals with CKD were successfully analyzed using the developed approach. The proposed automatic methodology can be described as an eco-friendly strategy, with a favorable score of 0.64 after greenness evaluation using the AGREE metric.

The Effect of Dietary Protein Concentration on the Fecal Microbiome and Serum Concentrations of Gut-Derived Uremic Toxins in Healthy Adult Cats.

The purpose of this study was to evaluate the effect of feeding healthy adult cats with foods containing variable protein concentrations on the fecal microbiome and serum concentrations of the gut-derived uremic toxins indoxyl sulfate, p-cresol sulfate (pCS), and trimethylamine-n-oxide. Twenty healthy young adult cats were randomized into two groups and fed either a low-protein diet (LPD; 7.4 g/100 kcal ME) or a high-protein diet (HPD; 11.0 g/100 kcal ME) for a 12-week period. Serum uremic toxin concentrations were measured via liquid chromatography tandem mass spectrometry, and the fecal microbiome was characterized using shallow sequence shotgun metagenomics. Cats that consumed the HPD had higher pCS concentrations at 8 weeks (p = 0.028) when compared to baseline. After 12 weeks, cats fed the HPD had higher fecal alpha diversity indices at both the taxonomic and functional levels and lower fecal Bifidobacterium relative abundance compared to those cats fed the LPD. In conclusion, a change in diet and dietary protein concentration shifted the fecal microbial community and microbial function. Feeding cats a high amount of protein increased serum concentrations of the uremic toxin pCS; however, the effect was short-lived.

Berberine ameliorates chronic kidney disease through inhibiting the production of gut-derived uremic toxins in the gut microbiota.

At present, clinical interventions for chronic kidney disease are very limited, and most patients rely on dialysis to sustain their lives for a long time. However, studies on the gut-kidney axis have shown that the gut microbiota is a potentially effective target for correcting or controlling chronic kidney disease. This study showed that berberine, a natural drug with low oral availability, significantly ameliorated chronic kidney disease by altering the composition of the gut microbiota and inhibiting the production of gut-derived uremic toxins, including p-cresol. Furthermore, berberine reduced the content of p-cresol sulfate in plasma mainly by lowering the abundance of g_Clostridium_sensu_stricto_1 and inhibiting the tyrosine-p-cresol pathway of the intestinal flora. Meanwhile, berberine increased the butyric acid producing bacteria and the butyric acid content in feces, while decreased the renal toxic trimethylamine N-oxide. These findings suggest that berberine may be a therapeutic drug with significant potential to ameliorate chronic kidney disease through the gut-kidney axis.

In-situ synchrotron imaging, experimental, and computational investigations on the efficiency of Trametes versicolor laccase on detoxification of P-Cresyl Sulfate (PCS) Protein Bound Uremic Toxin (PBUT).

Protein bound uremic toxins (PBUTs) are small substances binding to larger proteins, mostly human serum albumin (HSA), and are challenging to remove by hemodialysis (HD). Among different classes of PBUTs, p-cresyl sulfate (PCS) is the most widely used marker molecule and major toxin, as 95 % is bound to HSA. PCS has a pro-inflammatory effect and increases both the uremia symptom score and multiple pathophysiological activities. High-flux HD to clear PCS leads to serious loss of HSA, which results in a high mortality rate. The goal of the present study is to investigate the efficacy of PCS detoxification in serum of HD patients using a biocompatible laccase enzyme from Trametes versicolor. Molecular docking was used to gain an in-depth understanding of the interactions between PCS and the laccase to identify the functional group(s) responsible for ligand-protein receptor interactions. UV-Vis spectroscopy and gas chromatography-mass spectrometry (GC-MS) were used to assess the detoxification of PCS. GC-MS was used to identify the detoxification byproducts and their toxicity was assessed using docking commutations. In situ synchrotron radiation micro-computed tomography (SR-µCT) imaging available at the Canadian Light Source (CLS) was conducted to assess HSA binding with PCS before and after detoxification with laccase and undertake the corresponding quantitative analysis. GC-MS analyses confirmed the detoxification of PCS with laccase at a concentration of 500 mg/L. The potential pathway of PCS detoxification in the presence of the laccase was identified. Increasing laccase concentration led to the formation of m-cresol, as indicated by the corresponding absorption in the UV-Vis spectra and a sharp peak on the GC-MS spectra. Our analysis provides insight into the general features of PCS binding on Sudlow site II, as well as insights into PCS detoxification product interactions. The average affinity energy for detoxification products was lower than that of PCS. Even though some byproducts showed potential toxicity, the level was lower than for PCS based on toxicity indexes (e.g., LD50/LC50, carcinogenicity, neurotoxicity, mutagenicity). In addition, these small compounds can also be more easily removed by HD compared to PCS. SR-µCT quantitative analysis showed adhesion of the HSA to a significant reduced extent in the presence of the laccase enzyme in bottom sections of the polyarylethersulfone (PAES) clinical HD membrane tested. Overall, this study opens new frontiers for PCS detoxification.

