Puromycin aminonucleoside induces oxidant-dependent DNA damage in podocytes in vitro and in vivo

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1 originl rticle & 6 Interntionl Society of Nephrology Puromycin minonucleoside induces oxidnt-dependent DNA dmge in podocytes in vitro nd in vivo CB Mrshll 1, JW Pippin 1, RD Krofft 1 nd SJ Shnklnd 1 1 Division of Nephrology, University of Wshington, Settle, Wshington, USA A decline in podocyte number correltes with progression to glomerulosclerosis. A mechnism underlying reduced podocyte number is the podocyte s reltive inbility to proliferte in response to injury. Injury by the podocyte toxin puromycin minonucleoside () is medited vi rective oxygen species (ROS). The precise role of ROS in the pthogenesis of -induced glomerulosclerosis remins to be determined. We sought to exmine whether -induced ROS cused podocyte DNA dmge, possibly ccounting for the podocyte s inbility to proliferte in response to. In vitro, podocytes were exposed to, with or without the rdicl scvenger 1,3-dimethyl--thioure (DMTU). In vivo, mle Sprgue Dwley rts were divided into experimentl groups (n ¼ 6/group/time point):, with DMTU, nd control, killed t dys 1.5, 3, or 7. DNA dmge ws mesured by DNA precipittion, purinic/pyrimidinic site, Comet, nd 8-hydroxydeoxygunosine ssys. Cell cycle checkpoint protein upregultion (by immunostining nd Western blotting), histopthology, nd biochemicl prmeters were exmined. DNA dmge ws incresed in cultured podocytes tht received, but not with DMTU. exposure ctivted specific cell cycle checkpoint proteins, with ttenution by DMTU. DNA repir enzymes were ctivted, providing evidence for ttempted DNA repir. The -treted nimls developed worse proteinuri nd histopthologic disese nd exhibited more DNA dmge thn the DMTU pretreted group. No significnt poptosis ws detected by terminl deoxynucleotidyl trnsferse-medited dutp nick end-lbeling stining. A mechnism underlying the lck of podocyte prolifertion following -induced injury in vitro nd in vivo my be ROS-medited DNA dmge, with upregultion of specific cell cycle checkpoints leding to cell cycle rrest. Kidney Interntionl (6) 7, doi:.38/sj.ki.51965; published online 11 October 6 Correspondence: SJ Shnklnd, Division of Nephrology, University of Wshington, 1959 NE Pcific Street, Box 35651, Settle, Wshington 98195, USA. E-mil: sturtjs@u.wshington.edu Received 19 October 5; revised 17 August 6; ccepted 18 August 6; published online 11 October 6 KEYWORDS: podocyte; DNA dmge; puromycin minonucleoside nephropthy; rective oxygen species; prolifertion; glomerulosclerosis The glomerulr viscerl epithelil cell, or podocyte, is highly specilized nd terminlly differentited cell tht forms criticl prt of the glomerulr filtrtion brrier nd functions to prevent urinry protein lekge, mintin glomerulr cpillry loop integrity, oppose intrcpillry hydrosttic pressure, nd synthesize the glomerulr bsement membrne. 1 Common to mny humn kidney diseses nd experimentl niml models is strong ssocition between podocyte injury nd glomerulosclerosis. Studies hve demonstrted tht decline in podocyte number strongly correltes with, nd likely underlies, progression to glomerulosclerosis. 6 Two importnt mechnisms for podocyte depletion following injury re poptosis nd detchment. Podocyte loss leds to res of denuded bsement membrne, culminting in proteinuri, the development of focl glomerulosclerosis, nd progressive deteriortion in kidney function. Owing to their highly differentited stte, mture podocytes redily do not proliferte in vivo. Therefore, the pprent lck of prolifertion in response to injury contributes to reduced podocyte number. At the cell cycle level, reduced prolifertion is consequence of either n bnormlity in DNA synthesis, secondry to G 1 /S checkpoint rrest, or reduced mitosis, secondry to G /M checkpoint rrest. DNA dmge triggers ctivtion of cell cycle checkpoints, moleculr pthwys tht monitor pssge through the cell cycle nd generte puses in cell cycle progression when DNA dmge is present. Thus, DNA dmge leds to cell cycle rrest, permitting the cell sufficient time for DNA repir or other cell fte decisions, including poptosis or entry into permnent senescence-like stte, thereby limiting prolifertion. 7 Cell cycle rrest following DNA dmge is medited, in prt, by tumor suppressor p53 nd cyclin-dependent kinse inhibitor p1 WAF1/CIP1. It is unknown whether the presence of DNA dmge, with subsequent cell cycle rrest, ccounts for the podocyte s limited prolifertion in non-immune-medited podocyte injury, nmely puromycin minonucleoside nephropthy (N). 196 Kidney Interntionl (6) 7,

