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In patients with chronic kidney disease, the uremic toxin indoxyl sulfate (IS) accelerates kidney damage and the progression of cardiovascular disease. IS may contribute to vascular diseases by inducing inflammation in endothelial cells. Luteolin has documented antioxidant and anti‐inflammatory properties. This study aimed to investigate the effect of luteolin on IS‐mediated reactive oxygen species (ROS) production and intercellular adhesion molecule (ICAM‐1) and monocyte chemoattractant protein (MCP‐1) expression in EA.hy926 cells and the possible mechanisms involved. IS significantly induced ROS production (by 6.03‐fold, p < 0.05), ICAM‐1 (by 2.19‐fold, p < 0.05) and MCP‐1 protein expression (by 2.18‐fold, p < 0.05), and HL‐60 cell adhesion (by 31%, p < 0.05), whereas, luteolin significantly decreased IS‐induced ROS production, ICAM‐1 and MCP‐1 protein expression, and HL‐60 cell adhesion. Moreover, luteolin attenuated IS‐induced nuclear accumulation of p65 and c‐jun. Luteolin dose‐dependently increased heme oxygenase‐1 (HO‐1) expression and the maximum fold induction of HO‐1 by luteolin was 3.68‐fold (p < 0.05), whereas, HO‐1 knockdown abolished the suppression of ICAM‐1 and MCP‐1 expression by luteolin. Luteolin may protect against IS‐induced vessel damage by inducing HO‐1 expression in vascular endothelial cells, which suppresses nuclear factor kappa B (NF‐κB) and activator protein 1 (AP‐1) mediated ICAM‐1 and MCP‐1 expression. [ABSTRACT FROM AUTHOR]

Indoxyl sulfate (IS), a uremic toxin, is a harmful factor that damages kidneys. Chronic heat stress in laying hens causes renal injury; however, whether IS accumulation is involved in this injury remains unknown. We selected 20 Boris brown laying hens (27 weeks old) and randomly assigned them to two groups (n = 10), one group was exposed to chronic heat stress (32 °C for 4 weeks), whereas the other was maintained at 24 °C. Chronic heat exposure significantly increased plasma and renal IS concentrations (P < 0.05). Exposure to heat also increased renal expression of the aryl hydrocarbon receptor (AhR) and its target genes (CYP1A4 and CYP1B1). Furthermore, chronic heat exposure tended to increase the 2-thiobarbituric acid reactive substances content (P = 0.08) and significantly decreased the antioxidant capacity in the kidney, while increasing the transcription levels of nuclear factor κB and fibrosis-related genes (COLA1A1, αSMA, TGF-β, Smad3, and VCAM-1) and the area of renal fibrosis. Our results indicate that chronic heat exposure induces systemic and renal IS accumulation in laying hens. This accumulated IS may activate the AhR pathway and chronically disrupt the oxidative stress status and antioxidant activity, thus promoting renal fibrosis and dysfunction in laying hens. [ABSTRACT FROM AUTHOR]

Modern hemodialysis employs weak acids as buffers to prevent bicarbonate precipitation with calcium or magnesium. Acetate, the most used acid, is linked to chronic inflammation and poor dialysis tolerance. Citrate has emerged as a potential alternative, though its effect on dialysis efficiency is not clear. This study aims to compare the efficacy of acetate- and citrate-based dialysates, focusing on protein-bound uremic toxins and dialysis doses. This single-center prospective crossover study includes prevalent patients participating in a thrice-weekly online hemodiafiltration program. Four dialysates were tested: two acetate-based (1.25 and 1.5 mmol/L calcium) and two citrate-based (1.5 mmol/L calcium with 0.5 and 0.75 mmol/L magnesium). Pre- and post-dialysis blood samples of eighteen patients were analyzed for urea, creatinine, p-cresyl sulfate, indoxyl sulfate, and albumin. Statistical significance was assessed using paired t-tests and repeated measures of ANOVA. There were no significant differences in dialysis dose (Kt), urea, creatinine, or indoxyl sulfate reduction ratios between acetate- and citrate-based dialysates. However, a significant decrease in the reduction ratio of p-cresyl sulfate was observed with the acetate dialysate containing 1.25 mmol/L calcium and the citrate dialysate with 0.5 mmol/L magnesium compared to the acetate dialysate containing 1.5 mmol/L calcium and the citrate dialysate with 0.75 mmol/L magnesium (51.56 ± 4.75 and 53.02 ± 4.52 vs. 65.25 ± 3.38 and 58.66 ± 4.16, p 0.007). No differences in dialysis dose were found between acetate- and citrate-based dialysates. However, citrate dialysates with lower calcium and magnesium concentrations may reduce the albumin displacement of p-cresyl sulfate. Further studies are needed to understand the observed differences and optimize the dialysate composition for the better clearance of protein-bound uremic toxins. [ABSTRACT FROM AUTHOR]

Background: Diabetic retinopathy (DR), the principal cause of acquired blindness among the working-age population, is the most frequent microvascular complication of diabetes. Although metabolic disorders are hypothesized to play a role in its pathogenesis, the underlying mechanism remains largely elusive. Methods: To elucidate the mechanism, we initially compared metabolite profiles of vitreous fluid between 23 patients with DR and 12 non-diabetic controls using liquid chromatography/tandem mass spectrometry, identifying the distinct metabolite indoxyl sulfate (IS). Subsequently, streptozotocin (STZ)-induced diabetic and IS-injected rat models were established to examine the effects of IS on retinal microvasculature. RNA sequencing was conducted to identify potential regulatory mechanisms in IS-treated human retinal endothelial cells (HREC). Finally, target gene knockdown in HREC and treatment of IS-injected rats with inhibitors (targeting IS production or downstream regulators) were employed to elucidate the detailed mechanisms and identify therapeutic targets for DR. Results: Metabolomics identified 172 significantly altered metabolites in the vitreous humor of diabetics, including the dysregulated tryptophan metabolite indoxyl sulfate (IS). IS was observed to breach the blood-retinal barrier and accumulate in the intraocular fluid of diabetic rats. Both in vivo and in vitro experiments indicated that elevated levels of IS induced endothelial apoptosis and disrupted cell junctions. RNA sequencing pinpointed prostaglandin E2 (PGE2) synthetase-cyclooxygenase 2 (COX-2) as a potential target of IS. Validation experiments demonstrated that IS enhanced COX-2 expression, which subsequently increased PGE2 secretion by promoting transcription factor EGR1 binding to COX-2 DNA following entry into cells via organic anion transporting polypeptides (OATP2B1). Furthermore, inhibition of COX-2 in vivo or silencing EGR1/OATP2B1 in HREC mitigated IS-induced microcapillary damage and the activation of COX-2/PGE2. Conclusion: Our study demonstrated that indoxyl sulfate (IS), a uremic toxin originating from the gut microbiota product indole, increased significantly and contributed to retinal microvascular damage in diabetic retinopathy (DR). Mechanistically, IS impaired retinal microvascular integrity by inducing the expression of COX-2 and the production of PGE2. Consequently, targeting the gut microbiota or the PGE2 pathway may offer effective therapeutic strategies for the treatment of DR. [ABSTRACT FROM AUTHOR]

