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Pathogenesis of diabetic nephropathy: the role of oxidative stress and protein kinase C
Diabetes Research and Clinical Practice 45 (1999) 147 – 151 www.elsevier.com/locate/diabres
Pathogenesis of diabetic nephropathy: the role of oxidative stress and protein kinase C Hunjoo Ha *, Kyung Hwan Kim Department of Pharmacology, Yonsei Uni6ersity College of Medicine, 134 Sinchon-dong, Seodaemun-ku, Seoul 120 -752, South Korea
Abstract Hyperglycemia, a well recognized pathogenetic factor of long-term complications in diabetes mellitus, not only generates more reactive oxygen species but also attenuates antioxidative mechanisms through glycation of the scavenging enzymes. Therefore, oxidative stress has been considered to be a common pathogenetic factor of the diabetic complications including nephropathy. A causal relationship between oxidative stress and diabetic nephropathy has been established by observations that (1) lipid peroxides and 8-hydroxydeoxyguanosine, indices of oxidative tissue injury, were increased in the kidneys of diabetic rats with albuminuria; (2) high glucose directly increases oxidative stress in glomerular mesangial cells, a target cell of diabetic nephropathy; (3) oxidative stress induces mRNA expression of TGF-b1 and fibronectin which are the genes implicated in diabetic glomerular injury, and (4) inhibition of oxidative stress ameliorates all the manifestations associated with diabetic nephropathy. Proposed mechanisms involved in oxidative stress associated with hyperglycemia are glucose autooxidation, the formation of advanced glycosylation end products, and metabolic stress resulting from hyperglycemia. Since the inhibition of protein kinase C (PKC) effectively blocks not only phorbol ester-induced but also high glucose- and H2O2-induced fibronectin production, the activation of PKC under diabetic conditions may also have a modulatory role in oxidative stress-induced renal injury in diabetes mellitus. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Diabetic nephropathy; Extracellular matrix; High glucose; Oxidative stress; Protein kinase C
1. Introduction Tight glycemic control delays the onset [1,2] and slows the progression [2] of microvascular complications, including nephropathy in patients with insulin-dependent diabetes mellitus and those found in diabetic animals [3 – 6]. High glucose is * Corresponding author. Tel.: +82-2-3615233; fax: +82-23131894. E-mail address: [email protected] (H. Ha)
the main determinant of the initiation and progression of diabetic nephropathy. However, strict control of blood glucose in diabetics is difficult and sometimes dangerous. Therefore, an understanding of the distal pathways involved in high glucose-induced renal injury may have clinical significance. Of the several biochemical abnormalities that have been proposed as pathogenic mechanisms of diabetic complications [7–11], this review will focus only on the role of oxidative stress in renal injury found in diabetes mellitus.
0168-8227/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 8 2 2 7 ( 9 9 ) 0 0 0 4 4 - 3
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H. Ha, K.H. Kim / Diabetes Research and Clinical Practice 45 (1999) 147–151
2. Oxidative stress and renal injury The global term reactive oxygen species (ROS) includes both oxygen radicals such as superoxide (O2 − ), alkoxyl (RO), peroxyl (ROO), and hydroxyl radicals (OH), plus non-radical derivatives of oxygen, namely, hydrogen peroxide (H2O2) and ozone (O3). ROS is continuously generated in physiological conditions and effectively eliminated by several intracellular and extracellular antioxidative systems. When the generation of ROS exceeds cellular defence power, these unstable ROS will interact with essential biological cellular macromolecules such as lipids, proteins, and DNA which leads to histologic changes as well as functional abnormalities. The shift of balance between prooxidant and antioxidant activity in favor of the former, which results in potential damage, is defined as oxidative stress [12]. Numerous studies [13 – 17] on experimental models of both immune and nonimmune glomerular injury demonstrated ROS to be primary mediators in the pathogenesis of these disorders and showed that the kidney is, in fact, susceptible to oxidative stress.
