Uremic Plasma Research Articles

Endogenous ouabain is not ouabain.

The concept of a circulating digitalis-like inhibitor of the sodium pump, Na+, K+-ATPase, evolved from studies performed in the 1960s. De Wardener et al1 addressed the question of whether a small increase in the glomerular filtration rate together with changes in the concentration of the more recently discovered hormone aldosterone could explain the natriuresis that generally followed salt (sodium) loading. In their studies, dogs had their renal blood flow reduced significantly by constricting the aorta above the renal arteries and were given supramaximal doses of fludrocortisone, a synthetic analogue of aldosterone, and vasopressin before being challenged with intravenous saline. Their ability to develop a natriuresis clearly demonstrated that a third factor (beyond changes in glomerular filtration rate and aldosterone concentrations) was involved in the natriuresis of salt loading. Subsequent experiments demonstrated that the responsible agent could be transmitted by the plasma of the volume-expanded animal.2 Although we may now ask whether in these experiments the effect was mediated, at least in part, by the release of atrial and B-type natriuretic peptide from the heart, it was nearly 2 decades before those hormones were discovered during which time it was demonstrated that the plasma of volume-expanded animals had the ability to inhibit the sodium pump3—which is not a target of atrial natriuretic peptide and B-type natriuretic peptide. Essentially parallel studies were performed by Welt and colleagues4 in uremia where inhibition of the sodium pump of erythrocytes was demonstrated along with the ability of uremic plasma to induce such a defect in normal erythrocytes. In 1975, it was shown that patients with essential hypertension had, as a group, reduced activity of the sodium pump of leukocytes, with corresponding elevated values for intracellular sodium.5 This finding proved to be reproducible in various laboratories, …

Suicidal erythrocyte death in end-stage renal disease.

Anemia in end-stage renal disease (ESRD) results mainly from erythropoietin and iron deficiency. Anemia could be confounded, however, by accelerated clearance of circulating erythrocytes because of premature suicidal erythrocyte death or eryptosis characterized by phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increased cytosolic Ca(2+) concentration ([Ca(2+)]i), oxidative stress, and ceramide. The present study explored whether and how ESRD influences eryptosis. Blood was drawn from healthy volunteers (n = 20) as well as ESRD patients (n = 20) prior to and after hemodialysis. Phosphatidylserine exposure was estimated from annexin V binding, [Ca(2+)]i from Fluo3-fluorescence, reactive oxygen species (ROS) from 2',7'dichlorodihydrofluorescein fluorescence, and ceramide from fluorescein-isothiocyanate-conjugated antibody binding in flow cytometry. Measurements were made in erythrocytes from freshly drawn blood and in erythrocytes from healthy volunteers exposed in vitro for 24 h to plasma from healthy volunteers or ESRD patients prior to and following dialysis. The patients suffered from anemia (hemoglobin 10.1 ± 0.5 g/100 ml) despite 1.96 ± 0.34 % reticulocytes. The percentage of phosphatidylserine-exposing erythrocytes was significantly higher in ESRD patients (0.84 ± 0.09 %) than in healthy volunteers (0.43 ± 0.04 %) and was significantly increased immediately after dialysis (1.35 ± 0.13 %). The increase in phosphatidylserine exposure was paralleled by increase in [Ca(2+)]i, oxidative stress, and ceramide abundance. As compared to addition of plasma from healthy individuals, addition of predialytic but not of postdialytic plasma from ESRD patients increased phosphatidylserine exposure, [Ca(2+)]i, ROS, and ceramide abundance. In conclusion, both, dialyzable components of uremic plasma and dialysis procedure, trigger eryptosis at least in part by increasing erythrocyte [Ca(2+)]i, ROS, and ceramide formation. Anemia in uremia results in part from eryptosis, the suicidal erythrocyte death. Eryptosis in uremia is triggered in part by a dialyzable plasma component. Eryptosis in uremia is further triggered by dialysis procedure. Eryptosis in uremia is in part due to increased cytosolic Ca(2+) concentration. Eryptosis in uremia is further due to oxidative stress and ceramide formation.

