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Recent Advancement of Understanding Pathogenesis of Type 1 Diabetes and Potential Relevance to Diabetic NephropathyIchinose K.a · Kawasaki E.b · Eguchi K.a
aUnit of Translational Medicine, Department of Rheumatology, Graduate School of Biomedical Sciences, Nagasaki University, and bDepartment of Metabolism/Diabetes and Clinical Nutrition, Nagasaki University Hospital of Medicine and Dentistry, Nagasaki, Japan Corresponding Author
Eiji Kawasaki, MD, PhD
Department of Metabolism/Diabetes and Clinical Nutrition
Nagasaki University Hospital of Medicine and Dentistry, 1-7-1 Sakamoto
Nagasaki 852-8501 (Japan)
Tel. +81 95 849 7550, Fax +81 95 849 7552, E-Mail firstname.lastname@example.org
Type 1 diabetes mellitus is an autoimmune disease characterized by progressive destruction of pancreatic beta cells by genetic and environmental factors which leads to an absolute dependence of insulin for survival and maintenance of health. Although the majority of mechanisms of beta cell destruction remain unclear, many molecules, including proinflammatory cytokines and chemokines such as tumor necrosis factor alpha and monocyte chemoattractant protein-1, are implicated in the development of beta cell damage. Furthermore, beta cell destruction is enhanced by the Th1 and Th17 subsets of CD4+ T cells. In contrast, there are mechanisms involved in the maintenance of peripheral tolerance by regulatory T cells, the function of which depends on the pleiotropic cytokine transforming growth factor beta. Development and progression of renal injuries in patients with diabetic nephropathy are also associated with several growth factors and proinflammatory cytokines, including tumor necrosis factor alpha, insulin-like growth factor-1, monocyte chemoattractant protein-1, vascular endothelial growth factor, and transforming growth factor beta. Although the pathogenic mechanisms underlying type 1 diabetes and diabetic nephropathy are principally different, i.e., autoimmunity and inflammation, some common factors, including susceptibility genes and proinflammatory cytokines, are involved in both mechanisms, including infiltrating cell recruitment, upregulation of other cytokines and chemokines, or apoptosis.
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Diabetes mellitus is a complex syndrome characterized by absolute or relative insulin deficiency that leads to hyperglycemia and an altered glucose, fat, and protein metabolism. These metabolic dysfunctions are associated pathologically with specific microvascular diseases secondary to accelerated atherosclerosis and various other characteristic long-term complications, including diabetic retinopathy, nephropathy, and neuropathy. It has been clearly established that diabetes mellitus is a genetically and clinically heterogeneous disorder. Four types of diabetes mellitus have been defined by an Expert Committee on the Diagnosis and Classification of Diabetes Mellitus [1 ]and a WHO consultation , based on our current understanding of the pathogenesis rather than the requirement for insulin therapy: type 1 diabetes mellitus, type 2 diabetes mellitus, gestational diabetes mellitus, and diabetes secondary to other conditions.
Type 1 diabetes is an organ-specific autoimmune disease characterized by a progressive cell-mediated destruction of beta cells of the pancreas which leads to an absolute dependence on insulin for survival and maintenance of health. In contrast, type 2 diabetes is a nonautoimmune form of diabetes characterized by insulin resistance and relative (rather than absolute) insulin deficiency. Both forms of the disease may exist in the same family, and persons with either type are subject to the same long-term complications, including diabetic nephropathy which affects ∼40% of the patients. It has been reported  that hyperglycemia, increased blood pressure, and genetic predisposition are the main risk factors for the development of diabetic nephropathy. There is increasing evidence for the role of genetic factors in the development of diabetic nephropathy . Furthermore, recent studies suggest that inflammatory processes and immune system cells might be involved in development and progression of diabetic nephropathy. There is a growing body of evidence implicating inflammatory cells at every stage of diabetic nephropathy which produce various reactive oxygen species, proinflammatory cytokines, metalloproteinases, and growth factors that are also associated with the development of type 1 diabetes. The aim of this article is to discuss the current knowledge on the pathogenesis of type 1 diabetes and its potential relevance to the development of diabetic nephropathy.
