CD56+ T cells are increased in kidney transplant patients following cytomegalovirus infection.

BACKGROUND: CD56+ T cells previously have been identified as potentially cytotoxic lymphocytes, and relative numbers are increased in some infectious diseases. PATIENTS AND METHODS: Relative proportions of CD56+ T cells were measured by flow cytometry in groups of renal transplant patients differing in cytomegalovirus (CMV) status of donor (D) and recipient (R). These measurements were related to episodes of CMV viremia. RESULTS: Patient groups in which recipients (R+) or donors (D+/R-) were CMV+ had significantly higher proportions of CD56+ T cells (5.11 ± 0.69% and 5.42 ± 1.01%, respectively) than the D-/R- group (1.9 ± 0.35%; P = 0.0018 and 0.017, respectively). In the high-risk D+/R- group, it was found that patients who had post-transplant CMV viremia had higher levels than those who remained CMV negative (9.09 ± 2.34% vs. 3.16 ± 1.22%; P = 0.01). CD56+ T cells from R+ and D+/R- groups had higher proportions of both CD4+ and CD8+ cells than the D-/R- group. When activation markers were examined, some CD56+ T cells from both CMV+ groups had a TEM phenotype, with significantly more expressing CD45RO and NKG2C, and less expressing CD28, CD62L, CD127, and CD161 compared to the D-/R- group. Some CD56+ T cells showed specificity for CMV antigens and similar proportions of CD8+ cells were positive for class I HLA-CMV tetramers containing immunodominant CMV peptides compared to the majority CD56- T cells. CONCLUSION: The results show significant increases in proportions of CD56+ T cells in relation to CMV infection in renal transplant patients and suggest that these cells have a cytotoxic function against CMV-infected cells.

A 26-week, placebo- and pioglitazone-controlled monotherapy study of rivoglitazone in subjects with type 2 diabetes mellitus.

AIMS: To evaluate the efficacy and safety of rivoglitazone, a peroxisome proliferator-activated receptor γ agonist in the thiazolidinedione class, in subjects with suboptimally controlled type 2 diabetes mellitus (T2DM). METHODS: Subjects aged ≥18 years with T2DM and haemoglobin A1c (HbA1c) >7.0% and ≤8.5%, who were treatment naïve or receiving a non-thiazolidinedione antidiabetes monotherapy, entered a 2-week washout and single-blind placebo run-in period and were then randomized 2 : 4 : 11 : 11 to double-blind treatment with placebo, rivoglitazone 1.0 mg/day, rivoglitazone 1.5 mg/day, or pioglitazone 45 mg/day, for 26 weeks. RESULTS: A total of 1912 subjects received placebo (n = 137), rivoglitazone 1.0 mg (n = 274), rivoglitazone 1.5 mg (n = 750) or pioglitazone (n = 751). Rivoglitazone 1.5 mg was statistically superior (p = 0.0339) and rivoglitazone 1.0 mg was non-inferior (p = 0.0339) to pioglitazone in reducing HbA1c from baseline (changes of -0.7%, -0.4% and -0.6%, respectively). Rivoglitazone also significantly reduced fasting plasma glucose from baseline (p < 0.0001). Rivoglitazone significantly improved estimates of insulin sensitivity, high-density lipoprotein cholesterol levels, and other metabolic and inflammatory biomarkers. Rivoglitazone was generally well tolerated at both doses, with treatment-emergent adverse event (TEAE) rates similar to pioglitazone. The most common drug-related TEAEs were peripheral oedema (active, 5.2-6.2%; placebo 0.7%), increased weight (active, 1.6-3.1%; placebo, 0%) and pitting oedema (active, 1.3-2.2%; placebo, 0%). CONCLUSIONS: In subjects with suboptimally controlled T2DM, rivoglitazone 1.5 mg was associated with statistically superior glycaemic control to pioglitazone 45 mg, while rivoglitazone 1.0 mg was non-inferior; the safety profiles of the two drugs appeared similar.

Predictive population pharmacokinetic/pharmacodynamic model for a novel COX-2 inhibitor.

