| Purpose:Obstructive sleep apnea hypopnea syndrome(OSAHS)could cause recurrent apneas,and most of them were arterial desaturation.Studies have shown that cardiovascular and target organ damage caused by OSAHS results from intermittent hypoxia,which can lead to oxidative stress,inflammation,hemodynamic instability,increased sympathetic nerve activity,and ultimately organ damage.Clinical studies and animal experiments have shown that OSAHS can cause chronic intermittent hypoxia,renin-angiotensin system,sympathetic nervous system activity and activation of inflammatory response,thereby promoting the development of chronic kidney disease(CKD).Renal tissue fibrosis is the ultimate pathologic change in the development of CKD.So far,the specific mechanism of how OSAHS causes renal fibrosis has not been studied.Therefore,this study uses a chronic intermittent hypoxia rat model of OSAHS in the progression of renal fibrosis and its possible mechanism.Method:(1)Model establishment and detection of fibrosis-related indicators: Simulate the pathogenesis of OSAHS,establish a chronic intermittent hypoxia rat model,the experiment is divided into blank control group(NC group),mild intermittent hypoxia group(CIH 1 group),moderate intermittent hypoxia group(CIH 2 group),and severe intermittent hypoxia group(CIH 3 group).The concents of TNF-?,IL-1?,IL-6,and MCP-1 was researched by Elisa technology in the serum of rats.The pathological changes and collagen distribution for kidney of rats were observed by HE staining and Masson staining.Immunohistochemistry was used to discover the protein expression levels of CD3,CD68,Col I,and FN in renal tissues of rats.Immunofluorescence method was used to dected the expressions of ?-SMA and IL-1?.The m RNA expression levels of MCP-1,MIP-1?,Col I,FN,TNF-? and IL-1? in renal tissues of rats was detected by RT-PCR.The protein expression levels of MCP-1,MIP-1?,Caspase-1 and TNF-? was detected by western blot.(2)Transcriptome sequencing technology was performed on the renal tissues of rats in NC group and CIH group.According to the sequencing results,the differentially expressed genes were screened and carried on the GO function and KEEG Pathway enrichment analysis of the them.Then the genes related to fibrrosis were selected from the differentially expressed genes,and verified their expression level by RT-PCR.(3)Establish a hypoxic injury model of HK-2 cells,MTT method was used to analysis the cell viability for HK-2 cells,the protein expression levels of Aqp1 and HIF-1? were detected by western blot;immunohistochemistry was used to detect the expression of Aqp1 in rat kidney tissue after chronic intermittent hypoxia-induced.(4)Aqp1 was significantly down-regulated in the kidney tissue of intermittent hypoxia rats by sequencing analysis of the transcriptome.The target gene was selected for the following research: TGF-? was used to induce the fibrosis model of HK-2 cell,and the Aqp1 interferes with adenovirus was used to transfected the HK-2 cell.MTT was used to analysis the viability of HK-2 cell.the expression of E-cadherin,Fsp1 and Vimentin in HK-2 cell were analysised by the method of immunofluorescence.The m RNA expression levels of TNF-?,IL-1?,?-SMA and FN were used studied by RT-PCR.The relative protein expression levels of E-Cadherin,Vimentin,Erk,p-Erk,and Aqp1 in HK-2 cell was tested by western blot.Results:(1)The results of the Elisa assay found that compared with the NC group,the contents of IL-1?,TNF-?,IL-6,MCP-1,and IFN-? in the serum of rats in the CIH 1 group was not changed significantly.The levels of IL-1?,TNF-?,IL-6,MCP-1,and IFN-? in CIH 2 group and CIH 3 group increased significantly,and as the degree of hypoxia deepened,their contents increased more significantly.(2)The results of HE staining was indicated that the pathology of the renal tissues of the experimental rats in NC group and CIH 1 group were not occurred obvious damages.Compared with the NC group,the pathology of the renal tissues of the experimental rats in the CIH 2 group was slightly injured,and the pathology of the renal tissues in the CIH 3 group was severly damaged.The results of Masson staining was found that compared with the NC group,the collagen deposition in renal tissues of rats were significantly deposited in CIH 1 group,CIH 2group and CIH 3 group,and as the degree of hypoxia deepened,the more collagen was deposited;(3)The immunohistochemical staining results showed that compared with the NC group,the expression of CD3,CD68,FN and Col I in the kidney tissues of rats in the CIH 1,CIH 2 and CIH 3 groups all increased,and their expression levels increased more significantly as the degree of hypoxia increased.