Axl-inhibitor bemcentinib alleviates mitochondrial dysfunction in the unilateral ureter obstruction murine model

Abstract

Renal fibrosis is a progressive histological manifestation leading to chronic kidney disease (CKD) and associated with mitochondrial dysfunction. In previous work, we showed that Bemcentinib, an Axl receptor tyrosine kinase inhibitor, reduced fibrosis development. In this study, to investigate its effects on mitochondrial dysfunction in renal fibrosis, we analysed genome-wide transcriptomics data from a unilateral ureter obstruction (UUO) murine model in the presence or absence of bemcentinib (n = 6 per group) and SHAM-operated (n = 4) mice. Kidney ligation resulted in dysregulation of mitochondria-related pathways, with a significant reduction in the expression of oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), citric acid cycle (TCA), response to reactive oxygen species and amino acid metabolism-related genes. Bemcentinib treatment increased the expression of these genes. In contrast, AKT/PI3K signalling pathway genes were up-regulated upon UUO, but bemcentinib largely inhibited their expression. At the functional level, ligation reduced mitochondrial biomass, which was increased upon bemcentinib treatment. Serum metabolomics analysis also showed a normalizing amino acid profile in UUO, compared with SHAM-operated mice following bemcentinib treatment. Our data suggest that mitochondria and mitochondria-related pathways are dramatically affected by UUO surgery and treatment with Axl-inhibitor bemcentinib partially reverses these effects.

Keywords: AXL inhibition; UUO model; bemcentinib; mitochondria; oxidative stress; renal fibrosis.

FIGURE 1

Multivariate analysis of transcriptomics data. A, Principal component analysis (PCA) performed on the transcriptomics dataset. Ligation is the major contributing factor (PC1: 66.5%) in separating samples, followed by treatment with bemcentinib (PC2: 8.9%). B, Loading plot with an overlay of mitochondrial‐related genes shows that changes in mitochondrial gene expression contribute to the variance observed in the PCA 3 Pathway enrichment shows the top 30 enriched terms from the Reactome Pathway Database. Larger nodes represent larger enrichment. Red refers to significant in Bem‐L compared to Veh‐L and blue between L vs NL

FIGURE 2

Univariate analysis of transcriptomics data. A, Differential expression of genes using GO‐terms. Data are consistent with a significant down‐regulation of the expression of genes involved in mitochondrial‐related processes after ligation and a significant reversal of these effects upon bemcentinib treatment, whereas PI3K/AKT signalling genes were up‐regulated after ligation but this effect was reversed by treatment with bemcentinib. B, Differential expression of genes related to MAPK signalling pathway using the KEGG database. Veh‐L vs NL genes are displayed at the top, whereas Bem‐L vs Veh‐L at the bottom. Up‐regulated genes (FC > 1.15, adj P value < 0.05) are shown in red, down‐regulated (FC < −1.15, adj P value < .05) in blue and non‐significantly affected in grey

FIGURE 3

Multivariate analysis of mitochondria‐related genes. A, PCA based on the expression of significant genes (q < 0.05) filtered from MitoCarta v2. Database shows groups of samples clustering upon ligation and treatment. The variance seen in PC1 reflects ligation whereas PC2 represents the effect of bemcentinib treatment. B, Hierarchical clustering of top 50 loadings from PC1. C, Hierarchical clustering of top 50 loadings from PC2

FIGURE 4

Visualization and quantification of TOMM20 and SDHB staining. Immunohistochemical analysis of sections from ligated and non‐ligated kidneys with or without bemcentinib treatment from male C57BI/6 mice after 14 d of ureteral obstruction for (A) SDHB and (B) TOMM20 protein expression. Quantitative analysis of positive pixels (%) with fibrotic tissue subtracted from quantification is provided for (C) TOMM20, (D) SDHB, (E) SDHB/TOMM20 ratio (paired samples). F, Western blot analysis of SDHB and TOMM20. G, Western blot protein quantification SDHB/TOMM20 ratio (paired samples). H, mtDNA quantification. All data were analysed by Mann‐Whitney U test

FIGURE 5

Multivariate analysis of serum amino acids and related metabolites. A, PCA shows groups of samples clustering according to ligation and treatment. The variance seen in PC2 reflects ligation and how treatment with bemcentinib appears to reverse the effects of ligation whereas PC1 represents differences between samples. B, Loading plot from PC2 shows how serum levels of modified amino acids separate different treatment groups whereas PC1 mainly consists of essential amino acids. C, Fold changes and statistics of serum metabolites