The Mechanism Of “zhuo Qi” Blockade Of Lung And “Toxic Damage Of Kidney” Was Explored Based On CIH-induced Macrophage-myofibroblast Transition Participating In Renal Fibrosis

Part one Mechanism of lung and kidney injury by CIH-induced Zhou QiObjective:The purpose was to explore CIH-induced inhalation of Zhuo Qi affected the generation of Zong Qi.The lungs function was impaired by deficiency of Zong Qi,which caused renal injury.Methods:The literatures were finished refinedly to analyze CIH-induced kidney damage and the pathogenesis of Chinese medicine.Results:1.This paper demonstrated the differences between chronic intermittent hypoxia and obstructive sleep apnea syndrome from modern medicine and traditional Chinese medicine research.The purpose was to explore CIH-induced renal injury from the perspective of the formation of Zong Qi.2.Zong Qi can host the acquired gas.Zong Qi is not only the power of breath,but also the assistance of heart beat.Zong Qi is up to the chest to produce Pectoral Qi.Zong Qi and Primordial Qi are mutually beneficial.Lung and kidney maintain respiration and water metabolism together,so the lung and kidney are closely related.CIH-induced inhalation of Zhuo Qi affected the generation of Zong Qi.The lungs function was impaired by deficiency of Zong Qi,which caused renal injury.3.We speculated that Zhuo Qi entered the lung causing deficiency of Zong Qi and Pectoral Qi.Once deficiency of Lung-Qi happened,turbid toxin cannot be eliminated.It entered the kidney along the meridians,causing obstacle of blood-flow and producing blood stasis.The interaction between stasis and toxin could aggravate renal injury.We propose that the pathogenesis of traditional Chinese medicine is“Zhuo Qi”blockade of lung and“toxic damage of kidney”.Conclusion:The five internal organs are related,and the lungs and kidneys influence each other.Turbid toxin enters the kidney along the meridians,causing obstacle of blood-flow and producing blood stasis.Therefore,the pathogenesis of Chinese medicine that CIH causes kidney damage is"turbid qi obstructs lung and kidney damage."Part two The effect of“Zhuo Qi”in lung causes renal injury based on chronic intermittent hypoxiaObjective:The changes of renal structure and function was observed to verify the severity of turbid toxin and toxic damage of kidney.Methods:Male Wistar rats（n=30）were randomly divided into 3 groups:the control group（Cont group）,the chronic intermittent hypoxia group（CIH group）,and the eplerenone treatment group（EPL group）.Rats in CIH and EPL groups were exposed to hypoxic chambers from 8:00am-4:00pm.The rats in EPL group were given eplerenone(100 mg·kg^-1·d^-1)for 35 days.The serum and renal tissue specimens were collected for the detection.The blood pressure was detected to observe whether there was any abnormality of yin and yang in blood.The level of aldosterone and kidney function were detected to verify the severity of turbid toxin.The expression of HIF-1αwas observed by immunohistochemistry and western blot.Pathological changes were observed by HE,sirius red and masson staining to verify toxic damage of kidney.Results:1.General inspection:The body weight and kidney weight of rats increased in each group.there was no statistical difference between the groups（P>0.05）.2.Verification of kidney hypoxia:Compared with rats in Cont group,HIF-1αstaining was significantly increased in the nucleus of renal tubular epithelial cells in CIH-exposed rats.Western blot analysis showed a substantial increase for HIF-1αin kidneys of CIH rats（P<0.05）.The HIF-1αin the kidneys of the EPL group decreased（P<0.05）.3.Changes in blood pressure:Compared with rats in Cont group,the blood pressure of CIH rats increased gradually,and there was a statistical difference on the 14th day（P<0.05）,and then the blood pressure continued to rise（P<0.001,P<0.0001）.The blood pressure was restored with EPL and there was a statistical difference after 28 days of EPL administration（P<0.05）.4.Detection of aldosterone:Levels of ALD in kidneys,plasma and urine in CIH rats elevated than in the Cont group（P<0.05）;levels of ALD in kidneys of the EPL group decreased,which was statistically significant（P<0.05）,levels of ALD in plasma and urine were not statistically significant compared with CIH rats（P>0.05）5.Renal function:Compared with the Cont group,there was a significantly increased in level of BUN,Scr the CIH rats（P<0.05）,level of BUN and Scr decreased after EPL administration（P<0.05）.6.Pathological and morphological changes in the kidney:HE,sirius red,masson and immunofluorescence staining showed that the kidney morphological structure was normal with a small amount of collagen fiber deposition in Cont rats.There was a renal tubular structure disorder and a large number of inflammatory cells infiltrated,obvious collagen deposition in renal interstitial area in CIH rats.The pathological damage was improved after EPL administration.Conclusion:CIH contributes to secretion of aldosterone resulting in hypertension and renal fibrosis.EPL can reduce the hypertension and renal injury caused by CIH.Zhuo Qi enters the lung causing deficiency of Zong Qi and Primordial Qi,which affects the physiological functions of lung and kidney.Part three The mechanism of toxic damage kidney was explored based on CIH-induced renal macrophage-myofibroblast transitionObjective:The effect of macrophage infiltration and macrophage-myofibroblast transition（MMT）on renal injury was observed to explore the pathogenesis of“Zhuo Qi”blockade of lung and“toxic damage of kidney”.Methods:Animal model preparation and EPL administration were the same as the first part.The expression of MCP-1,M-CSF,IL-1βand TGF-β1was detected