Puromycin aminonucleoside induces oxidant-dependent DNA damage in podocytes in vitro and in vivo
2006; Elsevier BV; Volume: 70; Issue: 11 Linguagem: Inglês
10.1038/sj.ki.5001965
ISSN1523-1755
AutoresCaroline B. Marshall, Jeffrey W. Pippin, Ronald D. Krofft, Stuart J. Shankland,
Tópico(s)Renal and related cancers
ResumoA decline in podocyte number correlates with progression to glomerulosclerosis. A mechanism underlying reduced podocyte number is the podocyte's relative inability to proliferate in response to injury. Injury by the podocyte toxin puromycin aminonucleoside (PA) is mediated via reactive oxygen species (ROS). The precise role of ROS in the pathogenesis of PA-induced glomerulosclerosis remains to be determined. We sought to examine whether PA-induced ROS caused podocyte DNA damage, possibly accounting for the podocyte's inability to proliferate in response to PA. In vitro, podocytes were exposed to PA, with or without the radical scavenger 1,3-dimethyl-2-thiourea (DMTU). In vivo, male Sprague–Dawley rats were divided into experimental groups (n=6/group/time point): PA, PA with DMTU, and control, killed at days 1.5, 3, or 7. DNA damage was measured by DNA precipitation, apurinic/apyrimidinic site, Comet, and 8-hydroxydeoxyguanosine assays. Cell cycle checkpoint protein upregulation (by immunostaining and Western blotting), histopathology, and biochemical parameters were examined. DNA damage was increased in cultured podocytes that received PA, but not PA with DMTU. PA exposure activated specific cell cycle checkpoint proteins, with attenuation by DMTU. DNA repair enzymes were activated, providing evidence for attempted DNA repair. The PA-treated animals developed worse proteinuria and histopathologic disease and exhibited more DNA damage than the DMTU pretreated group. No significant apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. A mechanism underlying the lack of podocyte proliferation following PA-induced injury in vitro and in vivo may be ROS-mediated DNA damage, with upregulation of specific cell cycle checkpoints leading to cell cycle arrest. A decline in podocyte number correlates with progression to glomerulosclerosis. A mechanism underlying reduced podocyte number is the podocyte's relative inability to proliferate in response to injury. Injury by the podocyte toxin puromycin aminonucleoside (PA) is mediated via reactive oxygen species (ROS). The precise role of ROS in the pathogenesis of PA-induced glomerulosclerosis remains to be determined. We sought to examine whether PA-induced ROS caused podocyte DNA damage, possibly accounting for the podocyte's inability to proliferate in response to PA. In vitro, podocytes were exposed to PA, with or without the radical scavenger 1,3-dimethyl-2-thiourea (DMTU). In vivo, male Sprague–Dawley rats were divided into experimental groups (n=6/group/time point): PA, PA with DMTU, and control, killed at days 1.5, 3, or 7. DNA damage was measured by DNA precipitation, apurinic/apyrimidinic site, Comet, and 8-hydroxydeoxyguanosine assays. Cell cycle checkpoint protein upregulation (by immunostaining and Western blotting), histopathology, and biochemical parameters were examined. DNA damage was increased in cultured podocytes that received PA, but not PA with DMTU. PA exposure activated specific cell cycle checkpoint proteins, with attenuation by DMTU. DNA repair enzymes were activated, providing evidence for attempted DNA repair. The PA-treated animals developed worse proteinuria and histopathologic disease and exhibited more DNA damage than the DMTU pretreated group. No significant apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. A mechanism underlying the lack of podocyte proliferation following PA-induced injury in vitro and in vivo may be ROS-mediated DNA damage, with upregulation of specific cell cycle checkpoints leading to cell cycle arrest. The glomerular visceral epithelial cell, or podocyte, is a highly specialized and terminally differentiated cell that forms a critical part of the glomerular filtration barrier and functions to prevent urinary protein leakage, maintain glomerular capillary loop integrity, oppose intracapillary hydrostatic pressure, and synthesize the glomerular basement membrane.1.Pavenstadt H. Kriz W. Kretzler M. Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307Crossref PubMed Scopus (1117) Google Scholar Common to many human kidney diseases and experimental animal models is a strong association between podocyte injury and glomerulosclerosis. Studies have demonstrated that a decline in podocyte number strongly correlates with, and likely underlies, progression to glomerulosclerosis.2.Kriz W. Gretz N. Lemley K.V. Progression of glomerular diseases: is the podocyte the culprit?.Kidney Int. 1998; 54: 687-697Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, 3.Kim Y.H. Goyal M. Kurnit D. et al.Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat.Kidney Int. 2001; 60: 957-968Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 4.Pagtalunan M.E. Miller P.L. Jumping-Eagle S. et al.Podocyte loss and progressive glomerular injury in type II diabetes.J Clin Invest. 1997; 99: 342-348Crossref PubMed Scopus (808) Google Scholar, 5.Wharram B.L. Goyal M. Wiggins J.E. et al.Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene.J Am Soc Nephrol. 2005; 16: 2941-2952Crossref PubMed Scopus (521) Google Scholar, 6.Ichikawa I. Ma J. Motojima M. Matsusaka T. Podocyte damage damages podocytes: autonomous vicious cycle that drives local spread of glomerular sclerosis.Curr Opin Nephrol Hypertens. 2005; 14: 205-210Crossref PubMed Scopus (72) Google Scholar Two important mechanisms for podocyte depletion following injury are apoptosis and detachment. Podocyte loss leads to areas of denuded basement membrane, culminating in proteinuria, the development of focal glomerulosclerosis, and progressive deterioration in kidney function. Owing to their highly differentiated state, mature podocytes readily do not proliferate in vivo. Therefore, the apparent lack of proliferation in response to injury contributes to reduced podocyte number.2.Kriz W. Gretz N. Lemley K.V. Progression of glomerular diseases: is the podocyte the culprit?.Kidney Int. 1998; 54: 687-697Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar At the cell cycle level, reduced proliferation is a consequence of either an abnormality in DNA synthesis, secondary to G1/S checkpoint arrest, or reduced mitosis, secondary to G2/M checkpoint arrest. DNA damage triggers activation of cell cycle checkpoints, molecular pathways that monitor passage through the cell cycle and generate pauses in cell cycle progression when DNA damage is present. Thus, DNA damage leads to cell cycle arrest, permitting the cell sufficient time for DNA repair or other cell fate decisions, including apoptosis or entry into a permanent senescence-like state, thereby limiting proliferation.7.Shackelford R.E. Kaufmann W.K. Paules R.S. Oxidative stress and cell cycle checkpoint function.Free Radic Biol Med. 2000; 28: 1387-1404Crossref PubMed Scopus (441) Google Scholar Cell cycle arrest following DNA damage is mediated, in part, by tumor suppressor p53 and cyclin-dependent kinase inhibitor p21WAF1/CIP1. It is unknown whether the presence of DNA damage, with subsequent cell cycle arrest, accounts for the podocyte's limited proliferation in non-immune-mediated podocyte injury, namely puromycin aminonucleoside nephropathy (PAN). Puromycin aminonucleoside (PA), a podocyte toxin used to induce experimental minimal change disease progressing to focal segmental glomerulosclerosis in rats, leads to podocyte foot process effacement, and massive proteinuria within 7–10 days. In vivo studies have supported that, acutely, PA-induced glomerular injury is mediated via overproduction of reactive oxygen species (ROS),8.Diamond J.R. Bonventre J.V. Karnovsky M.J. A role for oxygen free radicals in aminonucleoside nephrosis.Kidney Int. 1986; 29: 478-483Abstract Full Text PDF PubMed Scopus (239) Google Scholar, 9.Thakur V. Walker P.D. Shah S.V. Evidence suggesting a role for hydroxyl radical in puromycin aminonucleoside-induced proteinuria.Kidney Int. 1988; 34: 494-499Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 10.Gwinner W. Landmesser U. Brandes R.P. et al.Reactive oxygen species and antioxidant defense in puromycin aminonucleoside glomerulopathy.J Am Soc Nephrol. 