Proteomics in extracorporeal blood purification and peritoneal dialysis

Extracorporeal blood purification and peritoneal dialysis are widely used in renal replacement therapy for patients with end-stage renal disease (ESRD) and acute kidney injury (AKI). Additionally, extracorporeal blood purification can be used also for treatment of non-renal disorders to remove endogenous or exogenous toxins from the blood circulation. Efforts have been made to characterize these toxins removed by diffusion (dialysis), convection (ultrafiltration), and/or adsorption (toxins are adsorbed onto the dialysis membrane and are thus removed) using different types of dialysis membrane. This review summarizes important findings obtained from recent proteomic studies applied to extracorporeal blood purification and peritoneal dialysis in settings of ESRD, AKI and hepatic failure.     

Intracerebral Dialysis and the Blood-Brain Barrier

Abstract: The aim of the study was to evaluate how implantation of a dialysis probe influences the blood-brain barrier. Leakage of endogenous serum albumin was evaluated by Evans blue/albumin staining and by immunohistochemistry. The passage from blood to dialysate of two substances that normally do not pass into the brain, [³H]inulin and glutamate, was studied 3 and 24 h after insertion of a dialysis probe. Evans blue, given 20 min before rats were killed, was observed around the probe and surrounding brain tissue. Albumin immunoreactivity was seen at considerable distance from the probe with larger spread at 24 h than at 3 h after probe insertion. Glutamate and [³H]inulin were detected in the dialysate with no significant further increase of radioactivity after intracarotid infusion of protamine sulfate that enhances the permeability over the blood-brain barrier. When protamine was followed by infusion of glutamate, the concentrations of taurine increased in the dialysate in four of eight rats. That plasma constituents have access to the brain around the dialysis probe is essential to consider, particularly in studies using substances and drugs that do not pass an intact blood-brain barrier.     

Oxidative damage of albumin in advanced liver disease

Albumin has a number of biological functions and the serum albumin level is related to prognosis in advanced liver disease. Oxidative stress is believed to play an important role in the pathogenesis of liver failure. The aim of the present study was to characterize oxidative modification of albumin in patients with various degrees of liver failure and to investigate implications for its binding function. Patients with liver cirrhosis (n=10), acute-on-chronic liver failure (n=8) and healthy controls (n=15) were included in the study. Three fractions of albumin were separated by HPLC according to the redox state of cysteine-34 and detected by fluorescence as well as UV absorption. Carbonyl groups were measured as a marker of oxidative modification in plasma proteins and, by western blotting, on albumin. Progressive oxidative modification of albumin was found with increasing severity of liver failure indicated by an increased content of carbonyl groups and oxidation of cysteine-34. Fluorescence properties of albumin were altered by oxidation and, in patients with acute-on-chronic liver failure, by high plasma levels of bilirubin. This alteration of albumin fluorescence by bilirubin provides evidence for a preferred binding of bilirubin to the fully reduced form of albumin.     

Scope and limitations of in vivo brain dialysis: a comparison of its application to various neurotransmitter systems

Brain dialysis is rapidly becoming a routine research method with a wide range of applications. Since 1982 this sampling technique is frequently used as a method to study the in vivo release of endogenous neurotransmitters such as dopamine, noradrenaline, serotonin, acetylcholine and certain amino acids. In this review most of the studies that have appeared in this field, are evaluated. Special attention was given to the question whether the neurotransmitter content in the dialysate is related to neurotransmission. Criteria such as the presence of a high tissue/dialysate concentration ratio, the sensitivity of the transmitters to membrane active compounds and the occurrence of receptor-mediated effects, are discussed. It is concluded that dopamine, noradrenaline and acetylcholine found in the dialysate are directly derived from neurotransmission, whereas the overflow of excitatory amino acid neurotransmitters is related to neurogenic as well as to metabolic events.     

