Free p-cresyl sulfate shows the highest association with cardiovascular outcome in chronic kidney disease.

BACKGROUND: Several protein-bound uraemic toxins (PBUTs) have been associated with cardiovascular (CV) and all-cause mortality in chronic kidney disease (CKD) but the degree to which this is the case per individual PBUT and the pathophysiological mechanism have only partially been unraveled. METHODS: We compared the prognostic value of both total and free concentrations of five PBUTs [p-cresyl sulfate (pCS), p-cresyl glucuronide, indoxyl sulfate, indole acetic acid and hippuric acid] in a cohort of 523 patients with non-dialysis CKD Stages G1-G5. Patients were followed prospectively for the occurrence of a fatal or non-fatal CV event as the primary endpoint and a number of other major complications as secondary endpoints. In addition, association with and the prognostic value of nine markers of endothelial activation/damage was compared. RESULTS: After a median follow-up of 5.5 years, 149 patients developed the primary endpoint. In multivariate Cox regression models adjusted for age, sex, systolic blood pressure, diabetes mellitus and estimated glomerular filtration rate, and corrected for multiple testing, only free pCS was associated with the primary endpoint {hazard ratio [HR]1.39 [95% confidence interval (CI) 1.14-1.71]; P = 0.0014}. Free pCS also correlated with a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (r = -0.114, P < 0.05), angiopoietin-2 (ANGPT2) (r = 0.194, P < 0.001), matrix metallopeptidase 7 (MMP-7; (r = 0.238, P < 0.001) and syndecan 1 (r = 0.235, P < 0.001). Of these markers of endothelial activation/damage, ANGPT2 [HR 1.46 (95% CI 1.25-1.70); P < 0.0001] and MMP-7 [HR 1.31 (95% CI 1.08-1.59); P = 0.0056] were also predictive of the primary outcome. CONCLUSIONS: Among PBUTs, free pCS shows the highest association with CV outcome in non-dialysed patients with CKD. Two markers of endothelial activation/damage that were significantly correlated with free pCS, ANGPT2 and MMP-7 were also associated with CV outcome. The hypothesis that free pCS exerts its CV toxic effects by an adverse effect on endothelial function deserves further exploration.

The urinary proteomics classifier chronic kidney disease 273 predicts cardiovascular outcome in patients with chronic kidney disease.

BACKGROUND: The urinary proteomic classifier chronic kidney disease 273 (CKD273) is predictive for the development and progression of chronic kidney disease (CKD) and/or albuminuria in type 2 diabetes. This study evaluates its role in the prediction of cardiovascular (CV) events in patients with CKD Stages G1-G5. METHODS: We applied the CKD273 classifier in a cohort of 451 patients with CKD Stages G1-G5 followed prospectively for a median of 5.5 years. Primary endpoints were all-cause mortality, CV mortality and the composite of non-fatal and fatal CV events (CVEs). RESULTS: In multivariate Cox regression models adjusting for age, sex, prevalent diabetes and CV history, the CKD273 classifier at baseline was significantly associated with total mortality and time to fatal or non-fatal CVE, but not CV mortality. Because of a significant interaction between CKD273 and CV history (P = 0.018) and CKD stages (P = 0.002), a stratified analysis was performed. In the fully adjusted models, CKD273 classifier was a strong and independent predictor of fatal or non-fatal CVE only in the subgroup of patients with CKD Stages G1-G3b and without a history of CV disease. In those patients, the highest tertile of CKD273 was associated with a >10-fold increased risk as compared with the lowest tertile. CONCLUSIONS: The urinary CKD273 classifier provides additional independent information regarding the CV risk in patients with early CKD stage and a blank CV history. Determination of CKD273 scores on a random urine sample may improve the efficacy of intensified surveillance and preventive strategies by selecting patients who potentially will benefit most from early risk management.

Increased urinary osmolyte excretion indicates chronic kidney disease severity and progression rate.

