Diabetes is on the rise and diabetic kidney disease has quench nitric oxide ... difficult, and some times dangerous. ... preliminary reports, the activation of NF-kB by AGEs .... occurrence of lipid peroxidation in diabetes. ... be responsible for the excessive induction of TGF-b in antioxidants on mitigating dibetic renal injury are.
Nephrol Dial Transplant ( 1997) 12: 664–668
Nephrology Dialysis Transplantation
Invited Comment
Pathogenesis of diabetic nephropathy: a radical approach A. K. Salahudeen1, V. Kanji1, J. F. Reckelhoff 2 and A. M. Schmidt3 1Department of Medicine, 2Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS; and 3Department of Medicine and Surgery, College of Physicians and Surgeons of Columbia University, NY, USA
Introduction Diabetes is on the rise and diabetic kidney disease has emerged as a leading cause for end-stage renal failure in certain parts of the world [1 ]. Although data from the Diabetes Control and Complication Trial establish a central role for hyperglycaemia in diabetic complications [2], strict control of glucose in diabetics can be difficult, and some times dangerous. Understanding the distal pathway of glucose toxicity therefore has clinical significance. Although during chronic hyperglycaemia multiple glucose metabolites and reaction products accumulate, accruing evidence supports a key role for advanced glycation end-products (AGEs) in diabetic complications [3–10]. There are several theoretical reasons and experimental evidence that suggest an important role for AGEs and oxidative stress in the pathogenesis of diabetic nephropathy.
AGEs in diabetic nephropathy AGEs are formed from the non-enzymatic glycation/ oxidation not only of amino acids of proteins, but also of lipids and lipoproteins [11,12]. In patients with diabetic nephropathy, high levels of AGEs accumulate in the plasma, and importantly, within the sclerosing glomeruli [3,4]. The latter has been noted to precede the onset of diabetic renal disease, and correlate with the course of the disease [10]. In animals, chronic administration of advanced glycated albumin to nondiabetic rats led to proteinuria and glomerular changes similar to those seen in diabetic nephropathy, and these changes were associated with activation of collagen, laminin, and TGF-b genes [9,11]. Tissue accumulation of AGEs is associated with a number of toxic effects. These include cross-linking of long-lived proteins such as collagens and other matrix proteins, increasing of vascular permeability, and promotion of mononuclear cell influx [8,9]. Additionally AGEs have been shown to induce genes for growth Correspondence and offprint requests to: Abdulla K. Salahudeen MD FRCP, Renal Division, Department of Medicine, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216–4505, USA.
factors, extracellular matrix (ECM ) proteins and inflammatory cytokines, as well as to stimulate cell proliferation [8,9]. AGEs have also been shown to quench nitric oxide, and have been implicated in the pathogenesis of atherosclerosis [11,12]. The cellular and molecular mechanisms responsible for AGE-mediated cellular effects have not yet been fully defined. Recent studies suggest that a central pathway through which AGEs mediate their cellular effects may involve the interaction of AGEs with their cellular binding sites, the best characterized is the Receptor for AGE (RAGE ) [13,14].
AGE–RAGE interaction RAGE is expressed on a number of cell types that are relevant to the pathogenesis of diabetic complications. These include endothelial, mesangial and vascular smooth muscle cells and macrophages [15]. Based on preliminary reports, the activation of NF-kB by AGEs appears to be mediated through a signalling mechanism involving p21ras and MAP kinase [16 ]. AGE–RAGE interactions may be a potential pathway for the extracellular protein gene activation during diabetes based on the following observation. AGE albumin when perfused in situ interacted with vascular wall, via binding to RAGE. AGE–RAGE interaction was accompanied by activation of IL-6 and VCAM-1 genes [17,18]; the latter may explain the elevated plasma soluble VCAM-1 (sVCAM ) reported in human diabetics [19]. Such upregulation of VCAM-1 expression, in theory, can contribute to the diabetic vascular lesions, since VCAM-1 is a cell–cell recognition protein on the endothelial cell surface, which promotes interaction between circulating monocytes and endothelium. Since many effects of AGEs such as cross-linking of proteins, increased vascular permeability, induction of lipid peroxidation, quenching of nitric oxide, and acceleration of atherosclerosis can also be duplicated by the effects of free radicals, attention has recently been focused on examining whether AGE–RAGE interaction can indeed lead to increased free radical production.