1997; 8: 1722-1731PubMed Google Scholar, 11.Beaman M. Birtwistle R. Howie A.J. et al.The role of superoxide anion and hydrogen peroxide in glomerular injury induced by puromycin aminonucleoside in rats.Clin Sci (London). 1987; 73: 329-332Crossref PubMed Scopus (64) Google Scholar, 12.Shibouta Y. Terashita Z. Imura Y. et al.Involvement of thromboxane A2, leukotrienes and free radicals in puromycin nephrosis in rats.Kidney Int. 1991; 39: 920-929Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 13.Ricardo S.D. Bertram J.F. Ryan G.B. Antioxidants protect podocyte foot processes in puromycin aminonucleoside-treated rats.J Am Soc Nephrol. 1994; 4: 1974-1986PubMed Google Scholar, 14.Nakamura K. Kojima K. Arai T. et al.Dipyridamole and dilazep suppress oxygen radicals in puromycin aminonucleoside nephrosis rats.Eur J Clin Invest. 1998; 28: 877-883Crossref PubMed Scopus (19) Google Scholar which then may interact with biomolecules, leading to modification and potentially deleterious cellular consequences.15.Cooke M.S. Evans M.D. Dizdaroglu M. Lunec J. Oxidative DNA damage: mechanisms, mutation, and disease.FASEB J. 2003; 17: 1195-1214Crossref PubMed Scopus (2110) Google Scholar This study was designed to examine whether DNA damage occurs in PAN, if DNA damage accounts for the inability of podocytes to proliferate in response to the toxin PA, and which cell cycle regulatory proteins may underlie this response. To determine whether PA induced DNA damage in vitro, growth-restricted podocytes were exposed to PA, and DNA damage was measured by three methods. DNA precipitation assay results are shown in Figure 1. As there is a basal level of oxidative DNA damage owing to non-pathogenic metabolic processes in healthy aerobic organisms,15.Cooke M.S. Evans M.D. Dizdaroglu M. Lunec J. Oxidative DNA damage: mechanisms, mutation, and disease.FASEB J. 2003; 17: 1195-1214Crossref PubMed Scopus (2110) Google Scholar,16.Halliwell B. Gutteridge J.M.C. Free radicals in biology and medicine. 3rd edn. Oxford University Press, Oxford New York, Clarendon Press2003Google Scholar the amount of single-stranded DNA (ssDNA) present in control cells at the earliest time point was considered the reference. This basal level of ssDNA was subtracted from subsequent values for all conditions and time points. At 6 h, there was a significant difference in the percent ssDNA between podocytes exposed to PA versus control conditions (control=22.43%, PA=37.08%; P<0.05). There was no significant difference between these two groups at 2 and 24 h. Podocytes exposed to PA+1,3-dimethyl-2-thiourea (DMTU) exhibited a lower percent ssDNA at 6 and 24 h than cells exposed to PA alone, with values falling below the basal level observed in control cells. To confirm that PA mediates DNA damage, the results of the AP (apurinic/apyrimidinic or abasic) site assay are shown in Figure 2. With PA exposure, there were an increased number of abasic sites in DNA at 6 and 18 h (values per 100 000 base pairs: 6 h: control=0.102, PA=14.649, P<0.05; 18 h: control=1.396, PA=2.689, P<0.05). In podocytes exposed to PA+DMTU, DNA damage was lessened, with no significant increase in abasic sites when compared to controls (6 h: 0.078, 18 h: 1.441). Cells exposed to H2O2, the positive control for oxidant-mediated DNA damage, exhibited levels of abasic sites intermediate between control and PA groups (data not shown). As further corroboration that PA mediates DNA damage in podocytes, the results of the Comet assay are shown in Figure 3. Following