The Expression Of Tim-3 In Diabetic Nephropathy

[Background]Diabetic nephropathy (DN) is a serious complication of diabetes. In 2015, the global prevalence of diabetes was 8.8%. China was list on the top in patients number. Despite of the new progress in diagnosis and treatment, diabetic nephropathy is still characterized by high incidence of disability and mortality. So it is very important to further study the mechanism of DN and to seek the new treatment target.DN patients have a long-term immune system activation and micro inflammatory state. Inflammatory cytokines and immune cells have been involved in the pathogenesis of DN. TNF-a, IL-1, IL-6 and IL-18 were significantly increased in the animal model of diabetic nephropathy. Elevated TNF-a, IL-6. IL-18 significantly related to the increase of index of kidney and urinary protein excretion rate. These cytokines are related to alterations in endothelial permeability, glomerular hypertrophy, glomerular basement membrane thickening and apoptosis of endothelial cells. There is growing evidence that even in the early stages of DN, immune cells including activated macrophages, T cells and B cells accumulate in the glomeruli and interstitium. Macrophages are the key inflammatory cells. Macrophage accumulation in diabetic kidneys predicts declining renal function and macrophage derived products can induce inflammation in the diabetic kidneys. Macrophages are highly versatile cells. Macrophage can differentiate into different phenotypes in different environments, and play different functions. As we all know, states of macrophage activation have been identified:the classically activated (Ml) macrophages and the alternatively activated (M2) macrophages. Ml macrophages that are characterized by high production of pro-inflammatory molecules (like IL-12, TNF-a, iNOS), promotion of Thl response. M2 macrophages produce high amounts of IL-10, CD206, IL-6 and arginase 1 (Arg-1). and exhibit anti-inflammatory, and reduce the infiltration of macrophages in STZ induced diabetic mice. Under certain conditions, the M1 andM2 subtypes can be converted to each other. Therefore, if we can find the molecules that promote the transformation of macrophages to M2, it is possible to find a new target for the treatment of DN.T cell immunoglobulin and mucin-domain containing protein-3 (Tim-3) is a new type of immune regulatory molecule found in 2002, and was initially discovered as a marker of differentiated Thl cells. Subsequent studies have found that Tim-3 is widely expressed in a variety of innate immune cells, such as NK, DC, macrophages, etc. Studies have demonstrated that Tim-3 can negative regulate Thl function,and induce the apoptosis of Thl cells,and mediate immune tolerance. Tim-3 is involved in a variety of autoimmune diseases. These studies have demonstrated the important role of Tim-3 in the induction of immune negative regulation and immune tolerance and its potential value in the treatment of immune diseases. TGF-β in liver tumor microenvironment enhances the transcription of Tim-3 in TAMs which in turn promotes the alternative activation of macrophages; Tim-3 on macrophages activated TAMs produce high level of inflammatory cytokines foster the HCC growth, including IL-6 via NF-kB pathway. The inhibition of the Gal-9-Tim-3 pathway on DC, upstream of T(H)1 response, may be a new target for the treatment of type 1 diabetes. However, the expression of Tim-3 and the related mechanisms in DN remain unclear. This study is aiming to explore the expression of Tim-3 in DN development.[Materials and results]1.Tim-3 expression was up-regulated in renal tissues from DN patients.Immunohistochemical (IHC) testing showed that Tim-3 expression was greatly enhanced in renal tissues of DN patients than healthy donors.IHC double staining data suggested a strong co-location between the expression of Tim-3 and CD68 in DN group and the number of Tim-3+CD68+cells was also higher in DN group than HD group.2. Tim-3 expression on peripheral macrophages from DN patients was higher than that from healthy donors.Peripheral blood of 18 DN patients and 16 healthy donors from Shandong University Qilu Hosipital were collected. Freshly isolated mononuclear cells were detected on the expression of Tim-3 on CD14+ subset. Results demonstrated that the expression of Tim-3 on CD14+ population was higher in DN patients than healthy donors (P<0.05). Moreover, Tim-3 expression on type 1 macrophages was largely up-regulated in DN patients compared with heathy donors (P＜0.05). Taken together, these results suggested that Tim-3 might be involved in DN development by regulating macrophages.3.Tim-3 expression was up-regulated on renal or peritoneal macrophages from DN mice.After one of the kidneys was removed, streptozocin (STZ,50mg/kg, ph=4.45) was intraperitoneal injected in five consecutive days to establish DN mouse model. This model was confirmed successful by detecting blood glucose, serum creatinine, urinary albumin creatinine ratio and pathological changes of renal tissues (PAS staining). Renal mononuclear cells was separated by using type Ⅰ collagenase. The expression of Tim-3 on F4/80high CD11b+ macrophages was analyzed by flow cytometry. Data showed that the blood glucose and urinary albumin creatinine ratio were increased in DN model mice, and glomerular mesangial matrix was also significantly increased. Importantly, the expression of Tim-3 on F4/80highCD11b+ macrophages was largely enhanced (p<0.05). Similarly,Tim-3 was also up-regulated on peritoneal macrophages (p<0.05). Using the method of Western Bolot, we found Tim-3 expression in renal tissues was obviously increased(P<0.05). Thus, these results further indicated that Tim-3 might be involved in DN development by affecting renal macrophages.4. The percentage of Tregs was decreased but the ratio of Th17 cells was increased in DN mice. Data from flow cytometry showed that the frequence of Tregs in the spleen of DN mice was down-regulated, but the ratio of Th17 cells was greatly up-regulated compared with control mice (P<0.05). Analyzing the serum cytokine levels by CBA kits, we found the production of IL-6 was relatively increased in DN mice.5. The expression of IL-1β, TNF-α and IL-6 on renal tissues was largely up-regulated.RNA was extracted by Trizol method, and the expression of IL-1, TNF-, IL-6 in mouse kidney was detected by RT-PCR technique. The data showed that the renal expression of IL-1β, TNF-a and IL-6 in DN mice was largely enhanced compared with control mice.6. Tim-3 blocking can regulate the function of macrophageSterile isolation of peritoneal macrophages in DN mice, then Pretreatment cells with Anti-Tim3 antibody. Data from flow cytometry showed that the expression of TNF-a was significantly increased.[Conclusions]Datas from DN patients and mice models suggested that Tim-3 expression on mononuclear/macrophages was significantly increased, especially on type 1 macrophages, which indicated that Tim-3 might play important roles in DN development by regulating macrophage polarization.