In the rat liver, Adenoviral gene transfer efficiency is comparable to AAV.

Adenoviral (AdV) and Adenovirus-associated viral (AAV) vectors both are used for in vivo gene therapy of inherited liver disorders, such as Crigler-Najjar syndrome type 1. In a relevant animal model, the Gunn rat, both vectors efficiently correct the severe hyperbilirubinemia characteristic of this liver disorder. Although the clinical use of AAV is more advanced, as demonstrated by the successful phase 1 trial in hemophilia B patients, because of its large cloning capacity AdV remains an attractive option. A direct comparison of the efficacy of these two vectors in the liver in a relevant disease model has not been reported. Aim of this study was to compare the efficiency of clinically applicable doses of both vectors in the Gunn rat. AdV or scAAV (self-complimentary AAV) ferrying identical liver-specific expression cassettes of the therapeutic gene, UGT1A1, were injected into the tail vein. As the titration methods of these two vectors are very different, a comparison based on vector titers is not valid. Therefore, their efficacy was compared by determining the amount of vector genomes delivered to the liver required for therapeutic correction of serum bilirubin. Like AAV, the liver-specific first-generation AdV also provided sustained correction in this relevant disease model. UGT1A1 mRNA expression provided per genome was comparable for both vectors. Flanking the expression cassette in AdV with AAV-ITRs (inverted terminal repeats), increased UGT1A1 mRNA expression eightfold which resulted in a significant improvement of efficacy. Compared with AAV, less AdV genomes were needed for complete correction of hyperbilirubinemia.

Absorption, distribution, and biliary excretion of cafestol, a potent cholesterol-elevating compound in unfiltered coffees, in mice.

Cafestol is a diterpene present in unfiltered coffees. It is the most potent cholesterol-elevating compound present in the human diet. However, the precise mechanisms underlying this effect are still unclear. In contrast, cafestol is also known as a hepatoprotective compound, which is likely to be related to the induction of glutathione biosynthesis and conjugation. In the present study, we investigated whole-body distribution, biliary excretion, and portal bioavailability of cafestol in mice. First, dissection was used to study distribution. Five hours after an oral dose with (3)H-labeled cafestol, most activity was found in small intestine, liver, and bile. These results were confirmed by quantitative whole-body autoradiography in a time course study, which also showed elimination of all radioactivity within 48 h after administration. Next, radiolabeled cafestol was dosed intravenously to bile duct-cannulated mice. Five hours after the dose 20% of the radioactivity was found in bile. Bile contained several metabolites but no parent compound. After intestinal administration of radioactive cafestol to portal vein-cannulated mice, cafestol was shown to be rapidly absorbed into the portal vein as the parent compound, a glucuronide, and an unidentified metabolite. From the presence of a glucuronide in bile that can be deconjugated by a bacterial enzyme and the prolonged absorption of parent compound from the gastrointestinal tract, we hypothesized that cafestol undergoes enterohepatic cycling. Together with our earlier observation that epoxidation of the furan ring occurs in liver, these findings merit further research on the process of accumulation of this coffee ingredient in liver and intestinal tract.

Carcinogen and anticancer drug transport by Mrp2 in vivo: studies using Mrp2 (Abcc2) knockout mice.

The ATP-binding-cassette (ABC) transporter multidrug resistance protein (MRP) 2 (ABCC2) forms a natural barrier and efflux system for various (conjugates of) drugs, other xenotoxins, and endogenous compounds. To obtain insight in the pharmacological and physiological functions of Mrp2, we generated Mrp2 knockout mice, which were viable and fertile but suffered from mild hyperbilirubinemia due to impaired excretion of bilirubin monoglucuronides into bile. The mice also had an 80-fold decreased biliary glutathione excretion and a 63% reduced bile flow. Levels of Mrp3 (Abcc3) in liver and Mrp4 (Abcc4) in kidney of Mrp2-/- mice were approximately 2-fold increased. After oral administration of the food-derived carcinogens [(14)C]PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and [14C]IQ (2-amino-3-methylimidazo[4,5-f]quinoline) plasma values were 1.9- and 1.7-fold higher in Mrp2-/- mice versus wild-type mice, respectively, demonstrating the role of Mrp2 in restricting exposure to these compounds. At a high dose of 50 mg/kg of the drug [3H]methotrexate, the plasma area under the curve for i.v. administration was 1.8-fold higher in Mrp2-/- mice (1345+/-207 versus 734+/-81 min.microg/ml). No clear plasma concentration difference arose at low dose (1 mg/kg). Subsequently, Mdr1a/b/Mrp2 knockout mice were generated. Their biliary excretion of doxorubicin after i.v. administration (5 mg/kg) was 54-fold decreased (0.32+/-0.13 versus 17.30+/-6.59 nmol/g liver in wild type), and a role for both Mdr1a/b and Mrp2 in this process was revealed. Our results demonstrate that the Mrp2-/- mouse provides a valuable tool for studies of the impact of Mrp2 on behavior of drugs and other toxins, especially when combined with other ABC transporter knockout mice.

Influence of biliary cirrhosis on the detoxification and elimination of a food derived carcinogen.