exposure to experimental conditions, podocytes underwent harvesting, immobilization in agarose, gentle lysis, and alkaline electrophoresis. With PA exposure, there was an increase in cells exhibiting long ‘comet tails’, when compared to control group (Figure 3a and c), at 2 and 6 h (values per 200 cells: 2 h: control=0.8, PA=7, P<0.05; 6 h: control=1.2, PA=22, P<0.05) (Figure 3d). In podocytes exposed to PA+DMTU, DNA damage was lessened, with a decrease in long ‘comet tails’ when compared to PA-exposed cells (2 h: PA+DMTU=1.8, P<0.05; 6 h: PA+DMTU=7, P<0.05) (Figure 3b). The results from the DNA precipitation, AP site, and Comet assays demonstrated that PA caused DNA damage in cultured podocytes. This DNA damage was attenuated by pre-exposure to DMTU, providing support that PA mediated oxidative DNA damage. In contrast to other glomerular cells, podocytes typically do not proliferate following injury. To test the hypothesis that following PA-mediated oxidative DNA damage, cell cycle checkpoint proteins are activated thereby limiting proliferation, Western blotting was performed on extracts from cultured podocytes (Figure 4). At 6 and 24 h after PA exposure, protein expression was increased for p21WAF1/CIP1 (ratio to control: 5.24 at 6 h, 2.28 at 24 h) and p53 (ratio to control: 2.27 at 6 h, 1.91 at 24 h). In podocytes pretreated with DMTU, the levels of p21WAF1/CIP1 (ratio to control: 0.98 at 6 h, 1.02 at 24 h) and p53 (ratio to control: 1.16 at 6 h, 0.89 at 24 h) were reduced compared to the PA-only group. The expression levels of checkpoint proteins checkpoint kinase 1 (CHK-1), checkpoint kinase 2 (CHK-2), and growth arrest, DNA damage-inducible-45α were not significantly different between PA and PA+DMTU groups at 6 and 24 h (data not shown). These results established that specific cell cycle checkpoint proteins were activated in response to PA-mediated oxidative DNA damage. Following pre-exposure to DMTU, PA did not induce the same level of cell cycle checkpoint protein activation. To determine if podocytes possessed the capacity to repair damaged DNA, specific DNA repair enzymes were evaluated. Figure 5 shows that, in addition to upregulation of cell cycle checkpoint proteins in response to PA-mediated oxidative DNA damage, there was also upregulation in DNA repair enzymes AP-endonuclease and DNA polymerase-β. By Western blotting, at 6 and 24 h, there was increased protein expression of AP-endonuclease (ratio to control: 3.24 at 6 h, 1.96 at 24 h) and DNA polymerase-β (ratio to control: 7.1 at 6 h, 1.34 at 24 h) following PA exposure. Upon exposure to PA with DMTU, the levels were decreased at 6 and 24 h (AP-endonuclease ratio to control: 1.51 at 6 h, 1.13 at 24h; DNA polymerase-β ratio to control: 1.65 at 6 h, 0.33 at 24 h). These results demonstrated that DNA repair enzymes AP-endonuclease and DNA polymerase-β were activated following PA exposure, providing indirect support that DNA damage was present and that the ‘appropriate’ response to injury was activated in podocytes. Decreased repair enzyme activation was seen in podocytes exposed to PA+DMTU, further supporting that DNA damage was oxidant-dependent. There was no significant difference in plasma blood urea nitrogen measurements in rats receiving PA versus PA+DMTU (data not shown). However, by day 7, rats receiving PA alone had significantly greater proteinuria than rats pretreated with DMTU (PA=28.73 mg/24h; PA+DMTU= 15.85 mg/24 h; P<0.05) (Figure 6a). To semiquantitatively assess the severity of glomerular injury, a glomerular sclerosis index was calculated by examining histopathologic sections stained with periodic acid-Schiff, as described previously,17.Raij L. Azar S. Keane W. Mesangial immune injury, hypertension, and progressive glomerular damage in Dahl rats.Kidney Int. 1984; 26: 137-143Abstract Full Text PDF PubMed Scopus (628) Google Scholar, 18.Saito T. Sumithran E. Glasgow E.F. Atkins R.C. The enhancement of aminonucleoside nephrosis