| Objective:According to the latest epidemiological statistics in 2020,the number of patients with obstructive sleep apnea syndrome(OSA)in China has reached 176 million,with a high incidence among middle-aged people aged 30-60 years.Studies at home and abroad have confirmed that OSA is one of the independent,correctable and important inducing factors leading to multiple organ injuries.It has a great negative impact on life and health.Most OSA patients will suffer serious organ damage,such as heart,cerebrovascular accident,diabetes,tumor and kidney diseases.Chronic intermittent hypoxia(CIH)is the main pathological feature of OSA,and it can cause serious damage to many target organs.Therefore,CIH is considered to be an independent risk factor for many diseases.Over the past 20 years,the increasing evidence indicates that patients with untreated OSA have an increased risk of chronic kidney disease(CKD).Recent studies have shown that OSA can promote the occurrence of CKD through intrarenal hypoxia.Therefore,the development of a new drug for OSA mediated CKD will have a significant impact on improving the clinical treatment effect and reducing the economic burden of patients.Recent studies have shown that CIH can cause target organ damage by regulating mitochondrial function.Due to the high energy demand,renal tubular epithelial cells are rich in mitochondria.Therefore,the role of mitochondria in the pathogenesis of renal diseases has become a research hotspot in recent years.Previous studies have shown that mitochondrial damage and mitochondrial oxidative stress could lead to renal tubular cell damage and induce apoptosis in renal diseases.Mitochondria are the main source of reactive oxygen species(ROS).Excessive ROS will lead to oxidative stress and mitochondrial dysfunction,causing cell aging,damage and apoptosis.Mitochondria are also dynamic organelles,which constantly divide and fuse to maintain a healthy mitochondrial pool.The Mitochondrial dynamics are finely regulated by Mitofusins 1and 2(Mfn1,Mfn2),optical atrophy(OPA1),and mitogenic proteins: dynamic related protein 1(DRP1)and mitochondrial fission 1(Fis1).P66 Shc is an adaptor protein belonging to Shc A family.Recent research have shown that p66 Shc activation and phosphorylation can induce mitochondrial rupture,increase the interaction between mitotic proteins and apoptotic factors,and activate the downstream apoptotic pathway.Therefore,identifying molecules that regulate mitochondrial dynamics in CIH and lead to kidney disease are essential for the implementation of therapeutic strategies.Recent studies have provided a new sight for cannabinoid receptor 1(CB1R),which may affect mitochondrial function by regulating renal mitochondrial dynamics.At present,CB1 R antagonists have been used to inhibit the overexpression of CB1 R,but it is unclear whether these antagonists can reduce or prevent CIH mediated renal injury.Our previous research have confirmed that CIH can increase the expression of CB1 R,so we speculate that CIH may cause abnormal mitochondrial dynamics by increasing the expression of CB1 R,resulting in renal injury.Therefore,we established the animal model of OSA-CIH to detect the expression of CB1 R,p66Shc and mitochondrial dynamics related proteins Fis1 and Mfn1 in rat renal tissue and human renal cortical proximal convoluted tubular epithelial cells(HK2)in vivo and in vitro respectively,and the expression of the above indexes after targeted blocking of CB1 R with CB1 R antagonist rimonaban(Ri).The renal histopathology,mitochondrial morphology,mitochondrial function and cell apoptosis of renal tubular epithelial cells were observed before and after treatment to verify whether Ri can reduce OSA induced renal injury by regulating mitochondrial dynamics.Methods:1.Male SD rats were randomly divided into 5 groups: normal control(NC)group,chronic intermittent hypoxia 4 weeks(4w CIH)group,chronic intermittent hypoxia 6weeks(6w CIH)group,chronic intermittent hypoxia 4 weeks + rimonabant(4w CIH +Ri)group and chronic intermittent hypoxia 6 weeks + rimonabant(6w CIH + Ri).NC group rats were exposed to room air,while the two CIH groups were exposed to advanced American Biospherix A84 intermittent low oxygen chamber for 4 and 6 weeks,respectively.The rats in the two CIH + Ri groups were given Ri of 1.5 mg / kg / day for4 weeks and 6 weeks respectively.Then,the rats were euthanized and their kidneys were removed for analysis.2.The pathological changes of kidney were observed by hematoxylin eosin(HE)staining and Schiff periodate(PAS)staining.The expressions of CB1 R,p66Shc,Fis1 and Mfn1 were evaluated by western blot and immunohistochemistry,and the ultrastructural changes of mitochondria in renal were evaluated by electron microscope.3.HK2 cells were divided into NC group,18 h intermittent hypoxia(IH)group and IH + Ri group.The morphological changes of mitochondria in HK2 were observed by laser confocal after mitotracker staining before and after Ri administration,the mitochondrial membrane potential was detected by JC-1,the cell apoptosis was detected by flow cytometry,and the expressions of CB1 R,p66Shc,Fis1 and Mfn1 in HK2 cells were analyzed by cellular immunofluorescence.3.Analyze the correlation between CB1 R and the expression of p66 Shc,Fis1 and Mfn1.Results:Results in vivo research:1.The expression of CB1 R was significantly increased by CIH,and further increased with the extension of hypoxia time.Ri intervention inhibited the increase of CB1 R expression induced by CIH.2.CB1 R increased the expression of p66 Shc and mitochondrial dynamics related protein Fis1,decreased the expression of Mfn1,and increased mitochondrial fragmentation in kidney.Ri targeted blocking of CB1 R decreased the expression of Fis1 and p66 Shc,increased the expression of Mfn1 and reduced the renal mitochondria fragmentation.3.CIH caused renal injury,and the renal injury was further aggravated with the extension of hypoxia time.After Ri intervention,the renal injury of rats was improved.4.The expression of CB1 R in kidney of rats was positively correlated with Fis1 and p66Shc,and negatively correlated with Mfn1.Results in vitro research:1.After 18 hours of intermittent hypoxia,the expression of CB1 R,p66Shc and Fis1 in HK2 cells increased,the expression of Mfn1 decreased,the mitochondrial fragmentation increased,the membrane potential decreased and cell apoptosis increased.Ri inhibited the expression of CB1 R,p66Shc and Fis1,increased the expression of Mfn1,improved the mitochondrial fragmentation,increased mitochondrial membrane potential and reduced the proportion of apoptotic cells.2.The expression of CB1 R in HK2 cells was positively correlated with Fis1 and p66 Shc,and negatively correlated with Mfn1.Conclusion:1.CB1 R is involved in renal injury mediated by chronic intermittent hypoxia.2.CIH could increase the expression of CB1 R,induces abnormal mitochondrial dynamics,leading to renal injury.3.CB1 R antagonist rimonabant can reduce CIH induced renal injury by inhibiting the dysregulated renal mitochondrial dynamics. |
