Indoxyl Sulfate Levels Research Articles

Models incorporating physical, laboratory and gut metabolite markers can be used to predict severe hepatic steatosis in MAFLD patients.

Metabolic-associated fatty liver disease (MAFLD) induced-severe hepatic steatosis poses significant health risks. Early prediction of this condition is crucial for prompt intervention. Short-chain fatty acids (SCFAs) and tryptophan are gut metabolites correlated with MAFLD pathogenesis in the gut-liver axis. This study aims to construct prediction models for severe hepatic steatosis by including SCFAs and tryptophan metabolites. This study enrolled 83 participants from the outpatient department in 2023. Physical measurements, serum metabolic and inflammatory markers, metabolites of serum SCFAs and tryptophan were collected. Severe hepatic steatosis was diagnosed using vibration-controlled transient elastography and abdominal sonography. All 40 (48.2%) participants diagnosed with severe hepatic steatosis had MAFLD, while approximately three-quarters of those without severe hepatic steatosis had MAFLD. In comparison to the non-severe hepatic steatosis group, individuals with severe hepatic steatosis exhibited higher levels of waist and arm circumference, serum triglyceride (TG), and lower levels of serum high-density lipoprotein cholesterol (HDL-C) and AST/ALT ratio. They also had higher serum levels of lipopolysaccharide-binding protein, isovaleric acid, and propionic acid, and lower levels of 3-methylvaleric acid, indole-3-propionic acid, and indoxyl sulfate. Models incorporating these markers predicted severe hepatic steatosis. One model additionally included waist circumference and triglyceride-glucose index, while the other incorporated arm circumference and TG/HDL-C ratio. The area under the curve reached 0.958 and 0.938, respectively (p < 0.001). SCFAs and tryptophan metabolites are valuable in predicting severe hepatic steatosis. Further research is needed to investigate the roles of these metabolites in MAFLD.

AST-120 Protects Cognitive and Emotional Impairment in Chronic Kidney Disease Induced by 5/6 Nephrectomy

Background: Uremic toxins resulting from chronic kidney disease (CKD) can cause cognitive and emotional disorders, as well as cardiovascular diseases. Indoxyl sulfate (IS) and p-cresol are notable uremic toxins found in patients with CKD. However, few studies have investigated whether reducing uremic toxins can alleviate cognitive and emotional disorders associated with CKD. Methods: We studied the effects of AST-120, which lowers IS levels, through behavioral tests, local field potentials, field excitatory postsynaptic potentials, and histological experiments in a 5/6 nephrectomy CKD model. Results: We confirmed AST-120’s effectiveness in CKD by measuring serum creatinine, blood urea nitrogen, and IS levels and performing renal tissue staining. Behavioral phenotypes indicated an alleviation of cognitive and anxiety disorders following AST-120 treatment in CKD-induced rats, which was further validated through local field potentials and field excitatory postsynaptic potential recordings. Double immunofluorescence staining for aquaporin-4 and glial fibrillary acidic protein in the hippocampus of CKD rats treated with AST-120 showed reduced coexpression. Conclusions: Our findings demonstrate the potential therapeutic effects of AST-120 in lowering IS levels and improving cognitive and emotional impairments associated with CKD.

Gut Microbiota and Metabolic Alterations Associated with Heart Failure and Coronary Artery Disease.

This study investigates the role of gut microbiota in cardiovascular diseases, with an additional focus on pro-atherogenic metabolites. We use advanced network analysis and machine learning techniques to identify key microbial features linked to coronary artery disease (CAD) and heart failure with reduced ejection fraction (HFrEF). This cross-sectional study included 189 participants divided into three groups: coronary artery disease (n = 93), heart failure with reduced ejection fraction (n = 43), and controls (n = 53). Assessments included physical exams, echocardiography, dietary surveys, blood analysis, and fecal analysis. Gut microbiota composition was analyzed using next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR). Statistical analysis methods for testing hypotheses and correlations, alpha and beta-diversity analyses, co-occurrence networks, and machine learning were conducted using Python libraries or R packages with multiple comparisons corrected using the Benjamini-Hochberg procedure. Significant gut microbiota alterations were observed, with higher Bacillota/Bacteroidota ratios in CAD and HFrEF groups compared to controls (p < 0.001). Significant differences were observed in α-diversity indices (Pielou, Chao1, Faith) between disease groups and controls (p < 0.001). β-diversity analyses also revealed distinct microbial profiles (p = 0.0015). Interestingly, trimethylamine N-oxide (TMAO) levels were lower in CAD and HFrEF groups compared to controls (p < 0.05), while indoxyl sulfate (IS) levels were comparable between the study groups. Co-occurrence network analysis and machine learning identified key microbial features linked to these conditions, highlighting complex interactions within the gut microbiota associated with cardiovascular disease.