Fate of parathyroid hormone during hemodialysis and ultrafiltration

In order to shed light on the discrepant changes in plasma immunoreactive parathyroid hormone (iPTH) during hemodialysis (HD) and ultrafiltration (UF) in end-stage renal failure, the influence of filtration of PTH fragments on the iPTH level in plasma was examined in 2 sets of experiments: in vitro dialysis of 125I-bPTH 1-84, 125I-hPTH 1-34 and 125I-hPTH 53-84 added to plasma was successively performed through a cuprophane membrane. Gel filtration on a Biogel P-100 column and subsequent counting of the eluate were performed with the plasma before and after dialysis, and with the dialysate fluid after dialysate fluid after dialysis. An ultrafiltrate obtained from a patient with renal failure was also analyzed for iPTH with a 'C-' and with an 'N-terminal' antiserum (GP 500 MA and AS 211/32), and so was his plasma before and after UF, and after a subsequent dialysis session. Fluid obtained by lavage of the filter with acetic acid after dialysis was also analyzed. Chromatography with measurements of iPTH in the eluate was performed in each case, and the procedure was repeated applying a different transmembrane pressure. Immunoreactive material found in the concentrated ultrafiltrate, but not in plasma, was characterized by means of dilution curves in different RIA performed with the C-terminal antiserum preincubated with various synthetic PTH fragments. Results showed that intact PTH does not cross the cuprophane membrane during both in vitro dialysis and in vivo UF. The 1-34 fragments are poorly dialyzed in vitro; either they stick to the membrane or they are disintegrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selenium,Lead, and Cadmium Levels in Renal Failure Patients in China

Whole blood and serum samples of Chinese stable chronic renal failure (CRF) patients (n = 81), hemodialysis patients (n = 135), posttransplant patients (n = 60), and subjects with normal renal function (NRF; N = 42) were collected, as well as water and dialysate samples from five dialysis centers. The concentration of selenium (Se), lead (Pb), and cadmium (Cd) was measured by atomic absorption spectrometry. The mean serum Se levels in patients with different degrees of renal failure were significantly lower than those of subjects with NRF (p < 0.01). Pb levels were not increased in renal failure patients, while the Cd levels in patients with various degrees of renal failure were higher than in subjects with NRF (p < 0.05). After correcting the results of Pb and Cd for hematocrit (Hct) however, Pb levels of dialysis patients were also increased. In the dialysis population under study, blood Pb and Cd levels were closely related to the time on dialysis, while contamination of the final dialysate may also contribute to the increased blood Cd and to a less extent Pb levels. Correction for Hct may be recommended to accurately compare blood Pb and Cd levels in dialysis patients and CRF patients with varying degrees of anemia to those of subjects with NRF.     

Experimental study of chronic ambulatory peritoneal dialysis. I. Inhibition of protein and amino acid losses by single amino acids

A successful dog model of the continuous ambulatory peritoneal dialysis patient was developed. These preparations were employed in initial studies of the effects of single amino acid-containing dialysis solutions on the losses of protein and amino acids into the dialysate. A decrease of about 40% in the loss of total amino acids into the dialysate was observed when DL-serine-containing dialysis solutions were employed. The addition of DL-serine, L-lysine, or DL-alanine to the dialysis solutions diminished protein loss into the dialysate by 27-55%. DL-Glutamic acid and DL-aspartic acid were ineffective in this regard.     

An experimental, non-uremic rabbit model of peritoneal dialysis

Peritoneal dialysis (PD) is a well established method of depuration in uremic patients. Standard dialysis solutions currently in use are not biocompatible with the peritoneal membrane. Studying effects of dialysate on peritoneal membrane in humans is still a challenge. There is no consensus on the ideal experimental model so far. We, therefore, wanted to develop a new experimental non-uremic rabbit model of peritoneal dialysis, which would be practical, easy to conduct, not too costly, and convenient to investigate the long-term effect of dialysis fluids. The study was done on 17 healthy Chinchilla male and female rabbits, anesthetized with Thiopental in a dose of 0.5 mg/kg body mass. A catheter, specially made from Tro-soluset (Troge Medical GMBH, Hamburg, Germany) infusion system, was then surgically inserted and tunneled from animals' abdomen to their neck. The planned experimental procedure was 4 weeks of peritoneal dialysate instillation. The presented non-uremic rabbit model of peritoneal dialysis is relatively inexpensive, does not require sophisticated technology and was well tolerated by the animals. Complications such as peritonitis, dialysis fluid leakage, constipation and catheter obstruction were negligible. This model is reproducible and can be used to analyze the effects of different dialysis solutions on the rabbit peritoneal membrane.     