Serum Level of Protein-Bound Uraemic Toxins in Haemodialysis Patients with Chronic Kidney Disease-Associated Pruritus: Myths and Facts.

Recent studies place great importance on Protein-Bound Uraemic Toxins (PBUT) in the context of etiopathogenesis of chronic kidney disease-associated pruritus (CKD-aP). This study aimed to investigate the possible contribution of free and total Indoxyl Sulfate (IS) and p-Cresol Sulfate (PCS) to the cause of CKD-aP. Group A included 64 patients on maintenance haemodialysis (HD) with CKD-aP. Group B included 62 patients on maintenance HD that did not report CKD-aP, and group C included 50 healthy controls. Pruritus severity was assessed using a Numerical Rating Scale (NRS). Moreover, other tools like UP-Dial, ItchyQoL, and the 4-Item Itch Questionnaire evaluating CKD-aP were completed by the patients. The serum levels of free and total IS and PCS concentrations were measured using the Ultra Performance Liquid Chromatography System. No significant difference in the serum level of free and total IS, or PCS, was observed between the patients who reported CKD-aP and those without pruritus. Moreover, there was no correlation between serum IS or PCS levels and the severity of the itch. Our study does not support earlier findings about higher levels of IS and PCS in patients reporting CKD-aP. Further studies will be needed to investigate these discrepancies as well as to understand the cause of CKD-aP.

Sample preparation and chromatographic methods for the determination of protein-bound uremic retention solutes in human biological samples: An overview.

Protein-bound uremic retention solutes, such as indole-3-acetic acid, indoxyl sulfate, p-cresol and p-cresol sulfate, are associated with the development of several pathologies, namely renal, cardiovascular, and bone toxicities, due to their potential accumulation in the human body, thus requiring analytical methods for monitoring and evaluation. The present review addresses conventional and advanced sample treatment procedures for sample handling and the chromatographic analytical methods developed for quantification of these compounds in different biological fluids, with particular focus on plasma, serum, and urine. The sample preparation and chromatographic methods coupled to different detection systems are critically discussed, focusing on the different steps involved for sample treatment, namely elimination of interfering compounds present in the sample matrix, and the evaluation of their environmental impact through the AGREEprep tool. There is a clear trend for the application of liquid-chromatography coupled to tandem mass spectrometry, which requires protein precipitation, solid-phase extraction and/or dilution prior to analysis of biological samples. Furthermore, from a sustainability point of view, miniaturized methods resorting to microplate devices are highly recommended.

Dietary strategies for gut-derived protein-bound uremic toxins and cardio-metabolic risk factors in chronic kidney disease: A focus on dietary fibers.

