| In recent years,the results from animal studies on organ damage have shown that exosomes released by a variety of stem cells,including mesenchymal stem cells(MSC),possess organ-protective,anti-inflammatory and tissue-reparative/regenerative abilities.In small animal models of acute kidney injury(AKI),infusion of MSC-derived exosomes has also shown a powerful renoprotective effect.However,so far,there are still no reports on the application of MSC-derived exosomes in a large animal model of AKI.In this study,we thus utilized miniature Bama pigs to establish a large animal model of I/R-induced AKI,and then infused the exosomes extracted from the supernatant of the culture of human umbilical cord MSC(h UC-MSC)to the pig via IV in order to explore the therapeutic effects of exosomes on AKI and mechanisms involved.The first part of this study was to establish a miniature pig model of I/R-induced AKI by open surgery.We randomly divided 16 Bama miniature pigs into 4 groups,with 4 pigs in each group.Miniature pigs received a procedure with ischemia-reperfusion on the left kidney and right nephrectomy to create an I/R-induced AKI model.The duration of ischemia was 0,60,90,and 120 minutes for each group,respectively.Blood was collected before operation and at 24,48,and 72hours after operation for determining renal function,and the miniature pigs were sacrificed at 72 hours after operation,and kidneys were collected for further investigations.Our results showed that 1)compared with the baseline values(serum creatinine 55.29±4.69μmol/L,blood urea nitrogen 4.77±0.63 mmol/L),the serum creatinine(SCr)and blood urea nitrogen(BUN)of pigs 72 hours after renal reperfusion were significantly higher;2)the pathological staining of kidney tissue exhibited the lesions mainly with tubular damage;3)the expression of the kidney injury marker neutrophil gelatinase-associated lipocalin(NGAL)was significantly up-regulated;4)the level of apoptosis and necroptosis was significantly increased.All the above-mentioned parameters were the most obvious in the group of mice with 120-minutes of ischemia.Therefore,120 minutes of ischemia followed by restoration of renal flow was selected as the modeling scheme for the follow-up study.In the second part of this study,ultracentrifugation was used to extract the exosomes from the culture supernatant of human umbilical cord MSCs,and the exosomes were identified and characterized by nanoparticle tracking analysis,transmission electron microscopy,protein concentration determination,biomarker identification and fluorescent labeling.The extracted exosomes from MSC culture supernatants expressed two exosome-specific biomarkers-CD9 and CD81.The particle density in the exosome solution extracted by ultracentrifugation was(4.7±0.2)×10~9 particles/m L,with the average diameter in 109.4 nm;the distribution of particle size was relatively concentrated,with approximately 96%of the particles being 110 nm in diameter.The electron microscopic examination of the exosome sample showed about a diameter of about 100 nm vesicle structures,with spherical,hemispherical or saucer-shaped.hu MSC-derived exosomes labeled with Di I staining were infused intravenously into the pigs,which were sacrificed at 72 hours after surgery and then the liver,lungs and kidneys were collected.A fluorescence microscope was used to examine the frozen section of tissues to ensure the distribution of exosomes.Our results showed that exosomes were most distributed in the lungs and also distributed in the liver and kidneys to a certain degree.In the kidney,exosomes are mainly observed in the renal interstitium and renal tubular epithelial cells.This suggests that exosomes remain in the body for at least 72 hours,being distributed mostly in major organs.In the third part of this study,human umbilical MSC-derived exosomes(1×10~9particles/kg)were injected intravenously into minipigs as a treatment for I/R-induced AKI.Sixteen Bama miniature pigs were randomly divided into 4 groups with 4 in each group:1)sham+PBS;2)sham+exosome;3)I/R+PBS;4)I/R+exosome.We firstly assessed the protective effect of exosomes on the kidneys by determining renal function,renal histopathology,and renal injury markers.The results showed that human umbilical MSC-derived exosomes could improve the renal function,alleviate renal pathological changes,reduce the upregulated NGAL in the kidney of pigs,suggesting a renoprotective effect of exosomes.Then,we explored the possible mechanisms of action of exosomes in terms of programmed cell death,inflammation,angiogenesis,and the expression of molecules related to renal protection and regeneration.Our results indicated that compared with the I/R+PBS group,the numbers of apoptotic and necroptotic cells and expression levels of pro-inflammatory factors were decreased in the porcine kidneys of the I/R+exosome group,while the expression of molecules associated kidney protection-Klotho and bone morphogenic protein 7(BMP-7)-and regeneration-proliferating cell nuclear antigen(PCNA)-as well as angiogenesis-vascular endothelial growth factor A,(VEGFA),vascular endothelial growth factor receptor(VEGFR)and CD31-were increased.This suggests that the exosomes may exert their renal protective effects through regulating those molecular mechanisms.Moreover,a proteomic analysis on the kidney tissue of pigs indicated that the protein expression profile of porcine kidney was significantly altered after treatment with h UC-MSC derived exosomes in mini pigs subjected to I/R-AKI,with 411 proteins up-regulated and 353 proteins down-regulated.Differential proteins were mainly enriched in signaling pathways as associated with cellular metabolism and RNA transport.In summary,h UC-MSC derived exosomes showed a strong renoprotective effect in the minipig model of I/R-induced AKI and revealed that exosomes may exert such an effect through inhibiting tubular cell death and proinflammatory responses,while promoting the production/expression of renal molecules associated with renoprotection and angiogenesis,as well as altering the renal protein expression profile.The results of this study provide reliable data for clinical research and application. |
PDF Full Text Request