Indoxyl Sulfate-Induced Macrophage Toxicity and Therapeutic Strategies in Uremic Atherosclerosis.

Cardiovascular disease (CVD) frequently occurs in patients with chronic kidney disease (CKD), particularly those undergoing dialysis. The mechanisms behind this may be related to traditional risk factors and CKD-specific factors that accelerate atherosclerosis and vascular calcification in CKD patients. The accumulation of uremic toxins is a significant factor in CKD-related systemic disorders. Basic research suggests that indoxyl sulfate (IS), a small protein-bound uremic toxin, is associated with macrophage dysfunctions, including increased oxidative stress, exacerbation of chronic inflammation, and abnormalities in lipid metabolism. Strategies to mitigate the toxicity of IS include optimizing gut microbiota, intervening against the abnormality of intracellular signal transduction, and using blood purification therapy with higher efficiency. Further research is needed to examine whether lowering protein-bound uremic toxins through intervention leads to a reduction in CVD in patients with CKD.

Hemodiafiltration with endogenous reinfusion for uremic toxin removal in patients undergoing maintenance hemodialysis: a pilot study.

OBJECTIVE: To delineate the efficacy and safety profile of hemodiafiltration with endogenous reinfusion (HFR) for uremic toxin removal in patients undergoing maintenance hemodialysis (MHD). METHODS: Patients who have been on MHD for a period of at least 3 months were enrolled. Each subject underwent one HFR and one hemodiafiltration (HDF) treatment. Blood samples were collected before and after a single HFR or HDF treatment to test uremic toxin levels and to calculate clearance rate. The primary efficacy endpoint was to compare uremic toxin levels of indoxyl sulfate (IS), λ-free light chains (λFLC), and ß2-microglobulin (ß2-MG) before and after HFR treatment. Secondary efficacy endpoints was to compare the levels of urea, interleukin-6 (IL-6), P-cresol, chitinase-3-like protein 1 (YKL-40), leptin (LEP), hippuric acid (HPA), trimethylamine N-oxide (TMAO), asymmetric dimethylarginine (ADMA), tumor necrosis factor-α (TNF-α), fibroblast growth factor 23 (FGF23) before and after HFR treatment. The study also undertook a comparative analysis of uremic toxin clearance between a single HFR and HDF treatment. Meanwhile, the lever of serum albumin and branched-chain amino acids before and after a single HFR or HDF treatment were compared. In terms of safety, the study was meticulous in recording vital signs and the incidence of adverse events throughout its duration. RESULTS: The study enrolled 20 patients. After a single HFR treatment, levels of IS, λFLC, ß2-MG, IL-6, P-cresol, YKL-40, LEP, HPA, TMAO, ADMA, TNF-α, and FGF23 significantly decreased (p < 0.001 for all). The clearance rates of λFLC, ß2-MG, IL-6, LEP, and TNF-α were significantly higher in HFR compared to HDF (p values: 0.036, 0.042, 0.041, 0.019, and 0.036, respectively). Compared with pre-HFR and post-HFR treatment, levels of serum albumin, valine, and isoleucine showed no significant difference (p > 0.05), while post-HDF, levels of serum albumin significantly decreased (p = 0.000). CONCLUSION: HFR treatment effectively eliminates uremic toxins from the bloodstream of patients undergoing MHD, especially protein-bound toxins and large middle-molecule toxins. Additionally, it retains essential physiological compounds like albumin and branched-chain amino acids, underscoring its commendable safety profile.

How renal toxins respond to renal function deterioration and oral toxic adsorbent in pH-controlled releasing capsule.

The number of patients with chronic kidney disease (CKD) is increasing. Oral toxin adsorbents may provide some value. Several uremic toxins, including indoxyl sulfate (IS), p-cresol (PCS), acrolein, per- and poly-fluoroalkyl substances (PFAS), and inflammation markers (interleukin 6 [IL-6] and tumor necrosis factor [TNF]-alpha) have been shown to be related to CKD progression. A total of 81 patients taking oral activated charcoal toxin adsorbents (AC-134), which were embedded in capsules that dissolved in the terminal ileum, three times a day for 1 month, were recruited. The renal function, hemoglobulin (Hb), inflammation markers, three PFAS (PFOA, PFOS, and PFNA), and acrolein were quantified. Compared with the baseline, an improved glomerular filtration rate (GFR) and significantly lower acrolein were noted. Furthermore, the CKD stage 4 and 5 group had significantly higher concentrations of IS, PCS, IL-6, and TNF but lower levels of Hb and PFAS compared with the CKD Stage 3 group at baseline and after the intervention. Hb was increased only in the CKD Stage 3 group after the trial (p = .032). Acrolein did not differ between the different CKD stage groups. Patients with improved GFR (responders) (about 77%) and nonresponders had similar baseline GFR. Responders had higher acrolein and PFOA levels throughout the study and a more significant reduction in acrolein, indicating a better digestion function. Both the higher PFOA and lower acrolein may be related to improved eGFR (and possibly to improvements in proteinuria, which we did not measure. Proteinuria is associated with PFAS loss in the urine), AC-134 showed the potential to improve the GFR and decrease acrolein, which might better indicate renal function change. Future studies are needed with longer follow-ups.

