Table 1.
Baseline characteristics of the study patients.
Fig 1.
ESRD patients aged ≥ 19 years were included if stable for at least two months on continuous ambulatory PD (CAPD) or continuous cyclic PD (CCPD) and without severe concomitant disease. Exclusion criteria included hypersensitivity to study medication, malignancy requiring chemotherapy or radiation, pregnancy, limited PD efficacy secondary to anatomical considerations, clinically significant inflammation or current immunosuppressive therapy. After a positive screening visit and an observation period of four weeks, subjects were randomly allocated on an alternating basis to either sequence “A-B” (Sequence 1) or “B-A” (Sequence 2), stratified by sex, age (< or ≥ 60 years), time on PD (< or ≥ 1 year) and peritonitis history (yes or no). Randomization occurred immediately before the start of treatment period 1. Treatment visits 1 and 2 consisted of one single PD exchange performed as a 4-hour-peritoneal equilibration test (PET) using either standard PDF (Dianeal® PD4 with 3.86% glucose, Baxter) or standard PDF supplemented with 8 mM alanyl-glutamine dipeptide (AlaGln; Dipeptiven, Fresenius Kabi). The two treatments occurred in a random order and were separated by a wash-out period of 28–35 days. At times 0 (immediately after completion of PDF infusion), 1 h, 2 h, and 4 h, peritoneal samples were withdrawn. At 4 h, the remaining effluent was drained and collected. Blood was drawn 60 min before and 60 min following the PET. Four patients were excluded for reasons of sub-ileus and abdominal surgery (n = 1), catheter-related infection (n = 1), screening failure (n = 1) and deviation from protocol (no last bag before PET) (n = 1).
Table 2.
Clinical routine and safety parameters in blood and dialysate.
Fig 2.
Temporal profile of dialysate glutamine and AlaGln dipeptide with standard PDF and with PDF containing 8 mM AlaGln.
To assess transfer kinetics, AlaGln dipeptide and glutamine were analyzed in dialysate specimens sampled at baseline (immediately after completion of infusion), 1 h, 2 h and 4 h after initiation of the PET. As shown in panel A, peritoneal fluid AlaGln dipeptide concentration declined from 7.5 mmol/l immediately after infusion to < 2 mmol/l within four hours. The first-order elimination half-life of AlaGln dipeptide from the dialysate was 1.07 h. The value at time 0 h was set to 8 mM (as added to standard PDF). Panel B shows that glutamine concentrations in standard PDF remained below normal (serum) levels after 2 hours, but were significantly higher after 1 h and 2 h in the presence of added AlaGln. Data are shown as means and standard errors. Asterisk indicates p<0.05 vs. standard PDF (without AlaGln).
Fig 3.
AlaGln treatment increased HSP expression in peritoneal effluent cells.
Peritoneal cellular HSP expression in the study samples was detected by combining saturation labeling two-dimensional difference gel electrophoresis (2D-DIGE) with 2-D Western blotting. In panel A the upper segment shows the fluorescently labeled protein pattern whereas the lower segment shows the signals from immune-blotting, superimposed by the gel warping capabilities of Delta 2D software. The first dimension separation according to the isoelectric point (pI) was carried out on a nonlinear gradient. Panel B shows the quantification of total abundance of Hsp72 spot volumes, normalized by the internal standard. Total abundance of Hsp72 increased from 2.12 (CI 1.46–3.09) without AlaGln to 3.20 (CI 2.20–4.66) with AlaGln (effect size 1.51 (CI 1.07–2.14), p = 0.022) (N = 20 in each group, Counts: up = 14; down = 6). Grey bars indicate the mean; red bars indicate the median in each group.
Table 3.
Metabolomic analysis of peritoneal effluents following treatment with standard PDF vs. PDF with 8 mM AlaGln.
Table 4.
Secondary outcome parameters in dialysate effluent at 4 hours.
Fig 4.
Lipopolysaccharide (LPS)-stimulated release of tumor necrosis factor alpha (TNF-α) by heterologous normal human peripheral blood mononuclear cells (PBMC) following a 4 h ex-vivo exposure to PD effluents obtained from the PET of patients treated with standard PDF or AlaGln-supplemented PDF.
In panel A exposure to effluent mixed 1:1 with fresh PDF (representing the situation of the “early dwell”) is shown. In panel B exposure to pure effluent (representing the situation of the “late dwell”) is shown. Each data point represents the mean value of TNF-α release by PBMC from 4 healthy donors exposed to each PET effluent (n = 20 in each group). The left part of the figures represents the control with exposure to patient effluents without stimulation by LPS (0 ng/ml). Differences between the presence and absence of AlaGln were statistically significant for PBMC in the presence of 10 or 100 ng/ml LPS in case of the early and the late dwell (p<0.001). (Panel A counts: 10 ng/ml: down = 6, up = 14; 100 ng/ml: down = 7, up = 13; Panel B counts: 10 ng/ml: down = 1, up = 19; 100 ng/ml: down = 4, up = 16). Red bars indicate the mean in each group.
Fig 5.
Intracellular glutamine of peripheral blood mononuclear cells (PBMC) following exposure to PD effluents obtained from the PET of patients treated with standard PDF or AlaGln-supplemented PDF.
Intracellular amino acid concentrations of PBMC after exposure to effluent mixed 1:1 with fresh PDF for 4 h (i.e. at the start time of LPS stimulation (see Fig 4, panel A) are shown. Of the 20 canonical amino acids, only alanine and glutamine show significantly higher intracellular concentrations when PBMC are exposed to effluents from patients treated with AlaGln-supplemented PDF (p<0.001, n = 80 in each box (= 4 biological PBMC experiments exposed to 20 PD effluents).
Fig 6.
Levels of interleukin 8 (IL-8) and interleukin 6 (IL-6) in PET effluents from patients treated with PDF in the absence or presence of 8mM AlaGln.
Each data point represents the IL-8 (A) or IL-6 (B) concentration in PD effluent of a single study patient treated with standard PDF lacking or containing added AlaGln. Among patients with history of peritonitis (Peritonitis Hx), IL-8 levels after exposure to PDF containing AlaGln were significantly lower than after exposure to standard PDF (p<0.05). History of peritonitis was not associated with differences in IL-6 levels (p>0.05). Statistical mixed model analysis for IL-6 was performed after excluding the single extremely high value in the standard PDF group. Red bars indicate the median in each group.
Fig 7.
Inflammation and immuno-competence in the mouse model of PD- associated peritonitis.
Mice were subjected for 9 days to twice-daily intraperitoneal PDF injection, in combination with 107 cfu Staphylococcus epidermidis on days 2 and 4. PDF was applied with (N = 10) or without (N = 10) added 8 mM AlaGln. Control mice (N = 4) received no treatment. As shown in Panel A and B, basal levels of IL-6 and TNF-α in peritoneal effluents were lower in the group treated with AlaGln than in the group treated without it, although not statistically significant, when p-values were Bonferroni-corrected for testing two outcomes. Values of controls are shown as dashed line. Ex-vivo LPS stimulation (10 ng/ml; 4 h) of peritoneal cells resulted in increased IL-6 and TNF-α release in control animals (ratio of stimulated/unstimulated is shown as dashed line C and D). Ex-vivo stimulated release of IL-6 and TNF-α was depressed in the group without AlaGln and restored in the group with AlaGln (raw p = 0.023 vs. without AlaGln for IL-6; Bonferroni-corrected p = 0.046). Data are shown as individual points (each representing an animal), means and standard errors. # P-values in the figure are given unadjusted for multiple testing.
Table 5.
Effluent and blood parameters of the mouse model of PD-related peritonitis.