Indoxyl sulfate, a gut microbiome-derived uremic toxin, is associated with psychic anxiety and its functional magnetic resonance imaging-based neurologic signature.

It is unknown whether indoles, metabolites of tryptophan that are derived entirely from bacterial metabolism in the gut, are associated with symptoms of depression and anxiety. Serum samples (baseline, 12 weeks) were drawn from participants (n = 196) randomized to treatment with cognitive behavioral therapy (CBT), escitalopram, or duloxetine for major depressive disorder. Baseline indoxyl sulfate abundance was positively correlated with severity of psychic anxiety and total anxiety and with resting state functional connectivity to a network that processes aversive stimuli (which includes the subcallosal cingulate cortex (SCC-FC), bilateral anterior insula, right anterior midcingulate cortex, and the right premotor areas). The relation between indoxyl sulfate and psychic anxiety was mediated only through the metabolite's effect on the SCC-FC with the premotor area. Baseline indole abundances were unrelated to post-treatment outcome measures, and changes in symptoms were not correlated with changes in indole concentrations. These results suggest that CBT and antidepressant medications relieve anxiety via mechanisms unrelated to modulation of indoles derived from gut microbiota; it remains possible that treatment-related improvement stems from their impact on other aspects of the gut microbiome. A peripheral gut microbiome-derived metabolite was associated with altered neural processing and with psychiatric symptom (anxiety) in humans, which provides further evidence that gut microbiome disruption can contribute to neuropsychiatric disorders that may require different therapeutic approaches. Given the exploratory nature of this study, findings should be replicated in confirmatory studies.Clinical trial NCT00360399 "Predictors of Antidepressant Treatment Response: The Emory CIDAR" https://clinicaltrials.gov/ct2/show/NCT00360399 .

Apoptosis signal-regulating kinase 1 inhibition reverses deleterious indoxyl sulfate-mediated endothelial effects.

AIMS: Indoxyl sulfate (IS), a protein-bound uremic toxin, is implicated in endothelial dysfunction, which contributes to adverse cardiovascular events in chronic kidney disease. Apoptosis signal regulating kinase 1 (ASK1) is a reactive oxygen species-driven kinase involved in IS-mediated adverse effects. This study assessed the therapeutic potential of ASK1 inhibition in alleviating endothelial effects induced by IS. MAIN METHODS: IS, in the presence and absence of a selective ASK1 inhibitor (GSK2261818A), was assessed for its effect on vascular reactivity in rat aortic rings, and cultured human aortic endothelial cells where we evaluated phenotypic and mechanistic changes. KEY FINDINGS: IS directly impairs endothelium-dependent vasorelaxation and endothelial cell migration. Mechanistic studies revealed increased production of reactive oxygen species-related markers, reduction of endothelial nitric oxide synthase and increased protein expression of tissue inhibitor of matrix metalloproteinase 1 (TIMP1). IS also increases angiopoietin-2 and tumour necrosis factor α gene expression and promotes transforming growth factor ß receptor abundance. Inhibition of ASK1 ameliorated the increase in oxidative stress markers, promoted autocrine interleukin 8 pro-angiogenic signalling and decreased anti-angiogenic responses at least in part via reducing TIMP1 protein expression. SIGNIFICANCE: ASK1 inhibition attenuated vasorelaxation and endothelial cell migration impaired by IS. Therefore, ASK1 is a viable intracellular target to alleviate uremic toxin-induced impairment in the vasculature.

The relationship of indoxyl sulfate and p-cresyl sulfate with target cardiovascular proteins in hemodialysis patients.

