Serum Indoxyl Sulfate Levels Research Articles

Meclofenamate elicits a nephropreventing effect in a rat model of ischemic acute kidney injury by suppressing indoxyl sulfate production and restoring renal organic anion transporters.

Indoxyl sulfate (IS), a putative low-molecular weight uremic toxin, is excreted in the urine under normal kidney function, but is retained in the circulation and tissues during renal dysfunction in acute kidney injury and chronic kidney disease. IS, which is one of the most potent inducers of oxidative stress in the kidney and cardiovascular system, is enzymatically produced in the liver from indole by cytochrome P450-mediated hydroxylation to indoxyl, followed by sulfotransferase-mediated sulfate conjugation. We used rat liver S9 fraction to identify inhibitors of IS production. After testing several compounds, including phytochemical polyphenols, we identified meclofenamate as a potent inhibitor of IS production with an apparent IC50 value of 1.34 μM. Ischemia/reperfusion (I/R) of rat kidney caused a marked elevation in the serum IS concentration 48 hours after surgery. However, intravenous administration of meclofenamate (10 mg/kg) significantly suppressed this increase in the serum level of IS. Moreover, IS concentrations in both kidney and liver were dramatically elevated by renal I/R treatment, but this increase was blocked by meclofenamate. Serum creatinine and blood urea nitrogen were markedly elevated in rats after renal I/R treatment, but these increases were significantly restored by administration of meclofenamate. Renal expression of both basolateral membrane-localized organic anion transporters rOAT1 and rOAT3 was downregulated by I/R treatment. However, expression of rOAT1 and rOAT3 recovered after administration of meclofenamate, which is associated with the inhibition of I/R-evoked elevation of prostaglandin E2. Our results suggest that meclofenamate inhibits hepatic sulfotransferase-mediated production of IS, thereby suppressing serum and renal accumulation of IS. Meclofenamate also prevents the prostaglandin E2-dependent downregulation of rOAT1 and rOAT3 expression. In conclusion, meclofenamate was found to elicit a nephropreventive effect in ischemic acute kidney injury.

Effects of chitosan on oxidative stress and related factors in hemodialysis patients

In recent world-wide studies, chitosans were tested as a dietary supplement for inhibiting the absorption of certain lipids and bile acids. We previously demonstrated the antioxidative and renoprotective potential of chitosan supplementation in chronic renal failure using 5/6 nephrectomized rats. In this study, we report the effects of chitosan on oxidative stress and related factors in hemodialysis patients. The ingestion of chitosan over a 12-week period resulted in a significant decrease in serum indoxyl sulfate and phosphate levels, compared with the levels prior to the start of the study. The ingestion of chitosan also resulted in a lowered ratio of oxidized to reduced albumin and a decrease in the level of advanced oxidized protein products. In in vitro studies, chitosan solutions were found to bind 38.5% of the indoxyl sulfate and 17.8% of the phosphate, respectively. Further, the oxidized albumin ratio was correlated with serum indoxyl sulfate levels in vivo. These results suggest that the ingestion of chitosan results in a significant reduction in the levels of pro-oxidants, which include uremic toxins, in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation. In addition, the long-term ingestion of chitosan has the potential for use in treating hyperphosphatemia in hemodialysis patients.

Heritability and clinical determinants of serum indoxyl sulfate and p-cresyl sulfate, candidate biomarkers of the human microbiome enterotype.

BackgroundIndoxyl sulfate and p-cresyl sulfate are unique microbial co-metabolites. Both co-metabolites have been involved in the pathogenesis of accelerated cardiovascular disease and renal disease progression. Available evidence suggests that indoxyl sulfate and p-cresyl sulfate may be considered candidate biomarkers of the human enterotype and may help to explain the link between diet and cardiovascular disease burden.Objective and DesignInformation on clinical determinants and heritability of indoxyl sulfate and p-cresyl sulfate serum is non-existing. To clarify this issue, the authors determined serum levels of indoxyl sulfate and p-cresyl sulfate in 773 individuals, recruited in the frame of the Flemish Study on Environment, Genes and Health Outcomes (FLEMENGHO study).ResultsSerum levels of indoxyl sulfate and p-cresyl sulfate amounted to 3.1 (2.4–4.3) and 13.0 (7.4–21.5) μM, respectively. Regression analysis identified renal function, age and sex as independent determinants of both co-metabolites. Both serum indoxyl sulfate (h2 = 0.17) and p-cresyl sulfate (h2 = 0.18) concentrations showed moderate but significant heritability after adjustment for covariables, with significant genetic and environmental correlations for both co-metabolites.LimitationsFamily studies cannot provide conclusive evidence for a genetic contribution, as confounding by shared environmental effects can never be excluded.ConclusionsThe heritability of indoxyl sulfate and p-cresyl sulfate is moderate. Besides genetic host factors and environmental factors, also renal function, sex and age influence the serum levels of these co-metabolites.

