Addition of Alanyl-Glutamine to Dialysis Fluid Restores Peritoneal Cellular Stress Responses – A First-In-Man Trial

Background: Peritonitis and ultrafiltration failure remain serious complications of chronic peritoneal dialysis (PD). Dysfunctional cellular stress responses aggravate peritoneal injury associated with PD fluid exposure, potentially due to peritoneal glutamine depletion. In this randomized cross-over phase I/II trial we investigated cytoprotective effects of alanyl-glutamine (AlaGln) addition to glucose-based PDF. Methods: In a prospective randomized cross-over design, 20 stable PD outpatients underwent paired peritoneal equilibration tests 4 weeks apart, using conventional acidic, single chamber 3.86% glucose PD fluid, with and without 8 mM supplemental AlaGln. Heat-shock protein 72 expression was assessed in peritoneal effluent cells as surrogate parameter of cellular stress responses, complemented by metabolomics and functional immunocompetence assays. Results: AlaGln restored peritoneal glutamine levels and increased the primary outcome heat-shock protein expression (effect 1.51-fold, CI 1.07–2.14; p = 0.022), without changes in peritoneal ultrafiltration, small solute transport, or biomarkers reflecting cell mass and inflammation. Further effects were glutamine-like metabolomic changes and increased ex-vivo LPS-stimulated cytokine release from healthy donor peripheral blood monocytes. In patients with a history of peritonitis (5 of 20), AlaGln supplementation decreased dialysate interleukin-8 levels. Supplemented PD fluid also attenuated inflammation and enhanced stimulated cytokine release in a mouse model of PD-associated peritonitis. Conclusion: We conclude that AlaGln-supplemented, glucose-based PD fluid can restore peritoneal cellular stress responses with attenuation of sterile inflammation, and may improve peritoneal host-defense in the setting of PD

WDR73 Mutations Cause Infantile Neurodegeneration and Variable Glomerular Kidney Disease

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Inflammation and immuno-competence in the mouse model of PD- associated peritonitis.

<p>Mice were subjected for 9 days to twice-daily intraperitoneal PDF injection, in combination with 10⁷ cfu <i>Staphylococcus epidermidis</i> 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. <i>Ex-vivo</i> 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). <i>Ex-vivo</i> 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 <i>vs</i>. 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.</p

AlaGln treatment increased HSP expression in peritoneal effluent cells.

<p>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.</p

Effluent and blood parameters of the mouse model of PD-related peritonitis.

<p>Effluent and blood parameters of the mouse model of PD-related peritonitis.</p

Temporal profile of dialysate glutamine and AlaGln dipeptide with standard PDF and with PDF containing 8 mM AlaGln.

<p>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 <i>vs</i>. standard PDF (without AlaGln).</p

Baseline characteristics of the study patients.

<p>Baseline characteristics of the study patients.</p

Clinical routine and safety parameters in blood and dialysate.

<p>Clinical routine and safety parameters in blood and dialysate.</p

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.

<p>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.</p