2 Puromycin minonucleoside (), podocyte toxin used to induce experimentl miniml chnge disese progressing to focl segmentl glomerulosclerosis in rts, leds to podocyte foot process effcement, nd mssive proteinuri within 7 dys. In vivo studies hve supported tht, cutely, -induced glomerulr injury is medited vi overproduction of rective oxygen species (ROS), 8 14 which then my interct with biomolecules, leding to modifiction nd potentilly deleterious cellulr consequences. 15 This study ws designed to exmine whether DNA dmge occurs in N, if DNA dmge ccounts for the inbility of podocytes to proliferte in response to the toxin, nd which cell cycle regultory proteins my underlie this response. RESULTS DNA dmge in cultured podocytes exposed to, oxidnt-dependent To determine whether induced DNA dmge in vitro, growth-restricted podocytes were exposed to, nd DNA dmge ws mesured by three methods. DNA precipittion ssy results re shown in Figure 1. As there is bsl level of oxidtive DNA dmge owing to non-pthogenic metbolic processes in helthy erobic orgnisms, 15,16 the mount of single-strnded DNA (ssdna) present in control cells t the erliest time point ws considered the reference. This bsl level of ssdna ws subtrcted from subsequent vlues for ll conditions nd time points. At 6 h, there ws significnt difference in the percent ssdna between podocytes exposed to versus control conditions (control ¼.43%, ¼ 37.8%; Po.5). There ws no significnt difference between these two groups t nd 4 h. Podocytes exposed to þ 1,3- dimethyl--thioure (DMTU) exhibited lower percent ssdna t 6 nd 4 h thn cells exposed to lone, with vlues flling below the bsl level observed in control cells. To confirm tht medites DNA dmge, the results of the AP (purinic/pyrimidinic or bsic) site ssy re shown in Figure. With exposure, there were n incresed number of bsic sites in DNA t 6 nd 18 h (vlues per bse pirs: 6 h: control ¼., ¼ , Po.5; 18 h: control ¼ 1.396, ¼.689, Po.5). In podocytes exposed to þ DMTU, DNA dmge ws lessened, with no significnt increse in bsic sites when compred to controls (6 h:.78, 18 h: 1.441). Cells exposed to H O, the positive control for oxidnt-medited DNA dmge, exhibited levels of bsic sites intermedite between control nd groups (dt not shown). As further corrobortion tht medites DNA dmge in podocytes, the results of the Comet ssy re shown in Figure 3. Following exposure to experimentl conditions, podocytes underwent hrvesting, immobiliztion in grose, gentle lysis, nd lkline electrophoresis. With exposure, there ws n increse in cells exhibiting long comet tils, when compred to control group (Figure 3 nd c), t nd 6 h (vlues per cells: h: control ¼.8, ¼ 7, Po.5; 6 h: control ¼ 1., ¼, Po.5) (Figure 3d). In podocytes exposed to þ DMTU, DNA dmge ws lessened, with decrese in long comet tils when compred to -exposed cells ( h: þ DMTU ¼ 1.8, Po.5; 6 h: þ DMTU ¼ 7, Po.5) (Figure 3b). The results from the DNA precipittion, AP site, nd Comet ssys demonstrted tht cused DNA dmge in cultured podocytes. This DNA dmge ws ttenuted by pre-exposure to DMTU, providing support tht medited oxidtive DNA dmge. Cell cycle checkpoint ctivtion in cultured podocytes exposed to, oxidnt-dependent In contrst to other glomerulr cells, podocytes typiclly do not proliferte following injury. To test the hypothesis tht following -medited oxidtive DNA dmge, cell cycle % ssdna Time (h) Figure 1 -induced DNA dmge in cultured podocytes, ttenuted by pre-exposure to DMTU: DNA precipittion ssy. The ssdna ws mesured by the DNA precipittion ssy, performed on cultured podocytes following exposure to 3 mg/ml t time points, 6, nd 4 h, with or without pre-exposure to the ntioxidnt DMTU mm. induced significnt DNA dmge t time points 6 nd 4 h, n effect reduced by DMTU. As there is bsl quntity of ssdna owing to endogenous processes, the level of ssdna present in the control cells t the erliest time point ws the reference point for ll other mesurements nd this level of bseline ssdna ws subtrcted from other vlues. *Po.5, for versus þ DMTU. Absic sites (per bp) Time (h) Figure -induced DNA dmge in cultured podocytes, ttenuted by pre-exposure to DMTU: AP site ssy. Absic sites were mesured by the AP site ssy in cultured podocytes following exposure to 3 mg/ml t time points, 6, 18, nd 4 h, with or without pre-exposure to DMTU mm. Following exposure to, t 6 nd 18 h, there ws sttisticlly significnt increse in bsic sites compred with time-mtched controls, n effect reduced by DMTU. *Po.5, for versus þ DMTU. Kidney Interntionl (6) 7,

3 b c d Positive cells/ Time (h) Figure 3 -induced DNA dmge in cultured podocytes, ttenuted by pre-exposure to DMTU: Comet (single-cell gel electrophoresis) ssy. Dmged DNA ws mesured by the Comet ssy in cultured podocytes following exposure to 3 mg/ml t time point, 6, nd 4 h, with or without pre-exposure to DMTU mm (originl mgnifiction ). () Representtive microgrph of fluorescent DNA stin of control cells, showing undmged nd supercoiled DNA remining within the nucler cell membrne. (b) Representtive microgrph of fluorescent DNA stin of þ DMTU-exposed cells, showing mild degree of dentured DNA frgments migrting out from cell. (c) Representtive microgrph of fluorescent DNA stin of -exposed cells, showing dentured DNA frgments migrting out from cell in long comet til. (d) Quntifiction of podocytes with long comet tils. Following exposure to, t, 6, nd 4 h, there ws sttisticlly significnt increse in the number of cells with long comet tils compred with time-mtched controls. DMTU significntly lessened -induced DNA dmge t time points nd 6 h. *Po.5, for versus þ DMTU. p1 protein expression c p1 Actin p53 GAPDH 6 h 4 h b p53 protein expression h 4 h 6 h 4 h Figure 4 Activtion of cell cycle checkpoint proteins in cultured podocytes, following exposure to, ttenuted by DMTU. () p1 WAF1/CIP1 protein expression. The protein expression of p1 WAF1/CIP1 ws incresed by rtio of 5.4 nd.8 in -exposed cells, compred to time-mtched controls, t time points 6 nd 4 h, respectively. With DMTU pre-exposure, the rtio decresed to.98 nd 1., t time points 6 nd 4 h, respectively. Vlues re verges of three