© 1997 European Renal Association–European Dialysis and Transplant Association
Pathogenesis of diabetic nephropathy: a radical approach
AGE–RAGE interaction: a potential source for oxidative stress and lipid peroxidation In a recent study by Yan et al., short-term infusion of AGE albumin into normal mice led to the appearance of malondialdehyde determinants in the vessel wall, activation of the pleotropic transcription factor NF-kB and induction of haeme oxygenase mRNA [20]. Malondialdehyde is one of the end-products of lipid peroxidation, and regulation of both NF-kB and haeme oxygenase is sensitive to oxidative stress. Pretreatment of animals with either antibodies to the AGE receptor/binding proteins or antioxidants abrogated the above-mentioned vascular effects. In an in vitro study, the antioxidant N-acetylcysteine inhibited AGE–RAGE-mediated increased VCAM-1 transcription in cultured human endothelial cells [19]. Treatment of endothelial cells with AGE led to induction of specific DNA binding activity for NF-kB in the VCAM-1 promoter and this was blocked by N-acetylcysteine. These data collectively suggest that interaction of AGEs with cellular targets leads to oxidative stress. The resultant cellular genotypic and phenotypic changes in response to oxidative stress may potentially contribute to the development of vascular lesions in diabetes. Similarly free radical mechanisms, with emphasis on lipid peroxidation, are widely implicated in the pathogenesis of atherosclerosis [21 ]. In a series of experiments, Wautier et al. [22 ] extended the role of AGE–RAGE interaction and oxidative stress in the mediation of diabetic vascular hyperpermeability. Rats rendered diabetic with streptozotocin exhibited increased vascular permeability on week 9–11, as determined by tissue–blood isotope ratio. Increased vascular permeability was threefold higher in intestine, skin, and kidneys. Administration of sRAGE with an intention to saturate the AGEs, and therefore to achieve RAGE blockade was followed by complete inhibition of hyperpermeability in the skin and gut and 90% in the kidneys. In further experiments in this study employing antioxidants, a role for free radicals was demonstrated in AGE–RAGE-mediated vascular hyperpermeability. Membrane unsaturated fatty acids are susceptible to oxidative damage and the resultant lipid peroxidation can alter membrane fluidity and permeability.
Lipid peroxidation in diabetes Data from a large number of studies demonstrate the presence of enhanced lipid peroxidation in diabetic animals and humans [23–28]. A recent report that plasma levels of F -isoprostanes, specific markers for 2 lipid peroxidation [26 ], are elevated in diabetic patients is highly supportive of the presence of heightened lipid peroxidation in diabetes. There are a number of potential pathways for the occurrence of lipid peroxidation in diabetes. In in vitro studies, glucose had been shown to undergo oxidation, producing protein reactive ketoaldehydes, hydrogen
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peroxide and highly reactive oxidants [28]. Attachment of these reaction products, particularly reactive aldehydes, to proteins is purported to contribute to protein fragmentation and cross-linking in diabetes [28 ]. Advanced glycation of phospholipids and lipoproteins has also been reported to induce lipid peroxidation. In an in vitro study, oxidation of unsaturated fatty acid residues occurred readily during the formation of advanced glycation end products of phospholipids [29]. Aminoguanidine inhibited the advanced glycation of lipids with attendant suppression of lipid peroxidation. Furthermore, incubation of low-density lipoprotein ( LDL) with glucose produced AGE moieties that were attached to both the lipid and the apoprotein components, and oxidized LDL formed concomitantly with AGE-modified LDL. That AGE oxidation may play a role in initiating lipid oxidation in vivo is suggested by the finding that LDL from diabetics revealed increased levels of both apoprotein- and lipidlinked AGEs. Moreover, circulating levels of oxidized LDL were elevated in diabetics and this correlated with lipid AGE levels [30]. Glucose has been shown to activate the glomerular protein kinase C ( PKC ) signalling system that enhances phospholipase A activity [31,32]. The 2 increased free arachidonic acid released by phospholipase A can fuel the enzymatic production of prosta2 glandins (PGs). Since PG synthesis is a well-recognized source for free radicals, the increased PG synthesis, that is known to occur in early stages of diabetes, could also contribute to concomitant occurrence of lipid peroxidation. Lipid peroxides, a product of lipid peroxidation, can in turn further catalyse PG production. Recent studies have identified novel groups of prostaglandins (F -isoprostanes) formed by free 2 radical-catalysed lipid peroxidation process. These are notably formed independent of cyclo-oxygenase enzymatic pathway [33,34] and were intensely vasoconstrictive when infused at nanomolar concentration into rat kidneys [35 ]. In diabetes, thus, prostanoid synthesis and lipid peroxidation can in theory enter into a self perpetuating cycle.
