Binding Affinity and Capacity for the Uremic Toxin Indoxyl Sulfate

Protein binding prevents uremic toxins from removal by conventional extracorporeal therapies leading to accumulation in maintenance dialysis patients. Weakening of the protein binding may enhance the dialytic elimination of these toxins. In ultrafiltration and equilibrium dialysis experiments, different measures to modify the plasma binding affinity and capacity were tested: (i), increasing the sodium chloride (NaCl) concentration to achieve a higher ionic strength; (ii), increasing the temperature; and (iii), dilution. The effects on the dissociation constant K_D and the protein bound fraction of the prototypical uremic toxin indoxyl sulfate (IS) in plasma of healthy and uremic individuals were studied. Binding of IS corresponded to one site binding in normal plasma. K_D increased linearly with the NaCl concentration between 0.15 (K_D = 13.2 ± 3.7 µM) and 0.75 M (K_D = 56.2 ± 2.0 µM). Plasma dilution further reduced the protein bound toxin fraction by lowering the protein binding capacity of the plasma. Higher temperatures also decreased the protein bound fraction of IS in human plasma. Increasing the NaCl concentration was effective to weaken the binding of IS also in uremic plasma: the protein bound fraction decreased from 89% ± 3% to 81% ± 3% at 0.15 and 0.75 M NaCl, respectively. Dilution and increasing the ionic strength and temperature enhance the free fraction of IS allowing better removal of the substance during dialysis. Applied during clinical dialysis, this may have beneficial effects on the long-term outcome of maintenance dialysis patients.     

Protein-Bound Uremic Toxins: New Culprits of Cardiovascular Events in Chronic Kidney Disease Patients

Chronic kidney disease (CKD) has been considered a major risk factor for cardiovascular diseases. Although great advances have recently been made in the pathophysiology and treatment of cardiovascular diseases, CKD remains a major global health problem. Moreover, the occurrence rates of cardiovascular events among CKD patients increase even in cases in which patients undergo hemodialysis, and the mechanisms underlying the so-called “cardiorenal syndrome” are not clearly understood. Recently, small-molecule uremic toxins have been associated with cardiovascular mortality in CKD and/or dialysis patients. These toxins range from small uncharged solutes to large protein-bound structures. In this review, we focused on protein-bound uremic toxins, such as indoxyl sulfate and p-cresyl sulfate, which are poorly removed by current dialysis techniques. Several studies have demonstrated that protein-bound uremic toxins, especially indoxyl sulfate, induce vascular inflammation, endothelial dysfunction, and vascular calcification, which may explain the relatively poor prognosis of CKD and dialysis patients. The aim of this review is to provide novel insights into the effects of indoxyl sulfate and p-cresyl sulfate on the pathogenesis of atherosclerosis.     

pH-Dependent Protein Binding Properties of Uremic Toxins In Vitro

Protein-bound uremic toxins (PBUTs) are difficult to remove using conventional dialysis treatment owing to their high protein-binding affinity. As pH changes the conformation of proteins, it may be associated with the binding of uremic toxins. Albumin conformation at pH 2 to 13 was analyzed using circular dichroism. The protein binding behavior between indoxyl sulfate (IS) and albumin was examined using isothermal titration calorimetry. Albumin with IS, and serum with IS, p-cresyl sulfate, indole acetic acid or phenyl sulfate, as well as serum from hemodialysis patients, were adjusted pH of 3 to 11, and the concentration of the free PBUTs was measured using mass spectrometry. Albumin was unfolded at pH < 4 or >12, and weakened interaction with IS occurred at pH < 5 or >10. The concentration of free IS in the albumin solution was increased at pH 4.0 and pH 11.0. Addition of human serum to each toxin resulted in increased free forms at acidic and alkaline pH. The pH values of serums from patients undergoing hemodialysis adjusted to 3.4 and 11.3 resulted in increased concentrations of the free forms of PBUTs. In conclusion, acidic and alkaline pH conditions changed the albumin conformation and weakened the protein binding property of PBUTs in vitro.     