BACKGROUND AND AIMS: The liver is the central organ for the detoxification of numerous xenobiotics, including carcinogens. We studied the influence of cholestasis and biliary cirrhosis on the detoxification, elimination, and tissue distribution of a model compound and food derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). METHODS: Wistar rats were injected with (14)C-PhIP into the portal vein one or six weeks after common bile duct ligation (CBDL). Bile flow was reconstituted, bile and urine were collected over 120 minutes, and metabolites were analysed using high performance liquid chromatograpy. Total tissue radioactivity levels in several organs as well as tissue bound (ethanol insoluble tissue fraction) radioactivity levels were determined. RESULTS: Significant downregulation of the transport proteins multidrug resistance associated protein 2 and breast cancer resistance protein was observed in biliary cirrhosis. Biliary excretion of radioactivity was significantly reduced in cholestasis and biliary cirrhosis compared with controls (15 (2.9)% and 3.2 (1)% of the dose v 36.5 (2)%, respectively). Phase II metabolism was severely reduced in cirrhotic rats, resulting in a twofold increase in tissue radioactivity levels in the liver, kidney, and colon. Biliary cirrhosis increased tissue binding of reactive metabolites, as expressed in cpm/100 mg tissue in the liver and the colon (3267 (1218) v 1191 (429) in the liver, 3044 (1913) v 453 (253) in the colon). CONCLUSIONS: Biliary cirrhosis induced by CBDL causes impaired metabolism and elimination of PhIP, and leads to higher tissue levels of potentially genotoxic metabolites in the liver and colon of rats. These data may explain the increased incidence of hepatic and extrahepatic cancers in cholestasis and liver cirrhosis.

Biliary anionic peptide fraction/calcium binding protein inhibits apolipoprotein A-I-mediated cholesterol efflux from cultured cells.

The ABC transporter ABCA1 has been implicated to control cholesterol efflux in a variety of cell types including macrophages, fibroblasts, and intestinal epithelial cells. In this study we have investigated whether the 6-kD protein anionic peptide fraction/calcium binding protein (APF/CBP) which has homology to apolipoprotein AI may regulate efflux mediated by lipoproteins. APF/CBP was purified from T-tube bile by ultracentrifugation and preparative reversed phase HPLC. Cholesterol efflux to a variety of acceptors was determined using cultured fibroblasts from controls and patients with Tangiers disease. APF/CBP (0.1 to 2.4 microg/ml) inhibited ApoA-1 (2 microg/ml) mediated cholesterol efflux from normal fibroblasts in a dose dependent manner but had no effect on aspecific efflux to methyl-beta-cyclodextrin or phosphatidylcholine liposomes. In ABCA1 deficient fibroblasts no effect of APF/CBP on efflux was seen. We conclude that APF/CBP specifically interferes with ApoA-I mediated cholesterol trafficking. We hypothesize that competitive binding to ABCA1 may explain the decreased ApoA-I mediated efflux from fibroblasts.

Role of MRP2 and GSH in intrahepatic cycling of toxins.

MRP2 is a canalicular transporter in hepatocytes mediating the transport of a wide spectrum of amphipathic compounds. This includes organic anions but also compounds complexed with GSH as, e.g. alpha-naphthylisothiocyanate (ANIT) and arsenite. These reversible complexes may fall apart in bile after MRP2-mediated transport, which induces high concentrations of the toxic compound in the biliary tree. To further investigate the role of MRP2 in transport and toxicity of both compounds, we conducted experiments in transduced polarized epithelial cells and in vivo, using the Mrp2-deficient TR(-) rat as a model. Our results show, that in MRP2-transduced MDCK II cells both compounds induce disproportionally strong apical GSH secretion. This induction of GSH secretion was not observed in the parent cells lacking MRP2 expression. This indicated that after transport via MRP2 both complexes released GSH upon which the compound could re-enter the cells. The resulting cycling of both toxins led to concentration dependent GSH depletion of the cells. To further test our hypothesis we administered arsenite (12.5 micromol absolute i.v.) to Wistar and Mrp2-deficient TR(-) rats and collected bile. While both arsenite and GSH secretion were absent in TR(-) rats, the total secretion of arsenite into Wistar bile (2.91 micromol) was accompanied by a excess secretion of 24 micromol GSH, indicating that arsenite undergoes multiple cycles of GSH complexation. We also administered ANIT to both animal models and could show that TR(-) rats are protected from ANIT induced cholestasis. This indicates that Mrp2-mediated biliary secretion of GS-ANIT is a prerequisite for development of cholestasis in rats. We hypothesize that the toxic parent compound ANIT is regenerated in the biliary tree where it can exert its toxic properties on bile duct epithelial cells.

Amphotericin B, mercury chloride and peritoneal transport in rabbits.