Beneficial effects of enalapril on chlorhexidine digluconate-induced liver peritoneal fibrosis in rats

Peritoneal fibrosis (PF) is a recognized complication of long-term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The present study was designed to investigate the protective effects of enalapril on chlorhexidine digluconate-induced liver PF by decreasing transforming growth factor-β1 (TGF-β1) production in rats. PF was induced in Sprague-Dawley rats by daily administration of 0.5 ml 0.1% chlorhexidine digluconate in normal saline via PD tube for one week. Rats received daily intravenous injections of low dose enalapril (1 mg/kg), or high dose enalapril (2.5 mg/kg), for one week. After 7 days, conventional 4.25% Dianeal (30 ml) was administered via a PD catheter with a dwell time of 4 h and assessment of peritoneal function. At the end of dialysis, the rats were sacrificed and liver peritoneum was harvested for microscopic examination and immunohistochemistry. There was no significant difference in mean arterial pressure and heart rate between groups. After 4 h of PD, the D?/P?(urea) level was reduced, the D?/D? glucose level, serum and the dialysate TGF-β1 level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-β1, alpha-smooth muscle actin (α-SMA), fibronectin, collagen and vascular endothelial growth factor (VEGF) were elevated in the PF group compared with the vehicle group. High dose of enalapril decreased the serum and dialysate TGF-β1 levels, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-β1, α-SMA, fibronectin, collagen and VEGF-positive cells in the liver peritoneum. Low dose of enalapril did not protect against chlorhexidine digluconate-induced PF in the rat. Enalapril protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-β1 production.     

Pathophysiological basis of therapy of raised intracranial pressure in acute liver failure

In acute liver failure (ALF) patients that have raised increased intracranial pressure (ICP), mortality remains unacceptably high. There has been an explosion in the knowledge about the pathophysiological basis of raised ICP but treatment modalities are limited. Current therapy is aimed at reducing the circulating ammonia levels and attempts to reduce brain swelling which are only moderately effective. More recently, cerebral hyperemia has been suggested as being of major importance in the pathogenesis of increased ICP providing a new look at interventions such as hyperventilation, N-acetylcysteine, thiopentone sodium and propofol. More recently studies have focused upon the role of systemic inflammatory response in the pathogenesis of increased ICP and support the use of antibiotics prophylactically. The application of moderate hypothermia to treat uncontrolled intracranial hypertension seems promising and its exact place will be decided in a large trial being planned in USA and Europe. Early data from studies in an animal model suggests that albumin dialysis is a promising new tool to treat intracranial hypertension in patients with ALF. The recent advance in our understanding of the pathophysiological basis of intracranial hypertension has provided the platform for the discovery of new treatments.     