The objectives of these analyses were to (1) develop a population pharmacokinetic/pharmacodynamic model for a novel COX-2 inhibitor (CS-706) using data from primarily Caucasian subjects, (2) predict responses in subpopulations of interest (including Japanese subjects), and (3) correlate pharmacodynamic parameters to safety outcomes. The model was developed using data from 130 healthy adults following single or multiple doses of CS-706. Serial plasma concentrations of CS-706 and ex vivo whole-blood cyclooxygenase-1 (COX-1) and COX-2 activity were determined up to 72 hours postdose. An E(max) model described relationships between CS-706 plasma concentrations and COX-1 and COX-2 inhibition. CS-706 potency (EC(50)) was 397 ng/mL for COX-1 and 20 ng/mL for COX-2. None of the tested covariates influenced the pharmacodynamics of CS-706. Japanese subjects are expected to show a slightly reduced response to CS-706, consistent with lower exposure following the same dose given to Caucasian subjects. Predictive pharmacokinetic/pharmacodynamic modeling for COX-1 and COX-2 inhibition indicates a 20-fold potency ratio that is expected to be similar in Japanese and Caucasians. There was good correlation between COX-1 inhibition and the incidence of 7-day gastroduodenal mucosal injury. A dose of less than 25 mg bid could be adequate to inhibit COX-2 activity with a low risk of gastrointestinal mucosal injury.

Development of a predictive pharmacokinetic model for a novel cyclooxygenase-2 inhibitor.

A predictive population pharmacokinetic model was developed for a novel cyclooxygenase-2 (COX-2) inhibitor CS-706, using data from 130 subjects in 3 phase 1 trials after single or multiple doses of CS-706 (2- to 800-mg doses daily, up to 14 days) and validated using sparse data from a separate study. A 2-compartment model described the data. Typical apparent clearance (CL/F) was 47.2 L/h and was reduced by 43% at doses greater than 200 mg. Apparent clearance was decreased by 38% in female subjects and by 64% and 15%, respectively, in poor/intermediate CYP 2D6 and poor CYP 2C9 metabolizers. Typical apparent volume of the central compartment was 166 L and increased with body weight. Bioavailability increased by 42% after nighttime doses and decreased saturably with increasing dose (50% reduction at 221 mg). Predicted exposures in Japanese subjects were reduced relative to whites because of a lower frequency of poor metabolizers. The model may aid in optimizing the design of future studies and predicting exposures in other subpopulations.

Oleic acid stimulation of apolipoprotein B secretion from HepG2 and Caco-2 cells occurs post-transcriptionally.

HepG2 and Caco-2 cells were studied to compare the regulation of liver and intestinal apolipoprotein (apo) biosynthesis and secretion by fatty acids. Incubation with fatty acid consistently stimulated apoB production by both HepG2 and Caco-2 cells. Media concentrations of apoB, determined by radioimmunoassay, were approx. 3-fold greater for cells incubated for 24 h in serum-free medium containing oleic acid bound to albumin than for cells incubated with albumin alone. Oleic acid also resulted in a 2-3-fold accumulation of cellular triacylglycerol for HepG2 cells and Caco-2 cells. Cellular apoB and media and cellular apoA-I concentrations were not affected by oleic acid. Immunoblotting with a monoclonal antibody against human apoB confirmed a greater mass of apoB in media from HepG2 and Caco-2 cells incubated with oleic acid. Radiolabeled apoB-100 was also increased in media from HepG2 and Caco-2 cells incubated with [35S]methionine for 24 h in the presence of oleic acid, suggesting enhanced apoB synthesis. However, apoB mRNA concentrations were unchanged in response to oleic acid. Gel filtration of media by fast protein liquid chromatography (FPLC) revealed a redistribution of apoB from LDL-sized particles to VLDL or chylomicrons in media from Caco-2 cells incubated with oleic acid, whereas apoB remained in LDL for HepG2 cells. ApoA-I in media from HepG2 and Caco-2 cells eluted as free or lipid-poor apoA-I, and the apoA-I distribution was unaltered by incubation with oleic acid. These data demonstrate that HepG2 and Caco-2 cells maintained in supplemented serum-free medium respond to oleic acid by a similar post-transcriptional increase in apoB synthesis, but different packaging of apoB into triacylglycerol-rich lipoproteins.