Enhanced platelet apoptosis in chronic uremic patients

Background: There is a paucity of research on platelet apoptosis and its contribution to platelet dysfunction in uremic patients. The present study sought to analyze platelets apoptosis in uremic patients who underwent different dialysis modalities. Methods: Sixteen chronic uremic patients (5 on hemodialysis, 6 on peritoneal dialysis and 5 on non-dialysis) and 16 controls were studied. Platelet-rich plasma was detected for apoptotic events including depolarization of mitochondrial inner membrane potential (ΔΨm), phosphatidylserine (PS) exposure, activation of caspases-3 and Bcl-2 family proteins variations by Flow Cytometry or by Western-Blot. Washed normal platelets were incubated with normal or uremic platelet poor plasma and then were detected apoptotic events. Platelets function was assessed by ristocetin induced aggregative function test. Results: Compared to controls, uremic platelets demonstrated greater apoptosis for the ΔΨm depolarization (43.48 ± 9.58 vs. 52.76 ± 15.36, p = 0.005) as well as PS exposure (1.36 ± 0.51 vs. 0.99 ± 0.27, p < 0.001). There was no significant difference among different treatment groups (for the ΔΨm depolarization f = 0.16, p = 0.85; for the PS exposure f = 1.06, p = 0.36). Western Blot analyses showed caspase-3 activation and pro-apoptotic Bcl-2 family proteins expression. Platelets exposed to uremic plasma exhibited distinct apoptosis phenomena. Ristocetin induced platelet aggregation was markedly diminished in uremic patients and treated platelets. Conclusions: These findings indicate that platelets are incurred apoptosis in uremia patients. Uremic plasma accelerates apoptosis of normal platelets, resulting in a dysfunctional pattern of platelets in uremia. Uremic platelets apoptosis has no relationship with dialysis modality.

Binding Affinity and Capacity for the Uremic Toxin Indoxyl Sulfate

Protein binding prevents uremic toxins from removal by conventional extracorporeal therapies leading to accumulation in maintenance dialysis patients. Weakening of the protein binding may enhance the dialytic elimination of these toxins. In ultrafiltration and equilibrium dialysis experiments, different measures to modify the plasma binding affinity and capacity were tested: (i), increasing the sodium chloride (NaCl) concentration to achieve a higher ionic strength; (ii), increasing the temperature; and (iii), dilution. The effects on the dissociation constant KD and the protein bound fraction of the prototypical uremic toxin indoxyl sulfate (IS) in plasma of healthy and uremic individuals were studied. Binding of IS corresponded to one site binding in normal plasma. KD increased linearly with the NaCl concentration between 0.15 (KD = 13.2 ± 3.7 µM) and 0.75 M (KD = 56.2 ± 2.0 µM). Plasma dilution further reduced the protein bound toxin fraction by lowering the protein binding capacity of the plasma. Higher temperatures also decreased the protein bound fraction of IS in human plasma. Increasing the NaCl concentration was effective to weaken the binding of IS also in uremic plasma: the protein bound fraction decreased from 89% ± 3% to 81% ± 3% at 0.15 and 0.75 M NaCl, respectively. Dilution and increasing the ionic strength and temperature enhance the free fraction of IS allowing better removal of the substance during dialysis. Applied during clinical dialysis, this may have beneficial effects on the long-term outcome of maintenance dialysis patients.

Direct Inhibition and Down-regulation by Uremic Plasma Components of Hepatic Uptake Transporter for SN-38, an Active Metabolite of Irinotecan, in Humans

Clinical study has previously revealed that plasma concentration of 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, was higher in patients with end-stage renal failure than those with normal kidney function although SN-38 is mainly eliminated in the liver. Here, we focused on inhibition by uremic toxins of hepatic SN-38 uptake and down-regulation of uptake transporter(s) by uremic plasma in humans. We evaluated SN-38 uptake and its inhibition by uremic toxins, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), indoxyl sulfate (Indox), hippuric acid (HA) and indole acetate (IA), with cryopreserved human hepatocytes and HEK293 cells stably expressing hepatic uptake transporters, organic anion transporting polypeptides (OATPs). We also collected plasma samples from patients with severe renal failure to examine their effects on mRNA level of OATPs in primary cultured human hepatocytes. SN-38 was taken up by hepatocytes, which showed biphasic saturation patterns. The SN-38 uptake by hepatocytes was significantly inhibited by a uremic toxin mixture including clinically relevant concentrations of CMPF, Indox, HA and IA. Kinetic analyses for OATP-mediated transport revealed that the uptake of SN-38 by OATP1B1 was the highest, followed by OATP1B3. Among the uremic toxins, CMPF exhibited most potent inhibition of OATP1B1-mediated SN-38 uptake and directly inhibited the uptake of SN-38 also in hepatocytes. In addition, gene expression of OATP1B1 and OATP1B3 in hepatocytes was significantly down-regulated by the treatment with the uremic plasma. OATP1B1-mediated hepatic uptake of SN-38 was inhibited by uremic toxins, and gene expression of OATP1B1 was down-regulated by uremic plasma.

