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.     

靶向肠道菌群调控肠源尿毒素代谢通路干预慢性肾病进展的治疗策略分析

慢性肾病(chronic kidney disease, CKD)是一种发病率高、预后差、并发症复杂的重大慢性疾病,对人类健康造成极大危害。硫酸吲哚酚(indoxyl-sulfate, IS)和硫酸对甲酚(p-cresol sulfate, PCS)是两种典型的肠源尿毒素,由肠道菌群与宿主共代谢生成。随着慢性肾病的进展,慢性肾病患者体内的IS和PCS等肠源尿毒素不断蓄积,并进一步促进CKD进展。肠道菌群与CKD密切相关,靶向肠道菌群调控肠源尿毒素合成及代谢通路很可能是延缓CKD进展的新思路及新策略。本文通过对肠道菌群及肠源尿毒素与慢性肾病进展之间的关系进行分析,提出基于肠源尿毒素代谢调控干预慢性肾病进展的治疗策略。     

Protein/Fiber Index Modulates Uremic Toxin Concentrations in Hemodialysis Patients

Background: Indoxyl sulfate (IS) and p-cresyl sulfate (PCS), two uremic toxins (UTs), are associated with increased mortality in patients with chronic kidney disease (CKD). These toxins are produced by the microbiota from the diet and excreted by the kidney. The purpose of this study was to analyze the effect of diet on IS and PCS concentration in hemodialysis (HD) patients. Methods: We performed a prospective monocentric study using a seven-day diet record and determination of serum IS and PCS levels in HD patients. We tested the association between toxin concentrations and nutritional data. Results: A total of 58/75 patients (77%) completed the diet record. Mean caloric intake was 22 ± 9.2 kcal/kg/day. The protein/fiber index was 4.9 ± 1.8. No correlation between IS or PCS concentration and protein/fiber index was highlighted. In the 18 anuric patients (31%) in whom residual renal function could not affect toxin concentrations, IS and PCS concentrations were negatively correlated with fiber intake and positively correlated with the protein/fiber index. In a multivariate analysis, IS serum concentration was positively associated with the protein/fiber index (p = 0.03). Conclusions: A low protein/fiber index is associated with low concentrations of uremic toxins in anuric HD patients. Diets with an increased fiber intake must be tested to determine whether they reduce PCS and IS serum concentrations.     

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.     

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.     

A molecular functional study on the interactions of drugs with plasma proteins

The binding of drugs to plasma proteins, such as albumin and alpha1-acid glycoprotein (AGP) is a major determinant in the disposition of drugs. A topology analysis of drug binding sites on HSA and AGP was determined using various methods, including spectroscopy, QSAR, photoaffinity labeling and site directed mutagenesis. Recombinant albumin was found to be useful for rapidly identifying drug binding sites. The binding sites on AGP are not completely separated but are partially overlapped, and Trp, Tyr, Lys and His residues in the drug binding pockets play important roles in this process. Drug displacement is somewhat complex, due to the involvement of multiple effects. The reduced binding in uremic patients may be explained by a mechanism that involves a combination of direct displacement by free fatty acids as well as cascade effects of free fatty acids and unbound uremic toxins for significant inhibition in serum binding. Albumin-containing dialysate is useful for the extracorporeal removal of endogenous toxins and in the treatment of drug overdoses. Oxidized albumin is a useful biomarker for the quantitative and qualitative evaluation of oxidative stress. Interestingly, AGP undergoes a structural transition to a unique structure that differs from the native and denatured states, when it interacts with membranes.     

Effect of protein-bound uraemic toxins on the thermodynamic characteristics of human albumin

The ability of albumin to bind drugs and other lipophilic organic acids is decreased in chronic renal failure by the accumulation of albumin-bound uraemic toxins such as hippuric acid, indoxyl sulphate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF). This furan acid is the most highly bound and is not removed by haemodialysis. The inhibitory effects of these three uraemic toxins on the interaction of three marker ligands sodium octanoate (for medium chain fatty acids), salicylic acid and phenol red (bilirubin site/site I) with albumin have been investigated by differential scanning microcalorimetry and flow microcalorimetry. CMPF was the most potent inhibitor and its binding site coincided with that of bilirubin (site I). Indoxyl sulphate binds to the site for medium-chain fatty acids and tryptophan (site II) and hippuric acid, the weakest inhibitor, inhibited binding to the salicylic acid site.     

