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

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

Figure 1

Characterization of human urine-derived stem cells (USCs). (A) Spindle-shaped morphology of USCs (scale bar = 50 µm). (B) Growth curves for USCs of different passages (P3 and P6). (C) After 3 passages, the majority of isolated USCs expressed MSC markers CD29, CD73, CD44, CD90, and CD146 but not CD31, C45, and HLA-DR. (D) Protein expression of renal markers Nephrin and WT-1 in USCs from two healthy people. (E) Relative expression levels of pluripotency markers OCT4 and NANOG in USCs.

Figure 2

The protective effects of USCs on renal function after renal IRI. (A) Diagram of the ischemia-reperfusion injury (IRI) animal model. (B) Survival curves for the control and USC-treated groups (Control group, n=19; USC-treated group, n=19; P= 0.0328). (C) Time-dependent changes in blood urea nitrogen (BUN) and serum creatinine (sCr) in control (n=25) and USC-treated groups (USCs, n=26) at days 1, 3, 5, 7 and 14 after IRI (BUN: P= 0.0028 on day 3, P= 0.0066 on day 5; sCr: P<0.0001 on day 3). Data represent the mean ± SEM, *P<0.05, **P<0.01, ***P<0.001. (D) Histopathological scores for control (n=10) and USC-treated groups (USCs, n=11) on day 7 (P= 0.0311). Scale bars =100 µm. Data represent the mean ± SEM, *P<0.05, **P<0.01.

Figure 3

USCs reduced the expression of apoptosis-related proteins, inflammatory cell infiltration, and oxidative stress level in the kidney after IRI. (A) TUNEL staining analysis of the apoptosis among renal tubular epithelial cells in the control (n=5) and USC-treated group (USCs, n=5) on days 3 and 7 after IRI. Scale bars = 100 µm, P<0.001 on day 3, P= 0.0139 on day 7. (B) Western blot analysis of cleaved-caspase-3 and Bcl2/Bax expression in the control group (n=5) and USC-treated group (n=5) on day 3 after IRI. GAPDH was used as a loading control. (C) MPO staining analysis of neutrophil infiltration in the kidney tissue on day 3. n=5 in each group. Scale bars = 200 µm in a,b; 100 µm in c,d; P<0.001. (D) SOD and MDA analysis of oxidative stress in kidney tissue on day 3 after IRI. n=4 in each group. P= 0.0378 for MDA, P= 0.0262 for SOD. Data represent the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.

Figure 4

USC-Exo protect renal function in vivo and HK2 cells after H/R injury in vitro. (A) Characterization of USC-Exo. TEM images of exosomes isolated from USC CM (scale bar = 200 nm). Size analysis of USC-Exo (mean diameter = 144.9 ± 41.6 nm). Western blot analysis of exosomal markers CD81, CD9 CD63, and HSP70. CM served as the control. (B) Diagram of IRI animal model with exosome treatment. (C) Survival curve analysis for the two groups (control group, n=19; USC-Exo-treated group, n=6; P=0.0201). (D) Time-dependent changes in BUN and sCr in the control group (n=25) and USC-Exo-treated group (USC-Exo, n=6) on days 1, 3, 5, and 7 after IRI (BUN: P= 0.0228 on day 3; sCr: P= 0.0589 on day 1, P<0.001 on day 3). (E) After incubation of HK2 cells with USC-Exo (PKH67, green fluorescence) for 30 min, 1 h, or 3 h, and washing with PBS, the fusion of exosomes and cells was observed fluorescence microscopy (scale bars = 50 µm). (F) Schematic diagram of H/R-induced injury in HK2 cells with or without USC-Exo treatment during the reoxygenation process. (G) Oxidative stress levels in HK2 cells after H/R in the two groups were determined by measuring MDA and SOD (P=0.0073 for MDA, P=0.0132 for SOD). Each experiment was repeated three times. (H) Protein expression of cleaved-Caspase-3 and BCL2/BAX in the indicated groups. After USC-Exo treatment, the expression levels of cleaved-Caspase-3 and BAX in HK2 cells were decreased significantly. Each experiment was repeated three times. GAPDH was used as loading control. Data represent the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.

