Yiqi Huayu Jiedu Recipe Attenuates Fibrosis In The Contralateral Kidney Of UUO Rats By Preventing Macrophage-to-myofibroblast Transition Based On The ALD/MR/SGK-1 Pathway

Part one To explore the mechanism of UUO induced chronic renal failure based on the theory of“holistic concept" and“treatment before disease"The holistic concept is the academic thought of Traditional Chinese Medicine(TCM)to understand itself and the connection between human and environment.According to traditional Chinese medicine,human is an organic whole composed of several viscera,body and organs,which coordinate with each other in physiological functions and influence each other in pathology.Therefore,kidney disease often affects other viscera and spreads."Treatment before disease" highlights the preventive thinking of traditional Chinese medicine,including prevention before disease and prevention of change of existing disease.Grasping the law of transmission and change of the disease location can grasp the development trend of the disease,so as to seize the opportunity to treat the disease to prevent the development of the disease and cure the disease at the initial stage.In the early stage of UUO,only the obstructive kidney damage,and the healthy side is damaged over time,eventually causing both kidneys to be injured.This process is complicated and involves many internal organs."Poison","Yu" pathogenesis theory had provided the theory basis for the clinical treatment of chronic kidney disease,and have important significance for the progress of chronic kidney disease.By giving UUO rats Yiqi Huayu Jiedu Recipe,we observe the protective effect on the contralateral kidney,to provide a basis for clinical treatment and to clarify the guiding significance of the theory of "holistic concept" and "preventive treatment".Part two The effect of Yiqi Huayu Jiedu Recipe on pathomorphology of contralateral kidney in UUO ratsObjective:To observe the pathological process of contralateral kidney in UUO rats and the protective effects of Yiqi Huayue Jiedu RecipeMethods:Male Sprague-Dawley rats were randomly divided into four groups:Sham group,UUO group,MRB group,and TCM group.In the rats with UUO,the left ureter was ligated,and the rats in the Sham group,the left ureter was exposed but not ligated.After UUO,the rats in the MRB group were orally administered mixed eplerenone(100mg·kg-1·d-1)with diet,and TCM(13g·kg-1·d-1)for the TCM group.All the animals were euthanized 180 days after the surgeries.The general conditions of rats were observed.The blood pressure,the kidney function(Scr,BUN)were measured.Pathological changes were demonstrated by HE staining,sirius red staining and masson staining.The expression of PCNA was detected by immunohistochemistry.Data were expressed as mean±standard deviation(SD).Statistical analyses using one-way analysis of variance(ANOVA)followed by SNK and LSD test,a P value<0.05 was considered statistically significant.Results:1.Detected general condition.The kidney weight and the ratio of kidney to body weight in the UUO group,the MRB group and the TCM group were significantly increased than those in the Sham group(P<0.05).The systolic blood pressure in the UUO group was higher than that in the Sham group at 2-6 months after UUO(P<0.05),and in the MRB group and the TCM group the pressure was declined obviously(P<0.05).2.Kidney pathology examination.HE staining,sirius red staining and masson staining showed that the kidney structure of the Sham group was basically normal,with a small amount of inflammatory cells and collagen deposition in the kidney interstitial.In UUO group,inflammatory cells infiltration was increased,the structures of renal tubular and glomerular were damaged,and collagen deposition was significantly increased than that in the Sham group.Compared with the UUO group,the degree of renal tissue injury in MRB group and TCM group was significantly reduced,and the collagen deposition was reduced.3.Renal function.Compared with the Sham group,there was a significant increase in the levels of Scr