In vivo monitoring of norepinephrine and its metabolites in skeletal muscle

Although skeletal muscle sympathetic nerve activity plays an important role in the regulation of vascular tone and glucose metabolism, relatively little is known about regional norepinephrine (NE) kinetics in the skeletal muscle. With use of the dialysis technique, we implanted dialysis probes in the adductor muscle of anesthetized rabbits and examined whether dialysate NE and its metabolites were influenced by local administration of pharmacological agents through the dialysis probes. Dialysate dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured as two major metabolites of NE. The skeletal muscle dialysate NE, DHPG and MHPG were 11.7+/-1.2, 38.1+/-3.2, and 266.1+/-28.7 pg/ml, respectively. Basal dialysate NE levels were suppressed by tetrodotoxin (Na(+) channel blocker, 10 microM) (5.1+/-0.6 pg/ml), and augmented by desipramine (NE uptake blocker, 100 microM) (25.8+/-3.2 pg/ml). Basal dialysate DHPG levels were suppressed by pargyline (monoamine oxidase blocker, 1mM) (24.3+/-4.6 pg/ml) and augmented by reserpine (vesicle NE transport blocker, 10 microM) (75.8+/-2.7 pg/ml). Basal dialysate MHPG levels were not affected by pargyline, reserpine, or desipramine. Addition of tyramine (sympathomimetic amine, 600 microM), KCl (100 mM), and ouabain (Na(+)-K(+) ATPase blocker, 100 microM) caused brisk increases in dialysate NE levels (200.9+/-14.2, 90.6+/-25.7, 285.3+/-46.8 pg/ml, respectively). Furthermore, increases in basal dialysate NE levels were correlated with locally administered desipramine (10, 100 microM). Thus, dialysate NE and its metabolite were affected by local administration of pharmacological agents that modified sympathetic nerve endings function in the skeletal muscle. Skeletal muscle microdialysis with local administration of a pharmacological agent provides information about NE release, uptake, vesicle uptake and degradation at skeletal muscle sympathetic nerve endings.     

Application of creatinine-sensitive biosensor for hemodialysis control

The highly sensitive and selective potentiometric biosensor for creatinine determination has been developed by us earlier. In it, pH-sensitive field effect transistors were used as transducer and immobilized creatinine deiminase (EC 3.5.4.21)--as a biosensitive element. In the work presented, we optimized this biosensor for creatinine analysis in real samples of dialysate in patients with renal failure. The optimized version of biosensor was applied for on-line monitoring of the level of creatinine in the patient's dialysate fluid in the course of dialysis session. High correlation between the biosensor analysis and traditional Jaffe method was demonstrated.     

Effects of moderate hypothermia on in situ cardiac sympathetic nerve endings

Although hypothermia is known to alter neuronal control of circulation, it has been uncertain whether clinically used hypothermia (moderate hypothermia) affects in situ cardiac sympathetic nerve endings. We examined the effects of moderate hypothermia on cardiac sympathetic nerve ending function in anesthetized cats. By use of a cardiac dialysis technique, we implanted dialysis probes in the midwall of the left ventricle and monitored dialysate norepinephrine (NE) levels as an index of NE output from cardiac sympathetic nerve endings. Hypothermia (27.0+/-0.5 degrees C) induced decreases in dialysate NE levels. Dialysate NE levels did not return to the control level at normothermia after rewarming. Dialysate NE response to inferior vena cava occlusion was attenuated at hypothermia but restored at normothermia after rewarming. Dialysate NE response to high K(+) (100 mM) was attenuated at hypothermia and was not restored at normothermia after rewarming. Hypothermia induced increases in dialysate dihydroxyphenylglycol (DHPG) levels. There were no differences in desipramine (neuronal NE uptake blocker, 10 microM) induced increment in dialysate NE level among control, hypothermia, and normothermia after rewarming. However, hypothermia induced an increase in DHPG/NE ratio. These data suggest that hypothermia impairs vesicle NE mobilization rather than membrane NE uptake. We conclude that moderate hypothermia suppresses exocytotic NE release via central mediated reflex and regional depolarization.     

Total Body Potassium in Non-Dialysed and Dialysed Patients with Chronic Renal Failure

Total body potassium was studied in 33 patients with chronic renal failure, 18 of whom had been receiving regular dialysis therapy for 1 to 48 months. In nondialysed patients body potassium was not significantly different from normal in the group as a whole, but was significantly greater than normal in three patients, and significantly less than normal in two patients. In 14 of the dialysed patients, both as individuals and as a group, body potassium was not significantly different from normal but in the remaining four it was less than normal.Potassium transfer during dialysis was studied in two patients. Uptake by these two patients of ⁴³K added to the dialysate (1 mEq K/litre) was measured by whole-body monitoring. Transfer of administered ⁴³K from the patients to the dialysate was measured by whole-body monitoring and by radioactive and chemical assay of the dialysate. A negative balance due to twice-weekly dialysis of 178 and 244 mEq K/week was found, which with weekly faecal and urine losses of 20-30 mEq K approximately equals the dietary intake of 210-315 mEq K.     