(Poly)phenol-rich grape and blueberry extract prevents LPS-induced disruption of the blood-brain barrier through the modulation of the gut microbiota-derived uremic toxins

The dynamic protective capacity of (poly)phenols, attributed to their potent antioxidant and anti-inflammatory properties, has been consistently reported. Due to their capacity to alter gut microbiome composition, further actions of (poly)phenols may be exerted through the modulation of the microbiota-gut-brain axis. However, the underlying mechanisms remain poorly defined. Here, we investigated the protective effect of a (poly)phenol-rich grape and blueberry extract (Memophenol™), on the microbiota-gut-brain axis in a model of chronic low-grade inflammation (0.5 mg/kg/wk lipopolysaccharide (LPS) for 8 weeks). Dietary supplementation of male C57BL/6 J mice with Memophenol™ prevented LPS-induced increases in the microbe-derived uremia-associated molecules, indoxyl sulfate (IS) and trimethylamine N-oxide (TMAO). These changes coincided with shifts in gut microbiome composition, notably Romboutsia and Desulfovibrio abundance, respectively. In the brain, LPS exposure disrupted the marginal localisation of the endothelial tight junction ZO-1 and downregulated ZO-1 mRNA expression to an extent closely correlated with TMAO and IS levels; a process prevented by Memophenol™ intake. Hippocampal mRNA sequencing analysis revealed significant downregulation in regulatory pathways of neurodegeneration with Memophenol™ intake. These findings may indicate a novel protective role of the (poly)phenol-rich grape and blueberry extract on the endothelial tight junction component ZO-1, acting through modulation of gut microbial metabolism.

Indoxyl sulfate induces retinal microvascular injury via COX-2/PGE2 activation in diabetic retinopathy

BackgroundDiabetic 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.MethodsTo 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.ResultsMetabolomics 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.ConclusionOur 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

Effects of Propolis Supplementation on Gut Microbiota and Uremic Toxin Profiles of Patients Undergoing Hemodialysis.

Propolis possesses many bioactive compounds that could modulate the gut microbiota and reduce the production of uremic toxins in patients with chronic kidney disease (CKD) undergoing hemodialysis (HD). This clinical trial aimed to evaluate the effects of propolis on the gut microbiota profile and uremic toxin plasma levels in HD patients. These are secondary analyses from a previous double-blind, randomized clinical study, with 42 patients divided into two groups: the placebo and propolis group received 400 mg of green propolis extract/day for eight weeks. Indole-3 acetic acid (IAA), indoxyl sulfate (IS), and p-cresyl sulfate (p-CS) plasma levels were evaluated by reversed-phase liquid chromatography, and cytokines were investigated using the multiplex assay (Bio-Plex Magpix®). The fecal microbiota composition was analyzed in a subgroup of patients (n = 6) using a commercial kit for fecal DNA extraction. The V4 region of the 16S rRNA gene was then amplified by the polymerase chain reaction (PCR) using short-read sequencing on the Illumina NovaSeq PE250 platform in a subgroup. Forty-one patients completed the study, 20 in the placebo group and 21 in the propolis group. There was a positive correlation between IAA and TNF-α (r = 0.53, p = 0.01), IL-2 (r = 0.66, p = 0.002), and between pCS and IL-7 (r = 0.46, p = 0.04) at the baseline. No significant changes were observed in the values of uremic toxins after the intervention. Despite not being significant, microbial evenness and observed richness increased following the propolis intervention. Counts of the Fusobacteria species showed a positive correlation with IS, while counts of Firmicutes, Lentisphaerae, and Proteobacteria phyla were negatively correlated with IS. Two months of propolis supplementation did not reduce the plasma levels of uremic toxins (IAA, IS, and p-CS) or change the fecal microbiota.