Role of Organic Anion Transporter NPT4 in Renal Handling of Uremic Toxin 3-indoxyl Sulfate.

Sodium-phosphate transporter NPT4 (SLC17A3) is a membrane transporter for organic anionic compounds localized on the apical membranes of kidney proximal tubular epithelial cells and plays a role in the urinary excretion of organic anionic compounds. However, its physiological role has not been sufficiently elucidated because its substrate specificity is yet to be determined. The present study aimed to comprehensively explore the physiological substrates of NPT4 in newly developed Slc17a3-/- mice using a metabolomic approach. Metabolomic analysis showed that the plasma concentrations of 11 biological substances, including 3-indoxyl sulfate, were more than two-fold higher in Slc17a3-/- mice than in wild-type mice. Moreover, urinary excretion of 3-indoxyl sulfate was reduced in Slc17a3-/- mice compared to that in wild-type mice. The uptake of 3-indoxyl sulfate by NPT4-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. The calculated Km and Vmax values for NPT4-mediated 3-indoxyl sulfate uptake were 4.52 ± 1.18 mM and 1.45 ± 0.14 nmol/oocyte/90 min, respectively. In conclusion, the present study revealed that 3-indoxyl sulfate is a novel substrate of NPT4 based on the metabolomic analysis of Slc17a3-/- mice, suggesting that NPT4 regulates systemic exposure to 3-indoxyl sulfate by regulating its urinary excretion.

Huang Gan Formula Alleviates Systemic Inflammation and Uremia in Adenine-Induced Chronic Kidney Disease Rats May Associate with Modification of Gut Microbiota and Colonic Microenvironment.

Purpose: This study aims to investigate the effects of Huang Gan formula (HGF), a Chinese herbal prescription used for chronic kidney disease (CKD), on the regulation of the gut microbiota and colonic microenvironment of CKD. Methods: CKD rats were induced by 150 mg/kg adenine gavage for 4 weeks, then orally treated with or without 3.6 g/kg or 7.2 g/kg of HGF for 8 weeks. The renal function and structure were analyzed by biochemical detection, hematoxylin and eosin, Masson's trichrome, Sirius red and immunochemical staining. Average fecal weight and number in the colon were recorded to assess colonic motility. Further, the changes in the gut microbiota and colonic microenvironment were evaluated by 16S rRNA sequencing, RT-PCR or immunofluorescence. The levels of inflammatory cytokines, uremic toxins, and NF-κB signaling pathway were detected by RT-PCR, ELISA, chloramine-T method or Western blotting. Redundancy analysis biplot and Spearman's rank correlation coefficient were used for correlation analysis. Results: HGF significantly improved renal function and pathological injuries of CKD. HGF could improve gut microbial dysbiosis, protect colonic barrier and promote motility of colonic lumens. Further, HGF inhibited systemic inflammation through a reduction of TNF-α, IL-6, IL-1ß, TGF-ß1, and a suppression of NF-κB signaling pathway. The serum levels of the selected uremic toxins were also reduced by HGF treatment. Spearman correlation analysis suggested that high-dose HGF inhibited the overgrowth of bacteria that were positively correlated with inflammatory factors (eg, TNF-α) and uremic toxins (eg, indoxyl sulfate), whereas it promoted the proliferation of bacteria belonging to beneficial microbial groups and was positively correlated with the level of IL-10. Conclusion: Our results suggest that HGF can improve adenine-induced CKD via suppressing systemic inflammation and uremia, which may associate with the regulations of the gut microbiota and colonic microenvironment.

Trimethylamine-N-oxide (TMAO) and predicted risk of cardiovascular events after partial nephrectomy.