by the co-administration of protamine.Kidney Int. 1987; 32: 691-699Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 19.Nakamura T. Obata J. Kimura H. et al.Blocking angiotensin II ameliorates proteinuria and glomerular lesions in progressive mesangioproliferative glomerulonephritis.Kidney Int. 1999; 55: 877-889Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar using the following scale: 0=normal glomerulus, 1=sclerotic area ≤25% of glomerular area, 2=25–50% of glomerular area, 3=50–75% of glomerular area, and 4=global sclerosis. The glomerular sclerosis index was calculated by multiplying the number of glomeruli with a sclerosis score of 1 by one, 2 by two, and so on. These values were summed to obtain the final glomerular sclerosis index. Consistent with the proteinuria data, there was a significant difference in glomerular sclerosis index among the groups (control=2, PA=34, PA+DMTU=7.8) (Figure 6b). The histopathology from the PA group was typified by marked acute tubular injury with proteinaceous casts and cytoplasmic protein absorption droplets, and evidence of early segmental glomerular sclerotic lesions (Figure 6d). These results provided supporting data that PA induced glomerular and tubular injury, mediated by ROS. To determine if oxidant-dependent DNA damage was induced in vivo, DNA damage was measured by the AP site assay on isolated glomeruli. At the earliest time point, there was a significant difference in the number of abasic sites between the PA and PA+DMTU groups (PA=17.13/100 000 bp, PA+DMTU=3.06/100 000 bp; P<0.05) (Figure 7a). Beyond this time point, the difference between the groups diminished and was no longer significant. To confirm the presence of DNA damage, immunostaining for 8-hydroxydeoxyguanosine (8-OHdG), an oxidized nucleotide commonly a byproduct of ROS-mediated DNA damage, was performed. In tissue from rats treated with PA (Figure 7c), there was a marked increase in 8-OHdG immunostaining compared to that in rats pretreated with DMTU (Figure 7d). Substantiating the previous findings, in addition to inducing DNA damage in vitro, PA also induced oxidant-mediated DNA damage in vivo. To determine if specific cell cycle checkpoint proteins were activated following PA-mediated oxidative DNA damage, immunostaining for checkpoint proteins in tissues harvested from experimental animals was performed. By immunohistochemistry, the number of p21WAF1/CIP1-positive cells by day 7 was increased in the PA group compared to control and PA+DMTU groups (p21WAF1/CIP1-positive cells per glomerulus: control 0.934, PA 4.061, PA+DMTU 2.026) (control versus PA, PA versus PA+DMTU: P<0.05) (Figure 8). Similarly, the number of p53-positive cells by day 7 was increased in the PA group compared to control and PA+DMTU groups (p53-positive cells per glomerulus: control 0.332, PA 5.673, PA+DMTU 2.373) (control versus PA, PA versus PA+DMTU: P<0.05) (Figure 9). Correlating with the in vitro data, cell cycle checkpoint proteins p21WAF1/CIP1 and p53 were activated following exposure to PA. With DMTU pretreatment, the activation of these specific checkpoint proteins was reduced.Figure 9Activation of cell cycle checkpoint protein p53 in PAN. By immunohistochemistry (original magnification × 40), the number of p53-positive cells was increased significantly in tissues from rats treated with PA compared with time-matched animals receiving PA+DMTU (arrows indicate positive cells). (a) Representative immunostaining for p53 in control tissues. (b and d) Representative immunostaining for p53 in tissues treated with PA only. (c and e) Representative immunostaining for p53 in tissues treated with PA and DMTU. (f) Quantification of p53-positive cells per glomerulus shown in graphic form (150 glomeruli counted per section × 6 animals in each group).View Large Image Figure ViewerDownload (PPT) Studies have shown that PA may induce podocyte apoptosis in a dose- and time-dependent manner.20.Sanwal V. Pandya M. Bhaskaran M. et al.Puromycin aminonucleoside induces glomerular epithelial cell apoptosis.Exp Mol Pathol. 