Chronic kidney disease (CKD) is associated with altered composition and function of gut microbiota. The cause of gut dysbiosis in CKD is multifactorial and encompasses the following: uremic state, metabolic acidosis, slow colonic transit, dietary restrictions of plant-based fiber-rich foods, and pharmacological therapies. Dietary restriction of potassium-rich fruits and vegetables, which are common sources of fermentable dietary fibers, inhibits the conversion of dietary fibers to short-chain fatty acids (SCFA), which are the primary nutrient source for the symbiotic gut microbiota. Reduced consumption of fermentable dietary fibers limits the population of SCFA-forming bacteria and causes dysbiosis of gut microbiota. Gut dysbiosis induces colonic fermentation of protein and formation of gut-derived uremic toxins. In this review, we discuss the roles and benefits of dietary fiber on gut-derived protein-bound uremic toxins and plant-based dietary patterns that could be recommended to decrease uremic toxin formation in CKD patients. Recent studies have indicated that dietary fiber supplementation may be useful to decrease gut-derived uremic toxin formation and slow CKD progression. However, research on associations between adherence of healthy dietary patterns and gut-derived uremic toxins formation in patients with CKD is lacking.

Rapid and sustainable HPLC method for the determination of uremic toxins in human plasma samples.

Protein-bound uremic toxins, mainly indoxyl sulfate (3-INDS), p-cresol sulfate (pCS), and indole-3-acetic acid (3-IAA) but also phenol (Pol) and p-cresol (pC), are progressively accumulated during chronic kidney disease (CKD). Their accurate measurement in biomatrices is demanded for timely diagnosis and adoption of appropriate therapeutic measures. Multianalyte methods allowing the establishment of a uremic metabolite profile are still missing. Hence, the aim of this work was to develop a rapid and sensitive method based on high-performance liquid chromatography with fluorescence detection for the simultaneous quantification of Pol, 3-IAA, pC, 3-INDS, and pCS in human plasma. Separation was attained in 12 min, using a monolithic C18 column and isocratic elution with acetonitrile and phosphate buffer containing an ion-pairing reagent, at a flow rate of 2 mL min-1. Standards were prepared in plasma and quantification was performed using the background subtraction approach. LOQ values were ≤ 0.2 µg mL-1 for all analytes except for pCS (LOQ of 2 µg mL-1). The method proved to be accurate (93.5-112%) and precise (CV ≤ 14.3%). The multianalyte application of the method, associated to a reduced sample volume (50 µL), a less toxic internal standard (eugenol) in comparison to the previously applied 2,6-dimethylphenol and 4-ethylphenol, and a green extraction solvent (ethanol), resulted in the AGREE score of 0.62 which is in line with the recent trend of green and sustainable analytical chemistry. The validated method was successfully applied to the analysis of plasma samples from control subjects exhibiting normal levels of uremic toxins and CKD patients presenting significantly higher levels of 3-IAA, pC, 3-INDS, and pCS that can be further investigated as biomarkers of disease progression.

Berberine ameliorates chronic kidney disease through inhibiting the production of gut-derived uremic toxins in the gut microbiota

At present, clinical interventions for chronic kidney disease are very limited, and most patients rely on dialysis to sustain their lives for a long time. However, studies on the gut-kidney axis have shown that the gut microbiota is a potentially effective target for correcting or controlling chronic kidney disease. This study showed that berberine, a natural drug with low oral availability, significantly ameliorated chronic kidney disease by altering the composition of the gut microbiota and inhibiting the production of gut-derived uremic toxins, including p-cresol. Furthermore, berberine reduced the content of p-cresol sulfate in plasma mainly by lowering the abundance of g_Clostridium_sensu_stricto_1 and inhibiting the tyrosine-p-cresol pathway of the intestinal flora. Meanwhile, berberine increased the butyric acid producing bacteria and the butyric acid content in feces, while decreased the renal toxic trimethylamine N-oxide. These findings suggest that berberine may be a therapeutic drug with significant potential to ameliorate chronic kidney disease through the gut-kidney axis.

The Clostridium Metabolite P-Cresol Sulfate Relieves Inflammation of Primary Biliary Cholangitis by Regulating Kupffer Cells.

