From the Departments of Medicine and Pathology, Veterans Affairs Pittsburgh Healthcare System and University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania
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ABSTRACT |
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Oxidative and glyco-oxidative stress have been implicated in the pathogenesis of diabetic complications, including nephropathy (16). There is evidence for multiple pathways of increased generation of reactive oxygen species (ROS) in diabetes. These include enhanced glucose auto-oxidation; increased mitochondrial superoxide production; protein kinase C-dependent activation of NADPH oxidase by angiotensin-II, glucose, and other stimuli; uncoupled endothelial nitric oxide synthase (eNOS) activity; formation of advanced glycation products (AGEs); stimulation of cellular ROS production by extracellular AGEs via their receptors; and others (14). Markers of oxidative stress and reduced levels of antioxidants have been found in blood and/or tissues in both human and experimental diabetes, including kidney (110). In addition to the potential cytotoxic effects of increased ROS generation, there is increasing evidence that ROS function as physiological cellular second messengers via redox sensitive gene transcription factors and cell signaling pathways (1,2). Thus, increases in ROS in the diabetic environment may alter several redox-sensitive genes and/or cellular signaling pathways (1,2). With respect to diabetic nephropathy, studies in cultured mesangial cells support a role for increased superoxide production in signaling increases in matrix protein synthesis induced by either high ambient glucose concentrations or angiotensin-II (11,12), two factors strongly linked to renal injury in vivo in diabetes (13,14). Similarly, studies in cultured endothelial cells have demonstrated increased superoxide and eNOS expression in response to high glucose (15) and have implicated increased superoxide from mitochondria (2) and uncoupled eNOS (16) in the activation of signaling pathways linked to cell injury.
Consistent with a role for oxidative mechanisms in the pathogenesis of diabetic renal injury in vivo, dietary antioxidant supplementation with vitamin E, N-acetyl-cysteine, oxerutin, taurine, or lipoic acid have all been found to attenuate nephropathy in experimental models of diabetes (5,8,1618). Recently, we demonstrated that transgenic mice that overexpress human cytoplasmic Cu2+/Zn2+ superoxide dismutase (SOD)-1 are protected from early diabetic renal injury compared with their nontransgenic diabetic littermates in a model of streptozotocin (STZ)-induced type 1 diabetes (11). These observations provided the first in vivo evidence for the involvement of superoxide in the pathogenesis of diabetic nephropathy.
In the current studies, we assessed the effects of overexpression of SOD-1 on renal injury in an experimental model of type 2 diabetes and the possibility of altered superoxide interaction with nitric oxide (NO) in diabetic mice that overexpress SOD-1.
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RESEARCH DESIGN AND METHODS |
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Animals were maintained in a 22°C room with a 12-h light/dark cycle and given free access to food (Purina pellet mouse chow) and water. Blood glucose levels were determined at monthly intervals on blood obtained from the retro-orbital venous plexus. Urine samples were collected in metabolic cages at age 5 months and frozen for subsequent determination of albumin and [14C]inulin when inulin clearance (CIN) was examined. CIN was determined in conscious unrestrained mice from the clearance of [14C]inulin given by osmotic minipumps (Durect, Cupertino, CA) implanted subcutaneously, as previously reported (5,11). CIN and fractional clearance of albumin (FCAlb) were calculated as previously described (5,11). At age 5 months, mice were weighed and then killed by exsanguination under isoflurane anesthesia. Their blood was collected in heparinized tubes and their kidneys were weighed and then processed for biochemical, histological, and immunohistochemical determinations, as described below or previously reported (5,11).
In separate studies using four additional experimental mouse groups analogous to those described above, CIN and 24-h urinary excretion of guanosine 3',5'-cyclic monophosphate (UcGMP) and NO2/NO3 (UNOx) were determined before and on the 3rd day of oral administration of N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS (5,11,19). Preliminary studies indicated that ingestion of
20 mg · kg body wt-1 · day-1 of L-NAME for 3 days yielded marked suppression of UNOx in NTg-db/db mice, and therefore this dosage was used in the current experiments. Average daily consumption of L-NAME per mouse was
23 mg/kg body wt and did not differ significantly among the four study groups. Mice were killed 1 week after completion of the L-NAME studies; their kidneys were removed and their glomeruli were harvested by differential sieving, as previously described (20). Glomeruli harvested from kidneys of five randomly selected mice from each study group were used for determination of SOD-1 activities.
Glomerular incubations.
