Regulatory role of FSTL1 in the differentiation of mesenchymal stem cells into myofibroblasts during renal fibrosis induced by unilateral renal ischemia-reperfusion injury in mice
10.3760/cma.j.cn421203-20250125-00019
- VernacularTitle:FSTL1对小鼠单侧肾缺血再灌注损伤诱导肾纤维化中间充质干细胞向肌成纤维细胞分化的调节作用
- Author:
Jingjing GUO
1
;
Yongsheng LUO
;
Ruiming RONG
Author Information
1. 复旦大学附属中山医院肾脏移植科 上海市器官移植重点实验室，上海 200032
- Publication Type:Journal Article
- Keywords:
Kidney transplantation;
Renal fibrosis;
Follistatin-like 1 protein;
Mesenchymal stem cell;
Ischemia reperfusion injury;
Myofibroblast
- From:
Chinese Journal of Organ Transplantation
2025;46(7):516-525
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the regulatory effect of follistatin-like 1 protein (FSTL1) on the differentiation of mesenchymal stem cells (MSCs) into myofibroblasts in renal tissue and on renal fibrosis induced by renal ischemia-reperfusion injury (IRI).Method:In animal experiments, a unilateral renal IRI-induced fibrosis model was established by clamping the left renal pedicle (IRI 7 d group, n=3), with a sham-operated group as control (Sham 7 d group, n=3). Twelve male C57BL/6 mice were randomly assigned to sham operation (Sham group, n=4), model (IRI group, n=4) and FSTL1 treatment (IRI+FSTL1 group, n=4) groups. The Sham group underwent laparotomy sham surgery, while the IRI group and IRI+FSTL1 group underwent unilateral renal IRI surgery. From 1 day before surgery to 7 days after surgery, mice received daily intraperitoneal injections of phosphate buffered saline (Sham group and IRI group) or recombinant FSTL1 protein (IRI+FSTL1 group). The mice were sacrificed 14 days after surgery. Renal histopathological damage was evaluated by hematoxylin-eosin (HE) staining. Collagen fiber deposition and fibronectin (FN) accumulation in renal interstitium were assessed by Sirius red staining, Masson’s trichrome staining and immunohistochemical staining. The co-expression level of stem cell antigen-1 (SCA-1) and α-smooth muscle actin (α-SMA) was detected by double immunofluorescence staining. The mRNA expression levels of fibrosis-related genes ( Col1a1, Col3a1, Fn1) and myofibroblast marker gene ( Acta2) were detected by real time quantitative polymerase chain reaction (RT-qPCR). In cell experiments, a differentiation model of mouse MSCs into myofibroblasts was established through TGF-β1 induction (MSC+TGF-β1 group, n=3), with a blank control group (MSC group, n=3) set up for comparison. Bone marrow MSCs from C57BL/6 mice were randomly divided into 4 groups: the TGF-β1 induction group (MSC+TGF-β1, n=6), the FSTL1 treatment group (MSC+TGF-β1+FSTL1, n=6), the negative control siRNA transfection group (MSC-siNC+TGF-β1, n=6), and the Fstl1-targeted siRNA transfection group (MSC-si Fstl1+TGF-β1, n=6). The mRNA and protein expression levels of Col1a1, Col3a1, Fn1 and Acta2 were detected by RT-qPCR and Western blot respectively. The concentration of FSTL1 protein in cell culture supernatant was measured by enzyme-linked immunosorbent assay. Result:The expression of Col1a1 and Acta2 mRNA in the kidney tissue of IRI 7 d group mice was higher than that of the Sham 7 d group (both P<0.05), successfully establishing a mouse model of renal fibrosis. The mRNA expression of Fstl1 in kidney tissues of the IRI 7 d group mice was upregulated ( P<0.01). Immunofluorescence double staining revealed increased co-expression of SCA-1/α-SMA and SCA-1/FSTL1 in the renal interstitium. At 14 days after surgery, HE staining results showed reduced pathological damage in kidney tissues of the IRI+FSTL1 group compared to the IRI group, and Sirius Red and Masson staining revealed decreased collagen fiber deposition in the renal interstitium, while immunohistochemical staining demonstrated reduced FN accumulation in the renal interstitium (all P<0.05). The mRNA expression levels of Col1a1, Col3a1, Fn1 and Acta2 in renal tissue of IRI+FSTL1 group were lower than IRI group (all P<0.05). Double immunofluorescence staining showed a decrease in the co-expression of SCA-1 and α-SMA in renal interstitium. In cell experiments, the mRNA expression levels of Col1a1, Col3a1, Fn1, and Acta2 in the MSC+TGF-β1 group were higher than those in the MSC group (all P<0.05), confirming successful TGF-β1-induced myofibroblast differentiation of MSCs. Additionally, both intracellular FSTL1 protein expression and FSTL1 protein concentration in the cell supernatant were elevated in the MSC+TGF-β1 group (both P<0.05). Compared with MSC+TGF-β1 group, the mRNA levels of Acta2 and Col1a1 in MSC+TGF-β1+FSTL1 group were decreased (both P<0.01), and the protein expression levels of collagen Ⅰ and α-SMA were significantly decreased (both P<0.001). Compared with MSC-siNC+TGF-β1 group, the protein level of FSTL1 in cell culture supernatant of MSC-si Fstl1+TGF-β1 group were decreased ( P<0.001), while the protein expression levels of FN and α-SMA were increased (both P<0.001), and the mRNA expression levels of Fn1 and Acta2 were increased (both P<0.05). Conclusions:During unilateral renal IRI-induced renal fibrosis, MSCs in renal interstitium may differentiate into myofibroblasts. FSTL1 may alleviate renal fibrosis after unilateral renal IRI by inhibiting the differentiation of MSCs into myofibroblasts.