Exploring Protein Binding of Uremic Toxins in Patients with Different Stages of Chronic Kidney Disease and during Hemodialysis

As protein binding of uremic toxins is not well understood, neither in chronic kidney disease (CKD) progression, nor during a hemodialysis (HD) session, we studied protein binding in two cross-sectional studies. Ninety-five CKD 2 to 5 patients and ten stable hemodialysis patients were included. Blood samples were taken either during the routine ambulatory visit (CKD patients) or from blood inlet and outlet line during dialysis (HD patients). Total (C_T) and free concentrations were determined of p-cresylglucuronide (pCG), hippuric acid (HA), indole-3-acetic acid (IAA), indoxyl sulfate (IS) and p-cresylsulfate (pCS), and their percentage protein binding (%PB) was calculated. In CKD patients, %PB/C_T resulted in a positive correlation (all p < 0.001) with renal function for all five uremic toxins. In HD patients, %PB was increased after 120 min of dialysis for HA and at the dialysis end for the stronger (IAA) and the highly-bound (IS and pCS) solutes. During one passage through the dialyzer at 120 min, %PB was increased for HA (borderline), IAA, IS and pCS. These findings explain why protein-bound solutes are difficult to remove by dialysis: a combination of the fact that (i) only the free fraction can pass the filter and (ii) the equilibrium, as it was pre-dialysis, cannot be restored during the dialysis session, as it is continuously disturbed.     

Targeting protein-bound uremic toxins in chronic kidney disease

Introduction: Protein-bound uremic toxins such as indoxyl sulfate cannot be removed efficiently by hemodialysis. These protein-bound uremic toxins have emerged as important risk factors for the progression of chronic kidney disease (CKD) as well as cardiovascular disease (CVD).

Areas covered: Indoxyl sulfate shows toxic effects on a variety of cells such as renal proximal tubular cells, glomerular mesangial cells, vascular smooth muscle cells, vascular endothelial cells, cardiomyocytes, cardiac fibroblasts, monocytes, osteoblasts and osteoclasts. This review overviews the cellular toxicity of indoxyl sulfate, its molecular mechanism and its role in the progression of CKD and CVD. Further, this review summarizes the clinical effects of AST-120 and the other strategies to reduce serum levels of indoxyl sulfate.

Expert opinion: Protein-bound uremic toxins such as indoxyl sulfate have emerged as target molecules for therapeutic intervention of not only CKD but also CVD. An oral sorbent AST-120 reduces serum level of indoxyl sulfate by adsorbing indole in the intestine. The modulation of intestinal bacteria by prebiotics/probiotics might be effective in reducing the production of indole in the intestine followed by reduced serum levels of indoxyl sulfate. An alternative approach might be antagonist which can counteract indoxyl sulfate-induced cellular effects and signaling pathways.     

Inhibitory effects of uraemic toxins 3‐indoxyl sulfate and p‐cresol on losartan metabolism in vitro

Objectives The purpose of this study was to clarify the cause of decreased metabolic clearance of losartan in patients with end‐stage renal failure. The influence of serum from haemodialysis patients (uraemic serum) and uraemic toxins on the metabolism of losartan to EXP‐3174 was investigated in vitro. Methods The formation of EXP‐3174 was estimated using pooled human liver microsomes. 3‐Carboxy‐4‐methyl‐5‐propyl‐2‐furanpropanoic acid, hippuric acid, indole‐3‐acetic acid, 3‐indoxyl sulfate and p‐cresol were used as uraemic toxins. Key findings Uraemic serum potently decreased the formation of EXP‐3174 in pooled human liver microsomes. In addition, 3‐indoxyl sulfate and p‐cresol significantly decreased the formation of EXP‐3174 in a concentration‐dependent manner. Furthermore, normal serum (10% v/v) with both 3‐indoxyl sulfate and p‐cresol (both 20 μmol/l) significantly decreased the formation of EXP‐3174 by 46%, which was similar to the level of inhibition with uraemic serum (10% v/v). Conclusions These results suggest that decreased the metabolic clearance of losartan in patients with end‐stage renal failure is partly due to high concentrations of 3‐indoxyl sulfate and p‐cresol.     

