Pentraxin-3 Attenuates Renal Damage In Diabetic Nephropathy By Promoting M2 Macrophage Differentiation

Background and ObjectiveAs one of the most important long-term complications of diabetes, diabetic nephropathy (DN) is the major cause of end-stage renal disease and high mortality in diabetic patients. The main clinical features of DN are persistent albuminuria and progressively declined glomerular filtration rate (GFR). Several mechanisms,including hyperglycemia, hypertension, hyperlipidemia, oxidative stress,inflammation, and immune system activation, are believed to contribute to the pathogenesis and development of DN. Inflammatory cytokines and immune cells are also involved in the pathogenesis of DN.The long pentraxin 3 (PTX3) is a member of a superfamily of conserved proteins characterized by a cyclic multimeric structure and a conserved C-terminal domain. PTX3 protein is expressed in vascular endothelial cells and macrophages.PTX3 play important roles in inflammatory response,innate immunity and therosclerosis. The long pentraxin PTX3 is an essential component of humoral innate immunity and plays a role in the regulation of inflammation. PTX3 has complex effects on the vasculature, including an interaction with the angiogenic growth factor FGF2 and the regulation of vessel wall tone. By modulating complement-driven inflammation, PTX3 acts as an oncosuppressor gene in mice and selected human tumors. By interacting with provisional matrix components, PTX3 contributes to the orchestration of wound healing and tissue repair/remodeling. Recently, several clinical investigations have demonstrated that elevated plasma PTX3 levels are associated with cardiovascular and chronic kidney diseases (CKD), Furthermore,plasma PTX3 levels are inversely associated with body mass index (BMI) suggesting that PTX3 may play a role in obesity and metabolic syndrome. Recently, Abu Seman N et al provided evidence that decreased plasma PTX3 levels were associated with type 2 diabetes and diabetic nephropathy. However, the current study only focused on endogenous PTX-3 levels rise or the relationship between silence and other diseases,not on the study of PTX3 treatment, only sporadic research found that PTX3 treatment can effectively prevent cytomegalovirus infection, and to prevent aspergillus infection, Kenji Daigo et al demonstrated that in mouse models of sepsis the intraperitoneal administration of PTX3 reduced mortality. It has been shown that in acute myocardial infarction PTX3 reduced no-reflow area, IL-6 level, neutrophil infiltration and C3 deposition,that demonstrates its cardioprotective function.Similarly endogenous and exogenous pentraxin-3 could protect against postischemic acute and chronic kidney injury. However, the therapeutic effect of PTX3 on DN has never been investigated,Soin our current study Part l,we show whether PTX3 plays a role in attenuating renal damage in diabetic nephropathy.Materials and Methods1 .Establishing DN mouse model: 8-12-week-old male C57BL/6 mice were given 50 mg/kg streptozotocin to induce hyperglycemia by intraperitoneal injection in five consecutive days. Blood glucose and urinary protein was monitored at regular intervals. At 4 weeks after streptozotocin treatment, DN mice models were successfully built with postfast blood glucose of 16.5 mmol/1 and urine microalbumin positive. Animals were divided into four groups, 6 normal mice without STZ injection were regarded as NC(normal control,n=6) group. 18 DN mice were divided into three groups,PTX3 group(n=6): Recombinant PTX3 was injected intraperitoneally once a day (0.5 mg/kg body weight) for 4 weeks after the induction of DN(after STZ 4 weeks); Anti-PTX3 group(n=6): For neutralization of endogenous PTX3, 0.2mg/kg of anti-mouse PTX3 mAb was injected intraperitoneally once a day for 4 weeks after the induction of DN(after STZ 4 weeks);Control group(n=6): As control,DN mice were ntraperitoneally injected of equal amounts of buffer. Mice were euthanized after 4 weeks (STZ 8 weeks). Blood,urine and kidneys were collected for urinary microalbumin detection,histological examination and molecularassays. The protein levels of Desmin and Nephrin were determined in NC(normal control,n=6) group and Control group by Western blotting.2. 18 DN mice were divided into three groups: PTX3 group(n=6), Anti-PTX3 group(n=6) and Control group(n=6). Immunohistochemial analysis of Desmin was performed in three groups. The protein levels of Nephrin and WT-1 were determined by Western blotting. The number of CD4+ T cells, CD8+ T cells, Ly6G+ neutrophils and CDllb+ macrophages in kidney were analyzed by FCM. The protein levels of IFN-??TNF-??IL-4 and IL-13 in kidney were determined by Western blotting.Results1. After STZ 4 weeks hyperglycemia significantly increased blood glucose level and urinary protein excretion (P<0.05). The expression of podocyte injury marker desmin was markedly increased in DN group,and nephrin was markedly decreased in Control group as control with NC group (P<0.05).2. PTX3-treated mice with DN showed significantly decrease in the expression of podocyte injury marker desmin. The urinary protein excretion was also markedly decreased in PTX3-treated mice when compared with control(P<0.05). Furthermore,PTX3 also significantly increased the levels of nephrin and Wilm's tumor-1 protein(WT-1) when compared with control (P<0.05). However, the Anti-PTX3 treatment showed opposite effect and aggravated renal damage