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Reversal of hyperglycemia and diabetic nephropathy
Journal of Diabetes and Its Complications 18 (2004) 282 – 288
Reversal of hyperglycemia and diabetic nephropathy Effect of reinstitution of good metabolic control on oxidative stress in the kidney of diabetic rats Renu A. Kowluru *, Saiyeda N. Abbas, Sarah Odenbach Kresge Eye Institute, Wayne State University, Detroit, MI 48201, USA Received 8 December 2003; received in revised form 11 February 2004; accepted 3 March 2004
Abstract Studies have shown that good metabolic control (GC) is beneficial in slowing the progression of nephropathy in diabetes, and if the duration of poor metabolic control (PC) is prolonged before reinstitution of GC, nephropathy is not easily reversed. This study is to identify the biochemical abnormalities that could contribute to the resistance of nephropathy to reverse after establishment of GC in rats. The effect of reinstitution of GC and its duration is evaluated on oxidative stress and nitric oxide (NO) levels in the renal cortex and urine of diabetic rats. The rats were maintained in GC (5% glycated hemoglobin, GHb) soon after or 6 months after induction of hyperglycemia, and were sacrificed 13 months after induction of diabetes. For rats in which GC was initiated soon after induction of diabetes, oxidative stress [as measured by the levels of lipid peroxides (LPOs), 8-hydroxy-2V-deoxyguanosine (8-OHdG), and reduced glutathione (GSH)] and NO in urine and renal cortex were not different from that observed in normal control rats, but when reinstitution of GC was delayed for 6 months after induction of diabetes, oxidative stress and NO remain elevated in both urine and renal cortex. This suggests that hyperglycemia-induced oxidative stress and NO can be prevented if GC is initiated very early, but are not easily reversed if PC is maintained for longer durations. Understanding the mechanisms responsible for this phenomenon could reveal novel means to reverse nephropathy in diabetic patients. D 2004 Elsevier Inc. All rights reserved. Keywords: Glycemic control; Nephropathy; Nitric oxides; Oxidative stress
1. Introduction Nephropathy, a major chronic complication of diabetes mellitus, is a leading cause of end-stage renal disease, and hyperglycemia is considered a major risk factor for the development of nephropathy. Diabetes Control and Complications Trial (DCCT) studies have shown that improved metabolic control is associated with decreased development and progression of nephropathy in diabetes (DCCT Research Group, 1993). Islet transplantation in insulindeficient diabetic rats, if started at earlier stages of diabetes, can prevent diabetes-induced structural and functional changes in glomerulus (including total proteinuria, mean glomerular and extraglomerular lesions), but if initiated after longer durations (8 months) of hyperglycemia cannot reverse these renal abnormalities (Pugliese et al., 1997).
* Corresponding author. Tel.: +1-313-993-6714; fax: +1-313-577-8884. E-mail address: [email protected] (R.A. Kowluru). 1056-8727/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jdiacomp.2004.03.002
Hyperglycemia-induced disorders are postulated to initiate the sequence of events leading to the development of nephropathy, but which abnormalities are critical in the etiology of diabetic nephropathy remains to be established. Several mechanisms have been postulated to be involved in the pathogenesis of diabetic nephropathy, including increased polyol pathway, activation protein kinase C, accumulation of advanced glycation end products and increased oxidative stress (Kikkawa, Koya, & Haneda, 2003). Diabetes is shown to increase oxidative stress in various tissues, and the increased oxidative stress is postulated to play a significant role in the development of diabetic complications (Armstrong et al., 1992; Baynes, 1991; Kowluru, Tang, & Kern, 2001). Increased oxidative stress in diabetes is shown to play a pivotal role in the pathogenesis of diabetic nephropathy, and inhibition of oxidative stress ameliorates the manifestations associated with diabetic nephropathy (Agardh, Stenram, Torrfvit, & Agardh, 2002; Ha & Kim, 1999; Hinokio et al., 2002; Melhem, Craven, Liachenko, & DeRubertis, 2002; Obrosova, Fathallah, Liu, & Nourooz-Zadeh,
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2003). An increase in cellular superoxide dismutase, the enzyme known to scavenge superoxides, is shown to attenuate diabetic renal injury (Craven, Melhem, & Phillips, 2001). Reactive oxygen species can damage cellular macromolecules and act as proapoptotic agents (BaumgartnerParzer et al., 1995; Du, Stocklauser-Farber, & Rosen, 1999). Furthermore, urinary nitric oxide (NO) levels are increased in diabetes, and are postulated to contribute to the pathogenesis of glomerular hyperfiltration (Goor et al., 1996; Hiragushi et al., 2001). Superoxides and NO can form peroxynitrite (Behar-Cohen, Heydolph, Droy-Lefaix, Courtois, & Goureau, 1996), and increased peroxynitrite levels are reported in the proximal tubules of patients with diabetic nephropathy (Thuraisingham, Nott, Dodd, & Yaqoob, 2000). The purpose of this study is to identify the biochemical abnormalities that could contribute to the resistance of nephropathy to reverse after establishment of good metabolic control (GC) in rats. The effect of reinstitution of GC soon after and 6 months after induction of diabetes in rats is investigated on renal cortex oxidative stress and NO levels.