seprte experiments nd re normlized for differences in protein loding. (b) p53 protein expression. The protein expression of p53 ws incresed by rtio of.7 nd 1.91 in -exposed cells, compred to time-mtched controls, t time points 6 nd 4 h, respectively. With DMTU pre-exposure, the rtio decresed to 1.16 nd.89, t time points 6 nd 4 h, respectively. Vlues re verges of three seprte experiments nd re normlized for differences in protein loding. (c) Western blot nlysis. Representtive Western blots for p1 WAF1/CIP1 nd p53 re shown. Actin nd glycerldehyde- 3-phosphte dehydrogense were used s housekeeping proteins to ensure equl protein loding. The results show tht p1 WAF1/CIP1 nd p53 protein levels incresed following exposure to. This ws reduced by DMTU. checkpoint proteins re ctivted thereby limiting prolifertion, Western blotting ws performed on extrcts from cultured podocytes (Figure 4). At 6 nd 4 h fter exposure, protein expression ws incresed for p1 WAF1/CIP1 (rtio to control: 5.4 t 6 h,.8 t 4 h) nd p53 (rtio to control:.7 t 6 h, 1.91 t 4 h). In podocytes pretreted with DMTU, the levels of p1 WAF1/CIP1 (rtio to control:.98 t 6 h, 1. t 4 h) nd p53 (rtio to control: 1.16 t 6 h,.89 t 4 h) were reduced compred to the -only group. The expression levels of checkpoint proteins checkpoint kinse 1 (CHK-1), checkpoint kinse (CHK-), nd growth rrest, DNA dmge-inducible-45 were not significntly different between nd þ DMTU groups t 6 nd 4 h (dt not shown). These results estblished tht specific cell cycle checkpoint proteins were ctivted in response to -medited oxidtive DNA dmge. Following pre-exposure to DMTU, did not induce the sme level of cell cycle checkpoint protein ctivtion. DNA repir enzyme ctivtion in cultured podocytes exposed to, oxidnt-dependent To determine if podocytes possessed the cpcity to repir dmged DNA, specific DNA repir enzymes were evluted. Figure 5 shows tht, in ddition to upregultion of cell cycle checkpoint proteins in response to -medited oxidtive DNA dmge, there ws lso upregultion in DNA repir enzymes AP-endonuclese nd DNA polymerse-b. By Western blotting, t 6 nd 4 h, there ws incresed protein 1964 Kidney Interntionl (6) 7,

4 APE protein expression c h 4 h DNA poly-β protein expression h 4 h 6 h 4 h APE Tubulin DNA poly-β Tubulin Figure 5 Induction of DNA repir enzymes purinic/pyrimidinic endonuclese (APE) nd DNA polymerse-b (DNA poly-b) following tretment with, ttenuted by DMTU. () APE protein expression. The protein expression of APE ws incresed by rtio of 3.4 nd 1.96 in -exposed cells, compred with timemtched controls, t time points 6 nd 4 h, respectively. With DMTU pre-exposure, the rtio decresed to 1.51 nd 1.13, t time points 6 nd 4 h, respectively. Vlues re verges of three seprte experiments nd re normlized for differences in protein loding. (b) DNA poly-b protein expression. The protein expression of DNA poly-b ws incresed by rtio of 7.1 nd 1.34 in -exposed cells, compred with time-mtched controls, t time points 6 nd 4 h, respectively. With DMTU pre-exposure, the rtio ws decresed to 1.65 nd.33, t time points 6 nd 4 h, respectively. Vlues re verges of three seprte experiments nd re normlized for differences in protein loding. (c) Western blot nlysis. Representtive Western blots for APE nd DNA poly-b re shown. Tubulin ws used s housekeeping protein to ensure equl protein loding. The results show tht APE nd DNA poly-b protein levels incresed following exposure to. This ws reduced by DMTU. Arrows indicte bnd of interest. b expression of AP-endonuclese (rtio to control: 3.4 t 6 h, 1.96 t 4 h) nd DNA polymerse-b (rtio to control: 7.1 t 6 h, 1.34 t 4 h) following exposure. Upon exposure to with DMTU, the levels were decresed t 6 nd 4 h (AP-endonuclese rtio to control: 1.51 t 6 h, 1.13 t 4h; DNA polymerse-b rtio to control: 1.65 t 6 h,.33 t 4 h). These results demonstrted tht DNA repir enzymes AP-endonuclese nd DNA polymerse-b were ctivted following exposure, providing indirect support tht DNA dmge ws present nd tht the pproprite response to injury ws ctivted in podocytes. Decresed repir enzyme ctivtion ws seen in podocytes exposed to þ DMTU, further supporting tht DNA dmge ws oxidnt-dependent. Proteinuri nd histopthology in N, oxidnt-dependent There ws no significnt difference in plsm blood ure nitrogen mesurements in rts receiving versus þ DM- TU (dt not shown). However, by dy 7, rts receiving lone hd significntly greter proteinuri thn rts pretreted with DMTU ( ¼ 8.73 mg/4h; þ DMTU ¼ mg/4 h; Po.5) (Figure 6). To semiquntittively ssess the severity of glomerulr injury, glomerulr sclerosis index ws clculted by exmining histopthologic sections stined with periodic cid-schiff, s described previously, using the following scle: ¼ norml glomerulus, 1 ¼ sclerotic re p5% of glomerulr re, ¼ 5 5% of glomerulr re, 3 ¼ 5 75% of glomerulr re, nd 4 ¼ globl sclerosis. The glomerulr sclerosis index ws clculted by multiplying the number of glomeruli with sclerosis score of 1 by one, by two, nd so on. These vlues were summed to obtin the finl glomerulr sclerosis index. Protein (mg/4 h) Sclerosis index Proteinuri b P=. * P=. P=.4 Bseline Dy 1.5 Dy 3 Dy 7 Time (dys) Degree of sclerosis/ glomeruli c d e Tretment group Figure 6 Protein excretion nd histopthologic chnges by periodic cid-schiff (S) stining following tretment with, with or without pre-exposure to DMTU. () Proteinuri. By dy 7, rts receiving lone hd sttisticlly significntly greter degree of protein excretion thn rts pretreted with DMTU. *Po.5. (b) Sclerosis index. Tissue from rts receiving lone exhibited greter disese severity s semiquntittively ssessed by sclerosis index. The number of erly glomerulr sclerotic lesions ws reduced in the þ DMTU group (Po.5). (c) Histopthology (originl mgnifiction 4). Representtive microgrphs of S stining of tissues re shown (c ¼ control, d ¼, nd e ¼ þ DMTU). Tissue from rts receiving lone exhibited greter disese severity thn rts tht received þ DMTU. Kidney Interntionl (6) 7,

