Anneleen Pletinck

The Uremic Toxicity of Indoxyl Sulfate and p-Cresyl Sulfate: A Systematic Review

A growing number of publications supports a biologic effect of the protein-bound uremic retention solutes indoxyl sulfate and p-cresyl sulfate. However, the use of unrealistically high free concentrations of these compounds and/or inappropriately low albumin concentrations may blur the interpretation of these results. Here, we performed a systematic review, selecting only studies in which, depending on the albumin concentration, real or extrapolated free concentrations of indoxyl sulfate and p-cresyl sulfate remained in the uremic range. The 27 studies retrieved comprised in vitro and animal studies. A quality score was developed, giving 1 point for each of the following criteria: six or more experiments, confirmation by more than one experimental approach, neutralization of the biologic effect by counteractive reagents or antibodies, use of a real-life model, and use of dose-response analyses in vitro and/or animal studies. The overall average score was 3 of 5 points, with five studies scoring 5 of 5 points and six studies scoring 4 of 5 points, highlighting the superior quality of a substantial number of the retrieved studies. In the 11 highest scoring studies, most functional deteriorations were related to uremic cardiovascular disease and kidney damage. We conclude that our systematic approach allowed the retrieval of methodologically correct studies unbiased by erroneous conditions related to albumin binding. Our data seem to confirm the toxicity of indoxyl sulfate and p-cresyl sulfate and support their roles in vascular and renal disease progression.

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 Toxins Stimulate Crosstalk between Leukocytes and Vessel Wall

Leukocyte activation and endothelial damage both contribute to cardiovascular disease, a major cause of morbidity and mortality in CKD. Experimental in vitro data link several protein-bound uremic retention solutes to the modulation of inflammatory stimuli, including endothelium and leukocyte responses and cardiovascular damage, corroborating observational in vivo data. However, the impact of these uremic toxins on the crosstalk between endothelium and leukocytes has not been assessed. This study evaluated the effects of acute and continuous exposure to uremic levels of indoxylsulfate (IS), p-cresylsulfate (pCS), and p-cresylglucuronide (pCG) on the recruitment of circulating leukocytes in the rat peritoneal vascular bed using intravital microscopy. Superfusion with IS induced strong leukocyte adhesion, enhanced extravasation, and interrupted blood flow, whereas pCS caused a rapid increase in leukocyte rolling. Superfusion with pCS and pCG combined caused impaired blood flow and vascular leakage but did not further enhance leukocyte rolling over pCS alone. Intravenous infusion with IS confirmed the superfusion results and caused shedding of heparan sulfate, pointing to disruption of the glycocalyx as the mechanism likely mediating IS-induced flow stagnation. These results provide the first clear in vivo evidence that IS, pCS, and pCG exert proinflammatory effects that contribute to vascular damage by stimulating crosstalk between leukocytes and vessels.

Protecting the peritoneal membrane: factors beyond peritoneal dialysis solutions.

Functional deterioration of the peritoneal membrane in patients on peritoneal dialysis has been described as being the result of a combination of neoangiogenesis and fibrosis. Glucose, glucose degradation products, and the unphysiological pH of the dialysate solution contribute to these changes. Although newer solutions clearly perform better in terms of their biocompatibility in an in vitro setting and in animal models, the benefit of such solutions over older solutions in the clinical setting is so far unproven. The difficulties in showing a benefit of the newer, more biocompatible solutions in the clinical setting can be explained by the fact that other factors also affect the properties of the peritoneal membrane. These factors are often neglected in clinical studies, which results in unnoticed differences in case-mix and blurs the potential impact of the novel solutions. However, many of these factors are modifiable, and attention should be paid to them in clinical practice to maintain the integrity of the peritoneal membrane. This Review focuses on factors that potentially influence the integrity of the peritoneal membrane, other than those associated with the peritoneal dialysis fluid itself.

