Analysis of the Mitochondrial Dynamics in NAFLD: Drp1 as a Marker of Inflammation and Fibrosis

Abstract

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, projected to affect 55% globally by 2040. Up to one-third of NAFLD patients develop non-alcoholic steatohepatitis (NASH), with 40% progressing to fibrosis. However, there are currently few reliable tools to predict disease progression. Impaired mitochondrial dynamics, characterized by dysregulated fission, fusion, and mitophagy, have emerged as key events in NAFLD pathophysiology, contributing to hepatocyte death and inflammation. This study explored the transition from steatosis to NASH through transcriptomic analyses, including data from patients with steatosis and those with NASH at different fibrosis stages. By identifying a transcriptomic signature associated with disease progression, the study revealed increased expression of genes involved in mitochondrial dynamics in NASH compared to steatosis and during NASH-related fibrosis. Histological analyses highlighted the central role of Dynamin-related protein 1 (Drp1), a dynamin GTPase essential for mitochondrial fission and mitophagy. In human liver biopsies, Drp1 expression progressively increased from NAFLD to NASH and NASH-related fibrosis and cirrhosis, predominantly in Kupffer cells. These finding suggest Drp1 is a potential driver of the transition to more severe liver damage, making it a promising biomarker for NASH development and progression and a potential therapeutic target in metabolic disorders.

Keywords: Drp1; Kupffer cells; cirrhosis; fibrosis; mitochondrial dynamics; mitochondrial fission; mitochondrial fusion; mitophagy; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis.

Figure 1

Mitochondrial turnover (fusion–fission–mitophagy) alterations in NASH (training dataset GSE48452): (A): unsupervised principal component analysis performed with mitochondrial fission differentially expressed genes between NASH and steatosis on training dataset GSE48452; (B): unsupervised principal component analysis performed with mitochondrial fusion differentially expressed genes between NASH and steatosis on training dataset GSE48452; (C): unsupervised principal component analysis performed with mitophagy differentially expressed genes between NASH and steatosis on training dataset GSE48452; (D): Venn diagram of differential expression genes. P-values for each functionality were calculated by principal component analysis; (E): Heatmap of the consensus matrix (100 iterations) from non-negative matrix factorization performed with the mitochondrial expression profile (fission-fusion-mitophagy) on the GSE48452 training dataset.

Figure 2

Mitochondrial turnover associated with advanced fibrosis in NAFLD: (A): heatmap of consensus matrix (100 iterations) from non-negative matrix factorization performed with mitochondrial expression profile (fission–fusion–mitophagy) on validation dataset GSE49541. Consensus coefficient was maximal with rank 2 of clustering matching with NAFLD with advanced fibrosis (blue) and (purple) NAFLD with mild fibrosis patient groups; unsupervised principal component analysis performed with mitochondrial expression profile between NAFLD with mild or advanced fibrosis on validation dataset GSE49541. (B): Representation of a principal component analysis performed with the mitochondrial expression profile (fission–fusion–mitophagy) on the validation dataset GSE49541, demonstrating a significant separation between “mild fibrosis” and “advanced fibrosis” patient samples. (C): Random Forest error plot created with mitochondrial expression profile on validation dataset GSE49541: a total misclassification error was evaluated at 13.9% with a learning of 500 trees. (D): Gene importance plot performed with Random Forest algorithm and mitochondrial gene expression profile on validation set: 10 most important discriminant genes taking account of fibrosis grades in NAFLD are highlighted in red. (E): Gene expression boxplots of 10 most important discriminant genes taking account of fibrosis grades in NAFLD: genes are classed by their mitochondrial function (blue: mitophagy; orange: fission; pink: fusion); green boxes represent downregulated genes in advanced fibrosis; red boxes represent upregulated genes in advanced fibrosis; p-values were calculated by two-sided t-test with Welch correction.

Figure 3

Immunohistochemical staining for Drp1 in liver biopsies diagnosed as normal, steatotic, cirrhotic, and non-alcoholic steatohepatitis (NASH): Kupffer cells within lobular inflammatory infiltrates (arrow) exhibit positive Drp1 expression, whereas the liver parenchyma expression remains negative in all conditions.