Role And Migration Mechanism Of Dendritic Cells In Experimental Diabetic Kidney Disease

Diabetic kidney disease (DKD) is one of the most common and serious complications of diabetes. Diabetic kidney disease is the secondly most important factor in patients requiring hemodialysis in China, following only primary glomerulonephritis. Therefore, it is important to find safe and effective methods to prevent or delay the occurrence and development of DKD, in order to improve the survival of patients with diabetes. However, the pathogenesis of the DKD is not entirely clear, and there is no effective method to prevent DKD. Despite of the strict control of blood glucose level and the application of ACEI and ARB, the occurrence and the progress of DKD can be only reduced or delayed but not prevented. In recent years, people have paied more and more attention on the inflammatory theory, in which DKD is considered as natural immunity disorder and a low grade inflammatary disease. It is claimed that inflammation can lead to DKD and play an important role throughout the process of the DKD. Hemodynamic disorder and high glucose can cause inherent kidney cells damage, which induces migration to the injury sites and activation of leukocytes through release of pro-inflammatory mediators. Locally infiltrated inflammatory cells and intrinsic renal cells can produce inflammatory cytokines, which are involved in the occurrence and development of DKD. A lot of researches have proven that the infiltrated inflammatory cells in DKD include macrophages and lymphocytes.Dendritic cells (DC) are known as the most powerful cells on specially antigen presenting, which play a role of regulating inflammation and immune defense. Immune regulatory function of DCs is related to their maturation status during the migration process. In the inflammatory environment, immature DCs migrate from blood circulation to peripheral tissues through mediation of the adhesion molecules and chemokines, follwed by capturing and processing antigens, and then start local inflammatory immune response by presenting antigens to the T cells and B cells after maturation of DCs. Some researchers have confirmed that DCs are involved in tubulointerstitial inflammation and that the migration of DCs to local tissues is mediated of the P-selectin in models of IgA nephropathy, lupus nephritis, acute renal damage and the UUO. However, there has been no report about the involment of DCs in DKD so far. We established the rat model of diabetes to investigate whether DCs participate in DKD pathological injury. We found that DCs played an important role in renal pathological injury in DKD. DCs were distributed only in the renal tubulointerstitial and related to tubulointerstitial damage. The distribution of DCs was related to the expression of P-selection. In order to further investigate the possible mechanisms of DCs infiltration in the DKD, diabetic mice were treated by ursolic acid and the activity of NF-κB was determined by EMSA. We found that DCs accumulated in the kidney through the nuclear factor-kappaB (NF-κB)-P-selectin pathway and the accumulation of DCs could be relieved by ursolic acid treatment through suppressing the expression of NF-κB. In vitro studies, we also found that high glouse could cause the phenotpye changes of DCs, which could be inhibited by ursolic acid directly.In summary, DCs are involved in the pathological injury in DKD by migrating to the kidney through the NF-κB-P-selectin pathway. High glucose could promote maturation of DC and Ursolic acid could inhibit local DC infiltration and maturation by blocking NF-κB-P-selectin pathway, resulting in amelioration of pathological damage in DKD. These discoveries may provide new theoretical evidence for the pathogenesis and treatment of DKD.Part 1. Distribution of dendritic cells in diabetic ratsObjective To explore whether the local dendritic cells (DCs) accumulate in the rat kidney with diabetic kidney, as well as to investigate the role of atorvastatin on DCs.Methods The SD rats were randomly divided into three groups as follows: normal control rats, diabetic rats, diabetic rats treated with atorvastatin(20mg/kg.d). The rats of latter two groups were intraperitoneally injected high-dose