Optimized Metabolomic Approach to Identify Uremic Solutes in Plasma of Stage 3–4 Chronic Kidney Disease Patients

BackgroundChronic kidney disease (CKD) is characterized by the progressive accumulation of various potential toxic solutes. Furthermore, uremic plasma is a complex mixture hampering accurate determination of uremic toxin levels and the identification of novel uremic solutes.MethodsIn this study, we applied 1H-nuclear magnetic resonance (NMR) spectroscopy, following three distinct deproteinization strategies, to determine differences in the plasma metabolic status of stage 3–4 CKD patients and healthy controls. Moreover, the human renal proximal tubule cell line (ciPTEC) was used to study the influence of newly indentified uremic solutes on renal phenotype and functionality.ResultsProtein removal via ultrafiltration and acetonitrile precipitation are complementary techniques and both are required to obtain a clear metabolome profile. This new approach, revealed that a total of 14 metabolites were elevated in uremic plasma. In addition to confirming the retention of several previously identified uremic toxins, including p-cresyl sulphate, two novel uremic retentions solutes were detected, namely dimethyl sulphone (DMSO2) and 2-hydroxyisobutyric acid (2-HIBA). Our results show that these metabolites accumulate in non-dialysis CKD patients from 9±7 µM (control) to 51±29 µM and from 7 (0–9) µM (control) to 32±15 µM, respectively. Furthermore, exposure of ciPTEC to clinically relevant concentrations of both solutes resulted in an increased protein expression of the mesenchymal marker vimentin with more than 10% (p<0.05). Moreover, the loss of epithelial characteristics significantly correlated with a loss of glucuronidation activity (Pearson r = −0.63; p<0.05). In addition, both solutes did not affect cell viability nor mitochondrial activity.ConclusionsThis study demonstrates the importance of sample preparation techniques in the identification of uremic retention solutes using 1H-NMR spectroscopy, and provide insight into the negative impact of DMSO2 and 2-HIBA on ciPTEC, which could aid in understanding the progressive nature of renal disease.

A regenerable potassium and phosphate sorbent system to enhance dialysis efficacy and device portability: an in vitro study

Continuous dialysis could provide benefit by constant removal of potassium and phosphate. This study investigates the suitability of specific potassium and phosphate sorbents for incorporation in an extracorporeal device by capacity and regenerability testing. Capacity testing was performed in uraemic plasma. Regenerability was tested for potassium sorbents, with adsorption based on cationic exchange for sodium, with 0.1 M and 1.0 M NaCl. To regenerate phosphate sorbents, with adsorption based on anionic exchange, 0.1 M and 1.0 M NaHCO3 and NaOH were used. Subsequently, sodium polystyrene divinylbenzene sulphonate (RES-A) and iron oxide hydroxide (FeOOH) beads were incorporated in a cartridge for testing in bovine blood using a recirculating blood circuit and a dialysis circuit separated by a high-flux dialyzer (dynamic setup). Preloading was tested to assess whether this could limit calcium and magnesium adsorption. In the batch-binding assays, zirconium phosphate most potently adsorbed potassium (0.44 ± 0.05 mmol/g) and RES-A was the best regenerable potassium sorbent (92.9 ± 5.7% with 0.1 M NaCl). Zirconium oxide hydroxide (ZIR-hydr) most potently adsorbed phosphate (0.23 ± 0.05 mmol/g) and the polymeric amine sevelamer carbonate was the best regenerable sorbent (85.7 ± 5.2% with 0.1 M NaHCO3). In the dynamic setup, a potassium adsorption of 10.72 ± 2.06 mmol in 3 h was achieved using 111 g of RES-A and a phosphate adsorption of 4.73 ± 0.53 mmol in 3 h using 55 g of FeOOH. Calcium and magnesium preloading was shown to reduce the net adsorption in 3 h from 3.57 ± 0.91 to -0.29 ± 1.85 and 1.02 ± 0.05 to -0.31 ± 0.18 mmol, respectively. RES-A and FeOOH are suitable, regenerizable sorbents for potassium and phosphate removal in dialysate regeneration. Use of zirconium carbonate and ZIR-hydr may further increase phosphate adsorption, but may compromise sorbent regenerability. Use of polymeric amines for phosphate adsorption may enhance sorbent regenerability. Calcium and magnesium preloading considerably reduced net adsorption of these ions.

