Cyclin I Protects Podocytes from Apoptosis
2006; Elsevier BV; Volume: 281; Issue: 38 Linguagem: Inglês
10.1074/jbc.m513336200
ISSN1083-351X
AutoresSiân Griffin, Jean-Frédéric Olivier, Jeffrey W. Pippin, James M. Roberts, Stuart J. Shankland,
Tópico(s)Phagocytosis and Immune Regulation
ResumoThe limited regenerative capacity of the glomerular podocyte following injury underlies the development of glomerulosclerosis and progressive renal failure in a diverse range of kidney diseases. We discovered that, in the kidney, cyclin I is uniquely expressed in the glomerular podocyte, and have constructed cyclin I knock-out mice to explore the biological function of cyclin I in these cells. Cyclin I knock-out (–/–) podocytes showed an increased susceptibility to apoptosis both in vitro and in vivo. Following induction of experimental glomerulonephritis, podocyte apoptosis was increased 4-fold in the cyclin I –/– mice, which was associated with dramatically decreased renal function. Our previous data showed that the Cdk inhibitor p21Cip1/Waf1 protects podocytes from certain apoptotic stimuli. In cultured cyclin I –/– podocytes, the level of p21Cip1/Waf1 was lower at base line, had a shorter half-life, and declined more rapidly in response to apoptotic stimuli than in wild-type cells. Enforced expression of p21Cip1/Waf1 reversed the susceptibility of cyclin I –/– podocytes to apoptosis. Cyclin I protects podocytes from apoptosis, and we provide preliminary data to suggest that this is mediated by stabilization of p21Cip1/Waf1. The limited regenerative capacity of the glomerular podocyte following injury underlies the development of glomerulosclerosis and progressive renal failure in a diverse range of kidney diseases. We discovered that, in the kidney, cyclin I is uniquely expressed in the glomerular podocyte, and have constructed cyclin I knock-out mice to explore the biological function of cyclin I in these cells. Cyclin I knock-out (–/–) podocytes showed an increased susceptibility to apoptosis both in vitro and in vivo. Following induction of experimental glomerulonephritis, podocyte apoptosis was increased 4-fold in the cyclin I –/– mice, which was associated with dramatically decreased renal function. Our previous data showed that the Cdk inhibitor p21Cip1/Waf1 protects podocytes from certain apoptotic stimuli. In cultured cyclin I –/– podocytes, the level of p21Cip1/Waf1 was lower at base line, had a shorter half-life, and declined more rapidly in response to apoptotic stimuli than in wild-type cells. Enforced expression of p21Cip1/Waf1 reversed the susceptibility of cyclin I –/– podocytes to apoptosis. Cyclin I protects podocytes from apoptosis, and we provide preliminary data to suggest that this is mediated by stabilization of p21Cip1/Waf1. Cyclins were originally discovered for their role in governing cell cycle progression and proliferation (1Evans T. Rosenthal T.E. Youngblom J. Distel D. Hunt T. Cell. 1983; 33: 389-396Abstract Full Text PDF PubMed Scopus (1009) Google Scholar). More recently it has been appreciated that cyclins may influence a wide range of additional cellular functions, including apoptosis, hypertrophy, and differentiation. The increasingly diverse members of the cyclin protein family are all characterized by the presence of a conserved domain through which they bind to cyclin-dependent kinases, the cyclin box (2Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (498) Google Scholar). Cyclin I, the focus of this manuscript, is most abundant in post-mitotic tissues. In contrast to the classical cyclins, its level does not fluctuate during the cell cycle (3Nakamura T. Sanokawa R. Sasaki Y.F. Ayusawa D. Oishi M. Mori N. Exp. Cell Res. 1995; 221: 534-542Crossref PubMed Scopus (73) Google Scholar, 4Jensen M.R. Audolfsson T. Factor V.M. Thorgeirsson S.S. Gene (Amst.). 