2001; 70: 54-64Crossref PubMed Scopus (42) Google Scholar,21.Fernandez L. Romero M. Soto H. Mosquera J. Increased apoptosis in acute puromycin aminonucleoside nephrosis.Exp Nephrol. 2001; 9: 99-108Crossref PubMed Scopus (17) Google Scholar Cleavage of genomic DNA during apoptosis by specific endonucleases may yield double- and single-stranded breaks.22.Hengartner M.O. The biochemistry of apoptosis.Nature. 2000; 407: 770-776Crossref PubMed Scopus (6001) Google Scholar To confirm that PA-induced apoptosis was not the etiology of the DNA damage detected under our experimental conditions, designed specifically to avoid apoptosis initiation by utilizing low doses and short time courses of PA, the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was performed. Figure 10 shows the in vitro results. Following UV-C irradiation, the positive control for apoptosis induction, there were 200.75 TUNEL-positive cells per 1000. However, there was no statistically significant difference in the number of TUNEL-positive cells among the control (9.5/1000), PA (18.5/1000), and PA+DMTU (12.5/1000) groups at 6 h, the time point at which our studies showed the greatest degree of DNA damage. Figure 11 shows the in vivo results. TUNEL staining in tissue sections showed no statistically significant difference in the number of TUNEL-positive cells among the control (0.6/200 glomeruli), PA (1.8/200 glomeruli), and PA+DMTU (1.83/200 glomeruli) groups. These data support that apoptosis did not account for the DNA damage observed under our experimental design.Figure 11Apoptosis detection following PA exposure, in vivo. TUNEL staining of tissue sections. Representative images of tissue sections from rats in (a) control group versus (b and d) groups treated with PA alone or (c and e) PA+DMTU. (d and e) TUNEL-positive cells in the tubulointerstitium adjacent to glomeruli without positive cells. (f) Quantification of TUNEL-positive cells per 200 glomeruli counted. There is no significant difference in the number of TUNEL-positive cells between control, PA, and PA+DMTU treatment groups.View Large Image Figure ViewerDownload (PPT) The highly specialized and terminally differentiated podocyte typically does not proliferate in response to injury. The detrimental decline in podocyte number post-injury leads to glomerulosclerosis.2.Kriz W. Gretz N. Lemley K.V. Progression of glomerular diseases: is the podocyte the culprit?.Kidney Int. 1998; 54: 687-697Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, 3.Kim Y.H. Goyal M. Kurnit D. et al.Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat.Kidney Int. 2001; 60: 957-968Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 4.Pagtalunan M.E. Miller P.L. Jumping-Eagle S. et al.Podocyte loss and progressive glomerular injury in type II diabetes.J Clin Invest. 1997; 99: 342-348Crossref PubMed Scopus (808) Google Scholar, 5.Wharram B.L. Goyal M. Wiggins J.E. et al.Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene.J Am Soc Nephrol. 2005; 16: 2941-2952Crossref PubMed Scopus (521) Google Scholar, 6.Ichikawa I. Ma J. Motojima M. Matsusaka T. Podocyte damage damages podocytes: autonomous vicious cycle that drives local spread of glomerular sclerosis.Curr Opin Nephrol Hypertens. 2005; 14: 205-210Crossref PubMed Scopus (72) Google Scholar Our group has focused on the mechanisms underlying the relative inability of podocytes to proliferate in response to most types of injury. Previously, we have shown that abnormalities in DNA synthesis, in part, underlie decreased podocyte number.23.Pippin J.W. Durvasula R. Petermann A. et al.DNA damage is a novel response to sublytic complement C5b-9-induced injury in podocytes.J Clin Invest. 