and BUN in the UUO group(P<0.05).Compared with the UUO group,the level of BUN reduced in the MRB group and the TCM group(P<0.05).4.Expression of PCNA in kidney of rats in each group.Immunohistochemistry was used to detect the expression of PCNA in the contralateral kidney of UUO rats.Compared with Sham,the positive expression of PCNA in the contralateral kidney of rats in the UUO group was significantly enhanced,mainly in the nuclei of renal tubular epithelial cells.Compared with the UUO group,the positive expression of PCNA in MRB group and TCM group was significantly reduced.Conclusion:Chronic obstructive kidney disease can induce contralateral kidney damage;In the contralateral kidney,the infiltration of inflammatory cells was increased and the index of cell proliferation,PCNA,was significantly increased;Eplerenone,an aldosterone receptor blocker,and Yiqi Huayue Jiedu Recipe can reduce kidney damage in UUO rats.Part three The effect of Yiqi Huayu Jiedu Recipe on inhibiting macrophage-to-myofibroblast transition and alleviating contralateral renal fibrosisObjective:To observe the effects of macrophage infiltration and macrophage-to-myofibroblast transition(MMT)on contralateral kidney fibrosis in UUO rats and the protective effects of Yiqi Huayu Jiedu Recipe.Methods:Animal grouping,model preparation,drug intervention and statistical analysis were performed as previously described in part two.Confocal microscopy identified collagen Ⅰ and collagen Ⅲ in the contralateral kidney.The expression of α-SMA and Vimentin in contralateral renal tissues were detected by immunohistochemistry and the expression of α-SMA mRNA was detected by Real time-PCR to observe the activation of myofibroblasts.The expression of α-SMA,F4/80 and CD68 were detected by immunofluorescence to observe the activation and proliferation of myofibroblasts and the infiltration of macrophages.The expressions of F4/80,CD68,α-SMA,iNOS,CD206,Collagen I and III were detected to identify whether the macrophages in the contralateral kidney underwent MMT and participated in renal fibrosis.Results:1.Detected collagen deposition.Immunofluorescence staining showed that compared with the Sham group,the expression of collagen I and collagen were substantially increased in the UUO group,especially in renal interstitium and vascular wall.The upregulation of these proteins was reversed in the MRB group and the TCM group.2.Detected activation and proliferation of myofibroblasts.The expression of α-SMA and Vimentin in kidney tissues of UUO rats was detected by immunohistochemical staining.Compared with the Sham group,the expression of α-SMA and Vimentin was increased in the UUO group(P<0.05),whereas it was suppressed in the MRB group and the TCM group.Real-time PCR results showed that compared with Sham group,the mRNA level of α-SMA in UUO group was significantly up-regulated,with statistical significance(P<0.05).However,compared with the UUO group,the expression of α-SMA mRNA was down-regulated in MRB group and TCM group,with statistical significance(P<0.05).3.Detected MMT cells.Results showed the presence of both CD68+macrophages and resident α-SMA+cells,but only a few cells were evident in the Sham group.In the UUO group,there were large numbers of both CD68+macrophages and α-SMA+myofibroblasts in areas of severe interstitial fibrosis with many double positive cells evident.In the MRB group and the TCM group,numerous CD68+macrophages were identified,but a few CD68+α-SMA+cells were evident.4.Detected collagen produced by MMT cells.Immunofluorescence staining showed that MMT cells could synthesize and secrete collagen.5.MMT cells have a predominant M2 phenotype.Analysis by two-color and three-color immunofluorescence staining showed that the majority of MMT cells were M2 phenotype identified by co-expressing CD68+,α-SMA+and CD206+,whereas a few M1 cells identified by co-expressing CD68+,α-SMA+and iNOS+.Conclusion:UUO induces macrophage infiltration and participates in the inflammatory response.MMT is involved in the contralateral kidney