Indoxyl sulfate (IS), a uremic toxin, is a physiologically active sulfated metabolite, specifically in kidney failure patients. Our previous studies have shown that IS downregulates phagocytic immune function in a differentiated HL-60 human macrophage cell model. However, it remains unclear whether IS exerts similar effects on macrophage function in other cell types or in lipopolysaccharide (LPS)-sensitive immune cell models. Therefore, this study aimed to investigate the effects of IS on intracellular oxidation levels and phagocytic activity in a differentiated U937 human macrophage cell model, both in the absence and presence of LPS. Our results demonstrated that IS significantly increases intracellular oxidation levels and decreases phagocytic activity, particularly in cells activated by LPS. Furthermore, we found that 2-acetylphenothiazine, an NADH oxidase inhibitor, attenuates the effects of IS in LPS-activated macrophage cells. Representative antioxidants, trolox, α-tocopherol, and ascorbic acid, significantly mitigated the effects of IS on the macrophages responding to LPS. [ABSTRACT FROM AUTHOR]

Abstract Indoxyl sulfate (IS), a uremic toxin, is a harmful factor that damages kidneys. Chronic heat stress in laying hens causes renal injury; however, whether IS accumulation is involved in this injury remains unknown. We selected 20 Boris brown laying hens (27 weeks old) and randomly assigned them to two groups (n = 10), one group was exposed to chronic heat stress (32 °C for 4 weeks), whereas the other was maintained at 24 °C. Chronic heat exposure significantly increased plasma and renal IS concentrations (P

Abstract Background Diabetic retinopathy (DR), the principal cause of acquired blindness among the working-age population, is the most frequent microvascular complication of diabetes. Although metabolic disorders are hypothesized to play a role in its pathogenesis, the underlying mechanism remains largely elusive. Methods To elucidate the mechanism, we initially compared metabolite profiles of vitreous fluid between 23 patients with DR and 12 non-diabetic controls using liquid chromatography/tandem mass spectrometry, identifying the distinct metabolite indoxyl sulfate (IS). Subsequently, streptozotocin (STZ)-induced diabetic and IS-injected rat models were established to examine the effects of IS on retinal microvasculature. RNA sequencing was conducted to identify potential regulatory mechanisms in IS-treated human retinal endothelial cells (HREC). Finally, target gene knockdown in HREC and treatment of IS-injected rats with inhibitors (targeting IS production or downstream regulators) were employed to elucidate the detailed mechanisms and identify therapeutic targets for DR. Results Metabolomics identified 172 significantly altered metabolites in the vitreous humor of diabetics, including the dysregulated tryptophan metabolite indoxyl sulfate (IS). IS was observed to breach the blood-retinal barrier and accumulate in the intraocular fluid of diabetic rats. Both in vivo and in vitro experiments indicated that elevated levels of IS induced endothelial apoptosis and disrupted cell junctions. RNA sequencing pinpointed prostaglandin E2 (PGE2) synthetase-cyclooxygenase 2 (COX-2) as a potential target of IS. Validation experiments demonstrated that IS enhanced COX-2 expression, which subsequently increased PGE2 secretion by promoting transcription factor EGR1 binding to COX-2 DNA following entry into cells via organic anion transporting polypeptides (OATP2B1). Furthermore, inhibition of COX-2 in vivo or silencing EGR1/OATP2B1 in HREC mitigated IS-induced microcapillary damage and the activation of COX-2/PGE2. Conclusion Our study demonstrated that indoxyl sulfate (IS), a uremic toxin originating from the gut microbiota product indole, increased significantly and contributed to retinal microvascular damage in diabetic retinopathy (DR). Mechanistically, IS impaired retinal microvascular integrity by inducing the expression of COX-2 and the production of PGE2. Consequently, targeting the gut microbiota or the PGE2 pathway may offer effective therapeutic strategies for the treatment of DR. Graphical Abstract

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.

Background: Uremic toxin indoxyl sulfate (IS) induces vascular inflammation, a crucial event in renal failure, and vascular complications in patients with chronic kidney disease (CKD). In endothelial cells, IS increases the production of inflammatory cytokines partially via the activation of the aryl hydrocarbon receptor (AhR), and several food flavonoids have been reported to act as antagonists of AhR. Objective: This study aimed to investigate whether antagonistic flavonoids can attenuate IS-induced inflammatory responses in vascular endothelial cells in vitro and renal failure in vivo. Design: Human umbilical vein endothelial cells (HUVECs) pretreated with the flavones apigenin, chrysin, or luteolin were stimulated with IS. Expression levels of genes involved in AhR signaling, inflammatory cytokine production, and reactive oxygen species (ROS) production were analyzed. Uninephrectomized mice were orally administered chrysin and received daily intraperitoneal injections of IS for 4 weeks. Results: In HUVECs, IS upregulated the mRNA expression of AhR-targeted genes (CYP1A1 and AhRR), and genes involved in inflammation (NOX4, MCP-1, IL-6, and COX2) and monocyte invasion/adhesion (ICAM1). All three flavones attenuated the IS-induced increase in the expression of these mRNAs. They also suppressed the IS-induced nuclear translocation of AhR and intracellular ROS production. Furthermore, IS-induced phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was inhibited by treatment with these flavones. The results of in-vivo experiments showed that administration with chrysin attenuated the elevation of blood urea nitrogen levels and AhR-target gene expression and the pathological impairment of renal tissues in mice, regardless of higher serum levels of IS. Conclusions: Natural food flavones antagonizing AhR exerted protective effects against IS-induced inflammation through the inhibition of the AhR–STAT3 pathway in HUVECs. Moreover, chrysin ameliorated IS-induced renal dysfunction in a mouse model of CKD. These flavonoids could be a therapeutic strategy for vascular inflammation in CKD.

Sarcopenia in chronic kidney disease （CKD） occurs mainly in patients with end-stage renal disease （ESRD） on hemodialysis. The potential causes are insufficient nutrition intake, protein and energy consumption, inflammatory cytokines, oxidative stress, insulin resistance and uremic toxins. Protein-bound uremic toxin phenol sulfate is an intestinal-derived uremic toxin accumulating in CKD patients. It is difficult to remove through dialysis. It affects the occurrence of sarcopenia through various ways. The review focused upon the mechanisms of sarcopenia caused by indoxyl sulfate.

Uremic toxins cause bone disorders in patients with chronic kidney disease (CKD). These disorders are characterized by low turnover osteodystrophy and impaired bone formation in the early stages of CKD. Evidence indicates that the aryl hydrocarbon receptor (AhR) mediates signals that suppress early osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). However, whether the AhR mediates the effects of indoxyl sulfate (IS), a uremic toxin, on BMSC osteogenesis remains unclear. We investigated whether IS affects osteogenesis through the AhR/Hes1 pathway. Expression levels of osteogenesis genes (Runx2, Bmp2, Alp, and Oc), AhR, and Hes1 were measured in mouse BMSCs (D1 cells). At concentrations of 2–50 μM, IS significantly reduced mineralization, particularly in the early stages of BMSC osteogenesis. Furthermore, IS significantly downregulated the expression of Runx2, Bmp2, Oc, and Alp. Notably, this downregulation could be prevented using an AhR antagonist and through Ahr knockdown. Mechanistically, IS induced the expression of Hes1 through AhR signaling, thereby suppressing the transcription of Runx2 and Bmp2. Our findings suggest that IS inhibits early osteogenesis of BMSCs through the AhR/Hes1 pathway, thus suppressing the transcription of Runx2 and Bmp2. Our findings may guide new therapeutic strategies against CKD-related bone disorders. [ABSTRACT FROM AUTHOR]