2. Methods Male Wistar rats (200 g) were randomly assigned to normal or diabetic groups. Diabetes was induced with streptozotocin (55 mg/kg BW). Diabetic rats were divided at random among three groups according to the intended degree and duration of GC as reported by us previously (Kowluru, 2003). The rats in Group 1 were allowed to remain in poor metabolic control for 13 months (PC group), and in Group 2 the rats were maintained in GC soon after establishment of hyperglycemia (3– 4 days after induction of diabetes) for the entire duration of 13 months (GC). The diabetic rats in Group 3 were allowed to be in PC for 6 months followed by GC for seven additional months (PC – GC). All diabetic rats received insulin (NPH) injections; the rats in the PC group were injected with 1 –2 units of insulin four to five times a week to prevent ketosis and weight loss, and the rats in the GC group were injected twice daily (a total of 8 –10 units of insulin) to maintain a steady gain in body weight, and urine glucose values below 150 mg/24 h. The rats were housed in metabolism cages: 24-h urine samples were tested for glycosuria daily with Keto-Diastix (Bayer, IN), and three to four times every week using quantitative methods (Glucose Kit, 510-A, Sigma). Blood glucose was measured once a week (Glucometer Elite, Bayer), and glycated hemoglobin (GHb, measured using a kit # 442-B, Sigma) every 2 months. The entire rat colony received powdered diet (Purina-5001), and the food consumption and body weights were measured two to three times every week as described by us. These experiments conformed to the Association for Research in Vision and Ophthalmology Resolution on Treatment of Animals in Research, as well as to specific institutional guidelines. Thirteen months after induction of diabetes the
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rats were sacrificed by an overdose of sodium pentobarbital, kidneys were removed immediately, weighed and multiple small portions of renal cortex were frozen in liquid nitrogen. To measure the parameters of oxidative stress and nitrative stress in the urine, 24-h urine samples from each rat were collected on three consecutive days and analyzed. 2.1. Lipid peroxides Lipid peroxides (LPOs) in the renal cortex were measured directly by redox reactions with ferrous ions using an assay kit from Cayman Chemicals (Ann Arbor, MI), and the resulting ferric ions were detected using thiocyanate ion as the chromogen (Kowluru, 2003; Kowluru & Koppolu, 2002; Kowluru, Koppolu, Chakrabarti, & Chen, 2003). 2.2. Glutathione Cytosolic reduced glutathione (GSH) was measured using Glutathione Assay Kit from Cayman Chemicals according to the manufacturer’s instructions. The sample (100 –150 Ag) was deproteinized using phosphoric acid, and the amount of 5-thio-2-nitrobenzoic acid produced was measured in the supernatant. 2.3. 8-Hydroxy-2V-deoxyguanosine 8-Hydroxy-2V-deoxyguanosine (8-OHdG) levels were measured in 24-h urine samples using the 8-OHdG ELISA kit from Oxis Research (Portland, OR). The 8-OHdG standards (0.5 – 80 ng/ml) or 35 – 50 Al of urine were allowed to incubate for 1 h with monoclonal antibody against 8-OHdG in a microtiter plate precoated with 8-OHdG. After washing the antibodies bound to 8-OHdG in the sample, enzymelabeled secondary antibody was added to each well. One hour of incubation with the antibody was followed by washing, and the color was developed by the addition of 3,3V,5,5V-tetramethylbenzidine. The absorbance was measured at 450 nm. 2.4. Nitrite levels Nitrite production was measured in 100- to 150-Ag sample using Greiss reagent as described previously (Tannous, Veluthakal, Amin, & Kowluru, 2002). The absorbance was measured at 540 nm, and the nitrite concentration was calculated from a sodium nitrite standard curve. 2.5. Protein To study the relationship between metabolic control and protein excretion, urine protein was measured in the urine sample dialyzed against 20 mM Tris– HCl buffer (pH 7.5) containing 0.01% sodium azide for 8 – 10 h using the bicinchoninic acid assay (Sigma). Bovine serum albumin was used as standard.
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Table 1 Body weight and glycated hemoglobin of the rats during various stages of metabolic control Number Duration Body of rats (months) weight (g) Normal PC GC PC – GC
7 8 7 7
13 13 13 6!7
GHb (%)
567F70 5.0F0.76 350F54* 12.1F0.72* 591F65 5.6F0.61 332F42*!493F54 11.9F1.0*!4.9F0.38
Values are meanFS.D. Body weight represents mean value during the entire duration of the intended glycemic control. * P < .05 compared to diabetes.
The results are reported as meanFS.D. and analyzed statistically using the nonparametric Kruskal – Wallis test followed by Mann – Whitney test for multiple group comparisons. Similar conclusions were reached also by using ANOVA with Fisher or Tukey test.
3. Results
Fig. 1. Effect of metabolic control on urinary 8-OHdG levels: 8-OHdG levels were measured in 24-h urine samples using the 8-OHdG ELISA kit from Oxis Research. The values are meanFS.D. of six rats each in normal, PC and PC – GC groups and five rats in the GC group. *P < .05 compared to normal and **P < .05 compared to PC.
3.1. Glycemia The GHb values in the rats maintained in PC were greater than 11% throughout the experiment (13 months duration), and were around 5% in the normal group and in the GC group. Before initiation of good control in the rats in the PC – GC group, the GHb values were around 12%, but the values dropped to less than 5.5% at 2 months after initiation of GC (the first measurement made after initiation of GC), and the values remained unchanged during the 7 months of GC (Table 1).
weight) in the GC group were, however, not different form those in the normal group (Table 2). Body weight of the rats in the PC – GC group before initiation of GC was similar to the age-matched rats in the PC group, but increased more than 40% after reinstitution of GC. The absolute kidney weight was similar, but the kidney weight in relation to body weight was slightly higher ( P > .05) in the PC – GC group compared to the agematched rats in normal and GGC groups (Table 2).