Association of Indoxyl Sulfate With Fibroblast Growth Factor 23 in Patients With Advanced Chronic Kidney Disease

BackgroundProtein-bound uremic toxins–indoxyl sulfate (IS) and p-cresyl sulfate (PCS)–can not only predict clinical outcomes but also may relate to bone-mineral disorders in patients with chronic kidney disease (CKD). However, the relationship between protein-bound uremic toxins and fibroblast growth factor 23 (FGF23) has not been studied before. The objective of this study was to explore the association of IS and PCS with FGF23 in a CKD-based cohort. MethodsThis is a cross-sectional study that enrolled 80 stable CKD stage 3 to 5 patients who met the inclusion criteria in a single medical center. Serum levels of IS, PCS and FGF23 were measured concurrently. General biochemistry and patient background were also investigated. ResultsSerum FGF23 and IS concentrations were elevated commensurately with deteriorating renal function. Pearson's analysis showed that FGF23 levels were significantly associated with blood urea nitrogen (r=0.381, P<0.05), creatinine (r=0.632, P<0.01), estimated glomerular filtration rate (r=–0.447, P<0.05), phosphate (r=0.543, P<0.01), intact parathyroid hormone (r=0.543, P<0.01), IS (r=0.432, P<0.01) and PCS (r=0.318, P<0.05). After adjusting other confounding factors by stepwise multiple linear regression analysis, only creatinine (β=0.82, P<0.01), phosphate (β=0.28, P=0.02) and IS (β=0.39, P=0.04) retained statistically significant associations with FGF23. Moreover, serum levels of IS were higher in patients with high FGF23 concentration (>90pg/mL, median value) than those with lower FGF23 (P<0.01). ConclusionsResults indicated that only IS but not PCS correlated independently with FGF23 in worsening CKD. IS may be an independent factor involved in regulation of bone-mineral metabolism.

Indoxyl Sulfate-Induced Activation of (Pro)Renin Receptor Is Involved in Expression of TGF-β1 and α-Smooth Muscle Actin in Proximal Tubular Cells

Activation of (pro)renin receptor (PRR) is involved in the progression of chronic kidney disease. However, the role of indoxyl sulfate, a uremic toxin, in the activation of PRR is not clear. The present study aimed to clarify the role of indoxyl sulfate in activation of PRR, in relation to renal expression of fibrotic genes. Renal expression of PRR and renin/prorenin was up-regulated in chronic kidney disease rats compared with normal rats, whereas AST-120 suppressed these expression by reducing serum levels of indoxyl sulfate. Furthermore, administration of indoxyl sulfate to normotensive and hypertensive rats increased renal expression of PRR and renin/prorenin. Indoxyl sulfate induced expression of PRR and prorenin in cultured human proximal tubular cells (HK-2 cells). Indoxyl sulfate-induced PRR expression was inhibited by small interfering RNAs of signal transducer and activator of transcription 3 (Stat3) and nuclear factor-κB p65 in proximal tubular cells. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed indoxyl sulfate-induced PRR expression in proximal tubular cells. N-acetylcysteine prevented indoxyl sulfate-induced phosphorylation of Stat3 in proximal tubular cells. PRR small interfering RNA inhibited indoxyl sulfate-induced expression of TGF-β1 and α-smooth muscle actin in proximal tubular cells. Taken together, indoxyl sulfate-induced up-regulation of prorenin expression and activation of PRR through production of reactive oxygen species and activation of Stat3 and nuclear factor-κB play an important role in the expression of TGF-β1 and α-smooth muscle actin in proximal tubular cells. Thus, indoxyl sulfate-induced activation of prorenin/PRR might be involved in renal fibrosis.

