The peritoneal surface proteome in a model of chronic peritoneal dialysis reveals mechanisms of membrane damage and preservation

Michael Boehm, Rebecca Herzog, Florian Klinglmüller, Anton M. Lichtenauer, Anja Wagner, Markus Unterwurzacher, Robert H. J. Beelen, Seth L. Alper, Christoph Aufricht, Klaus Kratochwill

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Peritoneal dialysis (PD) fluids are cytotoxic to the peritoneum. Recent studies have shown that alanyl-glutamine (AlaGln) modulates the cellular stress response, improves mesothelial cell survival, reduces submesothelial thickening in experimental models of PD, and in clinical studies improves PD effluent cell stress and immune responses. However, the mechanisms of AlaGln-mediated membrane protection are not yet fully understood. Here, we explore those mechanisms through application of a novel proteomics approach in a clinically relevant in vivo model in rats. Experimental PD was performed for 5 weeks using conventional single-chamber bag (SCB) or neutral dual-chamber bag (DCB), PD fluid (PDF), with or without AlaGln supplementation, via a surgically implanted catheter. Rats subjected to a single dwell without catheter implantation served as controls. The peritoneal surface proteome was directly harvested by detergent extraction and subjected to proteomic analysis by two-dimensional difference gel electrophoresis (2D-DiGE) with protein identification by mass spectrometry. An integrated bioinformatic approach was applied to identify proteins significantly affected by the treatments despite biological variation and interfering high abundance proteins. From 505 of 744 common spots on 59 gels, 222 unique proteins were identified. Using UniProt database information, proteins were assigned either as high abundance plasma proteins, or as cellular proteins. Statistical analysis employed an adapted workflow from RNA-sequencing, the trimmed mean of M-values (TMM) for normalization, and a mixed model for computational identification of significantly differentially abundant proteins. The most prominently enriched pathways after 5 weeks chronic treatment with SCB or DCB, PDFs belonged to clusters reflecting tissue damage and cell differentiation by cytoskeletal reorganization, immune responses, altered metabolism, and oxidative stress and redox homeostasis. Although the AlaGln effect was not as prominent, associated enriched pathways showed mostly regression to control or patterns opposite that of the PDF effect. Our study describes the novel peritoneal surface proteome through combined proteomic and bioinformatic analyses, and assesses changes elicited by chronic experimental PD. The biological processes so identified promise to link molecular mechanisms of membrane damage and protection in the in vivo rat model to pathomechanisms and cytoprotective effects observed in vitro and in clinical PD.
Original languageEnglish
Article number472
JournalFrontiers in Physiology
Volume10
Issue numberMAY
DOIs
Publication statusPublished - 2019

Cite this

Boehm, M., Herzog, R., Klinglmüller, F., Lichtenauer, A. M., Wagner, A., Unterwurzacher, M., ... Kratochwill, K. (2019). The peritoneal surface proteome in a model of chronic peritoneal dialysis reveals mechanisms of membrane damage and preservation. Frontiers in Physiology, 10(MAY), [472]. https://doi.org/10.3389/fphys.2019.00472
Boehm, Michael ; Herzog, Rebecca ; Klinglmüller, Florian ; Lichtenauer, Anton M. ; Wagner, Anja ; Unterwurzacher, Markus ; Beelen, Robert H. J. ; Alper, Seth L. ; Aufricht, Christoph ; Kratochwill, Klaus. / The peritoneal surface proteome in a model of chronic peritoneal dialysis reveals mechanisms of membrane damage and preservation. In: Frontiers in Physiology. 2019 ; Vol. 10, No. MAY.
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abstract = "Peritoneal dialysis (PD) fluids are cytotoxic to the peritoneum. Recent studies have shown that alanyl-glutamine (AlaGln) modulates the cellular stress response, improves mesothelial cell survival, reduces submesothelial thickening in experimental models of PD, and in clinical studies improves PD effluent cell stress and immune responses. However, the mechanisms of AlaGln-mediated membrane protection are not yet fully understood. Here, we explore those mechanisms through application of a novel proteomics approach in a clinically relevant in vivo model in rats. Experimental PD was performed for 5 weeks using conventional single-chamber bag (SCB) or neutral dual-chamber bag (DCB), PD fluid (PDF), with or without AlaGln supplementation, via a surgically implanted catheter. Rats subjected to a single dwell without catheter implantation served as controls. The peritoneal surface proteome was directly harvested by detergent extraction and subjected to proteomic analysis by two-dimensional difference gel electrophoresis (2D-DiGE) with protein identification by mass spectrometry. An integrated bioinformatic approach was applied to identify proteins significantly affected by the treatments despite biological variation and interfering high abundance proteins. From 505 of 744 common spots on 59 gels, 222 unique proteins were identified. Using UniProt database information, proteins were assigned either as high abundance plasma proteins, or as cellular proteins. Statistical analysis employed an adapted workflow from RNA-sequencing, the trimmed mean of M-values (TMM) for normalization, and a mixed model for computational identification of significantly differentially abundant proteins. The most prominently enriched pathways after 5 weeks chronic treatment with SCB or DCB, PDFs belonged to clusters reflecting tissue damage and cell differentiation by cytoskeletal reorganization, immune responses, altered metabolism, and oxidative stress and redox homeostasis. Although the AlaGln effect was not as prominent, associated enriched pathways showed mostly regression to control or patterns opposite that of the PDF effect. Our study describes the novel peritoneal surface proteome through combined proteomic and bioinformatic analyses, and assesses changes elicited by chronic experimental PD. The biological processes so identified promise to link molecular mechanisms of membrane damage and protection in the in vivo rat model to pathomechanisms and cytoprotective effects observed in vitro and in clinical PD.",
author = "Michael Boehm and Rebecca Herzog and Florian Klinglm{\"u}ller and Lichtenauer, {Anton M.} and Anja Wagner and Markus Unterwurzacher and Beelen, {Robert H. J.} and Alper, {Seth L.} and Christoph Aufricht and Klaus Kratochwill",
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Boehm, M, Herzog, R, Klinglmüller, F, Lichtenauer, AM, Wagner, A, Unterwurzacher, M, Beelen, RHJ, Alper, SL, Aufricht, C & Kratochwill, K 2019, 'The peritoneal surface proteome in a model of chronic peritoneal dialysis reveals mechanisms of membrane damage and preservation' Frontiers in Physiology, vol. 10, no. MAY, 472. https://doi.org/10.3389/fphys.2019.00472