Protein-bound uremic toxins (Indoxyl sulfate [IS] and p-cresyl sulfate [PCS]) are both associated with cardiovascular (CV) and all-cause mortality in subjects with chronic kidney disease (CKD). Possible mechanisms have not been elucidated. In hemodialysis patients, we investigated the relationship between the free form of IS and PCS and 181 CV-related proteins. First, IS or PCS concentrations were checked, and high levels were associated with an increased risk of acute coronary syndrome (ACS) in 333 stable HD patients. CV proteins were further quantified by a proximity extension assay. We examined associations between the free form protein-bound uremic toxins and the quantified proteins with correction for multiple testing in the discovery process. In the second step, the independent association was evaluated by multivariable-adjusted models. We rank the CV proteins related to protein-bound uremic toxins by bootstrapped confidence intervals and ascending p-value. Six proteins (signaling lymphocytic activation molecule family member 5, complement component C1q receptor, C-C motif chemokine 15 [CCL15], bleomycin hydrolase, perlecan, and cluster of differentiation 166 antigen) were negatively associated with IS. Fibroblast growth factor 23 [FGF23] was the only CV protein positively associated with IS. Three proteins (complement component C1q receptor, CCL15, and interleukin-1 receptor-like 2) were negatively associated with PCS. Similar findings were obtained after adjusting for classical CV risk factors. However, only higher levels of FGF23 was related to increased risk of ACS. In conclusion, IS and PCS were associated with several CV-related proteins involved in endothelial barrier function, complement system, cell adhesion, phosphate homeostasis, and inflammation. Multiplex proteomics seems to be a promising way to discover novel pathophysiology of the uremic toxin.

Resveratrol Rescue Indoxyl Sulfate-Induced Deterioration of Osteoblastogenesis via the Aryl Hydrocarbon Receptor /MAPK Pathway.

Indoxyl sulfate (IS), a uremic toxin derived from dietary tryptophan metabolism by the gut microbiota, is an endogenous aryl hydrocarbon receptor (AhR) agonist and a key player in bone remodeling. Resveratrol (RSV), an AhR antagonist, plays a protective role in shielding against AhR ligands. Our study explored the impact of IS on osteoblast differentiation and examined the possible mechanism of IS in controlling the expression of osteoblastogenesis markers through an in-depth investigation of AhR signaling. In vivo, we found histological architectural disruption of the femoral bones in 5/6 nephrectomies of young adult IS exposed mice, including reduced Runx2 antigen expression. RSV improved the diaphysis architecture, Runx2 expression, and trabecular quality. In vitro data suggest that IS at 500 and 1000 µM disturbed osteoblastogenesis through suppression of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways, which were found to be downstream of AhR. RSV proved to ameliorate the anti-osteoblastogenic effects of IS through the inhibition of AhR and downstream signaling. Taken together, we demonstrated that the IS/AhR/MAPK signaling pathway plays a crucial role in the inhibition of osteoblastogenesis, and RSV has a potential therapeutic role in reversing the IS-induced decline in osteoblast development and suppressing abnormal bone turnover in chronic kidney disease patients.

TRPV1 Hyperfunction Involved in Uremic Toxin Indoxyl Sulfate-Mediated Renal Tubular Damage.

Indoxyl sulfate (IS) is accumulated during severe renal insufficiency and known for its nephrotoxic properties. Transient receptor potential vanilloid 1 (TRPV1) is present in the kidney and acts as a renal sensor. However, the mechanism underlying IS-mediated renal tubular damage in view of TRPV1 is lacking. Here, we demonstrated that TRPV1 was expressed in tubular cells of Lilly Laboratories cell-porcine kidney 1 (LLC-PK1) and Madin-Darby canine kidney cells (MDCK). IS treatment in both cells exhibited tubular damage with increased LDH release and reduced cell viability in dose- and time-dependent manners. MDCK, however, was more vulnerable to IS. We, therefore, investigated MDCK cells to explore a more detailed mechanism. Interestingly, IS-induced tubular damage was markedly attenuated in the presence of selective TRPV1 blockers. IS showed no effect on TRPV1 expression but significantly increased arachidonate 12-lipoxygenase (ALOX12) protein, mRNA expression, and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) amounts in a dose-dependent manner, indicating that the ALOX12/12(S)-HETE pathway induced TRPV1 hyperfunction in IS-mediated tubulotoxicity. Blockade of ALOX12 by cinnamyl-3,4-dihydroxy-α-cyanocinnamate or baicalein attenuated the effects of IS. Since aryl hydrocarbon receptor (AhR) activation after IS binding is crucial in mediating cell death, here, we found that the AhR blockade not only ameliorated tubular damage but also attenuated ALOX12 expression and 12(S)-HETE production caused by IS. The uremic toxic adsorbent AST-120, however, showed little effect on ALOX12 and 12(S)-HETE, as well as IS-induced cell damage. These results clearly indicated that IS activated AhR and then upregulated ALOX12, and this induced endovanilloid 12(S)-HETE synthesis and contributed to TRPV1 hyperfunction in IS-treated tubular cells. Further study on TRPV1 may attenuate kidney susceptibility to the functional loss of end-stage kidney disease via IS.