Decreased activity of acyl-CoA:cholesterol acyltransferase by insulin in human intestinal cell line Caco-2.

Diabetes mellitus is accompanied by increased intestinal cholesterol synthesis and cholesterol esterification. Both are reversed by insulin therapy. To assess whether the action of insulin on cholesterol esterification by intestinal cells is direct or mediated by other effectors associated with diabetes, we investigated the effect of insulin on the activity of microsomal acyl-CoA:cholesterol acyltransferase (ACAT) and on the incorporation rate of [14C]oleic acid into cholesteryl oleate in the Caco-2 human intestinal cell line. Microsomal ACAT activity of cells that were incubated with insulin for 3 h at a concentration of 1, 10, and 100 microU/ml was decreased by 48, 58, and 74%, respectively, compared with cells cultured in insulinfree medium. This effect was evident as soon as 10 min after the addition of 10 microU/ml insulin. The inhibition by insulin was reversible. After incubation for 24 h, intracellular esterified-cholesterol content and the ratio of esterified to nonesterified cholesterol were significantly lower in the cells treated with insulin (100 microU/ml) than in those not treated with insulin (esterified cholesterol 48.6 +/- 2.0 vs. 74.2 +/- 4.3 nmol/mg protein, respectively, P less than .005; esterified to nonesterified ratio 0.280 +/- 0.008 vs. 0.359 +/- 0.059, respectively, P less than .05). Cells cultured on filters manifested physiologic polarity; greater than 90% of [14C]oleic acid-labeled cholesterol ester secreted by cells was secreted into the basolateral chambers. Incorporation of [14C]oleic acid into cholesteryl oleate over 24 h in the cells and in the medium of the basolateral chamber was suppressed by 100 microU/ml insulin by 23 and 40%, respectively. These findings indicate that insulin acts directly on the enterocytes to suppress intestinal cholesterol ester synthesis and secretion.

9-cis-retinoic acid increases apolipoprotein AI secretion and mRNA expression in HepG2 cells.

HepG2 cells were studied as a model for regulation of hepatic apolipoprotein AI (apo AI) secretion and gene expression by 9-cis-retinoic acid. HepG2 cells cultured on plastic dishes were exposed to 9-cis-retinoic acid (9-cis-RA) for 48 h with a complete media change at 24 h. Apo AI mass in cultured media was determined by ELISA, by quantitative immunoblotting and by steady-state 35S-methionine labeling. Messenger RNA levels were determined by RNase protection using probes for apo AI and the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). 9-cis-RA increased secretion of apo AI by 52% at doses of 10 and 1 microM (6.3 +/- 0.6 vs. 4.2 +/- 0.3; P < 0.005; 6.1 +/- 0.3 vs. 4.0 +/- 0.7 ng of apo AI/mg cell protein, P < 0.05) and by 35% at 0.1 microM (5.5 +/- 0.6 vs. 4.1 +/- 0.4 ng apo AI/mg protein, P < 0.05, n = 4). Immunoblotting results were consistent with results from ELISA (70% increase at 10 microM 9-cis-RA, P < 0.001; 34% increase at 1 microM, P < 0.005, n = 3). Metabolically labeled apoAI in the medium was increased by 39% following steady-state labeling in the presence of 10 microM 9-cis-RA (597 +/- 7 vs. 430 +/- 13 DPM/microliters media; P < 0.001; n = 4). 9-cis-RA (10 microM) also increased HepG2 cell apo AI mRNA expression by 76% (68 700 +/- 400 vs. 38 900 +/- 2700 DPM, P < 0.01, n = 4), whereas expression of G3PDH mRNA was slightly decreased (14%, P < 0.05). Thus, 9-cis-RA stimulates apo AI expression in HepG2 cells, suggesting a role for retinoids in activating endogenous apo AI gene expression.

Inhibition of cholesteryl ester transfer protein activity in hamsters alters HDL lipid composition.