Effect of fruits granola (Frugra®) consumption on blood pressure reduction and intestinal microbiome in patients undergoing hemodialysis

Cardiovascular diseases (CVDs) are a major cause of death in patients undergoing hemodialysis (HD). Blood pressure (BP) and uremic toxins are well-known risk factors for CVDs, which are influenced by diet. Dietary fiber supplementation in patients undergoing HD may reduce the risk of CVDs by improving lipid profiles and inflammatory status and lowering the levels of the uremic toxin indoxyl sulfate (IS). In this study, we investigated the relationship between the intestinal microbiota and risk factors for CVDs, such as BP and serum IS, in patients undergoing HD who consumed fruits granola (FGR). The study participants were selected from patients undergoing HD at the Izu Nagaoka Daiichi Clinic and consumed FGR for 2 months. Body composition and blood samples were tested at months 0, 1, 2 and fecal samples were collected at months 0 and 2 for intestinal microbiota analysis. FGR consumption decreased systolic and diastolic BP, estimated salt intake, and serum IS levels and improved the stool characteristics according to the Bristol Stool Form Scale (N = 24). Gut microbiota analysis showed an increase in the alpha diversity and abundance of Blautia and Neglecta. The abundance of lactic acid- and ethanol-producing bacteria also significantly increased, whereas the abundance of indole-producing bacteria significantly decreased. FGR consumption could be a useful tool for salt reduction, fiber supplementation, and improvement of the intestinal environment, thus contributing to improvement of BP and the reduction of other risk factors for CVDs in patients undergoing HD.

The simultaneous inhibition of SLC6A19 and BCRP transporters leads to an increase of indoxyl sulfate (a uremic toxin) in plasma and kidney.

SLC6A19 inhibitors are being studied as therapeutic agents for Phenylketonuria. In this work, a potent SLC6A19 inhibitor (RA836) elevated rat kidney uremic toxin indoxyl sulfate (IDS) levels by intensity (arbitrary unit) of 13.7{plus minus}7.7 compared to vehicle 0.3{plus minus}0.1 (P=0.01) as determined by tissue mass spectrometry imaging (tMSI) analysis. We hypothesized that increased plasma and kidney levels of IDS could be caused by the simultaneous inhibition of both Slc6a19 and a kidney IDS transporter responsible for excretion of IDS into urine. To test this, we first confirmed the formation of IDS through tryptophan metabolism by feeding rats a Trp-free diet. Inhibiting Slc6a19 with RA836 led to increased IDS in these rats. Next, RA836 and its key metabolites were evaluated in vitro for inhibiting kidney transporters OAT1, OAT3 and BCRP. RA836 inhibits BCRP with an IC50 of 0.045 µM but shows no significant inhibition of OAT1 or OAT3. Finally, RA836 analogs with either potent or no inhibition of SLC6A19 and/or BCRP were synthesized and administered to rats fed a normal diet. Plasma and kidney samples were collected to quantify IDS using LC-MS. Neither a SLC6A19 inactive but potent BCRP inhibitor nor a SLC6A19 active but weak BCRP inhibitor raised IDS levels, while compounds inhibiting both transporters caused IDS accumulation in rat plasma and kidney, supporting the hypothesis that rat Bcrp contributes to the excretion of IDS. In summary, we identified that inhibiting Slc6a19 increases IDS formation, while simultaneously inhibiting Bcrp results in IDS accumulation in the kidney and plasma. Significance Statement This is the first publication to decipher the mechanism for accumulation of IDS (a uremic toxin) in rats via inhibition of both Slc6a19 and Bcrp. Specifically, inhibition of Slc6a19 in the GI track increases IDS formation and inhibition of Bcrp in the kidney blocks IDS excretion. Therefore, we should avoid inhibiting both SLC6A19 and BCRP simultaneously in humans to prevent accumulation of IDS, a known risk factor for cardiovascular disease, psychic anxiety, and mortality in chronic kidney disease patients.

Reducing the levels of indoxyl sulfate in patients undergoing dialysis: a promising approach to managing inflammation and the redox state of human serum albumin.

Indoxyl sulfate (IS) is one of the most potent uraemic toxins involved in the progression of chronic kidney disease (CKD) through the induction of inflammation and oxidative stress. This study assessed the potential benefits of reducing IS concentrations through dialysis treatment to improve renal function, inflammation, and oxidative stress. A prospective, observational cohort study of 50 patients with CKD undergoing dialysis treatment was conducted. IS levels, inflammatory markers (IL-6 and hs-CRP), and oxidative status (Cu/Zn-SOD) were measured using immunoenzymatic methods, and the albumin ratio (HNA/HMA) was assessed using high-performance liquid chromatography. Blood samples were collected at baseline and, at 8 weeks and 16 weeks after treatment. At baseline, patients with CKD had elevated levels of IS, renal function indicators, inflammatory markers (IL-6 and CRP), and oxidative markers (Cu/Zn-SOD and albumin ratio HNA/HMA). Dialysis treatment reduced IS levels, and a correlation among IS, renal function, and SOD levels (P < 0.0001) at 8 and 16 weeks was observed. The reduction in IS levels was associated with improved inflammatory marker levels (CRP and IL-6; P < 0.0001) and a significant decrease in the HNA/HMA ratio (P <0.0001) at 8 and 16 weeks. These associations strengthened over time. The results of this study suggest that IS levels may be a therapeutic target for improving outcomes in patients with CKD by improving renal function, inflammation, and oxidative stress. More research is needed to understand how IS contributes to CKD complications.

