Effect of Bicarbonate-Based Dialysis Solutions on Intracellular Ph (Phi) and Tnfα Production by Peritoneal Macrophages

Abstract

Objective To compare the effect of Dianeal and two newly-formulated bicarbonate-based peritoneal solutions on intracellular pH (pHi), tumor necrosis factor-α (TNFα) mRNA level, and TNFα secretion by peritoneal macrophages (PMΦ). Design and Measurements Peritoneal macrophages were isolated from dialysates collected after overnight dwells in peritonitis -free continuous ambulatory peritoneal dialysis patients. Dialysis solutions contained 1.5% or 4.25% dextrose. HCO3 concentrations of bicarbonate(TB)and bicarbonate/lactate-buffered (TBL) solution were 38 mM and 25 mM, respectively. TBL also contained lactate at a concentration of 15 mM. pCO2 levels were 78 mmHg and 51 mmHg, respectively. In all experiments pC02 was carefully maintained at a stable level. The pHi was measured by spectrofluorometry in BCECF-Ioaded PMΦ exposed to different dialysis solutions or Hank's balanced salt solution. TNFα levels were measured by ELISA in the supernatant of lipopolysaccharide (LPS) stimulated PMΦ after their incubation in different solutions for 15 and 30 minutes. TNFα mRNA was measured by reverse transcriptase polymerase chain reaction (RT PCR) of total RNA extracted from LPS-stimulated PMΦ after their incubation in different solutions for 30 minutes. β-actin mRNA was used as the control. Results Dianeal caused a profound drop in pHi to below 6.2. Following an initial drop, pHi stabilized after 4 minutes at levels of 6.96 and 6.8 after incubation in TB and TBL, respectively. In comparison to the control solution, a fall of 11% and 21% in TNFα secretion was seen after incubation in TB for 15 and 30 minutes, respectively, and 15% and 26% after incubation in TBL. Under identical conditions, Dianeal (Baxter, McGaw Park, IL, U.S.A.) caused 59% and >95% suppression of TNFα secretion. Accordingly, TNFα mRNA level in PMΦ was severely depressed by Dianeal but no detectable inhibition was observed following incubation for 30 minutes in TB and TBL. When dextrose concentration in TB and TBL was increased from 1.5% to 4.25%, TNFα secretion by PMΦ was not suppressed by more than 49%, even after 30 minutes incubation. Moreover, suppression of TNFα mRNA levels could not be detected with TB or TBL even at high dextrose concentrations. Conclusions In contrast to Dianeal, both bicarbonate based solutions caused only a mild drop in pHi of PMΦ. We postulate this effect to be responsible for the improved capacity of PMΦ to secrete TNFα when incubated in bicarbonate-based solutions compared to Dianeal. Reflecting its known cytotoxicity, dextrose in high concentrations diminishes the protective effect of TB and TBL on immune function of PMΦ. TBL is as effective as TB in preventing the deleterious effect of Dianeal on PMΦ function.