Klotho alleviates indoxyl sulfate-induced heart failure and kidney damage by promoting M2 macrophage polarization.

Indoxyl sulfate (IS) is a protein-bound uremic toxin that can accumulate in patients with chronic kidney disease (CKD) or acute kidney injury (AKI) and cause kidney and cardiac dysfunction. Klotho is an anti-aging protein that has reno- and cardio-protective effects. We investigated whether Klotho could alleviate IS-induced heart failure and kidney damage by regulating macrophages, which play a key role in the inflammatory response in CKD and AKI. Treatment of THP-1-derived macrophages with IS induced the production of the pro-inflammatory cytokines TNFα, IL-6, and IL-1ß, and stimulated M1 polarization. Additionally, IS induced downregulation of Klotho expression in macrophages. Overexpression of Klotho suppressed the IS-induced inflammatory response in macrophages by stimulating M2 polarization. It also alleviated IS-induced cardiac hypertrophy and renal fibrosis in mice. A reduction in IS-induced phosphorylation of NF-kB p65 was observed in response to Klotho overexpression, suggesting that Klotho alleviates kidney and cardiac injury by inactivating NF-kB signaling and promoting macrophage M2 polarization.

Indoxyl sulfate impairs valsartan-induced neovascularization.

Studies revealed that the use of renin-angiotensin-aldosterone system antagonism is not associated with a statistically significant reduction in the risk of cardiovascular events in patients with chronic kidney disease (CKD) compared with that in the general population. We tested the hypothesis that indoxyl sulfate (IS) can interfere with the protective effect of valsartan-mediated on endothelial function in vitro and neovascularization in mice underwent subtotal nephrectomy. In human aortic endothelial cells, we first demonstrated that IS impaired the valsartan-mediated phosphorylation of eNOSThr495, nitric oxide production and tube formation via NADPH oxidase (NOX) and protein kinase C (PKC) phosphorylation, but this effect was suppressed by cotreatment with apocynin and calphostin C. In vivo, IS attenuated valsartan-induced angiogenesis in Matrigel plugs in mice. Moreover, in subtotal nephrectomy mice who underwent hindlimb ischemic surgery, valsartan significantly increased the mobilization of endothelial progenitor cells in circulation as well as the reperfusion of blood flow and density of CD31+ capillaries in ischemic limbs. However, IS attenuated the protective effect of valsartan-induced neovascularization and increased the expression of p-PKCαSer657 and p-eNOSThr497 in ischemic limbs. Cotreatment of apocynin and calphostin C reversed the IS impaired-neovascularization and decreased the expression of p-PKCαSer657 and p-eNOSThr497 in ischemic limbs. Our study suggests that the NOX/PKC/eNOS signaling pathway plays a pivotal role in the IS-mediated inhibition of valsartan-conferred beneficial effects on endothelial function in vitro and neovascularization in subtotal nephrectomy mice. We proposed a novel causative role for IS in cardiovascular complications in CKD patients.

Indoxyl Sulfate and p-Cresyl Sulfate Promote Vascular Calcification and Associate with Glucose Intolerance.