Background: Chronic kidney disease (CKD) is a recognized global health problem. While some CKD patients remain stable after initial diagnosis, others can rapidly progress towards end-stage renal disease (ESRD). This makes biomarkers capable of detecting progressive forms of CKD extremely valuable, especially in non-invasive biofluids such as urine. Screening for metabolite markers using non-targeted metabolomic techniques like nuclear magnetic resonance spectroscopy is increasingly applied to CKD research. Methods: A cohort of CKD patients (n = 227) with estimated glomerular filtration rates (eGFRs) ranging from 9.4-130 mL/min/1.73 m2 was evaluated and urine metabolite profiles were characterized in relation to declining eGFR. Nested in this cohort, a retrospective subset (n = 57) was investigated for prognostic metabolite markers of CKD progression, independent of baseline eGFR. A transcriptomic analysis of murine models of renal failure was performed to validate selected metabolomic findings. Results: General linear modeling revealed 11 urinary metabolites with significant associations to reduced eGFR. Linear modelling specifically showed that increased urine concentrations of betaine (P < 0.05) and myo-inositol (P < 0.05) are significant prognostic markers of CKD progression. Conclusions: Renal organic osmolytes, betaine and myo-inositol play a critical role in protecting renal cells from hyperosmotic stress. Kidney tissue transcriptomics of murine preclinical experimentation identified decreased expression of Slc6a12 and Slc5a11 mRNA in renal tissue consistent with defective tubular transport of these osmolytes. Imbalances in renal osmolyte regulation lead to increased renal cell damage and thus more progressive forms of CKD. Increases in renal osmolytes in urine could provide clinical diagnostic and prognostic information on CKD outcomes.

Hereditary polycystic kidney disease is characterized by lymphopenia across all stages of kidney dysfunction: an observational study.

Background: Polycystic kidney disease (PKD) is characterized by urinary tract infections and extrarenal abnormalities such as an increased risk of cancer. As mutations in polycystin-1 and -2 are associated with decreased proliferation of immortalized lymphoblastoid cells in PKD, we investigated whether lymphopenia could be an unrecognized trait of PKD. Methods: We studied 700 kidney transplant recipients with (n = 126) or without PKD at the time of kidney transplantation between 1 January 2003 and 31 December 2014 at Ghent University Hospital. We also studied 204 patients with chronic kidney disease (CKD) with PKD and 204 matched CKD patients without PKD across comparable CKD strata with assessment between 1 January 1999 and 1 February 2016 at three renal outpatient clinics. We compared lymphocyte counts with multiple linear regression analysis to adjust for potential confounders. We analysed flow cytometric immunophenotyping data and other haematological parameters. Results: Lymphocyte counts were 264/µL [95% confidence interval (CI) 144-384] and 345/µL (95% CI 245-445) (both P < 0.001) lower in the end-stage kidney disease (ESKD) and CKD cohort, respectively, after adjustment for age, sex, ln(C-reactive protein) and estimated glomerular filtration rate (in the CKD cohort only). In particular, CD8+ T and B lymphocytes were significantly lower in transplant recipients with versus without PKD (P < 0.001 for both). Thrombocyte and monocyte counts were lower in patients with versus without PKD in both cohorts (P < 0.001 for all analyses except P = 0.01 for monocytes in the ESKD cohort). Conclusion: PKD is characterized by distinct cytopenias and especially lymphopenia, independent of kidney function. This finding has the potential to alter our therapeutic approach to patients with PKD.

Cardiovascular disease after transplantation: an emerging role of the immune system.

Cardiovascular disease (CVD) after transplantation remains a major concern. Little is known about what drives the increased cardiovascular risk in transplant recipients apart from traditional risk factors. The immune system is involved in the pathogenesis of hypertension, atherosclerosis, and coronary artery disease in the general population. Recently, inhibition of interleukin 1 - ß by canakinumab versus placebo decreased the incidence of cardiovascular events. Emerging evidence points to a role of adaptive cellular immunity in the development of CVD. Especially, expansion of pro-inflammatory and antiapoptotic cytotoxic CD4+ CD28null T cells is closely associated with incident CVD in various study populations including transplant recipients. The association of cytomegalovirus exposure with increased cardiovascular mortality might be explained by its capacity to upregulate these cytotoxic cells. Also, humoral immunity seems to be relevant for cardiovascular outcome in transplant recipients. Panel-reactive antibodies at baseline and donor-specific antibodies are independently associated with poor cardiovascular outcome after kidney transplantation. Cardiovascular effects of immunosuppressive drugs and statins do not only imply indirect positive or negative effects on traditional cardiovascular risk factors but also intrinsic immunological effects. How immunosuppressive drugs modify atherosclerosis largely remains elusive.

