Effects Of Overexpression Of Rab7 In Renal Tubular Epithelial Cells On Peritubular Angiogenesis And Extracellular Matrix Metabolism And Targeted Intervention

Chronic kidney disease(CKD)has already been a serious public health issue due to high morbidity and mortality.The morphological characteristics of CKD are progressive renal interstitial fibrosis and sparse peritubular capillary(PTC).Therefore,it is one of the strategies to alleviate the progression of the disease or cure the disease to search for molecular targets related to both peritubular capillary homeostasis and extracellular matrix(ECM)metabolism.Renal tubular epithelial cells are the main cells of the kidney,which are vulnerable to injury under the action of pathogenic factors.Various inflammatory mediators produced after injury play a significant role in promoting the development of disease.Rab7 is a small GTPase that can regulate both endocytosis and autophagy.The previous study of the research group found that Rab7 was highly expressed in hypoxic renal tubular epithelial cells cultured in vitro and kidney tissue of UUO model mice.We hypothesized that under pathological conditions,Rab7 was overexpressed in renal tubular epithelial cells,and endocytosis and autophagy were active,which interfered with the activation of matrix metalloproteinase-2(MMP-2)at the cell membrane.And then influence the angiogenesis and extracellular matrix metabolism of MMP-2.That is to say,overexpression of Rab7 in renal tubular epithelial cells may not only inhibit peritubular angiogenesis,but also affect cell phenotype and ECM metabolism.To test the hypothesis,both of renal tubular epithelial cells and microvascular endothelial cells were used to simulate the hypoxic microenvironment of CKD tissue and the renal fibrosis model of Rab7 knockout mice to observe whether hypoxic renal tubular epithelial cells regulate peritubular angiogenesis through Rab7 / MMP-2 axis at first.Then,the relationship between Rab7 expression and cell phenotype and ECM metabolism was studied in vitro.Finally,taking advantage of the homing characteristics of mesenchymal stem cells(MSCs)and their exosomes to injured tissues,the exosomes were used as carriers to deliver miR-186-5p and target Rab7 to observe the pathological morphology of renal fibrosis and the changes of related molecules,judge the intervention effect and evaluate whether Rab7 can be a potential target for the treatment of renal fibrosis.Part I In vitro study of the effect of Rab7 over-expression on angiogenesis in hypoxic renal tubular epithelial cellsObjectives: To determine whether the over-expression of Rab7 could affects PTC through paracrine effect in hypoxic renal TECs.Methods: Three kinds of Rab7 shRNA lentivirus vectors were constructed respectively and transfected into human renal tubular epithelial cells(HK-2 cells)to select a stable cell line with Rab7 down-regulating expression(HK-2-Rab7-).HK-2cells and HK-2-Rab7-cells were cultured under normoxia and hypoxia,and the supernatants were collected and used as the conditioned media to cultivate wild-type human microvascular endothelial cells-1(HMEC-1 cells),respectively.The cells were allocated into four groups,normoxic wild-type group,hypoxia wild-type group,normoxic+Rab7 sh RNA1 CM group,and hypoxia +Rab7 sh RNA1 CM group.CCK8 and Ed U staining experiment were used to measure HMEC-1 cell survival rate and proliferation,respectively.HMEC-1 cell migration was observed by transwell assays.The cyclization of endothelial cells was measured by inverted microscope and image analysis technique.MMP-2 activity in cultured supernatant was detected by Gelatin zymography assay.Western blot assay was applied to measure protein levels of HIF-1α,RECK,MMP-14 and Caveolin-1.Results: Our results showed that hypoxic HK-2 cells presented high expression of Rab7,and the supernatants could inhibit HUMEC-1 cells viability,proliferation,and cyclization,in contrast,the HUMEC-1 cells in the normoxic HK-2-Rab7-supernatants had high viability,better proliferation and tubulation.HUMEC-1 cells in HK-2-Rab7-supernatants displayed high MMP-2 activity,markedly upregulated MMP-14 level,down-regulated RECK-1 and Caveolin-1 levels,compared to those in the normoxic wild-type group.Conclusion: Increased Rab7 expression in hypoxic renal TECs can inhibit peritubular angiogenesis through paracrine effect.The low-expression of Rab7 weakens endocytosis,which contributes to MMP-2 activation in hypoxic TECs,and it maybe underlying mechanism of active angiogenesis.Part II In vivo study of the effect of Rab7 on angiogenesisObjective: To explore the influence of Rab7 on renal fibrosis and angiogenesis systemically in mice models of renal fibrosis.Methods: Rab7 knockout mice ca were first constructed using CRISPR/Cas9 technology based on C57BL/6 mice,then mice