Indoxyl Sulfate Concentrations Research Articles

TO011HEALTH UTILITY BUT NOT UREMIC TOXINS ARE ASSOCIATED WITH ONE YEAR MORTALITY IN HD PATIENTS

Abstract Background and Aims Patient centred care is progressively gaining importance. Standardized outcomes should take into account patient relevant outcomes, such as mortality and quality of life. It is unknown whether the health utility index EQ5D, an objective assessment of quality of life, is associated with mortality in haemodialysis patients. Serum concentrations of different uremic toxins have been associated with survival. We intended to explore whether EQ5D rather than concentrations of representative uraemic toxins (UT) was associated with mortality. Method Prospective longitudinal multicentric cohort study of all haemodialysis patients at 5 representative dialysis centres in Flanders. Total and free concentrations of representative uraemic toxins indoxyl sulfate (IxS), p-cresyl sulfate (pCS), p-cresyl glucuronide (pCG), indole acetic acid (IAA), 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), hippuric acid (HA) and uric acid (UA) were determined at baseline. Mini Nutritional Assessment (MNA) and EQ5D were assessed by dedicated nurses. Mortality was assessed from administrative or medical records. A Cox regression model was built, both fixed and as conditional forward including clinical parameters, MNA, EQ5D and serum concentrations of the different uraemic toxins. Results 216 prevalent patients (62%male, age 67.2 ± 15.7) all on high flux haemodialysis were included. Over the observation period, 59 patients (27%) died. Health utility as assessed by EQ5D was 0.69±0.28; perceived health by visual analogue scale (VAS) (0-100) was 59.6±18.6. Adjusting for age, gender and MNA score, free but not total concentrations of IxS, pCS and IAA and EQ5D score were associated with overall survival in the forced entry model. However, neither in the fixed entry, the forward or backward model were serum concentrations of uraemic toxins retained, and only age, gender, BMI, EQ5D and VAS were consistently associated with survival. Conclusion Measures of health utility, such as EQ5D, and quality of life, such as a simple VAS score, are more predictive of survival in patients on regular haemodialysis than serum concentrations of a panel of relevant uraemic toxins. Health utility and QoL are thus not only directly relevant to patients, but also indirectly through an association with improved survival. The concept of dialysis adequacy has been challenged over the last decade, mainly because it was focusing on small solute clearance. Different authors reported that concentrations of middle molecular and protein bound solutes were more representative. In our study, free but not total solute concentrations were associated with survival. However, adjusting additionally for health utility and/or health perception and/or assessment of nutritional status abolishes this association. Studies assessing interventions to enhance solute removal should not only include solute concentrations as outcome, but also consider EQ5D, VAS and nutritional status as important outcomes to enhance patient centeredness of their findings.

TO015INTRADIALYTIC ON-LINE MULTICOMPONENT TOTAL REMOVED SOLUTE MONITORING IN SPENT DIALYSATE BY A NOVEL MINIATURIZED OPTICAL SENSOR

Abstract Background and Aims Urea is the most commonly exploited marker of dialysis adequacy. Plenty of other uremic retention solutes with a deleterious effect on morbidity and mortality of uremic patients accumulate in ESKD patients [1]. Commonly, the uremic solutes are divided into three physicochemical types [2] with the representative markers urea, uric acid (UA),indoxyl sulfate (IS), and β2-Microglobulin (B2M). Optical monitoring of the uremic marker molecules has been proposed [3]. Despite being cumbersome as a clinical routine, direct dialysis quantification from total dialysate collection (TDC) is as the ‘gold standard’ for measuring the total amount of uremic solutes removed [4]. A robust and compact optical on-line dialysis monitoring device is a step closer to automatic assessment of removal of uremic toxins in dialysis. The aim of this study was to evaluate intradialytic on-line multicomponent Total Removed Solute (TRS) monitoring in the spent dialysate by a novel miniaturized optical sensor during hemodialysis (HD) and hemodiafiltration (HDF) with different settings. Method Ten ESKD patients (6 male and 4 female, 60.2±16.8 years) on chronic HDF were enrolled into the study (fistula N=9, graft N=1). For each patient 5 midweek dialysis sessions (length 240min, HD: N=1, Qb=200ml/min, Qd=300ml/min, 1,5m2; HDF: N=4, Qb≥300ml/min, Qd≥500ml/min, Vsubst≥15l, 1,8m2 and 2,2m2) were included. Spent dialysate from the drain was monitored on-line by a miniaturized sensor prototype (Optofluid Technologies OÜ, Estonia). For the reference, the samples from the spent dialysate drain tube of the HD machine were taken as 7, 60, 120 and 180min after the start and at the end of the session (240min). The concentrations of urea and B2M in dialysate were determined in the clinical laboratory. Concentration of IS and UA was determined utilizing the HPLC. TRS values were calculated using the tank weight and the lab or optical tank solute concentrations. t-test was used to determine significant differences between the methods (P≤0.05). Results The laboratory and optical TRS values were 489±112mM and 512±87mM for urea (R2=0.870), 4232±712µM and 4331±756µM for UA (R2=0.985), 230±47mg and 231±40mg for B2M (R2=0.906), 606±339µM and 616±321µM for IS (R2=0.969) (Fig. 1), being not statistically different for any uremic solutes. The reason for higher correlation for UA and IS is direct measurements of UA and IS by the optical sensor whereas urea and B2M are estimated indirectly. Conclusion Novel miniaturized optical sensor successfully carried out intradialytic on-line multicomponent TRS monitoring for the uremic solutes urea, UA, B2M and IS in the spent dialysate.

