Upregulated IL-18 expression in type 2 diabetic subjects with nephropathy: TGF-β1 enhanced IL-18 expression in human renal proximal tubular epithelial cells
Introduction
During the last decade, considerable evidence demonstrated the molecular and cellular links between metabolism and inflammation, especially in the context of obesity and diabetes. Chronic inflammation, characterized by elevated circulating levels of inflammatory markers, appears to play a critical role in the pathogenesis of type 2 diabetes and its associated complications [1], [2]. Recent accumulating evidence indicates that chemokines, adhesion molecules, growth factors, and interleukins are involved in the development of diabetic nephropathy (DN) [1], [2]. Particularly, transforming growth factor beta (TGF-β1) has been shown to play an important role in kidney diseases, including DN [3], [4]. Previous studies have demonstrated the expressions of TGF-β mRNA and protein are markedly elevated in human and experimental diabetic kidney [5], [6], [7]. Moreover, it is known that excretion of urinary TGF-β1 increased in response to hyperglycemia [8] and in patients with DN [9]. TGF-β1 is known to have pleiotropic immunomodulatory effects, functioning as a proinflammatory cytokine in various epithelial types including the proximal tubular cells of the kidney [10]. Latest report also showed that TGF-β1 also induces chemokines, such as Interleukin (IL)-8 and Monocyte Chemotactic Protein 1 (MCP-1) in renal proximal tubular cells [11]. In addition, TGF-β1 regulates epithelial-mesenchymal transition (EMT), which appears to be a major pathway leading to tubulointerstitial fibrosis in chronic kidney diseases. A recent study has described that IL-18, as well as TGF-β1, is also able to initiate and complete the EMT process by itself [12]. Recently growing evidence suggests that IL-18 is also a pathogenic cytokine in DN [2], [13], [14], [15], [16]. IL-18 is structurally similar to IL-1β, and considered as a member of the IL-1 family [17], IL-1β has been shown to be upregulated in experimental models of DN [18], [19]. IL-1β is associated with the severity of tubulointerstitial lesions [20], and to induce human proximal tubule cell injury, mediated by a TGF-β1 dependent mechanism [21]. On the other hand, the localization or regulation of IL-18 expression in diabetic kidney has not been evaluated. On the basis of these considerations, in the present study, we thought to test the hypothesis that TGF-β1 stimulates the production of IL-18 in renal tubular cells. Recent studies clarified that serum IL-18 concentrations are increased by acute hyperglycemia in humans [22] and patients with type 2 diabetes have higher serum IL-18 levels than matched nondiabetic subjects [23], [24]. Moreover, results from the MONICA/KORA Augsburg study demonstrated that elevated levels of serum IL-18 could predict the development of type 2 diabetes [25]. Furthermore, increased serum or urinary levels of IL-18 has been described in patients with DN, indicating that IL-18 is one of significant pathogenic mediators in progression of DN [13], [14], [15], [16].
IL-18 was originally cloned from Kupffer cells of mice by the action of interferon-γ inducing factor [17]. Subsequently, IL-18 has been demonstrated to have a variety of biological functions. IL-18 acts to enhance productions of Th1 type cytokines (IFN-γ, IL-2, GM-CSF, TNF-α, IL-1β, and CC and CXC chemokines) on T cells and B cells. IL-18 also stimulates IFN-γ synthesis in NK cells, upregulates perfolin-mediated NK cell activity, and enhances Fas-FasL-mediated cytotoxicity by inducing FasL expression [26]. It has been shown that IL-18 is produced not only by various types of immunocompetent cells but also by non-immune cells, and inappropriate IL-18 production may be involved in the pathogenesis and may influence the clinical outcome of patients [27]. In view of the importance of IL-18 in the development of DN, we investigated the expression of IL-18 in diabetic kidney and the regulatory mechanism of IL-18 expression in human renal tubular cells. In present study, we have demonstrated significant increases in the expression of IL-18 in the diabetic kidney accompanied with overt proteinuria, compared to those in minimal change nephrotic syndrome (MCNS), which is also known to cause proteinuria without evident pathological abnormal changes on light microscopy. We also observed for the first time that treatment of HRPTECs with TGF-β1 could enhance the expression of IL-18, and mitogen-activated protein kinases (MAPK) play a major role on this induction.
Section snippets
Materials
d-Glucose was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan), and the specific kinase inhibitors were purchased from Cell Signaling Technology Inc. (Beverly, MA). Recombinant human IL-18, anti-human IL-18 monoclonal antibodies were purchased from MBL (Nagoya, Japan). The anti-human IL-18 receptor α (IL-18 Rα) antibody and anti-human CD-15 antibody were from R&D Systems Inc. (Minneapolis, MN, USA), and DakoCytomation (Tokyo, Japan), respectively. Smad2, Smad3 and Samd4
IL-18 production is increased in diabetic kidney
To localize the in vivo site of IL-18 synthesis in DN, IL-18 production was first assessed by immunohistochemistry in renal biopsy specimens from 7 patients with MCNS and 12 patients with type 2 diabetes and overt proteinuria (urinary protein: 0.2–9.7 g/day, mean ±S.D.; 3.86 ± 3.123 g/day) (Table 2). Strongly positive immunoreactivity for IL-18 was seen in tubular epithelial cells in patients with DN (Fig. 1B, C) whereas biopsy from MCNS patient was negative (Fig. 1A). Most part of specimens with
Discussion
The present study showed that IL-18, that is important as a regulator of innate and acquired immune response [26], [27], was overexpressed in the renal specimens from the patients with DN. IL-18 was originally cloned as an interferon-γ inducing factor secreted from macrophages [17]. Subsequently, IL-18 has been demonstrated to have a variety of biological functions, including productions of cytokines and chemokines from not only various types of immunocompetent cells but also non-immune cells
Conflict of interest
There are no conflicts of interest.
Acknowledgment
We are grateful to Dr. Hidetoshi Sato for help in preparation of the kidney sections.
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