INTRODUCTION: Emerging evidence suggests that uremic toxins, in particular trimethylamine-N-oxide(TMAO), indoxyl-sulfate(IS), and p-cresyl-sulfate(PCS), may associate with increased risk of cardiovascular events(CVe). However, whether uremic toxins increase after partial nephrectomy(PN) and their correlation with risk for CVe remains unknown. METHODS: 100 patients managed with PN were retrospectively reviewed. TMAO/IS/PCS levels were examined by liquid chromatography-mass-spectrometry. Renal-parenchymal-volume-preservation(RPVP) was estimated from CT scans. Predicted risks for CVe were obtained using the Framingham score. Linear regression assessed association between uremic toxins, GFR and risk of CVe. Logistic regression evaluated factors associated with post-PN TMAO. RESULTS: TMAO, IS and PCS increased from 1.7, 3.7 and 3.5 µmol/L before PN to 3.6, 5.4 and 7.4 µmol/L at latest follow-up, respectively, while GFR declined from 102 to 93 ml/min/1.73 m2 (all p<0.001). TMAO, IS and PCS levels all negatively correlated with GFR(all p<0.001). Predicted 10-year risk of CVe increased from 1.1% pre-PN to 1.7% post-PN(p<0.001), primarily due to increased age(p<0.001), blood pressure(p = 0.002) and total cholesterol(p = 0.003). TMAO(ß = 0.038) and GFR (ß = -0.02) were independent predictors for predicted 10-year CVe risk on multivariable-analysis. Increased TMAO was an early and sustained finding maintained through 5 years, unlike IS, PCS and eGFR. On multivariable analysis, increased pre-PN TMAO(OR = 2.79) and decreased RPVP(OR = 3.23) were identified as independent risk factors for higher post-PN TMAO, while ischemia type/duration failed to correlate. CONCLUSION: Uremic toxin levels increased after PN correlating with reduced GFR. Higher TMAO independently associated with greater predicted 10-year CVe risk. Parenchymal mass preserved rather than ischemia time or type associated with increased TMAO.

Predictive value of protein-bound uremic toxins for heart failure in patients with chronic kidney disease.

AIMS: This retrospective cohort study aimed to be the first to evaluate the association between plasma protein-bound uremic toxins (PBUTs) concentrations, echocardiographic parameters of heart failure (HF), and incident HF events in patients with chronic kidney disease (CKD) not on dialysis. METHODS AND RESULTS: Retrospective, single-centre, cohort study at the Ghent University Hospital, Belgium. Adults with CKD stages G1-G5, not on dialysis, could be included. Exclusion criteria were ongoing pregnancy, age <18 years, active acute infection, active malignancy, history of transplantation, or a cardiovascular event within 3 months prior to inclusion. Free and total concentrations of five PBUTs were quantified at baseline: indoxyl sulfate (IxS), p-cresyl sulfate (pCS), p-cresyl glucuronide (pCG), indole-3 acetic acid (IAA), and hippuric acid (HA). Patients were grouped into three echocardiographic categories: normal left ventricular ejection fraction (LVEF) and normal left ventricular end-diastolic pressure (LVEDP), normal LVEF and increased LVEDP, and reduced LVEF, based on available echocardiographic data in a time interval of ±6 months around the plasma sample collection. A total of 523 patients were included between January 2011 and January 2014. Echocardiographic data within the predefined timeframe were available for 210 patients (40% of patients). Levels of pCG and pCS were significantly higher in patients with reduced (<50%) versus normal LVEF (P < 0.05). After a median follow-up 5.5 years, 43 (8.4%) patients reached the composite endpoint of hospitalization or mortality due to HF. Free fractions of IxS, pCS, and pCG showed the strongest association with clinical outcome: free IxS: HR 1.71 (95% CI 1.11-2.63; P = 0.015), free pCS: HR 1.82 (95% CI 1.11-3.01; P = 0.019), and free pCG: HR 1.67 (95% CI 1.08-2.58; P = 0.020), and these results were independent of age, gender, body mass index, diabetes, and systolic blood pressure. In models that were also adjusted for serum creatinine, the free fractions of these PBUTs remained significant. CONCLUSIONS: Elevated free concentrations of IxS, pCG, and pCS were independently associated with an increased risk of HF events in non-dialysed CKD patients. Further research is necessary to confirm these findings and investigate the potential impact of PBUT-lowering interventions on HF events in this patient group.

Intracellular tacrolimus concentration correlates with impaired renal function through regulation of the IS-AHR-ABC transporter in peripheral blood mononuclear cells.