3. Hyperglycemia-induced oxidative stress Hyperglycemia, a well recognized pathogenetic factor of long-term complications in diabetes mellitus [1,2], not only generates more ROS but also attenuates antioxidative mechanisms through glycation of the scavenging enzymes. Therefore, oxidative stress has been considered to be a common pathogenic factor of diabetic complications [11,18,19] including nephropathy [20]. In order to establish a role for oxidative stress in diabetic nephropathy, it has to be demonstrated that (1) oxidative stress is increased in the diabetic kidney preferably before clinical signs of nephropathy, (2) high glucose can increase oxidative stress in target cells in vitro, (3) oxidative stress has a relevant effect on target cells in vivo and in vitro, and (4) inhibition of oxidative stress in vivo as well as in vitro blocks the manifestations of the disease. As will be discussed, these criteria have been fulfilled.
Because a direct quantification of ROS generated in tissue is difficult due to their extremely short half-lives and high reactivity (10 − 9 s for the hydroxyl radical to 1–10 s for peroxyl radicals or nitric oxide), the formation of endogenous endproducts of ROS has been measured to address the involvement of oxidative stress in a given pathologic state, as a rule. Lipid peroxidation of unsaturated fatty acids, one of the radical reactions in vivo, has been an index of increased oxidative stress and subsequent cytotoxicity [21]. Streptozotocin-induced diabetic rats exhibiting albuminuria, a marker of glomerular injury in diabetes [22], had significantly higher levels of lipid peroxides in plasma, urine, and renal proximal tubules [23], suggesting increased oxidative stress in diabetic kidneys. Yet, current data on the level of lipid peroxides in diabetic kidneys [24,25] are equivocal. This might result from inherent complexity in in vivo studies, such as the diabetic state under experimental conditions, or limitations of lipid peroxides as an index of oxidative tissue damage [26,27]. More recently, 8-hydroxydeoxyguanosine (8OHdG), which is an oxidized purine residue in DNA, has been accepted as an excellent marker of oxidative tissue damage [28]. Formation of 8-OHdG was also increased in the kidney, but not in the pancreas or the liver of diabetic rats [29]. This selective oxidative DNA damage provided additional evidence of the involvement of oxidative stress in diabetic nephropathy. Possible alterations in the repair system for 8-OHdG, in addition to an increased production of ROS, might play a role in such oxidative DNA damage in the kidneys of diabetic rats. Typical lesions of the diabetic kidney involve the glomeruli and include capillary basement membrane thickening with expansion of the mesangium, resulting in diffuse and nodular diabetic glomerulosclerosis. Among the structural lesions, expansion of the glomerular mesangium appears to be the one most closely associated with loss of filtration function [30]. Since high levels of glucose stimulate fibronectin laminin, and type IV collagen synthesis and mRNA expression in mesangial cell cultures [31,32], a mesangial cell cultured under high levels of glucose is accepted
H. Ha, K.H. Kim / Diabetes Research and Clinical Practice 45 (1999) 147–151
as an in vitro model for diabetic nephropathy. Glucose produced a dose-dependent increase in lipid peroxidation in cultured mesangial cells [33 – 35], which is highly supportive of the presence of increased lipid peroxidation in diabetic glomeruli. Lack of the effects of either L-glucose or mannitol on lipid peroxidation of mesangial cells suggests that high glucose-induced lipid peroxidation in these tissue is not related to an effect of high osmolarity of the media per se. Hyperglycemia-induced oxidative stress plays a major role in extracellular matrix expansion, since high glucose-induced collagen production in cultured rat mesangial cells was effectively prevented by two antioxidants, taurine [33] and vitamin E [34]. We also demonstrated that hydroxyl radical scavengers at concentrations which inhibited high glucose-induced lipid peroxidation, suppressed TGF-b1 and fibronectin mRNA expression and protein synthesis by mesangial cells cultured under high levels of glucose [35]. TGF-b is a final common mediator of the principal lesions of renal disease in diabetes mellitus such as renal/glomerular hypertrophy and extracellular matrix expansion [36]. Hydrogen peroxide increased TGF-b1 and fibronectin production in mesangial cells [37], further suggesting an important role of oxidative stress in the expansion of extracellular matrix seen in diabetic nephropathy. The protective effect which antioxidants have on renal injury in diabetic animals has been recently reported [38– 41]. Taurine [38] and vitamin C [40] effectively reduced glomerular hypertrophy, albuminuria, glomerular collagen, and TGF-b1 accumulation in rats with streptozotocin-induced diabetes. We [41] also observed the protective effect of two antioxidants, taurine and melatonin, on glomerular TGF-b1 mRNA expression and proteinuria in diabetic rats. Administration of vitamin E prevented glomerular hyperfiltration [39], albuminuria [39], renal PKC activity [39], glomerular hypertrophy [40], and TGF-b1 accumulation [40] in the early phase of glomerular injury in rats with streptozotocin-induced diabetes. However, studies of chronic treatment with vitamin E are clearly needed since chronic dietary administration of vitamin E to diabetic rats was attended by higher mortality [38].
