Temperature Responsive Polymer Conjugate Prepared by "Grafting from" Proteins toward the Adsorption and Removal of Uremic Toxin.

In this study, temperature-responsive polymer-protein conjugate was synthesized using a "grafting from" concept by introducing a chain transfer agent (CTA) into bovine serum albumin (BSA). The BSA-CTA was used as a starting point for poly(N-isopropylacrylamide) (PNIPAAm) through reversible addition-fragmentation chain transfer polymerization. The research investigations suggest that the thermally responsive behavior of PNIPAAm was controlled by the monomer ratio to CTA, as well as the amount of CTA introduced to BSA. The study further synthesized the human serum albumin (HSA)-PNIPAAm conjugate, taking the advantage that HSA can specifically adsorb indoxyl sulfate (IS) as a uremic toxin. The HSA-PNIPAAm conjugate could capture IS and decreased the concentration by about 40% by thermal precipitation. It was also revealed that the protein activity was not impaired by the conjugation with PNIPAAm. The proposed strategy is promising in not only removal of uremic toxins but also enrichment of biomarkers for early diagnostic applications.

Early elimination of uremic toxin ameliorates AKI-to-CKD transition.

Acute kidney injury (AKI)-related fibrosis is emerging as a major driver of chronic kidney disease (CKD) development. Aberrant kidney recovery after AKI is multifactorial and still poorly understood. The accumulation of indoxyl sulfate (IS), a protein-bound uremic toxin, has been identified as a detrimental factor of renal fibrosis. However, the mechanisms underlying IS-related aberrant kidney recovery after AKI is still unknown. The present study aims to elucidate the effects of IS on tubular damage and its involvement in the pathogenesis of AKI-to-CKD transition. Our results showed that serum IS started to accumulate associated with the downregulation of tubular organic anion transporter but not observed in the small-molecule uremic toxins of the unilateral ischemia-reperfusion injury (UIRI) without a contralateral nephrectomy model. Serum IS is positively correlated with renal fibrosis and binding immunoglobulin protein (BiP) and CAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) expression induction in the UIRI with a contralateral nephrectomy model (UIRI+Nx). To evaluate the effects of IS in the AKI-to-CKD transition, we administered indole, a precursor of IS, at the early stage of UIRI. Our results demonstrated IS potentiates renal fibrosis, senescence-associated secretory phenotype (SASP), and activation of endoplasmic reticulum (ER) stress, which is attenuated by synergistic AST-120 administration. Furthermore, we clearly demonstrated that IS exposure potentiated hypoxia-reperfusion (H/R) induced G2/M cell cycle arrest, epithelial-mesenchymal transition (EMT) and aggravated ER stress induction in vitro. Finally, the ER chemical chaperon, 4-phenylbutyric acid (4-PBA), successfully reversed the above-mentioned AKI-to-CKD transition. Taken together, early IS elimination in the early stage of AKI is likely to be a useful strategy in the prevention and/or treatment of the AKI-to-CKD transition.

Improved dialysis removal of protein-bound uremic toxins by salvianolic acids.

BACKGROUND: Indoxyl sulfate (IS) and p-cresyl sulfate (pCS) are two key protein-bound uremic toxins that accumulate in patients with end-stage renal disease. IS and pCS cannot be efficiently removed by conventional hemodialysis because they are highly bound to proteins. One promising means to optimize the removal of protein-bound uremic toxins involves using binding competitors to liberate uremic toxins from protein-binding partners. PURPOSE: In this study, we try to identify potential binding competitors that can enhance the dialysis removal of IS and pCS in natural compounds of phytomedicine. METHODS: We employed microdialysis to evaluate whether Danhong injection (DHI) and its salvianolic acids can increase the free fractions of IS and pCS and thus improve their dialysis efficiency in vitro. Furthermore, we confirmed the positive effects of DHI and salvianolic acids in vivo on chronic kidney disease model rats in which IS and pCS had heavily accumulated. RESULTS: DHI significantly increased the dialysis efficiency of IS and pCS by 99.13% and 142.00% in vitro (10-fold dilution), respectively, and by 135.61% and 272.13% in vivo (4.16 ml/kg). Salvianolic acids including lithospermic acid (LA), salvianolic acid A (SaA), tanshinol (DSS), caffeic acid (CA), salvianolic acid B (SaB), protocatechuic aldehyde (PA) and rosmarinic acid (RA) significantly enhanced the dialysis removal of IS and pCS in a concentration-dependent manner. LA, the best competitor of the tested salvianolic acids, increased dialysis efficiency levels of IS and pCS by 197.23% and 198.31% in vitro (400 µM), respectively, and by 119.55% and 127.56% in vivo (24.69 mg/kg). CONCLUSION: The removal of protein-bound uremic toxins IS and pCS using DHI or salvianolic acids as protein-bound competitors is superior to previously reported strategies and drugs and may contribute to clinical hemodialysis therapeutic practice.

