Therapeutic Effects And Potential Mechanisms Of Human Adipose-derived Mesenchymal Stem Cells On Murine AKI To CKD Progression

AKI is an increasingly clinical problem associated with high morbidity and mortality.It shows a 25 percent increase in the risk of dormant progression to CKD and a 50 percent increase in mortality over a ten year follow-up.Recent epidemiological study strongly supports the hypothesis that AKI and CKD are intrinsically tied syndromes.The pathophysiology of AKI is characterized as renal tubular epithelial cell damage,inflammatory infiltration and vascular dysfunction,which initiates the self-repair mechanism of the kidney.Nevertheless,the regenerative capacity of kidney is relatively limited compared to heart,liver and other powerful organs.Generally,the injured kidney generates an inflammatory milieu,which inhibits the migration,homing and paracrine capacity of stem and progenitor cells into kidney.Thus,maladaptive repair presents as the persistent proliferation,activation of myofibroblasts and excellular matrix deposited in the renal interstitium,which are the hallmarks of AKI to CKD transition.However the underlying mechanisms of AKI to CKD progression are still incompletely understood,making the search for new and efficient therapeutic strategies an urgent necessity.MSCs are adult stem cells with abilities of self-renewal and multi-potent differentiation into mesodermal lineage cells,which are used to attenuate AKI induced by cisplatin,hypertonic glycerol and folic acid.However,the mechanisms remain controversial.Fate tracing studies demonstrated that exogenous transplanted MSCs failed to directly differentiate into renal epithelial cells.Recent studies have shown that MSC-derived microvesicles or exosomes mediate the protective effect of MSCs in AKI by promoting the repair of TECs.However,almost all these studies focused solely on AKI,we know very little about the potential role of MSCs and exosomes in the prevention of progression from AKI to CKD and the subsequent renal fibrosis.Sox9 is a transcription factor that belongs to the Sex-determining region Y box family and plays a crucial role in the development of multiple tissues and organs including kidneys.Recent translating ribosome affinity purification(TRAP)studies showed that Sox9 expression upregulated significantly within the injuried tubular epithelium in early phase of I/R injury,and this activation persisted on day 28 in spite of the recovery of renal function.Further immunofluorescence research demonstrated that about 40% of Sox9-positive cells were proliferating and expanding following renal injury,suggesting that Sox9 might facilitate the intrinsic repair process of injuried renal TECs.However,these previous research did not concern the association of Sox9 with MSC treatment in AKI-CKD transition,both of which could promote the repair of renal tubules.Thus,we supposed that hAD-MSC treatment can activate tubular Sox9,thereby mitigating AKI and subsequent chronic kidney fibrosis via tubular dependent exosome shuttling.To verify this hypothesis,we injected hAD-MSCs or exosomes to mice subjected to unilateral I/R and detected the levels of Sox9.Our results showed that hAD-MSCs promoted Sox9 activation in TECs and markedly alleviated AKI and its progression to CKD,drugs which inhibited exosome release suppressed Sox9 activation and inhibited its protective effects.In conclusion,hAD-MSCs employed exosomes to alleviate AKI to CKD transition through tubular epithelial cell dependent Sox9 activation.Part 1 h AD-MSCs alleviated AKI and promoted renal repairObjective The aim of this part was to investigate the effects of h AD-MSCs on I/R-induced murine AKI and the possible mechanism of action.Methods Unilateral renal I/R model was established in C57BL/6 mice and h AD-MSCs were injected through tail vein after 24 hours.Samples of kidneys and blood were collected after 5 days.The detection parameters were as follows: Renal histopathology: The changes of ischemic zone at the junction of the pulp were observed and PAS staining was performed to observe the tissue pathological alterations.Damage-associated molecules: Kim-1 was analyzed through immunofluorescence staining.Repair-associated factors: Immunofluorescence staining for the evaluation of residual proximal tubule(LTL)numbers and tubular epithelial cell Sox9 expression.Inflammatory cell infiltration: Flow cytometry was conducted for measurements of variations in renal tissue inflammatory cells(macrophages and T-lymphocytes).Proliferation and apoptosis of TECs: Immunofluorescence double-staining(Ki67 and Vimentin)for observation of effective proliferation of TECs;TUNEL fluorescence for observation of apoptotic variations in TECs,western blot analysis of Bcl-2 and Bax expression.AKI-CKD hallmarks: Fluorescence double-staining for evaluation of G2/M cell cycle arrest of TECs(PH3 and Ki67),fluorescence staining for detection of peritubular capillary networks(CD31)and western blot analysis of renal hypoxia(Hif-1?)variations.Results Compared to the I/R group,the ischemiac bands were