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Objective To evaluate the effect of advanced glycation end-products (AGEs) and the inhibitor of their formation, aminoguanidine, on tumor necrosis factor-α (TNFα) production (as a functional marker) by rat peritoneal macrophages (PMΦ). Design Charles River rats underwent a daily intraperitoneal injection of peritoneal dialysis solution [(PDS), 4.25 g/dL dextrose; Dialine, Travenol, Ashdod, Israel] for a 2-month period (group E). Another group of rats was subjected to the same protocol with the addition of 25 mg/kg aminoguanidine (group A). Three control groups were utilized: ( 1 ) rats that were injected daily with aminoguanidine only (group AO), ( 2 ) rats that were injected with Dulbecco's phosphate-buffered saline (group D), and ( 3 ) rats in which no intervention was carried out (group C). After 2 months, PMΦ were isolated from rat peritoneal effluent and their TNFα production measured by ELISA in cell-free culture supernatants, in both the basal state and after 24-hour stimulation with lipopolysaccharide (LPS). The concentrations of AGEs in peritoneal effluent were assayed and correlated to TNFα levels. PMΦ obtained from normal rats were then incubated for 24 hours with ( 1 ) the peritoneal effluent of each of the above respective groups, with or without LPS; ( 2 ) increasing concentrations of AGEs (0 - 250 μg/mL); and ( 3 ) increasing concentrations of aminoguanidine (0 - 7.5 mg/mL), and TNFα secretion again determined. Results After 2 months of daily intraperitoneal injection of PDS, in the basal state, TNFα production was significantly higher in PMΦ isolated from the peritoneal effluent groups (groups E, A, and AO) compared to controls (group C). Following LPS stimulation, a further increase in TNFα secretion was seen, with a significantly greater response in group AO versus groups E, A, and D. Effluent AGEs were markedly elevated only in group E. No correlation was found between TNFα secretion by these PMΦ and the concentration of AGEs. On incubation with the respective peritoneal effluents (groups E, A, and AO), in both the basal and stimulated state, TNFα production by PMΦ from normal rats was significantly enhanced compared to group C. Incubation with increasing concentrations of AGEs or aminoguanidine resulted in an increase of TNFα secretion by these PMΦ. Conclusions Following intermittent intraperitoneal administration of glucose-based PDS, rat PMΦ are chronically activated, as evidenced by increased basal TNFα secretion. The peritoneal effluent of such treated animals is capable of stimulating TNFα production by normal rat PMΦ. These data suggest that glucose-based PDS acts as a primer of PMΦ, which retain their ability to further stimulation by LPS. Although, in vitro, AGEs promote TNFα secretion by normal rat PMΦ, in vivo, their influence is probably modulated by other factors. Aminoguanidine has a specific inducing effect on rat PMΦ, independent of glucose-based PDS.

Objective Glucose-based dialysate induces non enzymatic glycation within the peritoneal cavity. To evaluate the relationship between the formation of advanced glycation end-products (AGEs) and peritoneal transfer for small solutes and macromolecules, we developed a model of simulated peritoneal dialysis (PD) in normal rats. Methods Male albino rats of the Charles River strain were divided into two sets of 3 groups (15 – 25 rats in each group). In the experimental (E) group, the rats were intra-peritoneally (IP) injected daily with a commercially available 4.25% dextrose solution. In the control puncture (CP) group, the peritoneum was punctured daily, but no PD solution infused. In an age-matched control (CC) group, no intervention was given. Two study protocols were used. Protocol A (duration 20 weeks) consisted of a daily IP injection of 10 mL PD solution per 100 g body weight. In protocol B, a double volume of PD solution was introduced (20 mL per 100 g body weight). At 9, 16, and 20 weeks in protocol A, and at 9 weeks in protocol B, urea, creatinine, microalbumin [(MAL) measured using specific anti-rat albumin monoclonal antibody], and AGEs (measured by fluorescent assay with excitation at 370 nm and emission at 440 nm) were measured in peritoneal effluent and serum. Results At no time during the study were AGEs detected in serum from any group in either protocol. In both protocols, no differences were found between the control groups (CP, CC) with respect to all parameters. In protocol A, the dialysate-to-plasma ratio (D/P) of urea was significantly higher in the experimental group as compared with the control groups at 9, 16, and 20 weeks [9 weeks: 0.59 ± 0.03 (E) vs 0.39 ± 0.02 (CP) vs 0.46 ± 0.02 (CC), p < 0.0004 and p < 0.002, respectively; 16 weeks: 0.71 ± 0.08 (E) vs 0.42 ± 0.01 (CP) vs 0.46 ± 0.01 (CC), p < 0.0001 and p < 0.02, respectively; 20 weeks: 0.57 ± 0.03 (E) vs 0.39 ± 0.01 (CP) vs 0.41 ± 0.02 (CC), p < 0.002 and p < 0.004, respectively]. At 16 and 20 weeks, dialysate MAL levels were significantly increased in group E [16 weeks: 354.00 ± 80.35 μg/mL (E) vs 134.75 ± 14.36 μg/mL (CP) vs 110.69 ± 7.83 μg/mL (CC), p < 0.04 and p < 0.03, respectively; 20 weeks: 283.17 ± 14.71 μg/mL (E) vs 105.14 ± 12.11 μg/mL (CP) vs 135.50 ± 19.03 μg/mL (CC), p < 0.00001 and p < 0.0001, respectively]. In protocol B, at completion of the study (week 9), D/P urea, effluent MAL, and AGEs were significantly higher in the experimental group as compared with the control groups [D/P: 0.67 ± 0.04 (E) vs 0.46 ± 0.07 (CP) vs 0.41 ± 0.02 (CC), p < 0.0002 and p < 00001, respectively; MAL: 336.8 ± 63.30 μg/mL (E) vs 125.71 ± 16.77 μg/mL (CP) vs 119.00 ± 39.75 μg/mL (CC), p < 0.008 and p < 0.007, respectively; AGEs: 265.77 ± 33.49 U/mg creatinine (E) vs 163.10 ± 21.99 U/mg creatinine (CP) vs 83.17 ± 22.66 U/mg creatinine (CC), p < 0.02 and p < 0.001, respectively]. Peritoneal effluent AGEs were found to be significantly correlated with D/P urea and dialysate MAL ( r = 0.42, p < 0.04, and r = 0.7, p = 0.00001, respectively). Conclusions In situ generation of AGEs constitutes the chief origin of peritoneal AGEs. Advanced glycation end-products affect peritoneal permselectivity for both small and large solutes. The rat model of simulated peritoneal dialysis developed in this experiment provides a reliable method for studying peritoneal AGE formation and effect on peritoneal transfer of small solutes and macro-molecules.