After being isolated, glomeruli were washed with and then resuspended in Krebs-Ringer bicarbonate buffer containing 5 mmol/l glucose and 2 mmol/l 1-methyl-3-isobulylmethylxanthene, an inhibitor of phosphodiesterase activity. The suspension was then aliquoted (0.5 ml/well in 24-well plate) and incubated at 37°C for 30 min with shaking in a Plexiglas chamber in an atmosphere of 95%/5% CO2 in the presence or absence of carbamylcholine (CCh) or nitroprusside (NP), as indicated in RESULTS. Incubations were terminated by the addition of 0.5 ml sodium acetate (pH 6.0) at 95°C and cGMP extracted from glomeruli plus media.
cGMP was assayed in extracts of glomerular incubates and urine, as previously reported (20) using a commercial radioimmunoassay system (Amersham Pharmacia Biotech, Piscataway, NJ). The glomerular pellet was extracted in NaOH and used for determination of protein by the Bradford method (19).
RT-PCR.
DNA was extracted from tail snips using an Aquapure Genomic DNA Isolation Kit (Bio-Rad, Hercules, CA). The PCR protocol for detection of the human SOD-1 transgene was obtained from The Jackson Laboratories (Bar Harbor, ME). The primers used were CAT CAG CCC TAA TCC ATC TGA, forward, and CGC GAC TAA CAA TCA AAG TGA, reverse (21). Interleukin-2 (IL-2) was used as an internal control for human SOD-1. The primers used for IL-2 were CTA GGC CAC AGA ATT GAA AGA TCT, forward, and GTA GGT GGA AAT TCT AGC ATC ATC C, reverse. PCR amplification was performed with Taq polymerase (Invitrogen, Carlsbad, CA). The primers used amplified a 236-bp fragment of the human SOD-1 gene. Simultaneous amplification was performed with primers specific for a 324-bp fragment of the IL-2 gene as an internal control. A cycle number of 35 was chosen based on preliminary experiments to ensure that amplification was within the linear range.
The PCR protocol for detection of the db mutation has been previously described by Schreyer et al. (22). The primer sequences used were AGA ACG GAC ACT CTT TGA AGT CTC, forward, and CAT TCA AAC CAT AGT TTA GGT TTG TGT, reverse. The primers introduce a new Rsa I site into the mutant sequence. The PCR product is digested with Rsa for 1 h at 37°. The db mutant allele yields a 135-bp fragment that is not cut by Rsa I.
Additional assays.
Plasma and urinary albumin, GHb, UNOx, renal cortical activities of SOD-1, mitochondrial Mn2+ SOD, catalase and glutathione peroxidase (GPx), renal cortical malondialdehyde (TBARS), and reduced glutathione (GSH) content were determined as previously reported (5,11,19).
Immunohistochemical staining.
Immunohistochemical staining of renal cortical sections for transforming growth factor-ß (TGF-ß), collagen 1(IV), and human SOD-1 were performed using techniques and reagents previously described in detail (5,11). To assess nitrotyrosine content of glomeruli by immunohistochemistry, sections of formalin-fixed mouse renal cortex were pretreated for 30 min at 95° with Target Retrieval Solution (Dako, Carpinteria, CA). Sections were cooled and blocked with endogenous oxidation blocking solution (PBS plus 3% H202) for 10 min at room temperature. Sections were then blocked with Superblock (ScyTek, Logan, UT). The primary antibody, antinitrotyrosine (5 ug/ml, rabbit immunoaffinity purified IgG; Upstate Biotechnology, Lake Placid, NY) was then applied and the incubation was continued overnight at 0°C. Sections were incubated for 30 min with biotin-conjugated goat antirabbit IgG diluted 1/500 (The Jackson Laboratories) followed by a 10-min incubation with horseradish-conjugated streptavidin (Dako). Color was developed with diaminobenzidine. Nonspecific staining was assessed by prior exposure of the primary antinitrotyrosine antibody to 10 mmol/l nitrotyrosine for 1 h at room temperature and application of this pretreated antibody preparation to renal cortical sections.
Glomerular TGF-ß, collagen 1(IV), and nitrotyrosine immunostaining were assessed with a SAMBA 4000 image analyzer (Image Products, Chantilly, VA) using designed software (Microsoft, Richmond, VA), as previously described (5,11). A total of 30 glomeruli per mouse were examined. Results are expressed as the proportional area of positive staining of the glomerular tuft (labeling index), as previously reported (5,11).
Glomerular morphometrics.
Glomerular volume (VG) was calculated from the cross-sectional area of the glomerular tuft as determined by light microscopy (23). The fractional area of the glomerular tuft occupied by mesangial matrix or mesangial cells was determined by a modification of the procedure reported previously (23). Briefly, a 1 x 1 cm grid was placed over each photomicrograph of periodic acid-Schiff stains of renal cortical sections. The number of boxes or fraction of boxes that contained portions of extracellular mesangial matrix or mesangial cells were counted and expressed as a proportion of the total number of boxes containing glomerular tuft. Measurements were made on 30 glomeruli per mouse at a magnification of x257. For assessment of both quantitative immunohistochemical staining and glomerular morphometrics, all samples were randomized and examined in a coded fashion by a pathologist blinded to the treatment group from which the sample came.