Association between indoxyl sulfate and bone histomorphometry in pre-dialysis chronic kidney disease patients.

Experimental studies have suggested that indoxyl sulfate (IS), a protein-bound uremic toxin, may be involved in the development of renal osteodystrophy. evaluate the association between IS levels and biochemical parameters related to mineral metabolism and bone histomorphometry in a cohort of pre-dialysis chronic kidney disease (CKD) patients. This is a post-hoc analysis of an observational study evaluating the association between coronary calcification and bone biopsy findings in 49 patients (age: 52 ± 10 years; 67% male; estimated glomerular filtration rate: 36 ± 17 ml/min). Serum levels of IS were measured. Patients at CKD stages 2 and 3 presented remarkably low bone formation rate. Patients at CKD stages 4 and 5 presented significantly higher osteoid volume, osteoblast and osteoclast surface, bone fibrosis volume and bone formation rate and a lower mineralization lag time than CKD stage 2 and 3 patients. We observed a positive association between IS levels on one hand and the bone formation rate, osteoid volume, osteoblast surface and bone fibrosis volume on the other. Multivariate regression models confirmed that the associations between IS levels and osteoblast surface and bone fibrosis volume were both independent of demographic and biochemical characteristics of the study population. A similar trend was observed for the bone formation rate. Our findings demonstrated that IS is positively associated with bone formation rate in pre-dialysis CKD patients.

AST-120 for the management of progression of chronic kidney disease

Uremic toxins such as indoxyl sulfate contribute to the pathogenesis of chronic kidney disease (CKD) by promoting glomerulosclerosis and interstitial fibrosis with loss of nephrons and vascular damage. AST-120, an orally administered intestinal sorbent, adsorbs indole, a precursor of indoxyl sulfate, thereby reducing serum and urinary concentrations of indoxyl sulfate. AST-120 has been available in Japan since 1991, and subsequently Korea (2005), and the Philippines (2010) as an agent to prolong the time to initiation of hemodialysis and for improvement of uremic symptoms in patients with CKD. A Medline search was performed to identify data supporting clinical experience with AST-120 for managing CKD. Prospective open-label and double-blind trials as well as retrospective analyses were included. In prospective trials and retrospective analyses, AST-120 has been shown to prolong the time to initiation of hemodialysis, and slow decline in glomerular filtration rate and the increase serum creatinine. In an initial randomized, double-blind, placebo-controlled trial in the United States, AST-120 was associated with a significant dose-dependent reduction in serum indoxyl sulfate levels and a decrease in uremia-related malaise. The Evaluating Prevention of Progression in CKD (EPPIC) trials, two double-blind, placebo-controlled trials undertaken in North America/Latin America and Europe, are evaluating the efficacy of AST-120 for preventing the progression of CKD. The results of the EPPIC trials will further define the role of AST-120 in this debilitating condition.

The Uremic Toxin Adsorbent AST-120 Abrogates Cardiorenal Injury Following Myocardial Infarction

An accelerated progressive decline in renal function is a frequent accompaniment of myocardial infarction (MI). Indoxyl sulfate (IS), a uremic toxin that accumulates from the early stages of chronic kidney disease (CKD), is contributory to both renal and cardiac fibrosis. IS levels can be reduced by administration of the oral adsorbent AST-120, which has been shown to ameliorate pathological renal and cardiac fibrosis in moderate to severe CKD. However, the cardiorenal effect of AST-120 on less severe renal dysfunction in the post-MI setting has not previously been well studied. MI-induced Sprague-Dawley rats were randomized to receive either AST-120 (MI+AST-120) or were untreated (MI+Vehicle) for 16 weeks. Serum IS levels were measured at baseline, 8 and 16 weeks. Echocardiography and glomerular filtration rate (GFR) were assessed prior to sacrifice. Renal and cardiac tissues were assessed for pathological changes using histological and immunohistochemical methods, Western blot analysis and real-time PCR. Compared with sham, MI+Vehicle animals had a significant reduction in left ventricular ejection fraction (by 42%, p<0.001) and fractional shortening (by 52%, p<0.001) as well as lower GFR (p<0.05) and increased serum IS levels (p<0.05). A significant increase in interstitial fibrosis in the renal cortex was demonstrated in MI+Vehicle animals (p<0.001). Compared with MI+Vehicle, MI+AST-120 animals had increased GFR (by 13.35%, p<0.05) and reduced serum IS (p<0.001), renal interstitial fibrosis (p<0.05), and renal KIM-1, collagen-IV and TIMP-1 expression (p<0.05). Cardiac function did not change with AST-120 treatment, however gene expression of TGF-β1 and TNF-α as well as collagen-I and TIMP-1 protein expression was decreased in the non-infarcted myocardium (p<0.05). In conclusion, reduction of IS attenuates cardio-renal fibrotic processes in the post-MI kidney. KIM-1 appears to be a sensitive renal injury biomarker in this setting and is correlated with serum IS levels.