P0668UNILATERAL URETER OBSTRUCTION (UUO)-INDUCED RENAL FIBROSIS IS ATTENUATED BY SUPPRESSION OF INDOXYL SULFATE (IS) ACCUMULATION IN SULFOTRANSFERASE (SULT)1A1-DEFICIENT MICE

Abstract Background and Aims Obstructive nephropathy is the result of functional or anatomic lesions located in the urinary tract, and renal interstitial fibrosis is a common finding associated with long-term nephropathy. Many factors are suggested to be involved in the pathogenesis of renal fibrosis, such as infiltration of macrophages, growth factors, oxidative stress and cytokines. Indoxyl sulfate (IS), a typical sulfate-conjugated uremic solute, accumulates markedly in serum and renal tissue of cisplatin- or ischemia/reperfusion-induced acute kidney injury model animals, thereby inducing generation of oxidative stress. However, the relationship between IS and obstructive nephropathy or renal fibrosis remains unclear. IS is produced in the liver by CYP2A6/2E1-dependent oxidative metabolism of dietary protein-derived indole, followed by sulfotransferase 1a1 (SULT1A1)-mediated sulfate conjugation of indoxyl. IS in the blood circulation is efficiently taken up by renal proximal tubules via basolateral membrane-localized organic anion transporters, OAT1 and OAT3, and excreted into urine via unidentified apical membrane-located transporter. Thus, we established SULT1A1 gene-deficient (SULTKO) mice and developed UUO mice to investigate the pathological role of IS in UUO-induced renal fibrosis. Method The left ureter of C57BL/6J mice (wild type (WT), 8 weeks-old) and SULTKO mice (8 weeks-old) were obstructed last for 2 weeks. IS concentration in serum and kidney was determined by LC-MS/MS. Changes in histology and interstitial fibrosis were examined with PAS staining and Sirius red staining, respectively. Quantitative PCR was applied for determining expression levels of col1a1 encoding the major component of type I collagen, fibronectin, plasminogen activator inhibitor (PAI)-1, the activator of plasminogen and hence fibrinolysis, pro-inflammatory cytokine interleukin 6 (IL-6), Wnt4 encoding one protein of Wnt and Sfrp5, a gene that codes for antagonist of Wnt pathway. Renal fibrosis also evaluated through the expression of alpha smooth muscle actin (SMA) by Western blotting. Results By UUO treatment, the concentration of IS in serum, kidney and liver were elevated, which were suppressed in SULTKO mice. Ureter dilation was obviously observed in the obstructed kidney of WT mice, which was slightly prevented in SULTKO mice with UUO. Sirius red staining revealed that severe collagen deposition was found in the interstitium of WT kidney with UUO, but it was partly prevented in the kidney of KO mice with UUO along with the decrease in IS accumulation. The high expression of SMA, col1a1 and fibronectin in the kidney of WT mice with UUO were significantly suppressed in the kidney of SULTKO mice, 2.3-fold, 1.4-fold and 2.3-fold, respectively, suggesting that renal fibrotic responses were ameliorated in SULTKO mice. The expression of PAI-1, which was upregulated in WT mice with UUO, was also suppressed (1.8-fold) in SULTKO mice with UUO. The elevated expression of IL-6 in the kidney of WT mice with UUO was inhibited (1.8-fold) in SULTKO mice with UUO, indicating the possibility that inflammation-related signalling pathway also participated in the IS-exacerbated renal fibrosis. The enhanced expression of Wnt4 in the kidney of WT mice with UUO was suppressed (1.8-fold) in SULTKO mice with UUO, and the gene of Sfrp5 exhibited a higher expression level (3.2-fold) in the kidney of SULTKO mice with UUO compared with WT UUO mice. Conclusion Sult1a1-deficient mice showed the suppressed accumulation of IS in the kidney with UUO. Renal IS accumulation during pathological progression of obstructive nephropathy could enhance interstitial fibrosis through the activation of Wnt signalling pathway. Hepatic SULT1A1 could be a therapeutic target for preventing the progression of renal interstitial fibrosis by suppressing IS production during obstructive nephropathy.