BACKGROUND: The effect of glucose-induced ultrafiltration in peritoneal dialysis is dependent on the presence and function of ultrasmall transendothelial cell water channels. The mercury-sensitive aquaporin-1 was thought to represent these transcellular pores. Amphotericin B (ampho B) has been reported to increase ultrafiltration in both experimental and patient studies. The objective of this study was to investigate the hypothesis that intraperitoneal ampho B increases and mercury chloride inhibits aquaporin-1-mediated water transport in a chronic peritoneal dialysis model in the rabbit. MATERIAL AND METHODS: Eighteen female New Zealand White rabbits were included for peritoneal catheter implantation. Peritoneal transport parameters were determined in all rabbits by standard peritoneal permeability analysis (SPAR) with 3.86% glucose-based dialysis solution during a one-hour dwell prior the intervention SPARs, as a control. Ampho B (0.06 mg/kg body weight) was added to the dialysate for 3 (n = 9) or 5 consecutive days (n = 5) before investigation. Four rabbits were investigated after 3-day i.p. 0.6 mg/kg body weight ampho B. In 3 rabbits 0.06 mg/kg body weight liposomal ampho B was administered i.p. during 3 days before intervention SPAR. Fifteen rabbits were investigated during a one-hour dwell with 0.1 mM HgCl2 containing 3.86% glucose-based dialysis solution, while they were anesthetized. Three of these underwent in vivo fixation with glutaraldehyde prior to the HgCl2 SPAR to prevent toxic effects of mercury on peritoneal tissues. RESULTS: Intraperitoneal administration of ampho B did enhance the change in intraperitoneal volume during a one-hour dwell after 3-day i.p. treatment with the low dose (p < 0.02), but it did not affect peritoneal solute permeability. This was likely mediated by transcellular water channels, but not by aquaporin-1. No beneficial effects on the ultrafiltration were found with prolonged treatment or with the higher dose. Ultrafiltration decreased (8 ml/4 h to 1 ml/4 h, p < 0.03) after i.p. administration of HgCl2 with and without in vivo fixation, accompanied by a significant decrease in aquaporin-mediated water transport, estimated as the sieving of sodium (p < 0.001). Marked increases in the clearances of macromolecules were found after i.p. HgCl2 administration due to toxic effects: total protein clearance from 97 to 172 microl/min, p < 0.005, and albumin clearance from 59 to 158 microl/min, p < 0.005. These changes were less pronounced after in vivo fixation. CONCLUSION: Ampho B has likely no clinical relevance in treatment of ultrafiltration failure in PD patients. Aquaporin-mediated water transport could be inhibited and consequently ultrafiltration was reduced by i.p. administration of mercury chloride in our rabbit model.

Icodextrin degradation products in spent dialysate of CAPD patients and the rat, and its relation with dialysate osmolality.

OBJECTIVE: Peritoneal dialysis (PD) with a 7.5% icodextrin-containing dialysis solution provides prolonged ultrafiltration compared with glucose-based dialysis solutions. Colloid osmosis is the most likely mechanism, but studies in rats suggest it is caused by an increase in osmolality due to degradation of icodextrin. Therefore, human spent dialysate was analyzed with high-performance liquid chromatography (HPLC) using gel permeation size-exclusion chromatography. An increasing peak (with a low molecular weight, < 1000 Da) was observed during the dwell. The aim of this study was to quantitate breakdown products of icodextrin (which could explain this peak) and investigate whether there was a relationship with dialysate amylase concentration and dialysate osmolality. DESIGN: Long-dwell effluents (dwell time 9.15- 14.30 hours) obtained from 12 PD patients using a 7.5% icodextrin solution during the night were analyzed. The following icodextrin breakdown products were measured: maltotetraose (G4), maltotriose (G3), maltose (G2), and glucose (G1). In 6 of these patients, the sugars maltoheptaose (G7), maltohexaose (G6), and maltopentaose (G5) were also determined in both effluent and plasma. In addition, G4, G3, G2, and G1 were measured in four Wistar rats during a 6-hour dwell study. RESULTS: In the human studies, the median distribution of the sugars in the effluent was G4,6.7%; G3,16.5%; G2, 23.1%; and G1, 53.5%. The osmolality in spent dialysate ranged between 288 and 326 mOsm/kg H2O. The median contribution of the sugars G2 - G4 was 5.4 mOsm/kg H2O. No correlation was present between dialysate osmolality and duration of the dwell (r= -0.04, p= 0.91); nor was there a relation between the concentration of G2 and duration of the dwell (r = 0.50, p = 0.10). No relationship was found between the amount of amylase and the concentration of G2 in the effluent (r = 0.49, p = 0.10), nor between the total concentration of the sugars G2 - G4 in the spent dialysate and dialysate osmolality (r = -0.31, p = 0.33). However, a strong correlation was seen between urea concentration and osmolality (r= 0.85, p < 0.001), and also between sodium concentration and dialysate osmolality in the spent dialysate (r = 0.92, p < 0.0001). The levels of the sugars G2, G3, and G4 in effluent were higher than in unused dialysate, but lower than or similar to plasma levels. Concentrations of the sugars G5, G6, and G7 were lower in spent dialysate than in unused dialysate, and higher than in plasma. In the rat study, dialysate osmolality increased with the duration of the dwell. A clear relationship was present between osmolality and concentration of the sugars G2 - G4 in the effluent. The median amount of amylase in the effluent was 1252 U/L. CONCLUSION: A 7.5% icodextrin-based dialysis solution used during the long exchange caused only a slight increase in dialysate osmolality in humans. The osmolality at the end of the dwell in the human situation was dependent mainly on concentrations of the small solutes urea and sodium in the effluent. The contribution of icodextrin degradation products was marginal. In the rat, however, a clear relationship was present between osmolality and icodextrin degradation products in spent dialysate, explaining the increased dialysate osmolality at the end of the dwell. The difference between the two species can be explained by the very high amylase concentrations in the rat, leading to a rapid degradation of icodextrin. The rat is therefore not suitable to study peritoneal fluid kinetics using icodextrin as an osmotic agent.