5 Absic sites (per bp) Dy 1.5 Dy 3 Dy 7 Tretment group (n=6) b c d Figure 7 DNA dmge induced in N, ttenuted by pretretment with DMTU. () AP site ssy. The number of bsic sites, s determined by the AP site ssy performed on DNA hrvested from isolted glomeruli, ws significntly incresed in rts treted with compred with rts receiving þ DMTU, t dy 1.5. *Po.5. The difference in bsic sites ws not significnt t lter time points. (b d) 8-OHdG immunostining (originl mgnifiction 4): (b) Representtive immunostining for 8-OHdG in control tissues. (c nd d) Representtive immunostining for 8-OHdG in tissues from (c) rts treted with lone nd (d) rts treted with þ DMTU, respectively (rrows indicte positive cells). In tissue from rts treted with lone, there ws mrked increse in 8-OHdG immunostining compred to levels in tissues from rts exposed to þ DMTU or control conditions. Consistent with the proteinuri dt, there ws significnt difference in glomerulr sclerosis index mong the groups (control ¼, ¼ 34, þ DMTU ¼ 7.8) (Figure 6b). The histopthology from the group ws typified by mrked cute tubulr injury with proteinceous csts nd cytoplsmic protein bsorption droplets, nd evidence of erly segmentl glomerulr sclerotic lesions (Figure 6d). These results provided supporting dt tht induced glomerulr nd tubulr injury, medited by ROS. DNA dmge in N, oxidnt-dependent To determine if oxidnt-dependent DNA dmge ws induced in vivo, DNA dmge ws mesured by the AP site ssy on isolted glomeruli. At the erliest time point, there ws significnt difference in the number of bsic sites between the nd þ DMTU groups ( ¼ 17.13/ bp, þ DMTU ¼ 3.6/ bp; Po.5) (Figure 7). Beyond this time point, the difference between the groups diminished nd ws no longer significnt. To confirm the presence of DNA dmge, immunostining for 8-hydroxydeoxygunosine (8-OHdG), n oxidized nucleotide commonly byproduct of ROS-medited DNA dmge, ws performed. In tissue from rts treted with (Figure 7c), there ws mrked increse in 8-OHdG immunostining compred to tht in rts pretreted with DMTU (Figure 7d). Substntiting the previous findings, in ddition to inducing DNA dmge in vitro, lso induced oxidnt-medited DNA dmge in vivo. Cell cycle checkpoint ctivtion in N, oxidnt-dependent To determine if specific cell cycle checkpoint proteins were ctivted following -medited oxidtive DNA dmge, immunostining for checkpoint proteins in tissues hrvested from experimentl nimls ws performed. By immunohistochemistry, the number of p1 WAF1/CIP1 -positive cells by dy 7 ws incresed in the group compred to control nd þ DMTU groups (p1 WAF1/CIP1 -positive cells per glomerulus: control.934, 4.61, þ DMTU.6) (control versus, versus þ DMTU: Po.5) (Figure 8). Similrly, the number of p53-positive cells by dy 7 ws incresed in the group compred to control nd þ DMTU groups (p53-positive cells per glomerulus: control.33, 5.673, þ DMTU.373) (control versus, versus þ DMTU: Po.5) (Figure 9). Correlting with the in vitro dt, cell cycle checkpoint proteins p1 WAF1/ CIP1 nd p53 were ctivted following exposure to. With DMTU pretretment, the ctivtion of these specific checkpoint proteins ws reduced. Apoptosis detection, in vitro nd in vivo Studies hve shown tht my induce podocyte poptosis in dose- nd time-dependent mnner.,1 Clevge of genomic DNA during poptosis by specific endonucleses my yield double- nd single-strnded breks. To confirm tht -induced poptosis ws not the etiology of the DNA dmge detected under our experimentl conditions, designed specificlly to void poptosis initition by utilizing low doses nd short time courses of, the terminl deoxynucleotidyl trnsferse-medited dutp nick end-lbeling (TUNEL) ssy ws performed. Figure shows the in vitro results. Following UV-C irrdition, the positive control for poptosis induction, there were.75 TUNEL-positive cells per. However, there ws no sttisticlly significnt difference in the number of TUNEL-positive cells mong the control (9.5/), (18.5/), nd þ DMTU (1.5/ ) groups t 6 h, the time point t which our studies showed the gretest degree of DNA dmge. Figure 11 shows the in vivo results. TUNEL stining in tissue sections showed no sttisticlly significnt difference in the number of TUNEL-positive cells mong the control (.6/ glomeruli), 1966 Kidney Interntionl (6) 7,