BACKGROUND: Protein-bound uremic toxins indoxyl sulfate (IS) and p-cresyl sulfate (PCS) have been associated with cardiovascular morbidity and mortality in patients with CKD. However, direct evidence for a role of these toxins in CKD-related vascular calcification has not been reported. METHODS: To study early and late vascular alterations by toxin exposure, we exposed CKD rats to vehicle, IS (150 mg/kg per day), or PCS (150 mg/kg per day) for either 4 days (short-term exposure) or 7 weeks (long-term exposure). We also performed unbiased proteomic analyses of arterial samples coupled to functional bioinformatic annotation analyses to investigate molecular signaling events associated with toxin-mediated arterial calcification. RESULTS: Long-term exposure to either toxin at serum levels similar to those experienced by patients with CKD significantly increased calcification in the aorta and peripheral arteries. Our analyses revealed an association between calcification events, acute-phase response signaling, and coagulation and glucometabolic signaling pathways, whereas escape from toxin-induced calcification was linked with liver X receptors and farnesoid X/liver X receptor signaling pathways. Additional metabolic linkage to these pathways revealed that IS and PCS exposure engendered a prodiabetic state evidenced by elevated resting glucose and reduced GLUT1 expression. Short-term exposure to IS and PCS (before calcification had been established) showed activation of inflammation and coagulation signaling pathways in the aorta, demonstrating that these signaling pathways are causally implicated in toxin-induced arterial calcification. CONCLUSIONS: In CKD, both IS and PCS directly promote vascular calcification via activation of inflammation and coagulation pathways and were strongly associated with impaired glucose homeostasis.

The specific impact of uremic toxins upon cognitive domains: a review / O impacto específico de toxinas urêmicas em domínios cognitivos: uma revisão

ABSTRACT One of the mechanisms proposed for chronic kidney disease (CKD)-related cognitive impairment is the accumulation of uremic toxins due to the deterioration of the renal clearance function. Cognition can be categorized into five major domains according to its information processing functions: memory, attention, language, visual-spatial, and executive. We performed a review using the terms 'uric acid', 'indoxyl sulfate', 'p-cresyl sulfate', 'homocysteine', 'interleukins' and 'parathyroid hormone'. These are the compounds that were found to be strongly associated with cognitive impairment in CKD in the literature. The 26 selected articles point towards an association between higher levels of uric acid, homocysteine, and interleukin 6 with lower cognitive performance in executive, attentional, and memory domains. We also reviewed the hemodialysis effects on cognition. Hemodialysis seems to contribute to an amelioration of CKD-related encephalopathic dysfunction, although this improvement occurs more in some cognitive domains than in others.

RESUMO Um dos mecanismos propostos para explicar o comprometimento cognitivo relacionado à doença renal crônica (DRC) é o acúmulo de toxinas urêmicas devido à deterioração da função de depuração renal. A cognição pode ser categorizada em cinco domínios principais de acordo com suas funções de processamento de informações: memória, atenção, linguagem, visual-espacial e executiva. Realizamos uma revisão usando os termos "ácido úrico", "indoxil sulfato", "p-cresil sulfato", "homocisteína", "interleucinas" e "paratormônio". Estes são os compostos que se mostraram fortemente associados ao comprometimento cognitivo na DRC na literatura. Os 26 artigos selecionados apontam para uma associação entre níveis mais elevados de ácido úrico, homocisteína e interleucina-6 com menor desempenho cognitivo nos domínios executivo, atenção e de memória. Também revisamos os efeitos da hemodiálise na cognição. A hemodiálise parece contribuir para uma melhoria da disfunção encefalopática relacionada à DRC, embora essa melhora ocorra mais em alguns domínios cognitivos do que em outros.

The specific impact of uremic toxins upon cognitive domains: a review.

One of the mechanisms proposed for chronic kidney disease (CKD)-related cognitive impairment is the accumulation of uremic toxins due to the deterioration of the renal clearance function. Cognition can be categorized into five major domains according to its information processing functions: memory, attention, language, visual-spatial, and executive. We performed a review using the terms 'uric acid', 'indoxyl sulfate', 'p-cresyl sulfate', 'homocysteine', 'interleukins' and 'parathyroid hormone'. These are the compounds that were found to be strongly associated with cognitive impairment in CKD in the literature. The 26 selected articles point towards an association between higher levels of uric acid, homocysteine, and interleukin 6 with lower cognitive performance in executive, attentional, and memory domains. We also reviewed the hemodialysis effects on cognition. Hemodialysis seems to contribute to an amelioration of CKD-related encephalopathic dysfunction, although this improvement occurs more in some cognitive domains than in others.

Indoxyl sulfate induces platelet hyperactivity and contributes to chronic kidney disease-associated thrombosis in mice.