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4. Mechanisms involved in high glucose-induced oxidative stress One of the major biochemical pathways inducing diabetic nephropathy is the activation of diacylglycerol (DAG)-PKC [10]. PKC is activated in the glomeruli of diabetic rats [42] and glomeruli cultured under high levels of glucose [43] as a result of de novo synthesis of DAG. Activation of PKC has a modulatory role in oxidative stress-induced glomerular injury [44–46]. We observed phorbol ester, a PKC activator, increased TGF-b1 and fibronectin mRNA expression by mesangial cells in a preliminary study [37]. Furthermore, inhibition of PKC activity effectively blocked high glucose- and H2O2-induced TGF-b1 and fibronectin mRNA expression as well as phorbol ester-induced TGF-b1 and fibronectin mRNA expression in mesangial cells. This study clearly demonstrated the relationship between PKC and oxidative stress under hyperglycemia. While further studies are necessary to gain a better understanding of the precise role of glucose autooxidation, the formation of advanced glycosylation end products (AGE), and metabolic stress resulting from oxidative stress associated with hyperglycemia, AGE is accepted as an independent risk factor for diabetic nephropathy [8,47,48]. AGE interaction with its cognate receptors is also a potential source for oxidative stress in diabetes [49,50], and AGE-induced oxidative stress has been proposed to play a major role in the development and progression of diabetic nephropathy [51]. Since glycated low density lipoprotein-induced fibronectin mRNA expression and protein synthesis by mesangial cells is, in part, mediated by PKC activation [52], PKC may also have a modulatory role in oxidative stress involved in AGE-induced extracellular expansion. Thus, it can be summarized that oxidative stress associated with hyperglycemia plays an important part in the development and progression of diabetic nephropathy. Understanding the mechanisms involved in oxidative stress associated with hyperglycemia should help delineate the pathogenesis of diabetic nephropathy further.