Figure 5

miRNA sequencing of USC-Exo contents and potential targets of miR-146a-5p.(A) The top 20 most-enriched miRNAs in USCs-Exo. (B) qRT-PCR analysis of the expression levels of the top 6 most-enriched miRNAs in USCs-Exo. (C) StarBase analysis of the target genes of miR-146a-5p. (D) Sequence alignments of miR-146a-5p and its two candidate target sites in the 3'UTR of IRAK1. (E) Luciferase reporter assay of miR-146a-5p mimic-treated HEK293T cells, which overexpressed either IRAK1-wildtype 3'UTR (WT1 and WT2) or IRAK1-mutant 3'UTR (mut1 and mut2). Data represent the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.

Figure 6

USCs or USC-Exo upregulate miR-146a-5p expression, which targets the IRAK1 and NF-κB signaling in vivo and in vitro. Rat IRI was induced before treatment with or without USCs (annotated as USCs and control, respectively). (A) qRT-PCR analysis of the relative expression levels of miR-146a-5p (P=0.0004) and IRAK1 (P=0.0227). (B) qRT-PCR analysis of the relative expression levels of miR-146a-5p in rat serum exosomes on day 3 (n=10 in each group, P=0.0012). (C) FISH images of miR-146a-5p expression in kidney sections (scale bars = 100 µm). (D) Western blot analysis of the protein levels of IRAK1 and nuclear NF-κB p65. n=5 in each group. GAPDH and H3 were used as loading controls, respectively. H/R injury was induced in HK2 cells in the absence or presence of USC-Exo (annotated as HK2 medium and HK2 medium+exosomes, respectively). (E) qRT-PCR analysis of the relative expression levels of miR-146a-5p (P=0.0078) and IRAK1 (P=0.0358). (F) Western blot assay of the protein levels of IRAK1 and nuclear NF-κB p65. n=3 in each group. GAPDH and H3 were used as loading controls, respectively. (G) Immunofluorescence analysis showed that NF-κB p65 in HK2 cells was transferred from the cytoplasm to the nucleus after H/R treatment, and this nuclear translocation of NF-κB p65 could be inhibited by USC-Exo treatment (scale bars = 50 µm). Each experiment was repeated three times. Data represent the mean ± SEM. *P<0.05, **P<0.01.

Figure 7

miR-146a-5p mimic reduces oxidative stress and inhibits IRAK1 and NF-κB signaling in H/R-induced injury of HK2 cells. HK2 cells were transfected with miR-146a-5p mimic or its inhibitor before the H/R process. (A) qRT-PCR analysis of the relative expression levels of miR-146a-5p and IRAK1 in the mimic and mimic negative control (mimic NC) groups (P<0.001). (B) Analysis of SOD activity and MDA content in the mimic and mimic NC groups after H/R (P=0.0216 for SOD, P=0.0497 for MDA). (C) Western blot analysis of the protein levels of IRAK1 and nuclear translocation of NF-κB p65 in the mimic and mimic NC groups. n=3 in each group. GAPDH and H3 were used as loading controls, respectively. (D) Immunofluorescence analysis of the nuclear translocation of NF-κB p65 in the mimic and mimic NC groups after H/R treatment (scale bars=50 µm). (E) qRT-PCR analysis of the relative expression levels of IRAK1 in the inhibitor and inhibitor negative control (NC) groups (P=0.015). (F) Analysis of SOD activity and MDA content in the inhibitor and inhibitor NC groups after H/R (P=0.0228 for SOD, P=0.0192 for MDA). (G) Western blot analysis of the protein levels of IRAK1 and nuclear translocation of NF-κB p65 in the inhibitor and inhibitor NC groups. n=3 in each group. GAPDH and H3 were used as loading controls, respectively. Each experiment was repeated three times. Data represent the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.