We investigated the role of cholesteryl ester transfer protein (CETP) in hamsters by using a monoclonal antibody (MAb) that inhibited hamster CETP activity. MAbs were prepared against partially purified human CETP and screened for inhibiton of 3H-cholesteryl oleate (CE) transfer from LDL to HDL in the presence of human plasma bottom fraction (d > 1.21 g/ml). Antibody 1C4 inhibited CE transfer activity in both human plasma bottom fraction (IC50 = approximately 4 micrograms/ml) and in whole plasma from male Golden Syrian hamsters (IC50 = approximately 30 micrograms/ml). Purified MAb 1C4 was injected into chow- and cholesterol-fed hamsters, and blood was collected for analysis of plasma CETP activity and HDL lipid composition. Plasma CETP activity was inhibited by 70%-80% at all and HDL lipid composition. Plasma CETP activity was inhibited by 70%-80% at all times up to 24 h following injection of 500 micrograms MAb 1C4 (approximately 3.7 mg/kg). The amount of antibody required for 50% inhibition at 24 h post-injection was 200 micrograms (approximately 1.5 mg/kg). Inhibition of hamster CETP activity in vivo increased hamster HDL cholesterol by 33% (P < 0.0001), increased HDL-CE by 31% (P < 0.0001) and decreased HDL-triglyceride by 42% (P < 0.0001) (n = 36) as determined following isolation of HDL by ultracentrifugation. An increase in HDL cholesterol and a redistribution of cholesterol to a larger HDL particle were also observed following fast protein liquid chromatography (FPLC) gel filtration of plasma lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

l-2-oxothiazolidine-4-carboxylic acid modulates function of peritoneal mesothelial cells in vitro.

The influence of the glutathione precursor, l-2-oxothiazolidine-4-carboxylic acid (OTZ), on the function of human peritoneal mesothelial cells in vitro, in conditions that mimic the in vivo effect of peritoneal dialysis solutions on mesothelium, was studied. Mesothelial monolayers were exposed to dialysis fluids (Dianeal 1.36 or Dianeal 3.86; Baxter Healthcare Corp, Round Lake, IL) that were diluted gradually with pooled-effluent dialysate obtained from patients undergoing continuous ambulatory peritoneal dialysis. In vitro exposure of mesothelium to standard dialysis fluid enhances their susceptibility to injury by hydrogen peroxide. OTZ added to dialysis solution in concentrations of 1 mmol/L prevented the toxic effect of hydrogen peroxide, probably by increasing intracellular glutathione. Mesothelial cells exposed to dialysis fluid become activated, evidenced by increased release of interleukin-6 and hyaluronan. OTZ used in concentrations of 1 mmol/L reduced that effect. Furthermore, the addition of glucose to the culture medium in a concentration of 45 mmol/L inhibits the proliferation of mesothelial cells; the presence of OTZ, 1 mmol/L, partially prevents the inhibitory effect of glucose. The results presented in this report show that by augmenting the intracellular concentration of glutathione in mesothelial cells by the addition of OTZ to the dialysis fluid, we can increase their resistance to the acute toxicity of free radicals and long-term toxicity of glucose. In addition, mesothelial cells with an increased glutathione level are less activated after their exposure to dialysis fluid.

Hyaluronan modifies inflammatory response and peritoneal permeability during peritonitis in rats.

The effect of high-molecular-weight hyaluronan (HA) on peritoneal and systemic inflammation and peritoneal permeability to water and solutes was studied during endotoxin-induced peritonitis in rats. Acute peritonitis was induced by adding lipopolysaccharide (LPS) to the dialysis fluid (Dianeal 3.86; Baxter Healthcare, Ireland, Castlebar). HA was added to the dialysis solution in a concentration of 10 mg/dL. During 4- and 8-hour dwells of the dialysis fluid, we studied the intensity of peritoneal (dialysate) and systemic (blood) inflammation (dialysate cell count and differential, cytokine and HA levels), as well as the transperitoneal transport of solutes and water. In rats, the addition of LPS to the dialysis fluid induced changes in inflammatory reaction and transperitoneal transport similar to those seen in continuous ambulatory peritoneal dialysis patients with peritonitis. During peritonitis, the addition of HA to the dialysis fluid reduced the loss of ultrafiltration, which resulted in a greater peritoneal creatinine clearance during the 8 hours of dwell (29.9 +/- 6.7 mL/8 h in the HA-LPS group versus 19.7 +/- 7.8 mL/8 h in the LPS group; P < 0.05). Dialysate interferon-gamma (INF-gamma) levels during peritonitis were greater in HA-treated animals (536.8 +/- 296.6 pg/mL in the HA-LPS group versus 169.8 +/- 137.8 pg/mL in the LPS group; P < 0.05). Dialysate elastase activity increased during peritonitis (44.4 +/- 9.3 versus 14.2 +/- 4.1 U/mL in peritonitis-free rats); during peritonitis, the increase in dialysate elastase activity was less pronounced in the rats that had HA in the dialysate (27.3 +/- 4.1 U/mL versus the LPS group; P: < 0.01). We conclude that HA added to the dialysis fluid reduces loss of ultrafiltration during peritonitis in rats. In the presence of HA dialysate, INF-gamma levels during peritonitis increased, whereas elastase activity decreased; these changes might improve the peritoneal immune reaction during peritonitis and at the same time prevent peritoneal membrane injury.