Increasing the removal of protein-bound uremic toxins by liposome-supported hemodialysis.

Protein-bound uremic toxins (PBUTs) accumulate at high plasma levels and cause various deleterious effects in end-stage renal disease patients because their removal by conventional hemodialysis is severely limited by their low free-fraction levels in plasma. Here, we assessed the extent to which solute removal can be increased by adding liposomes to the dialysate. The uptake of liposomes by direct incubation in vitro showed an obvious dose-response relationship for p-cresyl sulfate (PCS) and indoxyl sulfate (IS) but not for hippuric acid (HA). The percent removal of both PCS and IS but not of HA was gradually increased with the increased concentration of liposomes in a rapid equilibrium dialysis setup. In vitro closed circulation showed that adding liposomes to the dialysate markedly increased the dialysances of PBUTs without greatly altering that of urea and creatinine. In vivo experiments in uremic rats demonstrated that adding liposomes to the dialysate resulted in higher reduction ratios (RRs) and more total solute removal (TSR) for several PBUTs compared to the conventional dialysate, which was approximately similar to the addition of bovine serum albumin to the dialysate. These findings highlight that as an adjunct to conventional hemodialysis, addition of liposomes to the dialysate could significantly improve the removal of protein-bound uremic solutes without greatly altering the removal of small, water-soluble solutes.

Removal of indoxyl sulfate by water-soluble poly-cyclodextrins in dialysis.

Indoxyl sulfate (IS) is a uremic toxin related to the progression of chronic kidney diseases. Removal of IS from the plasma would reduce the risk of cardiovascular disease. In this study, crosslinked poly-ß-cyclodextrins (PCDs) were used as a water-soluble adsorbent agent for IS in dialysis for the first time. The molecular weight of PCDs was found to be proportional to the crosslinking time between ß-cyclodextrin monomers and epichlorohydrin, yet the proportion of ß-cyclodextrin that reacted with epichlorohydrin decreased. It was observed that PCD after 2 h crosslinking yielded the best IS-binding capability in PBS, while reaching the binding equilibrium within 30 min and yielding a maximum binding capability of 45 mg g-1. Furthermore, the binding mechanism was investigated by two-dimensional nuclear magnetic resonance, Job's plot method, and salt treatments. To simulate the clinical removal of IS we established a macro-dialysis and added PCD obtained from 2 h crosslinking (PCD1) to the dialysate. The removal of plasma IS from the dialysate by PCD1 was about twice as much as that removed from the dialysate without PCD1. Therefore, crosslinked poly-ß-cyclodextrins may represent a simple, low-cost, and effective IS removal strategy with great potential for removing other hydrophobic plasma-bound toxins in dialysis. It could also serve as a supplement for the existing non-adsorbent added therapy.

Carbon Adsorbents With Dual Porosity for Efficient Removal of Uremic Toxins and Cytokines from Human Plasma.

The number of patients with chronic kidney disease increases while the number of available donor organs stays at approximately the same level. Unavoidable accumulation of the uremic toxins and cytokines for these patients comes as the result of malfunctioning kidneys and their high levels in the blood result in high morbidity and mortality. Unfortunately, the existing methods, like hemodialysis and hemofiltration, provide only partial removal of uremic toxins and/or cytokines from patients' blood. Consequently, there is an increasing need for the development of the extracorporeal treatments which will enable removal of broad spectrum of uremic toxins that are usually removed by healthy kidneys. Therefore, in this work we developed and tested ordered mesoporous carbons as new sorbents with dual porosity (micro/meso) that provide selective and efficient removal of a broad range of uremic toxins from human plasma. The new sorbents, CMK-3 are developed by nanocasting methods and have two distinct pore domains, i.e. micropores and mesopores, therefore show high adsorption capacity towards small water soluble toxins (creatinine), protein-bound molecules (indoxyl sulfate and hippuric acid), middle molecules (ß-2-microglobulin) and cytokines of different size (IL-6 and IL-8). Our results show that small amounts of CMK-3 could provide selective and complete blood purification.

