NOD2Promotes Renalinjury By Exacerbating Inflammation And Podocyte Insulin Resistance In Diabetic Nephropathy

Background:Diabetic nephropathy (DN) is one of the major micro-vascular complications of diabetes mellitus and is the most common cause of end-stage renal diseases. Although DN is traditionally considered as a non-immune disease, an increasing number of clinical and animal model studies have implicated that a lot of immune cells, inflammatory mediators, cytokines and extracellular basement in the kidney of the diabetic nephropathy was associated with the injury of renal cell showing that the activation of innate immune system and inflammatory mechanisms are of importance in the pathogenesis of DN.Innate immunity is the first frontier of immunity system to fight with outside invasion, which induce tissue in a state of continuous injury by recognizing highly conserved pathogen-associated molecular patterns,(PAMPs) or damage-associated molecular patterns,(DAMPs). Now, intracellular PRRs, nucleotide-binding oligomerization domain,(NOD) protein family, called NOD-like receptors,(NLRs), is the center of research. Nucleotide-binding oligomerization domain containing2(NOD2) is a well-characterized member of the NLR family containing CARD domain that detects muramyl dipeptide (MDP) from peptidoglycan. It plays important role in inflammatory homeostasis. The importance of NOD2in inflammatory homeostasis is underscored by the observation that mutations in NOD2gene are associated with susceptibility to Crohn’s disease and Blau syndrome.9In addition to being present in inflammatory cells, NOD2is also widely distributed in other cell types, such as adipocytes and epithelial cells, where it plays an important role in triggering the inflammatory response under pathological conditions.Insulin resistance is a major defect underlying the development of type2diabetes and the innate immune activation. It can cause hyperglycemia reaction for a long time, which can induce the activation of innate immunity and inflammation promoting insulin resistance. Further studies showed that chronic low-grade inflammation was found to be associated with insulin resistance. Podocytes are important component in the filtration barrier and insulin sensitive of the kidney. The podocyte injury are associated with the albuminuria with states of insulin resistance. The insulin response of the podocyte occurs via the facilitative glucose transporters GLUT1and GLUT4, and this process is dependent on the filamentous actin cytoskeleton. At the same time, the specific protein of podocyte, nephrin, allows the GLUT1-and GLUT4-rich vesicles to fuse with the membrane of podocyte. Nephrin plays very important role in podocyte insulin responsiveness, which may have central relevance for understanding of DN and for the association of albuminuria with states of insulin resistance.The innate immunity and insulin resistance contribute to the development of diabetic nephropathy together.It has been revealed that NOD2is highly expressed in the renal epithelial cells and that the absence of NOD2in mice results in protection from renal ischemia/reperfusion injury. However, so far, the expression patterns of NOD2in the kidney and the contribution of NOD2to the pathogenesis of DN is still unknown.Objective:First, to detect the expression of NOD2in human DN biopsies and HFD (high-fat diet)/STZ-induced diabetic mice, and the potential role of NOD2in the inflammatory pathogenesis.Second, to reveal the mechanism that how NOD2paticipate in diabetic nephropathy by exacerbating inflammation or podocyte insulin resistance.Methods:Animal studiesTwenty eight-week wild male C57BL/6J mice and twenty male NOD2-/-mice were divided into four groups randomly, normal-diet wild-type mice, HFD/STZ-induced wild-type mice, normal-diet NOD2-/-mice, HFD/STZ-induced NOD2-/-mice.The normal-diet groups were given normal diet, while the HFD/STZ-induced groups were given high-fat diet for14weeks and injection of STZ to make diabetic mice model. After the blood glucose, albuminuria and pathological test to make sure the success models, the change of weight, blood pressure, plasma triglyceride and plasma free fatty acid were test, and samples were collected by heart perfusion by cold normal saline. The pathological changes were observed by PAS staining and electron microscopy analysis; the expression of NOD2in wild mice organs and the kidney of diabetic mice were tested by Western blotting; immunohistochemical staining of continuous slices were used to observe the change of NOD2in the kidney and the infiltrating macrophage cells of diabetic mice; enzyme-linked immunoabsorbent assay and real time RT-PCR were taken to detect the level of inflammatory factors; the differences of nephrin among four groups were observed by immunofluorescence staining and Western blotting.Human renal biopsy samples studiesThe patient samples were obtained from the Department of Pathology, Shandong University School of Medicine including