2000; 256: 59-67Crossref PubMed Scopus (15) Google Scholar). Cyclin I shows highest sequence homology to cyclins G1 and G2, and these three proteins are considered to form a separate subgroup (5Bates S. Rowan S. Vousden K.H. Oncogene. 1996; 13: 1103-1109PubMed Google Scholar). However, the biological function of cyclin I is not known. Our data show that within the kidney cyclin I is specifically expressed by glomerular podocytes. These are terminally differentiated, post-mitotic, highly specialized epithelial cells, which serve as the major barrier to prevent the excretion of serum proteins into the urine. The inability to replace podocytes lost by apoptosis is thought to underlie the subsequent development of glomerulosclerosis and progressive renal impairment, regardless of the initiating injury (6Rennke H.G. Kidney Int. 1994; S45: S58-S63Google Scholar, 7Kriz W. Nephrol. Dial. Transplant. 1996; 11: 1738-1742Crossref PubMed Scopus (131) Google Scholar, 8Kriz W. Gretz N. Lemley K.V. Kidney Int. 1998; 54: 687-697Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 9Kriz W. Lemley K.V. Curr. Opin. Nephrol. Hypertens. 1999; 8: 489-497Crossref PubMed Scopus (148) Google Scholar, 10Kim Y.H. Goyal M. Kurnit D. Wharram B. Wiggins J. Holzman L. Kershaw D. Wiggins R. Kidney Int. 2001; 60: 957-968Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 11White K.E. Bilous R.W. Marshall S.M. El Nahas M. Remuzzi G. Piras G. De Cosmo S. Viberti G.C. Diabetes. 2002; 51: 3083-3089Crossref PubMed Scopus (273) Google Scholar, 12Lemley K.V. Lafayette R.A. Safai M. Derby G. Blouch K. Squarer A. Myers B.D. Kidney Int. 2002; 61: 1475-1485Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 13Schiffer M. Bitzer M. Roberts I.S.D. Kopp J.B. ten Dijke P. Mundel P. Bottinger E.P. J. Clin. Invest. 2001; 108: 807-816Crossref PubMed Scopus (553) Google Scholar, 14Schiffer M. Mundel P. Shaw A.S. Bottinger E.P. J. Biol. Chem. 2004; 279: 37004-37012Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Given their limited regenerative capacity, prevention of podocyte apoptosis is of critical importance for the maintenance of normal renal function. The restricted expression of cyclin I to the renal podocyte suggested that it might play a specialized biological role in these cells. We describe here the characterization of cyclin I expression in the normal kidney and the first analysis of its function using cyclin I knock-out mice. We report that cyclin I regulates podocyte apoptosis, both in vitro and in a model of glomerular disease in vivo. Previous work has shown that p21Cip1/Waf1 has an important role in preventing podocyte apoptosis (15Kim Y.-G. Alpers C.E. Brugarolas J. Johnson R.J. Couser W.G. Shankland S.J. Kidney Int. 1999; 55: 2349-2361Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and we show here that cyclin I may control p21Cip1/Waf1 abundance by regulating its stability. We propose a role for cyclin I in protecting terminally differentiated cells from apoptosis. An 11-kb NotI fragment of the cyclin I gene was obtained from a mouse 129/Sv λ genomic library and sequenced. The sequencing data were then assembled into contigs 4The abbreviations used are: contig, group of overlapping clones; +/+, wild-type; –/–, knock-out; ES, embryonic stem; MEF, mouse embryonic fibroblast; PAN, puromycin aminonucleoside; GFP, green fluorescent protein; UUO, unilateral ureteric obstruction; WT-1, Wilm's tumor protein 1; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; PBS, phosphate-buffered saline; RT, reverse transcription. using Sequencher software. The knock-in vector was constructed by cloning a 1.3-kb SpeI-SacI fragment that encompasses the intron sequence immediately 5′ to the second exon of cyclin I as the upstream arm and a 4.5-kb BstEII-HindIII fragment that contained a portion of the coding region of the last exon of cyclin I and intron sequence cloned into the SA-β gal vector as the downstream arm. The vector was linearized with ScaI and electroporated into XY AK7 ES cells. The ES cells were then