P0874THE IMPACT OF CHRONIC EXPOSURE TO INDOXYL SULFATE ON BONE TURNOVER MARKERS, PTH, VITAMIN D3, AND BIOMECHANICAL AND DENSITOMETRIC PROPERTIES OF BONES IN RAT MODEL

Abstract Background and Aims Chronic kidney disease (CKD) is a crucial risk factor for biochemical and bone disturbances, as well as non-skeletal calcifications known as Chronic Kidney Disease – Mineral and Bone Disorders (CKD-MBD). Etiopathogenesis of CKD-MBD still remains unknown and arouses much controversy. Indoxyl sulfate (IS) is one of the most potent protein-bound uremic toxin that accumulates during CKD, and exerts aggressive and multidirectional effect on the body. In vitro studies indicate that IS modulates the differentiation and activation of osteoblasts and osteoclasts, however IS impact on bone and mineral disorders still remains unclear. The purpose of the study was to assess IS effect on bone turnover markers, parathyroid hormone (PTH), vitamin D3, and densitometric and biomechanical properties of bones in rat model. Method Male Wistar rats weighting 180 – 210 g (n=48) were divided into three groups – control group that received pure water and two experimental groups. Experimental groups were exposed to IS in the doses of 100 or 200 mg/kg of b.w. (200 IS) in the drinking water during 4 weeks. At the end of experiment, rats were anesthetized and samples of blood, as well as tibiae, femurs and lumbar spines were collected. Next, the IS bone (cortical and trabecular), plasma and urine levels were determined by high-performance liquid chromatography (HPLC). We also assessed IS influence on bone turnover markers (alkaline phosphatase – ALP, and tartrate-resistant acid phosphatase - TRAP), and level of vitamin D3, and PTH. Furthermore, we evaluated densitometric parameters of tibiae, femurs and lumbar spines using Horizon QDR Series X-ray Bone Densitometer, and performed biomechanical parameters analysis of the cortical bone of the femoral diaphysis using three-point bending test, and the mixed cortico-trabecular structure of the proximal femur by the bending test of the femoral neck. Results Chronic exposure to IS led to statistically significant increase in plasma, urine, cortical and trabecular bone IS concentrations. We observed reduction in bone turnover markers – bone formation marker ALP in trabecular and cortical bone, and bone resorption marker TRAP in cortical bone. Moreover, the highest dose of IS increased PTH. None of tested IS doses affected vitamin D3. From data obtained due to biomechanical tests, the highest dose of IS decreased the main indicators of bone strength: yield load (Fy) and displacement at yield [dl(Fy)] of femoral diaphysis and femoral neck, and ultimate load (Fu) and displacement at ultimate [dl(Fu)] of femoral diaphysis. Furthermore, we observed the decrease in elasticity of femoral diaphysis (increased Young’s modulus of elasticity E). Additionally, densitometric measurements revealed reduction only in the bone mineral density (BMD) and BMD of diaphyseal area (BMD R2) of tibiae, BMD R2 of femurs in 200 IS group, as well as BMD and BMD R2 of tibiae in 100 IS group. Conclusion Obtained data confirm that IS affects bones. We observed reduced bone metabolism and strength after exposure to IS. On the basis of the results, we concluded that IS can be one of crucial uremic factors responsible for increased risk of fracture during CKD.

P1509SEVERITY OF PRURITUS IN HEMODIALYSIS PATIENTS: RELATIONSHIP TO UREMIC TOXINS AND DIALYSIS MODALITY

Abstract Background and Aims Pruritus is a complication that can impair the quality of life of patients with chronic kidney disease (CKD). A previous report has suggested that the severity of pruritus is correlated with high serum concentrations of β2-microglobulin, calcium, and phosphorus, however recent developments in hemodialysis (HD), especially use of high-flux membrane and online hemodiafiltration (OLHDF), may improve the severity of pruritus. Protein-bound uremic toxins (PBUTs), such as indoxyl sulfate and p-cresyl sulfate, are known to be factors associated with atherosclerosis and impaired bone metabolism in CKD and may also be associated with pruritus, especially in those undergoing dialysis treatments. The aim of our study was to assess the severity of pruritus using a new tool, the 5D-itch scale (5D), as well as its relation to circulating PBUTs and dialysis modality in patients with end-stage kidney disease. Method This was a prospective cohort multicenter study, wherein we analyzed the baseline data. The 5D score (5–25 points) was measured in maintenance dialysis patients. The serum concentrations of indoxyl sulfate, p-cresyl sulfate, phenyl sulfate and hippuric acid were measured with mass spectrometry before a dialysis session. The correlations of the 5D scores with serum PBUTs, dialysis modality (HD or OLHDF) and the other background factors were assessed with Spearman correlation coefficients and the generalized linear model. Results This study included 135 dialysis patients (98 HD and 37 OLHDF). The mean 5D score was 6.84 (±SD 3.14). The mean serum concentrations of indoxyl sulfate, p-cresyl sulfate, indole acetic acid, phenyl sulfate, and hippuric acid were 2.99 ± 1.40, 2.05 ± 1.54, 0.11 ± 0.08, 0.94 ± 0.71, and 4.52 ± 3.17 mg/dL, respectively. There was a significant correlation between the 5D score and dialysis modality (correlation coefficient = 0.24, p < 0.01). Dialysis method was associated with the 5D score by the generalized linear model, and OLHDF was associated with a lower 5D score. There were no significant associations between the 5D score and serum concentrations of the various PBUTs (e.g., for indoxyl sulfate, the correlation coefficient was 0.11, p = 0.20) Conclusion Pruritus, as assessed by the 5D score, was associated with dialysis modality, but not with the serum concentration of PBUTs. Further studies will be needed to understand the mechanism of the effect of OLHDF on pruritus as well as the role of uremic toxins in the itchy skin in CKD patients.