Potential consequences of lipid peroxidation in diabetes In addition to causing cell damage [36 ], the products of peroxidation such as lipid peroxides, hydroxynonenals, ketoaldehydes, and F -isoprostanes participate 2 further in the disease processes. Impairment of endothelium-dependent relaxation as well as increased release of vasoconstrictor prostanoids in arteries from animals and humans with long-standing diabetes have been reported [32]. This impairment is restored towards normal by prostaglandin PGH /TXA recep2 2 tor blockade or superoxide dismutase, indicating that the PGH and/or free radicals generated contributes 2 to the abnormality. In the kidney, vasodilatory prostaglandins are increased more than the vasoconstrictive TXA and this may partly account for the renal 2
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vasodilatation seen in the early stages of diabetes [32]. The elevated F -isoprostanes noted in the plasma of 2 diabetics is in agreement with the view that free radical generation and prostanoid pathways may be linked in the pathogenesis of diabetic vascular dysfunction [26 ]. Lipid peroxidation may also play a potential role in diabetic glomerulosclerosis. The initial hyperfiltering phase of diabetic kidney disease, heralded by the appearance of microalbuminuria, is followed by gross proteinuria, progressive reduction in GFR and excessive accumulation of ECM proteins in the glomerular capillaries and mesangium. The latter process leads the way to eventual glomerulosclerosis and renal failure. The following studies suggest that there may be a link between oxidative stress and ECM protein accumulation. In a fibroblast cell culture study, vitamin E prevented the ascorbic-acid-induced lipid-peroxidation-mediated activation of collagen genes at the transcriptional level [37 ], and more relevant to the diabetic kidney disease, vitamin E inhibited high glucoseinduced excessive collagen production by mesangial cells in culture [38]. Recent in vivo studies also provide support for a potential link between lipid peroxidation and renal fibrosis. Antioxidant therapy concurrent with cyclosporin A (CsA) administration in the rats resulted in the inhibition of CsA-induced renal and plasma lipid peroxidation and a striking reduction in CsAinduced chronic tubulointerstitial fibrosis and renal dysfunction [39 ]. Conversely, antioxidant deficiency, which exposes tissue to free radical injury and lipid peroxidation, leads to marked tubulointerstitial fibrosis of the kidney [40 ]. Although in diabetes, sclerosis is prominent in the glomeruli rather than tubulointerstitium, the underlying mechanism leading to excessive ECM proteins may still operate. Recent studies associate excessive growth factor TGF-b with increased ECM accumulation in diabetic kidneys, and speculatively, free radicals-induced cellular effects may partly be responsible for the excessive induction of TGF-b in diabetes [41,42]. All diabetic complications, including nephropathy, are essentially the manifestation of diabetic vascular injury. The early increased vascular permeability is later associated with progressive atherosclerosis. AGE–RAGE interaction and accompanying oxidative stress may account for the early vascular permeability. The combination of hyperlipidaemia and the presence of oxidative stress in diabetes can accelerate atherosclerosis through increased LDL oxidation. AGEs are also implicated in the large-vessel atherosclerosis through their ability to induce LDL oxidation [30]. Since LDL oxidation and atherosclerosis can be suppressed by vitamin E therapy, this has become an additional consideration to test vitamin E as a therapy to mitigate complications in diabetes. Interestingly, reduction in AGE levels with aminoguanidine administration in the diabetic human has also been associated with a reduction in plasma lipid abnormalities [43 ]. Based on the foregoing discussion, the following sequence of events can be considered to occur in the pathogenesis of diabetic nephropathy ( Figure 1).