Background/Objectives: Indoxyl sulfate, a uremic toxin, is associated with mortality and cardiovascular events in patients with chronic kidney disease (CKD). This study aimed to evaluate the prognostic implications of serum indoxyl sulfate levels in patients with heart failure and CKD. Methods and Results: This was a prospective multicenter observational study. Overall, 300 patients with chronic heart failure with a previous history of hospitalization and an estimated glomerular filtration rate (eGFR) of 45 mL/min/1.73 m2 or less (CKD stage G3b to G5) without dialysis were analyzed. The primary outcome assessed in a time-to-event analysis from the measurement of indoxyl sulfate was a composite of all-cause death, hospitalization for heart failure, nonfatal myocardial infarction, and nonfatal stroke. Clinical events were followed-up to one year after indoxyl sulfate measurement. The median patient age was 75 years, and 57% of the patients were men. We divided the cohort into low and high indoxyl sulfate categories according to a median value of 9.63 mg/mL. The primary outcome occurred in 27 of 150 patients (18.0%) in the low indoxyl sulfate group and 27 of 150 patients (18.0%) in the high indoxyl sulfate group (hazard ratio, 1.00; 95% confidence interval, 0.58 to 1.70, p = 0.99). In the post hoc exploratory analyses, the results were consistent across age, sex, body mass index, left ventricular ejection fraction, eGFR, and N-terminal pro b-type natriuretic peptide. Conclusions: Among heart failure patients with CKD stages G3b to 5G, serum indoxyl sulfate concentrations were not significantly associated with the subsequent occurrence of cardiovascular events. [ABSTRACT FROM AUTHOR]

Simple Summary: In cats with chronic kidney disease (CKD), harmful toxins can build up in their blood, causing health problems. Some of these toxins originate from molecules that are generated in the gut by the action of certain microbes on digested food. Renaltec is a supplement that binds to these molecules in the gut before they enter the bloodstream and are transformed into toxins by the liver. In a study with 13 cats with experimentally induced CKD, researchers tested how well Renaltec worked at reducing two specific toxins, indoxyl sulfate and p-cresol sulfate, in the blood. The cats were given Renaltec either once or twice daily for 56 days, and their toxin levels were measured at different times. The study found that Renaltec significantly lowered the levels of these toxins, with the twice-daily dosing showing better results. This suggests that Renaltec is effective at reducing harmful toxins in cats with CKD. Serum uremic toxins markedly increase in cats with chronic kidney disease (CKD) and have deleterious consequences. Renaltec is an oral adsorbent that binds uremic toxin precursors in the gut. In this prospective cohort study utilizing 13 purpose-bred cats with remnant kidney model-induced CKD (12 IRIS Stage 2, 1 IRIS Stage 3) eating a standardized renal diet, we aimed to assess the effect of Renaltec administration on serum indoxyl sulfate (IDS) and p-cresol sulfate (pCS) concentrations. Cats were sequentially treated with standard of care for 56 days, 500 mg Renaltec orally once daily for 56 days, and then three months later, 500 mg Renaltec orally twice daily for 56 days. Serum IDS and pCS concentrations were measured 28 and 56 days after the administration of Renaltec. Blood pressure and kidney function were measured before and 56 days after the administration of Renaltec. Significant decreases in serum IDS and pCS concentrations were observed for both once- and twice-daily dosing, particularly during the first 28 days of administration. More cats with BID dosing had clinically significant reductions in serum IDS and pCS concentrations than with SID dosing. Renaltec can reduce the serum concentrations of deleterious gut-derived uremic toxins in cats with CKD. [ABSTRACT FROM AUTHOR]

Objective: To analyze the relationship between Serum indoxyl sulfate (IS), lysyl oxidase-like protein 2 (LOXL2), glucose-regulated protein 94 (GRP94) and recurrence after radiofrequency catheter ablation in patients with paroxysmal atrial fibrillation (PAF). Methods: 155 patients with PAF who were underwent radiofrequency catheter ablation in our hospital from January 2020 to January 2022 were selected, and followed up for 1 year after operation. The patients were divided into non recurrence group and recurrence group according to whether PAF recurred, and the levels of serum IS, LOXL2 and GRP94 were compared between two groups, and the factors affecting recurrence after operation were analyzed by multivariate logistic regression analysis. The value of serum IS, LOXL2, GRP94 and their combination in predicting the recurrence of patients with PAF after radiofrequency catheter ablation were analyzed by ROC curve. Results: 155 patients with PAF were followed up for 1 year after operation, and 40 cases (25.81%) experienced recurrence. The duration of atrial fibrillation and left atrial diameter (LAD) in recurrence group were longer than those in non recurrence group, the proportion of New York Heart Association (NYHA) cardiac function grade III in recurrence group was higher than that in non recurrence group, and the left ventricular ejection fraction (LVEF) in recurrence group was lower than that in non recurrence group (P<0.05). The level of serum GRP94 in recurrent group was lower than that in the non recurrent group, and the levels of serum IS and LOXL2 were higher than those in non recurrent group (P<0.05). Multivariate logistic regression analysis showed that, NYHA cardiac function grade III, elevated serum IS levels, elevated serum LOXL2 levels, longer duration of atrial fibrillation, and increased LAD were risk factors for recurrence after operation (P<0.05), and elevated serum GRP94 levels and elevated LVEF were protective factors (P<0.05). The ROC curve showed that, the AUC of serum IS, LOXL2, GRP94 levels and their combination in predicting recurrence after operation were 0.764, 0.710, 0.806 and 0.853, respectively, and the AUC of the combination of the three was significantly higher than that of IS, LOXL2 and GRP94. Conclusion: The risk of recurrence after operation increased in patients with PAF with increased serum IS and LOXL2 levels and decreased GRP94 levels before radiofrequency catheter ablation, and the combination of the three can guide the clinical identification of high risk groups of recurrence after operation, and predict the risk of early recurrence after operation. [ABSTRACT FROM AUTHOR]

The characteristic accumulation of circulating uremic toxins, such as indoxyl sulfate (IS), in chronic kidney disease (CKD) further exacerbates the disease progression. The gut microbiota, particularly gut bacterial-specific enzymes, represents a selective and attractive target for suppressing uremic toxin production and slowing the progression of renal failure. This study investigates the role of 4-phenylbutyrate (PB) and structurally related compounds, which are speculated to possess renoprotective properties in suppressing IS production and slowing or reversing renal failure in CKD. In vitro enzyme kinetic studies showed that 7-phenylheptanoic acid (PH), a PB homologue, suppresses the tryptophan indole lyase (TIL)-catalyzed decomposition of tryptophan to indole, the precursor of IS. A hydroxypropyl β-cyclodextrin (HPβCD) inclusion complex formulation of PH was prepared to enhance its biopharmaceutical properties and to facilitate in vivo evaluation. Prophylactic oral administration of the PH-HPβCD complex formulation reduced circulating IS and attenuated the deterioration of renal function and tubulointerstitial fibrosis in adenine-induced CKD mice. Additionally, treatment of moderately advanced adenine-induced CKD mice with the formulation ameliorated renal failure, although tissue fibrosis was not improved. These findings suggest that PH-HPβCD can slow the progression of renal failure and may have implications for preventing or managing CKD, particularly in early-stage disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objectives Materials and Methods Results Conclusions The purpose of this study was to determine whether indoxyl sulfate (IS) is involved in alveolar bone deterioration and to elucidate the mechanism underlying alveolar bone loss in chronic kidney disease (CKD) patients.Mice were divided into the control group, CP group (ligature‐induced periodontitis), CKD group (5/6 nephrectomy), and CKD + CP group. The concentration of IS in the gingival crevicular fluid (GCF) was determined by HPLC. The bone microarchitecture was evaluated by micro‐CT. MC3T3‐E1 cells were stimulated with IS, and changes in mitochondrial morphology and ferroptosis‐related factors were detected. RT‐PCR, western blotting, alkaline phosphatase activity assays, and alizarin red S staining were utilized to assess how IS affects osteogenic differentiation.Compared with that in the other groups, alveolar bone destruction in the CKD + CP group was more severe. IS accumulated in the GCF of mice with CKD. IS activated the aryl hydrocarbon receptor (AhR) in vitro, inhibited MC3T3‐E1 cell osteogenic differentiation, caused changes in mitochondrial morphology, and activated the SLC7A11/GPX4 signaling pathway. An AhR inhibitor attenuated the aforementioned changes induced by IS.IS activated the AhR/SLC7A11/GPX4 signaling pathway, inhibited osteogenesis in MC3T3‐E1 cells, and participated in alveolar bone resorption in CKD model mice through ferroptosis. [ABSTRACT FROM AUTHOR]