Increased bioavailability of the food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in MRP2-deficient rats.

MRP2 is an apical transporter expressed in hepatocytes and the epithelial cells of the small intestine and kidney proximal tubule. It extrudes organic anions, conjugated compounds, and some uncharged amphipaths. We studied the transport of an abundant food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in vitro, using an MRP2 transfected epithelial cell line (MDCK II) and intestinal explants from Wistar and MRP2-deficient TR(-) rats in Ussing chambers. In the experiments with the transfected cell line, we could demonstrate more than 3-fold higher transport from basolateral to apical than vice versa, whereas the transport in the parent cell line was equal in both directions. These results were confirmed in studies using isolated pieces of small intestine from Wistar and TR(-) rats in the Ussing chamber. Subsequent in vivo experiments demonstrated that after oral administration, absorption of PhIP was 2-fold higher in the TR(-) rat than in the Wistar rat. Consequently, PhIP tissue levels in several organs (liver, kidney, lung, and colon) were 1.7- to 4-fold higher 48 h after oral administration. MRP2 mediated transport of unchanged PhIP probably involves intracellular GSH, because GSH depletion by BSO-treatment in Wistar rats reduced intestinal secretion in the Ussing chamber to the same level as in TR(-) rats. In accordance, BSO treatment increased oral bioavailability in intact Wistar rats. This study shows for the first time that MRP2-mediated extrusion reduces oral bioavailability of a xenobiotic and protects against an abundant food-derived carcinogen.

Mrp2-deficiency in the rat impairs biliary and intestinal excretion and influences metabolism and disposition of the food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo.

While metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant food-derived heterocyclic amine and carcinogen, has been studied extensively in several species, transport of this compound and its metabolites has not been defined yet. Therefore we studied metabolism and disposition of PhIP in Wistar and Mrp2-deficient TR(-) rats to determine the role of Mrp2 in the defence against this compound. In the first 2 h after intravenous dosing, total excretion of PhIP and its metabolites in bile was > 4-fold reduced in TR(-) rats compared with Wistar rats, while excretion in the urine of the TR(-) rat was 1.8-fold higher. This difference was the result of an almost complete absence of secretion of glucuronidated metabolites but also a reduced level of secretion of unchanged PhIP into bile of the TR(-) rat. Direct intestinal excretion of unmetabolized PhIP was 3-fold higher in Wistar versus TR(-) rats. As a consequence, PhIP tissue levels in the liver were 1.7-fold higher in TR(-) rats, and tissue binding of PhIP, determined after ethanol extraction, was elevated by a similar magnitude. Mrp2-mediated transport of the parent compound PhIP is glutathione (GSH)-dependent, because GSH depletion by L-buthionine-[S,R]-sulfoximine (BSO) treatment in Wistar rats reduced intestinal secretion to the same level as that in TR(-) rats. TR(-) rats produced less glucuronides and 4'-OH-PhIP in the 2 h following PhIP administration. We conclude that Mrp2 protects against the carcinogen PhIP by biliary excretion of the parent compound and all major phase-II metabolites, but, more importantly, also by direct extrusion of the parent compound from the gut mucosa.

Prostaglandin inhibition by intraperitoneal indomethacin has no effect on peritoneal permeability during stable CAPD.

BACKGROUND: Prostaglandins can affect the vascular response and are locally produced in the peritoneal cavity. Prostaglandin inhibition in continuous ambulatory peritoneal dialysis (CAPD) patients during peritonitis using indomethacin intraperitoneally was found to decrease the intrinsic permeability to macromolecules. METHODS: In the present study the effects of prostaglandin inhibition were studied during stable, uninfected CAPD. Two standard peritoneal permeability analyses (1.36% glucose) were performed in 10 stable CAPD patients within 1 week with and without addition of 12.5 mg/l indomethacin. Furthermore, possible effects on the parameters of nitric oxide synthesis were determined. In five other patients a high dose of indomethacin was tested. The night before the indomethacin test, 12.5 mg/l indomethacin was added to the nightdwell and the test was performed with 25 mg/l indomethacin. RESULTS: In the normal dose indomethacin group, the dialysate concentrations of prostaglandin (PG) 6-keto-PGF1alpha and thromboxane (Tx) TxB2 were significantly lower with indomethacin (IND) compared with the control dwell (C): 6-keto-PGF1alpha median 93 (C) vs 7.5 (IND) ng/l, P=0.006 and TxB2 12.3 (C) vs 9.0 (IND) ng/l, P=0.04. The dialysate concentration of PGE2 was not different during the control dwell (68.5 ng/l) compared with the indomethacin experiment (50.3 ng/l, P=0.5). The mass transfer area coefficients (MTAC) of nitrate and cGMP, and parameters of nitric oxide synthesis, were similar during both experiments. The MTAC of creatinine and urate were not different with indomethacin: creatinine median 9.5 (C) vs 10.2 ml/min (IND), P=0.2 and urate 7.2 (C) vs 7.3 ml/min (IND), P=0.3. Only the MTAC of urea was marginally higher with indomethacin: 16.0 (C) vs 16.6 ml/min (IND), P=0.04. No differences were found in the clearances of the macromolecules beta2-microglobulin, albumin, IgG and alpha2-microglobulin. With the high indomethacin dose no inhibition of PGE2 was found: 69 (C) vs 63 ng/l(IND), not significant. Furthermore, no differences were found in the transport rates of small solutes or proteins. This indicates no effect of indomethacin on the peritoneal surface area and the size-selective permeability to macromolecules. In both groups no effect was found on the transcapillary ultrafiltration and the effective lymphatic absorption rate during the 4-h dwell. Consequently, the net ultrafiltration, the difference between these, did not change. CONCLUSIONS: The indomethacin induced inhibition of the synthesis of 6-keto-PGF1alpha and TxB2 did not lead to alterations in functional parameters of the peritoneal surface area, the intrinsic permeability to macromolecules and fluid kinetics. Therefore, these prostaglandins are not likely to be involved in the regulation of peritoneal transport during stable CAPD.