Concentration and Duration of Indoxyl Sulfate Exposure Affects Osteoclastogenesis by Regulating NFATc1 via Aryl Hydrocarbon Receptor.

Indoxyl sulfate (IS) is a chronic kidney disease (CKD)-specific renal osteodystrophy metabolite that affects the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a transcription factor promoting osteoclastogenesis. However, the mechanisms underlying the regulation of NFATc1 by IS remain unknown. It is intriguing that the Aryl hydrocarbon receptor (AhR) plays a key role in osteoclastogenesis, since IS is an endogenous AhR agonist. This study investigates the relationship between IS concentration and osteoclast differentiation in Raw 264.7 cells, and examines the effects of different IS concentrations on NFATc1 expression through AhR signaling. Our data suggest that both osteoclastogenesis and NFATc1 are affected by IS through AhR signaling in both dose- and time-dependent manners. Osteoclast differentiation increases with short-term, low-dose IS exposure and decreases with long-term, high-dose IS exposure. Different IS levels switch the role of AhR from that of a ligand-activated transcription factor to that of an E3 ubiquitin ligase. We found that the AhR nuclear translocator may play an important role in the regulation of these dual functions of AhR under IS treatment. Altogether, this study demonstrates that the IS/AhR/NFATc1 signaling axis plays a critical role in osteoclastogenesis, indicating a potential role of AhR in the pathology and abnormality of bone turnover in CKD patients.

Transcriptome Profiling Reveals Indoxyl Sulfate Should Be Culpable of Impaired T Cell Function in Chronic Kidney Disease

Introduction: Chronic inflammation and immune system dysfunction have been evaluated as major factors in the pathogenesis of chronic kidney disease (CKD), contributing to the high mortality rates observed in these populations. Uremic toxins seem to be the potential “missing link.” Indoxyl sulfate (IS) is one of the protein-bound renal toxins. It participates in multiple pathologies of CKD complications, yet its effect on immune cell has not been studied. This study aimed to explore the genome-wide expression profile in human peripheral blood T cells under stimulation by IS.Methods: In this study, we employed RNA-sequencing transcriptome profiling to identify differentially expressed genes (DEGs) responding to IS stimulation in human peripheral T cells in vitro. Flow cytometry and western blot were used to verify the discovery in RNA-sequencing analysis.Results: Our results yielded a total of 5129 DEGs that were at least twofold up-regulated or down-regulated significantly by IS stimulation and half of them were concentration-specific. Analysis of T cell functional markers revealed a quite different transcription profile under various IS concentration. Transcription factors analysis showed the similar pattern. Aryl hydrocarbon receptor (AhR) target genes CYP1A1, CYP1B1, NQO1, and AhRR were up-regulated by IS stimulation. Pro-inflammatory genes TNF-α and IFN-γ were up-regulated as verified by flow cytometry analysis. DNA damage was induced by IS stimulation as confirmed by elevated protein level of p-ATM, p-ATR, p-BRCA1, and p-p53 in T cells.Conclusion: The toxicity of IS to T cells could be an important source of chronic inflammation in CKD patients. As an endogenous ligand of AhR, IS may influence multiple biological functions of T cells including inflammatory response and cell cycle regulation. Further researches are required to promulgate the underling mechanism and explore effective method of reserving T cell function in CKD.