Thrombosis is a common complication of chronic kidney disease (CKD), but the causes and mechanisms of CKD-associated thrombosis are not well clarified. Here, we show that platelet activity is remarkably enhanced in CKD mice, with increase of serum indoxyl sulfate (IS), a typical uremic toxin, which cannot be effectively cleared by routine dialysis. Ex vivo and in vitro experiments reveal that IS displays a distinct ability to enhance platelet activities, including elevated response to collagen and thrombin, increases in platelet-derived microparticles, and platelet-monocyte aggregates. The flow chamber assay and carotid artery thrombosis model demonstrate that IS-induced platelet hyperactivity contributes to thrombus formation. Further investigations disclose that reactive oxygen species (ROS)-mediated p38MAPK signaling plays a key role in IS-induced platelet hyperactivity. Moreover, we show that Klotho, which is expressed dominantly in the kidneys, has the capacity to counteract IS-induced platelet hyperactivity by inhibiting ROS/p38MAPK signaling, whereas Klotho reduction may aggravate the effect of IS on platelet activation in CKD and klotho+/- mice. Finally, we demonstrate that Klotho protein treatment can protect against IS-induced thrombosis and atherosclerosis in apoE-/- mice. Our findings uncover the mechanism of platelet hyperactivity induced by IS and provide new insights into the pathogenesis and treatment of CKD-associated thrombosis.

Indoxyl Sulfate Impairs Endothelial Progenitor Cells and Might Contribute to Vascular Dysfunction in Patients with Chronic Kidney Disease.

BACKGROUND/AIMS: Indoxyl sulfate (IS) is a protein-bound uremic toxin that accumulates in patients with chronic kidney disease (CKD). We explored the effect of IS on human early endothelial progenitor cells (EPCs) and analyzed the correlation between serum IS levels and parameters of vascular function, including endothelial function in a CKD-based cohort. METHODS: A cross-sectional study with 128 stable CKD patients was conducted. Flow-mediated dilation (FMD), pulse wave velocity (PWV), ankle brachial index, serum IS and other biochemical parameters were measured and analyzed. In parallel, the activity of early EPCs was also evaluated after exposure to IS. RESULTS: In human EPCs, a concentration-dependent inhibitory effect of IS on chemotactic motility and colony formation was observed. Additionally, serum IS levels were significantly correlated with CKD stages. The total IS (T-IS) and free IS (F-IS) were strongly associated with age, hypertension, cardiovascular disease, blood pressure, PWV, blood urea nitrogen, creatine and phosphate but negatively correlated with FMD, the estimated glomerular filtration rate (eGFR), hemoglobin, hematocrit, and calcium. A multivariate linear regression analysis also showed that FMD was significantly associated with IS after adjusting for other confounding factors. CONCLUSIONS: In humans, IS impairs early EPCs and was strongly correlated with vascular dysfunction. Thus, we speculate that this adverse effect of IS may partly result from the inhibition of early EPCs.

Indoxyl sulfate, not p-cresyl sulfate, is associated with cognitive impairment in early-stage chronic kidney disease.

BACKGROUNDS: Patients with chronic kidney disease (CKD) more commonly experience cognitive impairment, but the etiologies are not clear. Uremic toxins such as p-cresyl sulfate (PCS) and indoxyl sulfate (IS) have been shown to increase the risks of cardiovascular diseases and mortality; however, no study has investigated the associations of PCS and IS with cognitive function in patients with CKD. METHODS: Patients with CKD aged ≥50 years and age- and sex-matched non-CKD comparison subjects were recruited. CKD stage was defined according to the National Kidney Foundation guidelines. Cognitive function was evaluated using comprehensive neuropsychological tests. The associations between uremic toxins and cognitive function domains were examined using multiple linear regression analysis. The interaction between uremic toxins and CKD stages on cognitive functions were also examined. RESULTS: In total, 199 patients with CKD and 84 comparison subjects completed the study. The patients with CKD had poorer cognitive function and higher serum PCS and IS levels. A higher serum IS level was associated with poor executive function (ß=-0.31, P=0.003) only in stage 3 CKD patients after adjustment for age, sex and educational level. Serum PCS level was not associated with cognitive function in patients with CKD. CONCLUSIONS: Our study showed that a higher serum IS level was associated with poor executive function in the early stage of CKD. It would be worthwhile to investigate the effect of IS removal in early-stage CKD on the prevention of cognitive impairment in future studies.

