Eva Schepers

Free p-cresylsulphate is a predictor of mortality in patients at different stages of chronic kidney disease

BACKGROUND Uraemic toxins are considered to be emerging mortality risk factors in chronic kidney disease (CKD) patients. p-Cresol (a prototype protein-bound uraemic retention solute) has been shown to exert toxic effects in vitro. Recently, it has been demonstrated that p-cresol is present in plasma as its sulphate conjugate, p-cresylsulphate. The present study evaluated the distribution of free and total p-cresylsulphate and sought to determine whether these parameters were associated with vascular calcification, arterial stiffness and mortality risk in a cohort of CKD patients. METHODS One hundred and thirty-nine patients (mean +/- SD age: 67 +/- 12; males: 60%) at different stages of CKD (8% at Stage 2, 26.5% at Stage 3, 26.5% at Stage 4, 7% at Stage 5 and 32% at Stage 5D) were enrolled in this study. RESULTS Baseline total and free p-cresylsulphate presented an inverse relationship with renal function and were significantly associated with vascular calcification. During the study period (mean follow-up period: 779 +/- 185 days), 38 patients died [including 22 from cardiovascular (CV) causes]. In crude survival analyses, free (but not total) p-cresylsulphate was shown to be a predictor of overall and CV death. Higher free p-cresylsulphate levels (>0.051 mg/100 mL; median) were associated with mortality independently of well-known predictors of survival such as age, vascular calcification, anaemia and inflammation. CONCLUSIONS Serum levels of free and total p-cresylsulphate (the main in vivo circulating metabolites of p-cresol) were elevated in later CKD stages. However, only free p-cresylsulphate seems to be a predictor of survival in CKD.

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.

Role of symmetric dimethylarginine in vascular damage by increasing ROS via store-operated calcium influx in monocytes.

BACKGROUND The guanidines asymmetric dimethylarginine (ADMA), a marker of endothelial dysfunction, and its counterpart symmetric dimethylarginine (SDMA), considered inert, are accumulated in chronic kidney disease (CKD). The present study evaluates their effect on monocyte function, since previous data demonstrated leukocyte activation by other guanidino compounds. METHODS The effect of ADMA and SDMA on reactive oxygen species (ROS) production in human whole blood at baseline and after N-formyl-methionine-leucine-phenylalanine (fMLP) stimulation was evaluated. By using the fluorescent probe Fluo3-AM, the role of changes in monocytic cytoplasmic calcium ([Ca2+]i) was studied. Thapsigargin, and removal followed by addition of extracellular Ca2+ (Ca2+(ex)), was used to investigate the contribution of store-operated Ca2+-channels (SOCs). SKF96365 was used as a selective inhibitor of the SOCs. A pharmacologic intervention with captopril, known to affect Ca2+ influx, was tested. RESULTS SDMA enhanced ROS production in fMLP-stimulated monocytes using heparinized blood, and this effect was abolished in EDTA-anticoagulated blood. In the presence of SDMA, an increased Ca2+ entry from the extracellular milieu resulted in an elevated amplitude of the peak [Ca2+]i change triggered by fMLP. None of these effects were seen with ADMA. Depletion of the intracellular stores with thapsigargin in the absence of Ca2+(ex), followed by re-addition of Ca2+(ex) triggered a significantly larger Ca2+ entry after SDMA treatment versus saline. This effect was prevented with SKF96365, as was the SDMA-enhanced oxidative burst after fMLP. Pre-incubation with captopril also reduced the increased ROS production seen with SDMA. CONCLUSIONS SDMA, a uraemic retention solute considered inert, stimulates ROS production of monocytes by acting on Ca2+ entry via SOCs. This pro-inflammatory effect may trigger vascular pathology and may be involved in altering the prevalence of cardiovascular disease in CKD.

The gut: the forgotten organ in uremia?