Statistical analysis.
Significance of differences was determined by ANOVA followed by Fishers multiple comparison test using Statview software. Differences at P < 0.05 were considered significant (5,11).
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RESULTS |
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As shown in Table 2, renal cortical SOD-1-specific activity was significantly higher in both the SOD-Tg-ND and the SOD-Tg-db/db mice compared with values in the corresponding nontransgenic groups. Renal cortical SOD-1 activity was comparable in the SOD-Tg-ND and SOD-Tg-db/db mice. Renal cortical SOD-1-specific activity of NTg-ND and NTg-db/db also did not differ significantly (Table 2).
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As shown in Fig. 1, glomerular content of immunoreactive TGF-ß, collagen 1(IV), and nitrotyrosine, expressed as the percent of the area of the glomerular tuft that stained positively, was significantly increased in NTg-db/db mice compared with corresponding values in either of the nondiabetic groups or in the SOD-Tg-db/db mice. Glomerular TGF-ß and nitrotyrosine of SOD-Tg-db/db mice did not differ from corresponding values in either of the nondiabetic mice groups. However, glomerular content of collagen
1(IV) of SOD-Tg-db/db mice was higher than the value in the SOD-Tg-ND group (Fig. 1).
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DISCUSSION |
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The renoprotective effect of SOD-1 overexpression in the db/db mice was associated with increased renal cortical SOD-1 enzymatic activity, lower TBARS, and higher GSH levels compared with corresponding values in renal cortex from NTg-db/db mice, findings consistent with a renal antioxidant effect. However, in the transgenic model used, SOD-1 activity is increased in multiple organs as well as in erythrocytes and leukocytes (30). Thus it is uncertain whether the changes in renal cortical TBARS and GSH reflect consequences of higher renal SOD-1, higher SOD-1 activity at one or more extrarenal sites, or a combination of these changes. It seems likely that superoxide generated in or entering the cell cytosol was the predominant target of SOD-1, given its subcellular distribution (31). However, an effect of SOD-1 on superoxide of mitochondrial origin cannot be completely excluded. Recent studies have demonstrated that SOD-1 is present not only in the cell cytosol but also in the intermembrane region of mitochondria, and may participate in the degradation of superoxide of mitochondrial origin that gain access to that region (32). The potential participation of SOD-1 in the dismutation of superoxide of mitochondrial origin is of interest in view of the work of Brownlee and colleagues (2,33). These studies implicate enhanced mitochondrial superoxide generation induced by high ambient concentrations of glucose, fatty acids, or leptin in vascular endothelial and other cells in the activation of multiple signaling pathways leading to cell injury (2,33). All of these potential mediators of mitochondrial superoxide generation are relevant to the db/db model (24) and may have contributed to renal oxidative stress.
SOD-1 converts superoxide to H2O2, a potent oxidizing agent. Thus, enhanced formation of H2O2 might be expected in mice overexpressing SOD-1 and is a potential concern. Of interest, however, is that an increase in cellular SOD-1 activity has not been uniformly observed to increase H2O2 levels. Indeed, overexpression of SOD-1 in some cells has been associated with a decrease in H2O2 levels (34). As noted by Liochev and Fridovich (35), the net effects of SOD increases on cellular H2O2 formation and levels depend on the cellular targets available to superoxide as well as the activities of antioxidant enzymes involved in H2O2 metabolism, predominantly catalase and GPx (36,37). No differences were found in the activity of renal cortical catalase among the four mouse groups in the current study. However, consistent with prior assessments of kidney from rats with experimental diabetes (37), renal cortical GPx was higher in both diabetic groups compared with controls (Table 2). This enzymatic change may have attenuated any increase in the levels of renal H2O2 in the diabetic mouse groups.
Overexpression of SOD-1 in db/db mice prevented accumulation of nitrotyrosine in glomeruli and was associated with both an NO-dependent increase in the glomerular filtration rate and preservation of glomerular cGMP responses to NO ex vivo. These findings suggest that enhanced dismutation of superoxide by SOD-1 may have suppressed the reaction of superoxide with NO with a resultant decrease in peroxynitrite generation and a concurrent increase in the bioavailability of NO. An increase in nitrotyrosine accumulation in kidney and other tissues has previously been described in experimental diabetes (6,3840). Accumulation of this product, at least in part, reflects formation of peroxynitrite (6,3840) from the reaction of NO with superoxide, and provides a stable footprint of peroxynitrite formation (6,3841). Increased peroxynitrite generation may lead to modification of proteins, nucleic acids, and lipids, with consequent cell dysfunction or death (41). Of note, studies in cultured vascular endothelial cells have indicated that eNOS per se is a target for inactivation by peroxynitrite and that inhibition of eNOS in these cells is prevented by overexpression of SOD-1, but not SOD-2 (42). Thus, overexpression of SOD-1 in glomerular endothelial cells in db/db mice may have enhanced NO production by these cells.