Methods of reducing the level of indoxyl sulfate – one of the most potent protein-bound uremic toxins

Chronic kidney disease (CKD) is associated with accumulation of uremic toxins, especially indoxyl sulfate (IS). IS has multi-directional, adverse influence on the organism, and is connected with cardiovascular and bone diseases. These properties make reducing level of IS so important issue. This article reviews methods allowing to reduce concentration of IS. Authors present methods like dialysis, using albumin-binding competitors, trans-cellular transporters and adsorbents, facilitation of dissociation from albumin, as well as modification of diet and intestinal bacteria. Despite the continued improvement and invention of new strategies, development of effective and safety methods of IS removing is still a challenge for researchers.     

Protein-bound uremic toxins: new insight from clinical studies

The uremic syndrome is attributed to the progressive retention of a large number of compounds which, under normal conditions, are excreted by healthy kidneys. The compounds are called uremic toxins when they interact negatively with biological functions. The present review focuses on a specific class of molecules, namely the family of protein-bound uremic toxins. Recent experimental studies have shown that protein-bound toxins are involved not only in the progression of chronic kidney disease (CKD), but also in the generation and aggravation of cardiovascular disease. Two protein-bound uremic retention solutes, namely indoxyl sulfate and p-cresyl sulfate, have been shown to play a prominent role. However, although these two molecules belong to the same class of molecules, exert toxic effects on the cardiovascular system in experimental animals, and accumulate in the serum of patients with CKD they may have different clinical impacts in terms of cardiovascular disease and other complications. The principal aim of this review is to evaluate the effect of p-cresyl sulfate and indoxyl sulfate retention on CKD patient outcomes, based on recent clinical studies.     

Interaction between two sulfate-conjugated uremic toxins, p-cresyl sulfate and indoxyl sulfate, during binding with human serum albumin

Recently, p-cresyl sulfate (PCS) has been identified as a protein-bound uremic toxin. Moreover, the serum-free concentration of PCS, which is associated with its efficacy of hemodialysis, appears to be a good predictor of survival in chronic kidney disease (CKD). We previously found that PCS interacts with indoxyl sulfate (IS), another sulfate-conjugated uremic toxin, during renal excretion via a common transporter. The purpose of this study was to further investigate the interaction between PCS and IS on the binding to human serum albumin (HSA). Here, we used ultrafiltration to show that there is only one high-affinity binding site for PCS, with a binding constant on the order of 10(5) M(-1) (i.e., comparable to that of IS). However, a binding constant of the low-affinity binding site for PCS is 2.5-fold greater than that for IS. Displacement of a fluorescence probe showed that PCS mainly binds to site II, which is the high-affinity site for PCS, on HSA. This finding was further supported by experiments using mutant HSA (R410A/Y411A) that displayed reduced site II ligand binding. A Klotz analysis showed that there could be competitive inhibition between PCS and IS on HSA binding. A similar interaction between PCS and IS on HSA was also observed under the conditions mimicking CKD stage 4 to 5. The present study suggests that competitive interactions between PCS and IS in both HSA binding and the renal excretion process could contribute to fluctuations in their free serum concentrations in patients with CKD.     

Interaction Mechanism Between Indoxyl Sulfate,a Typical Uremic Toxin Bound to Site II,and Ligands Bound to Site I of Human Serum Albumin