fibrosis.MMT cells have a predominant M2 phenotype,which synthesizes and secretes collagen and participates in kidney fibrosis.Eplerenone and Yiqi Huayu Jiedu Recipe can inhibit MMT and inflammatory response to reduce kidney fibrosis.Part four The effects of Yiqi Huayu Jiedu Recipe on the ALD/MR/SGK-1 pathway and the macrophage-to-myofibroblast transition in contralateral kidney of UUO ratsObjective:To observe the role of ALD/MR/SGK-1 pathway in the macrophage-to-myofibroblast transition and the effect of Yiqi Huayu Jiedu RecipeMethods:Animal grouping,model preparation,drug intervention and statistical analysis were perfomned as previously described in part one.Serum aldosterone was examined by radioimmunoassay(RIA).The expression of SGK-1 and NGAL in kidney tissue was detected by immunohistochemistry.Confocal microscopy identified the expressions of F4/80 and M-CSF in the contralateral kdiney.The expressions of NR3C2 and HSP90 showed the activation of MR.The expression of SGK-1,NF-κB,MCP-1 and TGF-β1 were detected by Real-time PCR.The expressions of M-CSF,SGK-1,p-SGK-1,NF-κB,MCP-1,and TGF-β1 were detected by Western blot to clarify the role of ALD/MR/SGK-1 pathway in the contralateral renal fibrosis induced by UUO.Results:1.Detected serum aldosterone.The level of serum aldosterone in the UUO group elevated remarkably than that in the Sham group(P<0.05).Compared with the UUO group,the level of serum aldosterone in the MRB group and TCM group was significantly decreased(P<0.05).2.The expression of SGK-land NGAL in contralateral kidney of UUO rats.Compared with Sham group,the expression of SGK-1 and NGAL in UUO group was significantly increased(P<0.05).Compared with UUO group,SGK-1 and NGAL expressions in MRB group and TCM group were significantly decreased(P<0.05).3.Macrophage recruitment and infiltration.Macrophages co-expression of F4/80 and M-CSF were examined by immunofluorescence staining.Compared with the Sham group,the co-expression of F4/80 and M-CSF was increased markedly in the UUO group,and this effect was attenuated by the treatment of EPL and TCM.4.Detected MR activation.MR is activated by binding to aldosterone and moves from the cytoplasm into the nucleus.The co-expression of NR3C2 and HSP90 was observed by immunofluorescence staining,which showed that NR3C2 was mostly in the cytoplasm of the Sham group.In the UUO group,the expression of NR3C2 was increased and the depolymerized HSP90 was located outside the nucleus.Compared with UUO,the expression of NR3C2 in MRB group and TCM group was significantly reduced.5.The expressions of SGK-1,NF-κB,MCP-1 and TGF-β1 were detected by real-time PCR.Compared with Sham group,mRNA gene levels of SGK-1,NF-κB,MCP-1 and TGF-β1 in UUO group were significantly up-regulated(P<0.05).Compared with the UUO group,the mRNA gene levels of SGK-1,NF-κB,MCP-1 and TGF-β1 in MRB group and TCM group were significantly down-regulated(P<0.05).6.Protein expression in related pathways was detected by Western blot.Compared with Sham group,the protein expression levels of M-CSF,MCP-1,TGF-β1,SGK-1,p-SGK-1 and NF-κB in UUO group were significantly up-regulated(P<0.05).Compared with UUO group,protein expression levels in MRB group and TCM group were significantly down-regulated(P<0.05).Conclusion:The MMT and the development of interstitial fibrosis in contralateral kidney were reversed by eplerenone,which suggests that MMT is mediated by ALD/MR/SGK-1 pathway.Yiqi Huayu Jiedu Recipe can reduce contralateral kidney fibrosis through blocking MR activation.Part five Activation of MR stimulate Macrophage-to-Myofibroblast Transition(MMT)in macrophagesObjective:To further investigate the role of MR activation in MMT,we tested the effects of additional aldosterone and MR blockade on the expression of α-SMA in macrophages.Methods:Raw264.7 macrophages and bone marrow differentiated monocyte/macrophages were maintained in culture media with 10%heat-inactivated FBS at 37℃ in an incubator with a humidified atmosphere and 5%CO2.10-7 M aldosterone(Ald)was used for Ald treatment and in