selected in 400 μg/ml G418. ES cell colonies with homologous recombination were identified by PCR amplification of a 2-kb fragment with a primer from the SA-β gal gene (SARev, 5′-CATCAAGGAAACCCTGGACTACTG-3′) and a primer from cyclin I genomic DNA just 5′ to the SpeI site (1BR+, 5′-TAGGACATGGGTCTCAGC-3′). PCR reactions were performed for 40 cycles (93 °C for 30 s, 57 °C for 30 s, 65 °C for 2 min). Proper recombination within the cyclin I locus was also confirmed by Southern blot of PstI digested genomic DNA using a probe designed with cyclin I sequences not contained within the original knock-in vector. ES cells were introduced into 5 days post-coitus C57/B6J mouse embryos. Germ line transmission, as determined by PCR, was identified in chimeric males obtained from two independent clones that were used for subsequent experiments. The wild-type allele of cyclin I was amplified using the 2718 oligonucleotide (5′-GGTGTGACTCTATGGTATTTC-3′) and the 1BR primer described above using the same PCR conditions. Day 13 embryos were washed twice in PBS and then fixed in 2% formaldehyde, 0.2% gluteraldehyde in PBS containing 0.1% sodium deoxycholate and 0.2% Nonidet P-40 (Nonidet P-40) for 2 h at 4 °C. Fixed embryos were washed for 15 min three times in PBS. Embryos were incubated for 6–8 h at room temperature in staining solution (2.5 mm ferrocyanide, 2.5 mm ferricyanide, 2 mm MgCl2, 0.1% sodium deoxycholate, 0.2% Nonidet P-40 in PBS) containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (diluted from a 40× stock solution in N,N-dimethylformamide). After staining, embryos were extensively washed in PBS and photographed. Mouse Embryonic Fibroblasts—Mouse embryonic fibroblasts (MEFs) were isolated from 13-day-old embryos and maintained using standard procedures. To induce quiescence, confluent MEFs were washed twice with phosphate-buffered saline (PBS) and cultured in Dulbecco's modified Eagle's medium with 0.1% fetal bovine serum for 24 h. Quiescent cells were trypsinized, replated at low density, and stimulated with complete medium containing 10% fetal bovine serum to enter the cell cycle. Entry into S phase was monitored by estimating the DNA content of propidium iodide-stained nuclei using fluorescence-activated flow cytometry or by bromodeoxyuridine incorporation. Mouse Podocytes—Female cyclin I –/– mice were crossed with a male H-2Kb-tsA58 transgenic mouse (ImmortoMouse; Jackson Laboratory, Bar Harbor, ME) and the F1 generation intercrossed. Conditionally immortalized mouse podocytes were derived from cyclin I +/+ and –/– littermates as described previously (16Mundel P. Reiser J. Zuniga Mejia Borja A. Pavenstadt A. Davidson G.R. Kriz W. Zeller R. Exp. Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (775) Google Scholar). Briefly, proliferating podocytes were grown on collagen I (BD Biosciences, Bedford, MA) at 33 °C in medium supplemented with recombinant mouse γ-interferon (10 units/ml; Coulter, Hialeah, FL) to promote expression of the thermosensitive SV40 large T antigen. To induce quiescence and the differentiated phenotype, cells were grown at 37 °C in the same medium with no γ-interferon for 14 days and characterized by their expression of podocyte specific proteins. A similar strategy was used to generate p21Waf1/Cip1 –/– podocytes. The expression of cyclin I by cultured mouse podocytes was determined by RT-PCR. cDNA was amplified in a semiquantitative fashion using primer sets specific for the mouse cyclin I gene (forward primer, 5′-ATGAAGTTTCCAGGACCTTTG-3′; reverse primer, 5′-CTACATGACAGAAACAGGCTG-3′). The PCR reaction was performed as follows: 94 °C for 2 min, followed by 30 cycles of 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min. PCR products were resolved on a 2% agarose gel and normalized to expression of glyceraldehyde-3-phosphate dehydrogenase. Total cell protein was extracted using TG buffer (1% Triton, 10% glycerol, 20 mm HEPES, 100 mm NaCl) with protease