| Objective: Diabetic kidney disease(DKD)is a chronic progressive metabolic disease of high incidence and insidious onset,with prevention as the primary treatment.Human umbilical cord mesenchymal stem cells(huc MSCs)therapy has been shown to be effective in the repair of various types of acute and chronic kidney injury.Human umbilical cord mesenchymal stem cells derived extracellular vesicles(huc MSC-s EVs),as the main paracrine components of huc MSCs,have a comparable role in the field of kidney injury repair.This research focused on the intrinsic cell podocyte community within the glomerulus.This study was conducted using10×Genomics single cell RNA sequencing(10×Genomics sc RNA-seq)technology and LC-MS/MS spectrometry.To investigate the specific molecular mechanism by which huc MSC-s EVs increased the autophagy level of podocytes and inhibited YAP activity,thereby reducing podocyte apoptosis and delaying the process of DKD,suggesting a new strategy for the prevention and treatment of DKD.Methods: Original huc MSCs were isolated and cultured from fresh umbilical cord tissues by the applanation method,and identified by cell morphology,lipogenic osteogenic induced differentiation,and flow cytometry detection of cell surface marker proteins.The huc MSCs culture supernatant was collected,and huc MSC-s EVs were extracted by ultrafiltration combined with differential ultracentrifugation for subsequent experimental analysis.The size and morphology were observed by transmission electron microscopy,the particle size and concentration were detected by NTA,and the specific surface markers of huc MSC-s EVs were detected by Western blot analysis.A high-fat diet combined with streptozocin(STZ)was constructed in a rat model of DKD.Huc MSC-s EVs were intervened into DKD rats through the tail vein from the 8th week after moulding,during which the renal injury repair effect of huc MSC-s EVs was assessed on blood glucose,body weight,renal function and H-E staining of renal tissues during this period.Kidney tissues from control,DKD and huc MSC-s EVs model rats at 24 weeks were collected for 10×Genomics sc RNA-seq analysis.Differences in number and apoptosis in different cells were observed by cell descending clustering,Marker gene identification and cell type identification.Then,we focused on glomerular podocytes,one of the potential therapeutic target cells of huc MSC-s EVs.In vitro experiments revealed the critical regulatory role of huc MSC-s EVs on autophagy in podocytes.Podocytes were treated with low glucose,high glucose and high glucose combined with huc MSC-s EVs and the formation of autophagic vesicles was observed by flow cytometry and electron microscopy.The formation of autophagic streams was observed by double-labeled autophagic adenovirus after intervention with the autophagy inhibitor chloroquine,and podocyte apoptosis was detected by flow cytometry and Western blot.KEGG analysis and GSVA analysis were performed on the podiatocytes community using the 10×Genomics sc RNA-seq database.The differential gene enrichment pathway and differential pathway enrichment analysis were used to screen the key signaling pathways involved in the disease process,and the key target protein molecule YAP was verified.In vivo experiments were designed to reveal YAP as a key protein molecule involved in the DKD process and the critical regulatory role of huc MSC-s EVs on podocyte YAP.Tissue immunofluorescence assay was performed to quantify and localize YAP and NPHS2 in renal tissues.Western blot was performed to detect changes in YAP protein expression in renal tissues,and immunohistochemistry was performed to detect the expression of YAP in DKD rats.In vitro experiments were set up to treat podocytes with different concentrations of high glucose,and YAP expression in podocytes was detected by Western blot and cellular immunofluorescence to screen for the most suitable high glucose treatment concentration.YAP expression and nuclear translocation in kidney tissues were detected by cellular immunofluorescence and q RT-PCR.Molecular screening of huc MSC-s EVs protein by LC-MS/MS technique for 14-3-3ζ involved in regulating intracytoplasmic retention of YAP.Construction of sh RNA vectors to achieve knockdown of 14-3-3ζ in huc MSCs.Knockdown 14-3-3ζ-engineered huc MSC-s EVs were collected and their knockdown efficiency was verified.Then,the binding of 14-3-3ζ to p-YAP at Ser 127 site was verified by Co-IP assay.The YAP protein expression of pedunculated cells after nucleoplasmic isolation was detected by Western blot,the proliferation and apoptosis of pedunculated cells were observed by cellular immunofluorescence.The effects of 14-3-3ζ on autophagy were observed by double-labeled autophagic adenovirus transfection in podocytes by SIM.The level of autophagy in podocytes after knockdown of 14-3-3ζ was detected by Western blot.The proliferation and apoptosis of podocytes were observed by flow cytometry and PCNA immunochemical staining to verify that huc MSC-s EVs-derived 14-3-3ζ increased the level of autophagy in podocytes.The expression of YAP was found to be independent after chloroquine application by Western blot.SIM observed an increase in the number of autophagic vesicles in the huc MSC-s EVs treated group,which encapsulated the YAP protein molecules lingering in the cytoplasm.It was confirmed that huc MSC-s EVs derived14-3-3ζ bridges the gap between YAP and autophagy and is jointly involved in the injury repair process of diabetic kidney disease.Results: Huc MSCs were stably passaged and exhibited spindle-shaped appearance and fish-like growth with osteogenic and lipogenic differentiation potential.Huc MSCs were highly expressed in typical MSC surface markers including CD29,CD166 and CD44.Huc MSC-s EVs were extracted from huc MSCs culture supernatants and characterised for particle size,concentration and signature proteins.The DKD model was successfully constructed and huc MSC-s EVs were injected into the tail vein