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Acknowledgements This work was supported by Korea Science and Engineering Foundation (KOSEF) Grant 940403-07-01-3. We are grateful to Professor Hi Bahl Lee, Hyonam Kidney Laboratory, Soon Chun Hyang University, for his continued interest in our work and constructive criticism. References [1] P. Reichard, B.Y. Nilsson, U. Roseuqvist, The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus, New Engl. J. Med. 329 (1993) 304–309. [2] The Diabetes Control and Complications Trial Research Group, The effect of intensive treatment of diabetes on the development and progression of long-term complication in insulin-dependent diabetes mellitus, New Engl. J. Med. 329 (1993) 977–986. [3] S. Stackhous, P.L. Miller, S.K. Park, T.W. Meyer, Reversal of glomerular hyperfiltration and renal hypertrophy by blood glucose normalization in diabetic rats, Diabetes 39 (1990) 985 – 995. [4] M. Fukui, T. Nakamura, I. Ebihara, I. Shirato, Y. Tomino, H. Koide, ECM gene expression and its modulation by insulin in diabetic rats, Diabetes 41 (1992) 1520– 1527. [5] D.C. Han, Y.J. Kim, M.K. Cha, et al., Glucose control suppressed the glomerular expression of TGF-b1 and the progression of experimental diabetic nephropathy (abstract), J. Am. Soc. Nephrol. 7 (1996) 1870. [6] H. Grønbaek, I. Vogel, R. Østerby, I. Lancranjan, A. Flyvbjerg, H. Ørskov, Effect of octreotide, captopril or insulin on renal change and UAE in long-term experimental diabetes, Kidney Int. 53 (1998) 173–180. [7] D. Greene, S.A. Lattimer, A.A.F. Sima, Sorbitol, phosphoinositides and sodium-potassium-ATPase in the pathogenesis of diabetic complications, New Engl. J. Med. 316 (1987) 599–606. [8] M. Brownlee, A. Cerami, H. Vlassara, Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications, New Engl. J. Med. 318 (1988) 1315 – 1321. [9] J.R. Williamson, K. Chang, M. Frangos, et al., Hyperglycemic pseudohypoxia and diabetic complications, Diabetes 42 (1993) 801 –813. [10] G.L. King, H. Ishii, D. Koya, Diabetic vascular dysfunctions: A model of excessive activation of protein kinase C, Kidney Int. 52 (Suppl. 60) (1997) S77–S85. [11] J.W. Baynes, Role of oxidative stress in development of complications in diabetes, Diabetes 40 (1991) 405–412. [12] H. Sies, Oxidative stress: Introduction, in: H. Sies (Ed.), Oxidative Stress; Oxidants and Antioxidants, Academic Press, London, 1991, pp. xv–xxii.
[13] L. Baud, L. Ardaillou, Reactive oxygen species: Production and role in the kidney, Am. J. Physiol. 251 (1986) F765 – F776. [14] S.V. Shah, Role of reactive oxygen metabolites in experimental glomerular disease, Kidney Int. 35 (1989) 1093 – 1106. [15] J.R. Diamond, The role of reactive oxygen metabolites in animal models of glomerular disease, Am. J. Kidney Dis. 19 (1992) 292 – 300. [16] K.A. Nath, M. Fischereder, T.H. Hostetter, The role of oxidants in progressive renal injury, Kidney Int. 45 (Suppl. 45) (1994) S111 – S115. [17] K.A. Nath, A.K. Salahudeen, Introduction of renal growth and injury in the intact rat kidney by dietary deficiency of antioxidants, J. Clin. Invest. 86 (1990) 1179 – 1192. [18] S.P. Wolff, Z.Y. Jiang, J.V. Hunt, Protein glycation and oxidative stress in diabetes mellitus and aging, Free Radic. Biol. Med. 10 (1991) 339 – 352. [19] S.P. Wolff, Diabetes mellitus and free radicals, Br. Med. Bull. 49 (1993) 642 – 652. [20] R.G. Larkins, M.E. Dunlop, The link between hyperglycemia and diabetic nephropathy, Diabetologia 35 (1992) 499 – 504. [21] N. Haugarrd, Cellular mechanisms of oxygen toxicity, Physiol. Rev. 48 (1968) 311 – 373. [22] C.E. Morgensen, Natural history of renal function abnormalities in human diabetes mellitus: from normoalbuminuria to incipient and overt nephropathy, in: B.M. Brenner, J.H. Stein (Eds.), The Kidney in Diabetes Mellitus, Churchill Livingstone, New York, 1989, pp. 19 – 49. [23] H. Ha, K.H. Kim, Role of oxidative stress in the development of diabetic nephropathy, Kidney Int. 48 (Suppl. 