Volume 85, Issue 3, Pages 624-635 (March 2014) MiR-223 downregulation promotes glomerular endothelial cell activation by upregulating importin α4 and α5 in IgA nephropathy  Hao Bao, Hao Chen, Xiaodong Zhu, Minchao Zhang, Genhong Yao, Yusheng Yu, Weisong Qin, Caihong Zeng, Ke Zen, Zhihong Liu  Kidney International  Volume 85, Issue 3, Pages 624-635 (March 2014) DOI: 10.1038/ki.2013.469 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Change of microRNAs (miRNAs) in glomerular endothelial cells (GEnCs) after incubation with IgA-human mesangial cell (HMC) medium. (a) Cellular proliferation of GEnCs treated with IgA-HMC medium at different dilutions for 24h. (b) Cellular proliferation of GEnCs treated with a 10-fold dilution of IgA-HMC medium for different times. (c) Cell death of GEnCs treated with a 10-fold dilution of IgA-HMC medium for 24h. (d) The miRNA expression profile of GEnCs after incubation with a 10-fold dilution of IgA-HMC medium for 24h. (e) Expression of miR-223 in GEnCs treated with a 10-fold dilution of IgA-HMC medium for 24h. BrdU, 5-bromodeoxyuridine; IgAN, IgA nephropathy; IgAN-EP-mIgA, IgA-HMC medium prepared with monomeric IgA from patients with glomerular endothelial proliferation; IgAN-EP-pIgA, IgA-HMC medium prepared with polymeric IgA from patients with glomerular endothelial proliferation; Normal-mIgA, IgA-HMC medium prepared with mIgA from normal control subjects; Normal-pIgA, IgA-HMC medium prepared with pIgA from normal control subjects; IgAN-NEP-mIgA, IgA-HMC medium prepared with mIgA from patients without glomerular endothelial proliferation; IgAN-NEP-pIgA, IgA-HMC medium prepared with pIgA from patients without glomerular endothelial proliferation. *P<0.05 versus cells treated with control medium. Kidney International 2014 85, 624-635DOI: (10.1038/ki.2013.469) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 MicroRNA-223 (miR-223) levels in the glomerular tissues of patients or normal control subjects. (a) The isolation of glomerular tissues by laser capture microdissection and reverse transcriptase–PCR (RT–PCR) analysis of glomerular miR-223 levels in normal controls and patients. (b) In situ hybridization (ISH) analysis of miR-223 in renal tissues from normal control subjects and IgA nephropathy (IgAN) patients (original magnification × 400). IgAN-EP, IgAN patients with glomerular endothelial proliferation; IgAN-NEP, IgAN patients without glomerular endothelial proliferation. *P<0.05 versus normal control subjects. Kidney International 2014 85, 624-635DOI: (10.1038/ki.2013.469) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Role of microRNA-223 (miR-223) in the proliferation of glomerular endothelial cells (GEnCs) treated with IgA-human mesangial cell (HMC) medium. (a) The effects of neutralizing antibodies in modulating miR-223 expression in GEnCs incubated with IgA-HMC medium. (b) Enzyme-linked immunosorbent assay (ELISA) analysis of interleukin-6 (IL-6) level in the IgA-HMC medium. (c) The change of miR-223 level in GEnCs exposed to IL-6 (20ng/ml). (d) The change in the proliferation of GEnCs exposed to IgA-HMC medium or IL-6 (20ng/ml) plus anti-IL-6 (1μg/ml). (e) The change in cell proliferation in GEnCs exposed to IgA-HMC medium plus miR-223 mimics or miR-223 antisense oligonucleotide (ASO). BrdU, 5-bromodeoxyuridine; IgAN, IgA nephropathy; IgAN-NEP, IgA-HMC medium prepared with polymeric IgA from patients without glomerular endothelial proliferation; IgAN-EP, IgA-HMC medium prepared with pIgA from patients with glomerular endothelial proliferation; Normal, IgA-HMC medium prepared with pIgA from normal control subjects; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α. *P<0.05 versus normal control; #P<0.05 versus cells exposed to IgA-HMC medium prepared with pIgA from patients with glomerular endothelial proliferation or IL-6. Kidney International 2014 85, 624-635DOI: (10.1038/ki.2013.469) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Intercellular adhesion molecule-1 (ICAM-1) expression and monocyte–endothelial adhesion in glomerular endothelial cells (GEnCs) after treatment with IgA-human mesangial cell (HMC) medium. (a) Reverse transcriptase–PCR (RT–PCR) analysis of ICAM-1 in GEnCs exposed to IgA-HMC medium or interleukin-6 (IL-6; 20ng/ml) plus anti-IL-6 (1μg/ml). (b) Fluorescence analysis of monocyte–endothelial adhesion in GEnCs exposed to IgA-HMC medium or IL-6 plus anti-IL-6. (c) Changes in ICAM-1 mRNA levels in GEnCs exposed to IgA-HMC medium plus microRNA-223 (miR-223) mimics or miR-223 antisense oligonucleotide (ASO). (d) Changes in monocyte–endothelial adhesion in GEnCs