narrower,the pathological injury was alleviated,the expression of Kim-1 was reduced,the infiltration of inflammatory cells was reduced,the apoptosis of TECs was reduced,cell cycle arrest in G2/M was reduced after h AD-MSC treatment.The residual TECs was increased,the expression of tubular Sox9 was upregulated,the CD31-positive capillaries were increased,and the effective proliferation of TECs was also increased through h AD-MSC treatment.Conclusion h AD-MSC treatment alleviated I/R-induced AKI in mice and promoted renal repair.Part 2 h AD-MSCs alleviated I/R-induced chronic renal interstitial fibrosisObjective This part was designed to observe the beneficial effects of h AD-MSCs on renal fibrosis deposition and the expression of fibrosis-associated factors 28 days after I/R with or without h AD-MSC treatment.Methods Unilateral renal I/R model was established in C57BL/6 mice and h AD-MSCs were injected through tail vein after 24 hours.Samples of kidneys and blood were collected after 28 days.The detection parameters were as follows: Kidney size: macroscopic observation of variations in kidney sizes,calculation of kidney size difference(subtraction of left kidney weight by right kidney weight).Damage-associated molecules: analysis of renal damage molecule(Kim-1)expression through immunofluorescence staining.Sox9: immunofluorescence staining for observation of Sox9 expression in TECs.Collagen deposition: Masson and Sirius red staining for evaluation of renal collagen deposition.Fibrosis-associated factors: immunofluorescence staining for the evaluation of variations in ?-SMA and Col-IV expression,western blot analysis of ?-SMA and PDGFR-? expression,RT-PCR for analysis of ?-SMA,PDGFR-?,Col-IV and Fibronectin expression.Results Compared to the I/R group,the volume of left kidney was increased in group of h AD-MSC treatment.Chronic renal injury was reduced,collagen deposition was reduced and the expression of fibrotic associated factors was also decreased in h AD-MSC treatment mice.Conclusion h AD-MSCs treatment alleviated chronic renal interstitial fibrosis induced by I/R in mice.Part 3 h AD-MSCs employed exosomes to upregulate Sox9 expression in tubular epithelial cellsObjective Previous studies have shown that MSCs protected AKI through paracrine pathway.The aim of this part was to investigate the mechanisms of the therapeutic effects of h AD-MSCs on AKI to CKD transition.Methods In vivo: Establishment of unilateral renal I/R models in the left kidneys of C57BL/6 mice,tail vein injection of different concentrations of h AD-MSC-derived exosomes after 24 hours,with or without addition of the drug,GW4869,inhibiting the release of exosomes,collection samples of renal tissue and peripheral blood after 7 days.Cell tracking: PKH-26 stained h AD-MSCs and AD-MSCs harvested from GFP mice were separately injected through tail vein of I/R mice;detection of fluorescent cells.Differentiation: immunofluorescence double-staining(GFP and LTL),evaluation of the presence of LTL-positive GFP cells;Exosome identification: WB analysis of membrane surface molecule CD63 expression,electron microscope examination of exosome size and shape.Exosome concentration: tail vein injection of h AD-MSC-derived exosomes,determination of optimal exosome concentration through tail vein.Fibrosis-associated factors: WB analysis of variations in ?-SMA,PDGFR-? expression.Sox9: immunofluorescence determination of variations in renal tubular Sox9 expression;fluorescent double-labeling(Sox9 and Kim-1)for evaluation of repair in damaged tubules,fluorescent double-labeling(Sox9 and LTL)for observation of residual tubules.In vitro: h AD-MSCs and their exosomes were co-cultured with TGF-?1-processed primary TECs or NIH3T3 cell line,with or without addition of GW4869.Fibrosis-associated factors: WB analysis to evaluate the effect of h AD-MSCs on the expression of profibrotic factors(?-SMA,Col-I,Fibronectin)by TGF-?1-stimulated TECs,NIH3T3 cell line.PCR analysis to determine the effect of h AD-MSCs on the secretion of fibrosis-associated factors(TGF-?1,CTGF,?-SMA,Col-I,Fibronectin)by TGF-?1-stimulated TECs,NIH3T3 cell line.Sox9: PCR analysis for determination of the effect of h AD-MSCs on Sox9 expression in TGF-?1-stimulated TECs,NIH3T3.Results In vivo: after h AD-MSC treatment,only an insignificant number of fluorescent cells could be found in the kidneys,moreover these cells had not differentiated into TECs.Compared to the I/R group,treatment with exosomes from h AD-MSC led to increased Sox9 activation in TECs and decreased renal interstitial fibrosis in a dose-dependent manner.However,pharmacological inhibition the release of exosomes prevented Sox9 activation and aggravated renal interstitial fibrosis.In vitro: h AD-MSCs and its exosomes promoted Sox9 activation in TGF-?1-stimulated TECs,thus reducing transdifferentiation of TECs into a profibrotic phenotype,but had no significant effect on embryonic fibroblast activation into myofibroblasts.Conclusion h AD-MSCs utilized exosomes to upregulate expression of tubular Sox9 and alleviated renal interstitial fibrosis.