The Effect Of IKKa In Macrophages In The Progression Of AKI To CKD Transition

Objective:Acute kidney injury(AKI)is a common clinical disease,and the incidence of AKI is rising.Severe AKI often leads to incomplete recovery of renal function and structure which is known as AKI to chronic kidney disease(CKD)transition.The mechanism under the AKI to CKD transition is not clearly,however,the infiltration of inflammatory cells such as macrophages was thought play a key role of leading to AKI-to-CKD transition.IKK? is thought as activator of NF-?B canonical pathway and induce the activation of macrophages after AKI.But the role of IKKa which is the activator of NF-?B noncanonical pathway in macrophages is not clear.The purpose of this study is to investigate the role of IKKa in macrophage involved in AKI to CKD transition.There is no effective treatment for AKI to CKD transition at present.Bone marrow mesenchymal stem cells(BMSC)is thought as a prospective therapy for organ injury,but cell senece as a drawback of BMSC inhibit its further using in organ injury treatment.The second part of this study aimed to improve the therapy effect of BMSC by modifying gene expression in BMSC.Methods:1.By mating IKKa-floxed mice with Lyz-2-Cre transgenic mice,mice with IKKa gene specifically ablated in macrophage were created.Male and age-matched C57BL/6 wild type(WT)mice(aged 8-12 weeks old)were bred as controls.Both WT mice and Mac IKKa-/-mice were divided into sham treated group and IRI group(each group 24 mice).After dorsal incision,the left renal pedicle was clamped with a micro vascular clamp for 45 min,while the sham-operated mice underwent the same treatment except clamping renal pedicle.Left kidneys and blood serum were harvested at day3,day 7,day 14 and day 21 after IRI,6 mice at each time point of 4 groups were sacrificed.Gross kidney morphology and renal weight of WT and Mac IKKa-/-mice were recorded,renal function was tested.HE staining was used to investigate renal tubular injury and Masson staining was used to investigate renal interstitial fibrosis.Immunohistochemistry and Western blot were used to investigate the expression of fibrosis related proteins.We isolated peritoneal cells by injecting Thioglycollate Broth into the abdominal cavity of male WT mice or Mac IKKa-/-mice(21-30 days)for 3 days.Then we used FACS to detect the polarization of macrophages.Frozen kidney sections were stained with macrophage marker antibody and we used fluorescence microscope to detect the infiltration of M2 macrophage.We isolated primary TECs from male WT mice and added CoC12 to the culture medium to establish the cell hypoxia injury model.We cocultured the injured TECs with WT macrophage or IKKa-/-macrophage to investigate the variation of EMT and Wnt/?-catenin pathway in TECs.2.We isolated the BMSC from C57BL/6 mice and identificated it by inducing differentiation.We transfected BMSC with GFP-adenovirus to generate GFP-BMSC and transfected Klotho-GFP-adenovirus to generate Klotho-GFP-BMSC.The expression of Klotho of different BMSCs were detected by Western blot.We used CCK-8 to investigate the proliferative ability of different BMSCs and used qRT-PCR to investigate the level of mRNA of OCT4 and Nanog in different BMSCs.Mice were randomly divided into four groups(each group 8 mice):the sham operation group;IRI operation and transplanted with PBS group;IRI operation and transplanted with GFP-BMSC group;IRI operation and transplanted with GFP-Klotho-BMSC group.After IRI,5×105 Klotho-GFP-BMSCs or GFP-BMSCs were injected into the capsule of the left kidney.The left kidney and blood serum of mice were harvested at day 7 and day 21 after IRI.In order to measure the function of the injury kidney,the right kidney was excise 24 h before sacrificing the mice.Gross kidney morphology and renal function of mice were recorded.HE staining was used to investigate renal tubular injury at day 21.Masson staining was used to investigate renal interstitial fibrosis at day 21.Immunohistochemistry and Western blot were used to investigate the expression of Wnt/?-catenin related proteins.We used fluorescence microscope to account the infiltration of BMSCs in frozen sections and account the CD68+ macrophages in immunohistochemistry sections.We cocultured TGF-? treated mCCD cells with Klotho-GFP-BMSCs or GFP-BMSCs to investigate the variance of EMT progress.Results:1.Conditional knockout mice with Mac IKKa-/-were generated,genotype was validated by nucleic acid gel electrophoresis and Western blot.The Mac IKKa-/-mice were born normal and have normal renal function and normal gross appearance within 2 months after birth.Within 21 days after IRI,the renal function of WT mice was markedly worse than Mac IKKa-/-mice,and Mac IKKa-/-mice showed less renal pathological injury compared with WT mice.The expression of proteins involved in EMT progression in kidneys of Mac IKKa-/-mice was lower compared with the kidneys of WT mice at the same time point.At the third day after IRI,the kidneys of Mac IKKa-/-mice showed less infiltration of M2 macrophages compared with WT mice.We isolated the inflammatory cells from peritoneal cavity and analyzed the cells with flowcytometry.We found that the proportion of M2 macrophages of WT mice was higher than Mac IKKa-/-mice.In vitro experiments,TECs showed increased expression of proteins involved in EMT after hypoxia stimulation,and EMT of injured TECs was elevated after coculturing with WT macrophages,while TECs cocultured with IKKa-/-macrophages did not show the increased expression of EMT relavant proteins.2.We isolated BMSC from WT mice and transfected BMSC with green fluorescence protein(GFP)overexpressed adenovirus(GFP-Ad)or Klotho-GFP overexpressed adenovirus(Klotho-GFP-Ad)and generated Klotho-GFP-BMSC and GFP-BMSC.We found that the proliferative ability of Klotho-GFP-BMSC was elevated,and the expression of OCT4 and Nanog was increased.In animal experiment,we found that the mice transplanted with Klotho-GFP-BMSC have better renal function and lighter histological injury compare with mice transplanted with GFP-BMSC.In addition,kidney of mice transplanted with Klotho-GFP-BMSC showed less activation of Wnt/?-catenin pathway and decreased the infiltration of macrophage.We cocultured TGF-?treated mCCD with GFP-BMSC or Klotho-GFP-BMSC and found that the expression of ?-SMA was lower in mCCD cells cocultured with Klotho-GFP-BMSC compared with cocultured with GFP-BMSC.Conclusions:1.Macrophage involved in the repairmen of kidney after IRI.The fibrosis progress of kidney after IRI is obviously inhibited in Mac IKK?-/-mice.IKK? involved in macrophage polarization,IKKa deficiency decrease the M2 macrophage infiltration in kidney.WT macrophage enhance the EMT progress in injured TECs while IKK?-/-macrophage doesn't have this effect.2.Klotho overexpressed BMSC showed increased proliferate ability.Kidneys transplanted with Klotho overexpressed BMSCs after IRI showed better renal function and less fibrosis.Klotho overexpressed BMSC have better renal protective effect compared with BMSC.