Further Evidence Regarding the Important Role of Chlorine Atoms of Aripiprazole on Binding to the Site II Area of Human Albumin

Previously, we reported on the high-affinity binding of aripiprazole (ARP), an antipsychotic drug, to human albumin and the role of the chlorine atom of ARP on this binding. In this study, we investigated the binding mode of ARP to human albumin in detail using ARP derivatives and several animal-derived albumins. ARP bound strongly to human and dog albumin. The circular dichroism (CD) spectra of ARP bound to human and dog albumin were also similar. Deschloro-ARP bound less strongly to all of the albumin species compared to ARP, and the shapes of CD spectra were similar for all albumin species. CD spectra of dimethyl-ARP, for which chlorine atoms were substituted methyl groups, were quite similar to that of deschloro-ARP. In displacement experiments, competitive binding was observed between ARP and deschloro-ARP. These results suggest that the chlorine atoms in ARP are involved in the binding modes of ARP for human and dog albumins, whereas ARP and deschloro-ARP appear to share the same binding region in site II. The aforementioned results imply that compounds having a chlorine atom bind more strongly to plasma proteins, resulting in a long blood retention time. Therefore, findings reported here may provide the basically useful data for drug design.     

In-vitro study on the competitive binding of diflunisal and uraemic toxins to serum albumin and human plasma using a potentiometric ion-probe technique

The competitive binding of diflunisal and three well-known uraemic toxins (3-indoxyl sulfate, indole-3-acetic acid and hippuric acid) to bovine serum albumin (BSA), human serum albumin (HSA) and human plasma was studied by direct potentiometry. The method used the potentiometric drug ion-probe technique with a home-made ion sensor (electrode) selective to the drug anion. The site-oriented Scatchard model was used to describe the binding of diflunisal to BSA, HSA and human plasma, while the general competitive binding model was used to calculate the binding parameters of the three uraemic toxins to BSA. Diflunisal binding parameters, number of binding sites, n(i) and association constants for each class of binding site, K(i), were calculated in the absence and presence of uraemic toxins. Although diflunisal exhibits high binding affinity for site I of HSA and the three uraemic toxins bind primarily to site II, strong interaction was observed between the drug and the three toxins, which were found to affect the binding of diflunisal on its primary class of binding sites on both BSA and HSA molecules and on human plasma. These results are strong evidence that the decreased binding of diflunisal that occurs in uraemic plasma may not be solely attributed to the lower albumin concentration observed in many patients with renal failure. The uraemic toxins that accumulate in uraemic plasma may displace the drug from its specific binding sites on plasma proteins, resulting in increased free drug plasma concentration in uraemic patients.     

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.     

An Oral Adsorbent,AST-120 Protects Against the Progression of Oxidative Stress by Reducing the Accumulation of Indoxyl Sulfate in the Systemic Circulation in Renal Failure

Purpose The effect of AST-120, an oral adsorbent, on oxidative stress in the systemic circulation in chronic renal failure (CRF) was examined and the potential role of indoxyl sulfate (IS), an uremic toxin adsorbed by AST-120, in inducing the formation of reactive oxygen species (ROS) in the vascular system was studied, in vitro and in vivo. Materials and methods The level of oxidized albumin, a marker for oxidative stress in the systemic circulation was determined by HPLC, as previously reported. The mRNA levels of TGF-β ₁ and Oat1 were measured by quantitative RT-PCR. The IS induced ROS generation in cultured human umbilical vein endothelial cells (HUVECs) was estimated using a fluorescence microplate reader. Results An increase in the ratio of oxidized to unoxidized albumin was determined using 5/6 nephrectomized rats (CRF rats) compared to a control group. The ratio was significantly reduced in the group that received AST-120 of 4 weeks, suggesting that AST-120 inhibits oxidative stress in CRF. An anti-oxidative effect of AST-120 was also observed in CRF rats with a similar renal function. The ratio of oxidized albumin was correlated with serum IS levels in vivo. The same relationship was also observed in CRF rats with the continued administration of IS. In addition, IS dramatically increased the generation of ROS in both a dose- and time- dependent manner in HUVEC, suggesting that accumulated IS may play an important role in enhancing intravascular oxidative stress. Conclusion We propose that AST-120 reduces IS concentrations in the blood that induces ROS production in endothelial cells, thereby inhibiting the subsequent occurrence of oxidative stress in the systemic circulation in renal failure.     