Review of Protein-Bound Toxins, Possibility for Blood Purification Therapy

Protein-bound uremic retention solutes, i.e. phenolic compounds, such as p-cresylsulfate, and indolic compounds, such as indoxyl sulfate, have been intensively studied in recent years and have been shown to be associated especially with cardiovascular toxicity and adverse outcomes in chronic kidney disease. In this review, we will focus on their toxicity and their removal by dialysis strategies, which is hampered due to their protein binding. Hemodiafiltration slightly improves the removal of protein-bound solutes as compared to hemodialysis, although the clinical benefit on outcomes still needs to be demonstrated. Removal by means of absorption and interference with intestinal generation or renal tubular excretion are interesting alternative strategies under investigation.

Impact of the Uremic Milieu on the Osteogenic Potential of Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs), the precursors of osteoblasts during osteogenesis, play a role in the balance of bone formation and resorption, but their functioning in uremia has not been well defined. To study the effects of the uremic milieu on osteogenic properties, we applied an in vitro assay culturing hMSCs in osteogenic medium supplemented with serum from healthy donors and from uremic patients on hemodialysis. Compared to control, serum from uremic patients induces, in hMSC cultures, a modification of several key regulators of bone remodeling, in particular a reduction of the ratio Receptor Activator of Nuclear factor Kappa B Receptor (RANKL) over osteoprotegerin, indicating an adaptive response of the system to favor osteogenesis over osteoclastosis. However, the levels of osteopontin, osteocalcin, and collagen type I, are increased in cell medium, while BMP-2, and alizarin red staining were decreased, pointing to a reduction of bone formation favoring resorption. Selected uremic toxins, such as p-cresylsulfate, p-cresylglucuronide, parathyroid hormone, indoxyl sulfate, asymmetric dimethylarginine, homocysteine, were able to mimic some of the effects of whole serum from uremic patients. Serum from cinacalcet-treated patients antagonizes these effects. Hydrogen sulfide (H2S) donors as well as hemodialysis treatment are able to induce beneficial effects. In conclusion, bone modifications in uremia are influenced by the capability of the uremic milieu to alter hMSC osteogenic differentiation. Cinacalcet, H2S donors and a hemodialysis session can ameliorate the hampered calcium deposition.

Biochemical and Clinical Impact of Organic Uremic Retention Solutes: A Comprehensive Update

In this narrative review, the biological/biochemical impact (toxicity) of a large array of known individual uremic retention solutes and groups of solutes is summarized. We classified these compounds along their physico-chemical characteristics as small water-soluble compounds or groups, protein bound compounds and middle molecules. All but one solute (glomerulopressin) affected at least one mechanism with the potential to contribute to the uremic syndrome. In general, several mechanisms were influenced for each individual solute or group of solutes, with some impacting up to 7 different biological systems of the 11 considered. The inflammatory, cardio-vascular and fibrogenic systems were those most frequently affected and they are one by one major actors in the high morbidity and mortality of CKD but also the mechanisms that have most frequently been studied. A scoring system was built with the intention to classify the reviewed compounds according to the experimental evidence of their toxicity (number of systems affected) and overall experimental and clinical evidence. Among the highest globally scoring solutes were 3 small water-soluble compounds [asymmetric dimethylarginine (ADMA); trimethylamine-N-oxide (TMAO); uric acid], 6 protein bound compounds or groups of protein bound compounds [advanced glycation end products (AGEs); p-cresyl sulfate; indoxyl sulfate; indole acetic acid; the kynurenines; phenyl acetic acid;] and 3 middle molecules [β2-microglobulin; ghrelin; parathyroid hormone). In general, more experimental data were provided for the protein bound molecules but for almost half of them clinical evidence was missing in spite of robust experimental data. The picture emanating is one of a complex disorder, where multiple factors contribute to a multisystem complication profile, so that it seems of not much use to pursue a decrease of concentration of a single compound.