Purpose. The study was performed for clarifying the mechanism of interaction between indoxyl sulfate (IS), a typical uremic toxin bound to site II, and site I-ligands when bound to human serum albumin (HSA). The effect of the N to B transition on the interactions was also examined. Methods. Quantitative investigation of the relations between ligands bound to HSA was performed by equilibrium dialysis, and the binding data were analyzed on the basis of a theoretical model for simultaneous binding of two ligands. Results. The high-affinity binding constants for the site I-ligands warfarin (WF) and dansyl-L-asparagine (DNSA) increased with increasing pH, whereas those for the site II-ligands IS and dansylsarcosine (DNSS) were hardly affected by pH. Mutual displacement experiments showed that even though IS binds to site II it influenced binding of DNSA at the azapropazone binding area in site I. By contrast, it is unlikely that IS affects the WF binding area of site I. Furthermore, pH-profiles showed that the interaction between IS and DNSA was very sensitive to the N to B transition: competitive-like strong allosteric regulation was observed for binding of the two ligands to the N conformer (pH 6.5), whereas in the B conformation (pH 8.5) binding of these molecules was nearly independent. Conclusions. The present data provide useful information for elucidating a potential mechanism of interaction between drugs and endogenous substances including uremic toxins.     

Protein-Bound Uremic Toxins Lowering Effect of Sevelamer in Pre-Dialysis Chronic Kidney Disease Patients with Hyperphosphatemia: A Randomized Controlled Trial

P-cresyl sulfate and indoxyl sulfate are strongly associated with cardiovascular events and all-cause mortality in chronic kidney disease (CKD). This randomized controlled trial was conducted to compare the effects between sevelamer and calcium carbonate on protein-bound uremic toxins in pre-dialysis CKD patients with hyperphosphatemia. Forty pre-dialysis CKD patients with persistent hyperphosphatemia were randomly assigned to receive either 2400 mg of sevelamer daily or 1500 mg of calcium carbonate daily for 24 weeks. A significant decrease of total serum p-cresyl sulfate was observed in sevelamer therapy compared to calcium carbonate therapy (mean difference between two groups −5.61 mg/L; 95% CI −11.01 to −0.27 mg/L; p = 0.04). There was no significant difference in serum indoxyl sulfate levels (p = 0.36). Sevelamer had effects in terms of lowering fibroblast growth factor 23 (p = 0.01) and low-density lipoprotein cholesterol levels (p = 0.04). Sevelamer showed benefits in terms of retarding CKD progression. Changes in vascular stiffness were not found in this study.     

Contributions of hippurate, indoxyl sulfate, and o-hydroxyhippurate to impaired ligand binding by plasma in azotemic humans

We have evaluated pH, chloride, calcium and several endogenous aromatic acids as possible causes of the impaired binding of drugs by plasma albumin in renal failure. Changes in pH, chloride and calcium over the range found in renal failure had minimal or no effects on the binding of [14C]salicylate, a model probe which binds to both of the major drug-binding loci of human albumin. Hippurate and indoxyl sulfate were weak inhibitors of binding by normal plasma. Ortho-hydroxy-hippurate was undetectable or minimally elevated, except among patients with elevated plasma salicylate concentration. Although plasma hippurate and indoxyl sulfate concentrations were elevated markedly in patients with renal failure, inhibition of salicylate binding was significantly correlated only with the concentration of indoxyl sulfate. Addition of hippurate and indoxyl sulfate separately and together to normal plasma showed that these ligands could account for only 15% of the impaired binding of salicylate by azotemic plasma. The retained solutes which account for most of this binding defect remain to be identified. This uremic disorder (and perhaps others) is due not to a single chemical but to the additive effect of a family of chemicals.     

The Binding of Aripiprazole to Plasma Proteins in Chronic Renal Failure Patients

The binding of drugs to plasma protein is frequently altered in certain types of renal diseases. We recently reported on the effects of oxidation and uremic toxins on the binding of aripiprazole (ARP) to human serum albumin. In our continuing investigations, we examined the binding of ARP to plasma pooled from patients with chronic renal dysfunction. We examined the issue of the molecular basis for which factors affect the changes in drug binding that accompany renal failure. The study was based on the statistical relationships between ARP albumin binding and biochemical parameters such as the concentrations of oxidized albumin and uremic toxins. The binding of ARP to plasma from chronic renal patients was significantly lower than healthy volunteers. A rational relationship between the ARP binding rate and the concentration of toxins, including indoxyl sulphate (IS) and p-cresyl sulphate (PCS), was found, particularly for IS. Moreover, multiple regression analyses that involved taking other parameters such as PCS or oxidized albumin ratio to IS into account supports the above hypothesis. In conclusion, the limited data reported in this present study indicates that monitoring IS in the blood is a very important determinant in the dosage plan for the administration of site II drugs such as ARP, if the efficacy of the drug in renal disease is to be considered.     

