Publication

Saurin et al., 2023, "The macrocyclic lactone Oxacyclododecindione reduces fibrosis progression", Frontiers in Pharmacology, https://www.frontiersin.org/articles/10.3389/fphar.2023.1200164/abstract

Authors

Sabrina Saurin, Myriam Meineck, Markus Rohr, Wilfried Roth, Till Opatz, Gerhard Erkel, Andrea Pautz, Julia Weinmann-Menke

Background

Renal fibrosis is one of the most important triggers of chronic kidney disease and only a very limited number of therapeutic options are available to stop fibrosis progression. As fibrosis is characterized by inflammation, myofibroblast activation and extracellular matrix deposition, a drug that is able to address all these processes might be an interesting therapeutic option.

Methods

We tested in vivo in an ischemia reperfusion (I/R) model in C57BL/6 mice and in kidney tubular epithelial cells (HK2 cell line and primary cells) and whether the natural product oxacyclododecindione (Oxa) reduces fibrosis progression in kidney disease. This was evaluated by western blot, mRNA expression, and mass spectrometry secretome analyses as well as by immunohistochemistry.

Results

Indeed, Oxa blocked expression of epithelial-mesenchymal transition marker proteins, reduced renal damage, immune cell infiltration and collagen expression and deposition, both in vivo and in vitro. Remarkebly, the beneficial effects of Oxa were detected also when the natural product was administered at a time point of established fibrotic changes, a situation that is close to the clinical situation. Initial in vitro experiments demonstrated, that a synthetic Oxa derivative possess similar features.

Conclusion

Although open questions such as possible side effects need to be investigated, we believe that the combination of anti-inflammatory and anti-fibrotic effects of Oxa make the substance a promising candidate for a new therapeutif approach in fibrosis treatment and therefore the prevention of progression of keidney disease.

Sample processing protocol

HK2 cells were stimulated and supernatant prepared as described in the publication. Mass spectrometry analysis was performed on a high-resolution LC-MS system (Eksigent nanoLC 425 coupled to a Triple-TOF 6600, AB Sciex) in information dependent acquisition (IDA) mode. For detailed information, see supplemental methods.

Data processing

The analysis of MS runs was performed using MaxQuant version 1.6.0.16 (Cox and Mann, 2008). Library generation for peptide spectrum matching was based on Homo sapiens. Oxidation of methionine and acetylation of the N-terminus were considered as peptide modifications. Minimal peptide length was set to 6 amino acids, the maximal mass to 6000 Da. Thresholds for peptide spectrum matching and protein identification were set by a false discovery rate (FDR) of 0.01.

Statistics

Data represent means +/- SEM. Statistical differences were determined by factorial analysis of variance followed by Tukey's or Dunnett's multiple comparison test. In the case of two means, classical t test analyses were used. Two-way ANOVA followed by Bonferroni's multiple comparison tests were performed. All statistical analyses were performed using GraphPad Prism 9.0.

Data files

Overview of significantly Oxa-regulated proteins in cell culture supernatant of TGF-b induced HK2 cells can be found at "runs\Proteomics\Book1_Secretome_TGF_Oxa_log2Ratio_Er.xlsx".

Additional details

Lab

• Name: Gerhard Erkel
• Email: erkel@rptu.de
• Affiliation: Molecular Biotechnology & Systems Biology, RPTU Kaiserslautern, Kaiserslautern, Germany