Losartan Attenuated Renal Peritubular Capillary Loss And Renal Fibrosis Of Rats Induced By Intermittent Hypoxia

Background:Obstructive sleep apnea?OSA?is the most common type of sleep disordered breathing,and chronic intermittent hypoxia?CIH?is the central pathophysiological feature of OSA.In recent years,researches have demonstrated that simple OSA is an independent risk factor for chronic kidney disease?CKD?.The main characteristics of CKD are the loss of peritubular capillary?PTC?and renal fibrosis.A large number of studies have suggested that angiotensin II receptor blockers?ARBs?can reduce the loss of PTC,decrease renal cell apoptosis,inhibit renal fibrosis,and delay the progress of CKD beyond lowering blood pressure.However,the molecular mechanisms by which CIH induced PTC loss and renal fibrosis,and the protective effects of losartan on renal injury induced by CIH are not completely clear.Objectives:In the present study,we used OSA animal model to elucidate the mechanisms underlying CIH-induced PTC loss and renal fibrosis.Furthermore,we investigated the molecular mechanisms by which losartan exerted protective effects against renal injury.In addition,we explored the protective effects of losartan on the damage of renal tubular epithelial cells?TEC?induced by intermittent hypoxia?IH?in vitro experiment.There were three parts in this study.The first part was to explore the mechanisms of PTC loss of rats induced by CIH and the protective effects of losartan.The second part was to investigate the effects of losartan on apoptosis and epithelial-mesenchymal transition?EMT?of renal TEC and renal fibrosis induced by CIH in rats.The third part was that the mechanisms of apoptosis and injury of human kidney proximal tubular epithelial cell?HK-2?induced by IH and the effects of losartan intervention were explored in vitro.Methods:Part one:40 healthy male Wistar rats were randomly divided into the following four groups:control group,CIH group,CIH+losartan group,CIH+normal saline group?CIH+NS?.After exposure to intermittent hypoxic condition for 6 weeks,blood was collected from the abdominal aorta of each rat to detect renal function.The pathological damage of renal TEC was observed by hematoxylin and eosin?HE?staining.The ultrastructural changes of renal TEC and PTC were observed by transmission electron microscopy?TEM?.The PTC density in the renal cortex was assessed by immunohistochemical staining of CD34.Angiotensin II?Ang II?levels in the kidney cortex were determined using a commercial enzyme-linked immunosorbent assay?ELISA?.The expression of hypoxia inducible factor-1??HIF-1??,angiotensin II type 1 receptor?AT1R?,vascular endothelial growth factor?VEGF?,and thrombospondin-1?TSP-1?were detected respectively by immunohistochemistry,Real-time quantitative PCR?RT-qPCR?and Western blotting.Part two:Based on the above experiments,the degree of renal fibrosis was evaluated by Masson staining and Sirius red staining.The apoptosis of renal TEC was evaluated by TUNEL staining.Further mechanisms of cellular apoptosis was investigated by measuring B-cell lymphoma 2?Bcl-2?,apoptotic Bcl-2-associated X?Bax?,Caspase-3 by immunohistochemistry,RT-qPCR and Western blotting.Transforming growth factor-?1?TGF-?1?,connective tissue growth factor?CTGF?,E-cadherin?E-cad?and?-smooth muscle actin??-SMA?were semi-quantified by immunohistochemistry to evaluate the expression of renal profibrosis factors and EMT of renal TEC.The expression levels of TGF-?1,CTGF,E-cad and?-SMA were further quantified by RT-qPCR and Western blotting.Part three:There were five groups in vitro experiments:control group,IH group,IH+10^-7mol/L losartan group,IH+10^-6mol/L losartan group,IH+10^-5mol/L losartan group.Except the control group,HK-2 cells of the other four groups were cultured in IH condition for 15 hours.Cell viability was assessed by MTT method.Cell apoptosis was analyzed by flow cytometry?FCM?.The gene and protein expressions of Bcl-2,Bax and caspase-3 in HK-2 cells were measured by RT-qPCR and Western blotting.Results:Part one:Compared with control group,the level of serum cystatin C of rats was increased in CIH condition.The morphological examination showed that CIH damaged TEC,such as tubular cell vacuolar degeneration,significantly increased size of tubular cells,karyopyknosis,chromatin margination,and swelling of the mitochondria.The immunohistochemical staining of CD34 and TEM examination showed that CIH induced PTC loss and narrowed PTC lumen.CIH increased the expression of HIF-1?,Ang II,AT1R,VEGF,TSP-1 in the renal cortex of rats.The results suggested that the level of serum cystatin C of rats was decreased,PTC loss,TEC injury and narrowed PTC lumen were attenuated,and the expression of Ang II,AT1R,HIF-1?,VEGF and TSP-1 were decreased with losartan pretreatment.Part two:Compared with control group,CIH significantly increased the apoptosis of renal TEC of rats.The expression of Bcl-2 and ratio of Bcl-2/Bax were decreased,while the expression of Bax and Caspase-3 of rats kidney were increased in the CIH group.CIH significantly increased the expression of TGF-?1,CTGF and?-SMA,decreased the expression of E-cad of rats kidney,and subsequently promoted EMT of renal TEC and renal fibrosis.Losartan attenuated apoptosis of renal TEC of rats.The expression of Bcl-2 and ratio of Bcl-2/Bax were increased,while the expression of Bax and Caspase-3 of rats kidney were decreased with losartan treatment.Losartan treatment significantly decreased TGF-?1,CTGF,?-SMA expression,increased the expression of E-cad of rats kidney,and then attenuated EMT of renal TEC and renal fibrosis.Part three:Compared with control group,IH reduced cell viability and increased proportion of apoptic cells in HK-2 cell,while losartan treatment significantly reversed these effects in a dose-independent manner compared with the IH group.The expression of Bcl-2 and the Bcl-2/Bax ratio were decreased,while the expression of Bax and Caspase-3 in HK-2 cell were increased in IH group compared with control group.The expression of Bcl-2 and ratio of Bcl-2/Bax were increased,and the expression of Bax and Caspase-3 in HK-2 cell were decreased with losartan treatment in a dose-dependent manner compared with the IH group.Conclusion:1.CIH activated introrenal RAS,damaged renal TEC,induced imbalance of renal angiogenic factors,subsequently resulted in loss of PTC of rats.Losartan ameliorated CIH-induced PTC loss of rats by down-regulating renal RAS to improve the crosstalk between PTC endothelial cells and TEC,and subsequently regulated the balance of angiogenesis factors.2.CIH induced the apoptosis of renal TEC,promoted EMT of renal TEC and renal fibrosis in rats.Losartan can reduce the apoptosis of renal TEC,inhibit EMT of renal TEC,and then reduce the renal fibrosis induced by CIH in rats.3.The apoptosis of HK-2 cells induced by IH was increased.Losartan inhibited the apoptosis of HK-2 cells induced by IH in a dose-dependent manner by regulating the Bcl-2/Bax/Caspase-3 signaling pathway.