by western blot and real-time PCR to identify the severity of turbid toxin;Immunostaining was performed to identify cells undergoing MMT on the basis of macrophage（CD68）and myofibroblast（α-SMA）co-expression to verify toxic damage of kidney.Three-color confocal microscopy identified collagen I and collagen III production by CD68⁺α-SMA⁺cells in an area of interstitial fibrosis in CIH rats to verify the interaction between stasis and toxin.Results:1.Macrophages infiltration:The immunofluorescence staining was employed to confirm the substantial infiltration of macrophages（CD68）and the polarization of type M1（i NOS）and M2（CD206）in interstitial area with rats subjected to hypoxia.The number of macrophages reduced markedly after EPL administration.2.Detected pro-inflammatory and pro-fibrotic cytokines:The protein expression and m RNA transcription levels of M-CSF,MCP-1 and IL-1βwere significantly up-regulated compared with Cont group（P<0.05）;After EPL administration,the expression levels of other indicators decreased（P<0.05）.3.Detected MMT process by immunofluorescence:Immunostaining was performed to identify cells undergoing MMT on the basis of macrophage（CD68）and myofibroblast（α-SMA）co-expression.Rats in Cont group showed the appearance of both CD68⁺macrophages andα-SMA⁺myofibroblasts,but only a few double labeled cells were evident in controls.CIH-exposed rats had increased numbers of CD68⁺andα-SMA⁺double labeled cells in areas of interstitial fibrosis.Significantly reduced double positive MMT cells were detected in rats with EPL.4.Detected collagen production by MMT cells:Three-color confocal microscopy was performed to identify collagen I and collagen III production by CD68⁺α-SMA⁺cells in rats subjected to hypoxia which suggested collagen-producing MMT cells correlated with interstitial fibrosis.Conclusion:Infiltration of macrophages participated in renal inflammation and fibrosis by secreting cytokines and transforming into myofibroblast.EPL can reduce macrophage infiltration and MMT improves renal interstitial fibrosis.A markedly reduction in collagen-producing MMT cells has been described by EPL.The“toxicity”represented inflammatory and the"stasis"represented fibrosis.“Toxicity”and“stasis”can mutually promote and then aggravate renal injury.Part four The pathogenesis of“toxicity”and“stasis”was observed based on CIH-mediated MMT process by MR/SGK1/NF-κB pathwayObjective:To further investigate the role of MR activation in MMT,we tested the effects of additional aldosterone and intermittent hypoxia.The role of MR/SGK-1/NF-κB signaling pathway in MMT process was observed to clarify the transformation of“toxicity”.Methods:Animal model preparation and EPL administration were the same as the first part.Raw264.7 macrophages were maintained in culture media with 10%heat-inactivated FBS at 37℃in an incubator with a humidified atmosphere and 5%CO₂.Intermittent hypoxia was used for CIH rats.For cycling hypoxia,cells were exposed to 60 min hypoxia（1%O₂+5%CO₂+94%N₂）followed by30 min reoxygenation（air,21%O₂+5%CO₂）（24h）.cells in EPL group were pre-treated with 10u M eplerenone 1 hour prior to cycling hypoxia.Immunofluorescence was employed to confirm MR activity by the expression of NR3C2 and HSP90.The expression of MR,SGK-1 and NF-κB was observed by immunohistochemistry staining,western blot and real-time PCR to clarify the transformation of“toxicity”.Results:1.The co-expression of CD68 andα-SMA in RAW264.7 cells was detected by immunofluorescence double staining.In the cont group,α-SMA was less expressed.Compared with the cont group,the expressionα-SMA increased significantly in CIH group.The expression ofα-SMA decreased in EPL group.2.Detected MR activation:In its nonactivated state,MR（NR3C2）is localized in the cytosol and part of a multiprotein complex,including heat shock protein 90（HSP90）,which keeps it ligand-binding competent.MR combined with aldosterone migrates from the cytoplasm to the nucleus to exert its physiological effects.Immunohistochemistry and immunofluorescence showed that NR3C2 was mainly expressed in the nuclei of renal tubule cells in CIH rats,whereas there was little staining in the nuclei and main in the cytoplasm in controls.EPL effectively reversed hypoxia-induced nuclear translation of NR3C2.3.Detected the expression of SGK-1 and NF-κB in kidney:SGK1 and NF-κB staining was observed to be markedly increased in the renal distal tubule epithelial cells in CIH group than in Cont group;EPL decreased the expressions of SGK-1 and NF-κB.4.Detected the protein expression and m RNA transcription levels of SGK-1and NF-κB by western blot and real-time PCR:The expression of SGK-1 and NF-κB significantly increased in rats subjected to CIH as compared with controls by western blot analysis and real-time PCR（P<0.01）.EPL was used to detected a downregulation in the expression（P<0.01）.Conclusion:The MMT process and development of interstitial fibrosis in CIH was mediated by MR/SGK-1/NF-κB signaling.“Zhuo Qi”blockade of lung and“toxic damage of kidney”is the pathogenesis of CIH-induced renal injury.General conclusion:1.Hypoxic condition contributes to secretion of aldosterone resulting in pathological and morphological changes in the kidney.2.Infiltration of macrophages participated in renal inflammation and fibrosis by secreting cytokines and transforming into myofibroblast.3.CIH worked through MR/SGK-1/NF-κB signaling induced MMT process involving in renal fibrosis directly.4.MR activation as a critical player drove the progression of MMT.EPL efficiently alleviated renal fibrosis by blocking MR activity.5.The pathogenesis of CIH-induced renal injury was“Zhuo Qi”blockade of lung and“toxic damage of kidney”.