BACKGROUNDS: Tacrolimus (TAC) concentration in peripheral blood mononuclear cells (PBMCs) is regarded as a better predictor of its immunosuppressive effect than the TAC concentration in whole blood. However, whether the exposure of TAC in PBMCs or WB was altered in post-transplant recipients with renal impairment remains unclear. METHODS: We investigated the relationship of trough TAC concentration in WB and PBMCs with renal functions in post-transplant recipients. The pharmacokinetic profiles of TAC in PBMCs and WB in the two chronic kidney disease (CKD) rat models were examined using UPLC-MS/MS. Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to analyze the expression of proteins and mRNAs related to TAC metabolism and transport, respectively. In addition, the effects of uremic toxins on human PBMCs were investigated using whole-transcriptome sequencing (RNA sequencing [RNA-seq]). RESULTS: We observed a decrease in the trough TAC concentration in PBMCs in the recipients with estimated glomerular filtration rate (eGFR) < 90 mL/min, compared with those of recipients with eGFR > 90 mL/min, but there was no difference in blood based on TAC concentrations (C0Blood). In a 150-patient post-transplant cohort, no significant relationship was observed between PBMCs and WB concentrations of TAC, and the eGFR value was correlated with TAC C0PBMCs but not with TAC C0Blood. In two CKD rat models, the TAC pharmacokinetic profile in the PBMCs was significantly lower than that in the control group; however, the blood TAC pharmacokinetic profiles in the two groups were similar. Transcriptome results showed that co-incubation of human PBMCs with uremic toxins upregulated the expression of AHR, ABCB1, and ABCC2. Compared to control rats, plasma IS increased by 1.93- and 2.26-fold and the expression of AHR, P-gp, and MRP2 in PBMCs was higher in AD and 5/6 nephrectomy (NX) rats, without modifying the expression of other proteins related to TAC exposure. CONCLUSION: The pharmacokinetics of TAC in PBMCs changed with a decline in renal function. Uremic toxins accumulate during renal insufficiency, which activates AHR, upregulates the expression of P-gp and MRP2, and affects their intracellular concentrations. Our findings suggest that monitoring TAC concentrations in PBMCs is more important than monitoring WB concentrations in post-transplant recipients with renal impairment.

[Clinical significance of uremic toxin indoxyl sulfate and inflammation in the development of vascular calcification and cardiovascular complications in stage C3-C5D chronic kidney disease].

AIM: To clarify the role of the uremic toxin indoxyl sulfate (IS) and inflammation in the development of vascular calcification and cardiovascular complications in chronic kidney disease (CKD). MATERIALS AND METHODS: One hundred fifteen patients aged 25 to 68 years with CKD stage C3-C5D were examined. Serum concentrations of IS, interleukin 6 (IL-6), tumor necrosis factor (TNF-α), troponin I, parathyroid hormone were determined by enzyme immunoassay using kits from BluGene biotech (Shanghai, China), Cloud-Clone Corp. (USA), ELISA Kit (Biomedica, Austria). RESULTS: An increase in the serum concentration of IS, IL-6, TNF-α was revealed, which was significantly associated with a deterioration in renal function and changes in the morphological and functional parameters of the heart and aorta. CONCLUSION: High concentrations of IS, IL-6, TNF-α, which are closely associated with an increase in renal failure and cardiovascular complications, indicate their significant role in vascular calcification, which underlies the damage to the cardiovascular system in CKD.

Mapping cardiac remodeling in chronic kidney disease.

Patients with advanced chronic kidney disease (CKD) mostly die from sudden cardiac death and recurrent heart failure. The mechanisms of cardiac remodeling are largely unclear. To dissect molecular and cellular mechanisms of cardiac remodeling in CKD in an unbiased fashion, we performed left ventricular single-nuclear RNA sequencing in two mouse models of CKD. Our data showed a hypertrophic response trajectory of cardiomyocytes with stress signaling and metabolic changes driven by soluble uremia-related factors. We mapped fibroblast to myofibroblast differentiation in this process and identified notable changes in the cardiac vasculature, suggesting inflammation and dysfunction. An integrated analysis of cardiac cellular responses to uremic toxins pointed toward endothelin-1 and methylglyoxal being involved in capillary dysfunction and TNFα driving cardiomyocyte hypertrophy in CKD, which was validated in vitro and in vivo. TNFα inhibition in vivo ameliorated the cardiac phenotype in CKD. Thus, interventional approaches directed against uremic toxins, such as TNFα, hold promise to ameliorate cardiac remodeling in CKD.

Evaluating changes in firefighter urinary metabolomes after structural fires: an untargeted, high resolution approach.

Firefighters have elevated rates of urinary tract cancers and other adverse health outcomes, which may be attributable to environmental occupational exposures. Untargeted metabolomics was applied to characterize this suite of environmental exposures and biological changes in response to occupational firefighting. 200 urine samples from 100 firefighters collected at baseline and two to four hours post-fire were analyzed using untargeted liquid-chromatography and high-resolution mass spectrometry. Changes in metabolite abundance after a fire were estimated with fixed effects linear regression, with false discovery rate (FDR) adjustment. Partial least squares discriminant analysis (PLS-DA) was also used, and variable important projection (VIP) scores were extracted. Systemic changes were evaluated using pathway enrichment for highly discriminating metabolites. Metabolome-wide-association-study (MWAS) identified 268 metabolites associated with firefighting activity at FDR q < 0.05. Of these, 20 were annotated with high confidence, including the amino acids taurine, proline, and betaine; the indoles kynurenic acid and indole-3-acetic acid; the known uremic toxins trimethylamine n-oxide and hippuric acid; and the hormone 7a-hydroxytestosterone. Partial least squares discriminant analysis (PLS-DA) additionally implicated choline, cortisol, and other hormones. Significant pathways included metabolism of urea cycle/amino group, alanine and aspartate, aspartate and asparagine, vitamin b3 (nicotinate and nicotinamide), and arginine and proline. Firefighters show a broad metabolic response to fires, including altered excretion of indole compounds and uremic toxins. Implicated pathways and features, particularly uremic toxins, may be important regulators of firefighter's increased risk for urinary tract cancers.

