Hyperglycaemia And The Pathogenesis Of Diabetic Nephropathy

The main metabolic disorder occuring in diabetes is hyperglycaemia. Two landmark studies, the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) showed that intensive blood glucose control clearly reduces the development or progression of diabetic nephropathy [43,44]. Thus, the impact of extracellular high glucose concentrations on renal cell gene expression was investigated in detail:

Ayo and coworkers reported that prolonged exposure to high glucose concentrations leads to an increase in collagen IV, laminin and fibronectin synthesis on the protein and mRNA level in mesangial cells [45]. Furthermore, mesangial cells exposed to elevated glucose synthesize less HSPG [46]. Studies with epithelial, endothelial and mesangial cells revealed that all three cell types of the glomerulus produce more collagen type IV when exposed to elevated glucose levels [47]. Periodic changes in glucose concentration, simulating more closely disordered glucose homeostasis, lead to enhanced synthesis of collagen type III and IV compared to continuous low or high glucose environment. This data indicates the deleterious effects of fluctuating glucose levels on the development of diabetic glomerulosclerosis [48].

Similar effects of ambient high glucose conditions were shown on growth factor gene expression: Several groups reported the high glucose-induced upregulation of TGF-P1 gene expression in both, proximal tubular cells and glomerular mesangial cells [49,50]. Culture of mesangial cells in high glucose showed markedly increased CTGF mRNA levels [33]; PDGF and VEGF expression was also found to be elevated by ambient high glucose in mesangial cells [51,52]. Other components of the TGF-^1-system were also found to be induced by high glucose: increased expression of thrombospondin-1 contributes to the excessive TGF-^1 bioactivity by conversion of latent TGF-P1 protein to its biologically active form [31], and induction of TGF-^1 type 2 receptor amplifies the activation of the TGF-^1 cascade [32].

Recent data show that high glucose concentrations stimulate gene expression of matrix proteins, the extracellular matrix protease inhibitor plasminogen activator inhibitor-I (PAI-I) and TGF-^1 by increasing the activity of the corresponding promoters. These studies using promoter fragments fused to reporter genes such as luciferase indicate an activation of the murine and human TGF-^1 promoter [53,54] and of the TGF-^1 type II receptor promoter [32] by high glucose. PAI-I promoter activity is enhanced by high glucose [55]; the fibronectin promoter is stimulated additively by high glucose and TGF-^1 [56]. TGF-^1 itself is known to activate promoters of several genes, including the promoters for a2 collagen type I [57], type IV [58], laminin [59] and in an autocrine loop, transcription of its own gene [60]. Similarly TGF-^1 increases the activity of the HSPG promoter [61]. The most frequently found transcription factors which may mediate the high glucose- and TGF-^1-induced promoter activation are members of the AP-1 family and Sp1. Consensus sequences for AP-1 have been found in the promoters of the different collagen types, fibronectin and TGF-P1 and confirmed to be responsible for the high glucose-or TGF-^1-mediated transcriptional activation [54;56,57;59,60]. Sp1 binding sites have been shown to mediate the high glucose-induced PAI-I promoter upregulation [55].

How do these diverse transcription factor and gene activations all result from high glucose concentrations? A large amount of data have been accumulated, indicating the participation of four main biochemical pathways as mediators of the adverse effects of hyperglycaemia on gene regulation: the protein kinase C (PKC) pathway, the generation of advanced glycation end-products (AGEs), increased flux through the hexosamine biosynthetic pathway (HBP) and the aldose reductase pathway. The PKC and the AGE pathway are also illustrated in detail in other chapters of the book.