Factors affecting the removal of low-molecular-weight fractions (LMWF) from egg yolk and seminal plasma in extended semen by dialysis: effect on post-thaw sperm survival

Three factors affecting dialysis of bovine semen were studied. These factors were (1) dialysis rates of egg yolk, seminal plasma, and glycerol, (2) temperature (37 degrees C, 5 degrees C, and while cooling from 37 to 5 degrees C), and (3) dialysis ratios between retentate and dialysate (1:1, 1:10, 1:20, 1:50, and 1:100). Ninety percent of the low-molecular-weight fraction (LMWF) from seminal plasma, egg yolk, and glycerol was removed from the retentate in a 2-hr period at 5 degrees C, and only slight changes were detected after the third hour of dialysis. Temperature affected dialysis and was faster at 37 degrees C. It was also found that a 1:20 dialysis ratio was sufficient to obtain 90% clearance of the LMWF. The effect of sperm dilution ratio, dialysis ratio, and exchange of the LMWF from egg yolk and/or seminal plasma for buffer systems was also studied. An improvement in post-thaw motility of spermatozoa (P less than 0.05) was obtained when the LMWF from both seminal plasma and egg yolk were replaced. A third experiment was conducted to study the effect of different combinations between the buffer systems, TEST and Na citrate, in the dialysate. The results indicated that a 1:1 combination of iso-osmotic solutions (320-325 mOsm/Kg, pH 7.0) between these two buffers, with 5% glycerol (v/v), yielded significant (P less than 0.05) sperm post-thaw motility as compared with the individual use of TEST-glycerol or Na citrate-glycerol. Dialyzed samples also yielded sperm post-thaw motility higher than that of the nondialyzed samples. Colloidal materials in the dialysate did not affect survival of spermatozoa.     

Potassium Balance and Acid-Base Changes in Patients Undergoing Regular Haemodialysis Therapy

Serial measurements of total body potassium in 21 patients with chronic renal failure being treated with three 10-hour periods of dialysis per week, against a dialysate fluid containing 1·5 mEq of potassium per litre, showed no evidence of potassium depletion. Mild hyperkalaemia was found in some patients before dialysis, correlated with the pre-dialysis hydrogen ion concentration. Hypokalaemia occurred during dialysis in almost half of the studies made; the plasma potassium concentration, however, rose to normal levels within two to four hours of stopping dialysis. A delay in the movement of potassium from the cells into the extracellular fluid is suggested as a cause for the observed hypokalaemia.In all but one patient the pre-dialysis blood pH was normal, but rose to alkalaemic levels during dialysis. A pronounced degree of hypocapnia was noted before dialysis, and this was not altered by a rising blood pH during dialysis. It is suggested that a stimulus to respiration other than the hydrogen ion gradient between the brain cells and cerebral spinal fluid may produce the observed hypocapnia.     

平衡型氨基酸透析液对维持性血液透析患者血浆游离氨基酸含量的影响

目的:研究一种平衡型氨基酸透析液,探讨其对维持性血透患者血游离氨基酸含量的影响。方法:实验采用自身对照设计,应用蛋白水解法测定、分析应用氨基酸透析液血透前后患者血浆氨基酸含量,并同时测定应用碳酸盐透析液患者及健康人血浆游离氨基酸作为比较。结果:经碳酸盐透析后,患者血浆大多数游离氨基酸和总氨基酸含量显著降低。给予平衡氨基酸透析液能不同程度改善患者血浆游离氨基酸的含量。结论:平衡型氨基酸透析液能减少血中部分氨基酸的丢失。     