Synbiotics, prebiotics and probiotics for people with chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is a major public health problem affecting 13% of the global population. Prior research has indicated that CKD is associated with gut dysbiosis. Gut dysbiosis may lead to the development and/or progression of CKD, which in turn may in turn lead to gut dysbiosis as a result of uraemic toxins, intestinal wall oedema, metabolic acidosis, prolonged intestinal transit times, polypharmacy (frequent antibiotic exposures) and dietary restrictions used to treat CKD. Interventions such as synbiotics, prebiotics, and probiotics may improve the balance of the gut flora by altering intestinal pH, improving gut microbiota balance and enhancing gut barrier function (i.e. reducing gut permeability). OBJECTIVES: This review aimed to evaluate the benefits and harms of synbiotics, prebiotics, and probiotics for people with CKD. SEARCH METHODS: We searched the Cochrane Kidney and Transplant Register of Studies up to 9 October 2023 through contact with the Information Specialist using search terms relevant to this review. Studies in the Register are identified through searches of CENTRAL, MEDLINE, and EMBASE, conference proceedings, the International Clinical Trials Registry Platform (ICTRP) Search Portal and ClinicalTrials.gov. SELECTION CRITERIA: We included randomised controlled trials (RCTs) measuring and reporting the effects of synbiotics, prebiotics, or probiotics in any combination and any formulation given to people with CKD (CKD stages 1 to 5, including dialysis and kidney transplant). Two authors independently assessed the retrieved titles and abstracts and, where necessary, the full text to determine which satisfied the inclusion criteria. DATA COLLECTION AND ANALYSIS: Data extraction was independently carried out by two authors using a standard data extraction form. Summary estimates of effect were obtained using a random-effects model, and results were expressed as risk ratios (RR) and their 95% confidence intervals (CI) for dichotomous outcomes, and mean difference (MD) or standardised mean difference (SMD) and 95% CI for continuous outcomes. The methodological quality of the included studies was assessed using the Cochrane risk of bias tool. Data entry was carried out by one author and cross-checked by another. Confidence in the evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. MAIN RESULTS: Forty-five studies (2266 randomised participants) were included in this review. Study participants were adults (two studies in children) with CKD ranging from stages 1 to 5, with patients receiving and not receiving dialysis, of whom half also had diabetes and hypertension. No studies investigated the same synbiotic, prebiotic or probiotic of similar strains, doses, or frequencies. Most studies were judged to be low risk for selection bias, performance bias and reporting bias, unclear risk for detection bias and for control of confounding factors, and high risk for attrition and other biases. Compared to prebiotics, it is uncertain whether synbiotics improve estimated glomerular filtration rate (eGFR) at four weeks (1 study, 34 participants: MD -3.80 mL/min/1.73 m², 95% CI -17.98 to 10.38), indoxyl sulfate at four weeks (1 study, 42 participants: MD 128.30 ng/mL, 95% CI -242.77 to 499.37), change in gastrointestinal (GI) upset (borborymgi) at four weeks (1 study, 34 participants: RR 15.26, 95% CI 0.99 to 236.23), or change in GI upset (Gastrointestinal Symptom Rating Scale) at 12 months (1 study, 56 participants: MD 0.00, 95% CI -0.27 to 0.27), because the certainty of the evidence was very low. Compared to certain strains of prebiotics, it is uncertain whether a different strain of prebiotics improves eGFR at 12 weeks (1 study, 50 participants: MD 0.00 mL/min, 95% CI -1.73 to 1.73), indoxyl sulfate at six weeks (2 studies, 64 participants: MD -0.20 µg/mL, 95% CI -1.01 to 0.61; I² = 0%) or change in any GI upset, intolerance or microbiota composition, because the certainty of the evidence was very low. Compared to certain strains of probiotics, it is uncertain whether a different strain of probiotic improves eGFR at eight weeks (1 study, 30 participants: MD -0.64 mL/min, 95% CI -9.51 to 8.23; very low certainty evidence). Compared to placebo or no treatment, it is uncertain whether synbiotics improve eGFR at six or 12 weeks (2 studies, 98 participants: MD 1.42 mL/min, 95% CI 0.65 to 2.2) or change in any GI upset or intolerance at 12 weeks because the certainty of the evidence was very low. Compared to placebo or no treatment, it is uncertain whether prebiotics improves indoxyl sulfate at eight weeks (2 studies, 75 participants: SMD -0.14 mg/L, 95% CI -0.60 to 0.31; very low certainty evidence) or microbiota composition because the certainty of the evidence is very low. Compared to placebo or no treatment, it is uncertain whether probiotics improve eGFR at eight, 12 or 15 weeks (3 studies, 128 participants: MD 2.73 mL/min, 95% CI -2.28 to 7.75; I² = 78%), proteinuria at 12 or 24 weeks (1 study, 60 participants: MD -15.60 mg/dL, 95% CI -34.30 to 3.10), indoxyl sulfate at 12 or 24 weeks (2 studies, 83 participants: MD -4.42 mg/dL, 95% CI -9.83 to 1.35; I² = 0%), or any change in GI upset or intolerance because the certainty of the evidence was very low. Probiotics may have little or no effect on albuminuria at 12 or 24 weeks compared to placebo or no treatment (4 studies, 193 participants: MD 0.02 g/dL, 95% CI -0.08 to 0.13; I² = 0%; low certainty evidence). For all comparisons, adverse events were poorly reported and were minimal (flatulence, nausea, diarrhoea, abdominal pain) and non-serious, and withdrawals were not related to the study treatment. AUTHORS' CONCLUSIONS: We found very few studies that adequately test biotic supplementation as alternative treatments for improving kidney function, GI symptoms, dialysis outcomes, allograft function, patient-reported outcomes, CVD, cancer, reducing uraemic toxins, and adverse effects. We are not certain whether synbiotics, prebiotics, or probiotics are more or less effective compared to one another, antibiotics, or standard care for improving patient outcomes in people with CKD. Adverse events were uncommon and mild.