Acute central haemodynamic effects induced by intraperitoneal glucose instillation

BACKGROUND The supposed lack of a haemodynamic impact of peritoneal dialysis (PD) has been challenged recently by the finding of a mild increase of peripheral blood pressure (BP) during an acute dwell. It is not clear whether, besides the effect of changes in intraperitoneal (IP) volume and/or pressure, IP glucose instillation and absorption plays a role in this. Therefore, we tested the impact of IP instillation of glucose on the evolution of central haemodynamic parameters, using SphygmoCor, during an acute dwell with two different glucose concentrations. METHODS Stable, non-diabetic PD patients (N = 22) were treated consecutively in a randomized, cross-over design (A then B or B then A) with one 1.36% (A) and one 3.86% (B) physioneal dwell of 100 min. Central BP was measured with SphygmoCor and blood was sampled for serum glucose and insulin levels every 20 min. Insulin resistance was defined as a Homeostatic Model Assessment Index (HOMA-index) >1.4. RESULTS Serum glucose levels rose during both the 1.36% and the 3.86% dwell, whereas insulin levels rose only during the 3.86% dwell. The increase of both glucose and insulin levels was more pronounced in patients with insulin resistance (11/22 patients). There was, however, no accompanying change versus baseline in haemodynamic parameters (carotid systolic blood pressure, diastolic BP, heart rate or augmentation index). CONCLUSION Despite substantial increases in blood glucose and insulin levels, there was no accompanying change in central haemodynamic parameters during an acute PD dwell with low or high glucose concentrations.

Quantification of carbamylated albumin in serum based on capillary electrophoresis

Protein carbamylation, a nonenzymatic posttranslational modification promoted during uremia, is linked to a poor prognosis. In the present study, carbamylation of serum albumin was assayed using the symmetry factor on a capillary electrophoresis instrument (Helena V8). The symmetry factor has been defined as the distance from the center line of the peak to the back slope, divided by the distance from the center line of the peak to the front slope, with all measurements made at 10% of the maximum peak height. Serum albumin, creatinine, and urea concentrations were assayed using routine methods, whereas uremic toxins were determined using HPLC. In vitro carbamylation induced a marked albumin peak asymmetry. Reference values for the albumin symmetry factor were 0.69–0.92. In kidney patients, albumin peak asymmetry corresponded to the chronic kidney disease stage (p < 0.0001). The symmetry factor correlated well with serum urea (r = −0.5595, p < 0.0001) and creatinine (r = −0.5986, p < 0.0001) concentrations. Several protein‐bound uremic toxins showed a significant negative correlation with the symmetry factor. Morphology of the albumin fraction was not affected by presence of glycated albumin and protein‐bound antibiotics. In conclusion, the presented method provides a simple, practical way for monitoring protein carbamylation.

Binding of bromocresol green and bromocresol purple to albumin in hemodialysis patients

Abstract Background: Colorimetric albumin assays based on binding to bromocresol purple (BCP) and bromocresol green (BCG) yield different results in chronic kidney disease. Altered dye binding of carbamylated albumin has been suggested as a cause. In the present study, a detailed analysis was carried out in which uremic toxins, acute phase proteins and Kt/V, a parameter describing hemodialysis efficiency, were compared with colorimetrically assayed (BCP and BCG) serum albumin. Methods: Albumin was assayed using immunonephelometry on a BN II nephelometer and colorimetrically based on, respectively, BCP and BCG on a Modular P analyzer. Uremic toxins were assessed using high-performance liquid chromatography. Acute phase proteins (C-reactive protein and α1-acid glycoprotein) and plasma protein α2-macroglobulin were assayed nephelometrically. In parallel, Kt/V was calculated. Results: Sixty-two serum specimens originating from hemodialysis patients were analyzed. Among the uremic toxins investigated, total para-cresyl sulfate (PCS) showed a significant positive correlation with the BCP/BCG ratio. The serum α1-acid glycoprotein concentration correlated negatively with the BCP/BCG ratio. The BCP/BCG ratio showed also a negative correlation with Kt/V. Conclusions: In renal insufficiency, the BCP/BCG ratio of serum albumin is affected by multiple factors: next to carbamylation, uremic toxins (total PCS) and α1-acid glycoprotein also play a role.