Volume 84, Issue 2, Pages 265-276 (August 2013) NOD2 promotes renal injury by exacerbating inflammation and podocyte insulin resistance in diabetic nephropathy  Pengchao Du, Baoxia Fan, Huirong Han, Junhui Zhen, Jin Shang, Xiaojie Wang, Xiang Li, Weichen Shi, Wei Tang, Chanchan Bao, Ziying Wang, Yan Zhang, Bin Zhang, Xinbing Wei, Fan Yi  Kidney International  Volume 84, Issue 2, Pages 265-276 (August 2013) DOI: 10.1038/ki.2013.113 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Expression of NOD2 (nucleotide-binding oligomerization domain containing 2) in the kidney and renal cells. (a) Reverse transcriptase–PCR (RT–PCR) analysis of NOD2 mRNA levels in selected tissues including murine kidney, liver, spleen, and intestine. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (b) Western blot analysis of NOD2 protein levels in selected tissues including murine kidney, liver, spleen, and intestine. (c) Representative photomicrographs of NOD2 immunohistochemical staining with anti-NOD2 in the kidney and intestine from both wild-type (WT) and NOD2−/− mice. Intestine: bars=100μm; kidney: bars=20μm. (d) RT–PCR analysis of the expression of NOD2 in renal cells including murine glomerular mesangial cells (MCs), murine podocytes, human glomerular endothelial cells (GECs), and human proximal tubule epithelial cells (HK-2). Kidney International 2013 84, 265-276DOI: (10.1038/ki.2013.113) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Renal cortical NOD2 (nucleotide-binding oligomerization domain containing 2) was significantly elevated in human diabetic nephropathy (DN) biopsies. (a) Renal cortical expression of NOD2 and macrophage infiltration in human kidney biopsies. Representative photomicrographs of NOD2 and CD68 staining, which denotes infiltrating CD68+ macrophages in human renal cortical tissue from normal DN patients and diabetic patients without nephropathy (DM-NN). Negative control by omission of the corresponding primary antibodies demonstrated no nonspecific staining. Red and blue arrows indicate CD68+ infiltrating macrophages in the interstitium and glomeruli, which were colocalized with NOD2. Bars=50μm. (b) Relative mRNA levels of NOD2 in the renal biopsies from normal subjects (n=7) and patients with selected kidney diseases: DN (n=9); DM-NN (n=9); focal segmental glomerulosclerosis (FSGS, n=7); IgA nephropathy (IgA, n=7); membranous glomerulonephritis (MGN, n=6); lupus nephritis (LN, n=9), and minimal change disease (MCD, n=8). (c) Negative correlation between NOD2 mRNA levels and estimated glomerular filtration rate (GFR) in all subjects. (d) No correlation was found between NOD2 mRNA levels and 24-h urine protein excretion among all subjects with proteinuria. Kidney International 2013 84, 265-276DOI: (10.1038/ki.2013.113) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Renal cortical NOD2 (nucleotide-binding oligomerization domain containing 2) was upregulated in HFD/STZ-induced diabetic mice. (a) Representative western blot gel documents and summarized data showing the protein levels of NOD2 in the kidney from HFD/STZ-induced diabetic mice. (b) Renal cortical expression of NOD2 and macrophage infiltration in the kidney from HFD/STZ-induced diabetic mice. Red and blue arrows indicate CD68-positive infiltrating macrophages in the interstitium and glomeruli, which were colocalized with NOD2. Bars=50μm. *P<0.05 versus control (n=10). DN, diabetic nephropathy; HFD, high-fat diet; Norm-diet, normal diet; STZ, streptozotocin. Kidney International 2013 84, 265-276DOI: (10.1038/ki.2013.113) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Characterization of glomerular injury and related signaling molecules in HFD/STZ-induced diabetic nephropathy. (a) Photomicrographs showing typical glomerular structure in control and diabetic mice; bars=20μm. (b) Morphological changes in the podocyte foot process by electron microscopy analysis; bars=2μm. (c) Relative levels of proinflammatory factors (interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1)) in the kidney from different groups of mice. (d) Relative mRNA levels of the renal fibrosis-associated molecules including collagen IV and fibronectin by real-time, reverse transcriptase–PCR (RT–PCR) analysis in the kidney from different groups of mice. *P<0.05 versus control, #P<0.05 versus wild-type diabetic mice (n=10). HFD, high-fat diet; Norm-diet, normal diet; STZ, streptozotocin; WT, wild type. Kidney International 2013 84, 265-276DOI: (10.1038/ki.2013.113) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Effects of different stimuli on the expression of NOD2 (nucleotide-binding oligomerization