Type 1 diabetes is a heterogeneous disease and is subdivided into two groups, i.e., immune system mediated and idiopathic type 1 diabetes. It has been reported  that >80% of the patients with type 1 diabetes have the immune sytem mediated (type 1A) form. To date, the detection of autoantibodies against multiple islet autoantigens in the serum is a sole hallmark of type 1A diabetes . Among the increasing number of anti-islet autoantibodies in type 1A diabetes, autoantibodies against glutamic acid decarboxylase 65 (GAD65), insulinoma-associated antigen-2 (IA-2), and insulin have been shown to be relevant to the diagnosis. The detection of at least one autoantibody provides evidence of an ongoing autoimmune process. However, negative autoantibodies do not completely exclude the presence of type 1 diabetes. Some of these patients have permanent insulinopenia and are prone to ketoacidosis, but have no evidence of autoimmunity. This form of diabetes is classified as the idiopathic (type 1B) form which accounts for 10–20% of the type 1 diabetes patients.
Several disorders other than type 1 diabetes also lead to severe pancreatic islet beta cell dysfunction. Within the past decades, an increasing number of genetic disorders have been defined which are associated with insulin-deficient diabetes . These disorders often have characteristic inheritance patterns or extrapancreatic disease manifestations. Several of these disorders are listed in table 1. MODY (maturity-onset diabetes of the young) is caused by mutations in hepatocyte nuclear factor-4α gene , glucokinase gene , hepatocyte nuclear factor-1α gene , insulin promoter factor-1 gene , hepatic transcription factor-2 gene , NeuroD1 gene , and KLF11 gene . The disorder appears not to be progressive, and these individuals usually require no specific therapy. Their diabetes can manifest in childhood or later in life, and many of these individuals behave as insulin-dependent patients, even though they have no evidence of autoimmunity. With their insulin dependence and their severe hyperglycemia, patients with MODY are at risk of developing chronic microvascular and macrovascular complications.
In contrast to MODY, patients with several of the other syndromes listed in table 1 have some extrapancreatic disease manifestations. Patients with Wolfram’s syndrome have a median survival of 30 years and develop progressive neurological dysfunction (DIDMOAD: diabetes insipidus, diabetes mellitus, optic atrophy, and deafness) [12,13,14]. The disorder has been linked to mutations of the WSF1 gene on chromosome 4 which has an important function in maintaining the homeostasis of the endoplasmic reticulum in pancreatic beta cells [12, 14, 15]. The maternally inherited diabetes mellitus is characterized by a point mutation in the mitochondrial gene coding for tRNALEU(UUR) (especially at nucleotide position 3243) and is associated with nerve deafness and a lower insulin-secretory capacity .
Although the etiology of type 1 diabetes is only partially characterized, it is recognized that both genetic and environmental factors are involved in the development of the disease. Many studies have shown that the human leukocyte antigen (HLA) class II loci on chromosome 6q21.3, including HLA-DRB1,HLA-DQB1, and HLA-DQA1, are most strongly associated with diabetes risk and may account for nearly 40% of the familial aggregation of type 1 diabetes. These HLA region susceptibility genes are collectively referred to as IDDM1. Furthermore, more than 20 putative diabetes-predisposing genes have been identified by linkage and association studies (table 2). Recent combined analysis of four datasets using a large number of multiplex families provided evidence for linkage of type 1 diabetes to nine non-HLA-linked regions, including 6q21 (IDDM15), 2q31-q33 (IDDM12 and IDDM7), 10p14-q11 (IDDM10), 11p15 (IDDM2), and 16q22-q24 . The genes responsible for IDDM2 and IDDM12 have been identified based on genetic association studies as a variable number of tandem repeats (VNTR) in insulin gene and cytotoxic T lymphocyte antigen-4 (CTLA-4) gene, respectively. Additional previously reported non-HLA genes include PTPN22 gene (chromosome 1p13), SUMO4 gene (chromosome 6q25), and IL-18 gene (chromosome 11q22) .
