The Protective Effect And Mechanism Of ACE2 In Renal Ischemia-reperfusion Injury

BackgroundThe kidney is crucial for synthesizing renin-angiotensin system(RAS)components.Several recent studies have shown that the local imbalance of the renal RAS system plays a critical role in the genesis and progression of renal ischemia-reperfusion injury(RIRI).The RAS system consists of two main regulatory axes:the renin,angiotensin-converting enzyme(ACE),and angiotensin Ⅱ(Ang Ⅱ)regulatory axes,and the angiotensin-converting enzyme 2(ACE2),angiotensin 1-7(Ang 1-7),and Mas regulatory axes.As an ACE homolog,ACE2 can degrade and hydrolyze Ang II into a heptapeptide,Ang(1-7),counteracting the adverse effects caused by Ang Ⅱ.Moreover,Ang(1-7)can act on Mas receptors to exert anti-inflammatory and anti-apoptotic effects.Research shows that,the expression of ACE2 is higher in normal kidneys but significantly lower in the kidneys of RIRI mice and ACE2 knockout aggravated renal injury in RIRI mice.The pathophysiological mechanisms of RIRI include renal tubular injury,inflammation,oxidative stress,and vascular dysfunction.Oxidative stress and apoptosis of renal tubular epithelial cells are important factors that contributed to RIRI.ObjectiveTo investigated the effects of ACE2 on oxidative stress,inflammation,apoptosis,and the Nrf2/HO-l signaling pathway in HK-2 cells induced by hypoxia-reoxygenation.Methods1.HK-2 cells were infected with the ACE2 lentivirus.At 48 hours,the transfection efficiency was determined by the corresponding experiment.The experimental groups included the control group,H/R group,H/R-NC group,and H/R-ACE2 group.2.The levels of inflammatory cytokines in the cell supernatant were measured by ELISA.The levels of SOD and MDA were detected using a colorimetric method.The protein levels of ACE2,Caspase-3,Bcl-2,Bax,Nrf2,and HO-1 were detected by Western Blot.3.Nrf2 inhibitor ML385 and HO-1 inhibitor SnPPIX were used to inhibit the Nrf2/HO-1 pathway,the changes in the expression of Caspase-3,Bcl-2,Bax,Nrf2 and HO-1 were detected by Western Blot,and changes in SOD and MDA were detected by colorimetry.Results1.Expression of ACE2 in human tissuesWhen the ACE2 gene was searched in the HPA database,the results revealed that ACE2 was primarily expressed in the human gut and kidney tissues,with renal proximal tubular cells being the most abundant in kidney tissues.2.ACE2 lentivirus transfected HK-2 cells successfullyAfter 48 hours of incubation with NC and ACE2 lentiviruses expressing a green fluorescent protein,bright green fluorescence was visible under an inverted fluorescence microscope.ACE2 mRNA and protein expression detected by RT-qPCR and Western Blot were significantly increased in HK-2-ACE2,confirming the success of the HK-2-ACE2 construct.3.Overexpression of ACE2 improved the cell viability after H/RThe results of CCK-8 showed that the cell viability of HK-2 cells was significantly reduced in the H/R group and H/R-NC group,while cell viability in the H/R-ACE2 group was significantly increased.4.Overexpression of ACE2 down-regulated H/R-induced oxidative stress levelsThe results of MDA and SOD activity assays showed that the level of oxidative stress in HK-2 cells was significantly increased in the H/R group and H/R-NC group.In contrast,the H/R-ACE2 group was able to reverse such changes.5.Overexpression of ACE2 down-regulated H/R-induced inflammatory statusThe results of RT-qPCR and ELISA showed that the levels of inflammatory factors IL-6,TNF-α,and IL-1β were significantly increased in HK-2 cells of the H/R group and H/R-NC group.In contrast,the expression of inflammatory factors was significantly decreased in the H/R-ACE2 group.6.Overexpression of ACE2 inhibited the level of H/R-induced apoptosisThe Western Blot results showed that the expression of apoptosis-related proteins Bax and Caspase-3 was significantly increased in the H/R group and H/R-NC group,while the expression of anti-apoptotic proteins Bcl-2 and ACE2 was significantly decreased in the H/R group and H/R-NC group.The H/R-ACE2 group was able to reverse such protein changes.7.ACE2 protection was associated with the activation of the Nrf2/HO-1 signaling pathwayThe Western Blot results showed that the expression of Nrf2 and HO-1 was significantly decreased in the H/R group and H/R-NC group.In contrast,the H/R-ACE2 group could activate this signaling pathway,and the protein expression was significantly increased.Moreover,treatment with Nrf2/HO-l signaling pathway inhibitor ML385 and SnPPIX will inhibit the protective effect of ACE2 gene overexpression on HK-2 cells in the H/R.ConclusionThrough the present study,we confirmed that ACE2 inhibits oxidative stress,regulates inflammation,and ameliorates apoptosis in HK-2 cells under H/R,and the Nrf2/HO-1 signaling pathway may play an essential role in this progress.BackgroundHuman umbilical cord mesenchymal stem cells(HUC-MSCs)have self-renewal and multilineage differentiation potential,which can be home to the injury site and exert anti-inflammatory and anti-injury effects.The exosome is a 30-150 nm extracellular vesicle containing complex RNA,proteins,and a range of bioactive chemicals,enabling intercellular communication.As paracrine products of MSCs,exosomes are essential for MSCs to fulfill their therapeutic role.Studies have shown that MSCs derived exosomes can alleviate RIRI and play a protective role in the kidney.Although angiotensin-converting