Long noncoding RNA ENST00000436340 promotes podocyte injury in diabetic kidney disease by facilitating the association of PTBP1 with RAB3B

www.nature.com/cddis ARTICLE OPEN Long noncoding RNA ENST00000436340 promotes podocyte injury in diabetic kidney disease by facilitating the association of PTBP1 with RAB3B Jinxiu Hu1, Qimeng Wang1, Xiaoting Fan1, Junhui Zhen2, Cheng Wang3, Huimin Chen3, Yingxiao Liu3, Ping Zhou1, Tingwei Zhang3, ✉ ✉ Tongtong Huang3, Rong Wang 1,3 and Zhimei Lv 1,3 1234567890();,: © The Author(s) 2023 Dysfunction of podocytes has been regarded as an important early pathologic characteristic of diabetic kidney disease (DKD), but the regulatory role of long noncoding RNAs (lncRNAs) in this process remains largely unknown. Here, we performed RNA sequencing in kidney tissues isolated from DKD patients and nondiabetic renal cancer patients undergoing surgical resection and discovered that the novel lncRNA ENST00000436340 was upregulated in DKD patients and high glucose-induced podocytes, and we showed a significant correlation between ENST00000436340 and kidney injury. Gain- and loss-of-function experiments showed that silencing ENST00000436340 alleviated high glucose-induced podocyte injury and cytoskeleton rearrangement. Mechanistically, we showed that fat mass and obesity- associate gene (FTO)-mediated m6A induced the upregulation of ENST00000436340. ENST00000436340 interacted with polypyrimidine tract binding protein 1 (PTBP1) and augmented PTBP1 binding to RAB3B mRNA, promoted RAB3B mRNA degradation, and thereby caused cytoskeleton rearrangement and inhibition of GLUT4 translocation to the plasma membrane, leading to podocyte injury and DKD progression. Together, our results suggested that upregulation of ENST00000436340 could promote podocyte injury through PTBP1-dependent RAB3B regulation, thus suggesting a novel form of lncRNA-mediated epigenetic regulation of podocytes that contributes to the pathogenesis of DKD. Cell Death and Disease (2023)14:130 ; https://doi.org/10.1038/s41419-023-05658-7 INTRODUCTION Diabetic kidney disease (DKD), a progressive kidney disease, is a major complication associated with diabetes and has become the leading cause of chronic kidney disease in China [1, 2]. Although the disease was defined many years ago, there are no effective therapies to date. Overall, current treatments such as glucose or blood pressure control achieve only partial renoprotection [3], increasing the need for novel therapeutic approaches. Podocytes are terminally differentiated epithelial cells that are located outside the glomerular capillaries and are an important component of the glomerular filtration barrier. Studies have confirmed that podocyte dysfunction or injury is the core event in the occurrence and progression of DKD [4]. Thus, identifying the key factors that mediate podocyte injury will provide important insights into the understanding of DKD pathogenesis. Long noncoding RNAs (lncRNAs) are a class of RNAs longer than 200 nucleotides in length and have limited or no protein-coding potential [5]. Increasing evidence has demonstrated that lncRNAs play vital roles in kidney diseases, including DKD [6, 7]. To search for new lncRNAs involved in podocyte injury, we performed gene expression profiling in kidney tissues isolated from DKD patients and nondiabetic renal cancer patients undergoing surgical resection by RNA sequencing. We identified that ENST00000436340 was significantly upregulated in the kidney tissues of DKD patients. However, its function and detailed molecular mechanisms in DKD are completely undefined. In the present study, we identified an ENST00000436340-PTBP1RAB3B regulatory network involved in cytoskeleton rearrangement and GLUT4 translocation, which facilitates podocyte injury in DKD. We revealed a novel mechanism for DKD pathogenesis, which will provide insights into the prevention and treatment of DKD in the future. MATERIALS AND METHODS Human samples Tissues, including kidney biopsy tissues from DKD patients and normal kidney tissues from nondiabetic renal cancer patients undergoing surgical resection, were obtained from the Department of Nephrology, Shandong Provincial Hospital, Shandong University. Serum samples obtained from DKD patients and normal controls were also obtained from the Department of Nephrology, Shandong Provincial Hospital, Shandong University. Our study protocol was approved by the Clinical Research Ethics Committee of Shandong Provincial Hospital, Shandong University, in 1 Department of Nephrology, Shandong Provincial Hospital, Shandong University, Jinan, Shandong 250021, China. 2Department of Pathology, School of Medicine, Shandong University, Jinan, Shandong 250021, China. 3Department of Nephrology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250021, China. ✉email: ; Edited by Alessandro Finazzi-Agrò Received: 7 September 2022 Revised: 1 February 2023 Accepted: 3 February 2023 Official journal of CDDpress J. Hu et al. 2 agreement with the guidelines set forth by the Declaration of Helsinki. All participants signed informed consent forms prior to using the tissues and serum samples for scientific research. RNA sequencing and data analyses Three kidney biopsy tissues from DKD patients and 3 normal kidney tissues from nondiabetic renal cancer patients undergoing surgical resection were used for RNA sequencing. Total RNA was extracted from kidney tissues using TRIzol (Invitrogen, USA) according to the manufacturer’s protocol. RNA purity was assessed using an ND-1000 Nanodrop. Each RNA sample had an A260:A280 ratio above 1.8 and an A260:A230 ratio above 2.0. RNA integrity was evaluated using the Agilent 2200 TapeStation (Agilent Technologies, USA), and each sample had an RIN above 7.0. Ribosomal RNA was removed using EpicentreRibo-Zero rRNA Removal Kit (Illumina, USA) and fragmented to approximately 200 bp. Subsequently, the purified RNAs were subjected to first-strand and second-strand cDNA synthesis following by adaptor ligation and enrichment with a low cycle according to the instructions of the NEBNext® Ultra™ RNA Library Prep Kit for Illumina (NEB, USA). The purified library products were evaluated using the Agilent 2200 TapeStation and Qubit® 2.0 (Life Technologies, USA) and then diluted to 10 pM for cluster generation in situ on the pair-end flow cell followed by sequencing (2 × 150 bp) HiSeq3000. Raw fastq sequences were treated with Trimmomatic tools (v0.36), and sequencing read quality was inspected using FastQC software. The statistically significant DE genes were obtained by an adjusted P value threshold of <0.05 and |log2(fold change)| >1 using DEGseq2 software. The data of DEncRNAs and its adjacent (100KB) DEmRNAs were integrated to obtain the potential cisregulated target genes of lncRNAs. For the prediction of trans-regulation, sequences of DEncRNAs and DEmRNAs were extracted and preliminarily screened using BLAST software (e < 1E−5). Then, RNAplex software was used to screen again to identify possible target genes of lncRNAs. 5′ and 3′ rapid amplification of cDNA en (...truncated)