Uremic Toxin-Induced Exosome-like Extracellular Vesicles Contain Enhanced Levels of Sulfated Glycosaminoglycans which Facilitate the Interaction with Very Small Superparamagnetic Iron Oxide Particles.

Uremic toxins exert pathophysiological effects on cells and tissues, such as the generation of a pro-calcifying subtype of exosome-like extracellular vesicles (EVs) in vascular cells. Little is known about the effects of the toxins on the surface structure of EVs. Thus, we studied the effects of uremic toxins on the abundance of sulfated glycosaminoglycans (GAGs) in EVs, and the implications for binding of ligands such as very small superparamagnetic iron oxide particles (VSOPs) which could be of relevance for radiological EV-imaging. Vascular cells were treated with the uremic toxins NaH2PO4 and a mixture of urea and indoxyl sulfate. Uremia in rats was induced by adenine feeding. EVs were isolated from culture supernatants and plasma of rats. By proton T1-relaxometry, magnetic particle spectroscopy, and analysis of genes, proteins, and GAG-contents, we analyzed the roles of GAGs in the ligand binding of EVs. By influencing GAG-associated genes in host cells, uremic toxins induced higher GAG contents in EVs, particularly of sulfated chondroitin sulfate and heparan sulfate chains. EVs with high GAG content interacted stronger with VSOPs compared to control ones. This was confirmed by experiments with GAG-depleted EVs from genetically modified CHO cells and with uremic rat-derived EVs. Mechanistically, uremic toxin-induced PI3K/AKT-signaling and expression of the sulfate transporter SLC26A2 in host cells contributed to high GAG contents in EVs. In conclusion, uremic conditions induce enhanced GAG contents in EVs, which entails a stronger interaction with VSOPs. VSOPs might be suitable for radiological imaging of EVs rich in GAGs.

Uremic Toxins Induce THP-1 Monocyte Endothelial Adhesion and Migration through Specific miRNA Expression.

Atherosclerosis is initiated by the activation of endothelial cells that allows monocyte adhesion and transmigration through the vascular wall. The accumulation of uremic toxins such as indoxyl sulphate (IS) and p-cresol (PC) has been associated with atherosclerosis. Currently, miRNAs play a crucial role in the regulation of monocyte activation, adhesion, and trans-endothelial migration. The aim of the present study is to evaluate the effect of IS and PC on monocyte adhesion and migration processes in monocytes co-cultured with endothelial cells as well as to determine the underlying mechanisms. The incubation of HUVECs and THP-1 cells with both IS and PC toxins resulted in an increased migratory capacity of THP-1 cells. Furthermore, the exposure of THP-1 cells to both uremic toxins resulted in the upregulation of BMP-2 and miRNAs-126-3p, -146b-5p, and -223-3p, as well as the activation of nuclear factor kappa B (NF-κB) and a decrease in its inhibitor IĸB. Uremic toxins, such as IS and PC, enhance the migratory and adhesion capacity of THP-1 cells to the vascular endothelium. These toxins, particularly PC, contribute significantly to uremia-associated vascular disease by increasing in THP-1 cells the expression of BMP-2, NF-κB, and key miRNAs associated with the development of atherosclerotic vascular diseases.

The Effect of Synbiotic Supplementation on Uremic Toxins, Oxidative Stress, and Inflammation in Hemodialysis Patients-Results of an Uncontrolled Prospective Single-Arm Study.