enzyme 2(ACE2)is crucial in many inflammatory illnesses,there have been little data on renal ischemia-reperfusion injury(RIRI).In this study,we used in vivo and in vitro experiments to observe the protective effect and molecular mechanism of MSC-ACE2-Exos in renal ischemia-reperfusion injury.ObjectiveWe transfected MSCs by lentivirus-ACE2 and extracted their exosomes.In vitro MSC-ACE2-Exos were co-cultured with HK-2 cells.In vivo,MSC-ACE2-Exos were injected via the tail vein.To explore the protective effects and underlying molecular mechanisms of MSC-ACE2-Exos in renal ischemia-reperfusion injury.Methods(1)Cell experimentThe lentivirus-ACE2 was transfected into MSCs,the exosomes were isolated by ultracentrifugation,and the exosomes were identified by relevant experiments.In an in vitro experiment,HK-2 cells were exposed to 1%oxygen for 12 hours in hypoxic incubators before being reoxygenated for 24 hours in normoxic incubators.A control group,a H/R group,a H/R and MSC-NC-Exos co-culture group,and a H/R and MSC-ACE2-Exos co-culture group were formed.According to the experimental needs,the cell viability was measured by CCK-8.The expression levels of the inflammatory factors were measured by ELISA in each group.The levels of oxidative stress products were detected by MDA and SOD assay kits.The expression levels of ACE2,Bcl-2,Bax,Cleaved caspase-3,and Caspase-3 apoptotic proteins were determined by Western blot.The apoptosis of each group was measured by flow cytometry.(2)Animal experimentFifteen 8-week-old male C57BL/6J mice were divided into three groups:Sham,I/R+PBS,and I/R+MSC-ACE2-Exos.The levels of serum creatinine and blood urea nitrogen in mice were measured;In vitro imaging was used to observe the distribution of the exosome in vivo;HE and PAS staining were used to observe the kidney damage of mice.ELISA was used to measure the expression level of inflammatory factors in the kidney tissue of mice in each group;Determination of oxidative stress product levels in renal tissue using MDA and SOD kits.The expression levels of ACE2 and Caspase-3 in kidney was observed by immunohistochemistry.TUNEL was used to detect the apoptosis level of mice kidney;The expression level of apoptotic protein was determined by Western blot.Results1.Extraction and identification of MSCsUnder the light microscope,the MSCs grew in the shape of spindle and vortex.They have succeeded in the MSCs induction differentiation experiment.The cell surface markers detected by flow cytometry were consistent with the characteristics of MSCs.2.ACE2 modified MSCs were successfully constructedAfter 48 hours of incubation with NC and ACE2 lentiviruse expressing a green fluorescent protein,bright green fluorescence was visible under an inverted fluorescence microscope.ACE2 mRNA and protein expression detected by RT-qPCR and Western Blot were significantly increased in MSC-ACE2.3.MSC-Exos extraction succeededExosomes were extracted by ultracentrifugation.Transmission electron microscopy showed that the exosomes formed a bilayer membrane vesicle structure,and NTA showed that the particle size of exosomes ranged from 30-150 nm.MSC-Exos expressed both exosome-associated proteins(CD9,TSG101,CD63),while the expression of the Golgi marker(GM130)was not detectable4.Uptake of MSC-ACE2-Exos by HK-2 cells and kidneyConfocal microscopy and in vitro imaging revealed that MSC-ACE2-Exos could be absorbed by HK-2 cells and mice kidneys.5.MSC-ACE2-Exos protected HK-2 cells from H/R injury in vitroFirstly,CCK-8 results showed that MSC-ACE2-Exos co-culture could significantly improve the cell viability reduced by H/R.Secondly,the results of ELISA showed that the expression of inflammatory factors in HK-2 cells was significantly increased after H/R,the expression in MSC-Exos-treated group was significantly decreased.Futhermore,the expression was further significantly decreased after MSC-ACE2-Exos intervention.SOD activity and MDA results revealed that the oxidative stress products in HK-2 cells increased significantly after H/R,while the expression in MSC-Exos-treated group was decreased significantly.The expression was decreased further after MSC-ACE2-Exos intervention.Finally,flow cytometry and Western Blot results showed that the apoptosis rate in HK-2 cells was significantly increased after H/R,while the apoptosis rate in MSC-Exos-treated group was decreased significantly.The apoptosis rate was decreased further after MSC-ACE2-Exos intervention.6.MSC-ACE2-Exos protected against I/R-induced kidney injury in vivoFirstly,the results of blood biochemical analysis showed that the SCr and BUN in the I/R+MSC-ACE2-Exos group were significantly lower than those in the I/R+PBS group.Also,compare to I/R+PBS group,the I/R+MSC-ACE2-Exos group’s tubular damage score was lower and the renal pathological alterations were greatly reduced,according to the results of HE and PAS staining.Secondly,inflammation and oxidative stress indicators showed significant improvement in the I/R+MSC-ACE2 Exos group compared to I/R+PBS.Lastly,the I/R+MSC-ACE2-Exos group had a significant decrease in renal apoptosis as determined by TUNEL and immunohistochemistry data.ConclusionExosomes derived from ACE2-modified human umbilical cord mesenchymal stem cells can reduce the inflammatory state,oxidative stress level and apoptosis caused by H/R and can improve renal function,renal tissue lesions and renal cell apoptosis in RIRI mice.