Peritoneal Dialysis Fluid Supplementation with Alanyl-Glutamine Attenuates Conventional Dialysis Fluid-Mediated Endothelial Cell Injury by Restoring Perturbed Cytoprotective Responses.

Long-term clinical outcome of peritoneal dialysis (PD) depends on adequate removal of small solutes and water. The peritoneal endothelium represents the key barrier and peritoneal transport dysfunction is associated with vascular changes. Alanyl-glutamine (AlaGln) has been shown to counteract PD-induced deteriorations but the effect on vascular changes has not yet been elucidated. Using multiplexed proteomic and bioinformatic analyses we investigated the molecular mechanisms of vascular pathology in-vitro (primary human umbilical vein endothelial cells, HUVEC) and ex-vivo (arterioles of patients undergoing PD) following exposure to PD-fluid. An overlap of 1813 proteins (40%) of over 3100 proteins was identified in both sample types. PD-fluid treatment significantly altered 378 in endothelial cells and 192 in arterioles. The HUVEC proteome resembles the arteriolar proteome with expected sample specific differences of mainly immune system processes only present in arterioles and extracellular region proteins primarily found in HUVEC. AlaGln-addition to PD-fluid revealed 359 differentially abundant proteins and restored the molecular process landscape altered by PD fluid. This study provides evidence on validity and inherent limitations of studying endothelial pathomechanisms in-vitro compared to vascular ex-vivo findings. AlaGln could reduce PD-associated vasculopathy by reducing endothelial cellular damage, restoring perturbed abundances of pathologically important proteins and enriching protective processes.

Targeted Metabolomic Profiling of Peritoneal Dialysis Effluents Shows Anti-oxidative Capacity of Alanyl-Glutamine.

Readily available peritoneal dialysis (PD) effluents from PD patients in the course of renal replacement therapy are a potentially rich source for molecular markers for predicting clinical outcome, monitoring the therapy, and therapeutic interventions. The complex clinical phenotype of PD patients might be reflected in the PD effluent metabolome. Metabolomic analysis of PD effluent might allow quantitative detection and assessment of candidate PD biomarkers for prognostication and therapeutic monitoring. We therefore subjected peritoneal equilibration test effluents from 20 stable PD patients, obtained in a randomized controlled trial (RCT) to evaluate cytoprotective effects of standard PD solution (3.86% glucose) supplemented with 8 mM alanyl-glutamine (AlaGln) to targeted metabolomics analysis. One hundred eighty eight pre-defined metabolites, including free amino acids, acylcarnitines, and glycerophospholipids, as well as custom metabolic indicators calculated from these metabolites were surveyed in a high-throughput assay requiring only 10 µl of PD effluent. Metabolite profiles of effluents from the cross-over trial were analyzed with respect to AlaGln status and clinical parameters such as duration of PD therapy and history of previous episodes of peritonitis. This targeted approach detected and quantified 184 small molecules in PD effluent, a larger number of detected metabolites than in all previous metabolomic studies in PD effluent combined. Metabolites were clustered within substance classes regarding concentrations after a 4-h dwell. PD effluent metabolic profiles were differentiated according to PD patient sub-populations, revealing novel changes in small molecule abundance during PD therapy. AlaGln supplementation of PD fluid altered levels of specific metabolites, including increases in alanine and glutamine but not glutamate, and reduced levels of small molecule indicators of oxidative stress, such as methionine sulfoxide. Our study represents the first application of targeted metabolomics to PD effluents. The observed metabolomic changes in PD effluent associated with AlaGln-supplementation during therapy suggested an anti-oxidant effect, and were consistent with the restoration of important stress and immune processes previously noted in the RCT. High-throughput detection of PD effluent metabolomic signatures and their alterations by therapeutic interventions offers new opportunities for metabolome-clinical correlation in PD and for prescription of personalized PD therapy.