Albumin is the main plasma binding protein for indoxyl sulfate and p‐cresyl sulfate

Indoxyl sulfate and p‐cresyl sulfate are two uremic retention solutes implicated in the uremic syndrome. Removal during dialysis is limited, mainly due to protein binding. Binding characteristics to healthy albumin have recently been characterized. Whether uremia alters the binding characteristics of albumin is currently unknown. Moreover, protein binding values previously determined with ultrafiltration are in sharp contrast to recently reported values based on microcalorimetry. In the present study, indoxyl sulfate and p‐cresyl sulfate binding were therefore quantified using both equilibrium dialysis and ultrafiltration. Deming regression demonstrated good agreement between equilibrium dialysis and ultrafiltration. Free serum concentrations of indoxyl sulfate (+26.6%) and p‐cresyl sulfate (+19.7%) were slightly higher at body temperature compared with at room temperature. To investigate binding kinetics, the plasma of healthy individuals or hemodialysis patients was titrated with albumin solutions. Theoretical models of protein binding were fitted to observed titration curves. Binding coefficients of both toxins were highest in purified albumin, and were reduced from healthy to uremic plasma. In conclusion, the ultrafiltration–HPLC technique reliably measures free serum concentrations of indoxyl sulfate and p‐cresyl sulfate. Albumin is the main binding protein, both in health and in advanced stages of chronic kidney disease. Modeling suggests that albumin contains two binding sites for both toxins, a single high affinity binding site and a second low affinity binding site. The high affinity binding site accounts for at least 90% of overall binding. Competition for this binding site could be used to augment free solute concentrations during dialysis, thus improving epuration. Copyright © 2013 John Wiley & Sons, Ltd.     

Contribution of Gut Microbiota-Derived Uremic Toxins to the Cardiovascular System Mineralization

Chronic kidney disease (CKD) affects more than 10% of the world population and leads to excess morbidity and mortality (with cardiovascular disease as a leading cause of death). Vascular calcification (VC) is a phenomenon of disseminated deposition of mineral content within the media layer of arteries preceded by phenotypic changes in vascular smooth muscle cells (VSMC) and/or accumulation of mineral content within the atherosclerotic lesions. Medial VC results in vascular stiffness and significantly contributes to increased cardio-vascular (CV) morbidity, whereas VC of plaques may rather increase their stability. Mineral and bone disorders of CKD (CKD-MBD) contribute to VC, which is further aggravated by accumulation of uremic toxins. Both CKD-MBD and uremic toxin accumulation affect not only patients with advanced CKD (glomerular filtration rate (GFR) less than 15 mL/min/1.72 m², end-stage kidney disease) but also those on earlier stages of a disease. The key uremic toxins that contribute to VC, i.e., p-cresyl sulphate (PCS), indoxyl sulphate (IS) and trimethylamine-N-oxide (TMAO) originate from bacterial metabolism of gut microbiota. All mentioned toxins promote VC by several mechanisms, including: Transdifferentiation and apoptosis of VSMC, dysfunction of endothelial cells, oxidative stress, interaction with local renin–angiotensin–aldosterone system or miRNA profile modification. Several attractive methods of gut microbiota manipulations have been proposed in order to modify their metabolism and to limit vascular damage (and VC) triggered by uremic toxins. Unfortunately, to date no such method was demonstrated to be effective at the level of “hard” patient-oriented or even clinically relevant surrogate endpoints.     

Indoxyl Sulfate Contributes to mTORC1-Induced Renal Fibrosis via The OAT/NADPH Oxidase/ROS Pathway

Activation of mTORC1 (mechanistic target of rapamycin complex 1) in renal tissue has been reported in chronic kidney disease (CKD)-induced renal fibrosis. However, the molecular mechanisms responsible for activating mTORC1 in CKD pathology are not well understood. The purpose of this study was to identify the uremic toxin involved in mTORC1-induced renal fibrosis. Among the seven protein-bound uremic toxins, only indoxyl sulfate (IS) caused significant activation of mTORC1 in human kidney 2 cells (HK-2 cells). This IS-induced mTORC1 activation was inhibited in the presence of an organic anion transporter inhibitor, a NADPH oxidase inhibitor, and an antioxidant. IS also induced epithelial–mesenchymal transition of tubular epithelial cells (HK-2 cells), differentiation of fibroblasts into myofibroblasts (NRK-49F cells), and inflammatory response of macrophages (THP-1 cells), which are associated with renal fibrosis, and these effects were inhibited in the presence of rapamycin (mTORC1 inhibitor). In in vivo experiments, IS overload was found to activate mTORC1 in the mouse kidney. The administration of AST-120 or rapamycin targeted to IS or mTORC1 ameliorated renal fibrosis in Adenine-induced CKD mice. The findings reported herein indicate that IS activates mTORC1, which then contributes to renal fibrosis. Therapeutic interventions targeting IS and mTORC1 could be effective against renal fibrosis in CKD.     