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

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.

Assessment of the association between increasing membrane pore size and endotoxin permeability using a novel experimental dialysis simulation set-up.

BACKGROUND: Membranes with increasing pore size are introduced to enhance removal of large uremic toxins with regular hemodialysis. These membranes might theoretically have higher permeability for bacterial degradation products. In this paper, permeability for bacterial degradation products of membranes of comparable composition with different pore size was investigated with a new in vitro set-up that represents clinical flow and pressure conditions. METHODS: Dialysis was simulated with an AK200 machine using a low-flux, high-flux, medium cut-off (MCO) or high cut-off (HCO) device (n = 6/type). A polyvinylpyrrolidone-solution (PVP) was recirculated at blood side. At dialysate side, a challenge solution containing a filtrated lysate of two water-borne bacteria (Pseudomonas aeruginosa and Pelomononas saccharophila) was infused in the dialysate flow (endotoxin ≥ 4EU/ml). Blood and dialysate flow were set at 400 and 500 ml/min for 60 min. PVP was sampled before (PVPpre) and after (PVPpost) the experiment and dialysate after 5 and 55 min. Limulus Amebocyte Lysate (LAL) test was performed. Additionally, samples were incubated with a THP-1 cell line (24 h) and IL-1ß levels were measured evaluating biological activity. RESULTS: The LAL-assay confirmed presence of 9.5 ± 7.4 EU/ml at dialysate side. For none of the devices the LAL activity in PVPpre vs. PVPpost was significantly different. Although more blood side PVP solutions had a detectable amount of endotoxin using a highly sensitive LAL assay in the more open vs traditional membranes, the permeability for endotoxins of the 4 tested dialysis membranes was not significantly different but the number of repeats is small. None of the PVP solutions induced IL-1ß in the THP-1 assay. CONCLUSIONS: A realisitic in vitro dialysis was developed to assess membrane translocation of bacterial products. LAL activity on the blood side after endotoxin exposure did not change for all membranes. Also, none of the PVPpost solutions induced IL-1ß in the THP-1 bio-assay.

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.

Metabolic profiling of human plasma and urine in chronic kidney disease by hydrophilic interaction liquid chromatography coupled with time-of-flight mass spectrometry: a pilot study.

A typical characteristic of chronic kidney disease (CKD) is the progressive loss in renal function over a period of months or years with the concomitant accumulation of uremic retention solutes in the body. Known biomarkers for the kidney deterioration, such as serum creatinine or urinary albumin, do not allow effective early detection of CKD, which is essential towards disease management. In this work, a hydrophilic interaction liquid chromatography time-of-flight mass spectrometric (HILIC-TOF MS) platform was optimized allowing the search for novel uremic retention solutes and/or biomarkers of CKD. The HILIC-ESI-MS approach was used for the comparison of urine and plasma samples from CKD patients at stage 3 (n = 20), at stage 5 not yet receiving dialysis (n = 20) and from healthy controls (n = 20). Quality control samples were used to control and ensure the validity of the metabolomics approach. Subsequently the data were treated with the XCMS software for multivariate statistical analysis. In this way, differentiation could be achieved between the measured metabolite profile of the CKD patients versus the healthy controls. The approach allowed the elucidation of a number of metabolites that showed a significant up- and downregulation throughout the different stages of CKD. These compounds are cinnamoylglycine, glycoursodeoxycholic acid, 2-hydroxyethane sulfonate, and pregnenolone sulfate of which the identity was unambiguously confirmed via the use of authentic standards. The latter three are newly identified uremic retention solutes.

Pro-inflammatory cytokines and leukocyte oxidative burst in chronic kidney disease: culprits or innocent bystanders?