White adipose tissue overproduces the lipid-mobilizing factor zinc α2-glycoprotein in chronic kidney disease

Chronic kidney disease (CKD) is frequently associated with protein-energy wasting, a recognized strong predictive factor of mortality. Zinc α2-glycoprotein (ZAG) is a new adipokine involved in body weight control through its lipid-mobilizing activity. Here we tested whether the uremic environment in CKD could alter ZAG production by white adipose tissue and contribute to CKD-associated metabolic disturbances. Compared with normal plasma, uremic plasma induced a significant increase in ZAG synthesis (124%), was associated with a significant increase in basal lipolysis (31%), and significantly blunted lipogenesis (-53%) in 3T3-L1 adipocytes in vitro. In 5/6 nephrectomized rats and mice in vivo, there was a significant decrease in white adipose tissue accretion (-44% and -43%, respectively) and a significantly higher white adipose tissue content of ZAG protein than in sham-operated, pair-fed control animals (498% and 106%, respectively). Subcutaneous white adipose tissue biopsies from patients with end-stage renal disease exhibited a higher content of ZAG (573%) than age-matched controls. Thus, the ZAG content is increased in white adipose tissue from patients or animal models with CKD. Overproduction of ZAG in CKD could be a major contributor to metabolic disturbances associated with CKD.

Albumin is the main plasma binding protein for indoxyl sulfate and p‐cresyl sulfate

Indoxyl sulfate and p-cresyl sulfate are two uremic retention solutes implicated in the uremic syndrome. Removal during dialysis is limited, mainly due to protein binding. Binding characteristics to healthy albumin have recently been characterized. Whether uremia alters the binding characteristics of albumin is currently unknown. Moreover, protein binding values previously determined with ultrafiltration are in sharp contrast to recently reported values based on microcalorimetry. In the present study, indoxyl sulfate and p-cresyl sulfate binding were therefore quantified using both equilibrium dialysis and ultrafiltration. Deming regression demonstrated good agreement between equilibrium dialysis and ultrafiltration. Free serum concentrations of indoxyl sulfate (+26.6%) and p-cresyl sulfate (+19.7%) were slightly higher at body temperature compared with at room temperature. To investigate binding kinetics, the plasma of healthy individuals or hemodialysis patients was titrated with albumin solutions. Theoretical models of protein binding were fitted to observed titration curves. Binding coefficients of both toxins were highest in purified albumin, and were reduced from healthy to uremic plasma. In conclusion, the ultrafiltration-HPLC technique reliably measures free serum concentrations of indoxyl sulfate and p-cresyl sulfate. Albumin is the main binding protein, both in health and in advanced stages of chronic kidney disease. Modeling suggests that albumin contains two binding sites for both toxins, a single high affinity binding site and a second low affinity binding site. The high affinity binding site accounts for at least 90% of overall binding. Competition for this binding site could be used to augment free solute concentrations during dialysis, thus improving epuration.

Post-transcriptional nature of uremia-induced downregulation of hepatic apolipoprotein A-I production

Chronic kidney disease is associated with premature death from cardiovascular disease, which is, in part, driven by high density lipoprotein deficiency and dysfunction. One of the main causes of high density lipoprotein deficiency in chronic kidney disease is diminished plasma apolipoprotein (Apo)A-I level. Plasma ApoA-I is reduced in dialysis patients and hepatic ApoA-I messenger RNA (mRNA) is decreased in the uremic rats. This study explored the mechanism of uremia-induced downregulation of ApoA-I. Human hepatoma derived cells were incubated in media containing whole plasma or plasma subfractionation from normal subjects and patients with end stage renal disease pre- and posthemodialysis. Cells and culture media were isolated to measure ApoA-I protein and mRNA. ApoA-I promoter activity was measured using transfection with a luciferase promoter construct containing the -2096 to +293 segment of ApoA-I gene. Finally, effect of uremic and control plasma was assessed on ApoA-I RNA stability. Exposure to uremic plasma significantly reduced ApoA-I mRNA expression and ApoA-I protein production. These effects were reversed by replacing uremic plasma with normal plasma. Although no difference in ApoA-I promoter activity was found between cells exposed to uremic and normal plasma, uremic plasma significantly reduced ApoA-I RNA stability. Experiments using plasma subfractions revealed that the inhibitory effect of uremic plasma on ApoA-I mRNA expression resides in fractions containing molecules larger but not smaller than 30 kd. The pre- and postdialysis plasma exerted an equally potent inhibitory effect on ApoA-I mRNA abundance. Uremia lowers ApoA-I production by reducing its RNA stability. The inhibitory effect of uremic milieu on ApoA-I mRNA expression is mediated by non-dialyzable molecule(s) larger than 30 kd.

Erythrocyte Adhesion in Hemodialysis Patients – A Novel Potential Contributor to Inflammation in End-Stage Renal Disease.