Indoxyl sulfate (IxS) and p-cresyl sulfate (pCS) are toxic uremic compounds with documented pathological outcomes. This review critically and comprehensively analyzes the available liquid chromatography-mass spectrometry methods quantifying IxS and pCS in human matrices and the biological applications of these validated assays. Embase, Medline, PubMed, Scopus, and Web of Science were searched until December 2023 to identify assays with complete analytical and validation data (N = 23). Subsequently, citation analysis with PubMed and Scopus was utilized to identify the biological applications for these assays (N = 45). The extraction methods, mobile phase compositions, chromatography, and ionization methods were evaluated with respect to overall assay performance (e.g., sensitivity, separation, interference). Most of the assays focused on human serum/plasma, utilizing acetonitrile or methanol (with ammonium acetate/formate or formic/acetic acid), liquid–liquid extraction, reverse phase (e.g., C18) chromatography, and gradient elution for analyte separation. Mass spectrometry conditions were also consistent in the identified papers, with negative electrospray ionization, select multiple reaction monitoring transitions and deuterated internal standards being the most common approaches. The validated biological applications indicated IxS and/or pCS were correlated with renal disease progression and cardiovascular outcomes, with limited data on central nervous system disorders. Methods for reducing IxS and/or pCS concentrations were also identified (e.g., drugs, natural products, diet, dialysis, transplantation) where inconsistent findings have been reported. The clinical monitoring of IxS and pCS is gaining significant interest, and this review will serve as a useful compendium for scientists and clinicians. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Uremic cardiomyopathy (UCM) is the leading cause of chronic kidney disease (CKD) related mortality. Uremic toxins including indoxyl sulfate (IS) play important role during the progression of UCM. This study was to explore the underlying mechanism of IS related myocardial injury. UCM rat model was established through five-sixths nephrectomy to evaluate its effects on blood pressure, cardiac impairment, and histological changes using echocardiography and histological analysis. Additionally, IS was administered to neonatal rat cardiomyocytes (NRCMs) and the human cardiomyocyte cell line AC16. DHE staining and peroxide-sensitive dye 2′,7′-dichlorofluorescein diacetate (H2DCFDA) was conducted to assess the reactive oxygen species (ROS) production. Cardiomyocyte hypertrophy was estimated using wheat germ agglutinin (WGA) staining and immunofluorescence. Aryl hydrocarbon receptor (AhR) translocation was observed by immunofluorescence. The activation of AhR was evaluated by immunoblotting of cytochrome P450 1 s (CYP1s) and quantitative real-time PCR (RT-PCR) analysis of AHRR and PTGS2. Additionally, the pro-oxidative and pro-hypertrophic effects were evaluated using the AhR inhibitor CH-223191, the CYP1s inhibitor Alizarin and the ROS scavenger N-Acetylcysteine (NAC). UCM rat model was successfully established, and cardiac hypertrophy, accompanied by increased blood pressure, and myocardial fibrosis. Further research confirmed the activation of the AhR pathway in UCM rats including AhR translocation and downstream protein CYP1s expression, accompanied with increasing ROS production detected by DHE staining. In vitro experiment demonstrated a translocation of AhR triggered by IS, leading to significant increase of downstream gene expression. Subsequently study indicated a close relationship between the production of ROS and the activation of AhR/CYP1s, which was effectively blocked by applying AhR inhibitor, CYP1s inhibitor and siRNA against AhR. Moreover, the inhibition of AhR/CYP1s/ROS pathway collectively blocked the pro-hypertrophic effect of IS-mediated cardiomyopathy. This study provides evidence that the AhR/CYP1s pathway is activated in UCM rats, and this activation is correlated with the uremic toxin IS. In vitro studies indicate that IS can stimulate the AhR translocation in cardiomyocyte, triggering to the production of intracellular ROS via CYP1s. This process leads to prolonged oxidative stress stimulation and thus contributes to the progression of uremic toxin-mediated cardiomyopathy. • Uremic cardiomyopathy model built via five-sixths nephrectomy, showing cardiac hypertrophy and ROS production. • Increased ROS production in uremic cardiomyopathy is related to aryl hydrocarbon receptor activation. • Downstream cytochrome P450s 1A1 and 1A2 expression is elevated after IS treating in cardiomyocytes. • Block of aryl hydrocarbon receptor and cytochrome P450s inhibits ROS production and cardiomyocytes hypertrophy. [ABSTRACT FROM AUTHOR]

Calcific Aortic Valve Disease (CAVD) is a significant concern for cardiovascular health and is closely associated with chronic kidney disease (CKD). Aortic valve endothelial cells (VECs) play a significant role in the onset and progression of CAVD. Previous research has suggested that uremic toxins, particularly indoxyl sulfate (IS), induce vascular calcification and endothelial dysfunction, but the effect of IS on valve endothelial cells (VECs) and its contribution to CAVD is unclear. Our results show that IS reduced human VEC viability and increased pro-calcific markers RUNX2 and alkaline phosphatase (ALP) expression. Additionally, IS-exposed VECs cultured in pro-osteogenic media showed increased calcification. Mechanistically, IS induced endothelial-to-mesenchymal transition (EndMT), evidenced by the loss of endothelial markers and increased expression of mesenchymal markers. IS triggered VEC inflammation, as revealed by NF-kB activation, and decreased integrin-linked kinase (ILK) expression. ILK overexpression reversed the loss of endothelial phenotype and RUNX2, emphasizing its relevance in the pathogenesis of CAVD in CKD. Conversely, a lower dose of IS intensified some of the effects in EndMT caused by silencing ILK. These findings imply that IS affects valve endothelium directly, contributing to CAVD by inducing EndMT and calcification, with ILK acting as a crucial modulator. [ABSTRACT FROM AUTHOR]

Inadequate clearance of protein-bound uremic toxins (PBUTs) during dialysis is associated with morbidities in chronic kidney disease patients. The development of high-permeance membranes made from materials such as graphene raises the question whether they could enable the design of dialyzers with improved PBUT clearance. Here, we develop device-level and multi-compartment (body) system-level models that account for PBUT-albumin binding (specifically indoxyl sulfate and p-cresyl sulfate) and diffusive and convective transport of toxins to investigate how the overall membrane permeance (or area) and system parameters including flow rates and ultrafiltration affect PBUT clearance in hemodialysis. Our simulation results indicate that, in contrast to urea clearance, PBUT clearance in current dialyzers is mass-transfer limited: Assuming that the membrane resistance is dominant, raising PBUT permeance from 3 × 10−6 to 10−5 m s−1 (or equivalently, 3.3 × increase in membrane area from ~ 2 to ~ 6 m2) increases PBUT removal by 48% (from 22 to 33%, i.e., ~ 0.15 to ~ 0.22 g per session), whereas increasing dialysate flow rates or adding adsorptive species have no substantial impact on PBUT removal unless permeance is above ~ 10−5 m s−1. Our results guide the future development of membranes, dialyzers, and operational parameters that could enhance PBUT clearance and improve patient outcomes. [ABSTRACT FROM AUTHOR]