A. K. Salahudeen et al.
Hyperglycaemia leads to increased formation/accumulation of AGEs in the kidney. AGE–RAGE interaction, at least in part via enhanced free radical mechanisms, induces membrane peroxidation, which in turn increases membrane permeability. Activation of PLA through PKC leads to increased production 2 of predominantly vasodilatory prostaglandins. Increased glomerular permeability and increased glomerular blood flow and pressure initiate and maintain the early proteinuria and glomerular hyperfiltration. AGE–RAGE interaction mainly through the free radical-sensitive pleotropic NF-kB pathway may chronically induces several genes including TGF-b responsible for the production of extracellular matrix proteins, resulting in unregulated production and deposition of ECM in the kidney. Interestingly, TGF-b has also been shown to reduce the activity of proteinases that hydrolyse ECM proteins [44,45]. The AGE and reactive aldehyde-mediated cross linking of ECM proteins further compounds the problem of excessive ECM accumulation by making the protein less susceptible to degradation. Excessive accumulation of ECM proteins begins to interfere with the function of the kidney. In the face of uncontrolled hyperglycaemia, AGEs continue to form/accumulate. Free radical production is thus exacerbated, leading to enhanced lipid peroxidation which by now is fuelled by increasing hyperlipidaemia. F -isoprostanes in higher levels join forces with 2 TXA , turning the initial renal vasodilatation into 2 progressive vasoconstriction and contribute to the pathogenesis of systemic hypertension. Reduced blood flow and continued unruly deposition of ECM proteins in the glomeruli lead the way to glomerulosclerosis and renal failure. Although, as discussed, accruing evidence would suggest a role for AGEs and oxidative stress in diabetic nephropathy, the number of detailed experimental studies examining the effects of AGE inhibitors or antioxidants on mitigating dibetic renal injury are limited. In the streptozotocin-induced diabetic model, administration of aminoguanidine has been shown to reduce retinopathy and neuropathy [46,47]. Administration of aminoguanidine from the initiation of diabetes in this model, but not 16 weeks after the onset of diabetes, was associated with significant reduction in albuminuria [48]. This suggests that suppression of AGE formation subsequent to the initiation of renal injury may not be sufficient to reverse the injury process, and that other factors such as abnormal renal prostaglandin production and haemodynamic alterations may also contribute to the development of diabetic nephropathy. Answers to the critical question of whether aminoguanidine will prevent or attenuate diabetes-induced renal failure are also unavailable at the present time. In another recent study, chronic dietary administration of vitamin E to streptozotocin-induced diabetic rats was attended by higher mortality [49 ]. A small number of animals survived at the end of the study precluding any firm conclusion on the findings of worsening of diabetic nephropathy in the vitamin E group. In contrast, in a preliminary report, administra-
Pathogenesis of diabetic nephropathy: a radical approach
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Fig. 1
tion of a combination of vitamins E with C in rats was associated with reduced diabetes-induced proteinuria [50 ]. Since recycling of tocopheryl radical to tocopherol is catalysed by vitamin C, provision of excess vitamin C with E has been suggested to reduce the potential for vitamin E to become a pro-oxidant. Thus, the limited number of experimental studies examining the efficacy of these potentially useful agents raise more questions than answers at the present time. Yet, interestingly, clinical studies are already under way with vitamin E or aminoguanidine to test their therapeutic efficacy on diabetic complications [51]. Acknowledgements. Part of this work is supported by a grant from The Kidney Care Inc. Foundation of Jackson, Mississippi.
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