Identification of urinary extracellular vesicles differentially expressed RNAs in diabetic nephropathy via whole-transcriptome integrated analysis

Zihao Zhao; Qianqian Yan; Li Fang; Guangpu Li; Yong Liu; Jia Li; Shaokang Pan; Sijie Zhou; Jiayu Duan; Dongwei Liu; Zhangsuo Liu

doi:10.1016/j.compbiomed.2023.107480

Identification of urinary extracellular vesicles differentially expressed RNAs in diabetic nephropathy via whole-transcriptome integrated analysis

Comput Biol Med. 2023 Sep 16:166:107480. doi: 10.1016/j.compbiomed.2023.107480. Online ahead of print.

Authors

Zihao Zhao¹, Qianqian Yan¹, Li Fang¹, Guangpu Li¹, Yong Liu², Jia Li², Shaokang Pan³, Sijie Zhou⁴, Jiayu Duan², Dongwei Liu⁵, Zhangsuo Liu⁶

Affiliations

¹ Department of Integrated Traditional and Western Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, PR China; Institute of Nephrology, Zhengzhou University, Zhengzhou, 450052, PR China; Henan Province Research Center for Kidney Disease, Zhengzhou, 450052, PR China; Key Laboratory of Precision Diagnosis and Treatment for Chronic Kidney Disease in Henan Province, Zhengzhou, 450052, PR China; Academy of Medical Science, Zhengzhou University, Zhengzhou, 450052, PR China.
² Institute of Nephrology, Zhengzhou University, Zhengzhou, 450052, PR China; Henan Province Research Center for Kidney Disease, Zhengzhou, 450052, PR China; Key Laboratory of Precision Diagnosis and Treatment for Chronic Kidney Disease in Henan Province, Zhengzhou, 450052, PR China.
³ Department of Integrated Traditional and Western Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, PR China; Institute of Nephrology, Zhengzhou University, Zhengzhou, 450052, PR China; Henan Province Research Center for Kidney Disease, Zhengzhou, 450052, PR China; Key Laboratory of Precision Diagnosis and Treatment for Chronic Kidney Disease in Henan Province, Zhengzhou, 450052, PR China.
⁴ Department of Integrated Traditional and Western Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, PR China; Henan Province Research Center for Kidney Disease, Zhengzhou, 450052, PR China; Key Laboratory of Precision Diagnosis and Treatment for Chronic Kidney Disease in Henan Province, Zhengzhou, 450052, PR China.
⁵ Department of Integrated Traditional and Western Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, PR China; Institute of Nephrology, Zhengzhou University, Zhengzhou, 450052, PR China; Henan Province Research Center for Kidney Disease, Zhengzhou, 450052, PR China; Key Laboratory of Precision Diagnosis and Treatment for Chronic Kidney Disease in Henan Province, Zhengzhou, 450052, PR China. Electronic address: liu-dongwei@zzu.edu.cn.
⁶ Department of Integrated Traditional and Western Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, PR China; Institute of Nephrology, Zhengzhou University, Zhengzhou, 450052, PR China; Henan Province Research Center for Kidney Disease, Zhengzhou, 450052, PR China; Key Laboratory of Precision Diagnosis and Treatment for Chronic Kidney Disease in Henan Province, Zhengzhou, 450052, PR China. Electronic address: zhangsuoliu@zzu.edu.cn.

PMID: 37738894
DOI: 10.1016/j.compbiomed.2023.107480

Abstract

Background: Diabetic nephropathy (DN) is a common systemic microvascular complication of diabetes and a leading cause of chronic kidney disease worldwide. Urinary extracellular vesicles (uEVs), which are natural nanoscale vesicles that protect RNA from degradation, have the potential to serve as an invasive diagnostic biomarker for DN.

Methods: We enrolled 24 participants, including twelve with renal biopsy-proven T₂DN and twelve with T₂DM, and isolated uEVs using ultracentrifugation. We performed microarrays for mRNAs, lncRNAs, and circRNAs in parallel, and Next-Generation Sequencing for miRNAs. Differentially expressed RNAs (DE-RNAs) were subjected to CIBERSORTx, ssGSEA analysis, GO enrichment, PPI network analysis, and construction of the lncRNA/circRNA-miRNA-mRNA regulatory network. Candidate genes and potential biomarker RNAs were validated using databases and machine learning models.

Results: A total of 1684 mRNAs, 126 lncRNAs, 123 circRNAs and 66 miRNAs were found in uEVs in T₂DN samples compared with T₂DM. CIBERSORTx revealed the involvement of uEVs in immune activity and ssGSEA explored possible cell or tissue sources of uEVs. A ceRNA co-expression and regulation relationship network was constructed. Candidate genes MYO1C and SP100 mRNA were confirmed to be expressed in the kidney using Nephroseq database, scRNA-seq dataset, and Human Protein Atlas database. We further selected 2 circRNAs, 2 miRNAs, and 2 lncRNAs from WGCNAs and ceRNAs and demonstrated their efficacy as potential diagnostic biomarkers for T₂DN using machine learning algorithms.

Conclusions: This study reported, for the first time, the whole-transcriptome genetic resources found in urine extracellular vesicles of T₂DN patients. The results provide additional support for the possible interactions, and regulators between RNAs from uEVs themselves and as potential biomarkers in DN.

Keywords: Biomarker; Diabetic nephropathy; Extracellular vesicles; Urine; mRNA; ncRNA.