Lack of UGT1 isoforms in Gunn rats changes metabolic ratio and facilitates excretion of the food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo.

UDP-glucuronosyltransferases (UGTs) play an important role in detoxification of endo- and xenobiotics. Deficiencies of these enzymes can have serious consequences, for example, in Crigler-Najjar disease Type I. Recently it was shown that the activated form of the abundant food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is glucuronidated mainly by UGT1 isoforms. Therefore UGT1 deficiency may have an important impact on metabolism and excretion of PhIP in the body and consequently for the susceptibility toward carcinogenic effects through PhIP. To test this hypothesis we investigated fate and distribution of PhIP in the UGT1-deficient Gunn rat. In 2 h after intravenous injection of PhIP, Gunn rats excreted significantly more PhIP and metabolites than control animals, which were age- and weight-matched Wistar rats. In bile, both glucuronides of N-OH-PhIP were reduced but, in urine, only the N3-glucuronide was reduced while the N2-glucuronide was elevated. The metabolic pathway ratio between 4'-hydroxylation and N-hydroxylation was dramatically changed in the Gunn rat (five times higher in bile and doubled in urine, resulting in a four times higher ratio in total), mostly because of the doubled amount of 4'-PhIP-sulfate in Gunn rats compared to Wistar rats. Tissue levels of PhIP and metabolites were significantly lower in liver and colon of the Gunn rats. We conclude that, in Gunn rats, PhIP is alternatively metabolized through UGT2B enzymes and sulfotransferases, which adds another clue to the potential importance of sulfotransferases in detoxification of PhIP.

Expression of cancer antigen 125 by peritoneal mesothelial cells is not influenced by duration of peritoneal dialysis.

OBJECTIVE: To investigate the presence of cancer antigen 125 (CA125) on mesothelial cells in the effluent of peritoneal dialysis (PD) patients and to analyze the effect of duration of PD on the number of mesothelial cells in peritoneal effluent, the number of CA125-positive cells, and dialysate CA125 concentration. DESIGN: A cross-sectional study in which long-dwell peritoneal effluents were investigated for mesothelial cells and CA125. SETTING: A university hospital population of chronic PD patients. PATIENTS: 33 stable PD patients who were free of peritonitis during the investigation and during the 4 weeks prior to the study. METHODS: Examination of cytospin preparations of peritoneal effluent stained with May-Grünwald Giemsa, and also with an immunocytochemical double-staining method consisting of anticalretinin (pan-mesothelial cell marker) and OC125. RESULTS: A close relationship was present between the numbers of mesothelial cells counted with the two staining methods (r= 0.998, p < 0.001). On average, 92% of mesothelial cells were positive for CA125, ranging between 75% and 100% in 80% of the patients. Correlations were found between the effluent CA125 concentration and the total number of mesothelial cells (r = 0.64, p < 0.001), and also the number of CA125-positive cells (r = 0.66, p < 0.001). A negative effect of time was seen on the effluent CA125 concentration, the total number of mesothelial cells, and the number of CA125-positive mesothelial cells. However, no effect of time was present on the percentage CA125-positive cells. CONCLUSIONS: On average, 92% of mesothelial cells in peritoneal effluent are positive for CA125. This figure is not dependent on the duration of PD. Long-term PD is associated with low dialysate CA125 concentrations, a low number of mesothelial cells, and a low number of CA125-positive mesothelial cells in effluent. These results support the hypothesis that dialysate CA125 can be used as a marker of mesothelial cell mass in stable PD patients.

Peritoneal transport characteristics with glycerol-based dialysate in peritoneal dialysis.