AbstractAbstract 2092 Background:Widespread adhesion of erythrocytes in the vasculature would be incompatible with life. However, in illnesses such as sickle cell disease, malaria and diabetes, adhesive RBCs have been documented and are likely contributors to disease severity, inflammation and coagulation activation. End-stage renal disease is characterized by profound, global inflammation and relative thrombophilia. While we, and others, have reported the extensive exposure of RBC phosphatidylserine in patients on hemodialysis, the role of RBC adhesion in inflammation has never been examined in this condition. We performed an analysis of RBCs from hemodialysis patients to characterize their ability to adhere to other cells, elucidate the potential mechanisms of adhesion, and to relate RBC adhesion to the inflammatory state of hemodialysis. Methods:This study was conducted in a cohort of 20 African American patients receiving in-center hemodialysis at 2 separate dialysis clinics as part of a study of sickle cell trait in hemodialysis. There were 9 of 20 patients with documented sickle cell trait. Blood samples were drawn immediately pre-dialysis and prior to administration of heparin. Plasma samples were processed to minimize platelet activation, aliquoted, and frozen for batch analysis. Plasma factors (RANTES, CRP & CD40L) were analyzed via ELISA. Adhesion events in whole blood were detected via two color flow cytometry. Cause of renal failure, mode of venous access and relevant clinical data were obtained from patient charts. All adhesion assays with washed blood cells were conducted under static conditions using microvascular endothelial cells. All studies were compared to those results of healthy control patients (n =11). Spearman’s regression analysis was performed to analyze the correlation between continuous variables. Mann-Whitney U and Kruskal Wallis tests were used to compare continuous variables between groups. A p<0.05 was considered significant. Results:We found that RBCs from patients on hemodialysis were significantly more adhesive than those from healthy controls. In whole blood, we detected marked RBC adhesion to T-cells (median % of T-cells bound to RBCs: 67.9 [hemodialysis] vs. 10.55 [healthy control]) and platelets (median % of platelets bound to RBCs: 33.0 vs. 1.1%). We also noted significant RBC adhesion to neutrophils (median % of neutrophils bound to RBCs: 11.0% vs. 0.1%). Incubation of healthy RBCs with plasma from hemodialysis patients, but not healthy control plasma, was sufficient to induce RBC adhesion to cultured endothelial cells (median RBCs/mm2: 8.0 vs. 0.5) and to T-cells (median % T-cells bound to RBCs: 30.0 vs. 5.0). Plasma from hemodialysis patients also induced phosphatidylserine exposure on healthy RBCs. Phosphatidylserine exposure on the RBC appeared to mediate RBC adhesion to endothelial cells as annexin V significantly reduced RBC adherence (8.0 vs 3.8 RBC/mm2). The extent to which RBCs in hemodialysis patients adhered to T-cells directly correlated with both plasma RANTES (rspearman = 0.65, 95% CI: 0.247–0.864) and CD40L levels (rspearman= 0.60, 95% CI: 0.1645 – 0.847), but not plasma CRP levels. There was also no significant difference in adhesion of RBCs due to cause of renal failure (diabetes, hypertension, or glomerulonephritis) or presence of sickle cell trait. Conclusions:We describe for the first time a novel adhesion of RBCs in patients receiving hemodialysis and how this adhesion may relate to inflammation in these patients. Our findings also suggest that factors in uremic plasma are sufficient to induce phosphatidylserine exposure on RBCs. This exposure, in addition to providing a site for thrombin generation, also serves as an adhesive moiety on RBCs for endothelial cells. These data may describe an unrecognized etiology of inflammation in hemodialysis and end-stage renal disease. Disclosures:No relevant conflicts of interest to declare.

Uremic Plasma Impairs Barrier Function and Depletes the Tight Junction Protein Constituents of Intestinal Epithelium

Background: Chronic kidney disease (CKD) causes intestinal barrier dysfunction which by allowing influx of endotoxin and other noxious products contributes to the CKD-associated systemic inflammation and uremic toxicity. We have recently shown that intestinal barrier dysfunction in CKD animals is due to degradation of transcellular (claudin-1 and occludin) and intracellular (ZO1) constituents of epithelial tight junction (TJ). This study determined whether CKD-associated disruption of TJ is mediated by retained uremic toxins/metabolites and, if so, whether they are removed by hemodialysis. Methods: The TJ-forming human enterocytes (T84 cells) were seeded on the Transwell plates and utilized when transepithelial electrical resistance (TER) exceeded 1,000 mΩ/cm² to ensure full polarization and TJ formation. The cells were then incubated for 24 h in media containing 10% pre- or posthemodialysis plasma from end-stage renal disease (ESRD) patients or healthy individuals. TER was then measured and cells were processed for Western blot and immunohistological analyses. Results: Compared with the control plasma, incubation in media containing predialysis plasma from ESRD patients resulted in a marked drop in TER pointing to increased epithelial permeability. This was accompanied by significant reductions in claudin-1 (85%), occludin (15%), and ZO1 (70%) abundance. The severity of TJ damage and dysfunction was significantly less in cells exposed to the postdialysis in comparison to predialysis plasma. These findings point to the presence of as-yet unidentified product(s) in the uremic plasma capable of depleting epithelial TJ. Conclusions: Exposure to uremic milieu damages the intestinal epithelial TJ and impairs its barrier function, events which are mediated by agents which are partially removed by hemodialysis.