Protein-Bound Uremic Toxins in Hemodialysis Patients Relate to Residual Kidney Function, Are Not Influenced by Convective Transport, and Do Not Relate to Outcome

Protein-bound uremic toxins (PBUTs) are predominantly excreted by renal tubular secretion and hardly removed by traditional hemodialysis (HD). Accumulation of PBUTs is proposed to contribute to the increased morbidity and mortality of patients with end-stage kidney disease (ESKD). Preserved PBUT excretion in patients with residual kidney function (RKF) and/or increased PBUT clearance with improved dialysis techniques might improve the prognosis of patients with ESKD. The aims of this study are to explore determinants of PBUTs in HD patients, and investigate whether hemodiafiltration (HDF) lowers PBUT plasma concentrations, and whether PBUTs are related to the outcome. Predialysis total plasma concentrations of kynurenine, kynurenic acid, indoxyl sulfate, indole-3-acetic acid, p-cresyl sulfate, p-cresyl glucuronide, and hippuric acid were measured by UHPLC-MS at baseline and after 6 months of follow-up in the first 80 patients participating in the CONvective TRAnsport Study (CONTRAST), a randomized controlled trial that compared the effects of online HDF versus low-flux HD on all-cause mortality and new cardiovascular events. RKF was inversely related to kynurenic acid (p < 0.001), indoxyl sulfate (p = 0.001), indole-3-acetic acid (p = 0.024), p-cresyl glucuronide (p = 0.004) and hippuric acid (p < 0.001) plasma concentrations. Only indoxyl sulfate decreased by 8.0% (−15.3 to 34.6) in patients treated with HDF and increased by 11.9% (−15.4 to 31.9) in HD patients after 6 months of follow-up (HDF vs. HD: p = 0.045). No independent associations were found between PBUT plasma concentrations and either risk of all-cause mortality or new cardiovascular events. In summary, in the current population, RKF is an important determinant of PBUT plasma concentrations in HD patients. The addition of convective transport did not consistently decrease PBUT plasma concentrations and no relation was found between PBUTs and cardiovascular endpoints.

Indoxyl Sulfate, a Uremic Endotheliotoxin.

Chronic kidney disease (CKD) is associated with a high prevalence of cardiovascular diseases. During CKD, the uremic toxin indoxyl sulfate (IS)—derived from tryptophan metabolism—accumulates. IS is involved in the pathophysiology of cardiovascular complications. IS can be described as an endotheliotoxin: IS induces endothelial dysfunction implicated in cardiovascular morbidity and mortality during CKD. In this review, we describe clinical and experimental evidence for IS endothelial toxicity and focus on the various molecular pathways implicated. In patients with CKD, plasma concentrations of IS correlate with cardiovascular events and mortality, with vascular calcification and atherosclerotic markers. Moreover, IS induces a prothrombotic state and impaired neovascularization. IS reduction by AST-120 reverse these abnormalities. In vitro, IS induces endothelial aryl hydrocarbon receptor (AhR) activation and proinflammatory transcription factors as NF-κB or AP-1. IS has a prooxidant effect with reduction of nitric oxide (NO) bioavailability. Finally, IS alters endothelial cell and endothelial progenitor cell migration, regeneration and control vascular smooth muscle cells proliferation. Reducing IS endothelial toxicity appears to be necessary to improve cardiovascular health in CKD patients.

Serum Levels and Removal by Haemodialysis and Haemodiafiltration of Tryptophan-Derived Uremic Toxins in ESKD Patients.

Tryptophan is an essential dietary amino acid that originates uremic toxins that contribute to end-stage kidney disease (ESKD) patient outcomes. We evaluated serum levels and removal during haemodialysis and haemodiafiltration of tryptophan and tryptophan-derived uremic toxins, indoxyl sulfate (IS) and indole acetic acid (IAA), in ESKD patients in different dialysis treatment settings. This prospective multicentre study in four European dialysis centres enrolled 78 patients with ESKD. Blood and spent dialysate samples obtained during dialysis were analysed with high-performance liquid chromatography to assess uremic solutes, their reduction ratio (RR) and total removed solute (TRS). Mean free serum tryptophan and IS concentrations increased, and concentration of IAA decreased over pre-dialysis levels (67%, 49%, −0.8%, respectively) during the first hour of dialysis. While mean serum total urea, IS and IAA concentrations decreased during dialysis (−72%, −39%, −43%, respectively), serum tryptophan levels increased, resulting in negative RR (−8%) towards the end of the dialysis session (p < 0.001), despite remarkable Trp losses in dialysate. RR and TRS values based on serum (total, free) and dialysate solute concentrations were lower for conventional low-flux dialysis (p < 0.001). High-efficiency haemodiafiltration resulted in 80% higher Trp losses than conventional low-flux dialysis, despite similar neutral Trp RR values. In conclusion, serum Trp concentrations and RR behave differently from uremic solutes IS, IAA and urea and Trp RR did not reflect dialysis Trp losses. Conventional low-flux dialysis may not adequately clear Trp-related uremic toxins while high efficiency haemodiafiltration increased Trp losses.