(4)The results of immunofluorescence was found that compared with the NC group,the expressions of IL-1? and ?-SMA in the renal tissues of rats in the CIH 1 group,CIH 2 and CIH 3 groups were increased,and the expression level increased more significantly with the deepening of the hypoxia level.(5)The results of RT-PCR results was found that compared with the NC group,the m RNA expression levels of MCP-1,MIP-1?,Col I,FN,TNF-? and IL-1? in the renal tissues of rats in the CIH 1 group,CIH 2 group and CIH 3 group were increased.The deeper the degree of hypoxia,the more significant its expression level increased.And the results of western blot results showed that compared with the NC group,the expression levels of MCP-1,MIP-1?,Caspase-1,and TNF-? in CIH 1 group,CIH 2 group and CIH 3 group were increased CIH groups.And as the degree of hypoxia deepens,its expression level increases more significantly;(6)The RNA-seq results indicated that compared with the NC group,there were 20 differentially expressed genes which 13 were significantly up-regulated and 7 were significantly down-regulated in the kidney tissues of rats in the CIH group.Combined with existing literature,it was found that Fos,Erg1,and Aqp1 were related to renal fibrosis.The results of RT-PCR was consistented with the RNA-seq.The differentially expressed genes were mainly annotated to transcription regulation,vascular development,cell proliferation and cell differentiation and other GO functions;mainly to apoptosis,proximal tubule heavy carbon On the KEGG signaling pathways such as salt recovery,renin angiotensin system,calcium absorption,and renin secretion(7)The hypoxia model of HK-2 cells was successfully established.Compared with the normal control group,after 12 h,24 h and 48 h of hypoxia,the protein expression of HIF-1? and Aqp1 in 12 h hypoxia group,24 h hypoxia group,and 48 h hypoxia group were significantly increased in the HK-2 cells.And,compared with the NC group,the expression level of Aqp1 in the kidney tissue of rats in the CIH 1,CIH 2 and CIH 3 groups were also increased significantly,and as the degree of hypoxia deepened,the expression level increased significantly.(8)Establishing a model of HK-2 cells induced by TGF-? and transfecting the Ad-si Aqp1 adenovirus,the results show that compare with TGF-? model group,the proliferation of the HK-2 cell was significantly inhibited in the Ad-si Aqp1 group.The E-cadherin protein level was up-regulated,and the Fsp1,Vimentin,and Aqp1 proteins level were down-regulated in the Ad-si Aqp1 group.The m RNA expression levels of TNF-?,IL-1?,?-SMA,and,FN were significantly reduced after Aqp1 interferenced.(9)The results of western blot experiments was instructions that compared with the blank group,the protein expression of Erk,p-Erk and Aqp1 protein in HK-2 cells in the model group was significantly up-regulated.Compared with the model group,the expression of Erk and p-Erk protein in the Ad-si Aqp1 group wasn't changed significantly,the levels of Vimentin and Aqp1 were significantly down-regulated,and the levels of E-Caherin was significantly up-regulated.The protein expresion levels of Vimentin,Erk,p-Erk,and Aqp1 in the PD98059 group and Ad-si Aqp1+PD98059 group were down-regulated,and the E-Cadherin protein level in the PD98059 group and Ad-si Aqp1+PD98059 group was up-regulated.Compared with the Ad-si Aqp1 group,the E-Caherin protein level in the Ad-si Aqp1+PD98059 group was significantly increased,and the proteins expression of Erk,p-Erk,Aqp1,and Vimentin were significantly decreased.Compared with the Ad-si Aqp1+PD98059 group,the E-Caherin protein level in the PD98059 group was decreased,the protein expression levels of Aqp1 and Vimentin were increased,and the protein expression level of Erk and p-Erk were not changed significantly.Conclusion:(1)Chronic intermittent hypoxia could cause renal fibrosis in rats;(2)Chronic intermittent hypoxia could cause the changes of gene expression in rat kidney tissues,among the which Fos,Aqp1,and Egr1 genes were closely related to fibrosis;(3)Aqp1 was participated in the EMT process of HK-2 cells induced by TGF-?(4)Erk/Aqp1 signaling pathway was participated in the fibrosis process of HK-2 cells. |
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