51) (1995) S18 – S21. [24] K. Asayama, S. Yokota, K. Kato, Peroxisomal oxidases in various tissues of diabetic rats, Diabetes Res. Clin. Pract. 11 (1991) 89 – 94. [25] N.L. Parinandi, E.W. Thompson, H.H.O. Schmid, Diabetic heart and kidney exhibit increased resistance to lipid peroxidation, Biochim. Biophys. Acta 1047 (1990) 63 – 69. [26] J.M.C. Gutteridge, B. Halliwell, The measurement and mechanism of lipid peroxidation in biological system, Trends Biol. Sci. 15 (1990) 129 – 135. [27] D.R. Janero, Malondialdehyde and thiobarbituric acidreactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury, Free Radic. Biol. Med. 9 (1990) 515 – 540. [28] H. Kasai, S. Nishimura, Y. Kurokawa, Y. Hayashi, Oral administration of the renal carcinogen, potassium bromate, specifically produces 8-hydroxydeoxyguanosine in rat target organ DNA, Carcinogenesis 8 (1987) 1959 – 1961. [29] H. Ha, C. Kim, Y. Son, M.H. Chung, K.H. Kim, DNA damage in the kidneys of diabetic rats exhibiting microalbuminuria, Free Radic. Biol. Med. 16 (1994) 271 – 274. [30] M.W. Steffes, R. Osterby, B. Chavers, S.M. Mauer, Mesangial expansion as a central mechanism for loss of
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Pathogenesis of diabetic nephropathy: the role of oxidative stress and protein kinase C Hunjoo Ha *, Kyung Hwan Kim Department of Pharmacology, Yonsei Uni6ersity College of Medicine, 134 Sinchon-dong, Seodaemun-ku, Seoul 120 -752, South Korea
Abstract Hyperglycemia, a well recognized pathogenetic factor of long-term complications in diabetes mellitus, not only generates more reactive oxygen species but also attenuates antioxidative mechanisms through glycation of the scavenging enzymes. Therefore, oxidative stress has been considered to be a common pathogenetic factor of the diabetic complications including nephropathy. A causal relationship between oxidative stress and diabetic nephropathy has been established by observations that (1) lipid peroxides and 8-hydroxydeoxyguanosine, indices of oxidative tissue injury, were increased in the kidneys of diabetic rats with albuminuria; (2) high glucose directly increases oxidative stress in glomerular mesangial cells, a target cell of diabetic nephropathy; (3) oxidative stress induces mRNA expression of TGF-b1 and fibronectin which are the genes implicated in diabetic glomerular injury, and (4) inhibition of oxidative stress ameliorates all the manifestations associated with diabetic nephropathy. Proposed mechanisms involved in oxidative stress associated with hyperglycemia are glucose autooxidation, the formation of advanced glycosylation end products, and metabolic stress resulting from hyperglycemia. Since the inhibition of protein kinase C (PKC) effectively blocks not only phorbol ester-induced but also high glucose- and H2O2-induced fibronectin production, the activation of PKC under diabetic conditions may also have a modulatory role in oxidative stress-induced renal injury in diabetes mellitus. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Diabetic nephropathy; Extracellular matrix; High glucose; Oxidative stress; Protein kinase C
1. Introduction Tight glycemic control delays the onset [1,2] and slows the progression [2] of microvascular complications, including nephropathy in patients with insulin-dependent diabetes mellitus and those found in diabetic animals [3 – 6]. High glucose is * Corresponding author. Tel.: +82-2-3615233; fax: +82-23131894. E-mail address: [email protected] (H. Ha)
the main determinant of the initiation and progression of diabetic nephropathy. However, strict control of blood glucose in diabetics is difficult and sometimes dangerous. Therefore, an understanding of the distal pathways involved in high glucose-induced renal injury may have clinical significance. Of the several biochemical abnormalities that have been proposed as pathogenic mechanisms of diabetic complications [7–11], this review will focus only on the role of oxidative stress in renal injury found in diabetes mellitus.