inhibitors (Roche Applied Science). Protein concentration was determined by the BCA protein assay (Pierce). For Western blot analysis, 15–40 μg of protein extracts were separated under reduced conditions on 15% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated overnight at 4 °C with the following commercially available primary antibodies: mouse monoclonal anti-p21 (clone SX118, PharMingen), anti-human p21 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p27 (BD Transduction Laboratories, Lexington, KY), anti-p53 (clone PAb421, Oncogene Research Products, San Diego, CA), anti-glyceraldehyde-3-phosphate dehydrogenase (Abcam, Cambridge, MA), anti-Grb2 (Santa Cruz Biotechnology), and anti-actin (Chemicon International Inc., Temecula, CA). The cyclin I antibody was developed in house. Cyclin I was subcloned into pET16b (Novagen, Madison, WI) as a full-length coding sequence or the sequence encoding the amino terminus (amino acids 1–52). Protein inductions were performed in BL21 pLysS bacteria and purified under denaturing conditions with 8 m urea on nickel-nitrilotriacetic acid (Qiagen, Valencia, CA). Antibodies to the two cyclin I proteins were raised in New Zealand White rabbits and affinity-purified using antigen immobilized on nickel-nitrilotriacetic acid. Antibody was eluted from the column using 4 m MgCl2 and dialyzed extensively against PBS at 4 °C. Proteins were visualized using the chromagen 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma) or ECL reagents (Pierce). Apoptosis was induced in 80–90% confluent, differentiated cyclin I +/+ and –/–, and p21Cip1/Waf1 –/– podocytes grown in 24-well plates (Primaria, VWR, West Chester, PA). Each experimental condition was carried out in quadruplicate, and experiments were performed at least three times. Apoptosis was detected as described below. Three apoptotic stimuli were used. (i) uv-C irradiation, 0–25 J/m2 using a Hoefer cross-linker (Stratagene, La Jolla, CA). Cells were irradiated in the absence of media and apoptosis assessed after 6 h. Protein was harvested from similarly treated cells for Western blot analysis. (ii) Puromycin aminonucleoside (PAN) has previously been shown to induce podocyte apoptosis in culture (17Sanwal V. Pandya M. Bhaskaran M. Franki N. Reddy K. Ding G. Kapasi A. Exp. Mol. Pathol. 2001; 70: 54-64Crossref PubMed Scopus (43) Google Scholar, 18Suzuki T. Takemura H. Noiri E. Nosaka K. Toda A. Taniguchi S. Uchida K. Fujita T. Kimura S. Nakao A. Free Radic. Biol. Med. 2001; 31: 615-623Crossref PubMed Scopus (17) Google Scholar) and induces proteinuria and apoptosis in vivo (10Kim Y.H. Goyal M. Kurnit D. Wharram B. Wiggins J. Holzman L. Kershaw D. Wiggins R. Kidney Int. 2001; 60: 957-968Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 19Shiiki H. Sasaki Y. Nishino T. Kimura T. Kurioka H. Fujimoto S. Dohi K. Pathobiology. 1998; 66: 221-229Crossref PubMed Scopus (41) Google Scholar). Cells were cultured in the presence of 0–100 μg/ml PAN (Sigma) and apoptosis measured after 24 h. (iii) Anti-podocyte antibody induces podocyte injury in vitro and in vivo and as described below was also used to induce experimental glomerulonephritis in mice. Cells were exposed to media containing 0–5% nephrotoxic or normal sheep serum for 30 min at 37 °C. The cells were then washed in HBSS and fresh media applied. Apoptosis was assessed after 16 h. In separate experiments, cells were fixed overnight in ice-cold methanol prior to immunofluorescence to confirm equal antibody binding. At the end of each experiment, Hoechst 33342 (Sigma) at a final concentration of 10 mm was added to each well. At least 400 cells were counted for each well, and the number of apoptotic nuclei expressed as a percentage of the total. Apoptosis was also assessed by a caspase 3 activity assay according to the manufacturer's instructions (BD Biosciences). pBabe vectors encoding cyclin I, wild-type human p21Cip1/Waf1, lysineless (ΔK) human p21Cip1/Waf1, or GFP were transfected into Phoenix packaging cells to generate retrovirus. The retrovirus-containing media were harvested and filtered onto 50% confluent proliferating, undifferentiated cyclin I –/– podocytes. Following 48-h selection with puromycin (2.5 μg/ml), cells were passaged and transferred to growth restrictive conditions. Apoptotic susceptibility following uv-C irradiation was assessed as above. Crescentic Glomerulonephritis—Glomerulonephritis was induced in 10–12-week-old male cyclin I +/+ and –/– matched control mice by the intraperitoneal injection of sheep anti-rabbit glomerular antibody (0.5 ml/20 g of body weight) on 2 consecutive days, as described previously (20Ophascharoensuk V. Pippin J. Gordon K.L. Shankland S.J. Couser W.G. Johnson R.J. Kidney Int. 1998; 54: 416-425Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 21Ophascharoensuk V. Fero M.L. Hughes J. Roberts J.M. Shankland S.J. Nat. Med. 1998; 4: 575-580Crossref PubMed Scopus (181) Google Scholar). We have previously characterized this model in detail and demonstrated that the observed pathological changes are not due to the presence of infiltrating cells. Cyclin I +/+ and –/– mice (n > 6/group) were sacrificed on days 7 and 14 after the second injection of anti-glomerular antibody. Urine was collected from each animal before disease induction and just prior to sacrifice for quantification of proteinuria by the sulfosalicylic acid method. Blood was collected at sacrifice to measure serum creatinine (Sigma kit number 555-A). Renal tissue was embedded in OCT compound (Miles, Elkart, IN) and frozen at –70 °C or fixed in either 10% neutral buffered formalin or methyl-Carnoy's solution (60% methanol, 30% chloroform, 10% acetic acid) for immunostaining (see below). Unilateral Ureteric Obstruction (UUO)—Experimental UUO was performed on anesthetized 8–10-week-old cyclin I +/+ and –/– animals (n = 8/group) by ligation of the left ureter at the ureteropelvic junction (22Truong L.D. Petrusevska G. Yang G. Gurnipar T. Shappell S. Lechago J. Rouse D. Suki W.N. Kidney Int. 1996; 50: 200-207Abstract Full Text PDF PubMed Scopus (169) Google Scholar). Mice were sacrificed at day 7 and kidneys fixed as above for histological assessment. The contralateral non-obstructed kidney served as control. Indirect immunoperoxidase staining was performed on formalin or methyl-Carnoy's fixed tissue with antibodies against β-galactosidase (1:1000 dilution, Abcam), WT-1 (sc192, 1:1000 dilution, Santa Cruz Biotechnology), and fibronectin (1:500 dilution, Chemicon International Inc.). Measurement of apoptosis was by the TUNEL assay, performed as described previously (23Baker A.J. Mooney A. Lombardi D. Johnson R.J. Savill J. J. Clin. Invest. 1994; 94: 2105-2116Crossref PubMed Scopus (402) Google Scholar). Apoptosis was also quantified in the thymus of unmanipulated cyclin I +/+ and –/– mice. Frozen sections were rehydrated in PBS and stained with fluorescein isothiocyanate-conjugated antibodies to sheep IgG (Cappel, Durham, NC), to ensure comparable glomerular antibody deposition between the cyclin I +/+ and –/– mice. The autologous phase of the disease was similarly assessed by immunostaining with a fluorescein isothiocyanate-conjugated antibody to mouse IgG (Cappel). Double immunostaining for β-galactosidase (goat polyclonal antibody, 1:100 dilution, Biogenesis, Kingston, NH) and WT-1 (sc192 rabbit polyclonal antibody, 1:100 dilution, Santa Cruz Biotechnology) was performed. After washing, sections were incubated with a biotinylated goat anti-rabbit IgG (1:500 dilution, Vector laboratories, Burlingame, CA). Binding was detected using Alexa Fluor 488-conjugated donkey anti-goat IgG (1:100 dilution; Molecular Probes, Eugene, OR) and Alexa Fluor 594 streptavidin (Molecular Probes). Glomerulosclerosis was determined on periodic acid Schiff-stained sections for a minimum of 50 glomeruli in each animal and was graded