OBJECTIVE: To study the effect and mechanism of the Clostridium metabolite p-Cresol sulfate (PCS) in primary biliary cholangitis (PBC). METHODS: Gas chromatography-mass spectrometry (GC-MS) was used to detect differences in tyrosine, phenylalanine, tryptophan, PCS, and p-Cresyl glucuronide (PCG) between the serum of PBC patients and healthy controls. In vivo experiments, mice were divided into the normal control, PBC group, and PBC tyrosine group. GC-MS was used to detect PCS and PCG. Serum and liver inflammatory factors were compared between groups along with the polarization of liver Kupffer cells. Additionally, PCS was cultured with normal bile duct epithelial cells and Kupffer cells, respectively. PCS-stimulated Kupffer cells were co-cultured with lipopolysaccharide-injured bile duct epithelial cells to detect changes in inflammatory factors. RESULTS: Levels of tyrosine and phenylalanine were increased, but PCS level was reduced in PBC patients, with PCG showing a lower concentration distribution in both groups. PCS in PBC mice was also lower than those in normal control mice. After oral administration of tyrosine feed to PBC mice, PCS increased, liver inflammatory factors were decreased, and anti-inflammatory factors were increased. Furthermore, Kupffer cells in the liver polarized form M1 transitioned to M2. PCS can damage normal bile duct epithelial cells and suppress the immune response of Kupffer cells. But PCS protects bile duct epithelial cells damaged by LPS through Kupffer cells. CONCLUSIONS: PCS produced by Clostridium-metabolized tyrosine reduced PBC inflammation, suggesting that intervention by food, or supplementation with PCS might represent an effective clinical strategy for treating PBC.

Role of the Gut Microbiome in Skeletal Muscle Physiology and Pathophysiology.

PURPOSE OF REVIEW: This review aims to summarize the recent findings about the contribution of the gut microbiome to muscle pathophysiology and discuss molecular pathways that may be involved in such process. Related findings in the context of cancer cachexia are outlined. RECENT FINDINGS: Many bacterial metabolites have been reported to exert a beneficial or detrimental impact on muscle physiology. Most of the evidence concentrates on short-chain fatty acids (SCFAs), with an emerging role for bile acids, bacterial amino acid metabolites (bAAms), and bacterial polyphenol metabolites. Other molecular players worth considering include cytokines, hormones, lipopolysaccharides, and quorum sensing molecules. The current literature clearly establishes the ability for the gut microbiome to modulate muscle function and mass. The understanding of the mechanisms underlying this gut-muscle axis may lead to the delivery of novel therapeutic tools to tackle muscle wasting in cancer cachexia, chronic kidney disease, liver fibrosis, and age-related sarcopenia.

The Role of Bacterial-Derived Aromatic Amino Acids Metabolites Relevant in Autism Spectrum Disorders: A Comprehensive Review.

In recent years, the idea of the gut microbiota being involved in the pathogenesis of autism spectrum disorders (ASD) has attracted attention through numerous studies. Many of these studies report microbial dysregulation in the gut and feces of autistic patients and in ASD animal models. The host microbiota plays a large role in metabolism of ingested foods, and through the production of a range of metabolites it may be involved in neurodevelopmental disorders such as ASD. Two specific microbiota-derived host metabolites, p-cresol sulfate and 4-ethylphenyl sulfate, have been associated with ASD in both patients and animal models. These metabolites originate from bacterially produced p-cresol and 4-ethylphenol, respectively. p-Cresol and 4-ethylphenol are produced through aromatic amino acid fermentation by a range of commensal bacteria, most notably bacteria from the Clostridioides genus, which are among the dysregulated bacteria frequently detected in ASD patients. Once produced, these metabolites are suggested to enter the bloodstream, pass the blood-brain-barrier and affect microglial cells in the central nervous system, possibly affecting processes like neuroinflammation and microglial phagocytosis. This review describes the current knowledge of microbial dysbiosis in ASD and elaborates on the relevance and synthesis pathways of two specific ASD-associated metabolites that may form a link between the microbiota and the brain in autism. While the two discussed metabolites are promising candidates for biomarkers and (nutritional) intervention targets, more research into the role of these metabolites in ASD is required to causally connect these metabolites to ASD pathophysiology.