Relationship Between Arterial Stiffness, Measured by Cardio-Ankle Vascular Index, and Uremic Toxins, Vascular Calcification, and Inflammation Markers After Kidney Donation.

This study investigated whether kidney transplant donors experience increased arterial stiffness compared with the general population and how arterial stiffness changes over time. Our study included 59 kidney transplant donors and 27 healthy volunteers. All subjects underwent cardio-ankle vascular index measurements. We studied fibroblast growth factor23, klotho, monocyte chemoattractant protein-1, N-terminal pro-B-type natriuretic peptide, indoxyl sulfate, and p-cresyl sulfate levels. Cardio-ankle vascular index level was higher in donors 6 to 11 years after donation (8.02 ± 0.24 m/s) than in donors 2 to 6 years after donation (7.02 ± 0.27 m/s) and healthy volunteers (6.65 ± 0.22 m/s). Cardioankle vascular index level was positively correlated with age (r = 0.382, P < .001) and levels of triglyceride (r = 0.213, P = .049), blood urea nitrogen (r = 0.263, P = .014), creatinine (r = 0.354, P = .001), calcium (r = 0.228, P = .035), indoxyl sulfate (r = 0.219, P = .042), p-cresyl sulfate (r = 0.676, P ≤ .001), and monocyte chemoattractant protein-1 (r = 0.451, P ≤ .001) and negatively correlated with estimated glomerular filtration rate (r = -0.383, P < .001). Multiple linear regression analysis revealed that age (P = .026, B = 0.244), mean arterial blood pressure (P < .001, B = 0.446), blood urea nitrogen (P = .006, B = 0.302), creatinine (P = .032, B = 0.236), estimated glomerular filtration rate (P = .003, B = -0.323), fibroblast growth factor-23 (P = .007, B = 0.294), N-terminal pro-B-type natriuretic peptide (P = .005, B = 0.304), and monocyte chemoattractant protein-1 (P ≤ .001, B = 0.434) independently predicted cardio-ankle vascular index levels. Even without additional risk factors, kidney donors should be followed closely for arterial stiffness and cardiovascular disease, especially in the long-term (>5 years) after kidney transplant.

Natural antagonistic flavones for AhR inhibit indoxyl sulfate-induced inflammatory gene expression in vitro and renal pathological damages in vivo.

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. 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. 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. 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. 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.

Impact of Serum Indoxyl Sulfate on One-Year Adverse Events in Chronic Kidney Disease Patients with Heart Failure.

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.

Increased Blood-Brain Barrier Permeability and Cognitive Impairment in Patients With ESKD

Background and AimsChronic kidney disease (CKD) is associated with an increased risk of cognitive impairment. This cognitive impairment is associated with an increased permeability of blood-brain barrier (BBB) in rodents with CKD, linked to activation of aryl hydrocarbon receptor (AhR) by indoxyl sulphate (IS). The objective of BREIN study is to confirm the increased BBB permeability in humans with CKD. MethodThe BREIN comparative study (NCT04328415) prospectively included patients with end-stage kidney disease (ESKD), and healthy volunteers (Ctrl) matched in age, sex, and educational level to a patient. In all participants, BBB permeability was quantified by brain 99mTc-DTPA SPECT/CT as percentage of injected activity (%IA). A battery of neuro-cognitive tests was performed, serum uremic toxins accumulation and AhR activation were assessed. ResultsFifteen ESKD patients and 14 healthy volunteers were analysed. ESKD patients had higher BBB permeability compared to controls: 0.29±0.07 vs. 0.14±0.06 %IA, p=0.002. ESKD patients displayed lower MoCA score: 22.0±5.0 vs. 27.3 ± 2.8, p=0.008, impaired short-term memory (doors test): 12.5±3.4 vs. 16.5±3.4, p=0.005, higher Beck depression score 8.1±9.1 vs. 2.7±3.4, p=0.046, and slightly more daily cognitive complaints: 42.5±29.3 vs. 29.8±14.0 p=0.060. ESKD patients displayed higher IS levels (86.1±48.4 vs. 3.2±1.7 μmol/L, p=0.001) and AhR activating potential (37.7±17.8% vs. 24.7±10.4%, p=0.027). BBB permeability was inversely correlated with MoCA score (r=-0.60, 95%CI [-0.772; -0.339], p=0.001) in the overall population. ConclusionESKD patients display an increased BBB permeability compared to matched healthy volunteers. Association with uremic toxins and cognitive impairment needs to be assessed in larger cohorts of patients.