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.     

Structural component changes of erythrocytes caused by oxidative stress generated by indoxyl sulfate

Indoxyl sulfate (IS) belongs to groups of uremic toxins binding to proteins. This compound may contribute to the generation of oxidative stress in chronic kidney disease (CKD) patients. We hypothesized that a high concentration of IS in the blood may induce structural changes of erythrocyte components and thus may contribute to CKD progression.In the present study, we evaluated the influence of IS on hemolysate and membrane proteins' conformational state, lipid membrane fluidity, and internal viscosity in erythrocytes. We examined thiols, carbonyl groups, peroxides, and TBARS levels in erythrocyte incubated with IS.The treatment of erythrocytes with IS led to increase in lipid membrane fluidity, decrease in the internal viscosity of the cells and the motion of the spin labels attached to hemolysate proteins. We did not observe conformational changes in plasma membrane proteins; however, in the plasma membranes of erythrocytes incubated with IS, a decrease in the content of thiol groups and increase in the carbonyls levels and peroxides and TBARS in comparison with the control was observed.The obtained results indicate that IS induces the oxidative damage of erythrocyte components. This may be an important factor that affects the functional properties of erythrocytes in CKD patients.     

Effects of uremic serum and uremic toxins on hepatic uptake of digoxin

Not only the renal clearance but also the hepatic clearance of drugs varies with the progression of renal failure. The aim of this study was to investigate the effects of human uremic serum and various uremic toxins on the hepatic uptake of digoxin (DX), a drug mainly excreted into bile in patients with severe renal failure, using isolated rat and human hepatocytes as model systems. Uremic serum inhibited the uptake of DX into rat hepatocytes in a concentration-dependent manner, whereas normal serum did not affect the uptake. In addition, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), hippuric acid, indole-3-acetic acid, indoxyl sulfate, and p-cresol (PC) concentration dependently inhibited the uptake. CMPF and PC at the concentration of 400 microM, which is within the plasma concentration range attained in patients with renal failure, inhibited the uptake of DX into rat hepatocytes by 27% and 23%, respectively. In human hepatocytes, 10% uremic serum, 400 microM CMPF, and 400 microM PC inhibited the uptake of DX by 23.3%, 23.4%, and 28.2%, respectively. In conclusion, our results suggest that hepatic uptake of DX is likely to be inhibited by uremic toxins, such as CMPF and PC, present in the serum of patients with renal failure.     

Plasma protein binding of clonazepam in hepatic and renal insufficiency and after hemodialysis

The plasma protein binding of clonazepam was investigated in healthy volunteers, cirrhotic patients, chronic uremic patients maintained on hemodialysis, and patients with reduced renal function. Each group consisted of six subjects. The unbound fraction of clonazepam (mean +/- SEM) was 13.9 +/- 0.2% in volunteers. 17.1 +/- 1.0% in cirrhotic patients, 1.56 +/- 0.5% before and 12.2 +/- 0.4% after hemodialysis in chronic uremic patients, and 16.0 +/- 0.7% in patients with poor renal function. The figure for the healthy subjects was significantly different from that of cirrhotic patients only. Binding of clonazepam to albumin and alpha 1-acid glycoprotein was also studied. Clonazepam bound preferentially to albumin.     

The Role of Gut-Derived,Protein-Bound Uremic Toxins in the Cardiovascular Complications of Acute Kidney Injury

Acute kidney injury (AKI) is a frequent disease encountered in the hospital, with a higher incidence in intensive care units. Despite progress in renal replacement therapy, AKI is still associated with early and late complications, especially cardiovascular events and mortality. The role of gut-derived protein-bound uremic toxins (PBUTs) in vascular and cardiac dysfunction has been extensively studied during chronic kidney disease (CKD), in particular, that of indoxyl sulfate (IS), para-cresyl sulfate (PCS), and indole-3-acetic acid (IAA), resulting in both experimental and clinical evidence. PBUTs, which accumulate when the excretory function of the kidneys is impaired, have a deleterious effect on and cause damage to cardiovascular tissues. However, the link between PBUTs and the cardiovascular complications of AKI and the pathophysiological mechanisms potentially involved are unclear. This review aims to summarize available data concerning the participation of PBUTs in the early and late cardiovascular complications of AKI.