model of renal fibrosis induced by Cyclosporin A toxic nephropathy were established.Kidney tissues were taken from the euthanized mice to make tissue homogenates and paraffin sections.The mice were divided into the groups,wild-type blank control group(negative control),wild-type model group(positive control),and Rab7-/-model group.The expression levels of HIF-1α,Rab7,RECK and MMP-14 were detected by western blot in homogenates.The expression of CD34 was detected by immunohistochemical staining in paraffin sections.The level of fibrosis was analyzed by Sirius red staining combined with image analysis.Results: Cyclosporin A toxic nephropathy mice model was established using Rab7 knockout mice.Western blot results showed that HIF-1α,Rab7 and RECK were overexpressed,and MMP-14 was low expressed in positive control groups.Immunohistochemical staining with CD34 and Sirius red staining exhibited that positive control group presented decreased density of capillaries and severe renal fibrosis,which was reversed in Rab7-/-model group.Conclusion: Rab7 plays a key role in slowing renal fibrosis and promoting angiogenesis systemically.Part III The mechanism of Rab7 in regulating peritubular angiogenesis in hypoxic renal tubular epithelial cellsObjective: To determine whether Rab7 molecule from renal TECs affects angiogenesis through regulating MMP-2 activity in cultured hypoxic endothelial cells.。Methods: HK-2 cells and HK-2-Rab7-cells were cultured under normoxia and hypoxia,and the supernatants were collected and used as the conditioned media to cultivate wild-type HMEC-1 cells,respectively.Here,ARP100 was simultaneously applied in HK-2-Rab7-culture groups.The cells are classified into the groups,normoxic wild-type group,hypoxic wild-type group,normoxic+Rab7 sh RNA1CM+ARP100 group,and hypoxic+Rab7 sh RNA1 CM+ ARP100 group.The cell survival rate was measured by CCK8 experiment.Endothelial cell migration was investigated by transwell migration assay.The cyclization of endothelial cells was examined by inverted microscope and image analysis.MMP-2 activity in supernatants was detected by gelatin zymography assay.Results: Compared with the normoxic group,the conditioned medium of wild-type renal tubular epithelial cells in hypoxia inhibited the viability,migration ability,cyclization ability and MMP-2 activity of endothelial cells.Whether in hypoxic or normoxic state,there was no significant difference in endothelial cell activity,migration ability,cyclization ability,and MMP-2 activity in supernatant between the conditioned medium of renal tubular epithelial cells with Rab7 low expression added ARP100 group and the corresponding wild-type group.Conclusion: ARP100 can block the effect of Rab7 on angiogenesis in renal TECs,which preliminarily demonstrates that Rab7 does affect capillary neovascularization by regulating MMP-2 activity in our study.Part IV Effect of Rab7 on renal tubular epithelial cell phenotype and ECM metabolismObjective: To observe the effect of Rab7 down-regulation on cell phenotype and ECM metabolism in renal tubular epithelial cells.Methods: Renal tubular epithelial cells(NRK52E)were transfected with control sh RNA and Rab7 sh RNA and divided into control group,control sh RNA group and Rab7 sh RNA group.The expression levels of Rab7 m RNA and protein were detected by q RT-PCR and Western blot.NRK52 E cells transfected with control sh RNA and Rab7 sh RNA were treated with TGF-β 1 for 72 hours,divided into untreated,TGF-β1,control-sh RNA+TGF-β1,and Rab7-sh RNA+TGF-β1 groups.Collagen I,collagen III,fibronectin and α-SMA were detected by Western blot.Cell apoptosis was analyzed by Flow cytometry and Western blot.The activities of MMP-2 and MMP-9in the culture supernatant were measured by gelatin zymography.Results: The expression of Rab7 was significantly decreased in Rab7 shRNA group compared with control sh RNA group.Compared with untreated group,TGF-β1 after treatment,the expression levels of collagen I,collagen III,fibronectin α-SMA,Bax and caspase 3 were significantly increased,the expression of Bcl-2 was significantly decreased,the apoptosis was increased,and the activities of MMP-2 and MMP-9 in the supernatant were significantly decreased.While compared with control sh RNA +TGF-β1 group,the expression of collagen I,collagen III,fibronectin and α-SMA was significantly decreased in Rab7 sh RNA + TGF-β1 group,and Rab7 down-regulation inhibited apoptosis of NRK52 E cells induced by TGF-β 1 and increased the activity of MMP-2 and MMP-9.Conclusion: The results suggest that the increased expression of Rab7 during the development of renal fibrosis can promote the EMT(epithelial–mesenchymal transition)of renal tubular epithelial cells,produce more extracellular matrix,and inhibit the activities of MMP-2 and MMP-9,so as to reduce the degradation of