Oat Dietary Fiber Delays the Progression of Chronic Kidney Disease in Mice by Modulating the Gut Microbiota and Reducing Uremic Toxin Levels.

Chronic kidney disease (CKD) has emerged as a significant public health concern. In this article, we investigated the mechanism of oat dietary fiber in regulating CKD. Our findings indicated that the gut microbiota of CKD patients promoted gut microbiota dysbiosis and kidney injury in CKD mice. Intervention with oat-resistant starch prepared by ultrasonic combined enzymatic hydrolysis (ORSU) and oat β-glucan with a molecular weight of 5 × 104 Da (OBGM) elevated the levels of short-chain fatty acids (SCFAs) and regulated gut dysbiosis in the gut-humanized CKD mice. ORSU and OBGM also reduced CKD-related uremic toxins such as creatinine, indoxyl sulfate (IS), and p-cresol sulfate (PCS) levels; reinforced the intestinal barrier function of the gut-humanized CKD mice; and mitigated renal inflammation and fibrosis via the NF-κB/TGF-β pathway. Therefore, ORSU and OBGM might delay the progression of CKD by modulating the gut microbiota to reduce uremic toxins levels. Our results explain the mechanism of oat dietary fiber aimed at mitigating CKD.

#2224 Higher serum level of indoxyl sulfate is not associated with increased abdominal aorta calcification in patients on maintenance hemodialysis

Abstract Background and Aims Vascular calcification is strongly associated with an increase in all-cause and cardiovascular mortality, especially in patients with and stage renal disease. Results of numerous experimental studies have shown direct toxic effect of microbial-derived uremic toxins on vascular endothelial and smooth muscle cells which may contribute vascular calcification. Meanwhile, the effects of these metabolites on vascular remodeling in patients receiving dialysis treatment remains largely unclear. Here we aimed to investigate the relationship between serum level of the uremic toxin indoxyl sulfate and severity of abdominal aorta calcification (AAC) in patients on maintenance hemodialysis. Method This single-center, cross-sectional study included eighty (40 men, average age – 59,7±11,8 years) clinically stable patients receiving hemodialysis treatment for 52(21,3-120) months. 13(16,3%) participants had history of smoking, 27(33,8%) were diabetic, 63(78,8%) had high blood pressure, 17(21,3%) – previous history of ischemic heart disease, including 5(6,3%) suffered a myocardial infarction. Severity of AAC was determined by lateral lumbar radiography with Kauppila scoring. The total AAC score ranged from 0 to 24 points, and a higher AAC score indicated a more pronounced degree of calcification. Routine laboratory data were collected before hemodialysis session after fasting for 12 hours. Serum concentration of indoxyl sulfate was assessed by ELISA using commercially available kit (Cloud-Clone Corp., USA). Statistical analysis was performed with IBM SPSS Statistics 26 program. Non-parametric Spearman's rank correlation method was used to evaluate the associations between the studied parameters. Value of p &amp;lt;0.05 was considered statistically significant in all tests. Results Median serum indoxyl sulfate was 2,1(1,4-3,0) μmol/l (the same parameter in relatively healthy individuals without impaired renal function is 0,1(0-0,3) μmol/l, р˂0,001). Serum levels of calcium (Ca) was 2,27(2,1-2,5) mmol/l, ionized calcium (Ca++) – 1,15(1,1-1,3) mmol/l, phosphate (P) – 1,6(1,3-2,0) mmol/l, intact parathyroid hormone (iPTH) – 303(162,2-492,8) pg/ml, total cholesterol (TH) – 4,5(3,5-5,4) mmol/l, low-density lipoprotein (LDL) – 2,6(2,0-3,3) mmol/l. AAC score in hemodialysis participants was 4,5(0,0-9,0) and significantly correlated with age (r=0,693; р˂0,001), Ca (r=0,208; р=0,04) and iPTH (r=0,418; р=0,021) but not with other indicators, including indoxyl sulfate concentration (r=0,164; р=0,147). Conclusion Our preliminary results do not allow to consider the elevated level of indoxyl sulfate as a factor contributing to increased AAC in patients on maintenance hemodialysis. Further prospective studies in larger populations should clarify the true role of indoxyl sulfate in vascular remodeling in individuals with chronic kidney disease.