Rhubarb Enema Attenuates Renal Tubulointerstitial Fibrosis in 5/6 Nephrectomized Rats by Alleviating Indoxyl Sulfate Overload.

AIM: To investigate the effects of rhubarb enema treatment using a 5/6 nephrectomized rat model and study its mechanisms. METHODS: Twenty-eight Sprague Dawley rats were divided into three groups: sham operation group (n = 8), 5/6 nephrectomized (5/6Nx) (n = 10), and 5/6Nx with rhubarb enema treatment (n = 10). The rhubarb enema was continuous for 1.0 month. Serum creatinine, serum indoxyl sulfate (IS) level, renal pathology, tubulointerstitial fibrosis, and renal oxidative stress were assessed. RESULTS: 5/6Nx rats showed increasing levels of serum creatinine and severe pathological lesions. Their serum creatinine levels obviously decreased after rhubarb enema treatment (P < 0.05 vs 5/6Nx group). The administration of rhubarb enema attenuated the histopathological changes in 5/6Nx rats. In addition, 5/6Nx rats showed an enhanced extent of tubulointerstitial fibrosis compared with sham rats, and administration of rhubarb enema to 5/6Nx rats ameliorated tubulointerstitial fibrosis. 5/6Nx rats showed increased serum levels of IS, renal oxidative stress, and NF-κB compared with sham rats, whereas administration of rhubarb enema to 5/6Nx rats decreased serum levels of IS, renal oxidative stress, and NF-κB levels. CONCLUSION: Rhubarb enema treatment ameliorates tubulointerstitial fibrosis in the kidneys of 5/6Nx rats, most likely by alleviating IS overload and reducing kidney oxidative stress and inflammatory injury.

Deleterious vascular effects of indoxyl sulfate and reversal by oral adsorbent AST-120.

BACKGROUND: In chronic kidney disease (CKD), blood vessels are permanently exposed to uremic toxins such as indoxyl sulfate (IS). We hypothesized that IS could alter vascular tone and that reducing its serum concentration could be beneficial. DESIGN: We studied acute and longer-term effects of IS and AST-120, an oral charcoal adsorbent, on vascular reactivity, endothelium integrity and expression of adhesion molecules VCAM-1 and ICAM-1 in aortic rings of normal and uremic wild type (WT) mice in vitro, and the cardiovascular effects of AST-120 in both WT and apoE-/- mice with CKD in vivo. RESULTS: In vitro, 1.0 mM IS acutely reduced vascular relaxation (64% for IS 1.0 mM vs. 80% for control, p < 0.05). The effect was more marked after 4 days exposure (39% for IS 1.0 mM 4 days; p < 0.001, prolonged vs. acute exposure), and was associated with endothelial cell loss and upregulation of ICAM-1/VCAM-1 expression. In vitro, AST-120 restored normal vascular function and prevented IS induced endothelial cell loss and ICAM-1/VCAM-1 upregulation. In vivo, AST-120 treatment of CKD mice (1) improved vascular relaxation (72% vs. 48% maximal relaxation in treated vs. untreated mice, p < 0.001), (2) reduced aortic VCAM-1 and ICAM-1 expression, (3) decreased aorta systolic expansion rate (9 ± 3% CKD vs. 14 ± 3% CKD + AST-120, p < 0.02), and (4) prevented the increase in pulse wave velocity (3.56 ± 0.17 m/s CKD vs. 3.10 ± 0.08 m/s CKD + AST-120, p < 0.006). Similar changes were observed in apoE-/- mice. CONCLUSION: IS appears to be an important contributor to the vascular dysfunction associated with CKD. AST-120 treatment ameliorates this dysfunction, possibly via a decrease in serum IS concentration.

Indoxyl sulfate-induced activation of (pro)renin receptor is involved in expression of TGF-ß1 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-ß1 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-ß1 and α-smooth muscle actin in proximal tubular cells. Thus, indoxyl sulfate-induced activation of prorenin/PRR might be involved in renal fibrosis.

An update on uremic toxins.