BACKGROUND: Pro-inflammatory cytokines are elevated in chronic kidney disease (CKD), a condition characterized by microinflammation with oxidative stress as key feature. However, their role in the inflammatory response at uraemic concentrations has not yet been defined. In this study, the contribution of cytokines on induction of leukocyte oxidative stress was investigated. METHODS: Whole blood from healthy donors was incubated with 20-1400 pg/mL TNFα, 5-102.8 pg/mL IL-6, 20-400 pg/mL IL-1ß and 75-1200 pg/mL IL-18 separately or in combination. Oxidative burst was measured, at baseline and after stimulation with fMLP (Phagoburst™). The effect of the TNFα blocker, adalimumab (Ada), was evaluated on TNFα-induced ROS production. Finally, the association between TNFα and the composite end point all-cause mortality or first cardiovascular event was analysed in a CKD population stage 4-5 (n = 121). RESULTS: While interleukin (IL)-6, IL-1ß and IL-18 alone induced no ROS activation of normal leukocytes, irrespective of concentrations, TNFα induced ROS activation at baseline (P < 0.01) and after fMLP stimulation (P < 0.05), but only at uraemic concentrations in the high range (400 and 1400 pg/mL). A similar pattern was observed with all cytokines in combination, but already at intermediate uraemic concentrations (all P < 0.05, except for monocytes after fMLP stimulation: n.s.), suggesting synergism between cytokines. ROS production induced by TNFα (400 pg/mL) and the cytokine combination was blocked with Ada. Uraemia-related oxidative stress in leukocytes of haemodialysis patients was however not blocked by Ada. In patients, TNFα was not associated to adverse events (HR: 1.52, 95% CI 0.81-2.85, P = 0.13). CONCLUSION: Among several pro-inflammatory cytokines, TNFα alone was pro-oxidative but only at high-range uraemic concentrations. Adding a TNFα blocker, Ada, blocked this ROS production, but not the oxidative stress in blood samples from haemodialysis patients, suggesting that other uraemic toxins than TNFα are more crucial in this process. However, the lack of association between TNFα and mortality suggests that the role of TNFα-linked oxidative stress is limited.

New insights in molecular mechanisms involved in chronic kidney disease using high-resolution plasma proteome analysis.

BACKGROUND: The reduced glomerular filtration rate in the advanced stages of chronic kidney disease (CKD) leads to plasma accumulation of uraemic retention solutes including proteins. It has been hypothesized that these changes may, at least in part, be responsible for CKD-associated morbidity and mortality. However, most studies focused on the role of individual proteins, while a holistic, large-scale, integrative approach may generate significant additional insight. METHODS: In a discovery study, we analysed the plasma proteome of patients with stage 2-3 CKD (n = 14) and stage 5 CKD with haemodialysis (HD) (n = 15), using high-resolution LC-MS/MS analysis. Selected results were validated in a cohort of 40 patients with different CKD stages with or without HD, using ELISA. RESULTS: Of a total of 2054 detected proteins, 127 displayed lower, while 206 displayed higher abundance in the plasma of patients on HD. Molecular pathway analysis confirmed the modification of known processes involved in CKD complications, including decreased haemostasis and increased inflammation, complement activation and vascular damage. In addition, we identified the plasma increase during CKD progression of lysozyme C and leucine-rich alpha-2 glycoprotein, two proteins related to vascular damage and heart failure. High level of leucine-rich alpha-2 glycoprotein was associated with higher mortality in stage 5 CKD patients on HD. CONCLUSIONS: This study provides for the first time a comprehensive assessment of CKD plasma proteome, contributing to new knowledge and potential markers of CKD. These results will serve as a basis for future studies investigating the relevance of these molecules in CKD associated morbidity and mortality.

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.

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.

Uremia-related oxidative stress in leukocytes is not triggered by ß2-microglobulin.

BACKGROUND: Chronic kidney disease (CKD) is characterized by low-grade inflammation and increased risk for cardiovascular disease. The interest in ß2-microglobulin (B2M) as a marker for cardiovascular outcome with and without CKD has grown. Clinical studies suggested that B2M could be involved in the pathogenesis of vascular disease, for which chronic leukocyte activation is a pathogenic factor. We investigated whether B2M is proinflammatory by inducing oxidative burst in leukocytes. METHODS: Oxidative burst was measured at baseline and after stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP), Escherichia coli, or phorbol-12-myristate-acetate (PMA) in the whole blood of healthy volunteers in the absence (saline) and presence of human B2M (hB2M; 10 and 50 mg/L) versus uremic whole blood. Because of suspicion of contamination, hB2M was dialyzed for purification and purified B2M (dB2M) and dialysates were tested in the burst test. As a comparator, reactive oxygen species (ROS) in response to lipopolysaccharide (LPS) was measured. RESULTS: Unpurified hB2M strongly enhanced ROS in monocytes and granulocytes after E. coli and PMA and moderately after fMLP stimulation compared with control (P < .01) and uremia (P < .01) whereas at baseline hB2M only induced ROS in granulocytes (P < .05). After purification, dB2M no longer increased burst activity, suggesting that contamination was responsible for the initial effect. An endotoxin concentration of less than 1.5 EU/mL, as observed in hB2M, could not induce oxidative stress. CONCLUSION: This study suggests that B2M, a traditional marker for middle molecule retention and a novel marker for cardiovascular outcome, may not by itself cause vascular damage by influencing inflammatory response due to induction of leukocyte free radical production. However, an effect on other cell types involved cannot be excluded. Our data further reveal that this type of research might be skewed by non-LPS contaminants, and that care should be taken to exclude this bias.

