LRG1 enhanced the efficacy of mesenchymal stem cells in the treatment of acute kidney injury

doi:10.3877/cma.j.issn.2095-3216.2024.01.003

Abstract

Abstract:

Objective

To investigate the impact of leucine-rich α-2-glycoprotein 1 (LRG1) pretreatment of mesenchymal stem cells (MSCs) on the efficacy of MSCs in the treatment of acute kidney injury (AKI).

Methods

Eight-week-old male C57BL/6 mice were randomly divided into four groups: sham operation group (sham group), ischemia/reperfusion injury (IRI) group, MSCs group, and LRG1-MSCs group. The renal IRI mouse model was established by clamping of the left renal pedicle for 30 min and nephrectomy of the right kidney. The sham group did not receive any treatment, while the IRI group, MSCs group, and LRG1-MSCs group were injected through the tail vein with the same volume of phosphate buffer saline (PBS), MSCs, and LRG1-pretreated MSCs, respectively. These mice were sacrificed at 3 d after surgery, and the serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured. Renal pathological injury was evaluated by acute tubular necrosis (ATN) score after periodic acid-Schiff (PAS) staining. The protein expression of kidney injury molecule-1 (KIM-1) was detected by Western blot in kidney tissues. For the in vitro experiments, HK-2 cells were divided into four groups: control group (normal HK-2 cells), hypoxia/reoxygenation group (HK-2 cells with treatment of hypoxia/reoxygenation), MSCs group (hypoxia/reoxygenation-treated HK-2 cells co-cultured with MSCs), and LRG1-MSCs group (hypoxia/reoxygenation-treated HK-2 cells co-cultured with LRG1-pretreated MSCs). Western blot analysis was used to detect the expression levels of apoptosis-related proteins (Bax and Bcl-2) and inflammation-related proteins (TNF-α and IL-6) in HK-2 cells. Cell proliferation was assessed by the cell counting kit-8 (CCK-8) method. The effect of LRG1 on the migration of MSCs was analyzed by the wound healing assay. To explore the mechanism by which LRG1 enhanced the therapeutic effect of MSCs in IRI mice, RNA sequencing was performed. The content of prostaglandin E2 (PGE2) in the supernatant of cells was detected by enzyme linked immunosorbent assay (ELISA).

Results

The levels of Scr, BUN, ATN score, and KIM-1 expression were significantly higher in the IRI group than in the sham group (all P<0.05). In contrast, the Scr, BUN, ATN score, and KIM-1 expression levels in both the MSCs group and LRG1-MSCs group were all significantly lower than those in the IRI group (all P<0.05). Furthermore, the LRG1-MSCs group exhibited significantly lower levels of Scr, BUN, ATN score, and KIM-1 expression than the MSCs group (all P<0.05). In vitro experiments revealed that the protein levels of Bax, IL-6, and TNF-α of both the MSCs group and LRG1-MSCs group were significantly lower than those in the H/R group, while Bcl-2 protein level was higher than that in the H/R group (all P<0.05). Additionally, the expression levels of Bax, TNF-α, and IL-6 proteins in HK-2 cells of the LRG1-MSCs group were lower than those in the MSCs group, while the expression level of Bcl-2 protein was higher than that in the MSCs group. Pretreatment with 250 ng/ml of LRG1 for 24 h significantly promoted the proliferation and migration of MSCs (both P<0.05). RNA sequencing revealed upregulation of the prostaglandin-endoperoxide synthase 2 (PTGS2) gene expression in LRG1-pretreated MSCs, and significant increase of PGE₂ content in the culture supernatant of LRG1-MSCs group (both P<0.05).

Conclusion

LRG1 may promote the secretion of PGE₂ by MSCs, reduce apoptosis and inflammatory response, and enhance the efficacy of MSCs in the treatment of AKI.

Key words: Leucine-rich α-2-glycoprotein 1, Mesenchymal stem cells, Acute kidney injury

Yifan Zhang, Xiaodong Geng, Yuwei Ji, Keying Zhang, Shupeng Lin, Guangyan Cai, Xiangmei Chen, Quan Hong. LRG1 enhanced the efficacy of mesenchymal stem cells in the treatment of acute kidney injury[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2024, 13(01): 16-25.

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References 41

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