Trained immunity is the long-term functional reprogramming of innate immune cells, which results in altered responses toward a secondary challenge. Despite indoxyl sulfate (IS) being a potent stimulus associated with chronic kidney disease (CKD)-related inflammation, its impact on trained immunity has not been explored. Here, we demonstrate that IS induces trained immunity in monocytes via epigenetic and metabolic reprogramming, resulting in augmented cytokine production. Mechanistically, the aryl hydrocarbon receptor (AhR) contributes to IS-trained immunity by enhancing the expression of arachidonic acid (AA) metabolism-related genes such as arachidonate 5-lipoxygenase (ALOX5) and ALOX5 activating protein (ALOX5AP). Inhibition of AhR during IS training suppresses the induction of IS-trained immunity. Monocytes from end-stage renal disease (ESRD) patients have increased ALOX5 expression and after 6 days training, they exhibit enhanced TNF-α and IL-6 production to lipopolysaccharide (LPS). Furthermore, healthy control-derived monocytes trained with uremic sera from ESRD patients exhibit increased production of TNF-α and IL-6. Consistently, IS-trained mice and their splenic myeloid cells had increased production of TNF-α after in vivo and ex vivo LPS stimulation compared to that of control mice. These results provide insight into the role of IS in the induction of trained immunity, which is critical during inflammatory immune responses in CKD patients.

Serum myostatin and indoxyl sulfate (IS) levels increase with kidney function decline and may function as uremic toxins in chronic kidney disease (CKD)-related sarcopenia. Herein, we analyzed the association between serum myostatin and IS levels and sarcopenia in patients with CKD, by performing a post hoc analysis of baseline data extracted from the RECOVERY study (clinicaltrials.gov: NCT03788252) of 150 patients with CKD. We stratified patients into two groups according to the median value of myostatin (cutoff 4.5 ng/mL) and IS levels (cutoff 0.365 mg/dL). The proportion of patients with sarcopenia was higher in those with high IS levels but lower in those with high myostatin levels. The skeletal muscle mass index (SMI) and handgrip strength (HGS) were significantly lower in patients with high IS levels but significantly higher in patients with high myostatin levels. IS levels showed a negative correlation with glomerular filtration rate (GFR), SMI, and HGS. However, myostatin levels were positively correlated with SMI and HGS, but not with GFR. Sarcopenia was independently associated with age and IS level after adjustment. Increased levels of serum total IS might play a role in sarcopenia, while increased levels of serum myostatin are associated with muscle mass in patients with CKD.

Background: With societal and economic development, the annual incidence of chronic kidney disease (CKD) is increasing. Current treatments for CKD are limited, and once patients progress to the uraemic stage, it places a significant economic burden on families and society. Based on the "gut-kidney axis" theory and real-world research, this study aims to evaluate the clinical efficacy, safety, and potential mechanism of high-position colon dialysis combined with traditional Chinese medicine (TCM) retention enema in treating stage 3-5 chronic kidney disease (non-dialysis). Additionally, it seeks to identify new therapeutic targets and approaches for CKD treatment. Methods: The TCM decoction was analyzed using Ultra-Performance Liquid Chromatography-Quadrupole-Orbitrap-High Resolution Mass Spectrometry (UPLC-Q-Orbitrap-HRMS). Participants meeting the inclusion criteria were divided into a control group (n = 153) and a treatment group (n = 159) based on their preferences and physicians' recommendations. Both groups adhered to a high-quality low-protein, low-salt, low-phosphorus, and low-fat diet supplemented with essential amino acids, and were monitored for blood pressure, blood glucose, and blood lipids. The treatment group received high-position colon dialysis combined with TCM retention enemas (administered at least 12 times every other day). Results: Thirteen compounds were identified from the herbs by UPLC-QOrbitrap-HRMS. The CKD3-5 treatment group exhibited improvements in blood biochemistry and other laboratory indices, with significant enhancements in renal function-related indices for CKD4 and CKD5 stages (p < 0.05). Following treatment, indoxyl sulfate (IS), endotoxin, and D-lactic acid levels decreased to a certain extent in both groups, with a statistically significant difference observed within the treatment group (p < 0.05). The treatment group displayed a significant reduction in aerobic bacterial colonies, an increase in anaerobic bacterial colonies, a decrease in Escherichia coli colonies, and an increase in Bifidobacterium and Lactobacillus colonies (p < 0.05). No significant changes in colony numbers were observed in the control group. Conclusion: High-position colon dialysis combined with TCM retention enema may serve as an adjuvant treatment for CKD4-5 (non-dialysis), and its mechanism may be related to the reduction of uraemic toxins, improvement of intestinal mucosal barrier function, and regulation of intestinal microecology. [ABSTRACT FROM AUTHOR]

Background Chronic kidney disease (CKD) is associated with a significant decrease in muscle strength and mass, possibly related to muscle cell damage by uremic toxins. Here, we studied in vitro and in vivo the effect of indoxyl sulfate (IS), an indolic uremic toxin, on myoblast proliferation, differentiation and expression of myogenic regulatory factors (MRF)—myoblast determination protein 1 (MyoD1), myogenin (Myog), Myogenic Factor 5 (Myf5) and myogenic regulatory factor 4 (Myf6 /MRF4)—and expression of myosin heavy chain, Myh2. Methods C2C12 myoblasts were cultured in vitro and differentiated in myotubes for 7 days in the presence of IS at a uremic concentration of 200 µM. Myocytes morphology and differentiation was analyzed after hematoxylin-eosin staining. MRF genes' expression was studied using reverse transcription polymerase chain reaction in myocytes and 5/6th nephrectomized mice muscle. Myf6 /MRF4 protein expression was studied using enzyme-linked immunosorbent assay; MYH2 protein expression was studied using western blotting. The role of Aryl Hydrocarbon Receptor (AHR)—the cell receptor of IS—was studied by adding an AHR inhibitor into the cell culture milieu. Results In the presence of IS, the myotubes obtained were narrower and had fewer nuclei than control myotubes. The presence of IS during differentiation did not modify the gene expression of the MRFs Myf5, MyoD1 and Myog, but induced a decrease in expression of Myf6 /MRF4 and MYH2 at the mRNA and the protein level. AHR inhibition by CH223191 did not reverse the decrease in Myf6/ MRF4 mRNA expression induced by IS, which rules out the implication of the ARH genomic pathway. In 5/6th nephrectomized mice, the Myf6 /MRF4 gene was down-regulated in striated muscles. Conclusion In conclusion, IS inhibits Myf6/ MRF4 and MYH2 expression during differentiation of muscle cells, which could lead to a defect in myotube structure. Through these new mechanisms, IS could participate in muscle atrophy observed in CKD. [ABSTRACT FROM AUTHOR]

Background: Pruritus is a common complaint in patients with end-stage renal disease. Indoxyl sulfate (IS) is a tryptophan end metabolite extremely renal excreted. Activated charcoal can interfere with IS intestinal absorption. Objectives: To evaluate the serum level of IS and the effect of activated charcoal on uremic pruritus. Materials and Methods: In all, 135 participants were divided into 2 main groups. In total, 45 normal and healthy individuals as a control group and 90 patients on regular hemodialysis; 45 of these patients had uremic pruritus and the other 45 were not complaining of uremic pruritus. Serum IS was measured. Activated charcoal was used by patients with uremic pruritus. The severity of pruritus and Dermatology Life Quality Index (DLQI) were assessed. Results: The serum IS was significantly elevated in uremic patients than in control subjects (P <.001) and significantly elevated in uremic patients without pruritus (P <.001). Furthermore, there were positive significant correlations between the serum IS and both severity of pruritus (P <.001) and DLQI (P <.001). After activated charcoal usage, there was a significant decrease in IS level with the improvement of pruritus and quality of life of patients. Conclusions: IS may play a role in uremic pruritus. Activated charcoal could be considered a treatment for uremic pruritus. [ABSTRACT FROM AUTHOR]

