Exosomes Derived From Human Umbilical Cord Mesenchymal Stem Cells Inhibit YAP Activity To Delay Diabetic Kidney Disease And The Mechanism

Objective:Diabetic kidney diseases(DKD)are the microvascular complications caused by persistent hyperglycemia.It is characterized by progressive fibrosis and lead to the end-stage renal disease(ESRD).Recent studies have shown that mesenchymal stem cells(MSC)have obvious repair effects in acute and chronic kidney injury.Exosomes,which are nano-vesicles produced by most cell types,play an irreplaceable role in cell-cell communication.They are extracellular small vesicles that can delivery various cargos of DNA,RNAs,proteins,and lipids.Based on 10xGenomics single-cell sequencing technology,this article aims to explore the molecular mechanism of human umbilical cord mesenchymal stem cells derived exosomes(hucMSC-Ex)to inhibit renal fibrosis and delay DKD progression.Methods:A T2DM rat model was constructed with 45%high-fat diet combined with streptozotocin(streptozotocin,STZ,35mg/kg)via tail vein injection.After 12weeks,the blood glucose lowering effect of exosomes was observed by measuring postprandial blood glucose and oral glucose tolerance tests(OGTTs).The serum biochemical tester was used to detect the creatinine and urea nitrogen indicators,and the appearance of obvious kidney damage indicated that the DKD rat model was constructed success.Sucrose density gradient ultracentrifugation was used to extract exosomes from MSC culture supernatant.Western blot detect for exosomes markers,such as CD9,CD63,CD81.Nanosight track analysis(NTA)detects the diameter and particle concentration of exosomes.Then injected with PBS as control,the experimental group was injected with hucMSC-Ex(10mg/kg,every 3 days/time)through the tail vein to observe the therapeutic effect in DKD.First,observe the distribution of hucMSC-Ex in vivo by a small animal in vivo imaging instrument(IVIS,perkin Elmer),and evaluate the renal function by measuring blood glucose,body weight,24h urine total protein,and creatinine clearance in rats.The histopathological score is calculated by HE staining to assess the degree of kidney tissue damage.Masson staining,PAS staining and Sirius red staining were used to analyze renal interstitial fibrosis.Immunohistochemical staining was used to detect the expression and localization of YAP,?-SMA and Bax in renal tissue.The expression of YAP and CK1?/?-TRCP were detected by immunofluorescence in renal tissues,immunohistochemistry and Western blot to evaluate the mechanism of hucMSC-Ex inhibiting YAP activity and reducing renal interstitial fibrosis.Take 24 weeks of DKD kidney tissue and prepare a specific tissue dissociation solution into a kidney single cell suspension for 10xGenomics single cell sequencing analysis.Collect and separate kidney tissue single-cell suspensions for 10xGenomics single-cell sequencing to analyze the changes in renal cell communities,numbers and gene levels in the state of DKD.The number of neutrophil community has increased significantly,and the proportion of the total cell community has changed.The number of renal intrinsic cells such as tubular epithelial cells,endothelial cells and glomerular mesangial was significantly decreased.And explore the changes in the number of neutrophils after the intervention of hucMSC-Ex and the protective effect on the number of renal tubular epithelial cells and mesangial cells.In order to explored the role of YAP protein in the proliferation of glomerular mesangial cells.In vitro experiments,the phosphorylated Ser381YAP and Ser127YAP protein expression and nuclear and cytoplasmic localization changes by fluorescent and Western blot.In further searching for the key molecules that hucMSC-Ex regulates YAP protein,firstly,the proteins interacting with YAP were analyzed by co-immunoprecipitation.HucMSC-Ex could promote YAP ubiquitination and degradation.Then CK1?/?-TRCP was significantly high expression in hucMSC-Ex by LC-MS/MS analysis.Western blot and qRT-PCR were used to verify the knockdown efficiency of lentivirus,and the anti-fibrosis effect of hucMSC-Ex was reduced.Results:First,we successfully constructed a DKD rat model.The HE staining and Masson staining of kidney tissue pathological sections showed that the glomerular basement membrane was significantly thickened in the DKD group,and found a large amount of collagen fibers were deposited in the kidney interstitial area.Through the small animal in vivo imaging device,it was found that the hucMSC-Ex injected through the tail vein could migrate to the damaged kidney tissue of DKD rats,which significantly improved renal function and inhibited renal interstitial fibrosis.Through10xGenomics single-cell sequence analysis,it was found that the number of macrophage colony cells in the kidney tissue of DKD rats increased significantly.By activating the TGF-?₁/Smad2/3/YAP signal axis,it promotes the change of mesangial cells to myofibroblast-like cells,and tissue immunity Fluorescence and histochemical experiments found that YAP protein in kidney tissue was significantly activated,and the expression of?-SMA increased and the progression of fibrosis was aggravated.The hucMSC-Ex treatment inhibited the decrease of YAP level and?-SMA expression in mesangial cells.In vitro experiments confirmed that macrophages were stimulated by high glucose and co-cultured with mesangial cells.It was found that Smad2/3 and YAP in mesangial cells increased into the nucleus.HucMSC-Ex treatment resulted in significant up-regulation of Ser381-YAP and Ser127-YAP in the cytoplasm.YAP decreases.Co-IP experiments showed that the ubiquitin molecules bound to YAP increased significantly and ubiquitinated degradation occurred.Further analysis of mass spectrometry data revealed that hucMSC-Ex is rich in CK1?/?-TRCP kinase ubiquitin system,which promotes YAP ubiquitination degradation and inhibits YAP signaling pathway,improves renal fibrosis and delays the process of DKD.Conclusion:HucMSC-Ex could migrated to damaged kidney tissue to improve renal function,and reduce collagen deposition and inhibit the progression of renal interstitial fibrosis.Based on 10xGenomics single cell sequencing analysis,the DKD associated macrophages promoted the transformation of mesangial cells into myofibroblast-like cells by activating the TGF-?₁/Smad2/3/YAP signal axis to accelerate the progression of renal interstitial fibrosis.And hucMSC-Ex promotes the YAP ubiquitination and degradation by delivering the CK1?/?-TRCP kinase ubiquitin system to inhibit renal interstitial fibrosis,improve renal function and delay the progression of DKD.This experiment provided an interesting treatment strategy and experimental basis for hucMSC-Ex in the treatment of DKD.