#279 The impact of synbiotics on the uremic toxins, inflammation, and gut microbiome of CKD patients—randomized, double-blind, placebo-controlled trial

Abstract Background and Aims The microbiome represents a complex system of microorganisms coexisting within the human body. The intestinal tract is the most densely populated region and is essential in preserving health and developing diseases such as chronic kidney disease (CKD). The relationship between the gut microbiome and the kidney is complex. CKD can significantly affect the composition of the gut microbiome, which, through the fermentation process, participates in the production of over 90 different uremic toxins with the potential to aggravate systemic inflammation, oxidative stress, and decrease renal function. Indoxyl-sulfate (IS), p-cresyl-sulfate (PCS), and trimethylamine-N-oxide (TMAO) are the most closely studied. Supplementation with pre-, pro-, or synbiotics could reduce the production and improve the intestinal clearance of uremic toxins. Assessing the efficacy and safety of this potential strategy has been the aim of our randomized, double-blind, placebo-controlled study. Method A total of 84 patients, aged over 18 years, with an estimated glomerular filtration rate (eGFR) between 15 and 45 ml/min, were randomized into two groups, the one treated with synbiotic—64 billion colonies of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium lactis with 6.4 g of inulin in the form of capsules, and a control group treated with an identical number and appearance of capsules but with a total of 6.8 g of bioinert maltodextrin, over a period of 12 weeks. Strict control of the diet and the use of antibiotics and other probiotics was carried out. The study examined the effect of therapy on the serum level of the uremic toxins IS, PCS, and TMAO, the composition of the stool microbiome, the serum level of interleukin 6 (IL6), high-sensitivity C-reactive protein (hs-CRP), eGFR, albuminuria, degree of liver steatosis according to ultrasound elastomeric measurement, occurrence of gastrointestinal symptoms and gut transition time. The level of uremic toxins was determined using ultra-performance liquid chromatography. To determine the composition of the intestinal microbiome, we used 16s rRNA amplicon sequencing targeting the V3-V4 hypervariable region. Results The synbiotic treatment significantly reduced the levels of IS (−11% vs. 1%, p = 0.033) and PCS (−6% vs. 0%, p = 0.011) in the intervention arm. We also observed that treatment with the synbiotic significantly reduced hsCRP levels (ΔhsCRP 0 vs −29%, p = 0.03). Synbiotic treatment greatly enriched the intestinal microbiome with the genera Lactobacillus, Bifidobacterium, and Faecalibacterium by 81%, 55%, and 40%, respectively, with a reduction in the genera Ruminococcus and Enterobacterium by 35% and 40%. The use of synbiotics led to a significant reduction of liver steatosis according to the dynamics of the attenuation coefficient—ATT (ΔATT% −0.6 vs −2.3%, p = 0.33), with a decrease in the serum alanine aminotransferase—ALT (ΔALT −19% vs −28%, p = 0.035). A statistically significant shortening of the gut transition time—GTT was observed in the intervention arm (ΔGTT −5 h vs −10 h, p = 0.031), paralleled by a reduction in the intensity and prevalence of constipation symptoms. No side effects were observed. Conclusion Synbiotics could be an effective, safe, and affordable therapy that could be used to modify the intestinal microbiome of CKD patients and contribute to the reduction of the level of uremic toxins, inflammation, degree of fatty liver, shorten gut transit time and reduce the symptoms of constipation.

#1086 Blood-brain barrier permeability in patients with end-stage kidney disease: results from the BREIN study