Impact of Frequent Nocturnal Hemodialysis on Myocardial Mechanics and Cardiomyocyte Gene Expression

Regression of left ventricular mass with nocturnal hemodialysis has been observed. The influence of nocturnal hemodialysis on myocardial mechanics and cardiomyocyte gene expression is unknown. Forty-two patients (30 male:12 female; age, 44 ± 12 years [mean ± SD]) with end-stage renal disease were followed for 3.1 ± 1.8 years before and after conversion to nocturnal hemodialysis and were compared with 29 normal subjects (18 male:11 female; age, 48 ± 13 years). Myocardial mechanics were assessed by 2-dimensional velocity vector imaging. Uremic plasma (10%) was added to cultures of neonatal Sprague-Dawley rat ventricular myocytes. Total RNA was isolated from cell cultures and subjected to differential gene expression profiling with specific interest in genes affecting apoptosis and fibrosis. Left ventricular mass index and left atrial volume index decreased from 122.6 ± 42.6 to 98.5 ± 34.9 g/m(2) (P<0.001) and 25.9 ± 9.1 to 22.5 ± 9.6 cm(3)/m(2) (P=0.005), respectively. Left ventricular apical circumferential strain and basal rotation improved after conversion to nocturnal hemodialysis and approximated normal values. Nocturnal hemodialysis increased sessional dialysis dose and lowered parathyroid hormone levels (from 51 ± 67 to 24 ± 37 pmol/L, P<0.05) and phosphate. Under conventional hemodialysis conditions, there was an upregulation of genes leading to apoptosis and fibrosis in cardiomyocytes. The change in left ventricle rotation was associated with the change in parathyroid hormone values (r=0.37, P=0.02) and to the change in left ventricle mass (r=0.31, P=0.046). Frequent hemodialysis is associated with improvement in myocardial mechanics and cardiac gene expression profile, which warrants prognostic validation.

Synthesis and physicochemical properties of the furan dicarboxylic acid, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, an inhibitor of plasma protein binding in uraemia.

The furan dicarboxylic acid, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (5-propyl FPA) accumulates in the plasma of patients with chronic renal failure and is a major contributor to the drug binding defect of uraemic plasma. This acid has also been implicated in several other aspects of the uraemic syndrome: anaemia, irregularities of thyroid function, neurological symptoms and inhibition of active tubular secretion. The acid is not commercially available and its synthesis, starting with Meldrum's acid and methyl succinyl chloride, is described. The pKa values were measured by titration and values of 3.2 and 3.6 respectively were assigned to the carboxylic acid groups attached directly to the ring at position 3 and at position 2 (on the side-chain). The partition coefficient (log P) between hydrochloric acid and octanol was 1.2 and the distribution coefficient (log D; octanol-phosphate buffer pH 7.4) was -0.59. The pKa values and the degree of hydrophobic character of 5-propyl FPA are consistent with those of other protein-bound acids which undergo active tubular secretion by the kidney and this substance may serve as an endogenous marker for the effects of drugs and disease on this process.

Plasma protein carbonylation in chronic uremia

Carbonylation is an irreversible modification caused by the introduction into proteins of carbonyl derivatives (aldehydes and ketones), which can alter protein structure and function and lead to cellular dysfunction and tissue damage. Chronic uremia may be associated with an increased carbonyl overload ("carbonyl stress"), though carbonyl formation has been proposed so far for major plasma proteins only. In this study we looked for evidence and for the targets of plasma protein carbonylation in patients on hemodialysis. We also examined the effect of in vitro carbonylated albumin on mRNA levels of endothelial cell adhesion molecules involved in early atherogenesis. Carbonylated proteins in uremic plasma were detected by a covalent hydrazine bait strategy and identified by combining electrophoretic separation with mass spectrometry analysis of tryptic digests. Some plasma samples were first depleted of albumin and immunoglobulins to improve detection of lower abundance proteins. The functional impact of carbonylation was assessed in human vein endothelial cells by studying models of modified human serum albumin. Post-dialysis plasma carbonylated protein levels were significantly increased compared to pre-dialysis levels. Susceptibility to carbonyl formation was described on a open-platform investigation for a number of plasma proteins, albumin being the main scavenger of carbonyl reactive species. Incubation of endothelial cells with low doses of carbonylated albumin caused a significant increase in intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA levels. Chronic uremia appears as a state of "carbonyl stress" targeting several different plasma proteins. Carbonylated albumin displayed biological effects that may be relevant to uremic atherosclerosis.