Activation of protein kinase C

The involvement of PKC in diabetic nephropathy is in accordance with several reports, which provide evidence for a role of glucose-induced activation of PKC in the elevated synthesis of matrix components [62,63]. Application of a PKC P isoform specific inhibitor ameliorated the changes in glomerular filtration rate, albumin excretion rate and retinal circulation in diabetic rats in a dose-responsive manner, in parallel with its inhibition of PKC activities [64]. Moreover, inhibition of PKC activities abrogated the high glucose-induced TGF-P1 promoter activation in mesangial cells [54]. The high glucose-induced activation of PKC isoforms can subsequently upregulate MAPK pathways, thus inducing the transcriptional activity of AP-1 proteins by enhanced gene expression or posttranslational modifications. The transcripts and protein levels of the AP-1 family members c-Jun and c-Fos are elevated in mesangial cells cultured in high glucose [65]. Posttranslational activation is shown by increased DNA binding activity of AP-1 proteins derived from mesangial cells exposed to elevated glucose concentrations, which is not due to differences in the protein level [66] and phosphorylation of the AP-1 related transcription factor CREB after treatment of mesangial cells with high glucose and TGF-P1 [67]. Hyperglycaemia has been reported to activate MAP kinases including ERK1/2 and p38 MAPK in glomeruli of diabetic rodents [68,69] and in mesangial cells [70,71] and the increase in MAPK activity has been shown to be PKC-dependent [54].

Increased formation of advanced glycation end-products

Recent approaches suggest that the development of diabetic late complications may be linked to the formation of advanced glycation end-products (AGE-products). These AGE-products, such as carboxymethyl-lysine, pentosidine and malondialdehyde-lysine, accumulate in expanded mesangial matrix and nodular lesions as shown in renal tissue from patients with diabetic nephropathy [72]. The cellular effects of AGE-products are mediated by specific binding to cell surface molecules of which the receptor for advanced glycation end-products (RAGE) is well characterized [73]. Expression of RAGE is increased in kidneys from patients with diabetic nephropathy [74]. Furthermore, AGE-products and their receptors co-localize in the renal glomerulus of rats with experimental diabetes [75]. Although upregulation of mesangial TGF-P1 synthesis with concomitant increase of extracellular matrix production by AGEs has been shown [76], and AGEs induced VEGF expression [77], the main action of the AGE-RAGE system not only in terms of diabetic nephropathy is to cause chronic cellular activation and oxidative stress. RAGE expression is enhanced by a positive feedback loop via activation of the transcription factor NF-kB [73], perpetuating the stimulatory event and leading to cellular perturbation [78]. In this stage the cells are highly susceptible to further stress stimuli resulting in chronic inflammation and accelerated sclerosis.

Increased flux through the hexosamine biosynthetic pathway

Hyperglycaemia increases the flux through the HBP; fructose-6-phosphate from glycolysis is thereby converted to glucosamine-6-phosphate, using glutamine as amino-group donor. The reaction is catalysed by glutamine:fructose-6-phosphate aminotransferase (GFAT), the rate-limiting enzyme of this pathway. The product glucosamine-6-phosphate is rapidly further converted to uridine-5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc), the substrate for the nucleocytoplasmic enzyme O-GlcNAc transferase (OGT). The attachment of a single O-GlcNAc to serine or threonine residues of proteins is considered as a new regulatory modification important to signal transduction cascades [79]. The important function of the HBP in the initiation of the alteration of the glomerular matrix has been proven by inhibition of GFAT, which was shown to block hyperglycaemia-induced expression of TGF-^1 and PAI-I and subsequently the production of matrix proteins [80,81]. Moreover, overexpression of GFAT in mesangial cells enhances TGF-^1 gene activation and fibronectin accumulation [82]. Promoter-reporter gene-assays revealed that binding sites for the transcription factor Sp1 regulate the high glucose-induced promoter activation of PAI-I [55]. The mechanism by which increased flux through the HBP resulted in gene activation is not completely clarified, but the HBP-mediated, enhanced O-GlcNAc modification of Sp1 was suggested to activate its transcriptional activity [83] and DNA binding activity [84]. Thus, the promoter activity of Sp1-driven genes, e.g. PAI-I and TGF-^1, could be stimulated by the O-GlcNAc modification of this transcription factor.

Increased flux through the aldose reductase pathway The aldose reductase pathway involves intracellular formation of sorbitol from glucose catalysed by aldose reductase. Chronic hyperglycaemia leads to sorbitol accumulation in a variety of tissues such as peripheral neurons, lens and renal tubuli [85]. The initial hypothesis that sorbitol accumulation causes tissue damage is unlikely to operate in the kidney [86]. The inositol depletion theory suggested by Greene and coworkers explains tissue damages as impairment of myo-inositol uptake leading to a decrease of phosphatidyl-inositides in the cell membrane [85]. Although the cellular inositol uptake is competitively inhibited by D-glucose [87] and non-competitively inhibited by hyperosmolar intracellular sorbitol [88], recent studies showed that cells may counterregulate inositol depletion [89,90]. Thus, it is not generally agreed that the increase in intracellular sorbitol is the cause of the impaired function of the affected tissues in diabetes. Furthermore, after treatment of diabetic rats for six months with the aldose reductase inhibitor tolrestat only a slight reduction in the urinary albumin excretion rate was observed indicating that other mechanism are operating in diabetic nephropathy [91].