Abstract Background and Aims Chronic kidney disease (CKD) is associated with an increased risk of cognitive impairment. In several models of CKD in rodents, we highlighted that CKD was associated with cognitive impairment and increased blood-brain barrier (BBB) permeability, quantified using 99mTc-DTPA scintigraphy imaging (SPECT/CT). BBB permeability was both correlated with cognitive impairment and with indoxyl sulfate (IS) levels. Inactivation of Aryl Hydrocarbon Receptor (AhR), receptor of the IS, protected the CKD mouse from the IS-induced BBB permeability. The objective of the BREIN study is to confirm the existence of an increased BBB permeability in humans with CKD. Method The prospective cohort study BREIN (NCT04328415) prospectively included patients with end-stage kidney disease (ESKD), and healthy volunteers (Ctrl) matched in age (±5 years), gender, and educational level to a patient. In patients and volunteers, BBB permeability was quantified by brain 99mTc-DTPA SPECT/CT performed by a senior nuclear physician and as percentage of injected activity (%IA). A battery of neuro-cognitive tests was performed, serum uremic toxins accumulation and AhR activation were assessed in all participants. Results 15 ESKD patients and 14 healthy volunteers were included. In ESKD patients had higher BBB permeability compared to controls: 0.29 0.07 vs. 0.14 0.06%IA, p &amp;lt; 0.0001. ESKD patients displayed lower MoCA score: 22.0 ± 5.0 vs. 27.3 ± 2.8, p = 0.008, impaired short-term memory (doors test): 12.5 ± 3.4 vs. 16.5 ± 3.4, p = 0.009, higher Beck depression score 8.1 ± 9.1 vs. 2.7 ± 3.4, p = 0.046, and slightly more daily cognitive complaints: 42.5 ± 29.3 vs. 29.8 ± 14.0 p = 0.153. ESKD patients displayed higher IS levels (2.8 ± 2.3 mmol/L vs. 0.9 ± 0.7 mmol/L, p = 0.006) and AhR activation (37.5 ± 17.8% vs. 24.7 ± 10.4%, p = 0.027). In ESKD patients, no significant correlation was found between BBB permeability and scores in cognitive tests, or serum uremic toxins levels. Conclusion This study confirms that ESKD patients display an increased BBB permeability compared to matched healthy volunteers, as well as cognitive and impairment. Association with uremic toxins and cognitive impairment needs to be assessed in larger cohorts.

#3023 Dietary protein intake modulates the tubular handling of the uremic retention solute indoxyl sulfate

Abstract Background and Aims Nutritional intervention constitutes a key part of the clinical management of individuals with chronic kidney disease (CKD). The gut emerged as an important source of uremic retention solutes, including indoxyl sulfate, p-cresyl sulfate and TMAO among the others. Indoxyl sulfate (IS) is produced through the gut microbial fermentation of tryptophan into indole and its conversion into indoxyl sulfate in the liver. Previous authors hypothesised that higher amounts of gut microbial metabolites can be sensed by kidney's proximal tubule cells through the use of receptors and signaling pathways. Particular evidence points to the involvement of kidney Organic Anion Transporters’(OATs') in controlling the gut-kidney communication. According to the so-called remote sensing hypothesis, kidney tubular cells respond to uremic toxins plasma fluctuations altering the activity of OAT1. Few studies have investigated whether in CKD the protein content in the diet affects gut generation, plasma retention and excretion mechanisms of indolic uremic toxins including kidney cells OAT1 expression. Method Eighteen male Sprague-Dawley rats (Janvier, France) aged 7-8 weeks and weighing between 270 and 388 g were randomly assigned to a low protein (LP) diet (n = 10) or a high protein (HP) diet (n = 8) after undergoing a 5/6 nephrectomy to develop CKD. For each diet, a sham-operated control group (n = 7 and n = 8 correspondingly) was used. After 7 weeks, urine was collected, and 8 weeks after the disease was induced, euthanasia was performed. Samples of kidney and blood were taken. mRNA and protein expression of OAT1 were sought via qPCR and Western blot. Forty patients with CKD were recruited in a cross-sectional study investigating eating habits and uremic toxins levels. Eating habits were searched via a food diary. Protein and energy intake were therefore calculated. Blood and 48 h-urine were collected. Blood creatinine and urea were determined with standard laboratory techniques. Total plasmatic and urinary IS concentrations were measured using LC-MS/MS. The fractional excretion (FE) i.e. the percentage of IS excreted relatively to the kidney filtered load, was calculated to assess IS remote sensing. Results Rats with chronic kidney disease (CKD) had considerably higher plasmatic levels of IS (p &amp;lt; .001) than sham rats. Rats with CKD on an HP diet, however, did not differ in plasma IS from rats fed an LP diet (p = .63). On the other hand, CKD rats fed an HP diet showed a substantial rise in 24-hour urinary IS (p &amp;lt; .001). The FE of IS was significantly higher (p = .005) in CKD rats on a HP diet and correlated with 24 h-urinary IS (Spearman r = .51, p value=.03) and with protein intake (Spearman r = .52, p value=.03) in CKD rats. OAT1 mRNA and protein expression was significantly higher in CKD rats given a HP diet (p = .025 and p = .009, respectively). By dividing the CKD patients into two groups by the median of protein intake, the FE of IS was significantly higher (p = .018) in the patients consuming a larger portion of proteins. Conclusion A diet higher in protein content in 5/6 nephrectomized CKD rats as well as in CKD patients likely leads to an increased production of indole in the colon. This results in increased IS fractional excretion but unchanged IS plasma retention. These findings offer more evidence for the mechanisms behind the remote sensing and signaling of uremic toxins derived from indole. In summary, a low-protein diet is still advised for people with chronic kidney disease.