BACKGROUND: Glycerol is a low molecular weight solute (MW 92 D) that can be used as an osmotic agent in continuous ambulatory peritoneal dialysis (CAPD). Due to its low molecular weight, the osmotic gradient disappears rapidly. Despite the higher osmolality at the beginning of a dwell, ultrafiltration has been found to be lower for glycerol compared to glucose (MW 180 D) when equimolar concentrations are used. Previous studies have shown glycerol to be safe for long-term use, but some discrepancies have been reported in small solute transport and protein loss. OBJECTIVE: To assess permeability characteristics for a 1.4% glycerol dialysis solution compared to 1.36% glucose. DESIGN: Two standardized peritoneal permeability analyses (SPA), one using 1.4% glycerol and the other using 1.36% glucose, in random order, were performed within a span of 2 weeks in 10 stable CAPD patients. The length of the study dwell was 4 hours. Fluid kinetics and solute transport were calculated and signs of cell damage were compared for the two solutions. SETTING: Peritoneal dialysis unit in the Academic Medical Center, Amsterdam. RESULTS: Median values for the 1.4% glycerol SPA were as follows: net ultrafiltration 251 mL, which was higher than that for 1.36% glucose (12 mL, p < 0.01); transcapillary ultrafiltration rate 2.12 mL/min, which was higher than that for glucose (1.52 mL/min, p = 0.01); and effective lymphatic absorption rate 1.01 mL/min, which was not different from the glucose-based solution. Calculation of peritoneal reflection coefficients for glycerol and glucose showed lower values for glycerol compared to glucose (0.03 vs 0.04, calculated with both the convection and the diffusion models). A marked dip in dialysate-to-plasma ratio for sodium was seen in the 1.4% glycerol exchange, suggesting uncoupled water transport through water channels. Mass transfer area coefficients for urea, creatinine, and urate were similar for both solutions. Also, clearances of the macromolecules beta2-microglobulin, albumin, IgG, and alpha2-macroglobulin were not different for the two osmotic agents. The median absorption was higher for glycerol, 71% compared to 49% for glucose (p < 0.01), as could be expected from the lower molecular weight. The use of a 1.4% glycerol solution during a 4-hour dwell caused a small but significant median rise in plasma glycerol, from 0.22 mmol/L to 0.45 mmol/L (p = 0.02). Dialysate cancer antigen 125 and lactate dehydrogenase (LDH) concentrations during the dwell were not different for both solutions. CONCLUSIONS: These findings show that glycerol is an effective osmotic agent that can replace glucose in short dwells and show no acute mesothelial damage. The higher net ultrafiltration obtained with 1.4% glycerol can be explained by the higher initial net osmotic pressure gradient. This was seen especially in the first hour of the dwell. Thereafter, the osmotic gradient diminished as a result of absorption. The dip in dialysate-to-plasma ratio for sodium seen in the glycerol dwell can also be explained by this high initial osmotic pressure gradient, implying that the effect of glycerol as an osmotic agent is more dependent on intact water channels than is glucose.

Growth factors VEGF and TGF-beta1 in peritoneal dialysis.

The morphologic alterations in the kidney and the retina that can be present in patients with diabetic microangiopathy are mediated by growth factors. Vascular endothelial growth factor (VEGF) is a mediator of neoangiogenesis in diabetic retinopathy. Transforming growth factor-beta (TGF-beta) is involved in the extracellular matrix proliferation in diabetic nephropathy. The aim of the present study was to investigate the presence of VEGF and TGF-beta1 in peritoneal effluents of patients undergoing continuous ambulatory peritoneal dialysis who are being treated with glucose-containing dialysis solutions in relation to parameters of peritoneal transport. Standard peritoneal permeability analyses with 3.86% glucose dialysate were performed in 16 stable patients undergoing peritoneal dialysis (PD) (median duration of PD 39 months, range 1 to 104 months). The power relationship that is present between dialysate/serum (D/S) ratios of serum proteins that are transported only across the peritoneal membrane and their molecular weights was used to predict the D/S ratios when diffusion would be the only explanation for the measured dialysate concentration. It was assumed that all TGF-beta1 in the circulation was bound to alpha2-macroglobulin. The D/S ratios of VEGF (P < .0005) and TGF-beta1 (P < .015) were significantly higher than expected when VEGF and TGF-beta1 would have been transported from the circulation only by diffusion. No relationship was present between the effluent concentration attributed to the local production of VEGF (LVEGF) and that of TGF-beta1 (LTGF-beta1). LVEGF correlated with the mass transfer area coefficient (MTAC) creatinine value (r = 0.69, P < .007), MTAC urate value (r = 0.60, P < .02), and glucose absorption value (r = 0.75, P < .004), all reflections of the peritoneal vascular surface area. A negative correlation was observed between the transcapillary ultrafiltration (926 mL/4 h, 394 to 1262 mL/4 h) and LVEGF (r = -0.52, P < .045). This negative tendency was also observed between the net ultrafiltration (622 mL/4 h, -43 to 938 mL/4 h) and LVEGF (r = -0.48) but did not reach significance. LVEGF and the duration of treatment did not correlate, possibly because of the relatively small number of patients. LTGF-beta1 showed no relationship with transport parameters or duration of treatment. In conclusion, we found evidence for the local production of both VEGF and TGF-beta1 in the peritoneal membrane of patients undergoing long-term peritoneal dialysis with glucose-based dialysate solutions. The analogy with VEGF in diabetic retinopathy suggests a pathogenetic role of high dialysate glucose concentrations in the development of these alterations in the peritoneal membrane.

Analysis of non enzymatic glycosylation in vivo: impact of different dialysis solutions.