exposed to IgA-HMC medium plus miR-223 mimics or miR-223 ASO. (e) Microscopic analysis of monocyte–endothelial adhesion in GEnCs exposed to IgA-HMC medium plus miR-223 mimics or miR-223 ASO. IgAN, IgA nephropathy; IgAN-EP, IgA-HMC medium prepared with polymeric IgA from patients with glomerular endothelial proliferation; IgAN-NEP, IgA-HMC medium prepared with pIgA from patients without glomerular endothelial proliferation; Normal, IgA-HMC medium prepared with pIgA from normal control subjects. *P<0.05 versus normal control; #P<0.05 versus cells exposed to IgA-HMC medium prepared with pIgA from patients with glomerular endothelial proliferation or IL-6. Kidney International 2014 85, 624-635DOI: (10.1038/ki.2013.469) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Activation of the nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) signaling pathways in glomerular endothelial cells (GEnCs) treated with IgA-human mesangial cell (HMC) medium. (a) Western blot analysis of the NF-κB signaling pathway in GEnCs exposed to IgA-HMC medium or interleukin-6 (IL-6; 20ng/ml) plus anti-IL-6 (1μg/ml). (b) Chromatin immunoprecipitation (ChIP) analysis of p65 DNA binding in GEnCs exposed to IgA-HMC medium or IL-6 plus anti-IL-6. (c) Western blot analysis of the STAT3 signaling pathway in GEnCs exposed to IgA-HMC medium or IL-6 plus anti-IL-6. (d) ChIP analysis of STAT3 DNA binding in GEnCs exposed to IgA-HMC medium or IL-6 plus anti-IL-6. (e) Changes in cellular proliferation, intercellular adhesion molecule-1 (ICAM-1) expression, and monocyte–endothelial adhesion in GEnCs exposed to IgA-HMC medium plus the NF-κB inhibitor Bay 11-7085 and the STAT3 inhibitor cucurbitacin I. IgAN, IgA nephropathy; IgAN-NEP, IgA-HMC medium prepared with polymeric IgA from patients without glomerular endothelial proliferation; IgAN-EP, IgA-HMC medium prepared with pIgA from patients with glomerular endothelial proliferation; Normal, IgA-HMC medium prepared with pIgA from normal control subjects. *P<0.05 versus normal control; #P<0.05 versus cells exposed to IgA-HMC medium prepared with pIgA from patients with glomerular endothelial proliferation or IL-6. Kidney International 2014 85, 624-635DOI: (10.1038/ki.2013.469) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 MicroRNA-223 (miR-223) inhibits nuclear translocation of the p65 and signal transducer and activator of transcription 3 (STAT3) in glomerular endothelial cells (GEnCs) treated with IgA-human mesangial cell (HMC) medium. (a) Western blot analysis of the nuclear factor-κB (NF-κB) signaling pathway in GEnCs exposed to IgA-HMC medium plus miR-223 mimics or miR-223 antisense oligonucleotide (ASO). (b) Immunofluorescence staining of p65 in GEnCs exposed to IgA-HMC medium plus miR-223 mimics or miR-223 ASO. (c) Chromatin immunoprecipitation (ChIP) analysis of p65 DNA binding in GEnCs exposed to IgA-HMC medium plus miR-223 mimics or miR-223 ASO. (d) Western blot analysis of the STAT3 signaling pathway in GEnCs exposed to IgA-HMC medium plus miR-223 mimics or miR-223 ASO. (e) Immunofluorescence staining of p-STAT3 in GEnCs exposed to IgA-HMC medium plus miR-223 mimics or miR-223 ASO. (f) ChIP analysis of STAT3 DNA binding in GEnCs exposed to IgA-HMC medium plus miR-223 mimics or miR-223 ASO. IgAN, IgA nephropathy; IgAN-EP, IgA-HMC medium prepared with polymeric IgA from patients with glomerular endothelial proliferation; IgAN-NEP, IgA-HMC medium prepared with pIgA from patients without glomerular endothelial proliferation; Normal, IgA-HMC medium prepared with pIgA from normal control subjects; NS, nonsense oligonucleotide. *P<0.05 versus normal control; #P<0.05 versus cells exposed to IgA-HMC medium prepared with pIgA from patients with glomerular endothelial proliferation. Kidney International 2014 85, 624-635DOI: (10.1038/ki.2013.469) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 MicroRNA-223 (miR-223) inhibits nuclear factor-κB (NF-κB) nuclear translocation by targeting importin α4 (IPOA4) in glomerular endothelial cells (GEnCs). (a) Immunohistochemical (IHC) staining for IPOA4 (Fast Blue, blue) and CD31 (3,3'-Diaminobenzidine (DAB), brown) in the glomeruli of normal controls and IgA nephropathy (IgAN) patients. (b) Western blot analysis of IPOA4 in GEnCs exposed to IgA-human mesangial cell (HMC) medium plus miR-223 mimics or miR-223 antisense oligonucleotide (ASO). (c) Normalized luciferase activity of a reporter construct containing the 3′-untranslated region (UTR) of IPOA4 in cells cotransfected with miR-223 mimics. (d) Western blot analysis of IPOA4 expression in GEnCs transfected with miR-223 