Human Uremic Plasma and not Urea Induces Exuberant Secretion of Leptin in 3T3-L1 Adipocytes

Chronic kidney disease (CKD) is frequently associated with malnutrition, anorexia, and hyperleptinemia. This study was designed to test the hypothesis that a component of the uremic milieu may trigger leptin release by adipocytes. To this end, mouse 3T3-L1 adipocytes were incubated for 16 hours in culture medium containing urea (80 mM) or plasma from either healthy volunteers or patients with CKD (20%, v/v). Uremic plasma and not urea induced a large release of leptin (+557%, P < .01). These results suggest that the hyperleptinemia reported in patients with CKD, could be, at least in part, because of an overproduction of leptin by the adipose tissue.

Metabolite Profiling Identifies Markers of Uremia

ESRD is a state of small-molecule disarray. We applied liquid chromatography/tandem mass spectrometry-based metabolite profiling to survey>350 small molecules in 44 fasting subjects with ESRD, before and after hemodialysis, and in 10 age-matched, at-risk fasting control subjects. At baseline, increased levels of polar analytes and decreased levels of lipid analytes characterized uremic plasma. In addition to confirming the elevation of numerous previously identified uremic toxins, we identified several additional markers of ESRD, including dicarboxylic acids (adipate, malonate, methylmalonate, and maleate), biogenic amines, nucleotide derivatives, phenols, and sphingomyelins. The pattern of lipids was notable for a universal decrease in lower-molecular-weight triacylglycerols, and an increase in several intermediate-molecular-weight triacylglycerols in ESRD compared with controls; standard measurement of total triglycerides obscured this heterogeneity. These observations suggest disturbed triglyceride catabolism and/or beta-oxidation in ESRD. As expected, the hemodialysis procedure was associated with significant decreases in most polar analytes. Unexpected increases in several metabolites, however, indicated activation of a broad catabolic program, including glycolysis, lipolysis, ketosis, and nucleotide breakdown. In summary, this study demonstrates the application of metabolite profiling to identify markers of ESRD, provide perspective on uremic dyslipidemia, and broaden our understanding of the biochemical effects of hemodialysis.

In-vitro study on the competitive binding of diflunisal and uraemic toxins to serum albumin and human plasma using a potentiometric ion-probe technique

The competitive binding of diflunisal and three well-known uraemic toxins (3-indoxyl sulfate, indole-3-acetic acid and hippuric acid) to bovine serum albumin (BSA), human serum albumin (HSA) and human plasma was studied by direct potentiometry. The method used the potentiometric drug ion-probe technique with a home-made ion sensor (electrode) selective to the drug anion. The site-oriented Scatchard model was used to describe the binding of diflunisal to BSA, HSA and human plasma, while the general competitive binding model was used to calculate the binding parameters of the three uraemic toxins to BSA. Diflunisal binding parameters, number of binding sites, n(i) and association constants for each class of binding site, K(i), were calculated in the absence and presence of uraemic toxins. Although diflunisal exhibits high binding affinity for site I of HSA and the three uraemic toxins bind primarily to site II, strong interaction was observed between the drug and the three toxins, which were found to affect the binding of diflunisal on its primary class of binding sites on both BSA and HSA molecules and on human plasma. These results are strong evidence that the decreased binding of diflunisal that occurs in uraemic plasma may not be solely attributed to the lower albumin concentration observed in many patients with renal failure. The uraemic toxins that accumulate in uraemic plasma may displace the drug from its specific binding sites on plasma proteins, resulting in increased free drug plasma concentration in uraemic patients.