Uremia toxin helps to induce inflammation in intestines by activating the ATM/NEMO/NF-kB signalling pathway in human intestinal epithelial cells

During progressive chronic kidney disease, toxic substances known as uremic toxins accumulate in body fluids. Uremia toxin has been documented to be involved in most inflammatory reactions, and indoxyl-sulfate (IS) a major serum metabolite of uremia is a key player in this. The mechanism by which uremia toxin establishes it inflammatory activity is scarcely known; however, researchers believes that a clear understanding of this process can serve as a guide to combat the situation. The study was designed to investigate the role played by uremia toxin in intestinal inflammation. SW480 was used as cell lines for this study. Luciferase assay was used to detect the cell viability of different concentrations of IS. RT-qPCR was used to detect the effect of IS on the expression of inflammatory factors. The comet assay was used as a tool to detect DNA damage. Western blot was used to detect the phosphorylation level of ATM/NEMO/NF-kB protein. The IS of 0.09 nM was determined to be the best experimental concentration by luciferase assay. Result showed that IS promotes the expression of inflammatory factors TNF-α and IL-6. In addition, IS led to enhanced DNA damage in cells. IS promoted ATM phosphorylation leading to phosphorylation of NEMO to activate the NF-kB signalling pathway. In conclusion, uremia toxin facilitates inflammation in intestines by activating the ATM/NEMO/ NF-kB signalling pathway in human intestinal epithelial cells.

Preliminary evaluation of fecal fatty acid concentrations in cats with chronic kidney disease and correlation with indoxyl sulfate and p-cresol sulfate.

BackgroundStraight‐ and branched‐chain (BCFA) short‐chain fatty acids (SCFAs) are produced by colonic microbiota and have both beneficial and deleterious effects in humans with chronic kidney disease (CKD). Fecal SCFAs in cats with CKD have not been described.ObjectiveTo characterize fecal SCFA concentrations in cats with CKD as compared to healthy geriatric cats and correlate SCFA to serum indoxyl sulfate (IS) and p‐cresol sulfate (pCS) concentrations.AnimalsTwenty‐eight cats with CKD (International Renal Interest Society [IRIS] stages 2, 3, and 4) and 11 older (≥ 8 years) healthy geriatric cats.MethodsProspective, cross‐sectional study. Voided feces were analyzed using stable isotope dilution gas chromatography‐mass spectrometry to determine fecal concentrations of SCFAs. Serum concentrations of IS and pCS were measured using liquid chromatography tandem mass spectrometry.ResultsFecal isovaleric acid concentrations were significantly higher in CKD cats(P = .02) Cats with IRIS CKD stage 3 and 4 had significantly higher fecal isovaleric acid concentrations compared to healthy geriatric cats (P = .03), but not compared to IRIS CKD stage 2 cats. Total fecal concentrations of BCFAs were found to correlate weakly with serum creatinine concentration (rho, 0.33; P = .05), blood urea nitrogen concentration (rho, 0.40; P = .01), and pCS concentration (rho, 0.35; P = .04).Conclusions and Clinical ImportanceFecal isovaleric acid concentrations were higher in CKD cats, particularly in late stage disease, compared to healthy geriatric cats. Fecal BCFA concentrations correlated with pCS and were higher in cats with muscle wasting, providing evidence for malassimilation of protein in CKD cats.

Effects of antibiotics on the pharmacokinetics of indoxyl sulfate, a nephro-cardiovascular toxin

Indoxyl sulfate (IS), a highly protein-bound nephro-cardiovascular toxin, was poorly removed by hemodialysis. IS exists as anions in the body and the renal excretion is mediated by organic anion transporter 1 (OAT1) and OAT3. Acidic antibiotics such as cephalosporins and fluoroquinolones were putative substrates/inhibitors of OATs. We hypothesized that cephalosporins and fluoroquinolones might compete with IS for OAT1- and/or OAT3-mediated renal excretions.This study investigated the effects of ciprofloxacin, cefuroxime, cefotaxime, cefazolin and ofloxacin on the intravenous pharmacokinetics of IS in rats. IS was intravenously injected with and without each individual antibiotics, and the concentrations of IS in serum and lysate were determined by HPLC.The results showed that ciprofloxacin significantly increased AUC0-t and T1/2 of IS by 272% and 491%, respectively, and decreased the clearance by 71%. However, ofloxacin, cefuroxime, cefotaxime and cefazolin did not alter the pharmacokinetics of IS. Furthermore, cell line study showed that ciprofloxacin inhibited the OAT3-mediated transport of IS.This study indicates 30 mg/kg of ciprofloxacin decreased the clearance of IS through inhibition on the OAT3-mediated transport, whereas 50 mg/kg of ofloxacin, cefuroxime, cefotaxime and cefazolin did not show significant influence.

