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Theranostics 2020; 10(21):9561-9578. doi:10.7150/thno.42153 This issue Cite

Research Paper

Human urine-derived stem cells protect against renal ischemia/reperfusion injury in a rat model via exosomal miR-146a-5p which targets IRAK1

Xirui Li^1#, Jun Liao^1#, Xiaojun Su^1#, Weiqiang Li², Zirong Bi¹, Jiali Wang³, Qun Su¹, Huiting Huang¹, Yongcheng Wei¹, Yifang Gao^1,4,5, Jun Li¹, Longshan Liu^1✉, Changxi Wang^1,4,5✉

1. Organ Transplant Center, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, People's Republic of China.
2. Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-Sen University, Guangzhou, Guangdong, People's Republic of China.
3. Department of Nephrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, People's Republic of China.
4. Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation), The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, People's Republic of China.
5. Guangdong Provincial Key Laboratory on Organ Donation and Transplant Immunology, Guangzhou, Guangdong, People's Republic of China.
#These authors contributed equally to this work.

✉ Corresponding authors: Longshan Liu, M.D., Ph.D., Organ Transplant Center, The First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan 2nd Road, Guangzhou, Guangdong 510000, China, Telephone: 86-18928735981; Fax: 86-20-87333428; E-mail: liulshansysu.edu.cn. Changxi Wang, M.D., Ph.D., Organ Transplant Center, The First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan 2nd Road, Guangzhou, Guangdong 510000, China. Telephone: 86-13600450862; Fax: 86-20-87333428; E-Mail: wangchxsysu.edu.cn. More

Abstract

Rationale: Ischemia/reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) that is associated with high morbidity and mortality, and for which specific treatments are lacking. In this study, we investigated the protective effect of human urine-derived stem cells (USCs) and their exosomes against IRI-induced AKI to explore the potential of these cells as a new therapeutic strategy.

Methods: USCs were derived from fresh human urine. Cell surface marker expression was analyzed by flow cytometry to determine the characteristics of the stem cells. Adult male Sprague-Dawley rats were used to generate a lethal renal IRI model. One dose of USCs (2×10⁶ cells/ml) or exosomes (20 µg/1 ml) in the experimental groups or saline (1 ml) in the control group was administered intravenously immediately after blood reperfusion. Blood was drawn every other day for measurement of serum creatinine (sCr) and blood urea nitrogen (BUN) levels. The kidneys were harvested for RNA and protein extraction to examine the levels of apoptosis and tubule injury. In vitro, the hypoxia-reoxygenation (H/R) model in human kidney cortex/proximal tubule cells (HK2) was used to analyze the protective ability of USC-derived exosomes (USC-Exo). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), western blotting, superoxide dismutase activity, and malonaldehyde content analyses were used to evaluate oxidative stress in HK2 cells treated with USC-Exo after H/R. Exosomal microRNA sequencing techniques and bioinformatics analysis were used to search for enriched miRNAs in the exosomes and interacting genes. The interaction between miRNAs and the 3' untranslated region of the target gene was detected using a dual luciferase reporting system. The miRNA mimic and inhibitor were used to regulate the miRNA level in HK2 cells.

Results: Treatment with USCs led to reductions in the levels of sCr, BUN, and renal tubular cell apoptosis; inhibited the infiltration of inflammatory cells; and protected renal function in the rat IRI model. Additionally, USC-derived exosomes protected against IRI-induced renal damage. miR-146a-5p was the most abundant miRNA in exosomes obtained from the conditioned medium (CM) of USCs. miR-146a-5p targeted and degraded the 3'UTR of interleukin-1 receptor-associated kinase 1 (IRAK1) mRNA, subsequently inhibited the activation of nuclear factor (NF)-κB signaling, and protected HK2 cells from H/R injury. USC transplantation also upregulated miR-146a-5p expression, downregulated IRAK1 expression and inhibited nuclear translocation of NF-κB p65 in the kidney of the rat IRI model.

Conclusions: According to our experimental results, USCs could protect against renal IRI via exosomal miR-146a-5p, which could target the 3'UTR of IRAK1 and subsequently inhibit the activation of NF-κB signaling and infiltration of inflammatory cells to protect renal function. As a novel cell source, USCs represent a promising non-invasive approach for the treatment of IRI.

Keywords: urine-derived stem cells, ischemia/reperfusion injury, exosomes, miR-146a-5p, IRAK1

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