Excessive daytime sleepiness (EDS) is associated with quality of life and all-cause mortality in the end-stage renal disease population. This study aims to identify biomarkers and reveal the underlying mechanisms of EDS in peritoneal dialysis (PD) patients. A total of 48 nondiabetic continuous ambulatory peritoneal dialysis patients were assigned to the EDS group and the non-EDS group according to the Epworth Sleepiness Scale (ESS). Ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) was used to identify the differential metabolites. Twenty-seven (male/female, 15/12; age, 60.1 ± 16.2 years) PD patients with ESS ≥ 10 were assigned to the EDS group, while twenty-one (male/female, 13/8; age, 57.9 ± 10.1 years) PD patients with ESS < 10 were defined as the non-EDS group. With UHPLC-Q-TOF/MS, 39 metabolites with significant differences between the two groups were found, 9 of which had good correlations with disease severity and were further classified into amino acid, lipid and organic acid metabolism. A total of 103 overlapping target proteins of the differential metabolites and EDS were found. Then, the EDS–metabolite–target network and the protein–protein interaction network were constructed. The metabolomics approach integrated with network pharmacology provides new insights into the early diagnosis and mechanisms of EDS in PD patients. [ABSTRACT FROM AUTHOR]

Aims: Indoxyl sulfate and parathyroid hormone (PTH), which accumulate in chronic kidney disease (CKD), have been reported to reduce cytochrome P450(CYP)3A activity. Homozygotes of the CYP3A5*3 allele have reduced CYP3A5 activity compared to carriers of at least one CYP3A5*1 allele. 4β‐Hydroxycholesterol (4β‐OHC) has been established as an endogenous substrate reflecting CYP3A activity. 4β‐OHC is produced through hydroxylation by CYP3A4 and CYP3A5 and by autoxidation of cholesterol, whereas 4α‐hydroxycholesterol (4α‐OHC) is produced solely by autoxidation of cholesterol. This study focused on CKD patients and evaluated the effects of plasma indoxyl sulfate and intact‐PTH concentrations on plasma 4β‐OHC concentration, 4β‐OHC/total cholesterol ratio and 4β‐OHC–4α‐OHC, with consideration of the influence of CYP3A5 polymorphism. Methods: Sixty‐three CKD patients were analysed and divided into CYP3A5 carrier group (n = 26) and non‐carrier group (n = 37). Results: Plasma indoxyl sulfate significantly correlated inversely with 4β‐OHC concentration and with 4β‐OHC–4α‐OHC in both the CYP3A5*1 carrier group (r = −0.42, P =.034; r = −0.39, P =.050, respectively) and the non‐carrier group (r = −0.45, P =.0054; r = −0.39, P =.019, respectively). However, multiple regression analysis did not identify plasma indoxyl sulfate concentration as a significant independent factor associated with any of the CYP3A activity indices. There was no significant correlation between plasma intact‐PTH concentration and any of the CYP3A activity indices. Conclusions: The present results suggest that plasma indoxyl sulfate and intact‐PTH concentrations do not have clinically significant effects on CYP3A activity in patients with CKD. [ABSTRACT FROM AUTHOR]

Asperulosidic acid is a bioactive iridoid isolated from Hedyotis diffusa Willd. with anti-inflammatory and renal protective effects. However, its mechanism on renal interstitial fibrosis has not been elucidated yet. The present study aims to explore whether asperulosidic acid could retard renal fibrosis by reducing the circulating indoxyl sulfate (IS), which is a uremic toxin and accelerates chronic kidney disease progression by inducing renal fibrosis. In this paper, a unilateral ureteral obstruction (UUO) model of Balb/C mice was established. After the mice were orally administered with asperulosidic acid (14 and 28 mg/kg) for two weeks, blood, liver and kidney were collected for biochemical, histological, qPCR and Western blot analyses. Asperulosidic acid administration markedly reduced the serum IS level and significantly alleviated the histological changes in glomerular sclerosis and renal interstitial fibrosis. It is noteworthy that the mRNA and protein levels of the organic anion transporter 1 (OAT1), OAT3 and hepatocyte nuclear factor 1α (HNF1α) in the kidney were significantly increased, while the mRNA expressions of cytochrome P450 2e1 (Cyp2e1) and sulfotransferase 1a1 (Sult1a1) in the liver were not altered after asperulosidic acid administration. These results reveal that asperulosidic acid could accelerate the renal excretion of IS by up-regulating OATs via HNF1α in UUO mice, thereby alleviating renal fibrosis, but did not significantly affect its production in the liver, which might provide important information for the development of asperulosidic acid. [ABSTRACT FROM AUTHOR]

Introduction: Indoxyl sulfate (IS) is a protein‐bound uremic toxin that causes uremic sarcopenia. IS has poor dialysis clearance; however, the addition of a binding competitor improves its removal efficiency. Methods: Dialysis experiments were performed using N‐acetyl‐l‐tryptophan (L‐NAT) instead of l‐tryptophan (Trp) using pooled sera obtained from dialysis patients. The molecular structures of L‐NAT and Trp were similar to that of IS. Therefore, we examined whether Trp and L‐NAT were involved in muscle atrophy in the same manner as IS by performing culture experiments using a human myotube cell line. Results: The removal efficiency of L‐NAT was the same as that of Trp. However, L‐NAT concentrations in the pooled sera increased at the end of the experiment. Trp (1 mM) decreased the area of human myocytes, similar to IS, whereas L‐NAT did not. Conclusion: L‐NAT is a binding competitor with the ability to remove protein‐bound IS while preventing sarcopenia. [ABSTRACT FROM AUTHOR]

Indoxyl sulfates are uremic indolic toxins known to participate in the pathogenesis of cardiovascular diseases during chronic kidney disease in humans and some animal species. However, nothing is known about the indoxyl sulfate effect on the thyroid gland which is especially responsible for the general organism metabolism. This study determines the morpho-functional status of the thyroid gland after exposure to indoxyl sulfate (10, 25, and 50 mM) with the use of an ex vivo system and rabbit (n=10) as an experimental model thyroid gland histology, immunoexpression of thyrotropin receptor (TSHR), and concentrations of thyroxine (T4) and triiodothyronine (T3) were evaluated. Statistical analyses were performed using one-way analysis of the variance (ANOVA) followed by Tukey's post hoc comparison test. Minor alterations in thyroid tissue structure e.g. very rare exfoliated epithelial cells, condensed colloid fluid, or slight loosening of the epithelium were found. In addition, modulated dose dependent-expression of TSHR (p<0.01, p<0.001) together with a decreased level of T4 and T3 (p<0.001, p<0.01) exception of an increased level of T4 after the middle dose of indoxyl sulfate were revealed. We report here, for the first time, that indoxyl sulfate affects the thyroid gland mainly at the molecular level. The rabbit thyroid gland ex vivo system seems to be suitable for further studies on the thyroid gland in health and disease. However, the effect of TSH-TSHR signaling at ultrastructural, and epigenetic levels needs supplementary appraisal. [ABSTRACT FROM AUTHOR]

