Griet Glorieux

Serum Indoxyl Sulfate Is Associated with Vascular Disease and Mortality in Chronic Kidney Disease Patients

BACKGROUND AND OBJECTIVES As a major component of uremic syndrome, cardiovascular disease is largely responsible for the high mortality observed in chronic kidney disease (CKD). Preclinical studies have evidenced an association between serum levels of indoxyl sulfate (IS, a protein-bound uremic toxin) and vascular alterations. The aim of this study is to investigate the association between serum IS, vascular calcification, vascular stiffness, and mortality in a cohort of CKD patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS One-hundred and thirty-nine patients (mean +/- SD age: 67 +/- 12; 60% male) 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. RESULTS Baseline IS levels presented an inverse relationship with renal function and a direct relationship with aortic calcification and pulse wave velocity. During the follow-up period (605 +/- 217 d), 25 patients died, mostly because of cardiovascular events (n = 18). In crude survival analyses, the highest IS tertile was a powerful predictor of overall and cardiovascular mortality (P = 0.001 and 0.012, respectively). The predictive power of IS for death was maintained after adjustment for age, gender, diabetes, albumin, hemoglobin, phosphate, and aortic calcification. CONCLUSIONS The study presented here indicates that IS may have a significant role in the vascular disease and higher mortality observed in CKD patients.

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.

A Bench to Bedside View of Uremic Toxins

Reviewing the current picture of uremic toxicity reveals its complexity. Focusing on cardiovascular damage as a model of uremic effects resulting in substantial morbidity and mortality, most molecules with potential to affect the function of a variety of cell types within the vascular system are difficult to remove by dialysis. Examples are the larger middle molecular weight molecules and protein-bound molecules. Recent clinical studies suggest that enhancing the removal of these compounds is beneficial for survival. Future therapeutic options are discussed, including improved removal of toxins and the search for pharmacologic strategies blocking responsible pathophysiologic pathways.

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.

What is new in uremic toxicity

Uremic syndrome results from a malfunctioning of various organ systems due to the retention of compounds which, under normal conditions, would be excreted into the urine and/or metabolized by the kidneys. If these compounds are biologically active, they are called uremic toxins. One of the more important toxic effects of such compounds is cardio-vascular damage. A convenient classification based on the physico-chemical characteristics affecting the removal of such compounds by dialysis is: (1) small water-soluble compounds; (2) protein-bound compounds; (3) the larger “middle molecules”. Recent developments include the identification of several newly detected compounds linked to toxicity or the identification of as yet unidentified toxic effects of known compounds: the dinucleotide polyphosphates, structural variants of angiotensin II, interleukin-18, p-cresylsulfate and the guanidines. Toxic effects seem to be typically exerted by molecules which are “difficult to remove by dialysis”. Therefore, dialysis strategies have been adapted by applying membranes with larger pore size (high-flux membranes) and/or convection (on-line hemodiafiltration). The results of recent studies suggest that these strategies have better outcomes, thereby clinically corroborating the importance attributed in bench studies to these “difficult to remove” molecules.

Effective removal of protein-bound uraemic solutes by different convective strategies: a prospective trial

BACKGROUND Although different on-line convective removal strategies are available, there are no studies comparing the efficiency of solute removal for the three main options [post-dilution haemodiafiltration (post-HDF), pre-dilution haemodiafiltration (pre-HDF) and pre-dilution haemofiltration (pre-HF)] in parallel. METHODS In this study, we compared post-HDF (Polyflux 170), pre-HDF (Polyflux 170) and pre-HF (Polyflux 210) in 14 patients. Parallelism of the evaluation protocols consisted in applying the same blood flow, dialysis time and effective convection (22.9 +/- 1.7 versus 22.2 +/- 2.0 L, P = NS) in pre-HDF versus post-HDF, and the same blood flow and dialysis time while comparing pre-HDF and pre-HF (1:1 dilution). With pre-HF, ultrafiltration was maximized and resulted in an effective convective volume of 28.5 L. We studied water-soluble compounds (urea, creatinine, uric acid), protein-bound compounds (hippuric acid, indole acetic acid, indoxylsulfate and p-cresylsulfate) and beta(2)-microglobulin (beta(2)M). RESULTS Post-HDF was superior to pre-HDF for water-soluble compounds and beta(2)M, whereas there was no difference for protein-bound compounds. Pre-HDF was superior to pre-HF for water-soluble compounds and protein-bound compounds. In contrast, removal of beta(2)M for pre-HF was higher than for pre-HDF, but it did not differ from that obtained with post-HDF. CONCLUSIONS It is concluded that post-dilution is superior to pre-dilution HDF under conditions of similar convective volume, and that HDF is superior to HF in pre-dilution, with the exception of removal of beta(2)M. Overall, post-HDF is the most efficient convective strategy among those tested.

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®).

p-Cresyl Sulfate Promotes Insulin Resistance Associated with CKD

The mechanisms underlying the insulin resistance that frequently accompanies CKD are poorly understood, but the retention of renally excreted compounds may play a role. One such compound is p-cresyl sulfate (PCS), a protein-bound uremic toxin that originates from tyrosine metabolism by intestinal microbes. Here, we sought to determine whether PCS contributes to CKD-associated insulin resistance. Administering PCS to mice with normal kidney function for 4 weeks triggered insulin resistance, loss of fat mass, and ectopic redistribution of lipid in muscle and liver, mimicking features associated with CKD. Mice treated with PCS exhibited altered insulin signaling in skeletal muscle through ERK1/2 activation. In addition, exposing C2C12 myotubes to concentrations of PCS observed in CKD caused insulin resistance through direct activation of ERK1/2. Subtotal nephrectomy led to insulin resistance and dyslipidemia in mice, and treatment with the prebiotic arabino-xylo-oligosaccharide, which reduced serum PCS by decreasing intestinal production of p-cresol, prevented these metabolic derangements. Taken together, these data suggest that PCS contributes to insulin resistance and that targeting PCS may be a therapeutic strategy in CKD.

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.