3.2. Body weight and kidney weight The final body weight of the rats in the PC group was 70% lower; kidney weight, expressed as absolute weight, was slightly higher (30%), but expressed relative to body weight (due to significant differences in the body weights of normal and diabetic rats) was twofold higher compared to the age-matched normal rats. The body weight and kidney weight (expressed either as absolute or relative to body Table 2 Effect of intended metabolic control on kidney weight, urine volume and protein excretion Kidney weight Absolute kidney relative to Urine volume Urine protein weight (g) body weight (ml/24 h) (g/24 h) Normal PC GC PC – GC
1.9F0.02 2.6F0.6* 2.2F0.4 2.1F0.4
3.2F0.02 7.3F1.9* 3.6F0.5 4.4F0.9
19F5 84F16* 26F5 33F11*
0.5F0.2 2.7F1.0* 0.7F0.3 1.4F0.4*
Values are meanFS.D. of eight rats in normal GC and PC – GC groups and seven rats in the PC group. Urine samples from each rat were collected three consecutive days and analyzed for total protein content. * P < .05 compared to normal.
Fig. 2. Effect of metabolic control on urinary NO levels: Nitrite levels were measured in duplicate using Greiss reagent in the 24-h urine samples that were collected on three consecutive days. Each sample was analyzed in duplicate, and the values presented in the figure are meanFS.D. of seven rats each in PC, GC and PC – GC groups and six rats in the normal group. *P < .05 compared to normal, **P < .05 compared to PC and #P < .05 compared to GC.
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Fig. 3. Metabolic control and renal cortex oxidative stress: (A) LPO was measured in the renal cortex obtained directly by redox reactions with ferrous ions using a kit from Cayman Chemical. The values represent meansFS.D. of six rats each in normal and PC groups, seven rats each in PC and PC – GC groups, each measurement made in duplicate. *P < .05 compared to normal, **P < .05 compared to PC and #P < .05 compared to GC. (B) GSH was measured in the deproteinized renal cortex sample (in duplicate) by using Glutathione Assay Kit from Cayman Chemicals. The values in the graph are meansFS.D. of seven rats each in PC, GC and PC – GC groups and six rats in the normal group. *P < .05 compared to normal and **P < .05 compared to PC.
3.3. Urine volume, protein, 8-OHdG and NO Twenty-four-hour urine volumes were almost four times higher in the rats in PC for 13 months compared to agematched normal rats. Total urinary protein and 8-OHdG excretions were 8 – 10 times higher and NO almost three times higher compared to the normal rats (Table 2 and Figs. 1 and 2). Rats in which good glycemic control was instituted soon after the induction of diabetes (GC group) had small increase in 24-h urine volume compared to normal control group, and the total urinary protein excretion was elevated
by 50%, but these values did not achieve any statistical significance (Table 2). The total amounts of 8-OHdG and NO in the urine excreted in 24 h were also not different from the normal rats (Figs. 1 and 2). However, in rats with 7 months of GC that was initiated 6 months after induction of diabetes, 24-h urine volume was increased by 80% and total urinary protein by 2.8-fold (Table 2). Total 24-h NO excretion was significantly higher in these animals compared with their age-matched normal control rats and GC rats ( P <.05), and total urinary 8-OHdG excretion was about 50% higher than normal, but the values did not achieve any statistical significance ( P >.05 compared to normal or GC). 3.4. Parameters of oxidative stress and nitrative stress in the renal cortex
Fig. 4. Effect of metabolic control on NO levels in the renal cortex: Nitrite levels were measured in the renal cortex in duplicate using Greiss reagent. The values presented in the figure are meanFS.D. of seven rats each in PC, and PC – GC groups and six rats each in normal and GC groups. *P < .05 compared to normal, **P < .05 compared to PC and #P < .05 compared to GC.
In order to evaluate the status of the kidney, we measured LPOs, GSH and NO levels in the renal cortex of the rats in the four experimental groups described above. Thirteen months of PC in rats resulted in increased oxidative stress and NO levels in the renal cortex; LPO were 40% higher, GSH 20% lower and NO levels 85% higher compared to the values obtained from age-matched normal rats (Figs. 2 – 4). Maintenance of rats in GC for the entire duration of 13 months did not result in any significant elevation in the oxidative stress; LPO and GSH in the renal cortex were similar to those in the normal group (Fig. 3). NO levels in the renal cortex were increased by less than 20%, but the differences were not statistically significant (Fig. 4). The rats that were allowed to remain in PC for 6 months before reinstitution of GC for seven additional months (PC – GC group) had values of LPO and NO in the renal cortex
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that were significantly elevated compared to the agematched normal rats, and these values were not different from those obtained from the PC group (Figs. 3 and 4), but were significantly different ( P < .05) from those in the GC group. In the same animals, renal cortex GSH levels were decreased significantly ( P < .05) compared to the PC group; however, the values did not achieve any statistical significance compared to those obtained from the renal cortex of the rats in normal control or GC groups ( P > .05).
