The macrocyclic lactone oxacyclododecindione reduces fibrosis progression

Background: Renal fibrosis is one of the most important triggers of chronic kidney disease (CKD), 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 (ECM) deposition, a drug that can 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 (TEC) (HK2 cell line and primary cells) 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 the expression of epithelial–mesenchymal transition marker proteins and reduced renal damage, immune cell infiltration, and collagen expression and deposition, both in vivo and in vitro. Remarkably, the beneficial effects of Oxa were also detected when the natural product was administered at a time point of established fibrotic changes, a situation close to the clinical situation. Initial in vitro experiments demonstrated that a synthetic Oxa derivative possesses similar features. Conclusion: Although open questions such as possible side effects need to be investigated, our results indicate that the combination of anti-inflammatory and anti-fibrotic effects of Oxa make the substance a promising candidate for a new therapeutic approach in fibrosis treatment, and thus in the prevention of kidney disease progression.


Materials
All oligonucleotides were purchased from Sigma, Deisenhofen, Germany. All cell culture grade plastic materials were obtained from Greiner, Solingen, Germany. The High-Capacity cDNA Reverse Transcription Kit was purchased from Applied Biosystems, Darmstadt, Germany. The AMPLIFYME SG Universal Mix, was obtained from 7Bioscience, Neuenburg am Rhein, Germany.

Animal experiments
We purchased female C57BL/6 (B6) (6 weeks of age, 18g ±1g) mice from Charles River Laboratory. All mice were housed in accordance with standard animal care requirements. The animal studies were approved by the ethical board (23 177-07/ G 16-1-021) and were performed in accordance with the German animal protection law and the guidelines for the use of experimental animals as stipulated by the Guide of Care and Use of Laboratory Animals of the National Institutes of Health.

Ischemia/Reperfusion (I/R).
We anesthetized mice with 1.5-2.5% isoflurane being added to the respiratory air and exposed the right kidney through a flank incision. We induced unilateral ischemia of the right kidney by clamping the renal pedicle with nontraumatic microaneurysm clamps (Roboz Surgical Instrument Co.). Clamps were removed after 45 minutes. Body temperature was controlled at 36.8°C-37.2°C throughout the procedure. Oxa (1mg/kg) or dexamethasone (2.5mg/kg) application was performed every other day by intraperitoneal injection for seven days, starting immediately or after one week of I/R injury.
PBS/10% EtOH was used as solvent control. Seven or 20 days after I/R injury mice were sacrificed.

Compounds
Oxacyclododecindione (Oxa) was isolated from fermentations of the imperfect fungus Exserohilum rostratum by chromatographic methods as previously described (1). The purity of Oxa as estimated by HPLC-DAD/MS analysis was greater than 99 %. 14-Deoxy-14-methyloxacyclododecindione was prepared as recently described (2).

Renal histopathology
Kidney pathology was assessed as described before (3). Briefly, kidneys were fixed in 10% neutral buffered formalin for 24 h and embedded in paraffin. Stained paraffin sections (4 μm) with periodic acid-Schiff reagent, hematoxylin and eosin were assessed. At the end, the evaluated scores were summed and divided by the number of high power fields (hpf) counted and reported as the total score.

Sirius Red/Goldner Staining
The institute of pathology of the medical center of the Johannes Gutenberg University performed according to the routinely used protocols the Goldner, PAS and HE staining. Sirius Red staining was performed with 4 µm paraffin sections and picro Sirius Red in saturated aqueous solution of picric acid (Sigma, Deisenhofen, Germany). Evaluation of both stains was performed quantitatively using ImageJ (https://imagej.nih.gov/ij/index.html; version 1.53c). We analyzed the percentage of blue (Goldner staining) or red (Sirius red staining) stained parts of 10 randomly selected high-power fields (hpf) of cortex and 3 randomly selected hpf of medulla of each kidney. The percentage was averaged in each case and reported as total percentage.

Analysis of mRNA expression in kidney of C57BL/6 (B6) mice and human tubulus epithelial cells
To analyze the mRNA expression of different immune relevant genes in cells or mouse tissue, we prepared total RNA by homogenizing the sample in guanidiniumisothiocyanat-buffer and isolated the RNA as described. Gene expression in samples was quantified in a two-step real-time RT-PCR (qRT-PCR) as previously described (5)  Relative mRNA amounts were determined using the mathematical model for relative quantification in real-time PCR proposed by Pfaffl et al. (7).