Peripheral-type benzodiazepine binding sites in a renal epithelial cell line (MDCK).

Madin-Darby canine kidney (MDCK) cells express a high density of binding sites (Bmax = 0.67 pmol/10(6) cells) for [3H]RO 5-4864, a peripheral-type benzodiazepine (BZD) receptor ligand. Receptor affinity (Kd = 45 nM) in MDCK cells is 30-50 fold lower than in rat kidney, but its pharmacological specificity is identical to that of peripheral-type BZD receptors in the rat kidney (PK 11195 greater than RO 5-4864 greater than diazepam = flunitrazepam greater than clonazepam). The MDCK cell line should provide a useful model system for studying the role of peripheral type BZD receptors in renal function.

Intraperitoneal hyaluronan administration in conscious rats: absorption, metabolism, and effects on peritoneal fluid dynamics.

BACKGROUND: Hyaluronan (HA) is a major component of interstitial tissue that participates in fluid homeostasis, response to inflammation, and wound healing. Previous studies have shown that intraperitoneal administration of HA can affect peritoneal fluid transport during short peritoneal dialysis exchanges in anesthetized rats. We sought to investigate the effect of high molecular weight HA on peritoneal permeability in conscious rats during dialysis exchanges up to 8 hours in duration. In addition, we sought to investigate the absorption of HA from the peritoneal cavity, its accumulation in peritoneal tissues, and its metabolism in normal and uremic rats. METHODS: Experiments were performed on male Wistar rats infused with 30 mL peritoneal dialysis solution (Dianeal, Baxter Healthcare; Castelbar, Ireland) containing 10 mg/dL HA or with Dianeal alone (control). Peritoneal fluid removal (net ultrafiltration), permeability to glucose, creatinine, and total proteins, and tissue and blood levels of HA were determined in separate groups of rats at 1,2, 4, 6, and 8 hours after intraperitoneal infusion. Hyaluronan appearance and disappearance from plasma were also studied for 24 hours in separate groups of normal and uremic rats. RESULTS: Net ultrafiltration was significantly greater (27%) in rats infused with HA at 4, 6, and 8 hours (p < 0.01) compared to controls. Transperitoneal equilibration of protein was reduced by 27% (p < 0.001) at 4 hours and by 30% (p < 0.01) at 8 hours. During the 8-hour exchange, peritoneal clearance of creatinine increased by 27% (p < 0.01), whereas the clearance of total protein decreased by 27% (p < 0.005). After 8 hours, 25.7% +/- 3.1% of the administered HA was absorbed from the peritoneal cavity, peritoneal tissue HA concentration was increased by 117% (p < 0.001), and plasma HA levels increased by 435% (p < 0.001). Plasma HA levels returned to normal within 24 hours after intraperitoneal administration in both healthy and uremic rats. CONCLUSIONS: Hyaluronan added to dialysis fluid is absorbed from the peritoneal cavity and accumulates in peritoneal tissues. Hyaluronan supplementation produces changes in peritoneal permeability, leading to higher net ultrafiltration and peritoneal creatinine clearance, whereas total protein clearance decreases. The HA that is absorbed from the peritoneal cavity appears to be rapidly metabolized in both healthy and uremic rats.

Dyslipidemia in peritoneal dialysis--relation to dialytic variables.