The RNA-binding protein QKI governs a muscle-specific alternative splicing program that shapes the contractile function of cardiomyocytes.

AIMS: In the heart, splicing factors orchestrate the functional properties of cardiomyocytes by regulating the alternative splicing of multiple genes. Work in embryonic stem cells has shown that the splicing factor Quaking (QKI) regulates alternative splicing during cardiomyocyte differentiation. However, the relevance and function of QKI in adult cardiomyocytes remains unknown. In this study, we aim to identify the in vivo function of QKI in the adult mouse heart. METHODS AND RESULTS: We generated mice with conditional deletion of QKI in cardiomyocytes by the Cre-Lox system. Mice with cardiomyocyte-specific deletion of QKI died during the foetal period (E14.5), without obvious anatomical abnormalities of the heart. Adult mice with tamoxifen-inducible QKI deletion rapidly developed heart failure associated with severe disruption of sarcomeres, already 7 days after knocking out QKI. RNA sequencing revealed that QKI regulates the alternative splicing of more than 1000 genes, including sarcomere and cytoskeletal components, calcium-handling genes, and (post-)transcriptional regulators. Many of these splicing changes corresponded to the loss of muscle-specific isoforms in the heart. Forced overexpression of QKI in cultured neonatal rat ventricular myocytes directed these splicing events in the opposite direction and enhanced contractility of cardiomyocytes. CONCLUSION: Altogether, our findings show that QKI is an important regulator of the muscle-specific alternative splicing program that builds the contractile apparatus of cardiomyocytes.

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.

an Obituary for GFR as the main marker for kidney function?

This publication comments on the recently published findings of a study by Eloot et al. (cJASN, 6: 1266-1273, 2011) that evaluated the correlation between several formulae for calculating estimated GFR (eGFR) and different low molecular weight uremic toxins; eGFRs were based on serum creatinine (SCrea), cystatin C (Cys C), or a combination of both. Unexpectedly, the correlations for the different solutes were highly inconsistent, irrespective of the eGFR formula. On the other hand, the different eGFR formulae gave consistent results per solute. Correlation coefficients for some solutes were low (hippuric acid, p-cresylsulfate, indole acetic acid, uric acid, asymmetric dimethylarginine) to nonsignificant (carboxy-methyl-propyl-furanpropionic acid). These data point to the fact that eGFR is a deceiving predictor of uremic solute concentration and their biological action; this inconsistency is very likely the result of the impact of other factors affecting concentration, such as tubular secretion, generation by intestinal flora and metabolism.

An update on protein-bound uremic retention solutes.

Although protein-bound uremic retention solutes are recognized as 1 of the 3 main categories of uremic retention solutes, they only recently have been submitted to thorough analysis. In vitro and ex vivo data link both p-cresyl sulfate and indoxyl sulfate, two of the main compounds of this solute group, to negative impact on the cardiovascular system and progression of kidney failure. Recent in vivo observational data also relate concentration of these compounds to survival outcome, inflammation, and vascular disease in different, even moderate, stages of chronic kidney disease. Removal by different dialysis strategies, even high-flux hemodialysis, is difficult, and only by applying convection, some improvement of removal has been obtained. The other strategy with the potential to decrease concentration is by influencing intestinal generation and/or absorption. The sorbent Kremezin (AST-120) has been shown in controlled studies to decrease protein-bound solute concentration. In pilot controlled studies, AST-120 has been shown to be superior on outcome parameters to placebo. Results from large randomized trials are awaited, before these data can be considered as solid enough to warrant the recommendation to use these compounds for overall therapeutic purposes.

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) (ß(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.