Gastrointestinal-related Uremic Toxins in Peritoneal Dialysis: A Pilot Study with a 5-year Follow-up

P-cresyl sulfate (PCS) and indoxyl sulfate (IS) were not only novel but essential factors associated with cardiovascular disease and mortality in patients with chronic kidney disease and hemodialysis. However, little evidence exams the effect in peritoneal dialysis (PD) patients. This pilot study recruited 46 stable PD patients in a single medical center. Serum levels of IS, PCS and biochemistry were measured concurrently. Clinical outcomes including cardiovascular, all-cause mortality and PD failure event were recorded during a 5-year follow-up. Serum levels of free and total PCS were lower in patients with residual renal function (11.67 ± 6.92, p = 0.014, 0.77 ± 0.48, p = 0.046, respectively). Multivariate Cox regression analysis showed age (HR: 1.07, p = 0.01), serum CO2 (HR: 0.67, p = 0.02) and total PCS (HR: 1.05, p <0.01) were independently associated with cardiovascular events; only free PCS (HR: 1.42, p <0.01) reached significant correlation with all-cause mortality. Total IS (HR: 1.27, p = 0.03) significantly correlated with PD failure event after adjusting other confounding factors. Kaplan-Meier analysis revealed that patients with higher total and free PCS levels had higher cardiovascular events (log rank p <0.01, log rank p = 0.05, respectively) and mortality event (log rank p = 0.02, log rank p = 0.03, respectively) than those with lower levels. In addition, total IS (log rank p = 0.04), total PCS (log rank p = 0.01) and free PCS (log rank p <0.01) could independently predict PD failure event during the study period. Our findings suggest PCS and IS may be a valuable surrogate in predicting poor clinical outcomes in PD patients.

Accumulated uremic toxins attenuate bone mechanical properties in rats with chronic kidney disease

The prevalence of hip fracture is very high among patients with chronic kidney disease (CKD); however, the reason for this is unclear. We examined the effects of accumulated uremic toxins on bone chemical composition and elastic mechanical properties. Rats underwent thyroparathyroidectomy and progressive partial nephrectomy (TPTx-Nx), and were administered with vehicle or AST-120 to reduce serum indoxyl sulfate (IS) levels. Bone mechanical properties, bone mineral density (BMD), cortical bone chemical composition, and histomorphometry were determined. Storage modulus was reduced in TPTx-Nx rats compared with rats that underwent TPTx alone. BMD and histomorphometric parameters did not differ between the groups. In terms of cortical bone chemical composition, the mineral/matrix ratio and carbonate substitution was increased, whereas crystallinity was decreased in TPTx-Nx rats. The enzymatic crosslink ratio and pentosidine:matrix ratio were increased in TPTx-Nx rats. AST-120 abolished the effects of TPTx-Nx and decreased the serum IS concentration. Stepwise multiple regression analysis revealed that the pentosidine:matrix and mineral:matrix ratios were independent contributors to the storage modulus. In conclusion, the accumulated uremic toxins, including IS, seem to play an important role in deteriorating bone mechanical properties by altering the chemical composition of bone. This mechanism may account for the increased prevalence of hip fracture among patients with CKD.