The peritoneal surface proteome in a model of chronic peritoneal dialysis reveals mechanisms of membrane damage and preservation. / Boehm, Michael; Herzog, Rebecca; Klinglmüller, Florian; Lichtenauer, Anton M.; Wagner, Anja; Unterwurzacher, Markus; Beelen, Robert H. J.; Alper, Seth L.; Aufricht, Christoph; Kratochwill, Klaus.

In: Frontiers in Physiology, Vol. 10, No. MAY, 472, 2019.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - The peritoneal surface proteome in a model of chronic peritoneal dialysis reveals mechanisms of membrane damage and preservation

AU - Boehm, Michael

AU - Herzog, Rebecca

AU - Klinglmüller, Florian

AU - Lichtenauer, Anton M.

AU - Wagner, Anja

AU - Unterwurzacher, Markus

AU - Beelen, Robert H. J.

AU - Alper, Seth L.

AU - Aufricht, Christoph

AU - Kratochwill, Klaus

PY - 2019

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AB - Peritoneal dialysis (PD) fluids are cytotoxic to the peritoneum. Recent studies have shown that alanyl-glutamine (AlaGln) modulates the cellular stress response, improves mesothelial cell survival, reduces submesothelial thickening in experimental models of PD, and in clinical studies improves PD effluent cell stress and immune responses. However, the mechanisms of AlaGln-mediated membrane protection are not yet fully understood. Here, we explore those mechanisms through application of a novel proteomics approach in a clinically relevant in vivo model in rats. Experimental PD was performed for 5 weeks using conventional single-chamber bag (SCB) or neutral dual-chamber bag (DCB), PD fluid (PDF), with or without AlaGln supplementation, via a surgically implanted catheter. Rats subjected to a single dwell without catheter implantation served as controls. The peritoneal surface proteome was directly harvested by detergent extraction and subjected to proteomic analysis by two-dimensional difference gel electrophoresis (2D-DiGE) with protein identification by mass spectrometry. An integrated bioinformatic approach was applied to identify proteins significantly affected by the treatments despite biological variation and interfering high abundance proteins. From 505 of 744 common spots on 59 gels, 222 unique proteins were identified. Using UniProt database information, proteins were assigned either as high abundance plasma proteins, or as cellular proteins. Statistical analysis employed an adapted workflow from RNA-sequencing, the trimmed mean of M-values (TMM) for normalization, and a mixed model for computational identification of significantly differentially abundant proteins. The most prominently enriched pathways after 5 weeks chronic treatment with SCB or DCB, PDFs belonged to clusters reflecting tissue damage and cell differentiation by cytoskeletal reorganization, immune responses, altered metabolism, and oxidative stress and redox homeostasis. Although the AlaGln effect was not as prominent, associated enriched pathways showed mostly regression to control or patterns opposite that of the PDF effect. Our study describes the novel peritoneal surface proteome through combined proteomic and bioinformatic analyses, and assesses changes elicited by chronic experimental PD. The biological processes so identified promise to link molecular mechanisms of membrane damage and protection in the in vivo rat model to pathomechanisms and cytoprotective effects observed in vitro and in clinical PD.

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