BACKGROUND: Glucose-containing dialysis solutions in peritoneal dialysis (PD) patients induce non enzymatic glycosylation (NEG) within the peritoneal cavity. The subsequent formation of advanced glycosylation end-products (AGEs) may be implicated in the functional deterioration of the peritoneal membrane in long-term PD patients. AIM OF THE STUDY AND PARAMETERS: Measurement of NEG by the determination of percent glycation of albumin and IgG (GP), and of AGEs by measuring pentosidine content of protein in 4-hour effluents (Peff) and serum. SUBJECTS: In 5 patients each, a comparison was made between 3.86% glucose and 1.36% glucose (GP and Peff), and between 3.86% glucose and 7.5% icodextrin (Peff). Nine patients with clinically severe ultrafiltration failure (UFF) were compared to nine patients treated with PD for 1 month. Six of the patients with UFF were treated with non glucose dialysis solutions and Peff was studied again after 6 weeks. RESULTS: No difference was found between Peff comparing 3.86% glucose to either 1.36% glucose or icodextrin. GP were higher in 3.86% glucose than in 1.36%. Glycated/non glycated (G/NG) protein clearance ratios were 1.29 for albumin and 1.12 for IgG (p = 0.003). In contrast to GP, both Peff and serum pentosidine were higher in the UFF patients than in the recently started patients. Peff, but not GP, correlated with duration of PD (r = 0.67, p = 0.04). In 5 of 6 patients treated with non glucose dialysate, Peff decreased while serum pentosidine was stable. DISCUSSION: These data show that 4-hour Peff contents are not influenced by glucose concentration or osmolality, in contrast to GP. The relation between Peff and duration of PD, and the effect of non glucose dialysate on Peff, suggest that long-term glucose exposure is an important determinant of membrane glycosylation. Thus Peff probably reflects the long-term effects of intraperitoneal glycosylation of peritoneal membrane proteins. Treatment with non glucose dialysis solutions may result in "washout" of glycosylated proteins from the peritoneal membrane.

Substrate and inhibitor for nitric oxide synthase during peritoneal dialysis in rabbits.

OBJECTIVE: To investigate the possible influence of nitric oxide (NO) on peritoneal transport during non infected peritoneal dialysis. DESIGN: A chronic peritoneal dialysis model in New Zealand White rabbits (2624 g; range: 2251-3034 g) was used. In 13 rabbits, 250 mg/L L-arginine, a substrate for NO synthesis, was added to 3.86% glucose dialysate. N(G)-monomethyl-L-arginine (L-NMMA) 25 mg/L, an inhibitor of NO synthase, was added to the dialysate in 10 rabbits. Standard peritoneal permeability analyses in rabbits (SPAR) were performed to analyze the effects of these interventions on solute and fluid transport during 1-hour dwells. The addition of 4.5 mg/L nitroprusside to the dialysate in 5 separate experiments was used for validation of this model. MAIN OUTCOME: For the transport of urea and creatinine, mass transfer area coefficients (MTACs) were calculated. Furthermore, the glucose absorption, the peritoneal albumin clearance, peritoneal fluid kinetics, and the dialysate-to-plasma (D/P) ratio of nitrate were calculated. RESULTS: Nitroprusside caused an 86% (48%-233%) increase in albumin clearance, which is similar to the nitroprusside-induced increase found in humans. Contrary to the findings in human studies, no effect was found on the clearances of urea and creatinine, or on peritoneal fluid kinetics. This suggests a lower sensitivity of the rabbit peritoneal membrane for the effect of NO on small-solute transport. L-arginine affected neither the MTACs of urea and creatinine, nor the absorption of glucose. Also, peritoneal fluid kinetics were similar. Peritoneal albumin clearance increased 18% (-24%-609%). This result resembles the NO-mediated effects of nitroprusside. Addition of L-NMMA caused no change in the transport rate of small solutes, in albumin clearance, or in fluid profile. This result suggests that NO synthase is not induced during non infected peritoneal dialysis, which accords with previous studies. CONCLUSION: This rabbit dialysis model can be used for analyzing the effects of interventions on peritoneal permeability characteristics, although the rabbit peritoneal membrane is probably less sensitive to NO compared to that of humans. L-Arginine-induced effects are similar to those of nitroprusside, which suggests that these effects are possibly mediated by NO. Because L-NMMA did not affect peritoneal transport, it is unlikely that NO is involved in the regulation of peritoneal permeability during stable continuous ambulatory peritoneal dialysis.

Enterohepatic cycling of bilirubin: a putative mechanism for pigment gallstone formation in ileal Crohn's disease.

BACKGROUND & AIMS: Patients with ileal disease, bypass, or resection are at increased risk for developing gallstones. In ileectomized rats, bilirubin secretion rates into bile are elevated, most likely caused by increased colonic bile salt levels, which solubilize unconjugated bilirubin, prevent calcium complexing, and promote its absorption and enterohepatic cycling. The hypothesis that ileal disease or resection engenders the same pathophysiology in humans was tested. METHODS: Sterile gallbladder bile samples were obtained intraoperatively from 29 patients with Crohn's disease and 19 patients with ulcerative colitis. Bilirubin, total calcium, biliary lipids, beta-glucuronidase activities, and cholesterol saturation indices in bile were measured, and markers of hemolysis and ineffective erythropoiesis in blood were assessed. RESULTS: Bilirubin conjugates, unconjugated bilirubin, and total calcium levels were increased 3-10-fold in bile of patients with ileal disease and/or resection compared with patients with Crohn's colitis or ulcerative colitis. Biliary bilirubin concentrations correlated positively with the anatomic length and duration of ileal disease. Endogenous biliary beta-glucuronidase activities were comparable in all groups, and both the hemogram and serum vitamin B12 levels were normal. CONCLUSIONS: This study establishes that increased bilirubin levels in bile of patients with Crohn's disease are caused by lack of functional ileum, supporting the hypothesis that enterohepatic cycling of bilirubin occurs.