domain containing 2) in podocytes. (a) Representative western blot gel documents and summarized data showing the upregulation of NOD2 in response to high glucose (HG) in podocytes. (b) Representative western blot gel documents and summarized data showing the upregulation of NOD2 in response to advanced glycation end-product (AGE) in podocytes. (c) Representative western blot gel documents and summarized data showing the upregulation of NOD2 in response to tumor necrosis factor-α (TNF-α) in podocytes. (d) Representative western blot gel documents and summarized data showing the upregulation of NOD2 in response to transforming growth factor-β (TGF-β) in podocytes. *P<0.05 versus control (n=6). Kidney International 2013 84, 265-276DOI: (10.1038/ki.2013.113) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Muramyl dipeptide (MDP)-induced activation of mitogen-activated protein kinase (MAPK) signaling pathways, proinflammatory cytokine production, and podocyte apoptosis. Podocytes were treated with MDP (2μg/ml). Cellular lysates were immunoblotted with specific antibodies against phospho-ERK1/2, total ERK1/2, phospho-p38 MAPK, total p38 MAPK, phospho-JNK, and total JNK. (a) Representative western blot gel documents and summarized data showing the levels of phospho-ERK1/2. (b) Representative western blot gel documents and summarized data showing phospho-p38 (p-p38). (c) Representative western blot gel documents and summarized data showing the levels of phospho-JNK. (d) Representative western blot gel documents and summarized data showing the levels of IκBα. (e) Relative levels of proinflammatory factors in podocytes treated with MDP. (f) Summarized data showing podocyte apoptosis determined by flow cytometric analysis. *P<0.05 versus control (n=6). ERK1/2, extracellular signal–regulated kinase 1/2; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor-κB; p-ERK1/2; phospho-ERK1/2; p-JNK, phospho-JNK. Kidney International 2013 84, 265-276DOI: (10.1038/ki.2013.113) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 NOD2 (nucleotide-binding oligomerization domain containing 2)–mediated glucose uptake, glucose transporter type 4 (GLUT4) translocation, and insulin signaling in podocytes. (a) Podocytes were pretreated with muramyl dipeptide (MDP) or its respective inactive controls and 2-deoxyglucose (2-DOG) uptake was measured after 20min of stimulation with 100nmol/l insulin and it was found that activation of NOD2 by MDP decreased insulin-stimulated glucose uptake in podocytes. (b) Representative confocal microscopic images showing the different extent of GLUT4 on the membrane of pretreated with or without MDP in insulin-stimulated podocytes; bar=20μm. (c) Representative western blot gel documents and summarized data showing the relative NOD2 protein levels in the membrane fraction of podocytes. (d) Representative western blot gel documents and summarized data showing MDP-induced phosphorylation of IR substrate-1 (IRS-1) at Ser612 in podocytes. (e) MDP impaired insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-1/p85 association by immunoprecipitation. *P<0.05 versus control, #P<0.05 versus vehicle of insulin treatment (n=6). Kidney International 2013 84, 265-276DOI: (10.1038/ki.2013.113) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 8 NOD2 (nucleotide-binding oligomerization domain containing 2) activation reduced nephrin expression in hyperglycemia. (a) Representative immunofluorescent micrographs of glomeruli with staining of nephrin in HFD/STZ-induced diabetic mice; bar=20μm. (b) Representative western blot gel documents and summarized data showing the protein levels of NOD2 in HFD/STZ-induced diabetic mice. (c) Representative western blot gel documents and summarized data showing that high glucose (HG) reduced nephrin expression in a time-dependent manner in podocytes. (d). Representative western blot gel documents and summarized data showing that muramyl dipeptide (MDP) reduced nephrin expression in a time-dependent manner in podocytes. (e) Representative western blot gel documents and summarized data showing the efficiency of gene silencing of NOD2 by short hairpin RNA (shRNA)-NOD2 transfection. (f) Representative western blot gel documents and summarized data showing that gene silencing of NOD2 attenuated HG-reduced nephrin expression. *P<0.05 versus control, #P<0.05 versus wild-type diabetic mice (n=10) or vehicle of HG treatment (n=6). HFD, high-fat diet; Norm-diet, normal diet; STZ, streptozotocin; WT, wild type. Kidney International 2013 84, 265-276DOI: (10.1038/ki.2013.113) Copyright © 2013 International Society of Nephrology Terms and Conditions