Introduction: Numerous studies to date have shown that the development of dysbiotic gut microbiota is a characteristic finding in chronic kidney disease (CKD). A number of uremic toxins progressively accumulate in the course of CKD, some of them generated by the intestinal microbiome, such as indoxyl sulfate (IS) and p-cresyl sulfate (p-CS). They are found to be involved in the pathogenesis of certain complications of uremic syndrome, including low-grade chronic inflammation and oxidative stress. The aim of the present study is to research the serum concentration of IS and p-CS in end stage renal disease (ESRD) patients undergoing conventional hemodialysis, as well as to study the possibilities of influencing some markers of inflammation and oxidative stress after taking a synbiotic. Materials and Methods: Thirty patients with end-stage renal disease (ESRD) undergoing hemodialysis treatment who were taking a synbiotic in the form of Lactobacillus acidophilus La-14 2 × 1011 (CFU)/g and prebiotic fructooligosaccharides were included in the study. Serum levels of total IS, total p-CS, Interleukin-6 (IL-6), and Malondialdehyde (MDA) were measured at baseline and after 8 weeks. Results. The baseline values of the four investigated indicators in the patients were significantly higher-p-CS (29.26 ± 58.32 pg/mL), IS (212.89 ± 208.59 ng/mL), IL-6 (13.84 ± 2.02 pg/mL), and MDA (1430.33 ± 583.42 pg/mL), compared to the results obtained after 8 weeks of intake, as we found a significant decrease in the parameters compared to the baseline-p-CS (6.40 ± 0.79 pg/mL, p = 0.041), IS (47.08 ± 3.24 ng/mL, p < 0.001), IL-6 (9.14 ± 1.67 pg/mL, p < 0.001), and MDA (1003.47 ± 518.37 pg/mL, p < 0.001). Conclusions: The current study found that the restoration of the intestinal microbiota in patients with CKD significantly decreases the level of certain uremic toxins. It is likely that this favorably affects certain aspects of CKD, such as persistent low-grade inflammation and oxidative stress.

N-methyl-2-pyridone-5-carboxamide (N-Me-2PY) has potent anti-fibrotic and anti-inflammatory activity in a fibrotic kidney model: is it an old uremic toxin?

BACKGROUND: Uremic toxins accumulate in renal tissues and cells due to chronic kidney disease (CKD). Abnormalities in nicotinamide adenine dinucleotide (NAD +) metabolism lead to the progression of CKD. NAD + metabolites, such as N-methyl-2-pyridone-5-carboxamide (N-Me-2PY) and N-methyl-4-pyridone-5-carboxamide (N-Me-4PY), have been recognized as uremic toxins. However, no reports have validated whether they are actually harmful to the body. Therefore, we focused on the structural similarity of these metabolites to the anti-fibrotic drug pirfenidone and evaluated their effects on renal fibrosis. METHODS: Each NAD + metabolite was treated with TGFß1 to kidney fibroblasts or tubular epithelial cells, and quantitative RT-PCR and Western blot analysis were conducted. N-Me-2PY was orally administered to a ligated murine kidney fibrosis model (UUO) to evaluate its anti-fibrotic and toxic effects on the body. RESULTS: N-Me-2PY, N-Me-4PY, and nicotinamide N-oxide (NNO) inhibited TGFß1-induced fibrosis and inflammatory gene expression in kidney fibroblasts. N-Me-2PY strongly suppressed the expression of types I and III collagen, αSMA, and IL-6. N-Me-2PY also suppressed TGFß1-induced type I collagen and IL-6 expression in renal tubular epithelial cells. No toxic effect was observed with N-Me-2PY treatment, while attenuating renal fibrosis and tubular dilation in UUO mice. Suppression of various fibrosis- and inflammation-related genes was also observed. N-Me-2PY did not inhibit TGFß1-induced Smad3 phosphorylation but inhibited Akt phosphorylation, suggesting that N-Me-2PY exerts anti-fibrotic and anti-inflammatory effects through Akt inhibition, similar to pirfenidone. CONCLUSIONS: NAD + metabolites, such as N-Me-2PY, are not uremic toxins but are potential therapeutic agents that have anti-fibrotic effects in CKD.

Possible Effects of Uremic Toxins p-Cresol, Indoxyl Sulfate, p-Cresyl Sulfate on the Development and Progression of Colon Cancer in Patients with Chronic Renal Failure.