Background and Objectives: The present study aims to investigate the association between gut microbiota's oxalate-degrading activity (ODA) and the risk of developing cardiovascular disease (CVD) over a three-year follow-up period in a cohort of patients undergoing kidney replacement therapy (KRT). Additionally, various factors were examined to gain insight into the potential mechanisms underlying the ODA–CVD link. Materials and Methods: A cohort of 32 KRT patients and 18 healthy volunteers was enrolled in this prospective observational pilot study. Total fecal ODA, routine clinical data, plasma oxalic acid (POx), serum indoxyl sulfate, lipid profile, oxidative stress, and proinflammatory markers were measured, and the patients were followed up for three years to assess CVD events. Results: The results revealed that patients with kidney failure exhibited significantly lower total fecal ODA levels compared to the healthy control group (p = 0.017), with a higher proportion showing negative ODA status (≤−1% per 0.01 g) (p = 0.01). Negative total fecal ODA status was associated with a significantly higher risk of CVD events during the three-year follow-up period (HR = 4.1, 95% CI 1.4–16.3, p = 0.003), even after adjusting for potential confounders. Negative total fecal ODA status was significantly associated with elevated POx and indoxyl sulfate levels and linked to dyslipidemia, increased oxidative stress, and inflammation, which are critical contributors to CVD. Conclusions: The findings contribute novel insights into the relationship between gut microbiota's ODA and cardiovascular health in patients undergoing KRT, emphasizing the need for further research to elucidate underlying mechanisms and explore potential therapeutic implications of targeting gut microbiota's ODA in this vulnerable population. [ABSTRACT FROM AUTHOR]

Abstract Chronic kidney disease (CKD) involves progressive renal fibrosis, which gradually reduces kidney function and often causes various complications in extrarenal tissues. Therefore, we investigated fibrogenesis in extrarenal tissues (heart, liver, and lungs) in different experimental CKD models, such as the 5/6‐nephrectomy (5/6 Nx), unilateral ureteral obstruction (UUO), and a combination (2/3 Nx + UUO). We evaluated the degree of fibrogenesis in kidneys and extrarenal tissues by histological analysis and quantification of fibrosis‐related gene and protein expression. To elucidate the fibrosis mechanisms observed in 2/3 Nx + UUO mice, we evaluated the effect of indoxyl sulfate (IS), a typical uremic toxin accumulated in CKD, and transforming growth factor‐β (TGF‐β), a fibrosis‐related factor, on fibrosis using human hepatoma (HepG2) and RAW264.7 cells. A significant decline in renal function was observed in the 5/6 Nx and 2/3 Nx + UUO models, whereas a significant increase in renal fibrosis was observed only in the obstructed kidneys. Notable amount of fibrosis was induced in the liver and heart in the 2/3 Nx + UUO model, with the induction of macrophage infiltration and increased tissue IS and TGF‐β levels. In agreement with the results of in vivo experiments, co‐stimulation with IS, TGF‐β, and macrophage‐conditioned medium increased the expression of fibrogenic genes in HepG2 cells. We demonstrated that the 2/3 Nx + UUO model induced both loss of renal function and renal fibrosis in the earlier stages, providing a novel CKD model that induces remote organ fibrosis in a shorter time.

Uremic toxins cause bone disorders in patients with chronic kidney disease (CKD). These disorders are characterized by low turnover osteodystrophy and impaired bone formation in the early stages of CKD. Evidence indicates that the aryl hydrocarbon receptor (AhR) mediates signals that suppress early osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). However, whether the AhR mediates the effects of indoxyl sulfate (IS), a uremic toxin, on BMSC osteogenesis remains unclear. We investigated whether IS affects osteogenesis through the AhR/Hes1 pathway. Expression levels of osteogenesis genes (Runx2, Bmp2, Alp, and Oc), AhR, and Hes1 were measured in mouse BMSCs (D1 cells). At concentrations of 2–50 μM, IS significantly reduced mineralization, particularly in the early stages of BMSC osteogenesis. Furthermore, IS significantly downregulated the expression of Runx2, Bmp2, Oc, and Alp. Notably, this downregulation could be prevented using an AhR antagonist and through Ahr knockdown. Mechanistically, IS induced the expression of Hes1 through AhR signaling, thereby suppressing the transcription of Runx2 and Bmp2. Our findings suggest that IS inhibits early osteogenesis of BMSCs through the AhR/Hes1 pathway, thus suppressing the transcription of Runx2 and Bmp2. Our findings may guide new therapeutic strategies against CKD-related bone disorders.

Serum uremic toxins markedly increase in cats with chronic kidney disease (CKD) and have deleterious consequences. Renaltec is an oral adsorbent that binds uremic toxin precursors in the gut. In this prospective cohort study utilizing 13 purpose-bred cats with remnant kidney model-induced CKD (12 IRIS Stage 2, 1 IRIS Stage 3) eating a standardized renal diet, we aimed to assess the effect of Renaltec administration on serum indoxyl sulfate (IDS) and p-cresol sulfate (pCS) concentrations. Cats were sequentially treated with standard of care for 56 days, 500 mg Renaltec orally once daily for 56 days, and then three months later, 500 mg Renaltec orally twice daily for 56 days. Serum IDS and pCS concentrations were measured 28 and 56 days after the administration of Renaltec. Blood pressure and kidney function were measured before and 56 days after the administration of Renaltec. Significant decreases in serum IDS and pCS concentrations were observed for both once- and twice-daily dosing, particularly during the first 28 days of administration. More cats with BID dosing had clinically significant reductions in serum IDS and pCS concentrations than with SID dosing. Renaltec can reduce the serum concentrations of deleterious gut-derived uremic toxins in cats with CKD.

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 C3C5D were examined. Serum concentrations of IS, interleukin 6 (IL-6), tumor necrosis factor (TNF-), 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- 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-, 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.

Marcin Choroszy,1 Kamila &Sacute;roda-Pomianek,2 Magdalena Wawrzy&nacute;ska,3 Mateusz Chmielarz,1 Edyta Bo&zdot;emska,1 Beata Sobieszcza&nacute;ska1 1Department of Microbiology, Wroclaw Medical University, Wroclaw, Poland; 2Department of Biophysics and Neuroscience, Wroclaw Medical University, Wroclaw, Poland; 3Department of Preclinical Studies, Faculty of Health Sciences, Wroclaw Medical University, Wroclaw, PolandCorrespondence: Marcin Choroszy, Chalubinskiego 4 Street, Wroclaw, 51-657, Poland, Tel +48-71-7840-065, Fax +48-71-784-0117, Email marcin.choroszy@student.umw.edu.plIntroduction: Metabolic endotoxemia most often results from obesity and is accompanied by an increase in the permeability of the intestinal epithelial barrier, allowing co-absorption of bacterial metabolites and diet-derived fatty acids into the bloodstream. A high-fat diet (HFD) leading to obesity is a significant extrinsic factor in developing vascular atherosclerosis. In this study, we evaluated the effects of palmitic acid (PA) as a representative of long-chain saturated fatty acids (LCSFA) commonly present in HFDs, along with endotoxin (LPS; lipopolysaccharide) and uremic toxin indoxyl sulfate (IS), on human vascular endothelial cells (HUVECs).Methods: HUVECs viability was measured based on tetrazolium salt metabolism, and cell morphology was assessed with fluorescein-phalloidin staining of cells’ actin cytoskeleton. The effects of simultaneous treatment of endothelial cells with PA, LPS, and IS on nitro-oxidative stress in vascular cells were evaluated quantitatively with fluorescent probes. The expression of vascular cell adhesion molecule VCAM-1, E-selectin, and occludin, an essential tight junction protein, in HUVECs treated with these metabolites was evaluated in Western blot.Results: PA, combined with LPS and IS, did not influence HUVECs viability but induced stress on actin fibers and focal adhesion complexes. Moreover, PA combined with LPS significantly enhanced reactive oxygen species (ROS) production in HUVECs but decreased nitric oxide (NO) generation. PA also considerably increased the expression of VCAM-1 and E-selectin in HUVECs treated with LPS or IS but decreased occludin expression.Conclusion: Palmitic acid enhances the toxic effect of metabolic endotoxemia on the vascular endothelium.Keywords: metabolic endotoxemia, high-fat diet, palmitic acid, endotoxin, indoxyl sulfate

ABSTRACTp-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.

