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Exosomes derived from GDNF-modified human adipose mesenchymal stem cells ameliorate peritubular capillary loss in tubulointerstitial fibrosis by activating the SIRT1/eNOS signaling pathway.

ABSTRACT: Mesenchymal stem cells (MSCs) have emerged as ideal cell-based therapeutic candidates for the structural and functional restoration of the diseased kidney. Glial cell line-derived neurotrophic factor (GDNF) has been demonstrated to promote the therapeutic effect of MSCs on ameliorating renal injury. The mechanism may involve the transfer of endogenous molecules via paracrine factors to salvage injured cells, but these factors remain unknown. Methods: GDNF was transfected into human adipose mesenchymal stem cells via a lentiviral transfection system, and exosomes were isolated (GDNF-AMSC-exos). Using the unilateral ureteral obstruction (UUO) mouse model and human umbilical vein endothelial cells (HUVECs) against hypoxia/serum deprivation (H/SD) injury models, we investigated whether GDNF-AMSC-exos ameliorate peritubular capillary (PTC) loss in tubulointerstitial fibrosis and whether this effect is mediated by the Sirtuin 1 (SIRT1) signaling pathway. Additionally, by using SIRT1 activators or siRNAs, the roles of the candidate mRNA and its downstream gene in GDNF-AMSC-exo-induced regulation of endothelial cell function were assessed. PTC characteristics were detected by fluorescent microangiography (FMA) and analyzed by the MATLAB software. Results: The green fluorescent PKH67-labeled exosomes were visualized in the UUO kidneys and colocalized with CD81. GDNF-AMSC-exos significantly decreased PTC rarefaction and renal fibrosis scores in mice with UUO. In vitro studies revealed that GDNF-AMSC-exos exerted cytoprotective effects on HUVECs against H/SD injury by stimulating migration and angiogenesis as well as conferring apoptosis resistance. Mechanistically, GDNF-AMSC-exos enhanced SIRT1 signaling, which was accompanied by increased levels of phosphorylated endothelial nitric oxide synthase (p-eNOS). We also confirmed the SIRT1-eNOS interaction in HUVECs by immunoprecipitation. Furthermore, we observed a correlation of the PTC number with the SIRT1 expression level in the kidney in vivo. Conclusion: Our study unveiled a mechanism by which exosomes ameliorate renal fibrosis: GDNF-AMSC-exos may activate an angiogenesis program in surviving PTCs after injury by activating the SIRT1/eNOS signaling pathway.

SUBMITTER: Chen L

PROVIDER: S-EPMC7415791 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Exosomes derived from GDNF-modified human adipose mesenchymal stem cells ameliorate peritubular capillary loss in tubulointerstitial fibrosis by activating the SIRT1/eNOS signaling pathway.

Chen Lu L Wang Yanping Y Li Shulin S Zuo Bangjie B Zhang Xiangyu X Wang Fengzhen F Sun Dong D

Theranostics 20200725 20

Mesenchymal stem cells (MSCs) have emerged as ideal cell-based therapeutic candidates for the structural and functional restoration of the diseased kidney. Glial cell line-derived neurotrophic factor (GDNF) has been demonstrated to promote the therapeutic effect of MSCs on ameliorating renal injury. The mechanism may involve the transfer of endogenous molecules via paracrine factors to salvage injured cells, but these factors remain unknown. <b>Methods:</b> GDNF was transfected into human adipos ...[more]

PMID: 32802201

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Project description:Vascular endothelial growth factor (VEGF) and its cognate receptor (VEGFR2) system are crucial for cell functions associated with angiogenesis and vasculogenesis. Klotho contributes to vascular health maintenance in the kidney and other organs in mammals, but it is unknown whether renoprotection by Klotho is dependent on VEGF/VEGFR2 signaling. We used heterozygous VEGFR2-haploinsufficient (VEGFR2+/-) mice resulting from heterozygous knockin of green fluorescent protein in the locus of fetal liver kinase 1 encoding VEGFR2 to test the interplay of Klotho, phosphate, and VEGFR2 in kidney function, the vasculature, and fibrosis. VEGFR2+/- mice displayed downregulated VEGF/VEGFR2 signaling in the kidney, lower density of peritubular capillaries, and accelerated kidney fibrosis, all of which were also found in the homozygous Klotho hypomorphic mice. High dietary phosphate induced higher plasma phosphate, greater peritubular capillary rarefaction, and more kidney fibrosis in VEGFR2+/- mice compared with wild-type mice. Genetic overexpression of Klotho significantly attenuated the elevated plasma phosphate, kidney dysfunction, peritubular capillary rarefaction, and kidney fibrosis induced by a high-phosphate diet in wild-type mice but only modestly ameliorated these changes in the VEGFR2+/- background. In cultured endothelial cells, VEGFR2 inhibition reduced free VEGFR2 but enhanced its costaining of an endothelial marker (CD31) and exacerbated phosphotoxicity. Klotho protein maintained VEGFR2 expression and attenuated high phosphate-induced cell injury, which was reduced by VEGFR2 inhibition. In conclusion, normal VEGFR2 function is required for vascular integrity and for Klotho to exert vascular protective and antifibrotic actions in the kidney partially through the regulation of VEGFR2 function.NEW & NOTEWORTHY This research paper studied the interplay of vascular endothelial growth factor receptor type 2 (VEGFR2), high dietary phosphate, and Klotho, an antiaging protein, in peritubular structure and kidney fibrosis. Klotho protein was shown to maintain VEGFR2 expression in the kidney and reduce high phosphate-induced cell injury. However, Klotho cytoprotection was attenuated by VEGFR2 inhibition. Thus, normal VEGFR2 function is required for vascular integrity and Klotho to exert vascular protective and antifibrotic actions in the kidney.

Dataset Information

Exosomes derived from GDNF-modified human adipose mesenchymal stem cells ameliorate peritubular capillary loss in tubulointerstitial fibrosis by activating the SIRT1/eNOS signaling pathway.

Publications

Exosomes derived from GDNF-modified human adipose mesenchymal stem cells ameliorate peritubular capillary loss in tubulointerstitial fibrosis by activating the SIRT1/eNOS signaling pathway.

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