normal subjects, diabetic nephropathy patients, diabetic patients without nephropathy, focal segmental glomerulosclerosis patients, IgA nephropathy patients, membranous glomerulonephritis patients, lupus nephritis patients and and minimal change disease patients, immunohistochemical staining of continuous slices were used to observe the change of NOD2in the kidney biopsies and the infiltrating macrophage cells of normal kidney, DN patients and diabetic patients without nephropathy. Relative mRNA levels of NOD2in the renal biopsies from all the subjects were examed by real time RT-PCR. The correlation between NOD2mRNA levels and estimated glomerular filtration rate (GFR) in all subjects and the correlation between NOD2mRNA levels and24-h urine protein excretion among all subjects with proteinuria were estimated.Studies in vitroRenal cells were cultured in vitro, and the relative level of NOD2mRNA were carried out by RT-PCR; Effects of different stimuli on the expression of NOD2in podocytes, such as high glucose, advanced glycation end-product (AGE), tumor necrosis factor-a (TNF-α), transforming growth factor-β (TGF-β) were carried out by Western blotting. MDP activate NOD2in podocytes, then, the activation of MAPKs and NF-κB signalings were examined by Western blotting, including phospho-ERK1/2, phospho-p38, phospho-JNK, IκBα Relative levels of proinflammatory factors in podocytes treated with MDP were carried out by real time RT-PCR and the podocyte apoptosis were determined by flow cytometric analysis. To understand the effect of NOD2activation on insulin signaling pathwathys, insulin-stimulated glucose uptake test was taken and confocal microscopic technology was used to show the different extent of GLUT4and Western blotting to show the relative NOD2protein levels in the membrane fraction of podocytes. Western blotting was used to show the level of phosphorylation of IR substrate-1(IRS-1) at Ser612in podocytes induced by MDP and immunoprecipitation was used to show the effect of MDP on a insulin-stimulated tyrosine phosphorylation of IRS-1and IRS-1/p85association. The effect of high glucose and MDP on the neprin expression in podocytes by Western blotting, and short hairpin RNA (shRNA)-NOD2transfection was used to show that gene silencing of NOD2attenuated HG-reduced nephrin expression.Results:Expression of NOD2in the renal cellsWe first detected the expression of NOD2in the kidney and renal cells. Among adult murine kidney, lung, spleen, and small intestine, the kidney revealed a higher level of NOD2. These results are consistent with previous studies showing the organ-specific expression patterns of NOD2. Immuno-histochemical staining with anti-NOD2further revealed immuno-histochemical staining with anti-NOD2in the kidney and intestine from both wild-type (WT) and NOD2-/-mice, indicating the expression patterns of NOD2in the kidney and intestine from both wild-type (WT) and NOD2-/-mice, and the specificity of the NOD2immunostaining. Moreover, we found that NOD2was expressed in murine glomerular mesangial cells, murine podocytes, human glomerular endothelial cells, and human proximal tubule epithelial cells.Renal cortical NOD2was significantly elevated in human DN biopsies and HFD/STZ-induced diabetic miceUpregulation of NOD2was observed in paraffin-embedded sections of human diabetic kidney tissues by immunohistochemical staining. Heavy granular staining for NOD2was detected in the kidney from DN subjects, but weak staining in the kidney from normal controls and diabetic patients without nephropathy. Continuous slices were stained with CD68and NOD2individually, and CD68-positive infiltrating monocytes/macrophages in the interstitium and glomeruli were observed, which were colocalized with NOD2, indicating that except enhanced NOD2expression in renal parenchymal cells, infiltrating immune cells also contribute to the upregulation of NOD2expression in the kidney. By real-time, reverse transcriptase-PCR (RT-PCR) analysis, we further confirmed the upregulation of NOD2in renal biopsies from DN subjects. We also examined the expression of NOD2in other kidney diseases. Of note, among renal biopsies from patients with focal segmental glomerulosclerosis (n=7), IgA nephropathy (n=7), membranous glomerulonephritis (n=6), lupus nephritis (n=9), and minimal change disease (n=8), except for minimal change disease patients, NOD2mRNA levels were markedly increased in other patients compared with control subjects. In addition, the NOD2mRNA expression was negatively correlated with estimated glomerular filtration rate in all subjects (Spearman’s r=-0.7274, P<0.01). No correlation was found between proteinuria and NOD2in all subjects with proteinuria (Spearman’s r=0.1384, P>0.05).In the HFD/STZ-induced DN mice, HFD/STZ treatment led to hyperglycemia and increase in plasma triglyceride and free fatty acids, and blood pressures were not significantly different among these groups. Consistent with the changes in DN patients, renal NOD2level