OBJECTIVE: To investigate whether the specific lipoprotein (LP) abnormalities of peritoneal dialysis (PD) are associated with functional variables of this mode of dialysis. DESIGN: A survey of the LP profile in relation to peritoneal dialysis capacity (PDC) variables. The LP profile was compared to that of a group of age- and sex-matched controls. SETTING: The Peritoneal Dialysis Unit at Sahlgrenska University Hospital in Gothenburg, Sweden. PATIENTS: Twenty-two nondiabetic PD patients (5 women, 17 men) who had been on PD for at least 6 months. MAIN OUTCOME MEASURES: The LP profile included plasma lipids, apolipoproteins (Apo), and individual ApoA- and ApoB-containing LP. The PDC measurement determined peritoneal glucose uptake, protein losses, effective peritoneal surface area, and total weekly creatinine clearance. RESULTS: The patients had been on PD for 6 to 48 months (mean 15.3 months) and had a total weekly creatinine clearance of 69.7+/-13.3 L/1.73 m2 body surface area, an average peritoneal glucose uptake corresponding to 446+/-162 kcal/24 hour, and a protein loss of 8.1+/-2.5 g/24 hr. The patients had significantly higher total cholesterol (7.1 mmol/L),VLDL-cholesterol (1.0 mmol/L), LDL-cholesterol (4.7 mmol/L), and triglyceride levels (2.5 mmol/L); whereas the HDL-cholesterol level (1.2 mmol/L) was significantly lower than in controls. The PD patients had increased levels of ApoB-containing LPs, both of the cholesterol-rich LP-B and of the triglyceride-rich LP-B complex, reflected in higher plasma concentrations of ApoB, ApoC-III, and ApoE. Furthermore, they had significantly lower levels of LP-A-I:A-II, as well as of ApoA-I and ApoA-II. The LP-A-I:A-II and ApoA-II levels correlated inversely with the duration of PD treatment (r = 0.54, p < 0.01 and r = 0.52, p < 0.05, respectively). The ApoA-II level was inversely correlated with the peritoneal surface area (r = 0.53, p < 0.05). There were no other correlations between LP variables and PDC variables, nor did any of the LP variables correlate with peritoneal glucose uptake or protein losses. CONCLUSION: The proatherogenic lipoprotein profile of patients on PD is characterized by increased concentrations of cholesterol-rich and triglyceride-rich ApoB-containing LPs. While the duration of treatment appears to have some influence on the development of this type of dyslipidemia, the pathophysiological links to the dialysis mode must be further explored.

Effects of peritoneal rest on peritoneal transport and peritoneal membrane thickening in continuous ambulatory peritoneal dialysis rats.

OBJECTIVE: To evaluate the effects of peritoneal rest on peritoneal transport and morphology in a rat model of peritoneal dialysis. DESIGN: Twenty-four rats (Sprague-Dawley, male, 250-300 g) were divided into three groups: group 1 (control, n = 6) without dialysis, group 2 (n = 9) sacrificed immediately after 3 weeks of dialysis, and group 3 (n = 9) sacrificed after 4 weeks of peritoneal rest after 3 weeks of dialysis. Both dialysis groups were dialyzed twice daily with an intraperitoneal instillation volume of 25 mL of 3.86% dextrose solution for 3 weeks. Peritonitis was induced by supplementing the dialysis fluid with lipopolysaccharide (5 microg/mL) on days 8, 10, and 12 in both dialysis groups. Peritoneal equilibration tests were performed on each animal at baseline. The equilibration tests were repeated at the 4th and the 8th week of dialysis. Morphometric analyses of the peritoneal membrane were carried out in tissue specimens obtained at the time of sacrifice. RESULTS: The D/D0 ratio for glucose at two hours in groups 2 and 3 at the beginning of week 4 was significantly lower than at baseline, indicating an increase in peritoneal permeability to glucose after 3 weeks of dialysis. D/D0 in group 3 at the beginning of week 8, after 4 weeks of peritoneal rest, was significantly higher than at week 4. The drain volume in groups 2 and 3 at week 4 was significantly lower than at baseline; however, the drain volume in group 3 at week 8 was significantly higher than at week 4. The thickness of the parietal peritoneal membrane in group 3 was significantly greater than in group 1 and less than in group 2 (group 1, 11.4+/-7.6 microm; group 2, 37.5+/-18.4 microm; group 3, 21.4+/-12.1 microm). CONCLUSIONS: Peritoneal rest improves ultrafiltration in rats by decreasing the hyperpermeability of glucose and also reduces the degree of peritoneal thickening. These data suggest that dialysis-induced changes in peritoneal transport and morphology are reversible under the conditions of peritoneal rest in this experimental model.