Effects of p-Cresol on Oxidative Stress, Glutathione Depletion, and Necrosis in HepaRG Cells: Comparisons to Other Uremic Toxins and the Role of p-Cresol Glucuronide Formation.

The toxicological effects of p-cresol have primarily been attributed to its metabolism products; however, very little human data are available in the key organ (i.e., liver) responsible for the generation of these metabolites. Experiments were conducted in HepaRG cells utilizing the following markers of cellular toxicity: 2'-7'-dichlorofluorescein (DCF; oxidative stress) formation, total cellular glutathione (GSH) concentration, and lactate dehydrogenase (LDH; cellular necrosis) release. Concentrations of p-cresol, p-cresol sulfate, and p-cresol glucuronide were determined using validated assays. p-Cresol exposure resulted in concentration- and time-dependent changes in DCF (EC50 = 0.64 ± 0.37 mM at 24 h of exposure) formation, GSH (EC50 = 1.00 ± 0.07 mM) concentration, and LDH (EC50 = 0.85 ± 0.14 mM) release at toxicologically relevant conditions. p-Cresol was also relatively more toxic than 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid, and hippuric acid on all markers. Although the exogenous administration of p-cresol sulfate and p-cresol glucuronide generated high intracellular concentrations of these metabolites, both metabolites were less toxic compared to p-cresol at equal-molar conditions. Moreover, p-cresol glucuronide was the predominant metabolite generated in situ from p-cresol exposure. Selective attenuation of glucuronidation (without affecting p-cresol sulfate formation, while increasing p-cresol accumulation) using independent chemical inhibitors (i.e., 0.75 mM l-borneol, 75 µM amentoflavone, or 100 µM diclofenac) consistently resulted in further increases in LDH release associated with p-cresol exposure (by 28.3 ± 5.3%, 30.0 ± 8.2% or 27.3 ± 6.8%, respectively, compared to p-cresol treatment). These novel data indicated that p-cresol was a relatively potent toxicant, and that glucuronidation was unlikely to be associated with the manifestation of its toxic effects in HepaRG cells.

Thrombolome and Its Emerging Role in Chronic Kidney Diseases.

Patients with chronic kidney disease (CKD) are at an increased risk of thromboembolic complications, including myocardial infarction, stroke, deep vein thrombosis, and pulmonary embolism. These complications lead to increased mortality. Evidence points to the key role of CKD-associated dysbiosis and its effect via the generation of gut microbial metabolites in inducing the prothrombotic phenotype. This phenomenon is known as thrombolome, a panel of intestinal bacteria-derived uremic toxins that enhance thrombosis via increased tissue factor expression, platelet hyperactivity, microparticles release, and endothelial dysfunction. This review discusses the role of uremic toxins derived from gut-microbiota metabolism of dietary tryptophan (indoxyl sulfate (IS), indole-3-acetic acid (IAA), kynurenine (KYN)), phenylalanine/tyrosine (p-cresol sulfate (PCS), p-cresol glucuronide (PCG), phenylacetylglutamine (PAGln)) and choline/phosphatidylcholine (trimethylamine N-oxide (TMAO)) in spontaneously induced thrombosis. The increase in the generation of gut microbial uremic toxins, the activation of aryl hydrocarbon (AhRs) and platelet adrenergic (ARs) receptors, and the nuclear factor kappa B (NF-κB) signaling pathway can serve as potential targets during the prevention of thromboembolic events. They can also help create a new therapeutic approach in the CKD population.