was significantly increased by Western blot and immunochemical analyses from HFD/STZ mice. The infiltration of inflammatory cells into the kidney also contributed to the upregulation of NOD2expression in DN.NOD2deficiency ameliorated renal injury in diabetic miceCompared with WT diabetic mice, albuminuria was significantly less in NOD2-/-diabetic mice accompanied by decreased mesangial expansion and ameliorated podocyte injury. Furthermore, NOD2-/-diabetic mice showed decreased levels of proinflammatory cytokines and chemokines including IL-1β, IL-6, IL-8, TNF-α, MCP-1and ICAM-1in both kidney and serum. Moreover, the renal fibrosis-associated molecules including collagen IV and fibronectin were also decreased in the kidney from NOD2-/-diabetic mice.NOD2was upregulated in podocytes in hyperglycemiaWe found that high glucose concentration dependently enhanced NOD2expression in podocytes, and mannitol control had no effects on NOD2expression. We further found that podocytes treated with AGE, TNF-α or TGF-β significantly increased NOD2expression in a concentration-dependent manner.MDP induced activation of MAPK signaling pathways, production of proinflammatory mediators, and podocyte apoptosisWe treated podocytes with MDP for the activation of NOD2, and we found that MDP induced activation of MAPKs as assessed by increasing the levels of phospho-specific extracellular signal-regulated kinase1/2, p38MAPK, and c-Jun N-terminal kinase, and the degradation of IκBα was mediated by MDP in a time-dependent manner, which is the key upstream event of NF-κB activation. We also observed that MDP enhanced the production of proinflammatory mediators in podoctes and the flow cytometry showed that MDP induced podocyte apoptosis.NOD2-mediated glucose uptake, GLUT4translocation, and insulin signaling in podocytesWe found that podocytes significantly increased glucose uptake in response to insulin and MDP selectively reduced insulin stimulation of2-deoxyglucose uptake. Immunofluorescent staining and Western blot showed that MDP disrupted insulin-induced GLUT4translocation to the plasma membrane, which was consistent with a loss of insulin-induced glucose uptake in podocytes. We further found that NOD2activation induced serine phosphorylation of IRS-1, followed by a reduction in IRS-1tyrosine phosphorylation in response to insulin. By immunoprecipitation, it was found that MDP reduced insulin-stimulated binding of p85to IRS-1.NOD2activation reduced nephrin expression in hyperglycemiaFinally, nephrin staining was detected as a fine, linear-like pattern along the glomerular capillary loop in normal-diet mice and nephrin level was significantly reduced in WT diabetic mice, which was improved in NOD2-/-diabetic mice by immunefluorescent and Western bolt analysis. In vitro, we found that high glucose decreased nephrin expression in podocytes and MDP reduced nephrin expression, which was also confirmed by immunofluorescent staining. Moreover, we found that the effect of MDP on nephrin was associated with changes in cytoskeleton distribution as evidenced by the loss of actin filaments and a granular cytoplasmic pattern of actin distribution. Further studies showed that silencing of NOD2expression by shRNA-NOD2transfection blocked high glucose-reduced nephrin expression.Conclusion:1. For the first time, the upregulation of NOD2was explored in the human diabetic nephropathy and diabetic mice. The expression of NOD2in renal cells and higher in diabetic mouse models and diabetic nephropathy patients, while the markedly attenuated severity of the glomerular changes, improved albuminuria and less proinflammatory mediators in NOD2-/-diabetic mice indicated that NOD2is important in diabetic nephropathy.2. The higher expression in kidney diseases and the negative correlation between NOD2expression and estimated glomerular filtration rate in inflammation-associated kidney diseases, and no correlation between NOD2and urine albumin indicated that the upregulation of NOD2could be a common feature of human inflammation-associated kidney diseases.3. CD68-positive infiltrating monocytes/macrophages in the interstitium and glomeruli, which were colocalized with NOD2, indicated that except enhanced NOD2expression in renal parenchymal cells, infiltrating immune cells also contributed to the upregulation of NOD2expression in the kidney.4. A significant positive correlation between NOD2expression and hyperglycemia in podocytes, along with increased proinflammatory cytokines; the NOD2-dependent decrease in insulin signaling coincided with a reduction in insulin-dependent GLUT4translocation and glucose uptake indicated the contribution of NOD2in the regulation of inflammation and the insulin resistance in podocyte.5. The blockade of hyperglycemia-reduced nephrin expression was observed in NOD2-/-diabetic mice, and the silence of Nod2gene reduce the high glucose-induced nephrin expression in podocytes indicated that NOD2mediated high glucose-induced podocyte dysfunction by nephrin.