CRISPR/Cas9 based knockout of lncRNA MALAT1 attenuates TGF-β1 induced Smad 2/3 mediated fibrosis during AKI-to-CKD transition

Abstract

Acute kidney injury (AKI) is a significant clinical issue with potential long-term consequences, as even a single episode can progress to chronic kidney disease (CKD). The AKI-to-CKD transition involves complex pathophysiology, including persistent inflammation, apoptosis, and fibrosis. Long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been recognized as a potential therapeutic target for various kidney diseases, including AKI and CKD. In our previous study, we conducted the transcriptomic analysis of lncRNAs in-vitro and animal models of AKI-to-CKD transition and found several dysregulated lncRNAs such as MALAT1, MEG3, NEAT1, MIAT, and H19 in this transition. Among these, we have selected lncRNA MALAT1 to further validate its role in AKI-to-CKD transition as a therapeutic target via a cluster regularly intercept short palindromic protein (CRISPR) associated protein 9 (Cas9)-mediated knockout approach in NRK52E cells. Guide RNAs (gRNAs) were designed to target MALAT1, and the PX459 turbo green fluorescence protein (GFP) plasmid containing MALAT1 gRNA1&2 was transfected into NRK52E cells using CRISPRMAX. Results demonstrated that MALAT1 knockout significantly reduced MALAT1 expression and attenuated Smad2/3-mediated fibrosis by decreasing pSmad2, pSmad2/3, Smad4, vimentin, fibronectin, collagen-I, and α-SMA expression levels, while increasing Smad7, Smurf2, and E-cadherin levels. These findings suggest that targeting the MALAT1/Smad2/3 pathway could be a potential therapeutic target for mitigating fibrosis to prevent AKI-to-CKD transition.