Chronic kidney disease (CKD) induces several systemic effects, including the accumulation and production of uremic toxins responsible for the activation of various harmful processes. Gut dysbiosis has been widely described in CKD patients, even in the early stages of the disease. The abundant discharge of urea and other waste substances into the gut favors the selection of an altered intestinal microbiota in CKD patients. The prevalence of bacteria with fermentative activity leads to the release and accumulation in the gut and in the blood of several substances, such as p-Cresol (p-C), Indoxyl Sulfate (IS) and p-Cresyl Sulfate (p-CS). Since these metabolites are normally eliminated in the urine, they tend to accumulate in the blood of CKD patients proportionally to renal impairment. P-CS, IS and p-C play a fundamental role in the activation of various pro-tumorigenic processes, such as chronic systemic inflammation, the increase in the production of free radicals and immune dysfunction. An up to two-fold increase in the incidence of colon cancer development in CKD has been reported in several studies, although the pathogenic mechanisms explaining this compelling association have not yet been described. Based on our literature review, it appears likely the hypothesis of a role of p-C, IS and p-CS in colon cancer development and progression in CKD patients.

Uremic Toxin Indoxyl Sulfate Promotes Macrophage-Associated Low-Grade Inflammation and Epithelial Cell Senescence.

In this study, we investigated the impact of the uremic toxin indoxyl sulfate on macrophages and tubular epithelial cells and its role in modulating the response to lipopolysaccharide (LPS). Indoxyl sulfate accumulates in the blood of patients with chronic kidney disease (CKD) and is a predictor of overall and cardiovascular morbidity/mortality. To simulate the uremic condition, primary macrophages and tubular epithelial cells were incubated with indoxyl sulfate at low concentrations as well as concentrations found in uremic patients, both alone and upon LPS challenge. The results showed that indoxyl sulfate alone induced the release of reactive oxygen species and low-grade inflammation in macrophages. Moreover, combined with LPS (proinflammatory conditions), indoxyl sulfate significantly increased TNF-α, CCL2, and IL-10 release but did not significantly affect the polarization of macrophages. Pre-treatment with indoxyl sulfate following LPS challenge induced the expression of aryl hydrocarbon receptor (Ahr) and NADPH oxidase 4 (Nox4) which generate reactive oxygen species (ROS). Further, experiments with tubular epithelial cells revealed that indoxyl sulfate might induce senescence in parenchymal cells and therefore participate in the progression of inflammaging. In conclusion, this study provides evidence that indoxyl sulfate provokes low-grade inflammation, modulates macrophage function, and enhances the inflammatory response associated with LPS. Finally, indoxyl sulfate signaling contributes to the senescence of tubular epithelial cells during injury.

Effects of resveratrol on inflammation and oxidative stress induced by the uremic toxin indoxyl sulfate in Murine macrophage-like RAW 264.7.

Indoxyl sulfate (IS) is a uremic toxin produced by the gut microbiota that commonly accumulates in patients with chronic kidney disease (CKD) and can be harmful. Resveratrol is a polyphenol with properties that attenuate oxidative stress and inflammation. This study aims to evaluate the effect of resveratrol against the damage caused by IS in RAW 264.7 murine macrophages. Cells were treated with 0, 250, 500 and 1000 µmol/L of IS, in the presence of 50 µmol/L of resveratrol. The mRNA and protein expressions of erythroid-related nuclear factor 2 (Nrf2) and nuclear factor kappa-B (NF-κB) were measured using rt-PCR and Western blot analysis, respectively. Malondialdehyde (MDA) and reactive oxygen species (ROS) levels were also analyzed. As a result, it was demonstrated that resveratrol induces the activation of the Nrf2 pathway that enhances cytoprotective response. IS upregulated the NF-κB expression and downregulated the Nrf2 expression. In contrast, resveratrol treatment significantly reduced the MDA and ROS production and inhibited the IS-induced expression of NF-κB in macrophage-like RAW 264.7. In conclusion, resveratrol can mitigate inflammation and oxidative stress caused by uremic toxins produced by the gut microbiota, such as IS.

The Uremic Toxin Indoxyl Sulfate Accelerates Senescence in Kidney Proximal Tubule Cells.

Kidney fibrosis is the common final pathway of nearly all chronic and progressive nephropathies. One cause may be the accumulation of senescent cells that secrete factors (senescence associated secretory phenotype, SASP) promoting fibrosis and inflammation. It has been suggested that uremic toxins, such as indoxyl sulfate (IS), play a role in this. Here, we investigated whether IS accelerates senescence in conditionally immortalized proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1), thereby promoting kidney fibrosis. Cell viability results suggested that the tolerance of ciPTEC-OAT1 against IS increased in a time-dependent manner at the same dose of IS. This was accompanied by SA-ß-gal staining, confirming the accumulation of senescent cells, as well as an upregulation of p21 and downregulation of laminB1 at different time points, accompanied by an upregulation in the SASP factors IL-1ß, IL-6 and IL-8. RNA-sequencing and transcriptome analysis revealed that IS accelerates senescence, and that cell cycle appears to be the most relevant factor during the process. IS accelerates senescence via TNF-α and NF-ĸB signalling early on, and the epithelial-mesenchymal transition process at later time points. In conclusion, our results suggest that IS accelerates cellular senescence in proximal tubule epithelial cells.