Heat Treatment Increases the Level of AGEs in Human Blood

92 Levels of plasma pentosidine, which is one of the advanced glycation end-products (AGEs) of the Maillard reaction, are elevated by the pathogenesis of diabetes 1) and end-stage renal failure 2). A recent study suggested that because plasma pentosidine levels in patients with mild renal dysfunction increase before plasma creatinine levels increase, the measurement of plasma pentosidine level can be a useful clinical marker for the early diagnosis of beginning renal failure 3). Several medical laboratories in Japan have measured the levels of blood AGEs, such as Ne-(carboxymethyl)lysine (CML) and pentosidine, by a competitive enzyme-linked immunosorbent assay (ELISA). According to the authors’ protocol, plasma samples are incubated with proteases for anti-AGE antibodies to assess the intramolecular AGE structures, and are incubated again at 100°C for 15 min 3) to inactivate the protease. However, our previous study demonstrated that CML, a major antigenic AGE structure, is generated from the Amadori products by short-term heating in vitro, demonstrating that CML generated from Amadori products are an artifact of immunochemical detection by heating process 4). Furthermore, Miyata et al. 5) reported that substance(s) with a molecular weight of less than 5,000 Da that are abundantly present in uremic plasma exhibit enhanced pentosidine formation during in vitro incubation of uremic plasma at 37°C, thus strongly demonstrating that pentosidine is also generated by incubation in vitro. To confirm this notion, we conducted additional experiments to clarify whether the pentosidine content measured by high-performance liquid chromatography (HPLC) 6) in blood increases using the same procedure as that used by medical laboratories. As a result, the levels of pentosidine in patients with nondiabetic hemodialysis increased 1.2to 3-fold by heating at 100°C for 15 min compared to unheated samples. Moreover, the increased rate of pentosidine levels generated by the heating process was found to differ between patients. Therefore, the heating process as a pretreatment for pentosidine measurement by competitive ELISA appears to compromise the accuracy of the pentosidine concentration present in plasma. Taken together, previous reports 4,5) and the present study indicate that the level of pentosidine in clinical samples is overestimated by using a heating process. Heating enhances both the oxidative cleavage of Amadori products and the production of α-oxoaldehydes such as glucosone, 3-deoxyglucosone, glyoxal, and methylglyoxal, which were indicated to be important precursors for AGE formation 4). We next measured the inhibitory effects of reduction of Amadori products on CML formation. Because CML is generated from the Amadori compounds by oxidative cleavage, whereas the Amadori compounds reduced into hexitol lysine and glucitol lysine are stable and do not easily generate CML, sodium cyanoborohydride (NaBH3CN) is used for the blood CML analysis using a competitive ELISA 7) to prevent CML formation during the heating process. However, the present study demonstrated that preincubation of glycated bovine serum albumin (glycated BSA), a model Amadori proteins, in the presence of 100 mM sodium borohydride (NaBH4), a stronger reducing agent than NaBH3CN, for 1 h did not inhibit CML formation induced by heating at 100°C for 15 min. From these data, it is likely that the conventional protocol used by medical laboratories which measures plasma pentosidine and CML after pretreatment may be in fact quantifying artifacts of heat treatment. Therefore, the establishment of a more reliable method for the quantification of AGEs in patient blood samples will clarify the physiological significance as well as the clinical usefulness of AGEs. Masako Nakano, Ryoji Nagai

Human uraemic plasma stimulates release of leptin and uptake of tumour necrosis factor- in visceral adipocytes

End-stage renal disease (ESRD) is commonly associated with anorexia, malnutrition and inflammation. In addition to serving as the primary reservoir for energy storage, adipocytes produce numerous pro- and anti-inflammatory mediators and regulate food intake by releasing the appetite-suppressing (leptin) and appetite-stimulating (adiponectin) hormones. Under normal conditions, release of leptin is stimulated by feeding to prevent excess intake, and release of adiponectin is stimulated by fasting to induce feeding. However, under certain pathological conditions such as inflammation, maladaptive release of these hormones leads to anorexia, wasting and malnutrition and simultaneously intensifies inflammation. Anorexia, malnutrition and inflammation in ESRD are frequently accompanied by hyper-leptinaemia. This study was designed to test the hypothesis that uraemic plasma may stimulate leptin release and suppress adiponectin release in normal adipocytes. Visceral adipose tissue was harvested from normal rats, and adipocytes were isolated and incubated for 2-4 h in media containing 90% plasma from 12 ESRD patients (before and after haemodialysis) and 12 normal control subjects. The ESRD group had a marked elevation of plasma TNF-alpha, IL-6, IL-8 and leptin concentrations before and after haemodialysis. Incubation in media containing plasma from the ESRD group elicited a much greater leptin release by adipocytes than that containing normal plasma. Post-dialysis plasma evoked an equally intense leptin release. The rise in leptin release was coupled with a parallel fall in TNF-alpha concentration in the incubation media. In contrast to leptin, adiponectin release in the presence of uraemic plasma was similar to that found with the control plasma. Exposure to uraemic plasma induces exuberant release of leptin that is coupled with avid uptake of TNF-alpha by visceral adipocytes. These observations confirm the role of TNF-alpha, formerly known as cachexin, in the over-production and release of leptin in patients with ESRD.