2003; 111: 877-885Crossref PubMed Scopus (113) Google Scholar This is largely owing to specific cyclin-dependent kinase inhibitors.24.Shankland S.J. Floege J. Thomas S.E. et al.Cyclin kinase inhibitors are increased during experimental membranous nephropathy: potential role in limiting glomerular epithelial cell proliferation in vivo.Kidney Int. 1997; 52: 404-413Abstract Full Text PDF PubMed Scopus (108) Google Scholar A major finding in this study was that PA induced podocyte DNA damage, both in vitro and in vivo, accompanied by an increase in cell cycle checkpoint proteins p53 and p21WAF1/Cip1. We also showed that increased ROS mediated this effect. In PAN, the glomerular injury acutely is mediated via overproduction of ROS,8.Diamond J.R. Bonventre J.V. Karnovsky M.J. A role for oxygen free radicals in aminonucleoside nephrosis.Kidney Int. 1986; 29: 478-483Abstract Full Text PDF PubMed Scopus (239) Google Scholar, 9.Thakur V. Walker P.D. Shah S.V. Evidence suggesting a role for hydroxyl radical in puromycin aminonucleoside-induced proteinuria.Kidney Int. 1988; 34: 494-499Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 10.Gwinner W. Landmesser U. Brandes R.P. et al.Reactive oxygen species and antioxidant defense in puromycin aminonucleoside glomerulopathy.J Am Soc Nephrol. 1997; 8: 1722-1731PubMed Google Scholar, 11.Beaman M. Birtwistle R. Howie A.J. et al.The role of superoxide anion and hydrogen peroxide in glomerular injury induced by puromycin aminonucleoside in rats.Clin Sci (London). 1987; 73: 329-332Crossref PubMed Scopus (64) Google Scholar, 12.Shibouta Y. Terashita Z. 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Antioxidants protect podocyte foot processes in puromycin aminonucleoside-treated rats.J Am Soc Nephrol. 1994; 4: 1974-1986PubMed Google Scholar and grape seed extract.27.Mattoo T.K. Kovacevic L. Effect of grape seed extract on puromycin-aminonucleoside-induced nephrosis in rats.Pediatr Nephrol. 2003; 18: 872-877Crossref PubMed Scopus (8) Google Scholar These studies have shown beneficial effects of antioxidants, with reduction in proteinuria and improvement in glomerular morphologic changes including foot process effacement. ROS are also important mediators in the pathogenesis of glomerular injury in other experimental animal models including passive Heymann nephritis,28.Neale T.J. Ojha P.P. Exner M. et al.Proteinuria in passive Heymann nephritis is associated with lipid peroxidation and formation of adducts on type IV collagen.J Clin Invest. 1994; 94: 1577-1584Crossref PubMed Scopus (127) Google Scholar anti-Thy1 mesangial proliferative glomerulonephritis,29.Budisavljevic M.N. Hodge L. Barber K. et al.Oxidative stress in the pathogenesis of experimental mesangial proliferative glomerulonephritis.Am J Physiol Renal Physiol. 2003; 285: F1138-F1148Crossref PubMed Scopus (54) Google Scholar and Mpv17 gene-inactivated steroid-resistant focal segmental glomerulosclerosis.30.Binder C.J. Weiher H. Exner M. Kerjaschki D. Glomerular overproduction of oxygen radicals in Mpv17 gene-inactivated mice causes podocyte foot process flattening and proteinuria: a model of steroid-resistant nephrosis sensitive to radical scavenger therapy.Am J Pathol. 1999; 154: 1067-1075Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar A major source of ROS in PA-induced injury is the podocyte itself, with generation of H2O2, OH•, superoxide anion radical, and lipid peroxidation products.20.Sanwal V. Pandya M. Bhaskaran M. et al.Puromycin aminonucleoside induces glomerular epithelial cell apoptosis.Exp Mol Pathol. 2001; 70: 54-64Crossref PubMed Scopus (42) Google Scholar,31.Kawaguchi M. Yamada M. Wada H. Okigaki T. Roles of active oxygen species in glomerular epithelial cell injury in vitro caused by puromycin aminonucleoside.Toxicology. 1992; 72: 329-340Crossref PubMed Scopus (55) Google Scholar Increased ROS are associated with amplification in the activities of antioxidant enzymes including catalase, superoxide dismutase, and glutathione peroxidase.32.Vega-Warner V. Ransom R.F. Vincent A.M. et al.Induction of antioxidant enzymes in m
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