4. Discussion Evidence is provided for the first time demonstrating that institution of GC soon after induction of diabetes in rats can prevent proteinuria, kidney hypertrophy and increases in oxidative stress and nitrative stress, but if the intervention with GC is delayed for 6 months, these abnormalities are not reversed. This suggests that these abnormalities that occur early in the development of nephropathy are not easily reversed by reinstitution of GC. In support of the results presented here, microalbuminuria in diabetic patients is shown to disappear and the risk of developing nephropathy is reduced by GC (Bojestig, Arnqvist, Karlberg, & Ludvigsson, 1996), and in diabetic dogs, renal abnormalities, including glomerular volume and thickness of basement membrane, are significantly inhibited if good control is initiated soon after induction of diabetes in dogs, but are not reversed if the dogs are allowed to maintain poor control for 2.5 years before initiation of good control (Kern & Engerman, 1990). DCCT studies have documented the beneficial effects of GC on the development of albuminuria and progression of nephropathy in diabetic patients (DCCT Research Group, 1993), and the Epidemiology of Diabetes Interventions and Complications (EDIC) studies that followed DCCT have clearly demonstrated that the previous glycemic control of diabetes during DCCT is important in delaying progression of diabetic nephropathy (Writing Team for the DCCT, 2003). Microalbuminuria is associated with PC in diabetic patients (Lepore, Bruttomesso, Nosari, Tiengo, & Trevisan, 2002), and glycemic control is considered a predictor of survival for the patients on hemodialysis with end-stage renal disease (Morioka et al., 2001). Here we provide data to identify the abnormalities that could contribute to the resistance of nephropathy to reverse; we provide evidence showing that hyperglycemia-induced oxidative stress and NO in the urine and renal cortex are prevented if GC is initiated soon after induction of diabetes, the urinary excretion of 8-OHdG and NO, and renal cortex LPO, GSH and NO in the rats that were maintained in GC from very initial stages of hyperglycemia are similar to those in the normal control rats. The results are supported by others showing that if islet transplantation is started at earlier stages of diabetes in insulin-deficient diabetic rats, it can prevent metabolic and functional changes in glomerulus (Pugliese et al., 1997).
Hyperglycemia is a major factor in the development and progression of diabetic nephropathy, and hyperglycemiainduced metabolic abnormalities, including increased oxidative stress, renal polyol formation, activation of protein kinase C, advanced glycation end products and hemodynamic factors (systemic hypertension and increased intraglomerular pressure) are important contributors in the development of nephropathy (Kikkawa et al., 2003). Oxidative stress is considered as a common pathogenic factor in the development of nephropathy, and the conversion of deoxyguanosine to 8-OHdG in DNA is increased. LPOs and 8-OHdG levels are elevated in the kidney and urine of diabetic rats, and inhibition of oxidative stress ameliorates these manifestations associated with diabetic nephropathy (Ha & Kim, 1999). In rats the levels of LPO and intracellular antioxidants (GSH), are decreased in the renal cortex at a very early stage of experimental diabetes (Obrosova et al., 2003; Reddi & Bollineni, 1997), and others have shown that LPO levels are elevated for 7 –8 months after induction of diabetes (Agardh et al., 2002; Sugimoto, Tsuruoka, & Fujimura, 2001). Increased urinary NO levels in diabetes are reported by others (Goor et al., 1996) and increased NO is postulated to contribute to the pathogenesis of glomerular hyperfiltration (Hiragushi et al., 2001). Superoxides and NO can react to form peroxynitrite, a highly reactive intermediate, which can increase DNA damage, deplete intracellular GSH levels and initiate lipid peroxidation (Behar-Cohen et al., 1996). In addition, peroxynitrite is reported to modify tyrosine in proteins to form nitrotyrosine, and nitration of proteins can inactive mitochondrial and cytosolic proteins and damage cellular constituents (Halliwell, 1997). Increased peroxynitrite levels are reported in the proximal tubules of patients with diabetic nephropathy (Thuraisingham et al., 2000). Our study is the first to show that the levels of LPO and NO remain elevated and of GSH remain decreased for 13 months of diabetes in rats (PC group). This suggests that the kidney experiences increased oxidative stress in diabetic patients conceivably for as long as the hyperglycemia is maintained. Furthermore, these abnormalities in oxidative stress and NO are not easily reversible after reinstitution of GC that has followed duration of PC. The possible mechanism(s) for the failure of oxidative stress and NO to reverse after reinstitution of GC is not clear, but may include oxidative modification or nitration of some of the proteins involved in the maintenance of balance between production and removal of free radicals. Islet transplantation in diabetic rats initiated after 8 months of hyperglycemia does not reverse renal abnormalities; glomerular volume and gene expression for extracellular matrix and TGF-h remain elevated (Pugliese et al., 1997). Intervention by insulin treatment starting at 8 weeks of diabetes in rats does not reverse diabetes-induced increase in the frequency of deleted mitochondrial DNA, suggesting that mitochondrial DNA mutations are not easily reversed by good glycemic control after they have been established (Kakimoto et al., 2002). In diabetic dogs, glomerular volume,
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the fractional volume of mesangium, thickness of glomerular basement membrane and frequency of glomerular obliteration are not reversed if the dogs are allowed to maintain poor control for 2.5 years that is followed by 2.5 years of good control (Writing Team for the DCCT, 2003). Similarly in diabetic patients 8-OHdG levels in urine are significantly lower in the multiple insulin injection therapy group (Nishikawa et al., 2003). Furthermore, EDIC studies have shown that previous intensive treatment with nearnormal glycemia during the DCCT has an extended benefit in delaying progression of diabetic nephropathy (Lepore et al., 2002). Here we provide convincing data showing that if GC is initiated after some duration of PC in rats (6 months) and sustained for seven additional months, metabolic abnormalities do not completely reverse; urinary 8-OHdG and NO levels, and renal cortex LPO and NO remain elevated. This suggests that the duration of PC before institution of GC has a significant effect on the outcome of the beneficial effect of GC. These results clearly strengthen the importance of early and sustained metabolic control in diabetic patients to prevent the progression of nephropathy. Thus, our study shows that oxidative stress and NO remain elevated in the renal cortex of rats diabetic for over a year and clearly suggests that hyperglycemia-induced oxidative stress and NO that are early events in the development of diabetic nephropathy can be prevented if GC is initiated very clearly, but resist reversal if GC is reinstituted after longer durations of PC. Understanding the mechanisms responsible for the resistance of nephropathy to reverse after reinstitution of GC in diabetes might reveal novel means to reverse nephropathy in diabetic patients.
Acknowledgments This work was supported in part by grants from Juvenile Diabetes Research Foundation, the Thomas Foundation, and Research to Prevent Blindness. Technical assistance of Prashant Koppolu and Xiaohua Zhou is sincerely appreciated.