at the corresponding time.Blood and urine samples were collected every week from rats in each group.The data of blood glucose,body weight,serum creatinine level and kidney/body weight ratio were statistically analyzed.In DKD rats,blood glucose increased,body weight decrease,kidney function decreased significantly,kidney/body weight ratio increased gradually,and H-E staining results showed typical microstructure damage.Huc MSC-s EVs intervention could play a good hypoglycemic effect and improved kidney function.A total of 27424 cells of 10×Genomics sc RNA-seq from 24 week normal group,DKD group and huc MSC-s EVs group were analyzed and divided into 13 cell communities.Significant infiltration of neutrophils was observed in the renal tissue of DKD rats,and the number of neutrophils decreased sharply after huc MSC-s EVs intervention.In addition,it was observed that the podocyte community maintained a certain number in the kidney tissue of normal rats,while the number of podocyte in the kidney tissue of DKD rats decreased sharply,suggesting that the podocyte loss occurred in DKD rats.The number of podocytes increased after huc MSC-s EVs intervention.WT1 immunohistochemical staining showed that the number of podocyte cells in the glomerulus decreased significantly in the DKD group,but increased in the normal group and huc MSC-s EVs group.Next,the physiological process related to podocyte apoptosis was explored: autophagy played a key role in this study.It was confirmed by flow cytometry and transmission electron microscopy that high glucose led to a decrease in the autophagy level of podocytes,a decrease in the number of autophagic vesicles and a decrease in the expression of autophagyrelated proteins,which was elevated after the huc MSC-s EVs intervention.To further confirm the key role of autophagy in this study,further validation was performed using an autophagy inhibitor,chloroquine,and it has been observed that chloroquine significantly inhibited the formation of autophagic streams and high glucose also inhibited autophagy production in podocytes.Huc MSC-s EVs intervention resulted in some recovery of the inhibited autophagy levels in both.It was then confirmed that huc MSC-s EVs increased the level of autophagy in podocytes and reduced apoptosis.The key role of the Hippo signaling pathway in this study was concluded by using biological signal analysis,and YAP was a key transcription factor of the Hippo signaling pathway.The high expression of YAP protein in the kidney tissues of DKD rats was confirmed by Western blot,tissue immunofluorescence assay and immunohistochemistry.The expression of YAP was reduced after huc MSC-s EVs treatment.In vitro experiments confirmed that high glucose caused podocyte injury and loss of podocyte function.At the RNA level,the expression of YAP1 and its downstream genes KM472＿gp063 and Tead1 were significantly increased by high glucose.cellular immunofluorescence experiments showed that YAP protein expression increased with increasing glucose concentration.In addition,high glucose increased YAP entry into the nucleus,while huc MSC-s EVs reduced YAP expression and inhibited its entry into the nucleus.YAP appeared cytoplasmic retention.LC-MS/MS mass spectrometry was used to identify 14-3-3ζ,an apoptosisrelated protein within huc MSC-s EVs.sh14-3-3ζ lentiviral vector(sh14-3-3ζ)was constructed and transfected into huc MSCs.Huc MSC-s EVs were obtained after transfection,and the knockdown efficiency was verified.A significant decrease in pYAP at serine 127 site bound by sh14-3-3ζ-s EVs was confirmed by Co-IP assay.A decrease in nuclear YAP expression and an increase in cytoplasmic YAP in podocytes after sh14-3-3ζ-s EVs treatment was confirmed by nucleoplasmic separation assay.Caspase3 expression was increased in podocytes after sh14-3-3ζ-s EVs intervention and Ki67-positive cells were reduced.Western blot experiments confirmed that knockdown of 14-3-3ζ in huc MSC-s EVs inhibited the autophagy level of podocytes,suggesting that huc MSC-s EVs promote podocyte autophagy by carrying 14-3-3ζ and inhibit podocyte apoptosis.The increased level of autophagy inhibited podocyte apoptosis.The expression of YAP did not decrease after intervention with the autophagy inhibitor chloroquine.Subsequently,an increase in the number of yellow colocalization sites in the cytoplasm of huc MSC-s EVs was observed by Structure Illumination Microscopy,suggesting that YAP is encapsulated in autophagic vesicles and is degraded,and clarifying the role of the 14-3-3ζ protein molecule delivered by huc MSC-s EVs as a "bridge" between YAP and autophagy.Conclusion: The type 2 DKD rat model was successfully established.It was confirmed that huc MSC-s EVs could reduce blood glucose,improve renal function,and reduce renal injury in DKD rats.Glomerular podocytes played an important role in this study.10×Genomics sc RNA-seq results confirmed that huc MSC-s EVs could reduce podocyte apoptosis in DKD rats,and the effector target molecule of huc MSCs EVs intervention in podocytes was excavated to be YAP.In vivo experiments confirmed that high glucose stimulated YAP protein expression in renal tissues,and huc MSC-s EVs attenuated YAP expression and promoted its cytosolic retention.High glucose promoted the high expression of YAP and increased apoptosis in podocytes,while autophagy was inhibited in vitro.Huc MSC-s EVs exerted cytoprotective effects by inhibiting YAP nuclear entry,reducing YAP expression,increasing autophagy level,and reducing podocyte apoptosis.Huc MSC-s EVs delivered 14-3-3ζ protein,which on the one hand inhibited YAP nuclear translocation and promoted its cytosolic retention,and on the other hand promoted the formation of autophagosomes in the cytoplasm.Finally,YAP retained in the cytoplasm was phagocytosed and cleared by autophagosomes.Huc MSC-s EVs can be considered as a novel therapeutic tool for the prevention and treatment of DKD. |
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