Activation of the T-cell-mediated immune system in genetically susceptible individuals leads to a lymphocytic infiltration within the islets (insulitis) as well as to a humoral (B cell) response with production of antibodies against one or more beta cell autoantigens. The model of the natural history of type 1 diabetes suggests that there is a long prodromal phase preceding the onset of clinical symptoms in type 1 diabetes (fig. 1). Overt diabetes clinically manifests only after destruction of approximately 90% of the beta cells . The initial interaction of genes and environmental factors, such as viral infections, trigger an immune response to islet autoantigens, with the emergence of autoantibodies as the first sign of beta cell destruction, followed by progressive loss of the first-phase insulin secretion . To date, the best autoantibody predictor of a high type 1 diabetes risk is the expression of multiple anti-islet autoantibodies. Among autoantibodies against insulin, GAD65 and IA-2, expression of a single autoantibody was associated with an approximate 20% risk of diabetes within 10 years of follow-up. In contrast, expression of multiple autoantibodies was associated with a very high risk of progression. The ‘combinatorial’ analysis allowing more than two autoantibodies to be defined, independent of which two autoantibodies are expressed, gives approximately an 80% sensitivity for progression to diabetes with a very high specificity [20, 21]. The progression to overt diabetes resulting in a significant beta cell destruction is triggered by the development of a more aggressive T cell phenotype and a change in the Th1-to-Th2 balance towards a more proinflammatory milieu (Th1 dominant). Furthermore, evidence demonstrating the association of the Th17 subset, the recently discovered CD4+ effector T cell lineage distinct from Th1 and Th2, with pathogenesis of type 1 diabetes is rapidly accumulating [22,23,24].
T-cell-mediated beta cell destruction is induced by the release of cytotoxic molecules, including cytokines, granzyme B, or perforin, or by direct delivery of cell death signals via the Fas pathway [25, 26]. Activated CD4+ and CD8+ T cells act in unison to activate beta cell death via apoptosis. Apoptosis is introduced by activation of the caspase pathway which, in turn, is activated by a number of alternative mechanisms such as Fas interaction with Fas ligand, action of nitric oxide and oxygen-derived free radicals, and membrane disruption by perforin and granzyme B produced by cytotoxic T cells. T cell cytokines, including IL-1, IFN-γ, and TNF-α, exacerbate beta cell death by upregulation of Fas and Fas ligand and stimulation of nitric oxide and free radical production. Various cytokines are involved in the enhancement beta cell damage in type 1 diabetes . Beta cell destruction is enhanced by the Th1 and Th17 subsets of CD4+ T cells and cytokines, such as INF-γ, TNF-α, and IL-2, IL-12, IL-17, and IL-18 (fig. 2). In patients with type 1 diabetes, infiltration of mononuclear cells consisting of CD4+ and CD8+ T cells, B cells, and macrophages is observed in islets of pancreas biopsy specimens .
In contrast, there are mechanisms involved in the maintenance of peripheral tolerance by a specialized subset of regulatory T cells (Tregs). CD4+ Tregs that constitutively coexpress the IL-2Rα chain (CD4+CD25+) have been shown to play a critical role in controlling undesired immune responses to self-antigens . A member of the forkhead family of transcription factors, FOXP3, has been shown to be expressed in murine and human CD4+CD25+ Tregs and appears to be a master gene controlling CD4+CD25+ Treg development . CD4+CD25+ Tregs with a reduced in vitro suppressive function were found in some studies performed on patients with type 1 diabetes [31, 32]. Treg development and function depend on the pleiotropic cytokine TGF-β which is also linked to Th17 cell development [22, 23].