AbstractExcessive daytime sleepiness (EDS) is associated with quality of life and all-cause mortality in the end-stage renal disease population. This study aims to identify biomarkers and reveal the underlying mechanisms of EDS in peritoneal dialysis (PD) patients. A total of 48 nondiabetic continuous ambulatory peritoneal dialysis patients were assigned to the EDS group and the non-EDS group according to the Epworth Sleepiness Scale (ESS). Ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) was used to identify the differential metabolites. Twenty-seven (male/female, 15/12; age, 60.1 ± 16.2 years) PD patients with ESS ≥ 10 were assigned to the EDS group, while twenty-one (male/female, 13/8; age, 57.9 ± 10.1 years) PD patients with ESS < 10 were defined as the non-EDS group. With UHPLC-Q-TOF/MS, 39 metabolites with significant differences between the two groups were found, 9 of which had good correlations with disease severity and were further classified into amino acid, lipid and organic acid metabolism. A total of 103 overlapping target proteins of the differential metabolites and EDS were found. Then, the EDS–metabolite–target network and the protein–protein interaction network were constructed. The metabolomics approach integrated with network pharmacology provides new insights into the early diagnosis and mechanisms of EDS in PD patients.

Chronic kidney disease (CKD) patients have a high risk of cardiovascular disease. Indoxyl sulfate (IS) is a uremic toxin that has been shown to inhibit nitric oxide production and cause cell senescence by inducing oxidative stress. High-density lipoprotein (HDL) has a protective effect on the cardiovascular system; however, its impacts on IS-damaged endothelial cells are still unknown. This study aimed to explore the effects of exogenous supplement of HDL on vascular endothelial cells in a uremia-mimic environment. Tube formation, migration, adhesion, and senescence assays were used to evaluate the cell function of human aortic endothelial cells (HAECs). Reactive oxygen species generation was measured by using Amplex red assay. L-NAME and MCI186 were used as a nitric oxide synthase inhibitor and a free radical scavenger, respectively. HDL exerted anti-inflammatory and antioxidant effects via HIF-1α/HO-1 activation and IL-1β/TNF-α/IL-6 inhibition in IS-stimulated HAECs. HDL improved angiogenesis ability through upregulating Akt/eNOS/VEGF/SDF-1 in IS-stimulated HAECs. HDL decreased endothelial adhesiveness via downregulating VCAM-1 and ICAM-1 in IS-stimulated HAECs. Furthermore, HDL reduced cellular senescence via upregulating SIRT1 and downregulating p53 in IS-stimulated HAECs. Importantly, the above beneficial effects of HDL were mainly due to its antioxidant ability. In conclusion, HDL exerted a comprehensive protective effect on vascular endothelial cells against damage from IS through its antioxidant ability. The results of this study might provide a theoretical basis for potential HDL supplementation in CKD patients with endothelial damage. [ABSTRACT FROM AUTHOR]

The accumulation of the uremic toxin indoxyl sulfate (IS) is a key pathological feature of chronic kidney disease (CKD). The effect of IS on ferroptosis and the role of IS-related ferroptosis in CKD are not well understood. We used a renal tubular cell model and an adenine-induced CKD mouse model to explore whether IS induces ferroptosis and injury and affects iron metabolism in the renal cells and the kidneys. Our results showed that exposure to IS induced several characteristics for ferroptosis, including iron accumulation, an impaired antioxidant system, elevated reactive oxygen species (ROS) levels, and lipid peroxidation. Exposure to IS triggered intracellular iron accumulation by upregulating transferrin and transferrin receptors, which are involved in cellular iron uptake. We also observed increased levels of the iron storage protein ferritin. The effects of IS-induced ROS generation, lipid peroxidation, ferroptosis, senescence, ER stress, and injury/fibrosis were effectively alleviated by treatments with an iron chelator deferoxamine (DFO) in vitro and the adsorbent charcoal AST-120 (scavenging the IS precursor) in vivo. Our findings suggest that IS triggers intracellular iron accumulation and ROS generation, leading to the induction of ferroptosis, senescence, ER stress, and injury/fibrosis in CKD kidneys. AST-120 administration may serve as a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) involves progressive renal fibrosis, which gradually reduces kidney function and often causes various complications in extrarenal tissues. Therefore, we investigated fibrogenesis in extrarenal tissues (heart, liver, and lungs) in different experimental CKD models, such as the 5/6‐nephrectomy (5/6 Nx), unilateral ureteral obstruction (UUO), and a combination (2/3 Nx + UUO). We evaluated the degree of fibrogenesis in kidneys and extrarenal tissues by histological analysis and quantification of fibrosis‐related gene and protein expression. To elucidate the fibrosis mechanisms observed in 2/3 Nx + UUO mice, we evaluated the effect of indoxyl sulfate (IS), a typical uremic toxin accumulated in CKD, and transforming growth factor‐β (TGF‐β), a fibrosis‐related factor, on fibrosis using human hepatoma (HepG2) and RAW264.7 cells. A significant decline in renal function was observed in the 5/6 Nx and 2/3 Nx + UUO models, whereas a significant increase in renal fibrosis was observed only in the obstructed kidneys. Notable amount of fibrosis was induced in the liver and heart in the 2/3 Nx + UUO model, with the induction of macrophage infiltration and increased tissue IS and TGF‐β levels. In agreement with the results of in vivo experiments, co‐stimulation with IS, TGF‐β, and macrophage‐conditioned medium increased the expression of fibrogenic genes in HepG2 cells. We demonstrated that the 2/3 Nx + UUO model induced both loss of renal function and renal fibrosis in the earlier stages, providing a novel CKD model that induces remote organ fibrosis in a shorter time. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Indoxyl sulfate (IS) is a uremic toxin produced by the gut microbiota. • IS is accumulated in chronic kidney disease (CKD) patients and can be harmful. • Resveratrol is a polyphenol found in foods and exerts antioxidant and anti-inflammatory activities. • IS upregulated the inflammation and oxidative stress in macrophage-like RAW 264.7 cells. • Resveratrol treatment significantly reduced the inflammation and oxidative stress induced by IS in macrophage-like RAW 264.7. [ABSTRACT FROM AUTHOR]

Protein-bound uremic toxins (PBUTs) are associated with the progression of chronic kidney disease (CKD) and its associated morbidity and mortality. The conventional dialysis techniques are unable to efficiently remove PBUTs due to their plasma protein binding. Therefore, novel approaches are being developed, but these require validation in animals before clinical trials can begin. We conducted a systematic review to document PBUT concentrations in various models and species. The search strategy returned 1163 results for which abstracts were screened, resulting in 65 full-text papers for data extraction (rats (n = 41), mice (n = 17), dogs (n = 3), cats (n = 4), goats (n = 1), and pigs (n = 1)). We performed descriptive and comparative analyses on indoxyl sulfate (IS) concentrations in rats and mice. The data on large animals and on other PBUTs were too heterogeneous for pooled analysis. Most rodent studies reported mean uremic concentrations of plasma IS close to or within the range of those during kidney failure in humans, with the highest in tubular injury models in rats. Compared to nephron loss models in rats, a greater rise in plasma IS compared to creatinine was found in tubular injury models, suggesting tubular secretion was more affected than glomerular filtration. In summary, tubular injury rat models may be most relevant for the in vivo validation of novel PBUT-lowering strategies for kidney failure in humans. [ABSTRACT FROM AUTHOR]