6 b c d e f Number of p1-positive cells Number of p1-positive cells per glomerulus P=.1 Tretment group P=.3 Figure 8 Activtion of cell cycle checkpoint protein p1 WAF1/CIP1 in N. By immunohistochemistry (originl mgnifiction 4), the number of p1 WAF1/CIP1 -positive cells ws incresed significntly in tissues from rts treted with compred with time-mtched nimls receiving þ DMTU (rrows indicte positive cells). () Representtive immunostining for p1 WAF1/CIP1 in control tissues. (b nd d) Representtive immunostining for p1 WAF1/CIP1 in tissues treted with only. (c nd e) Representtive immunostining for p1 WAF1/CIP1 in tissues treted with þ DMTU. (f) Quntifiction of p1 WAF1/CIP1 -positive cells per glomerulus shown in grphic form (15 glomeruli counted per section 6 nimls in ech group). (1.8/ glomeruli), nd þ DMTU (1.83/ glomeruli) groups. These dt support tht poptosis did not ccount for the DNA dmge observed under our experimentl design. DISCUSSION The highly specilized nd terminlly differentited podocyte typiclly does not proliferte in response to injury. The detrimentl decline in podocyte number post-injury leds to glomerulosclerosis. 6 Our group hs focused on the mechnisms underlying the reltive inbility of podocytes to proliferte in response to most types of injury. Previously, we hve shown tht bnormlities in DNA synthesis, in prt, underlie decresed podocyte number. 3 This is lrgely owing to specific cyclin-dependent kinse inhibitors. 4 A mjor finding in this study ws tht induced podocyte DNA dmge, both in vitro nd in vivo, ccompnied by n increse in cell cycle checkpoint proteins p53 nd p1 WAF1/ Cip1. We lso showed tht incresed ROS medited this effect. In N, the glomerulr injury cutely is medited vi overproduction of ROS, 8 13 which then my interct with biomolecules including DNA, leding to potentilly deleterious cellulr consequences. Indirect support for the importnce of ROS in the pthogenesis of N comes from interventionl studies utilizing therpy with ntioxidnts, including llopurinol, 8 vitmin E, 5,6 selenium, 6 DMTU, 9,13 nd grpe seed extrct. 7 These studies hve shown beneficil effects of ntioxidnts, with reduction in proteinuri nd improvement in glomerulr morphologic chnges including foot process effcement. ROS re lso importnt meditors in the pthogenesis of glomerulr injury in other experimentl niml models including pssive Heymnn nephritis, 8 nti- Thy1 mesngil prolifertive glomerulonephritis, 9 nd Mpv17 gene-inctivted steroid-resistnt focl segmentl glomerulosclerosis. 3 A mjor source of ROS in -induced injury is the podocyte itself, with genertion of H O,OH *, superoxide nion rdicl, nd lipid peroxidtion products.,31 Incresed ROS re ssocited with mplifiction in the ctivities of ntioxidnt enzymes including ctlse, superoxide dismutse, nd glutthione peroxidse. 3 my lso induce podocyte poptosis in dose- nd time-dependent mnner, with poptosis prtilly inhibited by ROS scvengers. Podocyte necrosis hs been observed when high concentrtions of re used.,1 Furthermore, in vivo studies hve demonstrted incresed poptosis in the glomeruli of nephrotic nimls, ccompnied by incresed expression of poptosis-ssocited proteins. 33 Kidney Interntionl (6) 7,

7 b c d e f Number of p53-positive cells Number of p53-positive cells per glomerulus P=.1 P=.4 Tretment group Figure 9 Activtion of cell cycle checkpoint protein p53 in N. By immunohistochemistry (originl mgnifiction 4), the number of p53-positive cells ws incresed significntly in tissues from rts treted with compred with time-mtched nimls receiving þ DMTU (rrows indicte positive cells). () Representtive immunostining for p53 in control tissues. (b nd d) Representtive immunostining for p53 in tissues treted with only. (c nd e) Representtive immunostining for p53 in tissues treted with nd DMTU. (f) Quntifiction of p53-positive cells per glomerulus shown in grphic form (15 glomeruli counted per section 6 nimls in ech group). Prior studies hve estblished tht induces ultrstructurl chnges in podocytes including desqumtion from the glomerulr bsement membrne, cytoskeletl disggregtion, 36 dmge to glomerulr bsement membrne chrge nd size brriers, 37,38 nd ltertion of dhesion molecule 3 b 1 integrin distribution nd cellulr content, 39,4 ll thought to be medited, t lest in prt, by overproduction of ROS. The present study introduced previously undescribed mechnism of -induced podocyte injury nd dysfunction, nmely DNA dmge. In cultured podocytes, medited DNA dmge, mesured by the DNA precipittion, AP site, nd Comet ssys. The ttenution of DNA dmge by DMTU provided evidence tht the DNA dmge ws medited vi ROS. -induced DNA dmge ws gretest t time point 6 h. Upregultion of DNA repir enzymes, with subsequent repir of dmged DNA, my ccount for the lower levels of DNA dmge t lter time points. Cell cycle checkpoints re signling pthwys tht mke up the eukryotic cell s response to dmged DNA nd function to coordinte cell cycle progression with DNA repir, chromtin remodeling, nd trnscriptionl progrms. DNA dmge-induced cell cycle rrest provides time for DNA repir or other cell fte decisions, including poptosis or permnent exit from the cell cycle by cellulr senescence. 41 In this study, -induced DNA dmge led to ctivtion of specific checkpoint proteins, notbly p53 nd p1 WAF1/CIP1, in cultured podocytes nd in experimentl N. A possible explntion for why CHK-1 nd CHK- expression did not substntilly differ between podocytes treted with versus þ DMTU is tht diversity exists in the vilble rry of checkpoint pthwys between cells depending on their distinct cell cycle phse. Bsed on the expression, subcellulr locliztion, nd stbility of specific proteins, the vilbility of mny checkpoint components my be limited or lcking t some cell cycle stges. 41 Although CHK-1 nd CHK- potentilly could be upstrem meditors of p53, p53 my lso be ctivted by other checkpoint-trnsducing kinses including mutted in txi telngiectsi nd mutted in txi telngiectsi nd Rd3- relted. AP-endonuclese nd DNA polymerse-b, two DNA repir enzymes importnt in the process of bse excision repir, were incresed in cultured podocytes following exposure. Activtion of repir enzymes suggested tht DNA dmge ws present nd tht the pproprite responses to injury were triggered in podocytes. With pre-exposure to DMTU, this ctivtion ws lessened significntly, providing further support tht DNA dmge ws medited by ROS. DNA dmge ws significntly incresed in glomeruli isolted from -treted nimls versus the glomeruli of nimls receiving þ DMTU. The difference in the number 1968 Kidney Interntionl (6) 7,