quantitatively based on the percentage of glomerular tuft area involvement as follows: grade 1 = <25%; grade 2 = 25–50%; grade 3 = 50–75%; grade 4 = 75–100%. Slides were viewed using a Leica confocal microscope (Leica, Deerfield, IL) using either bright-field or appropriate epifluorescent optics. All results are expressed as mean + S.D. Statistical significance was evaluated using the Student's t test. Targeted Disruption of the Cyclin I Gene Creates a Null Mutation—We constructed a cyclin I knock-out (–/–) mouse in which the cyclin I coding exons were replaced with the bacterial β-galactosidase gene (Fig. 1A). Staining of day 13 embryos for β-galactosidase activity confirmed expression of the transgene (Fig. 1B). Viable cyclin I –/– mice were obtained from intercrosses of cyclin I +/– heterozygotes at the normal Mendelian frequency of 25% (142/512) and showed no apparent developmental defects. The genotype of the offspring was confirmed by Southern blotting (Fig. 1C). Protein extracts from various tissues from one month old pups underwent Western blot analysis, and highest expression was seen in the brain, followed by testis (data not shown), although cyclin I –/– mice were fertile and displayed no behavioral abnormalities. The lack of cyclin I protein expression in the homozygous knock-out mice was confirmed by immunoprecipitation and Western blotting with an anti-cyclin I antibody (Fig. 1D). To test whether cyclin I –/– cells had detectable defects in cell proliferation, we isolated MEFs from 13-day-old embryos (Fig. 1E). Cyclin I –/– MEFs responded to serum stimulation and progressed through the cell cycle at the same rate as control MEFs (Fig. 1F). Population doubling times were also unaffected by the absence of cyclin I (data not shown). Taken together, these results suggested that cyclin I was not required for cell proliferation, and its abundant expression in some post-mitotic cells suggested that it may have a role distinct from the cell cycle. Cyclin I Is Expressed by Podocytes in Vitro and in Vivo —We used immunohistochemical analysis of β-galactosidase to determine the pattern of cyclin I expression in the kidney, as this gene has been introduced in place of cyclin I. As expected, no β-galactosidase expression was detected in the cyclin I +/+ mice (Fig. 2A). However, in the cyclin I –/– mice, distinct staining was seen in the glomerulus in a podocyte distribution (Fig. 2B). Weaker and variable expression was seen in tubular cells (data not shown). Wilm's tumor protein 1 (WT-1) was used as a podocyte specific marker (24Mundlos S. Pelletier J. Darveau A. Bachmann M. Winterpacht A. Zabel B. Development (Camb.). 1993; 119: 1329-1341Crossref PubMed Google Scholar) and by immunofluorescence co-localized with β galactosidase (Fig. 2, C–E), confirming within the glomerulus the exclusive expression of β-galactosidase (and therefore the cyclin I gene) by podocytes in vitro. To confirm the podocyte-specific expression of the endogenous cyclin I protein, we used RT-PCR (Fig. 2F) and Western blot analysis (Fig. 2G) for cyclin I using RNA and protein from proliferating, undifferentiated and from post-mitotic, differentiated podocytes. Cyclin I expression was similar in proliferating and quiescent cells. Similarly to MEFs, cyclin I –/– podocytes showed no detectable defects in cell proliferation. For our further studies we focused on the role of cyclin I in renal podocytes. Cultured Cyclin I –/– Podocytes Are More Susceptible to Apoptosis—The expression of cyclin I in post-mitotic cells suggested involvement in pathways other than the cell cycle. We therefore induced apoptosis in cultured cyclin I +/+ podocytes using uv-C irradiation. Western blot analysis demonstrated down-regulation of cyclin I protein levels in podocytes following irradiation (Fig. 3A). We then hypothesized that cyclin I might be important for determining the threshold at which podocytes undergo apoptosis following