0168-8227/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 8 2 2 7 ( 9 9 ) 0 0 0 4 4 - 3
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2. Oxidative stress and renal injury The global term reactive oxygen species (ROS) includes both oxygen radicals such as superoxide (O2 − ), alkoxyl (RO), peroxyl (ROO), and hydroxyl radicals (OH), plus non-radical derivatives of oxygen, namely, hydrogen peroxide (H2O2) and ozone (O3). ROS is continuously generated in physiological conditions and effectively eliminated by several intracellular and extracellular antioxidative systems. When the generation of ROS exceeds cellular defence power, these unstable ROS will interact with essential biological cellular macromolecules such as lipids, proteins, and DNA which leads to histologic changes as well as functional abnormalities. The shift of balance between prooxidant and antioxidant activity in favor of the former, which results in potential damage, is defined as oxidative stress [12]. Numerous studies [13 – 17] on experimental models of both immune and nonimmune glomerular injury demonstrated ROS to be primary mediators in the pathogenesis of these disorders and showed that the kidney is, in fact, susceptible to oxidative stress.
3. Hyperglycemia-induced oxidative stress Hyperglycemia, a well recognized pathogenetic factor of long-term complications in diabetes mellitus [1,2], not only generates more ROS but also attenuates antioxidative mechanisms through glycation of the scavenging enzymes. Therefore, oxidative stress has been considered to be a common pathogenic factor of diabetic complications [11,18,19] including nephropathy [20]. In order to establish a role for oxidative stress in diabetic nephropathy, it has to be demonstrated that (1) oxidative stress is increased in the diabetic kidney preferably before clinical signs of nephropathy, (2) high glucose can increase oxidative stress in target cells in vitro, (3) oxidative stress has a relevant effect on target cells in vivo and in vitro, and (4) inhibition of oxidative stress in vivo as well as in vitro blocks the manifestations of the disease. As will be discussed, these criteria have been fulfilled.
Because a direct quantification of ROS generated in tissue is difficult due to their extremely short half-lives and high reactivity (10 − 9 s for the hydroxyl radical to 1–10 s for peroxyl radicals or nitric oxide), the formation of endogenous endproducts of ROS has been measured to address the involvement of oxidative stress in a given pathologic state, as a rule. Lipid peroxidation of unsaturated fatty acids, one of the radical reactions in vivo, has been an index of increased oxidative stress and subsequent cytotoxicity [21]. Streptozotocin-induced diabetic rats exhibiting albuminuria, a marker of glomerular injury in diabetes [22], had significantly higher levels of lipid peroxides in plasma, urine, and renal proximal tubules [23], suggesting increased oxidative stress in diabetic kidneys. Yet, current data on the level of lipid peroxides in diabetic kidneys [24,25] are equivocal. This might result from inherent complexity in in vivo studies, such as the diabetic state under experimental conditions, or limitations of lipid peroxides as an index of oxidative tissue damage [26,27]. More recently, 8-hydroxydeoxyguanosine (8OHdG), which is an oxidized purine residue in DNA, has been accepted as an excellent marker of oxidative tissue damage [28]. Formation of 8-OHdG was also increased in the kidney, but not in the pancreas or the liver of diabetic rats [29]. This selective oxidative DNA damage provided additional evidence of the involvement of oxidative stress in diabetic nephropathy. Possible alterations in the repair system for 8-OHdG, in addition to an increased production of ROS, might play a role in such oxidative DNA damage in the kidneys of diabetic rats. Typical lesions of the diabetic kidney involve the glomeruli and include capillary basement membrane thickening with expansion of the mesangium, resulting in diffuse and nodular diabetic glomerulosclerosis. Among the structural lesions, expansion of the glomerular mesangium appears to be the one most closely associated with loss of filtration function [30]. Since high levels of glucose stimulate fibronectin laminin, and type IV collagen synthesis and mRNA expression in mesangial cell cultures [31,32], a mesangial cell cultured under high levels of glucose is accepted
H. Ha, K.H. Kim / Diabetes Research and Clinical Practice 45 (1999) 147–151