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Reversal of hyperglycemia and diabetic nephropathy Effect of reinstitution of good metabolic control on oxidative stress in the kidney of diabetic rats Renu A. Kowluru *, Saiyeda N. Abbas, Sarah Odenbach Kresge Eye Institute, Wayne State University, Detroit, MI 48201, USA Received 8 December 2003; received in revised form 11 February 2004; accepted 3 March 2004
Abstract Studies have shown that good metabolic control (GC) is beneficial in slowing the progression of nephropathy in diabetes, and if the duration of poor metabolic control (PC) is prolonged before reinstitution of GC, nephropathy is not easily reversed. This study is to identify the biochemical abnormalities that could contribute to the resistance of nephropathy to reverse after establishment of GC in rats. The effect of reinstitution of GC and its duration is evaluated on oxidative stress and nitric oxide (NO) levels in the renal cortex and urine of diabetic rats. The rats were maintained in GC (5% glycated hemoglobin, GHb) soon after or 6 months after induction of hyperglycemia, and were sacrificed 13 months after induction of diabetes. For rats in which GC was initiated soon after induction of diabetes, oxidative stress [as measured by the levels of lipid peroxides (LPOs), 8-hydroxy-2V-deoxyguanosine (8-OHdG), and reduced glutathione (GSH)] and NO in urine and renal cortex were not different from that observed in normal control rats, but when reinstitution of GC was delayed for 6 months after induction of diabetes, oxidative stress and NO remain elevated in both urine and renal cortex. This suggests that hyperglycemia-induced oxidative stress and NO can be prevented if GC is initiated very early, but are not easily reversed if PC is maintained for longer durations. Understanding the mechanisms responsible for this phenomenon could reveal novel means to reverse nephropathy in diabetic patients. D 2004 Elsevier Inc. All rights reserved. Keywords: Glycemic control; Nephropathy; Nitric oxides; Oxidative stress
1. Introduction Nephropathy, a major chronic complication of diabetes mellitus, is a leading cause of end-stage renal disease, and hyperglycemia is considered a major risk factor for the development of nephropathy. Diabetes Control and Complications Trial (DCCT) studies have shown that improved metabolic control is associated with decreased development and progression of nephropathy in diabetes (DCCT Research Group, 1993). Islet transplantation in insulindeficient diabetic rats, if started at earlier stages of diabetes, can prevent diabetes-induced structural and functional changes in glomerulus (including total proteinuria, mean glomerular and extraglomerular lesions), but if initiated after longer durations (8 months) of hyperglycemia cannot reverse these renal abnormalities (Pugliese et al., 1997).
* Corresponding author. Tel.: +1-313-993-6714; fax: +1-313-577-8884. E-mail address: [email protected] (R.A. Kowluru). 1056-8727/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jdiacomp.2004.03.002
Hyperglycemia-induced disorders are postulated to initiate the sequence of events leading to the development of nephropathy, but which abnormalities are critical in the etiology of diabetic nephropathy remains to be established. Several mechanisms have been postulated to be involved in the pathogenesis of diabetic nephropathy, including increased polyol pathway, activation protein kinase C, accumulation of advanced glycation end products and increased oxidative stress (Kikkawa, Koya, & Haneda, 2003). Diabetes is shown to increase oxidative stress in various tissues, and the increased oxidative stress is postulated to play a significant role in the development of diabetic complications (Armstrong et al., 1992; Baynes, 1991; Kowluru, Tang, & Kern, 2001). Increased oxidative stress in diabetes is shown to play a pivotal role in the pathogenesis of diabetic nephropathy, and inhibition of oxidative stress ameliorates the manifestations associated with diabetic nephropathy (Agardh, Stenram, Torrfvit, & Agardh, 2002; Ha & Kim, 1999; Hinokio et al., 2002; Melhem, Craven, Liachenko, & DeRubertis, 2002; Obrosova, Fathallah, Liu, & Nourooz-Zadeh,
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2003). An increase in cellular superoxide dismutase, the enzyme known to scavenge superoxides, is shown to attenuate diabetic renal injury (Craven, Melhem, & Phillips, 2001). Reactive oxygen species can damage cellular macromolecules and act as proapoptotic agents (BaumgartnerParzer et al., 1995; Du, Stocklauser-Farber, & Rosen, 1999). Furthermore, urinary nitric oxide (NO) levels are increased in diabetes, and are postulated to contribute to the pathogenesis of glomerular hyperfiltration (Goor et al., 1996; Hiragushi et al., 2001). Superoxides and NO can form peroxynitrite (Behar-Cohen, Heydolph, Droy-Lefaix, Courtois, & Goureau, 1996), and increased peroxynitrite levels are reported in the proximal tubules of patients with diabetic nephropathy (Thuraisingham, Nott, Dodd, & Yaqoob, 2000). The purpose of this study is to identify the biochemical abnormalities that could contribute to the resistance of nephropathy to reverse after establishment of good metabolic control (GC) in rats. The effect of reinstitution of GC soon after and 6 months after induction of diabetes in rats is investigated on renal cortex oxidative stress and NO levels.