Update on the pharmacokinetics and redox properties of protein-bound uremic toxins

Protein-bound uremic toxins, such as indoxyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, p-cresyl sulfate, hippuric acid, and indoleacetic acid, have been the subjects of extensive investigations. In this review, we summarized the recent works providing the new insight on the pharmacokinetics and redox properties of these uremic toxins. They have a common characteristic of being difficult to remove by conventional dialysis because they all bind tightly to serum albumin. They are transported via organic anion transporters to various tissues, and accumulate not only in the kidney but also in other tissues including vascular endothelial cells, smooth muscle cells, osteoblasts, and the central nervous system. Accumulated uremic toxins alter nonrenal drug clearance. Intracellular accumulated uremic toxins have been linked to the induction of oxidative stress and the stimulation of proinflammatory cytokines through the production of reactive oxygen species, which play a role in the progression of chronic kidney disease and the development of complications. Unfortunately, despite the massive amount of information on the undesirable effects of uremic toxins, methods for improving the detoxification of these toxins appear to be lacking.     

The Interplay between Uremic Toxins and Albumin,Membrane Transporters and Drug Interaction

Uremic toxins are a heterogeneous group of molecules that accumulate in the body due to the progression of chronic kidney disease (CKD). These toxins are associated with kidney dysfunction and the development of comorbidities in patients with CKD, being only partially eliminated by dialysis therapies. Importantly, drugs used in clinical treatments may affect the levels of uremic toxins, their tissue disposition, and even their elimination through the interaction of both with proteins such as albumin and cell membrane transporters. In this context, protein-bound uremic toxins (PBUTs) are highlighted for their high affinity for albumin, the most abundant serum protein with multiple binding sites and an ability to interact with drugs. Membrane transporters mediate the cellular influx and efflux of various uremic toxins, which may also compete with drugs as substrates, and both may alter transporter activity or expression. Therefore, this review explores the interaction mechanisms between uremic toxins and albumin, as well as membrane transporters, considering their potential relationship with drugs used in clinical practice.     

Uremic Toxins in the Progression of Chronic Kidney Disease and Cardiovascular Disease: Mechanisms and Therapeutic Targets

Chronic kidney disease (CKD) is a progressive loss of renal function. The gradual decline in kidney function leads to an accumulation of toxins normally cleared by the kidneys, resulting in uremia. Uremic toxins are classified into three categories: free water-soluble low-molecular-weight solutes, protein-bound solutes, and middle molecules. CKD patients have increased risk of developing cardiovascular disease (CVD), due to an assortment of CKD-specific risk factors. The accumulation of uremic toxins in the circulation and in tissues is associated with the progression of CKD and its co-morbidities, including CVD. Although numerous uremic toxins have been identified to date and many of them are believed to play a role in the progression of CKD and CVD, very few toxins have been extensively studied. The pathophysiological mechanisms of uremic toxins must be investigated further for a better understanding of their roles in disease progression and to develop therapeutic interventions against uremic toxicity. This review discusses the renal and cardiovascular toxicity of uremic toxins indoxyl sulfate, p-cresyl sulfate, hippuric acid, TMAO, ADMA, TNF-α, and IL-6. A focus is also placed on potential therapeutic targets against uremic toxicity.     