118 Altered intestinal barrier in psoriasis is associated with disease severity, gastrointestinal symptoms and presence of bacteria metabolites in the blood

Growing body of evidence suggests a functional interplay between gut microbiome, intestinal barrier and immune system. That so-called “gut-skin axis” has been considered as a key factor in the etiology of psoriasis. The aim of this study was to compare psoriasis severity, intensity of self-reported gastrointestinal symptoms and concentration of gut microbiota-derived metabolites (trimethylamine N-oxide; TMAO and indoxyl sulfate) between psoriatic patients with and without altered intestinal barrier. One hundred twenty consecutive patients with chronic plaque psoriasis were enrolled. To examine gut barrier integrity we investigated serum concentrations of claudin-3, a modulator of intestinal tight junctions and intestinal fatty acid-binding protein, a marker of enterocyte damage. Gastrointestinal symptoms were evaluated with a validated questionnaire – Gastrointestinal Symptom Rating Scale (GSRS). Concentration of TMAO and indoxyl sulfate was measured through high performance liquid chromatography. Patients with altered gut barrier showed significantly higher score in GSRS (3.09 vs 1.23; p<0.05), especially in “indigestion” section. Moreover, patients with psoriasis and disrupted intestinal barrier demonstrated higher disease activity (PASI: 18.9±1.9 vs 10.1±2.5, p<0.05). The inflammatory parameters and markers of bacterial translocation were significantly higher in a subgroup of patients with damaged gut integrity (TMAO: 375.7±51.9 vs 119.4±27.5 ng/mL; indoxyl sulfate: 1016.4±85.3 vs 654.2±69.1 ng/mL). Our results support the hypothesis that alterations of the intestinal barrier may play an essential role in the pathogenesis of psoriasis. Further study should focus on developing therapies that enhance intestinal barrier function.

Dysbiosis of the gut microbiome is associated with CKD5 and correlated with clinical indices of the disease: a case\u2013controlled study

BackgroundChronic kidney disease (CKD) is a universal chronic disease in China. The balance of the gut microbiome is highly crucial for a healthy human body, especially for the immune system. However, the relationship between the gut microbiome and CKD has not yet been clarified.MethodsA total of 122 patients were recruited for this study. Among them, 24 patients were diagnosed with CKD5 but did not receive hemodialysis therapy, 29 patients were diagnosed with CKD5 and received hemodialysis therapy and 69 were matched healthy controls. The gut microbiome composition was analyzed by a 16S rRNA (16S ribosomal RNA) gene-based sequencing protocol. High-performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS) technology was used to evaluate the levels of microbiome-related protein-binding uremic toxins level, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), in the patients.ResultsWe compared the gut microbiome results of 122 subjects and established a correlation between the gut microbiome and IS and PCS levels. The results indicated that alpha and beta diversity were different in patients with CKD5 than in the healthy controls (p < 0.01). In comparison to healthy controls, CKD5 patients exhibited a significantly higher relative abundance of Neisseria (p < 0.001), Lachnoclostridium (p < 0.001) and Bifidobacterium (p < 0.001). Faecalibacterium (p < 0.001) displayed a notably lower relative abundance for CKD5 patients both with and without hemodialysis than for controls. It was also found that the concentrations of IS and PCS were correlated with the gut microbiome.ConclusionsOur results indicate that CKD5 patients both with and without hemodialysis had dysbiosis of the gut microbiome and that this dysbiosis was associated with an accumulation of IS and PCS. These results may support further clinical diagnosis to a great extent and help in developing potential probiotics to facilitate the treatment of CKD5.

Performance evaluation of an Indoxyl Sulfate Assay Kit "NIPRO".

Background The relationship between renal disease and cardiovascular disease (CVD) is currently known as cardiorenal syndrome. Indoxyl sulfate (IS) is one of the uremic toxins that accelerates the progression of cardiorenal syndrome. This report presents a new method for measuring IS in a simpler way. Methods We evaluated the analytical performance of an IS Assay Kit "NIPRO" loaded on LABOSPECT 008. The evaluated analytical performances included accuracy, precision, dilution linearity, limit of detection (LOD), limit of quantitation (LOQ), recovery test, interference test and comparison against assays performed by high-performance liquid chromatography (HPLC). Results Total precision showed a <5.3% coefficient of variation at IS concentrations of 3.57-277.73 μmol/L, and satisfactory results were observed in the dilution linearity, LOD, LOQ, recovery and interference tests. The IS Assay Kit "NIPRO" showed a high correlation with the HPLC conventional method (r = 0.993). Conclusions The IS Assay Kit "NIPRO" demonstrated satisfactory analytical performance, and this suggests it could shortly become another common method to measure circulating IS.