The uremic toxin adsorbent AST-120 abrogates renal injury post-MI evidenced by normalization of KIM-1 protein expression and reduced collagen turnover

Introduction: A progressive decline in renal function is a frequent accompaniment of myocardial infarction (MI). Wehave previously shown that post-MI the uraemic toxin, indoxyl sulphate (IS), kidney injurymolecule (KIM)-1 and cardiac fibrosis are increased. In chronic kidney disease (CKD) we have reported increased IS and cardiac fibrosis; both were reduced following treatment with the oral carbonic adsorbent AST-120. However, the effect of AST-120 on renal injury post-MI has not been fully explored. Methods: MI-induced rats received either AST-120 (8% in rat chow) or were untreated (MI+Veh) for 16 weeks. Sham operated rats served as controls. Serum IS level was measured at baseline and 16 weeks. Echocardiography and glomerular filtration rate (GFR) was assessed prior to sacrifice at 16 weeks. Tissues were assessed for changes using immunohistological and western blot methods. Results: Heart: Ejection fraction and fractional shortening were reduced by 39% and 49% post-MI respectively (P< 0.001 vs. sham).Cardiac collagen I andTIMP-1 expression was elevated post-MI (10 and 5 fold respectively, P< 0.01) and following AST-120 treatment reduced back to sham levels (P< 0.01). No change in MMP-2 expression was detected; and no difference observed in blood pressure betweenMI groups. Kidney:MI+Veh increased delta serum IS levels, renal fibrosis and KIM-1 expression; and reduced GFR (P< 0.05 vs sham). Compared to MI+Veh, AST-120 treatment reduced serum IS, renal fibrosis and KIM-1 expression (P< 0.05). KIM-1 expression positively correlated with serum IS levels (r= 0.56; P= 0.002). Conclusions: AST-120 reduced cardiac collagen by suppressing TIMP-1 expression. KIM-1 appears to be a sensitive renal biomarker for post-MI kidney that may be influenced by elevated IS levels. AST-120 treatment post-MI improves renal fibrosis and normalises KIM-1 and IS levels. Thus AST-120 has beneficial cardiac and renal effects post-MI, and may be a potential treatment for cardiorenal syndrome.

In acute kidney injury, indoxyl sulfate impairs human endothelial progenitor cells: modulation by statin

Renal ischemia rapidly mobilizes endothelial progenitor cells (EPCs), which provides renoprotection in acute kidney injury (AKI). Indoxyl sulfate (IS) is a protein-binding uremic toxin with a potential role in endothelial injury. In this study, we examined the effects and mechanisms of action of IS on EPCs in AKI. Forty-one consecutive patients (26 male; age, 70.1±14.1years) diagnosed with AKI according to the AKIN criteria were enrolled. The AKI patients had higher serum IS levels than patients with normal kidney function (1.35±0.94×10(-4)M vs. 0.02±0.02×10(-4)M, P<0.01). IS levels were negatively correlated to the number of double-labeled (CD34(+)KDR(+)) circulating EPCs (P<0.001). After IS stimulation, the cells displayed decreased expression of phosphorylated endothelial nitric oxide (NO) synthase, vascular cell adhesion molecule-1, increased reactive oxygen species, decreased proliferative capacity, increased senescence and autophagy, as well as decreased migration and angiogenesis. These effects of IS on EPCs were reversed by atorvastatin. Further, exogenous administration of IS significantly reduced EPC number in Tie2-GFP transgenic mice and attenuated NO signaling in aortic and kidney arteriolar endothelium after kidney ischemia-reperfusion injury in mice, and these effects were restored by atorvastatin. Our results are the first to demonstrate that circulating IS is elevated in AKI and has direct effects on EPCs via NO-dependent mechanisms both in vitro and in vivo. Targeting the IS-mediated pathways by NO-releasing statins such as atorvastatin may preempt disordered vascular wall pathology, and represent a novel EPC-rescued approach to impaired neovascularization after AKI.

Levels of indoxyl sulfate are associated with severity of coronary atherosclerosis

Indoxyl sulfate (IS) is linked to endothelial damage, NF-κB activation and induced development of atherosclerosis. The purpose of this study was to investigate the relationship between serum IS levels and the severity of coronary artery stenosis. In addition, the relationship among IS and various cardiovascular risk factors was also explored. Serum IS concentrations were measured using ultra performance liquid chromatography in 191 consecutive patients presenting with stable angina. The associations between serum IS levels and angiographic indexes of the number of diseased vessels, modified Gensini scores and calcium scores were determined. Patients with significant coronary artery stenosis were found to have higher serum IS levels than patients with normal coronary arteries. Using multivariate analysis, serum IS levels were found to be independently associated with the presence and severity of coronary artery disease (CAD). Furthermore, statistically significant correlation was observed between the serum IS levels and age, Agatston calcium score, volume calcium score, modified Gensini score, coronary lesions, coronary disease and Framingham-10 year risk score. This study indicates that serum IS levels are significantly higher in the presence of CAD and correlate with the severity of the disease and coronary atherosclerosis scores, which suggest that increased serum IS may be involved in the pathogenesis of coronary atherosclerosis.

