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.

A novel UPLC-MS-MS method for simultaneous determination of seven uremic retention toxins with cardiovascular relevance in chronic kidney disease patients.

Chronic kidney disease (CKD) is a devastating illness characterized by accumulation of uremic retention solutes in the body. The objective of this study was to develop and validate a simple, rapid, and robust UPLC-MS-MS method for simultaneous determination, in serum, of seven organic acid uremic retention toxins, namely uric acid (UA), hippuric acid (HA), indoxylsulfate (IS), p-cresylglucuronide (pCG), p-cresylsulfate (pCS), indole-3-acetic acid (IAA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF). Isotopically labeled internal standards (d(5)-HA; 1,3-(15)N(2)-UA, and d(5)-IAA) were used to correct for variations in sample preparation and system performance. Separation on a C18 column was followed by negative electrospray ionization and tandem mass spectrometric detection. Accuracy was below the 15 % threshold. Within-day precision varied from 0.60 to 4.54 % and between-day precision was below 13.33 % for all compounds. The applicability of the method was evaluated by analyzing 78 serum samples originating both from healthy controls and from patients at different stages of CKD. These results were compared with those obtained by use of conventional HPLC-PDA-FLD methods. A good correlation was obtained between both methods for all compounds.

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.

Oral supplementation with sulodexide inhibits neo-angiogenesis in a rat model of peritoneal perfusion.

BACKGROUND: Peritoneal dialysis (PD) is associated with functional and morphological alterations of the peritoneal membrane (PM). It is hypothesized that vascular endothelial growth factor (VEGF) plays a role in this process. Sulodexide is a glycosaminoglycan with effects on vascular biology. Therefore, the impact of oral sulodexide on PM function and morphology in a rat model of peritoneal perfusion was evaluated. METHODS: Rats received 10 mL peritoneal dialysate fluid (PDF) twice daily via a tunnelled PD catheter. The test-PD group (Sul) received 15 mg/kg/day oral sulodexide versus none in the control-PD group (Con). A third group received no PDF (Sham). After 12 weeks, a peritoneal equilibration test was performed and the PM was sampled. Neo-angiogenesis was evaluated using immunostaining with von Willebrand, and epithelial-to-mesenchymal transition (EMT) using co-localization of cytokeratin and α-smooth muscle actin. VEGF was determined in the dialysate by enzyme-linked immunosorbent assay. RESULTS: PD induced loss of ultrafiltration, also in the sulodexide group. Creatinine and glucose transport were better preserved, and sodium dip was more pronounced in the sulodexide group versus control. Submesothelial thickness, neo-angiogenesis and EMT were more pronounced in the Con versus Sul versus Sham group. VEGF in the dialysate, corrected for diffusion was higher in Con and Sul versus Sham. CONCLUSION: Oral sulodexide administration diminishes neo-vascularization, submesothelial thickening and EMT induced by exposure to PDF in a rat model. As there was no difference in VEGF at the protein level in the dialysate, we hypothesize that oral sulodexide inhibits VEGF locally by binding.

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.

Salt intake induces epithelial-to-mesenchymal transition of the peritoneal membrane in rats.

BACKGROUND: Dietary salt intake has been linked to hypertension and cardiovascular disease through volume-mediated effects. Accumulating evidence points to direct negative influence of salt intake independent of volume overload, such as cardiac and renal fibrosis, mediated through transforming growth factor beta (TGF-beta). Epithelial-to-mesenchymal transition (EMT) has been implicated as a key process in chronic fibrotic diseases, such as chronic kidney disease or heart failure. The potential role of dietary salt intake on cell transdifferentiation has never been investigated. This study analysed the effect of dietary salt intake on EMT and fibrosis in the peritoneal membrane (PM) in a rat model. METHODS: Twenty-eight Wistar rats were randomized to a normal salt (NS) or a high salt (HS) intake. NS and HS rats had free access to tap water or NaCl 2% as drinking water, respectively. After 2 weeks, samples of peritoneum were taken, and TGF-beta(1), Interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) mRNA expression were quantified with qRT-PCR. Fibrosis and submesothelial PM thickness were scored. EMT was evaluated using fluorescence staining with cytokeratin and alpha smooth muscle actin (alpha-SMA). RESULTS: Dietary salt intake caused peritoneal fibrosis and thickening of the submesothelial layer and induced EMT as identified by colocalization of cytokeratin and alpha-SMA in cells present in the submesothelial layer. Peritoneal TGF-beta(1) and IL-6 mRNA expression were upregulated in the HS group. CONCLUSION: High dietary salt intake induces EMT and peritoneal fibrosis, a process coinciding with upregulation of TGF-beta1.

Prospective evaluation of the change of predialysis protein-bound uremic solute concentration with postdilution online hemodiafiltration.

Although protein-bound uremic compounds have been related to outcome in observational studies, few current dialysis strategies provide more removal of those compounds than standard hemodialysis. We evaluated the evolution of protein-bound uremic solutes after a switch from high-flux hemodialysis to postdilution hemodiafiltration (n = 13). We compared predialysis solute concentration at 4, 5, and 9 weeks versus baseline for several protein-bound compounds and water-soluble solutes, as well as for beta(2)-microglobulin. After 9 weeks of postdilution hemodiafiltration, a significant decrease versus baseline could be detected for total concentration of protein-bound solutes: p-cresylsulfate (3.98 +/- 1.51-3.17 +/- 1.77 mg/dL, -20%, P < 0.01) and 3-carboxyl-4-methyl-5-propyl-2-furanpropionic acid (0.72 +/- 0.52-0.64 +/- 0.46 mg/dL, -11%, P < 0.01). For the other protein-bound solutes, hippuric acid, indoleacetic acid, and indoxylsulfate, no change in total concentration could be detected. The concentration of the middle molecule, beta(2)-microglobulin, decreased as well after 9 weeks of postdilution hemodiafiltration (24.7 +/- 9.3-18.1 +/- 6.7 mg/L, -27%, P < 0.01). For water-soluble compounds, no significant change of concentration was found. Postdilution hemodiafiltration in comparison to high-flux hemodialysis provided significant reduction of predialysis concentration of protein-bound compounds, especially those with the highest protein binding, and of beta(2)-microglobulin, by -11 to -27% in 9 weeks.

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.