2. Methods Male Wistar rats (200 g) were randomly assigned to normal or diabetic groups. Diabetes was induced with streptozotocin (55 mg/kg BW). Diabetic rats were divided at random among three groups according to the intended degree and duration of GC as reported by us previously (Kowluru, 2003). The rats in Group 1 were allowed to remain in poor metabolic control for 13 months (PC group), and in Group 2 the rats were maintained in GC soon after establishment of hyperglycemia (3– 4 days after induction of diabetes) for the entire duration of 13 months (GC). The diabetic rats in Group 3 were allowed to be in PC for 6 months followed by GC for seven additional months (PC – GC). All diabetic rats received insulin (NPH) injections; the rats in the PC group were injected with 1 –2 units of insulin four to five times a week to prevent ketosis and weight loss, and the rats in the GC group were injected twice daily (a total of 8 –10 units of insulin) to maintain a steady gain in body weight, and urine glucose values below 150 mg/24 h. The rats were housed in metabolism cages: 24-h urine samples were tested for glycosuria daily with Keto-Diastix (Bayer, IN), and three to four times every week using quantitative methods (Glucose Kit, 510-A, Sigma). Blood glucose was measured once a week (Glucometer Elite, Bayer), and glycated hemoglobin (GHb, measured using a kit # 442-B, Sigma) every 2 months. The entire rat colony received powdered diet (Purina-5001), and the food consumption and body weights were measured two to three times every week as described by us. These experiments conformed to the Association for Research in Vision and Ophthalmology Resolution on Treatment of Animals in Research, as well as to specific institutional guidelines. Thirteen months after induction of diabetes the
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rats were sacrificed by an overdose of sodium pentobarbital, kidneys were removed immediately, weighed and multiple small portions of renal cortex were frozen in liquid nitrogen. To measure the parameters of oxidative stress and nitrative stress in the urine, 24-h urine samples from each rat were collected on three consecutive days and analyzed. 2.1. Lipid peroxides Lipid peroxides (LPOs) in the renal cortex were measured directly by redox reactions with ferrous ions using an assay kit from Cayman Chemicals (Ann Arbor, MI), and the resulting ferric ions were detected using thiocyanate ion as the chromogen (Kowluru, 2003; Kowluru & Koppolu, 2002; Kowluru, Koppolu, Chakrabarti, & Chen, 2003). 2.2. Glutathione Cytosolic reduced glutathione (GSH) was measured using Glutathione Assay Kit from Cayman Chemicals according to the manufacturer’s instructions. The sample (100 –150 Ag) was deproteinized using phosphoric acid, and the amount of 5-thio-2-nitrobenzoic acid produced was measured in the supernatant. 2.3. 8-Hydroxy-2V-deoxyguanosine 8-Hydroxy-2V-deoxyguanosine (8-OHdG) levels were measured in 24-h urine samples using the 8-OHdG ELISA kit from Oxis Research (Portland, OR). The 8-OHdG standards (0.5 – 80 ng/ml) or 35 – 50 Al of urine were allowed to incubate for 1 h with monoclonal antibody against 8-OHdG in a microtiter plate precoated with 8-OHdG. After washing the antibodies bound to 8-OHdG in the sample, enzymelabeled secondary antibody was added to each well. One hour of incubation with the antibody was followed by washing, and the color was developed by the addition of 3,3V,5,5V-tetramethylbenzidine. The absorbance was measured at 450 nm. 2.4. Nitrite levels Nitrite production was measured in 100- to 150-Ag sample using Greiss reagent as described previously (Tannous, Veluthakal, Amin, & Kowluru, 2002). The absorbance was measured at 540 nm, and the nitrite concentration was calculated from a sodium nitrite standard curve. 2.5. Protein To study the relationship between metabolic control and protein excretion, urine protein was measured in the urine sample dialyzed against 20 mM Tris– HCl buffer (pH 7.5) containing 0.01% sodium azide for 8 – 10 h using the bicinchoninic acid assay (Sigma). Bovine serum albumin was used as standard.
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Table 1 Body weight and glycated hemoglobin of the rats during various stages of metabolic control Number Duration Body of rats (months) weight (g) Normal PC GC PC – GC
7 8 7 7
13 13 13 6!7
GHb (%)
567F70 5.0F0.76 350F54* 12.1F0.72* 591F65 5.6F0.61 332F42*!493F54 11.9F1.0*!4.9F0.38
Values are meanFS.D. Body weight represents mean value during the entire duration of the intended glycemic control. * P < .05 compared to diabetes.
The results are reported as meanFS.D. and analyzed statistically using the nonparametric Kruskal – Wallis test followed by Mann – Whitney test for multiple group comparisons. Similar conclusions were reached also by using ANOVA with Fisher or Tukey test.
3. Results
Fig. 1. Effect of metabolic control on urinary 8-OHdG levels: 8-OHdG levels were measured in 24-h urine samples using the 8-OHdG ELISA kit from Oxis Research. The values are meanFS.D. of six rats each in normal, PC and PC – GC groups and five rats in the GC group. *P < .05 compared to normal and **P < .05 compared to PC.
3.1. Glycemia The GHb values in the rats maintained in PC were greater than 11% throughout the experiment (13 months duration), and were around 5% in the normal group and in the GC group. Before initiation of good control in the rats in the PC – GC group, the GHb values were around 12%, but the values dropped to less than 5.5% at 2 months after initiation of GC (the first measurement made after initiation of GC), and the values remained unchanged during the 7 months of GC (Table 1).
weight) in the GC group were, however, not different form those in the normal group (Table 2). Body weight of the rats in the PC – GC group before initiation of GC was similar to the age-matched rats in the PC group, but increased more than 40% after reinstitution of GC. The absolute kidney weight was similar, but the kidney weight in relation to body weight was slightly higher ( P > .05) in the PC – GC group compared to the agematched rats in normal and GGC groups (Table 2).