New developments in peritoneal dialysis solutions.

Existing peritoneal dialysis (PD) solutions were formulated mainly for the maintenance of fluid and electrolyte balance, correction of metabolic acidosis, and the removal of metabolic waste products. New solutions in development, and recently approved in some countries, are designed to improve ultrafiltration during long dwells (polyglucose or icodextrin solutions), to treat malnutrition (amino-acids solutions), and to improve peritoneal biocompatibility (bicarbonate-buffered solutions). Other new solutions under investigation are designed to address unmet clinical needs, including cardiovascular disease and sodium balance, through the use of a low-sodium PD solution; long-term peritoneal viability, through improvements in sterilization processes and the use of nonglucose osmotic agents; and PD adequacy, through the use of solution additives (such as glycosaminoglycans) and tailored PD prescriptions using APD. Future concepts for PD include remodeling of the peritoneum, perhaps using mesothelial gene therapy to introduce metabolic and anabolic machinery to remove or perpetually recycle metabolic wastes.

Lack of interference of icodextrin on creatinine measurements.

Glucose has been reported to interfere in the analysis of creatinine by the Jaffe method. The potential interference of icodextrin and its primary metabolites (maltose, maltotriose, maltotetraose) on creatinine measurements has not previously been addressed. We evaluated the potential interference of icodextrin and its metabolites at various concentrations using both the Jaffe and Creatinine Plus methods. Interference was determined in samples containing 0.6-20 mg/dL creatinine in saline solution or in plasma (n = 6), and in dialysate samples (n = 6) spiked with icodextrin, maltose, maltotriose, and maltotetraose at concentrations up to twofold the level found in plasma and dialysate from patients treated using icodextrin. Results confirm that no interference occurs when using either the colorimetric Jaffe method or the enzymatic Creatinine Plus method at levels up to 65 g/L icodextrin, 2 g/L maltose, 2 g/L maltotriose, and 1 g/L maltotetraose, levels representing worst-case clinical concentrations. In addition, our results confirm that comparable values can be obtained using either the Jaffe or the Creatinine Plus method for the analysis of creatinine in uremic plasma and in dialysate samples.

Decreased in vitro formation of AGEs with extraneal solution compared to dextrose-containing peritoneal dialysis solutions.

Extraneal peritoneal dialysis (PD) solution (Baxter Healthcare, Deerfield, Illinois, U.S.A.) contains glucose polymer (icodextrin) as an osmotic agent in place of dextrose. We investigated the ability of Extraneal to form advanced glycation end products (AGEs) in vitro compared to standard PD solutions containing dextrose. Extraneal, Dianeal PD-2 [1.5%, 2.5%, or 4.25% dextrose (Baxter Healthcare)], or phosphate buffered saline (PBS) were incubated for 45 days with human serum albumin (HSA) or type IV collagen. AGE formation was measured by spectrofluorometry using excitation at 350 nm and emission at 430 nm. Solutions were also incubated with collagen affixed to plastic, simulating matrix collagen in the peritoneal membrane. In addition, AGE formation was assessed using icodextrin metabolites (maltose, maltotriose, and maltotetraose) at concentrations normally found in the plasma of patients treated using icodextrin. For PD solutions incubated with albumin, the relative order of AGE formation was: 4.25% dextrose > 2.5% dextrose > 1.5% dextrose > Extraneal. For incubations with collagen (in solution or affixed to plastic), AGE formation was greatest for 4.25% dextrose, intermediate for Extraneal and 2.5% dextrose, and lowest for 1.5% dextrose. Incubation of icodextrin metabolites with albumin for 45 days did not result in appreciable AGE formation. These results confirm that solutions containing icodextrin result in less in vitro AGE formation than do high dextrose solutions. The results also suggest that Extraneal may lead to improved solution biocompatibility in vivo.