Hyperglycemia is a most important factor in the progression of diabetic nephropathy. Early alterations in diabetic nephropathy include glomerular hyperfiltration, glomerular and tubular epithelial hypertrophy, and the development of microalbuminuria, followed by the development of glomerular basement membrane thickening, accumulation of mesangial matrix, and overt proteinuria, eventually a leading cause of glomerulosclerosis and end-stage renal disease . The accumulation of matrix in the mesangial area reduces the capillary surface area available for filtration, thereby contributing to the progressive loss of the renal function . Hyperglycemia-induced metabolic and hemodynamic factors are thought to be mediators of this injury. The hemodynamic factors implicated in the pathogenesis of diabetic nephropathy include increased systemic and intraglomerular pressure and activation of various vasoactive hormone pathways, including the renin-angiotensin system and endothelins. Multiple biochemical pathways may interact with the metabolic pathway: activated advanced glycation end products, protein kinase C, acceleration of the polyol pathway, and overexpression of TGF-β (fig. 3).
Recent data suggest that the podocytes, specialized visceral epithelial cells, are important for the maintenance of the dynamic functional barrier , and the number of podocytes may be reduced in the glomeruli of both type 1 and type 2 diabetic patients [36, 37]. Furthermore, it has been reported that nephrin, a recently found podocyte protein, is crucial for maintaining the integrity of the interpodocyte slit membrane structure and for maintenance of an intact filtration barrier. In diabetic nephropathy, the protein level of nephrin decreases, possibly via loss into the urine due to synthesis of splice variant isoforms of the nephrin lacking a transmembrane domain [38, 39]. Several studies have been performed on the angiotensin II activity which is involved in podocyte injury in diabetes. Angiotensin-converting enzyme inhibitors prevented loss of podocytes and podocyte injury in the streptozotocin-induced diabetic rat. In addition to angiotensin-converting enzyme inhibitors, angiotensin II type 1 receptor antagonism attenuated podocyte foot process broadening in the streptozotocin-induced diabetic rat [40, 41].
Although mesangial cells and podocytes are proposed as the major mediators of diabetic nephropathy, several growth factors and cytokines, including insulin-like growth factor-1, monocyte chemoattractant protein-1 (MCP-1), and IL-6, also play a key role in the pathogenesis, but most importantly vascular endothelial growth factor (VEGF) and TGF-β. VEGF, a potent stimulator of angiogenesis, promotes endothelial cell proliferation and migration and endothelial tube formation . The protein and mRNA levels of VEGF and its receptor flk-1/KDR are upregulated in experimental diabetic nephropathy [43,44,45] (fig. 3).
There is also growing evidence that the genetic background determines the risk of nephropathy in patients with diabetes. Epidemiologic studies have shown that 35% of the patients with diabetes develop nephropathy, irrespective of glycemic control [46, 47]. Since only 1 in 3 individuals with type 1 diabetes ever developed diabetic nephropathy, both environmental and genetic factors have been postulated as the mechanisms that determine who develops hyperglycemia-related glomerular injury. Familial clustering of diabetic nephropathy has also been demonstrated by several investigators [48,49,50].
There are only a few descriptions of genomewide scans for diabetic nephropathy, and the number of the analyses would not be enough. In the genomewide scans for microvascular complications in Pima Indians, four loci on chromosomes 3, 7, 9, and 20 were identified . A candidate gene study of type 1 diabetic nephropathy also identified a 63-cM region on chromosome 3q, containing the angiotensin II type 1 receptor gene . Furthermore, other linkage studies identified additional loci on chromosomes 7q21.3, 10p15.3, 14q23.1, and 18q22.3 as diabetic nephropathy susceptibility genes [53, 54]. Recently, a high-throughput system for genotyping single-nucleotide polymorphisms has been developed, and genomewide association studies using more than 80,000 single-nucleotide polymorphisms to pinpoint loci involved in the susceptibility to diabetic nephropathy were performed in the Japanese population. Using this system, several candidate genes, including solute carrier family 12 member 3 (SLC12A3) gene on chromosome 16q13 and engulfment and cell motility-1 (ELMO1) gene on chromosome 7p14, were identified [55, 56].