Ald+Spir group cells were pre-treated with 10 μM spirolactone 1 hour prior to Ald treatment.24 hours after the treatment,all cells were harvested and stained with PE-conjugated anti-moue F4/80(Biolegend),APC-conjugated anti-α-SMA(Abcam)for 1 hour in dark tubes.Cells were analyzed in a BD Accuri C6 flow cytometer,viable singlet cells were selected by FSC/SSC gating,and data were further analyzed using FlowJo 10 software.Full protocol of mouse bone marrow-derived macrophages was obtained from Dr.Tiffany Weinkopff in University of Arkansas for Medical Sciences.Briefly,fresh bone marrow(BM)cells were harvested from mice in cRPMI media and cultured in MCSF conditioned macrophage media for 7 days(fresh media added at day 3).After days 7,cells were harvested and used for experiments.Flow cytometry using F4/80 antibody confirmed most cells used for experiments are macrophages.Total RNA was isolated using RNeasy plus mini kit(Qiagen)following the protocol recommended by the manufacturer.Superscript III kit(Thermo Fisher)was used for reverse transcription of RNA,and realtime PCR was performed using Taqman reaction system(Thermo Fisher)on an Mx3005p realtime PCR instrument.GAPDH was used as a housekeeping transcript for normalization,and relative realtime PCR result were calculated using the 2-ΔΔCT method.The co-expression of CD68 and α-SMA was detected by double immunofluorescence staining.All data were expressed as mean±SEM.The statistical comparisons between groups were determined using student t-test for two groups or one-way ANOVA followed by Tukey’s post hoc tests for multi-groups,with P<0.05 being considered statistically significant.Results:1.The co-expression of CD68 and α-SMA in RAW264.7 cells was detected by immunofluorescence double staining.In the control group,α-SMA was less expressed,and the co-expression of CD68 and α-SMA was less.Compared with the control group,the co-expression of CD68 and α-SMA increased significantly in ALD group,and the number of α-SMA expression in macrophages increased.Compared with ALD group,the co-expression of CD68 and α-SMA decreased in MRB group,and the number of α-SMA decreased significantly.2.Taqman Realtime PCR shows that addition of aldosterone stimulated mRNA expression of α-SMA in macrophage cell line RAW 264.7 cells,and this effect was blocked by specific MR inhibitor spironolactone.3.Flow cytometer shows that consistent with realtime-PCR results,aldosterone stimulation also upregulated protein level expression of α-SMA in RAW 264.7 cells,and this upregulation was attenuated by MR blocker spironolactone as well.4.As expected,similar data detected by flow cytometer was also observed in mouse bone marrow-derived macrophages,confirmed above results using cell line.Conclusion:MR activation plays an important role in MMT.The stimulating effects of aldosterone on the expression of α-SMA in macrophages were clearly blocked by MR specific blocker spironolactone,suggesting activation of MR contributes to MMT in macrophages.General conclusion:1.Unilateral ureteral obstruction can induce kidney structure and function changes,then lead to contralateral kidney damage and fibrosis.2.The mechanism of contralateral kidney fibrosis induced by unilateral ureteral obstruction is related to aldosterone,and the activation of mineralocorticoid receptor plays a key role in this process.3.Aldosterone induces inflammatory response and macrophage-to-myofibroblast transition to regulate the contralateral kidney fibrosis by MR/SGK-1 signaling pathway.4.Eplerenone and Yiqi Huayu Jiedu Recipe can inhibit macrophage-to-myofibroblast transition,inflammatory response to protect kidney effectively by ALD/MR/SGK-1 signaling pathway.5.In the chronic phase of obstructive kidney disease,activated MR stimulate MMT for renal infiltrated macrophages to transdifferentiate into myofibroblasts,producing excessive collagen,exacerbating renal fibrosis in the contralateral kidney.Moreover,MR blockers diminish MMT and protect the contralateral kidney from fibrosis and further chronic renal injury.