The standard peritoneal permeability analysis in the rabbit: a longitudinal model for peritoneal dialysis.

OBJECTIVE: The development of an experimental peritoneal dialysis (PD) model in rabbits to investigate peritoneal transport characteristics during a longitudinal follow-up and to assess normal values of these peritoneal transport parameters. DESIGN: Peritoneal transport parameters were determined in conscious, unrestrained rabbits by standard peritoneal permeability analysis adjusted for rabbits (SPAR). In this test a 1-hour dwell with 3.86% glucose dialysate is used. Dextran 70 (1g/L) was added to the dialysate to allow calculation of fluid kinetics. Dialysate samples were taken before, 10, and 40 minutes after instillation and at the end of the dwell. Blood was drawn at the end of the dwell. EXPERIMENTAL ANIMALS: Eighteen female New Zealand White rabbits (2565 g) were included for catheter implantation. SPARs were performed in 15 animals; the other 3 were excluded due to complications. MAIN OUTCOME: The mass transfer area coefficients (MTACs) of the low molecular weight solutes urea (MTAC(urea)) and creatinine (MTACcr) were calculated. The clearances of albumin (CIalb) and IgG (CI(IgG)), glucose absorption, and fluid transport were computed. Coefficients of intraindividual variation (Vc) were calculated for these parameters. RESULTS: The main complications were catheter obstruction and/or dislocation. Five rabbits underwent uncomplicated PD during a 4-week period. Fifteen SPARs in 15 stable rabbits were performed and analyzed to obtain normal values. Means and standard deviations of the transport parameters were as follows: MTAC(urea) 2.24+/-0.57 mL/min, MTACcr 1.61+/-0.30 mU/min, CI(alb) 52.9+/-17.2 microL/min, CI(IgG) 44.5+/-22.9 UL/min. The transcapillary ultrafiltration rate was 0.66+/-0.13 mL/min and the lymphatic absorption rate 0.47+/-0.26 mL/min. The parameters of solute transport were upscaled to those in humans using two different methods. MTACs of low molecular weight solutes in rabbits and patients were of the same order of magnitude, but the clearance of albumin was approximately four times higher in rabbits than in patients, and that of IgG eight times. In all rabbits sieving of sodium was observed. The dialysate/plasma (D/P) of sodium decreased to a minimum at 40 min (p<0.003 vs the initial value), followed by a rise to 60 min. The minimal value was 0.884+/-0.002. The coefficients of variation calculated on 7 rabbits that underwent two or more SPARs were similar to those assessed from the patient data. This indicates stability of the model and reproducibility of the SPAR. CONCLUSION: The conscious rabbit model for PD can be used for repeated studies on peritoneal transport.

Are phospholipase A2 and nitric oxide involved in the alterations in peritoneal transport during CAPD peritonitis?

The alterations in peritoneal permeability characteristics during peritonitis can only partly be explained by the increased concentrations of prostaglandins and cytokines in the dialysate. Fifteen patients undergoing continuous ambulatory peritoneal dialysis (CAPD) with 16 peritonitis episodes were examined in the acute phase of the infection by using standard peritoneal permeability analyses (SPAs). In 9 of these patients, a control SPA could be performed. The contribution of nitric oxide (NO), prostaglandins, and the acute phase reactants C-reactive protein (CRP) and secretory phospholipase A2 (sPLA2) were analyzed. The mass transfer area coefficients (MTACs) of low-molecular-weight solutes increased during peritonitis: urea 26%, creatinine 45%, and urate 45%. The MTAC of CO2, calculated to estimate peritoneal blood flow, was 71 mL/min (34 to 254 mL/min) during peritonitis and 55 mL/min (42 to 63 mL/min) after recovery, P < or = .05. The peritoneal protein clearances were also greater during peritonitis, but this increase was not related to the molecular weight of the protein. Therefore the restriction coefficients to macromolecules were not different. The net ultrafiltration in all peritonitis episodes was lower as compared with the control dwells: -97 mL (-196 to 19 mL) versus 25 mL (-132 to 216 mL), P = .03. The prostaglandin concentrations in dialysate were greater during peritonitis than after recovery. The median increase was 199% for prostaglandin E2 (PGE2), 68% for 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), and 44% for thromboxane B2 (TxB2). Plasma sPLA2 values were 22.7 microg/L (7.3 to 407.6) during peritonitis and 8.9 microg/L (5.5 to 11.5) after recovery, P < .01. The increased plasma sPLA2 during peritonitis correlated with plasma CRP (r = .6; P = .02). The peritoneal clearances of sPLA2 were greater during peritonitis, but this could be attributed completely to the increased peritoneal transport. Both during peritonitis and after recovery, the sPLA2 clearances did not exceed the predicted values based on transport from the circulation to the dialysate. No evidence was found for local production of nitrite or nitrate. However, the MTAC of cyclic guanosine monophosphate (cGMP) was greater during the experiments performed 48 to 72 hours after the onset of peritonitis, which suggests the synthesis of NO. It can be concluded that peritonitis does not induce detectable local release of sPLA2 and that the inflammation-induced increase in the vascular surface area could not be attributed to NO in the acute phase. The activation of inducible NO synthase may occur after 48 hours.