Candidate-gene-based association studies have been the most common approaches employed to identify susceptibility genes for diabetic nephropathy. The genes encoding for angiotensin-converting enzyme, angiotensin II receptor, glucometabolism, lipids, extracellular matrix, and cytokines have been selected to test for an association with diabetic nephropathy based on our current understanding of the pathogenesis of the disease. To date, a number of single-nucleotide polymorphisms are reported as diabetic nephropathy susceptibility genes . Candidate genetic determinants for diabetic nephropathy are provided in table 3.
Recent studies suggest that an inflammatory mechanism mediated by macrophages and angiogenesis may play important roles in the pathogenesis of diabetic nephropathy. Relatively recent reports [58,59,60]described that the degree of neovascularization was significantly increased in patients with diabetic nephropathy and correlates with the expression of VEGF and angiopoietin which likely contribute to diabetic nephropathy by promoting vessel leakage and reducing transendothelial electrical resistance. The angiogenic growth factor VEGF induces the activation of matrix-degrading protease represented by matrix metalloproteases and migration and proliferation of endothelial cells . Recent animal studies utilizing a neutralizing anti-VEGF antibody further demonstrated the involvement of this factor in early glomerular hypertrophy and mesangial matrix accumulation in the progressive stage of diabetic nephropathy [61, 62]. We have previously reported the therapeutic efficacy of endostatin peptide, a potent inhibitor of VEGF, which ameliorates renal alterations in the early stage of type 1 diabetic nephropathy. Increased accumulation of monocytes/macrophages in glomeruli has been reported in diabetic nephropathy [63,64,65]. Considering the effect of VEGF in promoting vascular permeability, it is possible that this may be partially mediated via stimulation of VEGF. In our study, increased expression of IL-6 and MCP-1 in diabetic glomeruli was suppressed by antiangiogenic treatment, suggesting that the cytokines may mediate monocyte/macrophage infiltration. It has also been shown that VEGF increases the survival of pancreatic islets and thus beta cell sparing after islet transplantation by stimulating angiogenesis and improving islet revascularization [66, 67]. Moreover, transgenic mice that overexpress VEGF are characterized by islet hyperplasia, suggesting that VEGF modulates endocrine pancreatic differentiation . Whether this cytokine also protects from the autoimmune destruction of beta cells is currently unknown.
Another angiogenesis-associated factor, angiopoietin-1, is involved in the attachment of mesenchymal cells to endothelial tubes and in the differentiation to mature pericytes, so-called ‘nonleaky’ blood vessels . Angiopoietin-2 is the natural antagonist of angiopoietin-1 and loosens the attachment of pericytes, resulting in the promotion of sprouting angiogenesis in the presence of VEGF. Angiopoietin-1 is an apparent endogenous VEGF inhibitor, and angiopoietin-2 synergizes with VEGF and is upregulated in diabetic microvascular complications . However, the involvement of angiopoietin-1 and angiopoietin-2 in the progression of diabetic nephropathy has yet to be elucidated.
TGF-β has been recognized as a profibrotic growth factor involved in the expansion of mesangial matrix and renal hypertrophy in diabetic nephropathy . Elevated levels of TGF-β have been measured in the glomeruli of streptozotocin-diabetic rats . It was reported  that neutralizing TGF-β antibody prevented diabetic renal atrophy, mesangial matrix expansion, and the development of renal insufficiency in type 2 db/db mice. Certain TGF-β-inducible genes, such as connective tissue growth factor and heat shock protein 47, appear to exert fibrogenic effects on diabetic kidneys [74,75,76,77]. Moreover, serum and urinary TGF-β levels have been found to correlate with the severity of microalbuminuria.