Alterations of intraperitoneal inflammation by the addition of L-2-oxothiazolidine-carboxylate.

The authors studied the effect of L-2-oxothiazolidine-carboxylate (OTZ), a substrate for intracellular glutathione synthesis, in an in vivo model of lipopolysaccharide (LPS)-induced peritonitis in rats. The addition of LPS to dialysis fluid increased the white blood cell (WBC) count and the nitrite (index of NO synthesis) level in the dialysate. The simultaneous addition of OTZ to the dialysis fluid prevented an increase of WBCs but not of nitrites in the dialysate. Intraperitoneal inflammation was accompanied by a decrease in net transperitoneal ultrafiltration, an increase in the absorption of glucose, and a loss of protein into the dialysate. OTZ partially reversed the effect of peritonitis on net ultrafiltration. Peritoneal leukocytes from rats exposed to LPS showed a reduced concentration of glutathione, an effect that was reversed in the presence of OTZ. These results show that the supplementation of dialysis fluid with OTZ modified the peritoneal reaction to acute inflammation.

Maltose and isomaltose in uremic plasma following icodextrin administration.

The presence of mixed disaccharides (maltose and isomaltose) in plasma from uremic patients has been previously investigated using gel-permeation chromatography. However, this method is unable to separate maltose (linked alpha-1-4) from isomaltose (linked alpha-1-6). We describe an alternative method using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) for the direct determination of maltose and isomaltose in uremic plasma. We measured maltose and isomaltose using HPAE-PAD in 6 normal subjects and in 15 uremic patients before and after once-daily icodextrin administration for at least 4 weeks. Both maltose and isomaltose were below limits of detection (< 1.0 mg/L) in plasma from normal controls. Patients with end-stage renal disease treated by continuous ambulatory peritoneal dialysis had elevated levels of isomaltose (23.6 +/- 8.3 mg/L) but low levels of maltose (< 3.0 mg/L). Treatment with icodextrin resulted in elevated plasma levels of maltose (range: 500-1600 mg/L), while levels of isomaltose declined to 9.8 +/- 5.2 mg/L (P < 0.0001 vs. baseline levels). We conclude that isomaltose (not maltose) is the primary disaccharide isomer that is elevated in the plasma of uremic patients, whereas maltose is the primary disaccharide isomer that is elevated following icodextrin administration. Furthermore, icodextrin administration results in an apparent reduction of isomaltose. Additional investigation will be required to address the mechanism for the reduction of isomaltose in patients treated by icodextrin.

A monoclonal antibody that recognizes a basolateral membrane protein in A6 epithelial cells.

The A6 cell line is a model for tight epithelia and studies of epithelial polarity. Monoclonal antibodies (MAbs) were produced by immunization of mice with intact A6 cells and fusion of spleen cells to generate hybridomas. Hybridoma supernatants were screened by ELISA to select MAbs binding to the apical membrane of confluent A6 cells. Localization of MAb binding was examined by indirect immunofluorescence using cross sections of A6 monolayers grown on collagen coated filters. One MAb, designated 13F12, was positive by apical surface ELISA but localized specifically to the basolateral membrane of cross sections of A6 monolayers on filters. Immunofluorescence labeling of confluent A6 cells grown on glass cover slips revealed that MAb 13F12 does not bind to the apical membrane, but binds to basolateral determinants in the regions of domes, where it appears able to penetrate cellular junctions. Subconfluent A6 cells express the antigen all over the cell surface. Cells approaching confluency express the antigen on the apical membrane of some cells but not others, and as the cells reach confluency, the antigen disappears from the apical surface, and the cells become fully polarized. A6 cells at confluency on glass cover slips are equally polarized as cells grown on filters with respect to this antigen. The antigen has been identified by immunoprecipitation as a 22 kDa protein. High concentrations of MAb 13F12 did not inhibit cell plating, indicating that the antigenic site is not directly involved in cell adhesion to the substrate. MAb 13F12 should prove to be a useful tool to study many aspects of epithelial polarity, including the signals involved in sorting of proteins to specific membrane domains.