Association Between Indoxyl Sulfate and Cardiac Dysfunction and Prognosis in Patients With Dilated Cardiomyopathy

Serum indoxyl sulfate (IS) is a uremic toxin that accelerates the progression of chronic kidney disease (CKD). The aim of this study was to determine whether serum IS is associated with hemodynamic parameters or cardiac events in patients with nonischemic dilated cardiomyopathy (DCM). The 76 patients with DCM had their serum IS and plasma brain natriuretic peptide (BNP) levels measured, and underwent echocardiographic examination. Mean (± standard deviation) left ventricular ejection fraction (LVEF) and BNP levels in the patients were 32.5 ± 10.7% and 204 ± 219 pg/ml, respectively. Patients were divided into 2 groups, low IS (<0.9 µg/ml) and high IS (≥ 0.9 µg/ml), based on the median value of serum IS. Although there were no significant differences in LVEF and BNP between the groups, E/e' was significantly greater in the high IS group than in the low IS group. Furthermore, E/e' was an independent determinant of serum IS level. The risk of a cardiac event was significantly higher in the high IS group than in the low IS group (P=0.014). Moreover, serum IS was a significant predictor of cardiac events even after adjustment for BNP. Cardiac dysfunction is associated with the serum IS level, which might serve as a new prognostic marker in DCM patients with normal renal function or mild to moderate CKD.

Usefulness of Parameters of Right Ventricular Function by Echocardiography to Predict Cardiac Events in Patients with Chronic Heart Failure

Introduction: A progressive decline in renal function is a frequent accompaniment of myocardial infarction (MI). Wehave previously shown that post-MI the uraemic toxin, indoxyl sulphate (IS), kidney injurymolecule (KIM)-1 and cardiac fibrosis are increased. In chronic kidney disease (CKD) we have reported increased IS and cardiac fibrosis; both were reduced following treatment with the oral carbonic adsorbent AST-120. However, the effect of AST-120 on renal injury post-MI has not been fully explored. Methods: MI-induced rats received either AST-120 (8% in rat chow) or were untreated (MI+Veh) for 16 weeks. Sham operated rats served as controls. Serum IS level was measured at baseline and 16 weeks. Echocardiography and glomerular filtration rate (GFR) was assessed prior to sacrifice at 16 weeks. Tissues were assessed for changes using immunohistological and western blot methods. Results: Heart: Ejection fraction and fractional shortening were reduced by 39% and 49% post-MI respectively (P< 0.001 vs. sham).Cardiac collagen I andTIMP-1 expression was elevated post-MI (10 and 5 fold respectively, P< 0.01) and following AST-120 treatment reduced back to sham levels (P< 0.01). No change in MMP-2 expression was detected; and no difference observed in blood pressure betweenMI groups. Kidney:MI+Veh increased delta serum IS levels, renal fibrosis and KIM-1 expression; and reduced GFR (P< 0.05 vs sham). Compared to MI+Veh, AST-120 treatment reduced serum IS, renal fibrosis and KIM-1 expression (P< 0.05). KIM-1 expression positively correlated with serum IS levels (r= 0.56; P= 0.002). Conclusions: AST-120 reduced cardiac collagen by suppressing TIMP-1 expression. KIM-1 appears to be a sensitive renal biomarker for post-MI kidney that may be influenced by elevated IS levels. AST-120 treatment post-MI improves renal fibrosis and normalises KIM-1 and IS levels. Thus AST-120 has beneficial cardiac and renal effects post-MI, and may be a potential treatment for cardiorenal syndrome.