Effects of Alanyl-Glutamine Treatment on the Peritoneal Dialysis Effluent Proteome Reveal Pathomechanism-Associated Molecular Signatures.

Peritoneal dialysis (PD) is a modality of renal replacement therapy in which the high volumes of available PD effluent (PDE) represents a rich source of biomarkers for monitoring disease and therapy. Although this information could help guide the management of PD patients, little is known about the potential of PDE to define pathomechanism-associated molecular signatures in PD.We therefore subjected PDE to a high-performance multiplex proteomic analysis after depletion of highly-abundant plasma proteins and enrichment of low-abundance proteins. A combination of label-free and isobaric labeling strategies was applied to PDE samples from PD patients (n = 20) treated in an open-label, randomized, two-period, cross-over clinical trial with standard PD fluid or with a novel PD fluid supplemented with alanyl-glutamine (AlaGln).With this workflow we identified 2506 unique proteins in the PDE proteome, greatly increasing coverage beyond the 171 previously-reported proteins. The proteins identified range from high abundance plasma proteins to low abundance cellular proteins, and are linked to larger numbers of biological processes and pathways, some of which are novel for PDE. Interestingly, proteins linked to membrane remodeling and fibrosis are overrepresented in PDE compared with plasma, whereas the proteins underrepresented in PDE suggest decreases in host defense, immune-competence and response to stress. Treatment with AlaGln-supplemented PD fluid is associated with reduced activity of membrane injury-associated mechanisms and with restoration of biological processes involved in stress responses and host defense.Our study represents the first application of the PDE proteome in a randomized controlled prospective clinical trial of PD. This novel proteomic workflow allowed detection of low abundance biomarkers to define pathomechanism-associated molecular signatures in PD and their alterations by a novel therapeutic intervention.

Functional and Transcriptomic Characterization of Peritoneal Immune-Modulation by Addition of Alanyl-Glutamine to Dialysis Fluid.

Peritonitis remains a major cause of morbidity and mortality during chronic peritoneal dialysis (PD). Glucose-based PD fluids reduce immunological defenses in the peritoneal cavity. Low concentrations of peritoneal extracellular glutamine during PD may contribute to this immune deficit. For these reasons we have developed a clinical assay to measure the function of the immune-competent cells in PD effluent from PD patients. We then applied this assay to test the impact on peritoneal immune-competence of PD fluid supplementation with alanyl-glutamine (AlaGln) in 6 patients in an open-label, randomized, crossover pilot trial (EudraCT 2012-004004-36), and related the functional results to transcriptome changes in PD effluent cells. Ex-vivo stimulation of PD effluent peritoneal cells increased release of interleukin (IL) 6 and tumor necrosis factor (TNF) α. Both IL-6 and TNF-α were lower at 1 h than at 4 h of the peritoneal equilibration test but the reductions in cytokine release were attenuated in AlaGln-supplemented samples. AlaGln-supplemented samples exhibited priming of IL-6-related pathways and downregulation of TNF-α upstream elements. Results from measurement of cytokine release and transcriptome analysis in this pilot clinical study support the conclusion that suppression of PD effluent cell immune function in human subjects by standard PD fluid is attenuated by AlaGln supplementation.

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.

Expression of the polycystin-1 C-terminal cytoplasmic tail increases Cl channel activity in Xenopus oocytes.