In the last decade, uremic toxicity as a potential cause for the excess of cardiovascular disease and mortality observed in chronic kidney disease gained more and more interest. This review focuses on uremic toxins with known cardiovascular effects and their removal. For protein-bound solutes, for example, indoxylsulfate and the conjugates of p-cresol, and for small water-soluble solutes, for example, guanidines, such as ADMA and SDMA, there is a growing evidence for a role in cardiovascular toxicity in vitro (e.g., affecting leukocyte, endothelial, vascular smooth muscle cell function) and/or in vivo. Several middle molecules (e.g., beta-2-microglobulin, interleukin-6, TNF-alpha and FGF-23) were shown to be predictors for cardiovascular disease and/or mortality. Most of these solutes, however, are difficult to remove during dialysis, which is traditionally assessed by studying the removal of urea, which can be considered as a relatively inert uremic retention solute. However, even the effective removal of other small water-soluble toxins than urea can be hampered by their larger distribution volumes. Middle molecules (beta-2-microglobulin as prototype, but not necessarily representative for others) are cleared more efficiently when the pore size of the dialyzer membrane increases, convection is applied and dialysis time is prolonged. Only adding convection to diffusion improves the removal of protein-bound toxins. Therefore, alternative removal strategies, such as intestinal adsorption, drugs interfering with toxic biochemical pathways or decreasing toxin concentration, and extracorporeal plasma adsorption, as well as kinetic behavior during dialysis need further investigation. Even more importantly, randomized clinical studies are required to demonstrate a survival advantage through these strategies.

Protein-bound uremic retention solutes.

Protein-bound uremic retention solutes are molecules with low molecular weight (MW) but should be considered middle or high MW substances. This article describes the best known substances of this group, which include p-cresol, indoxyl sulfate, hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furan-propionic acid (CMPF), and homocysteine. At concentrations encountered during uremia, p-cresol inhibits phagocyte function and decreases leukocyte adhesion to cytokine-stimulated endothelial cells. CMPF has been implicated in anemia and neurologic abnormalities of uremia. CMPF could alter the metabolism of drugs of inhibiting their binding to albumin and their tubular excretion. Indoxyl sulfate administrated to uremic rats increases the rate of progression of renal failure. Hippuric acid inhibits glucose utilization in the muscle, and its serum concentration is correlated with neurologic symptoms of uremia. Homocysteine predisposes uremic patients to cardiovascular disease through impairment of endothelial and smooth muscle cell functions. The removal of protein-bound compounds by conventional hemodialysis is low. Other strategies to decrease their concentrations include increase in dialyze pore size, daily hemodialysis, peritoneal dialysis, reduction of production or acceleration of degradation, and preservation of residual renal function.

Preventive effects of an oral sorbent on nephropathy in rats.

Circulating uremic substances are thought to be involved in the progression of chronic renal failure (CRF). An oral adsorbent AST-120 (Kremezin) is effective in removing circulating uremic toxins from the gastrointestinal tract, and retards the progression of CRF. AST-120 is widely used as an approved drug in Japan for the treatment of undialyzed uremic patients to delay the progression of CRF. AST-120 attenuates the progression of glomerular sclerosis and interstitial fibrosis in a variety of experimental rat models of CRF. However, the mechanism by which AST-120 delays the progression of CRF had not been clear. We have demonstrated that indoxyl sulfate, a dietary protein metabolite, is a circulating uremic toxin stimulating glomerular sclerosis and interstitial fibrosis, and that AST-120 decreases the serum and urine levels of indoxyl sulfate by adsorbing its precursor, indole, in the intestine. The administration of indoxyl sulfate to uremic rats stimulated the expression of transforming growth factor (TGF)-beta1, tissue inhibitor of metalloproteinase (TIMP)-1 and pro-alpha1(I)collagen in the kidneys. Further, the administration of AST-120 to uremic rats reduced the extent of glomerular sclerosis and interstitial fibrosis as well as the renal expression of TGF-beta1 and TIMP-1, by reducing the serum and urine levels of indoxyl sulfate. We propose the protein metabolite hypothesis that endogenous protein metabolites such as indoxyl sulfate play an important role in the progression of CRF, and that AST-120 is effective in retarding the progression of CRF by removing these protein metabolites through intestinal absorption.