8 c b UV-C d contribute to reduced podocyte number nd res of denuded bsement membrne, ultimtely leding to the development of glomerulosclerosis nd the progressive deteriortion in kidney function. Whether ntioxidnt strtegies will be useful therpeutic interventions in the setting of injury induced by oxidizing podocyte toxins is uncler. There re mny potentil ntioxidnt therpies, rnging from endogenous enzymes/ compounds (glutthione peroxidse-like molecules, epoetin) nd trce elements (selenium) to drugs (ngiotensinconverting enzyme inhibitors, b-blockers, sttins, N-cetylcysteine, nd metl ion cheltors) nd dietry ntioxidnts (phytoestrogens). 4 Further studies re needed to clrify the therpeutic potentil of ntioxidnts in non-immunemedited podocyte injury. e TUNEL-positive cells/ cells TUNEL stining, in vitro P=.1 P=.1 UV-C Tretment group Figure Apoptosis detection following exposure, in vitro. TUNEL stining of cultured podocytes. Representtive imges of podocytes exposed to () negtive control conditions (b) positive control, UV-C irrdition (c), nd (d) þ DMTU. Arrows indicte TUNEL-positive cells. (originl mgnifiction ). (e) Quntifiction of TUNEL-positive cells per cells counted. There is no significnt difference in the number of TUNEL-positive cells between negtive control,, nd þ DMTU tretment groups (time point 6 h shown). of bsic sites between the two groups ws most notble t the erliest time point. By dy 7, the level of bsic sites hd declined in both groups. This decline my be ttributble to repir of dmged DNA. TUNEL stining confirmed tht induced poptosis ws not significnt mechnism of injury under our experimentl conditions. We conclude tht podocyte injury in the N model is, in prt, medited by the oxidizing properties of. In ddition to the DNA dmge found in the pssive Heymnn nephritis model of immune-medited podocyte injury, 3 we demonstrted tht DNA dmge my be more generlized response to injury. The overproduction of ROS induced podocyte DNA dmge, leding to ctivtion of checkpoint pthwys to rrest or dely cell cycle progression. The resulting cell cycle rrest my be mechnism underlying the podocyte s reltive inbility to proliferte following induced injury. In combintion with -induced podocyte loss from other mechnisms, including poptosis,31,33 nd detchment, 35 the lck of podocyte prolifertion my MATERIALS AND METHODS Primry ntibodies Antibodies for Western blotting nd immunostining included mouse monoclonl ntibodies: nti-p1 WAF1/CIP1 (BD Biosciences, Sn Diego, CA, USA), nti-p53 (BD Biosciences; Oncogene Reserch Products, Sn Diego, CA, USA), nti-dna polymerse-b (Alph Dignostic Interntionl, Sn Antonio, TX, USA), nti-ap-endonuclese (Trevigen, Githersburg, MD, USA), nti-8-ohdg (Trevigen), nti-b-tubulin (Sigm-Aldrich, St Louis, MO, USA), nti-ctin (Chemicon Interntionl, Temecul, CA, USA), nd nti-glycerldehyde-3-phosphte dehydrogense (Abcm, Cmbridge, MA, USA). Mouse podocytes in culture A conditionlly immortlized mouse podocyte cell line isolted from H-K b -tsa58 trnsgenic mice kidneys (Jckson Lbortory, Br Hrbor, ME, USA) ws used in vitro In this cell line, g-interferon-inducible H-K b promoter controls temperturesensitive SV4 lrge T-cell ntigen. To induce prolifertion, cells were grown on Primri pltes (VWR Interntionl, West Chester,, USA) coted with collgen I (BD Biosciences) t 331C in Rosewell Prk Memoril Institute 164 medi (Invitrogen, Grnd Islnd, NY, USA) supplemented with % fetl clf serum (Hyclone, Logn, UT, USA), with dded recombinnt mouse g-interferon 5 U/ml (Roche, Indinpolis, IN, USA). 46 To induce the quiescent, differentited phenotype, cells were grown t 371C without g-interferon (growth-restrictive condition). Experiments were performed t lest three times, on growth-restricted dys 1 14, utilizing more thn one cell clone to verify reproducibility of results. The cell lines were derived in our lb, identified nd chrcterized by immunostining nd reverse trnscription-polymerse chin rection, s described previously. 47 DNA dmge detection in vitro following exposure to Following preliminry dose-defining studies, the DNA precipittion ssy 48 ws used to detect -induced DNA dmge. After incubtion with.5 mci/ml 3 H-thymidine to llow for DNA incorportion, podocytes were exposed to 3 mg/ml for h. Cells were wshed with Hnk s Blnced Slt Solution (Irvine Scientific, Snt An, CA, USA) nd incubted in medium until series of time points. At time of hrvest, podocytes were lysed, incubted with 1 M KCl t 651C for min, iced for 5 min, nd Kidney Interntionl (6) 7,

