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Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic strategy for acute kidney injury (AKI), but still suffer from the drawbacks of low survival rate, low homing rate, and uncertain differentiation. In search of better therapeutic strategies, we explored all-trans retinoic acid (ATRA) pretreatment of MSCs to observe whether it could improve the therapeutic efficacy on AKI.
We established a renal ischemia/reperfusion (I/R) injury model induced by clamping of left renal artery for 30 minutes and contralateral nephrectomy. Twenty-four male C57BL/6 mice were randomly divided into 4 groups: sham group, IRI group, MSCs group, and ATRA-MSCs group. MSCs or ATRA-pretreated MSCs were injected via tail vein after surgery (1 × 106 per mouse). These mice were sacrificed 3 days after surgery, and the serum creatinine and blood urea nitrogen levels were measured. The renal pathological changes were examined by Periodic Acid-Schiff staining. Western blot and quantitative real time-PCR (qRT-PCR) were used to detect inflammation-related indicators (TNF-α, IL-6, and IL-1β). Western blot analyses of apoptosis-related proteins (Bcl-2, Bax, and Cleaved caspase-3) and proliferation-related protein (PCNA) expression in kidney tissues. Immunohistochemistry was used to detect the expression level of Ki67 in each group. The in vitro hypoxia/reoxygenation (H/R) model was established using human proximal tubular epithelial cells (HK-2 cells). Co-culturing HK-2 cells with MSCs or ATRA-pretreated MSCs. The protein expression levels of TNF-α, IL-6, IL-1β, Bax, Bcl‐2, Cleaved caspase-3, and PCNA in HK-2 cells were assessed using western blot analysis. To explore the mechanisms by which ATRA-MSCs may enhance the efficacy of treatment in mice with IRI, we performed RNA sequencing analysis. GO and KEGG pathway enrichment analysis demonstrated that the hyaluronan biosynthetic process may be a functional region enriched and significantly expressed under specific conditions. Hyaluronan synthase (HAS1、HAS2、HAS3) mRNA expression level in ATRA-MSCs assessed by qRT-PCR analysis. The content of hyaluronic acid (HA) in the supernatant of ATRA-MSCs culture was detected by ELISA. The HAS2 promoter‐binding transcription factors were forecasted by the JASPAR Transcription Factor Binding Site database. We knocked down HAS2 or used a neutralizing antibody to CD44, a specific receptor for HA, to validate the mechanism of this effect and to confirm whether HA improves renal repair by activating the PI3K/AKT pathway. Statistical analyses were performed using SPSS 27.0 and GraphPad 9.3.0, and two-tailed p <0.05 was considered statistically significant.
In the in vivo experiments, the levels of Scr, BUN, and acute tubular necrosis score were significantly higher in the IRI group compared to the sham group. In contrast, the levels of Scr, BUN and ATN scores in the MSCs group and ATRA-MSCs group were significantly lower than those in the IRI group. Additionally, the ATRA-MSCs group exhibited significantly lower levels of Scr, BUN, and ATN scores, compared to the MSCs group. The protein expression levels of TNF-α, IL-6, IL-1β, Bax, Cleaved caspase-3, IL-6 and, TNF-α in kidney tissues were significantly lower in ATRA-MSCs group compared to the MSCs group. Conversely, the Bcl-2 and PCNA protein expression levels were significantly higher in the ATRA-MSCs group compared to the MSCs group. Immunohistochemistry staining showed a higher expression of Ki67 in the ATRA-MSCs group than in the MSCs group. In the in vivo experiments, the ATRA-MSCs group exhibited significantly lower expression of TNF-α, IL-6, IL-1β, Bax, and Cleaved caspase-3 proteins compared to the MSCs group, while the Bcl-2 and PCNA protein expression was significantly higher in the ATRA-MSCs group compared to the MSCs group. We found that the mRNA expression of HAS2 was significantly higher than that of HAS1 and HAS3. The HA content is significantly increased after the addition of ATRA, and this effect is greater than in the untreated group. Analysis for potential transcription factor binding sites revealed that the HAS2 promoter region has multiple potential binding sites for the retinoic acid receptor (RAR) and retinoid X receptor (RXR) of transcription factors. Based on the predictions from the JASPAR database, the RXR-RAR heterodimer had the highest prediction score, and then we identified possible binding sites for the RXR-RAR heterodimer of the HAS2 promoter. We further demonstrated that ATRA elevated the expression of HAS2 at both the mRNA and protein levels and activated the Akt signaling pathway in MSCs. However, we found that ATRA-elicited anti-inflammatory, anti-apoptosis, and pro-proliferation effects were substantially reversed following HAS2 knockdown or CD44 antibody neutralization.
ATRA-pretreated MSCs attenuated the inflammatory response and apoptosis after ischemia/reperfusion injury, and also promoted the proliferation of renal tubular epithelial cells for renal repair. ATRA promoted the secretion of HA by MSCs, activated the PI3K/AKT pathway, and enhanced the therapeutic efficacy of MSCs in AKI.