#2194 Targeted metabolomics analysis identifies a specific metabolomic profile in patients with early chronic kidney disease

Abstract Background and Aims The complex interconnections between various metabolites and the kidney play a major role in the pathogenesis of chronic kidney disease (CKD). Hence, by the assessment of the relationship between metabolites and the kidney, more accurate biomarkers could be discovered to prevent and detect CKD at an early stage. The primary goal of this study was to find new serum and urine biomarkers for the diagnosis of early CKD by conducting a metabolomic profiling through targeted metabolomics of both blood and urine in patients with CKD. Method This cross-sectional study comprised 80 patients with CKD who were categorized into six subgroups (G1-G6) based on their eGFR according to the KDIGO Guidelines. Additionally, 20 healthy individuals were included as healthy control participants (group C). Ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry was used to perform serum and urine metabolomic profiling. The 6 metabolites evaluated by this study were selected based on the results of a previous untargeted analysis research. Both blood and urine samples underwent multivariate analysis, which was subsequently followed by univariate analysis and quantitative analysis. Initially, the PLSDA score plot and VIP score were utilized. The application of the cross-validation procedure to the initial set of 6 molecules provided outstanding results, including high accuracy, high R² values, and a significant Q2 value. Hence, the model might be deemed as predictive. Random Forest analysis was utilized to conduct biomarker analysis and prediction. The specificity and sensitivity of the compounds identified as possible biomarkers were assessed using the Receiver Operating Characteristic (ROC) curve and the area under the curve (AUC). Results There were strong positive correlations between eGFR and serum levels of Arginine (p &amp;lt; 0.05) and L-Acetylcarnitine (p &amp;lt; 0.05). Of note, the amounts of L-Methionine, L-Phenylalanine, Kynurenic acid, and Indoxyl sulfate in serum showed a progressive increase from group C to subgroups G1-G5. Furthermore, L-Methionine, L-Phenylalanine, Kynurenic acid, and Indoxyl sulfate exhibited a negative correlation with eGFR (p &amp;lt; 0.05). In addition, there was a positive correlation between the levels of urine Arginine, L-Methionine, and L-Phenylalanine and eGFR (p &amp;lt; 0.05). Urine levels of Kynurenic acid, Indoxyl sulfate, and L-Acetylcarnitine were higher in groups G4 and G5, as compared to groups G1-G3b and C, respectively. The urinary concentrations of Indoxyl sulfate, Kynurenic acid, Indoxyl sulfate, and L-Acetylcarnitine exhibited a negative correlation with eGFR (p &amp;lt; 0.05). Conclusion The current study shows that serum and urinary metabolites levels in patients with CKD display an alteration in production, secretion, and excretion in all CKD stages. There was demonstrated a distinct metabolomic profile in the course of CKD which points to novel biomarkers useful for the diagnosis and progression evaluation of CKD from its early stages.

Determination of indoxyl sulfate by spectrofluorimetric method in human plasma through extraction with deep eutectic solvent

A rapid and efficient analytical method was established to quantify indoxyl sulfate (IS) in plasma through extraction technique with a deep eutectic solvent (DES) and spectrofluorimetric method. DES (choline chloride: urea) was mixed with plasma samples for the extraction of IS, followed by the addition of dipotassium hydrogen phosphate (K2HPO4) solution to form an aqueous two-phase system. The fluorescence intensity of IS which was first extracted to the DES-rich-phase and then back-extracted into the salt-rich-phase, was measured by spectrofluorimetric method. Some key factors such as pH, centrifugation speed and time, the volume ratio of DES/salt, and salt concentration were optimized. Under the optimized conditions, the suggested method had a dynamic range between 20 and 160 µg/mL with a coefficient of determination (R2) of 0.99. Precision (relative standard deviation) was less than 15% and accuracy (% relative recovery) was ± 15% at the nominal concentration level. In addition, results showed that IS levels in real samples were higher than 40 µg/mL which was compatible with reported IS levels in end-stage renal disease (ESRD) patients. Overall, all the results reflect the fact that the presented analytical method can potentially be used for the determination of IS in real plasma samples.