as an in vitro model for diabetic nephropathy. Glucose produced a dose-dependent increase in lipid peroxidation in cultured mesangial cells [33 – 35], which is highly supportive of the presence of increased lipid peroxidation in diabetic glomeruli. Lack of the effects of either L-glucose or mannitol on lipid peroxidation of mesangial cells suggests that high glucose-induced lipid peroxidation in these tissue is not related to an effect of high osmolarity of the media per se. Hyperglycemia-induced oxidative stress plays a major role in extracellular matrix expansion, since high glucose-induced collagen production in cultured rat mesangial cells was effectively prevented by two antioxidants, taurine [33] and vitamin E [34]. We also demonstrated that hydroxyl radical scavengers at concentrations which inhibited high glucose-induced lipid peroxidation, suppressed TGF-b1 and fibronectin mRNA expression and protein synthesis by mesangial cells cultured under high levels of glucose [35]. TGF-b is a final common mediator of the principal lesions of renal disease in diabetes mellitus such as renal/glomerular hypertrophy and extracellular matrix expansion [36]. Hydrogen peroxide increased TGF-b1 and fibronectin production in mesangial cells [37], further suggesting an important role of oxidative stress in the expansion of extracellular matrix seen in diabetic nephropathy. The protective effect which antioxidants have on renal injury in diabetic animals has been recently reported [38– 41]. Taurine [38] and vitamin C [40] effectively reduced glomerular hypertrophy, albuminuria, glomerular collagen, and TGF-b1 accumulation in rats with streptozotocin-induced diabetes. We [41] also observed the protective effect of two antioxidants, taurine and melatonin, on glomerular TGF-b1 mRNA expression and proteinuria in diabetic rats. Administration of vitamin E prevented glomerular hyperfiltration [39], albuminuria [39], renal PKC activity [39], glomerular hypertrophy [40], and TGF-b1 accumulation [40] in the early phase of glomerular injury in rats with streptozotocin-induced diabetes. However, studies of chronic treatment with vitamin E are clearly needed since chronic dietary administration of vitamin E to diabetic rats was attended by higher mortality [38].
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4. Mechanisms involved in high glucose-induced oxidative stress One of the major biochemical pathways inducing diabetic nephropathy is the activation of diacylglycerol (DAG)-PKC [10]. PKC is activated in the glomeruli of diabetic rats [42] and glomeruli cultured under high levels of glucose [43] as a result of de novo synthesis of DAG. Activation of PKC has a modulatory role in oxidative stress-induced glomerular injury [44–46]. We observed phorbol ester, a PKC activator, increased TGF-b1 and fibronectin mRNA expression by mesangial cells in a preliminary study [37]. Furthermore, inhibition of PKC activity effectively blocked high glucose- and H2O2-induced TGF-b1 and fibronectin mRNA expression as well as phorbol ester-induced TGF-b1 and fibronectin mRNA expression in mesangial cells. This study clearly demonstrated the relationship between PKC and oxidative stress under hyperglycemia. While further studies are necessary to gain a better understanding of the precise role of glucose autooxidation, the formation of advanced glycosylation end products (AGE), and metabolic stress resulting from oxidative stress associated with hyperglycemia, AGE is accepted as an independent risk factor for diabetic nephropathy [8,47,48]. AGE interaction with its cognate receptors is also a potential source for oxidative stress in diabetes [49,50], and AGE-induced oxidative stress has been proposed to play a major role in the development and progression of diabetic nephropathy [51]. Since glycated low density lipoprotein-induced fibronectin mRNA expression and protein synthesis by mesangial cells is, in part, mediated by PKC activation [52], PKC may also have a modulatory role in oxidative stress involved in AGE-induced extracellular expansion. Thus, it can be summarized that oxidative stress associated with hyperglycemia plays an important part in the development and progression of diabetic nephropathy. Understanding the mechanisms involved in oxidative stress associated with hyperglycemia should help delineate the pathogenesis of diabetic nephropathy further.
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