Binding of lysophosphatidylcholine to the rat liver fatty acid binding protein

In this study, lysophosphatidylcholine (lysoPC) was shown to bind to a fatty acid binding protein isolated from rat liver. To demonstrate the binding, lysoPC was incorporated into multilamellar liposomes and incubated with protein. For comparison, binding of both lysoPC and fatty acid to liver fatty acid binding protein, albumin, and heart fatty acid binding protein were measured. At conditions where palmitic acid bound to liver fatty acid binding protein and albumin at ligand to protein molar ratios of 2:1 and 5:1, respectively, lysoPC binding occurred at molar ratios of 0.4:1 and 1:1. LysoPC did not bind to heart fatty acid binding protein under conditions where fatty acid bound at a molar ratio of 2:1. Competition experiments between lysoPC and fatty acid to liver fatty acid binding protein indicated separate binding sites for each ligand. An equilibrium dialysis cell was used to demonstrate that liver fatty acid binding protein was capable of transporting lysoPC from liposomes to rat liver microsomes, thereby facilitating its metabolism. These studies suggest that liver fatty acid binding protein may be involved in the intracellular metabolism of lysoPC as well as fatty acids, and that functional differences may exist between rat liver and heart fatty acid binding protein.     

Calcitriol decreases TGF-β1 and angiotensin II production and protects against chlorhexide digluconate-induced liver peritoneal fibrosis in rats

Peritoneal fibrosis is a major complication of peritoneal dialysis that can lead to ultrafiltration failure. This study investigates the protective effects of calcitriol on chlorhexidine digluconate-induced peritoneal fibrosis in rats. Peritoneal fibrosis was induced in Sprague-Dawley rats by daily administration of 0.5 mL 0.1% chlorhexidine digluconate in normal saline via peritoneal dialysis for 1 week. Rats received daily intravenous injections of calcitriol (low-dose, 10 ng/kg; or high-dose, 100 ng/kg) for 1 week. After 7 days, conventional 4.25% Dianeal (30 mL) was administered via peritoneal dialysis over 4 h. Peritoneal solute transport was calculated from the dialysate concentration relative to its concentration in the initial infused dialysis solution (D₄/D_{0 glucose}) for glucose, and the dialysate-to-plasma concentration ratio (D₄/P_{4 urea}) at 4 h for urea. Rats were then sacrificed and the liver peritoneum was harvested for immunohistochemical analysis via microscopy. After dialysis, the D₄/P_{4 Urea} level was reduced; increases were observed in the D₄/D_{0 glucose} level and the levels of active transforming growth factor-β1 and angiotensin II in serum and dialysate; the liver peritoneum and muscle peritoneum was markedly thickened, and the expression of α-SMA, fibronectin, collagen, vascular endothelial growth factor, angiotensin II, transforming growth factor-β1, and phosphorylated Smad2/3 (P-Smad2/3)-positive cells in the liver peritoneum was elevated in the peritoneal fibrosis group compared with the vehicle group. Calcitriol decreased the serum and dialysate active transforming growth factor-β1 and angiotensin II level, decreased the thickness of the liver peritoneum and muscle peritoneum, and decreased the expression of α-SMA, fibronectin, collagen, vascular endothelial growth factor, angiotensin II, transforming growth factor-β1, and P-Smad2/3-positive cells in liver peritoneum cells. High-dose calcitriol exhibited better protective effects against peritoneal fibrosis than did the lower dose. Calcitriol protected against chlorhexidine digluconate-induced peritoneal fibrosis in rats by decreasing transforming growth factor-β1 and angiotensin II production.     

蛋白结合尿毒症毒素及其清除技术的研究进展

尿毒症毒素是一大组体内代谢的产物,在肾功能衰竭患者体液中水平明显升高,并与尿毒症毒素代谢紊乱或临床表现密切相关。部分毒素可与蛋白结合,形成大分子复合物,称为蛋白结合毒素。它们具有多种生物学作用,产生一系列尿毒症并发症,如心血管疾病、免疫功能紊乱、脏器纤维化等。研究发现:血浆分离吸附、高通量血液透析、服用肠道吸附剂等方法可增加蛋白结合毒素的清除。评价尿毒症患者的透析充分性时,也应考虑到蛋白结合毒素。