extracellular matrix and promote the development of renal fibrosis.Part Ⅴ Study on the treatment of renal fibrosis with Rab7 as targetObjective: After confirming the relationship between mir-186-5p and Rab7,MSCs exosomes were used to deliver mir-186-5p to target Rab7,observe the pathological morphology of renal fibrosis and the changes of related molecules,to clarify whether targeted delivery of miR-186-5p by MSCs exosomes plays a therapeutic effect on renal fibrosis and underlying mechanism.Methods:Firstly,to characterize exosomes from MSCs and identify the role of miR-186-5p.Exosomes were extracted from MSCs conditioned media,characterized by transmission electron microscopy and nanoparticle tracer analysis,and the levels of specific markers of exosomes were detected by Western blot.miR-186-5p agonist(MSCs/miR-186-5p agomir),miR-186-5p antagomir(MSCs/ anti-miR-186-5p)and empty vector(MSCs/NC)were transferred into MSCs,respectively.The RNA levels of miR-186-5p were detected by q RT-PCR in MSCs and exosomes.Secondly,the upstream and downstream relationship between miR-186-5p and Rab7 was predicted by bioinformatics software,and verified by double luciferase reporter gene assay,RT-q PCR,and western blot assay.Thirdly,to study the effect of targeted delivery of miR-186-5p by MSCs exosomes on renal fibrosis.Rat renal TECs NRK52 E were stimulated by TGF-β1 to simulate renal fibrosis in vitro.miR-186-5p agomir was delivered into NRK52 E cells by exosomes from MSCs.The level of miR-186-5p was detected by fluorescence in situ hybridization(FISH)in NRK52 E cells.Flow Cytometry(FCM)was used to examine the effect of miR-186-5p delivered by MSCs exosomes on apoptosis of NRK52 E cells.Western blot was chose to measure the expression of Collagen III,fibronectin and α-SMA in NRK52 E cells to clarify ECM synthesis and epithelial mesenchymal transformation(EMT)in detail.Fourthly,to study on the effect of miR-186-5p targeted delivery of MSCs exosomes on Rab7 expression in vitro.miR-186-5p agomir,miR-186-5p inhibitor and miRNA negative control were transfected into MSCs.The conditioned medium was collected and the exosomes were extracted.NRK52 E cells in TGF-β1,and then treated with the above exosomes or conditioned medium for 72 h.Then,the exosomes were identified by Western blot,the expression levels of Rab7 m RNA and protein were detected by q RT-PCR and Western blot in NRK52 E cells respectively.Fifthly,to study on the effect of Rab7 expression plasmid reversing miR-186-5p in exosomes on EMT,apoptosis and gelatinase activity of NRK52 E cells induced by TGF-β1.Rab7 expression plasmid or control plasmid was transfected into NRK52 E cells and identified.Then,the cells were treated with TGF-β1 to make a renal fibrosis model in vitro.The exosomes were extracted from MSCs transfected with miR-186-5p agomir for 72 h.The cells were divided into MSCs/miR-186-5p-Exo+TGF-β 1,MSCs/miR-186-5p-Exo+control-plasmid+TGF-β 1,MSCs/miR-186-5p-Exo+Rab7-plasmid + TGF-β1groups.Expression of Collagen I,collagen III,fibronectin andα-SMA in the cells was detected by Western blot.Cell apoptosis was detected by flow cytometry and Western blot.Gelatinase(MMP-2,MMP-9)activities in the supernatant were measured by gelatinase zymography.Finally,to study the effect of targeted delivery of miR-186-5p by MSCs exosomes on renal fibrosis in vivo.MSCs/ NC-EXO or MSCs/ miR-186-5p-EXO were injected into the tail veins of mice,respectively,and the mice were divided into four groups,Sham,UUO,UUO+MSCs/NC-EXO,and UUO+ MSCs/miR-186-5pEXO.MSCs-EXO was labeled with PKH67 to evaluate the bio-distribution in mice.The level of miR-186-5p in kidneys was detected by RT-q PCR.The levels of BUN and Cr in sera were detected by ELISA to define kidney injury,HE staining and Masson staining were performed to determine collagen deposition and renal fibrosis,and TUNEL assay was used to detect apoptosis in renal tissues.Results: The study showed that miR-186-5p was significantly down-regulated in both TGF-β1 treated NRK52 E cells and fibrotic renal tissues.The binding of miR-186-5p with Rab7 inhibited Rab7 expressions.MSCs exosomes could transfer miR-186-5p into TGF-β1-treated NRK52 E cells,and significantly reduce ECM deposition,inhibit EMT,and promote cell apoptosis.Rab7 expression plasmid can reverse the effects of miR-186-5p in exosomes on EMT,apoptosis and gelatinase activity of NRK52 E cells induced by TGF-β1.After UUO mice treated by miR-186-5p,renal ECM deposition was reduced,and EMT was inhibited,therefore,renal injury and fibrosis were alleviated.Conclusion: miR-186-5p delivered by MSCs exosomes can inhibit renal fibrosis in vivo and in vitro,and miR-186-5p-mediated down-regulation of Rab7 may be an important mechanism.These findings can highlight the understanding how MSCs exosomes prevent renal fibrosis in CKD patients.