Serum Concentration of Indoxyl Sulfate in Peritoneal Dialysis Patients and Low-Flux Hemodialysis Patients

Background/Aims: To compare the serum concentration of Indoxyl sulfate (IS) in patients on continuous ambulatory peritoneal dialysis (CAPD) and low-flux hemodialysis (HD), and analyze the risk factors associated with IS. Methods: We performed a single-center, cross-sectional observational study including 169 patients on CAPD and 115 patients on low-flux HD. Patients were divided into the anuric HD group, anuric peritoneal dialysis (PD) group, and non-anuric PD group on the basis of dialysis modality and residual urinary output. Serum concentration of IS was determined by high-performance liquid chromatography electrospray tandem mass spectrometry. Results: After matching the urinary volume and dialysis vintage, 58 anuric patients on PD and 58 anuric patients on HD were enrolled. The serum level of IS was significantly lower in patients on PD than that in those on HD (28.05 ± 13.98 vs. 39.64 ± 18.25 μg/mL; p < 0.001). This result persisted even after adjustment for confounding risk factors including nutritional status (β = 0.338, p < 0.001). In addition, the serum level of IS was significantly lower in non-anuric PD patients than that anuric PD patients (18.70 ± 11.21 vs. 28.05 ± 13.98 μg/mL; p < 0.001). After the adjustment for risk factors such as dialysis vintage, IS serum concentration in patients on PD was still significantly correlated with residual renal function (RRF; β = –0.355, p < 0.001). Conclusions: Dialysis modality is the independent risk factor of IS serum concentration and it is substantially lower in patients on CAPD than that in those on low-flux HD. Additionally, RRF was independently associated with IS serum concentration in CAPD patients, and the better the RRF is, the lower IS serum concentration.

Prognostic Value Serum Concentration of Indoxyl Sulfate During Acute Kidney Injury in Septic Shock Patients.

Prognostic Value Serum Concentration of Indoxyl Sulfate During Acute Kidney Injury in Septic Shock Patients.

Indoxyl Sulfate Induces Renal Fibroblast Activation through a Targetable Heat Shock Protein 90-Dependent Pathway.

Indoxyl sulfate (IS) accumulation occurs early during chronic kidney disease (CKD) progression and contributes to renal dysfunction by inducing fibrosis, inflammation, oxidative stress, and tissue remodeling. Renal toxicity of high IS concentrations (250 μM) has been widely explored, particularly in resident tubular and glomerular cells, while the effect of a moderate IS increase on kidneys is still mostly unknown. To define the effects of IS accumulation on renal fibroblasts, we first analyzed kidneys of C57BL/6 mice receiving IS (0.1%) in drinking water for 12 weeks. As a next step, we treated renal fibroblasts (NRK-49F) with IS (20 μM) with or without the HSP90 inhibitor 17-AAG (1 μM). In mouse kidneys, IS increased the collagen deposition and HSP90 and α-SMA expression (immunohistochemistry) in interstitial fibroblasts and caused tubular necrosis (histological H&E and picrosirius red staining). In NRK-49F cells, IS induced MCP1, TGF-β, collagen I, α-SMA, and HSP90 gene/protein expression and Smad2/3 pathway activation. IS had no effects on fibroblast proliferation and ROS production. 17-AAG counteracted IS-induced MCP1, TGF-β, collagen I, and α-SMA expression and Smad2/3 phosphorylation. Our study demonstrates that the IS increase promotes renal fibroblast activation by a HSP90-dependent pathway and indicates HSP90 inhibition as a potential strategy to restrain IS-induced kidney inflammation and fibrosis in CKD.

The association of indoxyl sulfate with fibroblast growth factor-23 in cats with chronic kidney disease.

BackgroundIndoxyl sulfate (IS) has been reported not only to increase with the severity of impaired renal function, but also possibly to be a factor associated with bone abnormalities linked to fibroblast growth factor‐23 (FGF‐23) in humans with chronic kidney disease (CKD). It is not yet known whether this correlation between IS and FGF‐23 holds true for cats with CKD.HypothesisAccumulation of IS is related to FGF‐23 secretion in cats with CKD.AnimalsTwenty clinically healthy cats and 73 cats with CKD cases were evaluated retrospectively.MethodsThe concentrations of IS and FGF‐23 in plasma were determined by high‐performance liquid chromatography and ELISA, respectively. Progression was defined as an increment of 0.5 mg/dL of serum creatinine concentration within 3 months.ResultsPlasma IS and FGF‐23 concentrations were significantly increased concurrently with decreasing renal function. Higher concentration of FGF‐23 was significantly associated with higher concentration of IS after adjusting for various confounding factors including creatinine and phosphate. Furthermore, the correlation between IS and phosphate was higher than that between FGF‐23 and phosphate. When the renal progression group was compared with the non‐progression group, both IS and FGF‐23 were found to be significantly increased (P < .05). In addition, the area under receiver operator curve of the combination of IS and FGF‐23 predicted renal progression at a level >0.9.Conclusions and Clinical ImportanceBoth FGF‐23 and IS are associated with phosphate metabolism and CKD progression.