9 b c d e f TUNEL-positive cells/ glomeruli TUNEL stining, in vivo P=.11 P=.91 Tretment group Figure 11 Apoptosis detection following exposure, in vivo. TUNEL stining of tissue sections. Representtive imges of tissue sections from rts in () control group versus (b nd d) groups treted with lone or (c nd e) þ DMTU. (d nd e) TUNEL-positive cells in the tubulointerstitium djcent to glomeruli without positive cells. (f) Quntifiction of TUNEL-positive cells per glomeruli counted. There is no significnt difference in the number of TUNEL-positive cells between control,, nd þ DMTU tretment groups. centrifuged (3 g) to seprte precipitted chromtin DNA from dmged DNA (superntnt). Superntnts nd DNA pellets were dissolved seprtely in scintilltion fluid nd counted in Beckmn 35 scintilltion counter. The percentge of ssdna ws clculted. ROS commonly induce depurintion nd depyrimidintion. 7 Therefore, the AP site ssy ws the second detection method of induced DNA dmge, using the DNA dmge quntifiction kit (Dojindo Moleculr Technologies, Githersburg, MD, USA). Briefly, DNA ws isolted from podocytes using the DNesy isoltion kit (Qigen, Vlenci, CA, USA). AP sites were detected using n ldehyde-rective probe regent. AP sites were tgged with biotin, nd the DNA ws bound to microtiter plte long with ldehyderective probe-tgged DNA stndrds. The sites were visulized using peroxidse streptvidin. The number of AP sites in the smple DNA ws determined by compring bsorbnce t 65 nm to the stndrd curve. To confirm -induced DNA dmge, the single-cell gel electrophoresis ssy (Comet ssy) (Trevigen), ws utilized Briefly, podocytes were gently hrvested nd embedded in n grose lyer on microscope slide. Cells were lysed to remove cellulr proteins nd DNA ws uncoiled under lkline conditions. After unwinding, DNA ws electrophoresed for min under lkline conditions nd stined with fluorescent dye. In contrst to undmged nd supercoiled DNA tht remins within the nucler cell membrne, the podocytes with dmged DNA, which migrted wy from the nucleus, demonstrted the chrcteristic long comet til. The extent of DNA liberted from the hed of the comet ws directly proportionl to DNA dmge. DNA dmge detection in vitro following exposure to 7DMTU Studies hve highlighted the importnce of hydroxyl rdicl (OH * ) in ROS-medited injury during the induction phse of N. Highly rective nd toxic, OH * cn generte modifictions in DNA including sugr/bse ltertions, strnd breks, nd DNA protein cross-links. 15,16,43 To determine the role of oxidnts in -induced DNA dmge, podocytes were pretreted with the OH * scvenger DMTU (Sigm-Aldrich). One hour before exposure, podocytes were incubted in medium with DMTU mm. One-hour incubtion with.5 mm H O served s the positive control for oxidnt-induced DNA dmge. Although H O itself is considered reltively non-rective towrd DNA, much of the H O -medited DNA dmge is secondry to OH * genertion vi the Fenton rection. 7 Western blot nlysis Becuse ctivted checkpoints induce cell cycle rrest following exposure to DNA-dmging gents, 7 Western blotting ws performed to mesure the expression of specific cell cycle checkpoint proteins in podocytes exposed to 7DMTU. 5 Cells were hrvested by trypsin digestion nd pelleted by centrifugtion (1 r.p.m. for 5 min). Protein ws extrcted using TG buffer, 46 in the presence of protese inhibitor cocktil (Roche) nd phosphtse inhibitors sodium orthovndte nd sodium fluoride (Sigm-Aldrich). Following freeze thw cycle, lystes were clered by centrifugtion (16 r.p.m. for min), nd protein concentrtions were determined by the bicinchoninic cid 197 Kidney Interntionl (6) 7,

10 protein ssy (Pierce, Rockford, IL, USA). Protein extrcts of 5 15 mg were seprted under reduced conditions on 1 15% sodium dodecyl sulfte-polycrylmide gels nd trnsferred to polyvinyl difluoride membrnes (PerkinElmer, Boston, MA, USA). 4,53,54 Membrnes were incubted with ntibodies to p1 WAF1/CIP1, p53, growth rrest, DNA dmge-inducible-45, APendonuclese, DNA polymerse-b, CHK-1, nd CHK-. To ensure equl protein loding, ntibodies to housekeeping proteins tubulin, ctin, nd glycerldehyde-3-phosphte dehydrogense were used. Bnds were detected with chromgen 5-bromo-4-chloro-3-inodyl phosphte/nitro blue tetrzolium (Sigm-Aldrich). Densitometry ws performed with the NIH imge nlysis progrm ImgeJ. Three seprte experiments were performed for ech Western blot nlysis nd the densitometry results were verged, with normliztion for differences in protein loding. N model To test the hypothesis tht induced podocyte DNA dmge in vivo, the N model 55 ws induced in mle Sprgue Dwley rts (Chrles River Lbortories, Wilmington, MA, USA). After preliminry studies to define the optiml dose to chieve mild-tomoderte disese, identifying dose tht would cuse DNA dmge without significnt ccompnying poptosis or necrosis, rts ged 6 dys, weighing 3 g, were divided into tretment groups (n ¼ 6/group/time point):, þ DMTU, nd control, killed t dys 1.5, 3, nd 7. 8,9,56,57 The groups received single injection of dissolved in.9% NCl, t 6 mg/ g body weight vi til vein. The þ DMTU groups received DMTU dissolved in.9% NCl, 5 mg/kg intrperitonelly one hour before injection, then 15 mg/kg intrperitonelly twice dy until killing. The control nimls were injected with equl volume of.9% NCl substituted for both nd DMTU. Ech niml ws nesthetized with % ethyl ether (VWR) in n inhltion chmber before til vein injections. Animls were nesthetized in n isoflurne (Hlocrbon Lbs, North August, SC, USA) nd oxygen vporizer chmber before intrperitonel injections. Before killing, urine collections were obtined in metbolic cges to determine 4-h protein nd cretinine excretion. Urine protein ws mesured by the sulfoslicylic cid method (stndrds from Dde Dignostics, Brisbne, Austrli). 