Release of uremic retention solutes from protein binding by hypertonic predilution hemodiafiltration.

Protein-bound uremic retention solutes accumulate in patients suffering from chronic kidney disease, and the removal of these solutes by hemodialysis is hampered. Therefore, we developed a dialysis technique where the protein-bound uremic retention solutes are removed more efficiently under high ionic strength. Protein-bound uremic solutes such as phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate were combined with plasma in the presence of increased ionic strength. The protein integrity of proteins and enzymatic activities were analyzed. In vitro dialysis of albumin solution was performed to investigate the clearance of the bound uremic retention solutes. In vitro hemodiafiltrations of human blood were performed to investigate the influence of increased ionic strength on blood cell survival. The protein-bound fraction of phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate was significantly decreased from 59.4% ± 3.4%, 95.7% ± 0.6%, 96.9% ± 1.5% to 36.4% ± 3.7%, 87.8% ± 0.6%, and 90.8% ± 1.3%, respectively. The percentage of phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate released from protein was 23.0% ± 5.7%, 7.9% ± 1.1%, and 6.1% ± 0.2%, respectively. The clearance during in vitro dialysis was increased by 13.1% ± 3.6%, 68.8% ± 15.1%, and 53.6% ± 10.2%, respectively. There was no difference in NaCl concentrations at the outlet of the dialyzer using isotonic and hypertonic solutions. In conclusion, this study forms the basis for establishing a novel therapeutic approach to remove protein-bound retention solutes.

Effects of AST-120 on blood concentrations of protein-bound uremic toxins and biomarkers of cardiovascular risk in chronic dialysis patients.

BACKGROUND: Removal of protein-bound uremic toxins by dialysis therapy is limited. The effect of oral adsorbent AST-120 in chronic dialysis patients has rarely been investigated. METHODS: AST-120 was administered 6.0 g/day for 3 months in 69 chronic dialysis patients. The blood concentrations of indoxyl sulfate, p-cresol sulfate and biomarkers of cardiovascular risk were determined before and after AST-120 treatment. RESULTS: AST-120 significantly decreased both the total and free forms of indoxyl sulfate and p-cresol sulfate ranging from 21.9 to 58.3%. There were significant simultaneous changes of the soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK, 24% increase), malondialdehyde (14% decrease) and interleukin-6 (19% decrease). A significant association between the decrease of indoxyl sulfate and changes of sTWEAK and interleukin-6 was noted. CONCLUSIONS: AST-120 effectively decreased indoxyl sulfate and p-cresol sulfate levels in both total and free forms. AST-120 also improved the profile of cardiovascular biomarkers.

An adsorbent monolith device to augment the removal of uraemic toxins during haemodialysis.

Adsorbents designed with porosity which allows the removal of protein bound and high molecular weight uraemic toxins may improve the effectiveness of haemodialysis treatment of chronic kidney disease (CKD). A nanoporous activated carbon monolith prototype designed for direct blood contact was first assessed for its capacity to remove albumin bound marker toxins indoxyl sulphate (IS), p-cresyl sulphate (p-CS) and high molecular weight cytokine interleukin-6 in spiked healthy donor studies. Haemodialysis patient blood samples were then used to measure the presence of these markers in pre- and post-dialysis blood and their removal by adsorbent recirculation of post-dialysis blood samples. Nanopores (20-100 nm) were necessary for marker uraemic toxin removal during in vitro studies. Limited removal of IS and p-CS occurred during haemodialysis, whereas almost complete removal occurred following perfusion through the carbon monoliths suggesting a key role for such adsorbent therapies in CKD patient care.

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.

Mixed matrix hollow fiber membranes for removal of protein-bound toxins from human plasma.