Characterization of human sulfotransferases catalyzing the formation of p-cresol sulfate and identification of mefenamic acid as a potent metabolism inhibitor and potential therapeutic agent for detoxification.

p-Cresol sulfate, the primary metabolite of p-cresol, is a uremic toxin that has been associated with toxicities and mortalities. The study objectives were to i) characterize the contributions of human sulfotransferases (SULT) catalyzing p-cresol sulfate formation using multiple recombinant SULT enzymes (including the polymorphic variant SULT1A1*2), pooled human liver cytosols, and pooled human kidney cytosols; and ii) determine the potencies and mechanisms of therapeutic inhibitors capable of attenuating the production of p-cresol sulfate. Human recombinant SULT1A1 was the primary enzyme responsible for the formation of p-cresol sulfate (Km = 0.19 ±â€¯0.02 µM [with atypical kinetic behavior at lower substrate concentrations; see text discussion], Vmax = 789.5 ±â€¯101.7 nmol/mg/min, Ksi = 2458.0 ±â€¯332.8 µM, mean ±â€¯standard deviation, n = 3), while SULT1A3, SULT1B1, SULT1E1, and SULT2A1 contributed negligible or minor roles at toxic p-cresol concentrations. Moreover, human recombinant SULT1A1*2 exhibited reduced enzyme activities (Km = 81.5 ±â€¯31.4 µM, Vmax = 230.6 ±â€¯17.7 nmol/mg/min, Ksi = 986.0 ±â€¯434.4 µM) compared to the wild type. The sulfonation of p-cresol was characterized by Michaelis-Menten kinetics in liver cytosols (Km = 14.8 ±â€¯3.4 µM, Vmax = 1.5 ±â€¯0.2 nmol/mg/min) and substrate inhibition in kidney cytosols (Km = 0.29 ±â€¯0.02 µM, Vmax = 0.19 ±â€¯0.05 nmol/mg/min, Ksi = 911.7 ±â€¯278.4 µM). Of the 14 investigated therapeutic inhibitors, mefenamic acid (Ki = 2.4 ±â€¯0.1 nM [liver], Ki = 1.2 ±â€¯0.3 nM [kidney]) was the most potent in reducing the formation of p-cresol sulfate, exhibiting noncompetitive inhibition in human liver cytosols and recombinant SULT1A1, and mixed inhibition in human kidney cytosols. Our novel findings indicated that SULT1A1 contributed an important role in p-cresol sulfonation (hence it can be considered a probe reaction) in liver and kidneys, and mefenamic acid may be utilized as a potential therapeutic agent to attenuate the generation of p-cresol sulfate as an approach to detoxification.

Biological variation of major gut-derived uremic toxins in the serum of healthy adult cats.

BACKGROUND: Biological variation of serum indoxyl sulfate (IS), p-cresol sulfate (pCS), and trimethylamine-n-oxide (TMAO) concentrations in cats is unknown. OBJECTIVES: To determine short- and medium-term biological variation, index of individuality (II), and reference change values for serum IS, pCS, and TMAO concentrations in healthy adult cats. To determine the effect of feeding on serum concentrations. ANIMALS: Twelve healthy adult cats. METHODS: Prospective, cohort study. Seven serum samples over a 12-hour period (short-term) and 5 serum samples over a 19-day period (medium-term) were collected. Serum concentrations of total IS, pCS, and TMAO were measured every 2 hours in a 12-hour period (hours 0-12) after a meal in 9 cats and compared to concentrations in a nonfed state. RESULTS: For IS, the II was high using short-term (1.96) and low using medium-term (0.65) biological variation estimates. Individuality was intermediate for pCS (short-term, 0.98; medium-term, 1.17) and TMAO (short-term, 1.47; medium-term, 0.83). Serum IS, pCS, and TMAO concentrations were significantly lower in a fed state compared to a nonfed state at hours 4, 6, 8, and 12; at hours 4 and 6; and at hours 2, 4, 6, 8, 10, 12, respectively. CONCLUSION AND CLINICAL IMPORTANCE: Population-based reference intervals with reference to the subject-based interval can be used to monitor serum pCS and TMAO concentrations. For IS, a subject-based and a population-based reference interval is best for short-term and medium-term monitoring, respectively. To compare serial measurements, it would be prudent to collect samples at the same time of day and consistently in either a fed or nonfed state.