The hallmark of type 1 diabetes is the selective destruction of insulin-producing islet beta cells by activated T cells. It has been reported [78, 79 ]that activated T cells are also associated with diabetic nephropathy. T cell accumulation is found in the juxtaglomerular apparatus of patients with type 1 diabetes. T cell influx would become the factor to exacerbate diabetes and correlates with glomerular filtration surface and albumin excretion rate. Expression of the adhesion molecules lymphocyte-function-associated antigen-1 and ICAM-1 is found on renal endothelial, epithelial, and mesangial cells, and T cell migration into kidney needs these interactions. IFN-γ secretion by T cells can initiate and induce further inflammation and oxidative stress within renal tissues . There are several cytokines associated with the development of the pancreatic beta cell destruction and diabetic nephropathy. It has been reported [81, 82] that the serum levels of IL-18 and TNF-α are elevated in patients with diabetic nephropathy. IL-18 is a potent proinflammatory cytokine that induces IFN-γ and TNF-α, and these cytokines are also associated with the beta cell destruction in type 1 diabetes. Furthermore, as described above, a profibrotic growth factor, TGF-β, is an important pleiotropic cytokine associated with the development of Tregs and Th17 cells. Collectively, in diabetes, induction of proinflammatory and profibrogenic molecules is responsible for dysregulation of both systemic and renal structural and function abilities.
Furthermore, many genetic determinants involved in both type 1 diabetes and diabetic nephropathy are also reported (table 3). One of the loci identified as diabetic nephropathy susceptibility genes, 18q22.3, is located near the type 1 diabetes susceptibility loci (IDDM6). Among the genetic variants listed in table 3, ICAM-1 gene, VEGF gene, MBL2 gene, SUMO4 gene, TNF-α gene, TGF-β gene, and MCP-1 gene have been reported as common genetic determinants for the development of both type 1 diabetes and diabetic nephropathy [83,84,85,86,87].
Mannose-binding lectin (MBL; also known as mannan-binding lectin) is associated with overt diabetic nephropathy. MBL can activate the complement system independent of antibodies via MBL-associated serine proteases . Through this pathway, MBL plays an important role in the innate immune system which leads to autoimmunity either by priming or by promoting aggressive immune responses. Recent reports have suggested increased levels of serum MBL and MBL complex activity in type 1 diabetic patients  and in diabetic patients with diabetic nephropathy [; for references [91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106] see table 3]. Therefore, it leads to the speculation that MBL is involved in the pathogenesis of type 1 diabetes by assisting the autoimmune process of insulitis as well as the development of diabetic nephropathy.
The development of both type 1 diabetes (beta cell injury) and diabetic nephropathy (glomerular injury) is determined by environmental and genetic factors. A vast amount of information has been collected through the years regarding the molecular mechanisms involved in developing type 1 diabetes or diabetic nephropathy. Although the pathogenic mechanisms underlying type 1 diabetes and diabetic nephropathy are principally different, i.e., autoimmunity and inflammation, some common factors, including susceptibility genes and proinflammatory cytokines, are involved in both mechanisms including infiltrating cell recruitment, upregulation of other cytokines and chemokines, or apoptosis. These include SUMO4, MCP-1, TGF-β, TNF-α, IL-18, and MBL, etc. Therefore, the research for clarifying the pathogenesis of microvascular complications in diabetes may lead to the unexpected new findings for the understanding the etiology of type 1 diabetes.
We would like to thank Yohei Maeshima, MD, PhD, for his detailed comments, suggestions, and constant support. This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports and Technology of Japan.
Eiji Kawasaki, MD, PhD
Department of Metabolism/Diabetes and Clinical Nutrition
Nagasaki University Hospital of Medicine and Dentistry, 1-7-1 Sakamoto
Nagasaki 852-8501 (Japan)
Tel. +81 95 849 7550, Fax +81 95 849 7552, E-Mail email@example.com
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