Expression of the polycystin-1 C-terminal cytoplasmic tail increases Cl(-) channel activity in Xenopus oocytes. Background. Cyst expansion in autosomal-dominant polycystic kidney disease (ADPKD) is characterized by active Cl(-) secretion in excess of solute reabsorption. However, the connections between elevated epithelial Cl(-) secretion and loss-of-function or dysregulation of either ADPKD gene polycystin-1 (PC1) or polycystin-2 (PC2) remain little understood. Methods. Cl(-) transport in Xenopus oocytes expressing the CD16.7-PKD1 (115-226) fusion protein containing the final 112 amino acid (aa) of the PC1 C-terminal cytoplasmic tail, or in oocytes expressing related PC1 fusion protein mutants, was studied by isotopic flux, two-electrode voltage clamp, and outside-out patch clamp recording. Results. Expression in oocytes of CD16.7-PKD1 (115-226) increased rates of both influx and efflux of (36)Cl(-), whereas CD16.7-PKD1 (1-92) containing the initial 92 aa of the PC1 C-terminal cytoplasmic tail was inactive. The increased Cl(-) transport resembled CD16.7-PKD1 (115-226)-stimulated cation current in its sensitivity to ADPKD-associated missense mutations, to mutations in phosphorylation sites, and to mutations within or encroaching upon the PC1 coiled-coil domain, as well as in its partial suppression by coexpressed PC2. The NS3623- and 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive (36)Cl(-) flux was not blocked by injected ethyleneglycol tetraacetate (EGTA) or by the cation channel inhibitor SKF96365, and was stimulated by the cation channel inhibitor La(3+), suggesting that CD16.7-PKD1 (115-226)-associated cation conductance was not required for (36)CI(-) flux activation. Outside-out patches from oocytes expressing CD16.7-PKD1 (115-226) also exhibited increased NS3623-sensitive Cl(-) current. Conclusion. These data show that CD16.7-PKD1 (115-226) activates Cl(-) channels in the Xenopus oocyte plasma membrane in parallel with, but not secondary to, activation of Ca(2+)-permeable cation channels.

Functional comparison of mouse slc26a6 anion exchanger with human SLC26A6 polypeptide variants: differences in anion selectivity, regulation, and electrogenicity.

The unusually low 78% amino acid identity between the orthologous human SLC26A6 and mouse slc26a6 polypeptides prompted systematic comparison of their anion transport functions in Xenopus oocytes. Multiple human SLC26A6 variant polypeptides were also functionally compared. Transport was studied as unidirectional fluxes of (36)Cl(-), [(14)C]oxalate, and [(35)S]sulfate; as net fluxes of HCO(3)(-) by fluorescence ratio measurement of intracellular pH; as current by two-electrode voltage clamp; and as net Cl(-) flux by fluorescence intensity measurement of relative changes in extracellular and intracellular [Cl(-)]. Four human SLC26A6 polypeptide variants each exhibited rates of bidirectional [(14)C]oxalate flux, Cl(-)/HCO(3)(-) exchange, and Cl(-)/OH(-) exchange nearly equivalent to those of mouse slc26a6. Cl(-)/HCO(3)(-) exchange by both orthologs was cAMP-sensitive, further enhanced by coexpressed wild type cystic fibrosis transmembrane regulator but inhibited by cystic fibrosis transmembrane regulator DeltaF508. However, the very low rates of (36)Cl(-) and [(35)S]sulfate transport by all active human SLC26A6 isoforms contrasted with the high rates of the mouse ortholog. Human and mouse orthologs also differed in patterns of acute regulation. Studies of human-mouse chimeras revealed cosegregation of the high (36)Cl(-) transport phenotype with the transmembrane domain of mouse slc26a6. Mouse slc26a6 and human SLC26A6 each mediated electroneutral Cl(-)/HCO(3)(-) and Cl(-)/OH(-) exchange. In contrast, whereas Cl(-)/oxalate exchange by mouse slc26a6 was electrogenic, that mediated by human SLC26A6 appeared electroneutral. The increased currents observed in oocytes expressing either mouse or human ortholog were pharmacologically distinct from the accompanying monovalent anion exchange activities. The human SLC26A6 polypeptide variants SLC26A6c and SLC26A6d were inactive as transporters of oxalate, sulfate, and chloride. Thus, the orthologous mouse and human SLC26A6 proteins differ in anion selectivity, transport mechanism, and acute regulation, but both mediate electroneutral Cl(-)/HCO(3)(-) exchange.