Streptozotocin(STZ, 65mg/kg). These groups of rats were killed on 4,8,12 weeks, respectively. CD1a+CD80+DCs were observed by the double immunostaining method and the images were analyzed with Axioplan 2 microscopy; P-selectin was analysed by immunohistochemistry method and Western blot..Results CD1a+CD80+DCs weren't found in healthy control rats group whereas in diabetic rats group, they were gradually found in the renal tubules, the interstitium and vessels, especially in the interstitium, and the number of infiltrated DCs in 12 weeks was much more than 4 week and 8 week. Comparing with healthy control rats group, the expression of P-selectin in diabetic rats group was up-regtlated among tubulointerstitium mainly on renal tubular epithelial cell. The accumulation of DCs and expression of P-selectin are both correlated with the degree of renal tubulointerstitial fibrosis closely. However, these changes became less conspicuous in rats treated with atorvastatin.Conclusion There were infiltration of DC in DKD kidney, and only distributed in the tubularinterstitium, not in the glomeruli. The accumulation of DCs and expression of P-selectin are both correlated with the degree of renal tubulointerstitial fibrosis closely. The renal protection of atorvastatin is partly associated with an anti-inflammatory action through a reduction of P-selectin expression and DCs accmulation in DKD, independent of the cholesterol-lowering effects.Part 2. Mechanism of infiltrative dendritic cells in diabetic miceObjective To investigate the infiltration mechanism of local dendritic cells (DCs) accumulate in the mice kidney with diabetic kidney.Methods The male mice were randomly divided into three groups as follows: normal control rats, diabetic rats, diabetic rats treated with urslic acid(40mg/kg.d). The mice of latter two groups were continuous intraperitoneally injected lowe-dose Streptozotocin(STZ,40mg/kg) 5 days. Mice were killed on 8 weeks, CDllc+DCs were observed by the immunostaining method and the images were analyzed with Axioplan 2 microscopy; P-selectin was analysed by RT-PCR and Western blot, the activity of NF-κB were measured by EMSA.Results CD11c+DCs weren't found in normal control rats group whereas in diabetic rats group, they were gradually found in the renal tubules, the interstitium and vessels. Comparing with healthy control rats group, the expression of P-selectin,the activity of NF-κB in diabetic rats group were up-regtlated. After 8 weeks of the therapy, ursolic acid ameliorated significantly the activity of NF-κB, the expression of P-selection, the distribution of DCs, improved the renal fuction and the renal tissue injury.Conclusion Dendritic cells through NF-κB-P-selection pathway accumulated in DKD kidney, ursolic acid ameliorated renal injury of diabetic mice possibly in part via suppression of actiovation of the NF-κB, reduction of P-selection mRNA and protein levels, inhibition of dendritic cells accumulation.Part 3. Effects of hyperglycemia on development and maturation of mouse bone marrow-derivd dendritic cellsObjective To investigate the effects of hyperglycemia on development and maturation of mouse bone marrow-derivd dendritic cells.Methods Bone marrow-derived DCs were induction by low-dose rmGM-CSF (recombinant mouse granulocyte-macrophage colony stimulating factor,20ng/ml) and rmIL-4 (recombinant mouse interleukin-4, lOng/ml) for 7 days. DCs were cultured in RPMI1640 medium with either 5.5mmol/L D-glucose (NG),30mmol/L D-glucose(HG) or 30mmol/L D-glucose+20μmol/L ursolic acid. Morphological changes were observed by inverted microscope, the DCs immunophenotypic expression was investigated by FACS, the activity of NF-κB were measured by EMSAResults (1), immature DCs that rugged, muti-round, burr-like protrusions lss, and high glucose stimulated the growth of DC suspension, more branch-like protrusions surface and shapes; (2), detected by flow cytometry in comparions with immature DCs, mature DCs stimulated by hige glucose, CD11c+CD80,MHCⅡ+CD86+ positive rates were as high as 94.58%,89.96%, uroslic acid intervention in high glucose stimulated DCs, CD11c+CD80,MHCⅡ+CD86+ positive rates were 60.63% and 40.94%.Conclusion High glucose can promote maturation of DCs, ursolic acid can inhibit this effect.