mimics. (e) Two binding sites in the 3′-UTR of IPOA4 are targeted by miR-223. (f) Normalized luciferase activity of reporter constructs containing Site 1 sequence, Site 2 sequence, or mutant sequence in cells cotransfected with miR-223 mimics. (g) Western blot analysis of p65 in GEnCs exposed to IgA-HMC medium plus IPOA4 small interfering RNA (siRNA). (h) Chromatin immunoprecipitation (ChIP) analysis of p65 DNA binding in GEnCs exposed to IgA-HMC medium plus IPOA4 siRNA. (i) Change of cell proliferation, intercellular adhesion molecule-1 (ICAM-1) expression, and monocyte–endothelial adhesion in GEnCs exposed to IgA-HMC medium plus IPOA4 siRNA. IgAN-EP, IgA-HMC medium prepared with polymeric IgA from patients with glomerular endothelial proliferation; IgAN-NEP, IgA-HMC medium prepared with pIgA from patients without glomerular endothelial proliferation; Normal, IgA-HMC medium prepared with pIgA from normal control subjects; RLU, relative light units. *P<0.05 versus normal control; #P<0.05 versus cells exposed to IgA-HMC medium prepared with pIgA from patients with glomerular endothelial proliferation. Kidney International 2014 85, 624-635DOI: (10.1038/ki.2013.469) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 8 MicroRNA-223 (miR-223) inhibits signal transducer and activator of transcription 3 (STAT3) nuclear translocation by targeting importin α5 (IPOA5) in glomerular endothelial cells (GEnCs). (a) Immunohistochemical (IHC) staining for IPOA5 (Fast Blue, blue) and CD31 (3,3'-Diaminobenzidine (DAB), brown) in the glomeruli of normal controls and IgA nephropathy (IgAN) patients. (b) Western blot analysis of IPOA5 in GEnCs exposed to IgA-human mesangial cell (HMC) medium plus miR-223 mimics or miR-223 antisense oligonucleotide (ASO). (c) Normalized luciferase activity of a reporter construct containing the 3′-untranslated region (UTR) of IPOA5 in cells cotransfected with miR-223 mimics. (d) Western blot analysis of IPOA5 expression in GEnCs transfected with miR-223 mimics. (e) Two binding sites in the 3′-UTR of IPOA5 are targeted by miR-223. (f) Normalized luciferase activity of reporter constructs containing Site 1 sequence, Site 2 sequence, or mutant sequence in cells cotransfected with miR-223 mimics. (g) Western blot analysis of STAT3 and p-STAT3 in GEnCs exposed to IgA-HMC medium plus IPOA5 small interfering RNA (siRNA). (h) Chromatin immunoprecipitation (ChIP) analysis of STAT3 DNA binding in GEnCs exposed to IgA-HMC medium plus IPOA5 siRNA. (i) Change of cell proliferation, intercellular adhesion molecule-1 (ICAM-1) expression, and monocyte–endothelial adhesion in GEnCs exposed to IgA-HMC medium plus IPOA5 siRNA. IgAN-EP, IgA-HMC medium prepared with polymeric IgA from patients with glomerular endothelial proliferation; IgAN-NEP, IgA-HMC medium prepared with pIgA from patients without glomerular endothelial proliferation; Normal, IgA-HMC medium prepared with pIgA from normal control subjects. *P<0.05 versus normal control; #P<0.05 versus cells exposed to IgA-HMC medium prepared with pIgA from patients with glomerular endothelial proliferation. Kidney International 2014 85, 624-635DOI: (10.1038/ki.2013.469) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 9 MicroRNA-223 (miR-223) levels in the circulating endothelial cells (CECs) of patients with IgA nephropathy (IgAN). (a) CECs were isolated through the combined use of anti-CD146, anti-CD45, and anti-CD133 beads. (b) The isolated CECs were validated with immunofluorescence staining for CD31, CD146, CD45, and CD133 (original magnification × 400). (c) Reverse transcriptase–PCR (RT–PCR) analysis of miR-223 in the CECs of IgAN patients and healthy control subjects. IgAN-EP, IgAN patients with glomerular endothelial proliferation; IgAN-NEP, IgAN patients without glomerular endothelial proliferation. *P<0.05 versus normal control. Kidney International 2014 85, 624-635DOI: (10.1038/ki.2013.469) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 10 Nonreducing western blot analysis of purified IgA fractions. IgAN, IgA nephropathy; IgAN-EP-mIgA, monomeric IgA from patients with glomerular endothelial proliferation; IgAN-EP-pIgA, polymeric IgA from patients with glomerular endothelial proliferation; IgAN-NEP-mIgA, mIgA from patients without glomerular endothelial proliferation; IgAN-NEP-pIgA, pIgA from patients without glomerular endothelial proliferation; Normal mIgA, mIgA from normal control subjects; Normal pIgA, pIgA from normal control subjects. Kidney International 2014 85, 624-635DOI: (10.1038/ki.2013.469) Copyright © 2014 International Society of Nephrology Terms and Conditions