P-Cresyl sulfate and indoxyl sulfate in pediatric patients on chronic dialysis

PurposeIndoxyl sulfate and p-cresyl sulfate are important protein-bound uremic retention solutes whose levels can be partially reduced by renal replacement therapy. These solutes originate from intestinal bacterial protein fermentation and are associated with cardiovascular outcomes and chronic kidney disease progression. The aims of this study were to investigate the levels of indoxyl sulfate and p-cresyl sulfate as well as the effect of probiotics on reducing the levels of uremic toxins in pediatric patients on dialysis.MethodsWe enrolled 20 pediatric patients undergoing chronic dialysis; 16 patients completed the study. The patients underwent a 12-week regimen of VSL#3, a high-concentration probiotic preparation, and the serum levels of indoxyl sulfate and p-cresyl sulfate were measured before treatment and at 4, 8, and 12 weeks after the regimen by using fluorescence liquid chromatography. To assess the normal range of indoxyl sulfate and p-cresyl sulfate we enrolled the 16 children with normal glomerular filtration rate who had visited an outpatient clinic for asymptomatic microscopic hematuria that had been detected by a school screening in August 2011.ResultsThe baseline serum levels of indoxyl sulfate and p-cresyl sulfate in the patients on chronic dialysis were significantly higher than those in the children with microscopic hematuria. The baseline serum levels of p-cresyl sulfate in the peritoneal dialysis group were significantly higher than those in the hemodialysis group. There were no significant changes in the levels of these uremic solutes after 12-week VSL#3 treatment in the patients on chronic dialysis.ConclusionThe levels of the uremic toxins p-cresyl sulfate and indoxyl sulfate are highly elevated in pediatric patients on dialysis, but there was no significant effect by probiotics on the reduction of uremic toxins in pediatric dialysis patients. Therefore, studies for other medical intervention to reduce uremic toxins are also necessary in pediatric patients on dialysis.

Activation of Aryl Hydrocarbon Receptor Mediates Indoxyl Sulfate-Induced Monocyte Chemoattractant Protein-1 Expression in Human Umbilical Vein Endothelial Cells

Indoxyl sulfate (IS) is a uremic toxin that causes renal injury, but little is known about its adverse effects on the cardiovascular system. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor that mediates adaptive and toxic responses in cells. Recent studies identified IS as an endogenous agonist for AhR, as well as other tryptophan metabolites. The aim of the study was to investigate whether IS activates AhR, with subsequent inflammatory responses contributing to the development of atherogenesis, in human umbilical vein endothelial cells (HUVECs). We demonstrated that IS stimulates the expression of AhR target genes, including cytochromes P450 1A1 and 1B1 mRNA, in a time-dependent manner, as well as translocation of AhR into the nucleus from the cytoplasm, indicating AhR activation. IS-stimulated AhR activation was accompanied by an increase in oxidative stress, proven by enhanced NADPH oxidase 4 expression and dihydroethidium staining. Additionally, AhR inhibitors abolished the IS-induced increase in monocyte chemoattractant protein-1 (MCP-1) expression in a dose-dependent manner. Taken together, these results suggest that IS activates AhR as an endogenous agonist and induces MCP-1 expression through reactive oxygen species production in HUVECs. Our findings give a novel understanding of the physiological effect of IS on the cardiovascular system and indicate possibilities for preventing cardiorenal syndrome by regulating serum IS levels.

Stat3 Contributes to Indoxyl Sulfate-Induced Inflammatory and Fibrotic Gene Expression and Cellular Senescence

Background/Aim: Increased phosphorylation (activation) of signal transducer and activator of transcription 3 (Stat3) on tyrosine 705 leads to renal fibrosis. Indoxyl sulfate, a uremic toxin, induces renal fibrosis through expression of transforming growth factor-β₁ (TGF-β₁) in proximal tubular cells. The present study aimed to determine whether Stat3 is involved in indoxyl sulfate-induced dysfunction of proximal tubular cells. Methods: Localization of phosphorylated Stat3 in the kidneys of normal, subtotally nephrectomized, and AST-120-treated subtotally nephrectomized rats was examined by immunohistochemistry. The effect of indoxyl sulfate on phosphorylation of Stat3 and the role of Stat3 on indoxyl sulfate-induced cellular effects were examined using human proximal tubular cells (HK-2 cells). Results: Subtotally nephrectomized rats showed increased immunostaining of phosphorylated Stat3 in the renal tubules compared with normal rats. Administration of AST-120, which reduces serum level of indoxyl sulfate, to subtotally nephrectomized rats reduced the immunostaining of phosphorylated Stat3 in the renal tubules. Indoxyl sulfate induced phosphorylation of Stat3 in HK-2 cells. Stat3 small interfering RNA suppressed indoxyl sulfate-induced expression of an inflammation marker gene (monocyte chemotactic protein-1), fibrosis marker genes (TGF-β₁, α-smooth muscle actin) and a subunit of nuclear factor-ĸB (p65), and attenuated a cellular senescence marker, senescence-associated β-galactosidase activity. Conclusions: Stat3 is involved in indoxyl sulfate-induced inflammatory and fibrotic gene expression and cellular senescence in proximal tubular cells.