In end stage renal disease (ESRD) waste solutes accumulate in body fluid. Removal of protein bound solutes using conventional renal replacement therapies is currently very poor while their accumulation is associated with adverse outcomes in ESRD. Here we investigate the application of a hollow fiber mixed matrix membrane (MMM) for removal of these toxins. The MMM hollow fiber consists of porous macro-void free polymeric inner membrane layer well attached to the activated carbon containing outer MMM layer. The new membranes have permeation properties in the ultrafiltration range. Under static conditions, they adsorb 57% p-cresylsulfate, 82% indoxyl sulfate and 94% of hippuric acid from spiked human plasma in 4 h. Under dynamic conditions, they adsorb on average 2.27 mg PCS/g membrane and 3.58 mg IS/g membrane in 4 h in diffusion experiments and 2.68 mg/g membrane PCS and 12.85 mg/g membrane IS in convection experiments. Based on the dynamic experiments we estimate that our membranes would suffice to remove the daily production of these protein bound solutes.

Removal of protein-bound, hydrophobic uremic toxins by a combined fractionated plasma separation and adsorption technique.

Protein-bound uremic toxins, such as phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate, contribute substantially to the progression of chronic kidney disease (CKD) and cardiovascular disease (CVD). However, based on their protein binding, these hydrophobic uremic toxins are poorly cleared during conventional dialysis and thus accumulate in CKD-5D patients. Therefore, we investigated whether hydrophobic and cationic adsorbers are more effective for removal of protein-bound, hydrophobic uremic toxins than conventional high-flux hemodialyzer. Five CKD-5D patients were treated using the fractionated plasma separation, adsorption, and dialysis (FPAD) system for 5 h. A control group of five CKD patients was treated with conventional high-flux hemodialysis. Plasma concentrations of phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate were measured. Removal rates of FPAD treatment in comparison to conventional high-flux hemodialysis were increased by 130% for phenylacetic acid, 187% for indoxyl sulfate, and 127% for p-cresol. FPAD treatment was tolerated well in terms of clinically relevant biochemical parameters. However, patients suffered from mild nausea 2 h after the start of the treatment, which persisted until the end of treatment. Due to the high impact of protein-bound, hydrophobic uremic toxins on progression of CKD and CVD in CKD-5D patients, the use of an adsorber in combination with dialysis membranes may be a new therapeutic option to increase the removal rate of these uremic toxins. However, larger, long-term prospective clinical trials are needed to demonstrate the impact on clinical outcome.

Electrophoretic analysis of biomarkers using capillary modification with gold nanoparticles embedded in a polycation and boron doped diamond electrode.

Field-amplified sample stacking using a fused silica capillary coated with gold nanoparticles (AuNPs) embedded in poly(diallyl dimethylammonium) chloride (PDDA) has been investigated for the electrophoretic separation of indoxyl sulfate, homovanillic acid (HVA), and vanillylmandelic acid (VMA). AuNPs (27 nm) exhibit ionic and hydrophobic interactions, as well as hydrogen bonding with the PDDA network to form a stable layer on the internal wall of the capillary. This approach reverses electro-osmotic flow allowing for fast migration of the analytes while retarding other endogenous compounds including ascorbic acid, uric acid, catecholamines, and indoleamines. Notably, the two closely related biomarkers of clinical significance, HVA and VMA, displayed differential interaction with PDDA-AuNPs which enabled the separation of this pair. The detection limit of the three analytes obtained by using a boron doped diamond electrode was approximately 75 nM, which was significantly below their normal physiological levels in biological fluids. This combined separation and detection scheme was applied to the direct analysis of these analytes and other interfering chemicals including uric and ascorbic acids in urine samples without off-line sample treatment or preconcentration.

Plasma protein binding of frusemide in renal failure rabbits: investigation of endogenous protein binding inhibitors.

The reduction mechanism of frusemide-protein binding in the plasma of renal failure was investigated. The drug-albumin binding was inhibited by the low molecular-weight fraction obtained from acute renal failure rabbits, suggesting the presence of the inhibitors in the plasma. Further, this fraction was divided into six subfractions by Bio-Gel P-2. Fractions II and V2 showed significant inhibition of the protein binding of frusemide. Among uraemic toxins, four indole derivatives markedly inhibited the protein binding. Analysis by hplc confirmed that the concentration of indican was markedly increased in acute renal failure rabbit plasma. It is suggested that this compound could be one of the major inducers of the protein binding defect.