in DN.3.We investigated the inflammatory cell infiltration in DN. The results showed that the numbers of CD4+ T,CD8+ T,Ly6G+ neutrophils and CD 11 b+ macrophages in PTX3-treated mice were significantly lower than that in control group (P<0.05).The proinflamatory immune response was also regulated by PTX3, as shown by lower induction of IFN-? and TNF-?, and higher induction of IL-4 and IL-13(P<0.05).Conclusion1.PTX3 ameliorates proteinuria and prevents podocytes impairment in streptozocin (STZ)-induced DN mice. PTX3 attenuates renal damage in diabetic nephropathy.2. PTX3 regulates proinflamatory immune response by lower induction of IFN-y and TNF-?, and higher induction of IL-4 and IL-13,and reduces inflammatory cells infiltration.Background and ObjectiveDiabetic nephropathy is the most common pathological disorder predisposing patients to end-stage renal disease. In the early stage of diabetic nephropathy,glomerular hyperfiltration, glomerular and tubular hypertrophy, microalbuminuria and thickening of the glomerular basement membrane (GBM) are observed. Thereafter,the expansion of the mesangial extracellular matrix and overt proteinuria emerge, thus eventually leading to glomerulosclerosis and tubulointerstitial fibrosis. Several mechanisms, including hyperglycemia, hypertension, hyperlipidemia, oxidative stress,inflammation, and immune system activation, are believed to contribute to the pathogenesis and development of DN. Inflammatory cytokines and immune cells take part in the pathogenesis of DN.The infiltration of macrophages is associated with diabetic nephropathy.Macrophages exhibit a range of phenotypes, a phenomenon that has been described as macrophage polarization or heterogeneity. The "classically" activated M1 macrophages, which are induced by IFN-?, LPS, TNF-?, express proinflammatory cytokines such as iNOS, TNF-?, CD16/32, IL-12 and MCP-1 ,play a pathogenic role in promoting renal inflammation. In contrast, exposure of macrophages to IL-4 or IL-13 inhibits the expression of these proinflammatory cytokines, and instead activates the expression of Arg-1, CD206 and IL-10. These �alternatively� activated M2 macrophages involved in the resolution of inflammation and repair of injury.Under certain conditions, the Ml and M2 subtypes can be converted to each other.In our study Part 1 ,we demonstrate that PTX3 plays a role in attenuating renal damage in diabetic nephropathy.The number of CD4+ T cells, CD8+ T cells, Ly6G+neutrophils and CD11b+ macrophages were all significantly lower in the PTX3-treated group than that in the control group in DN. The IL-4 and IL-13 levels in PTX3-treated group were markedly higher than that in control group in DN. PTX3 may be involved in promoting M2 macrophage differentiation. Therefore, in our current study Part 2, we will show whether PTX3 promotes M2 macrophages differentiation in vivo and in vitro.Materials and Methods1. To clarify whether PTX3-mediated attenuation of DN results from a phenotypic regulation of renal M1/M2 macrophages, we analyzed several specific markers of M1(iNOS?CD 16/32) and M2(CD206?Arg-1) macrophages phenotype by western blot and FCM using protein extracted from kidney tissue.2. RAW264.7 cells were maintained in RPMI 1640 media (containing 11.1 mM glucose) . In order to mimic macrophages phenotype in the state of DN in vivo,RAW264.7 derived macrophages were stimulated with glucose in a dose (11.1 mM,20 mM, 30 mM, 35 mM) and time (Oh, 6h, 12h, 24h, 48 h) dependent manner.Activity of intracellular iNOS were quantitated. Regular concentration of glucose(11.1 mM) in RPMI 1640 media was used as control. Thirdly, to examine the effect of PTX3 on macrophages polarization, RAW264.7 derived macrophages were preincubated with 30 mM high glucose in the presence or in the absence of PTX3 for 12h. The cells were washed twice with PBS followed by RNA harvest for quantitative RT-PCR. The mRNA expression of iNOS. CD16/32, Argl and CD206 of RAW264.7 cells were analyzed by RT-PCR.Results1.The PTX3-treated group showed decreased levels of iNOS and CD16/32, and increased levels of Argl and CD206(P<0.05), when compared with the control group by western blotting. PTX3-treated group showed a significant decrease in the number of iNOS-positive and CD16/32-positive macrophage, a substantial increase in the number of Arg 1-positive and CD206-positive macrophage in compared with the control group(P<0.05). And the Anti-PTX3 treatment showed opposite effect and aggravated renal damage in DN(P<0.05).2. The iNOS activity was increased in a glucose-dose-dependent manner(P<0.05).Particularly,30mM glucose gave the maximum response(48.1 �6.6 U/mgprot,P<0.05).From 0 to 12h, iNOS activity was increased in a time-dependent-manner (P<0.05).The peak level of iNOS activity achieved at 12h (44.6 � 10.7 U/mgprot,P<0.05) after high glucose stimulation. The PTX3-treated 30mM glucose group showed decreased mRNA levels of iNOS and CD16/32, and increased mRNA levels of Argl and CD206(P<0.05), compared with 30mM glucose group by RT-PCR. Above data demonstrated that PTX3 switched high glucose-induced inflammatory M1 macrophages toward M2 phenotype.Conclusion1. PTX3 in vivo and in vitro inhibits M1 macrophages activation, enhances M2 macrophages phenotype to protect against podocyte injury.2. The iNOS activity was increased in a glucose-dose-dependent manner and time-dependent manner after high glucose stimulation, High glucose promotes M1 macrophages differentiation.