58 Urine cretinine levels were determined vi colorimetric microplte ssy (Oxford Biomedicl, Oxford, MI, USA). Deth ws induced by exsnguintion following nesthetiztion with % ethyl ether. Blood smples, obtined t time of killing vi ven cvl puncture, were centrifuged (1 g for 5 min) nd plsm ws collected for mesurement of blood ure nitrogen (Sigm-Aldrich). Tissues were obtined for renl biopsies nd glomerulr isoltion. All niml procedures were conducted in ccord with the Institutionl Animl Cre nd Use Committee. Isoltion of glomeruli Preprtion of isolted glomeruli ws performed t 41C, with kidney tissue from one rt used per preprtion. 5 After discrding medull nd ppill, finely minced cortices were pssed through three differentil sieves (, 14, nd 8 mm) using ml of norml sline. Glomeruli were wshed nd resuspended in 5 ml of sline. Quntittion of glomeruli ws performed in liquots of glomerulr suspension by phse microscopy, with purity 495% for ll preprtions. Following centrifugtion ( r.p.m. t 41C for min), superntnt ws decnted, nd DNA isoltion nd purifiction ws performed from the pelleted glomeruli. Mesuring DNA dmge in vivo DNA ws isolted nd purified from glomerulr isoltes vi the DNesy tissue kit. DNA dmge ws detected by counting bsic sites using the DNA dmge quntifiction kit. Immunohistochemistry Indirect immunoperoxidse immunostining ws performed on formlin-fixed prffin-embedded kidney specimens. 53 Briefly, 4- mm tissue sections were deprffinized in Histo-Cler (Ntionl Dignostics, Atlnt, GA, USA) nd rehydrted in grded ethnol. Endogenous peroxidses were blocked with 3% H O, followed by overnight incubtion with primry ntibody diluted in 1% bovine serum lbumin in phosphte-buffered sline (PBS). After wshing in PBS, sections were incubted with biotinylted secondry ntibody, diluted in 1% bovine serum lbumin in PBS, for 1 h t room temperture. ABC (Avidin nd Biotinylted horserdish peroxidse mcromoleculr Complex)-Elite regent (Vector Lbortories, Burlingme, CA, USA) ws used for signl mplifiction nd 3,3 - diminobenzidine (Sigm-Aldrich) ctlyzed by NiCl ws used s chromgen. Slides were counterstined with methyl green, dehydrted, nd coverslipped. Immunostining for 8-OHdG, ws performed on methcrnfixed tissue. 59 Briefly, following deprffiniztion nd rehydrtion, slides were pretreted with proteinse K (Roche) nd RNse (Qigen). DNA ws dentured with 4 N HCl, followed by Trisbuffered sline neutrliztion. % fetl clf serum in mm Tris- HCl ws used to block unspecific sites. Sections were incubted overnight t 41C with primry ntibody diluted in mm Tris-HCl, % fetl clf serum. After wshing with PBS, sections were incubted with the secondry ntibody t 371C for 3 min. ABC-Elite regent ws used for signl mplifiction. 3,3 -diminobenzidine with 3% H O ws used s chromgen. Slides were counterstined with hemtoxylin, dehydrted, nd coverslipped. Apoptosis detection TUNEL stining ws utilized to exclude the possibility of significnt poptosis under our experimentl conditions. The protocol ws modifiction of methods described previously. 6 Briefly, cultured podocytes were fixed with % buffered formlin overnight t 41C. Following rehydrtion with PBS, endogenous peroxidses were inctivted with 3% H O. Cells were treted with citric cid to enhnce ntigen retrievl. Nuclei were permebilized by incubtion with proteinse K for min t room temperture. Following incubtion in One-Phor-All buffer (GE Helthcre, Pisctwy, NJ, USA), frgmented DNA ws lbeled by exposure of cells to diluted terminl deoxynucleotidyl trnsferse (GE Helthcre) nd biotin- 14-dATP (Invitrogen) for 6 min. The rection ws terminted with PBS. ABC-Elite regent ws used for signl mplifiction. 3,3 - diminobenzidine with NiCl ws used s chromgen. Cells were counterstined with Cmco Quik stin (Cmbridge Dignostic, Fort Luderdle, FL, USA). Similrly, in vivo, TUNEL stining ws performed on 4-mm formlin-fixed prffin-embedded tissue sections. Following deprffiniztion nd rehydrtion, the bove protocol ws followed. Slides were counterstined with methyl green, dehydrted, nd coverslipped. Sttisticl nlysis For comprison of men vlues between two groups, the unpired t test ws used. All vlues re mens7s.d. except where otherwise Kidney Interntionl (6) 7,

11 indicted. The experimentl findings were considered sttisticlly significnt if Po.5. All photomicrogrphs were mde t similr intensity nd bckground. During dt nlysis, the observer ws blinded to the tretment ctegories. ACKNOWLEDGMENTS This work ws supported by Ntionl Institutes of Helth grnts to SJS (DK655, DK56799, nd DK 596), by Ntionl Institutes of Helth grnt to CBM (3 RO1 DK596-6S1), nd by the Americn Dibetes Assocition. SJS is lso n Estblished Investigtor of the Americn Hert Assocition. REFERENCES 1. Pvenstdt H, Kriz W, Kretzler M. Cell biology of the glomerulr podocyte. Physiol Rev 3; 83: Kriz W, Gretz N, Lemley KV. Progression of glomerulr diseses: is the podocyte the culprit? Kidney Int 1998; 54: Kim YH, Goyl M, Kurnit D et l. Podocyte depletion nd glomerulosclerosis hve direct reltionship in the N-treted rt. Kidney Int 1; 6: Pgtlunn ME, Miller PL, Jumping-Egle S et l. Podocyte loss nd progressive glomerulr injury in type II dibetes. 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