Similarly, the SOD-Tg-db/db mice displayed increases in CIN, UcGMP, and UNOx, which were dependent on NO, as reflected by the suppression of these increases by administration of L-NAME. NO-dependent increases in CIN and UcGMP imply enhanced production and/or bioavailability of NO in SOD-Tg-db/db compared with NTg-db/db mice. In the latter group, basal CIN and UcGMP were modestly, albeit insignificantly, increased. However, these parameters were not altered by L-NAME in NTg-db/db mice. In contrast, basal UNOx was clearly elevated in NTg-db/db mice and suppressed by L-NAME. UNOx has been used as an index of renal NO production (4245). Previous studies in nontransgenic STZ diabetic rats have found increases in UNOx and UcGMP associated with glomerular hyperfiltration, and have suggested a role for NO in mediating the latter two changes (19,45). In the current studies, the dissociation between the actions of L-NAME on UNOx versus CIN and UcGMP suggests that the increase in UNOx in NTg-db/db mice was not reflective of a corresponding enhancement in vivo of the renal bioactivity of NO. To the extent that increases in UNOx reflected changes in renal NO production in the diabetic mice, one possible explanation for the failure to observe concurrent NO-dependent increases in CIN and UcGMP in NTg-db/db mice is accelerated inactivation of NO in this group compared with the SOD-Tg-db/db mice. Consistent with this possibility, increased oxidant quenching of NO has been implicated in the impairment of endothelial dependent vasodilation that is characteristic of diabetes (46), an impairment that is reversed by antioxidants, including superoxide dismutase (46,47). Thus, in the SOD-Tg-db/db model used in the current study, inactivation of NO by superoxide may have been attenuated by enhanced SOD-1 activity.
Evidence for reduced bioactivity of NO in the NTg-db/db mice compared with either the SOD-Tg-db/db group or nondiabetic mice was also provided by ex vivo studies of the cGMP responses of isolated glomeruli to NO (Fig. 4). These observations suggest increased quenching of NO by glomeruli from the NTg-db/db mice, analogous to earlier findings in glomeruli from STZ-diabetic rats (19,48). Although the influence of an increase in SOD activity on NO depends on multiple variables and is complex (35), the current data imply that in glomeruli of SOD-Tg-db/db mice, the net effect of overexpression of SOD-1 was preservation of the biological action of NO relative to that observed in glomeruli from the nontransgenic diabetic mice. In addition to renal hemodynamic effects of NO (19,4345), studies in cultured glomerular mesangial cells support direct actions of NO to suppress activation of protein kinase C, increases in TGF-ß, and accumulation of matrix protein induced by high concentrations of glucose, angiotensin II, or thromboxane-A2 (49,50). Thus, enhanced expression of NO actions in glomeruli of SOD-Tg-db/db mice relative to the NTg-db/db mice may be linked to the suppression of glomerular TGFß1 and mesangial matrix accumulation observed in the transgenic group.
In summary, the current findings demonstrated that overexpression of SOD-1 prevents biochemical, functional, and structural changes induced in kidney in a model of type 2 diabetes. They imply a role for superoxide in vivo in the mediation of these changes, possibly in part via reduced superoxide interaction with NO in the SOD-Tg-db/db mouse.
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ACKNOWLEDGMENTS |
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The authors are grateful for the excellent technical assistance of Julia Liachenko and Mark Barsic and to JoAnn Orbin for typing the manuscript and revisions.
Address correspondence and reprint requests to Frederick R. DeRubertis, MD, VA Pittsburgh Healthcare System, University Drive C, Pittsburgh, PA 15240. E-mail: frederick.derubertis{at}med.va.gov
Received for publication September 4, 2003 and accepted in revised form December 2, 2003
AGE, advanced glycation product; CCh, carbamylcholine; CIN, inulin clearance; cGMP, guanosine 3',5'-cyclic monophosphate; eNOS, endothelial nitric oxide synthase; FCAlb, fractional clearance of albumin; GPx, glutathione peroxidase; GSH, reduced glutathione; IL-2, interleukin-2; L-NAME, N-nitro-L-arginine methyl ester; NP, nitroprusside; ROS, reactive oxygen species; SOD, superoxide dismutase; STZ, streptozotocin; TBARS, renal cortical malondialdehyde; TGF-ß, transforming growth factor-ß; UAE, urinary albumin excretion; UcGMP, urinary excretion of cGMP; UNox, urinary clearance of NO2/NO3; VG, glomerular volume
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REFERENCES |
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