3.2. Body weight and kidney weight The final body weight of the rats in the PC group was 70% lower; kidney weight, expressed as absolute weight, was slightly higher (30%), but expressed relative to body weight (due to significant differences in the body weights of normal and diabetic rats) was twofold higher compared to the age-matched normal rats. The body weight and kidney weight (expressed either as absolute or relative to body Table 2 Effect of intended metabolic control on kidney weight, urine volume and protein excretion Kidney weight Absolute kidney relative to Urine volume Urine protein weight (g) body weight (ml/24 h) (g/24 h) Normal PC GC PC – GC
1.9F0.02 2.6F0.6* 2.2F0.4 2.1F0.4
3.2F0.02 7.3F1.9* 3.6F0.5 4.4F0.9
19F5 84F16* 26F5 33F11*
0.5F0.2 2.7F1.0* 0.7F0.3 1.4F0.4*
Values are meanFS.D. of eight rats in normal GC and PC – GC groups and seven rats in the PC group. Urine samples from each rat were collected three consecutive days and analyzed for total protein content. * P < .05 compared to normal.
Fig. 2. Effect of metabolic control on urinary NO levels: Nitrite levels were measured in duplicate using Greiss reagent in the 24-h urine samples that were collected on three consecutive days. Each sample was analyzed in duplicate, and the values presented in the figure are meanFS.D. of seven rats each in PC, GC and PC – GC groups and six rats in the normal group. *P < .05 compared to normal, **P < .05 compared to PC and #P < .05 compared to GC.
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Fig. 3. Metabolic control and renal cortex oxidative stress: (A) LPO was measured in the renal cortex obtained directly by redox reactions with ferrous ions using a kit from Cayman Chemical. The values represent meansFS.D. of six rats each in normal and PC groups, seven rats each in PC and PC – GC groups, each measurement made in duplicate. *P < .05 compared to normal, **P < .05 compared to PC and #P < .05 compared to GC. (B) GSH was measured in the deproteinized renal cortex sample (in duplicate) by using Glutathione Assay Kit from Cayman Chemicals. The values in the graph are meansFS.D. of seven rats each in PC, GC and PC – GC groups and six rats in the normal group. *P < .05 compared to normal and **P < .05 compared to PC.
3.3. Urine volume, protein, 8-OHdG and NO Twenty-four-hour urine volumes were almost four times higher in the rats in PC for 13 months compared to agematched normal rats. Total urinary protein and 8-OHdG excretions were 8 – 10 times higher and NO almost three times higher compared to the normal rats (Table 2 and Figs. 1 and 2). Rats in which good glycemic control was instituted soon after the induction of diabetes (GC group) had small increase in 24-h urine volume compared to normal control group, and the total urinary protein excretion was elevated
by 50%, but these values did not achieve any statistical significance (Table 2). The total amounts of 8-OHdG and NO in the urine excreted in 24 h were also not different from the normal rats (Figs. 1 and 2). However, in rats with 7 months of GC that was initiated 6 months after induction of diabetes, 24-h urine volume was increased by 80% and total urinary protein by 2.8-fold (Table 2). Total 24-h NO excretion was significantly higher in these animals compared with their age-matched normal control rats and GC rats ( P <.05), and total urinary 8-OHdG excretion was about 50% higher than normal, but the values did not achieve any statistical significance ( P >.05 compared to normal or GC). 3.4. Parameters of oxidative stress and nitrative stress in the renal cortex
Fig. 4. Effect of metabolic control on NO levels in the renal cortex: Nitrite levels were measured in the renal cortex in duplicate using Greiss reagent. The values presented in the figure are meanFS.D. of seven rats each in PC, and PC – GC groups and six rats each in normal and GC groups. *P < .05 compared to normal, **P < .05 compared to PC and #P < .05 compared to GC.
In order to evaluate the status of the kidney, we measured LPOs, GSH and NO levels in the renal cortex of the rats in the four experimental groups described above. Thirteen months of PC in rats resulted in increased oxidative stress and NO levels in the renal cortex; LPO were 40% higher, GSH 20% lower and NO levels 85% higher compared to the values obtained from age-matched normal rats (Figs. 2 – 4). Maintenance of rats in GC for the entire duration of 13 months did not result in any significant elevation in the oxidative stress; LPO and GSH in the renal cortex were similar to those in the normal group (Fig. 3). NO levels in the renal cortex were increased by less than 20%, but the differences were not statistically significant (Fig. 4). The rats that were allowed to remain in PC for 6 months before reinstitution of GC for seven additional months (PC – GC group) had values of LPO and NO in the renal cortex
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that were significantly elevated compared to the agematched normal rats, and these values were not different from those obtained from the PC group (Figs. 3 and 4), but were significantly different ( P < .05) from those in the GC group. In the same animals, renal cortex GSH levels were decreased significantly ( P < .05) compared to the PC group; however, the values did not achieve any statistical significance compared to those obtained from the renal cortex of the rats in normal control or GC groups ( P > .05).
4. Discussion Evidence is provided for the first time demonstrating that institution of GC soon after induction of diabetes in rats can prevent proteinuria, kidney hypertrophy and increases in oxidative stress and nitrative stress, but if the intervention with GC is delayed for 6 months, these abnormalities are not reversed. This suggests that these abnormalities that occur early in the development of nephropathy are not easily reversed by reinstitution of GC. In support of the results presented here, microalbuminuria in diabetic patients is shown to disappear and the risk of developing nephropathy is reduced by GC (Bojestig, Arnqvist, Karlberg, & Ludvigsson, 1996), and in diabetic dogs, renal abnormalities, including glomerular volume and thickness of basement membrane, are significantly inhibited if good control is initiated soon after induction of diabetes in dogs, but are not reversed if the dogs are allowed to maintain poor control for 2.5 years before initiation of good control (Kern & Engerman, 1990). DCCT studies have documented the beneficial effects of GC on the development of albuminuria and progression of nephropathy in diabetic patients (DCCT Research Group, 1993), and the Epidemiology of Diabetes Interventions and Complications (EDIC) studies that followed DCCT have clearly demonstrated that the previous glycemic control of diabetes during DCCT is important in delaying progression of diabetic nephropathy (Writing Team for the DCCT, 2003). Microalbuminuria is associated with PC in diabetic patients (Lepore, Bruttomesso, Nosari, Tiengo, & Trevisan, 2002), and glycemic control is considered a predictor of survival for the patients on hemodialysis with end-stage renal disease (Morioka et al., 2001). Here we provide data to identify the abnormalities that could contribute to the resistance of nephropathy to reverse; we provide evidence showing that hyperglycemia-induced oxidative stress and NO in the urine and renal cortex are prevented if GC is initiated soon after induction of diabetes, the urinary excretion of 8-OHdG and NO, and renal cortex LPO, GSH and NO in the rats that were maintained in GC from very initial stages of hyperglycemia are similar to those in the normal control rats. The results are supported by others showing that if islet transplantation is started at earlier stages of diabetes in insulin-deficient diabetic rats, it can prevent metabolic and functional changes in glomerulus (Pugliese et al., 1997).