The role of oxidative stress

Many studies have shown that diabetes and hyperglycaemia increase oxidative stress, but it was not known if it is an important early mediator of the high glucose effects on renal structures and functions. A recent study showed that high glucose increased the production of superoxide by the mitochondrial electron-transport chain in endothelial cells [81,92]. Inhibition of the generation of these reactive oxygen species (ROS) prevented the activation of PKC pathways, the increased flux through the HBP and the aldose reductase pathway, and the enhanced synthesis of AGEs. Whether this interference would also block high glucose-induced derranged production of glomerular matrix production is currently under investigation. It was demonstrated that mesangial cells grown in ambient high glucose produced ROS [93], thus leading to activation of PKC, AP-1 and NF-kB, and the up-regulation of TGF-^1 and of matrix protein expression. The existence of glucose-induced oxidative stress in mesangial cells was also shown by decreased glutathione (GSH) and elevated malondialdehyde (MDA) levels [94]. Addition of antioxidants caused the restorage of GSH and a clear reversal of fibronectin and collagen IV expression.

A scheme of the pathways mentioned above is depicted in figure 1.

Glucose transporter-1 as a permissive factor

Glucose transport is rate-limiting for glucose metabolism and the main glucose transporter (GLUT) on mesangial cells, GLUT1, is a high-affinity, low-capacity transporter [95]. Thus, mesangial glucose uptake appears to be essentially determined by the number of GLUT1 on the cell surface rather than by ambient glucose concentrations [96]. Accordingly, experimental work has recognized the upregulation of mesangial GLUT1 expression by ambient high glucose and IGF-1 [97]. The hyperglycaemia-induced TGF-^1 [98] and, similiarly, angiotensin II stimulate GLUT1 expression [99]. Furthermore, recent studies demonstrated a significant increase in GLUT1 protein in the renal cortex of diabetic animals [100] and in mesangial cells isolated from diabetic subjects

Fig. 1 Proposed molecular mechanism of the hyperglycaemia-induced matrix synthesis in mesangial cells. Elevated glucose concentrations entering the cell via the glucose transporter 1 (GLUT 1) activates the production of reactive oxygen species (ROS), protein kinase C (PKC), generation of advanced glycated end-products (AGEs) and the hexosamine biosynthetic pathway (HBP). Subsequently induced mitogen-activated protein kinases (MAPK)-dependent pathways or increases in UDP-GlcNAc are leading to enhanced expression and/or phosphorylation of proteins of the activating protein 1 complex (AP-1) or activation of Sp1. Since the promoters of extracellular matrix (ECM) proteins and of cytokines contain AP-1 and Sp1 binding sites the expression of these genes is induced.

The essential role of elevated GLUT1 levels in the development of the pathological changes in diabetic nephropathy was proven by a mesangial cell model stably transfected with human GLUT1 (GT1) [102]. These cells showed a five fold increased glucose uptake, a 2.1-fold increase in lactate production, and enhanced expression and net deposition of matrix proteins, e.g. fibronectin

glucose glucose

Diabetic Nephropathy Pathogenesis

when cultured in physiological levels of extracellular glucose. In a recent report we provide evidence for the activation of different intracellular signaling pathways in GT1 cells cultured in normal glucose concentrations and mesangial cells exposed to high glucose concentrations [103]. Our investigation of the molecular mechanism of the enhanced fibronectin production in these cells has revealed a protein kinase C-dependent, activated AP-1-mediated pathway, which acts independently of TGF-P1. In contrast to mesangial cells cultured in ambient high glucose, no production of reactive oxygen species, no activation of the p38 or ERK1/2 MAPK pathways nor any increase in TGF-P1 synthesis could be detected.

Diabetes Sustenance

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