Part of the uremic retention solutes are generated in the intestine, but this option is rarely discussed in the literature. In this publication, we describe consecutively the role of the intestine in generating uremic retention solutes, the pathophysiological importance of the generated solutes and therapeutic options that are inspired by this knowledge. Apart from its role as a route via which uremic toxins or their precursors enter the body, the intestine also acts as an active player by presenting more precursors for fermentation due to disturbances in assimilation caused by uremia, followed by alterations in further processing related to changes in the composition of the fermenting flora. Many of the toxins generated or introduced into the body via the intestine (advanced glycation end products, indoles, phenols) play an active role in vascular damage. Intestinal therapeutic interventions that could help decrease solute concentration are restriction of dietary intake, however at the expense of increasing the risk of malnutrition, rerouting of intestinal metabolism by administration of prebiotics or probiotics and/ or the administration of active sorbents such as AST-120 (Kremezin®).

Symmetric Dimethylarginine as a Proinflammatory Agent in Chronic Kidney Disease

BACKGROUND & OBJECTIVES Chronic kidney disease (CKD) is characterized by chronic inflammation, considered a nontraditional risk factor for cardiovascular disease, the major cause of death in CKD. Symmetric dimethylarginine (SDMA) was recently demonstrated to induce reactive oxygen species in monocytes. The present study further investigates the inflammatory character of SDMA compared with its structural counterpart asymmetric dimethylarginine (ADMA). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In vitro, the effect of SDMA on intracellular monocytic expression of IL-6 and TNF-α was studied followed by an evaluation of nuclear factor (NF)-κB activation. Additionally, an association of SDMA with inflammatory parameters in consecutive stages of CKD was evaluated in vivo. RESULTS Monocytes incubated with SDMA showed increased IL-6 and TNF-α expression and a rise in active NF-κB. N-acetylcysteine abrogated both these effects. No significant effects were observed with ADMA. In vivo, 142 patients (67 ± 12 years) at different stages of CKD showed an inverse association between serum SDMA and ADMA and renal function. Correlations between SDMA and IL-6, TNF-α, and albumin were more significant than for ADMA, while multiple regression analysis only retained TNF-α at a high significance for SDMA (P < 0.0001). In receiver operating characteristic analysis for inflammation, defined as an IL-6 level above 2.97 pg/ml (median), the discriminative power of SDMA (area under the curve [AUC]: 0.69 ± 0.05) directly followed that of C-reactive protein (AUC: 0.82 ± 0.04) and albumin (AUC: 0.72 ± 0.05; for all, P < 0.0001) and preceded that of ADMA (P = 0.002). CONCLUSIONS The present study shows that SDMA is involved in the inflammatory process of CKD, activating NF-κB and resulting in enhanced expression of IL-6 and TNF-α, which is corroborated by the clinical data pointing to an in vivo association of SDMA with inflammatory markers in CKD at different stages.

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.

Novel method for simultaneous determination of p-cresylsulphate and p-cresylglucuronide: clinical data and pathophysiological implications

BACKGROUND The uraemic retention solutes p-cresylsulphate (pCS) and p-cresylglucuronide (pCG), two conjugates of p-cresol, were never determined simultaneously. In the present paper, a high-performance liquid chromatography (HPLC) method was developed and used to quantify both compounds in parallel in an in vivo observational study and their in vitro effect was evaluated by flow cytometry. METHODS pCS and pCG were determined in serum. For the validation specificity, linearity, recovery, precision and the quantification limit were evaluated. In vivo, concentrations of both compounds were determined in 15 controls and 77 haemodialysis patients, as well as protein binding in the dialysed group and the reduction ratios during haemodiafiltration. In addition, the in vitro effect of the solutes on leucocyte free radical production at measured concentrations was assessed. RESULTS A fast and accurate HPLC method was developed to simultaneously quantify pCS and pCG. Both conjugates are retained in uraemia with a substantially higher total serum pCS in comparison to pCG (31.4 ± 15.8 versus 7.3 ± 6.5 mg/L) but also a substantial difference in protein binding (92.4 ± 3.0 versus 8.3 ± 4.4%) and in reduction ratio during post-dilution haemodiafiltration (37.4 ± 7.1 versus 78.6 ± 6.4%). pCG per se has no effect on leucocyte oxidative burst activity, whereas in combination with pCS, a synergistic activating effect was observed. CONCLUSIONS Serum concentrations of pCS and pCG are elevated in uraemia. Both conjugates show a different protein binding, resulting in a different dialytic behaviour. Biologically, both conjugates are synergistic in activating leucocytes.