Hyperglycemia is a major factor in the development and progression of diabetic nephropathy, and hyperglycemiainduced metabolic abnormalities, including increased oxidative stress, renal polyol formation, activation of protein kinase C, advanced glycation end products and hemodynamic factors (systemic hypertension and increased intraglomerular pressure) are important contributors in the development of nephropathy (Kikkawa et al., 2003). Oxidative stress is considered as a common pathogenic factor in the development of nephropathy, and the conversion of deoxyguanosine to 8-OHdG in DNA is increased. LPOs and 8-OHdG levels are elevated in the kidney and urine of diabetic rats, and inhibition of oxidative stress ameliorates these manifestations associated with diabetic nephropathy (Ha & Kim, 1999). In rats the levels of LPO and intracellular antioxidants (GSH), are decreased in the renal cortex at a very early stage of experimental diabetes (Obrosova et al., 2003; Reddi & Bollineni, 1997), and others have shown that LPO levels are elevated for 7 –8 months after induction of diabetes (Agardh et al., 2002; Sugimoto, Tsuruoka, & Fujimura, 2001). Increased urinary NO levels in diabetes are reported by others (Goor et al., 1996) and increased NO is postulated to contribute to the pathogenesis of glomerular hyperfiltration (Hiragushi et al., 2001). Superoxides and NO can react to form peroxynitrite, a highly reactive intermediate, which can increase DNA damage, deplete intracellular GSH levels and initiate lipid peroxidation (Behar-Cohen et al., 1996). In addition, peroxynitrite is reported to modify tyrosine in proteins to form nitrotyrosine, and nitration of proteins can inactive mitochondrial and cytosolic proteins and damage cellular constituents (Halliwell, 1997). Increased peroxynitrite levels are reported in the proximal tubules of patients with diabetic nephropathy (Thuraisingham et al., 2000). Our study is the first to show that the levels of LPO and NO remain elevated and of GSH remain decreased for 13 months of diabetes in rats (PC group). This suggests that the kidney experiences increased oxidative stress in diabetic patients conceivably for as long as the hyperglycemia is maintained. Furthermore, these abnormalities in oxidative stress and NO are not easily reversible after reinstitution of GC that has followed duration of PC. The possible mechanism(s) for the failure of oxidative stress and NO to reverse after reinstitution of GC is not clear, but may include oxidative modification or nitration of some of the proteins involved in the maintenance of balance between production and removal of free radicals. Islet transplantation in diabetic rats initiated after 8 months of hyperglycemia does not reverse renal abnormalities; glomerular volume and gene expression for extracellular matrix and TGF-h remain elevated (Pugliese et al., 1997). Intervention by insulin treatment starting at 8 weeks of diabetes in rats does not reverse diabetes-induced increase in the frequency of deleted mitochondrial DNA, suggesting that mitochondrial DNA mutations are not easily reversed by good glycemic control after they have been established (Kakimoto et al., 2002). In diabetic dogs, glomerular volume,
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the fractional volume of mesangium, thickness of glomerular basement membrane and frequency of glomerular obliteration are not reversed if the dogs are allowed to maintain poor control for 2.5 years that is followed by 2.5 years of good control (Writing Team for the DCCT, 2003). Similarly in diabetic patients 8-OHdG levels in urine are significantly lower in the multiple insulin injection therapy group (Nishikawa et al., 2003). Furthermore, EDIC studies have shown that previous intensive treatment with nearnormal glycemia during the DCCT has an extended benefit in delaying progression of diabetic nephropathy (Lepore et al., 2002). Here we provide convincing data showing that if GC is initiated after some duration of PC in rats (6 months) and sustained for seven additional months, metabolic abnormalities do not completely reverse; urinary 8-OHdG and NO levels, and renal cortex LPO and NO remain elevated. This suggests that the duration of PC before institution of GC has a significant effect on the outcome of the beneficial effect of GC. These results clearly strengthen the importance of early and sustained metabolic control in diabetic patients to prevent the progression of nephropathy. Thus, our study shows that oxidative stress and NO remain elevated in the renal cortex of rats diabetic for over a year and clearly suggests that hyperglycemia-induced oxidative stress and NO that are early events in the development of diabetic nephropathy can be prevented if GC is initiated very clearly, but resist reversal if GC is reinstituted after longer durations of PC. Understanding the mechanisms responsible for the resistance of nephropathy to reverse after reinstitution of GC in diabetes might reveal novel means to reverse nephropathy in diabetic patients.
Acknowledgments This work was supported in part by grants from Juvenile Diabetes Research Foundation, the Thomas Foundation, and Research to Prevent Blindness. Technical assistance of Prashant Koppolu and Xiaohua Zhou is sincerely appreciated.
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