Tissue preparation and cell culture.
Human TECs were isolated from a piece of kidney (discarded healthy tissue from nephrectomies) in the same manner as described before (8). Stimulation was performed with a cytokine mix (CM) (hu IFN-γ (300 U/mL), IL-1β (600 U/mL) and TNFα (37 ng/mL)) for 2 or 24 hours. Ethics approval number 837.467.13 and 2019-14695.

Transient Transfection and Cell Viability
The reporter plasmid (AGCCAGACA)9MLP-Luc contains nine tandem copies of the CAGA Smad binding element upstream of the adenovirus major late promoter driving luciferase expression (9). The control reporter vector pRL-EF1α for data normalization was purchased from Promega (Dual-Luciferase-Reporter-Assay). Luciferase-based reporter gene expression was thereby normalized for transfection variability and cytotoxicity against renilla expression of the constitutively active vector control (pRL-EF1α) assayed in the same sample. Transient transfections of HK2 cells were performed in 24 well cell culture plates using the transfection reagent jetPrime (Polyplus-transfection SA, Strasbourg, France) as described by the manufacturer. For induction of luciferase expression the cells were treated with 5 ng/mL TGF-β for 24 h. Luciferase activity was measured with a luminometer, using the Dual-Glo Luciferase assay system (Promega, Mannheim, Germany) according to the manufacturer´s instructions. To evaluate the effect of Oxa on cellular proliferation and viability, a Giemsa stain-based cell viability assay was performed after 48 h as previously described (10).

HK2 cell stimulation
As cellular model the human tubule epithelial cell line human kidney 2 (HK2, ATCC CRL-2190) was used.
HK 2cells were seeded in petri dishes with a diameter of 10 cm with a density of 1 x 10 6 cells/mL DMEM containing 10 % FCS and grown to 70-80 % confluency. The cells were then starved in medium containing 0.5 % FCS for 24 hours, pretreated for 1 h with or without different concentrations of test compound and induced with 5 ng/mL TGF-β as indicated. Total cell extracts were obtaining by washing the cells two times in ice cold PBS and resuspending the cell pellet in 200 µl ice cold RIPA buffer (150 mM NaCl, 5 mM EDTA (pH 8.0), 50 mM Tris (pH 8.0), 1.0% V/V NP-40, 0.5% V/V sodium deoxycholate, 0.1% SDS, 5 mM sodium orthovanadate, 10 mM sodium fluoride). The protein content was determined using the PierceTM BCA Protein assay kit (Thermo Fisher Scientific, Waltham, USA). conjugated with horseradish peroxidase were used and signals were visualized by the enhanced chemoluminescence detection system (New England Biolabs GmbH, Frankfurt, Germany).

Zymography
For the analysis of secreted proteins, serum starved HK2 cells were pretreated with or without the compounds for 1 h and stimulated with 5 ng/ mL TGF-β. The cell culture supernatant was collected, centrifuged at 5000 x g for 10 min at 4 °C to remove cellular debris and precipitated with 10% V/V TCA at -20°C. The samples were thawed on ice, centrifuged at 12000 x g at 4 °C for 30 min and washed with 9 volumes ice cold acetone. After centrifugation at max speed and 4°C the pellet was resuspended in a buffer containing 1% W/V SDS, 60 mM Tris-HCL, pH 6.8. 25 µL of 10-fold concentrated supernatant was analyzed for activity of matrix metalloproteinases by gelatin-zymography as described (11).

Mass Spectometry
The HK2 cells were seeded in petri dishes with a diameter of 10 cm with a density of 1 x 10 6 cells/mL DMEM containing 10 % FCS and grown to 70-80 % confluency. The cells were then starved in DMEM medium without FCS for 24 hours. Then the medium was replaced, and the cells were pretreated for 1 h with or without different concentrations of test compound and induced with 5 ng/mL TGF-β for 48 h. The cell culture supernatant was collected, centrifuged at 5000 x g for 10 min at 4 °C to remove cellular debris and precipitated with 10% V/V TCA at -20°C. The samples were thawed on ice, centrifuged at 12000 x g at 4 °C for 30 min and washed with 9 volumes ice cold acetone. After centrifugation at max speed and 4°C the pellet was resuspended in 100 µL buffer containing 25 mM ammonium bicarbonate and 8 M urea, pH 7.9. Proteins were digested and peptides desalted as

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 analysis followed by Bonferroni's multiple comparisons test was performed. All statistical analyses were performed using GraphPad Prism 9.0. Individual rows represent single proteins and graduated scale color codes from red (increased expression levels) to blue (decreased expression levels) Each column represents mean normalized data from n = 4 independent experiments.  (6)