Estimated glomerular filtration rate is a poor predictor of concentration for a broad range of uremic toxins.

BACKGROUND AND OBJECTIVES The degree of chronic kidney disease (CKD) is currently expressed in terms of GFR, which can be determined directly or estimated according to different formulas on the basis of serum creatinine and/or cystatin C measurements (estimated GFR [eGFR]). The purpose of this study was to investigate whether eGFR values are representative for uremic toxin concentrations in patients with different degrees of CKD. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Associations between eGFR based on serum cystatin C and different uremic solutes (mol wt range 113 to 240 D; determined by colorimetry, HPLC, or ELISA) were evaluated in 95 CKD patients not on dialysis (CKD stage 2 to 5). The same analysis was also applied for six other eGFR formulas. RESULTS There was a substantial disparity in fits among solutes. In linear regression, explained variance of eGFR was extremely low for most solutes, with eGFR > 0.4 only for creatinine. The other eGFR formulations gave comparably disappointing results with regard to their association to uremic solutes. Relative similarity in R(2) values per solute for the different eGFR values and the strong disparity in values between solutes suggest that the differences in R(2) are mainly due to discrepancies in solute handling apart from GFR. CONCLUSIONS eGFR is poorly associated with concentrations of all studied uremic toxins in patients with different degrees of CKD, correlates differently with each individual solute, and can thus not be considered representative for evaluating the accumulation of solutes in the course of CKD.

Warning: the unfortunate end of p-cresol as a uraemic toxin

The cresols are protein-bound uraemic retention solutes that have become a frequent subject of both in vivo and in vitro research. These investigations essentially focused on the mother compound p-cresol. Five years ago, two research groups independently demonstrated that not p-cresol but its conjugates, and among these especially p-cresylsulfate, predominate in the body [1,2]. Nevertheless, even nowadays, studies are performed whereby only the effects of p-cresol are evaluated, in spite of the likelihood that such an approach is pathophysiologically irrelevant. The present publication aims to explain the origins for this persisting misunderstanding, warns against emanating conceptual error and outlines a correct approach for the future.

Comparison of removal capacity of two consecutive generations of high-flux dialysers during different treatment modalities

BACKGROUND Innovative modifications have been introduced in several types of dialyser membranes to improve adequacy and permselectivity. Which aspects of removal are modified and how this relates to different diffusive or convective strategies has, however, been insufficiently investigated. METHODS In a prospective cross-over study, 14 chronic kidney disease (Stage 5D) patients were dialysed with a second-generation high-flux dialyser (Polynephron) in comparison to a first-generation type (DIAPES-HF800). Both dialysers were assessed in haemodialysis, in online pre-dilution and in post-dilution haemodiafiltration. Reduction ratio (RR, %) of small water-soluble compounds (urea and uric acid), low-molecular weight proteins (LMWPs) (β(2)-microglobulin, cystatin C, myoglobin and retinol-binding protein) and protein-bound solutes (hippuric acid, indole acetic acid, indoxylsulphate and p-cresylsulphate) was assessed, together with albumin losses into the dialysate. RESULTS Comparing the two types of membranes, the second-generation dialyser demonstrated a higher RR for LMWPs, whilst at the same time exhibiting lower albumin losses but only during post-dilution haemodiafiltration. No differences in RR were detected for both the small water-soluble and the protein-bound compounds. Comparing dialysis strategies, convection removed the same amount of solute or more as compared to diffusion. CONCLUSIONS The second-generation membrane resulted in a higher removal of LMWPs compared to the first-generation membrane, but for the other solutes, differences were less prominent. Convection was superior in removal of a broad range of uraemic retention solutes especially with the first-generation membrane.