Indoxyl Sulfate Predicts Cardiovascular Disease and Renal Function Deterioration in Advanced Chronic Kidney Disease

Indoxyl sulfate, a protein-bound uremic toxin, was found to be accumulated in kidney tissues with a reduction in renal function. This, in turn, not only leads to kidney fibrosis and endothelial dysfunction but also to adverse clinical effects. We investigated the adverse effects of indoxyl sulfate on clinical outcomes in a study involving human subjects. Seventy pre-dialysis patients were enrolled from a single medical center. Serum indoxyl sulfate and biochemistry data were measured concurrently. Clinical outcomes including dialysis event, cardiovascular event and all-cause mortality were recorded during a 36-month follow-up. Multivariate Cox regression analysis showed that age (HR: 0.95, p=0.05), serum creatinine (HR: 1.29, p=0.04) and indoxyl sulfate (HR: 1.06, p=0.02) were independently associated with dialysis event; age (HR: 1.16, p=0.01), serum phosphate (HR: 3.03, p=0.05) and indoxyl sulfate level (HR: 1.11, p=0.04) reached significant correlation with cardiovascular events after adjusting for other confounding factors. Kaplan-Meier analysis revealed that indoxyl sulfate level was significantly associated with cardiovascular and dialysis event (log rank p <0.01, log rank p=0.01, respectively). In addition, serum indoxyl sulfate concentration was significantly increased in patients with dialysis and cardiovascular event (p <0.01, p <0.01, respectively). Our results suggest that serum indoxyl sulfate level was a valuable marker in predicting cardiovascular disease and renal function decline in patients with advanced chronic kidney disease.

Chronic Kidney Disease-Induced Cardiac Fibrosis Is Ameliorated by Reducing Circulating Levels of a Non-Dialysable Uremic Toxin, Indoxyl Sulfate

Cardiovascular death commonly occurs in patients with chronic kidney disease. Indoxyl sulfate (IS), a uremic toxin, has been demonstrated in vitro as a contributory factor in cardiac fibrosis, a typical pathological finding in uremic cardiomyopathy. This study aimed to determine if cardiac fibrosis is reversible by lowering serum IS levels using an oral charcoal adsorbent, AST-120. Subtotal-nephrectomized (5/6-STNx) Sprague-Dawley rats were randomized to receive either AST-120 (AST-120, n = 13) or no treatment (vehicle, n = 17) for 12 weeks. Sham operated rats (n = 12) were used as controls. Early left ventricular (LV) diastolic dysfunction was demonstrated by an increase in peak velocity of atrial filling [A and A’ waves] and a decrease of E/A and E’/A’ ratios obtained by echocardiography. This was accompanied by a 4.5-fold increase in serum IS (p<0.001) as well as elevated tail-cuff blood pressure (p<0.001) and heart weight (p<0.001). Increased LV fibrosis (p<0.001), gene expression of pro-fibrotic (TGF-β, CTGF) and hypertrophic (ANP, β-MHC and α-skeletal muscle actin) markers, as well as TGF-β and phosphorylated NF-κB protein expression were observed in STNx + vehicle rats. Treatment with AST-120 reduced serum creatinine (by 54%, p<0.05) and urine total protein (by 27%, p<0.05) vs vehicle whilst having no effect on blood pressure (AST-120 = 227±11 vs vehicle = 224±8 mmHg, ns) and heart weight. The increase in serum IS was prevented with AST-120 (by 100%, p<0.001) which was accompanied by reduced LV fibrosis (68%, p<0.01) and TGF-β and phosphorylated NF-κB protein expression (back to sham levels, p<0.05) despite no significant change in LV function. In conclusion, STNx resulted in increased cardiac fibrosis and circulating IS levels. Reduction of IS with AST-120 normalizes cardiac fibrosis, in a blood pressure independent manner.