Protein binding of drugs in uremic and normal serum: The role of endogenous binding inhibitors

The protein binding of diazepam, indomethacin, salicylic acid, sulfadimetoxine and warfarin in serum of uremic patients has been studied by equilibrium dialysis and circular dichroism measurements and compared with that in normal serum. Comparisons have also been made with isolated human serum albumin (HSA) from uremic patients and healthy individuals. The binding of diazepam, salicylic acid, sulfadimetoxine and warfarin is impaired in the uremic sera, while the binding of indomethacin is apparently unchanged. The apparent binding constants of salicylic acid and warfarin in both uremic and normal sera are affected by dilution of the sera in buffer. The binding constants obtained with isolated albumins, however, are unaffected by dilution. The albumin isolated from uremic serum shows lower binding affinity for salicylic acid and warfarin than normal HSA, but the affinity was normalized by charcoal treatment at pH 3.0. It is shown that the binding both in normal and uremic sera is impaired compared with isolated defatted serum albumin due to the presence of competitive inhibitors. The inhibition is more pronounced in uremic serum. In addition, the binding to albumin in uremic sera is impaired by strongly bound allosteric inhibitors. It is also emphasized that determinations of association constants have to be related to the dilution of the serum, plasma or blood, respectively.     

Indoxyl sulfate, a uremic toxin, is biotransformed from indoxyl-beta-d-glucoside (indican) in rats

Indican (Indoxyl-beta-d-glucoside) is present in many Chinese herbs such as Isatis indigotica, Clerodendrum crytophyllum, Glehnia littoralis, Polygonum tinctorium and P. perfoliatum. This study aims to investigate whether indoxyl sulfate, a uremic toxin, would be biotransformed from indican in rats. Indican was administered intravenously and orally to Sprague-Dawley rats. The blood samples were withdrawn via cardiopuncture at specific time points and the serum concentrations of indican and indoxyl sulfate were assayed by HPLC method. The results showed that indican was rapidly and extensively metabolized to indoxyl sulfate either given intravenously or orally. Indoxyl sulfate showed markedly higher systemic exposure than indican. Because indoxyl sulfate is a harmful uremic toxin, we suggest that the content of indican in the aforementioned medicinal plants be quantitated and well controlled to ensure the safety for clinical use.     

Displacement by anionic drugs of endogenous ligands bound to albumin in uremic serum

Impaired binding of anionic drugs to serum albumin in patients with uremia is thought to be due to the accumulation of endogenous substances that bind to albumin. In this study the displacement by the anionic drugs diazepam, warfarin, and salicylic acid, which are known to be representative drugs for the binding sites on the albumin molecule, of several endogenous ligands that bind to albumin in uremic serum was examined. The free fractions of the ligands bound to albumin were separated by ultrafiltration in the presence and the absence of test drugs and assayed by high-performance liquid chromatography. Diazepam displaced indoxyl sulfate (IS), hippuric acid (HA), and indole-3-acetic acid (IAA), and warfarin displaced IS, HA, ISAA, and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid from serum albumin. However, salicylic acid did not displace the substance examined. The methods reported here are useful for determining the binding sites of the endogenous ligands on albumin and to clarify the drug-ligand interaction on albumin molecule in uremic serum.     

Removal of Protein-Bound Uremic Toxins Using Binding Competitors in Hemodialysis: A Narrative Review

Removal of protein-bound uremic toxins (PBUTs) during conventional dialysis is insufficient. PBUTs are associated with comorbidities and mortality in dialysis patients. Albumin is the primary carrier for PBUTs and only a small free fraction of PBUTs are dialyzable. In the past, we proposed a novel method where a binding competitor is infused upstream of a dialyzer into an extracorporeal circuit. The competitor competes with PBUTs for their binding sites on albumin and increases the free PBUT fraction. Essentially, binding competitor-augmented hemodialysis is a reactive membrane separation technique and is a paradigm shift from conventional dialysis therapies. The proposed method has been tested in silico, ex vivo, and in vivo, and has proven to be very effective in all scenarios. In an ex vivo study and a proof-of-concept clinical study with 18 patients, ibuprofen was used as a binding competitor; however, chronic ibuprofen infusion may affect residual kidney function. Binding competition with free fatty acids significantly improved PBUT removal in pre-clinical rat models. Based on in silico analysis, tryptophan can also be used as a binding competitor; importantly, fatty acids or tryptophan may have salutary effects in HD patients. More chemoinformatics research, pre-clinical, and clinical studies are required to identify ideal binding competitors before routine clinical use.