stimulation and explored this using cyclin I +/+ and –/– cultured podocytes. As determined by Hoechst 33342 staining, apoptosis occurred earlier, and was of a greater magnitude, in the cyclin I –/– podocytes following induction by three different stimuli: (i) uv-C (Fig. 3B), (ii) PAN (Fig. 3C), and (iii) anti-podocyte antibody (Fig. 3D). Apoptosis induced by uv-C activates both the intrinsic and extrinsic pathways of apoptotic signaling (25Scoltock A.B. Cidlowski J.A. Exp. Cell Res. 2004; 297: 212-223Crossref PubMed Scopus (52) Google Scholar), whereas PAN principally activates the intrinsic pathway (26Wada T. Pippin J.W. Marshall C.B. Griffin S.V. Shankland S.J. J. Am. Soc. Nephrol. 2005; 16: 2615-2625Crossref PubMed Scopus (163) Google Scholar), and the mechanism by which the anti-podocyte antibody causes apoptosis is unknown. The increased susceptibility of the cyclin I –/– podocytes to apoptosis induced by all three stimuli suggests that cyclin I acts distally in the pathways converging to cause cell death. To further confirm the increased apoptosis in cyclin I –/– cells, we performed an activity assay for caspase 3 using uv-C-irradiated cyclin I +/+ and –/– podocytes (Fig. 3E). The increased caspase 3 activity in the irradiated cyclin I –/– podocytes validates the results using Hoechst 33342 staining, showing that apoptosis was significantly increased in cyclin I –/– cells compared with cyclin I +/+ cells receiving the same stimulus. Apoptosis of Cyclin I –/– Podocytes Is Rescued by Reconstitution of Cyclin I—To verify that the lowered apoptotic threshold observed in cyclin I –/– podocytes is indeed due to a deficiency in cyclin I itself (rather than due to altered expression of an adjacent gene that might have been affected by the deletion of cyclin I) we stably reintroduced cyclin I into cyclin I –/– podocytes by retroviral transduction. GFP served as a negative control. Following cyclin I reconstitution in cyclin I –/– cells, apoptosis was measured 6 h following irradiation with 25 J/m2 (Fig. 3F). The reconstitution of cyclin I completely reversed the apoptotic susceptibility of the cyclin I –/– cells. Podocyte Number and Renal Function Are Normal in Unmanipulated Cyclin I –/– Mice—We next studied the role of cyclin I in vivo. The cyclin I –/– mouse is phenotypically normal under physiological conditions. Renal function and histology were evaluated in detail from 12-week-old male cyclin I +/+ and –/– mice (n = 6/group). There was no difference in serum creatinine (+/+, 0.23 + 0.06 mg/dl; –/–, 0.28 + 0.08 mg/dl, p = not significant), urine protein:creatinine ratio (+/+, 8.2 + 4.6 mg/mg; –/–, 8.3 + 7.8 mg/mg, p = not significant) or podocyte number (+/+, 8.1 + 0.3; –/–, 7.9 + 1.0 per glomerular cross-section, p = not significant) between the two groups. These results indicate that cyclin I is not required for normal glomerular development nor for maintenance of normal renal function. Apoptosis Is Increased in Cyclin I –/– Nephritic Mice—We reasoned that a critical role for cyclin I might be revealed following injury, as suggested by the in vitro data. To test this hypothesis, experimental glomerulonephritis was induced in cyclin I +/+ and –/– mice with a sheep anti-podocyte antibody (20Ophascharoensuk V. Pippin J. Gordon K.L. Shankland S.J. Couser W.G. Johnson R.J. Kidney Int. 1998; 54: 416-425Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 21Ophascharoensuk V. Fero M.L. Hughes J. Roberts J.M. Shankland S.J. Nat. Med. 1998; 4: 575-580Crossref PubMed Scopus (181) Google Scholar). We have previously demonstrated that this model is not characterized by the presence of infiltrating leukocytes, and the observed rates of apoptosis are in resident glomerular cells (20Ophascharoensuk V. Pippin J. Gordon K.L. Shankland S.J. Couser W.G. Johnson R.J. Kidney Int. 1998; 54: 416-425Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). By immunofluorescence, equal deposition of sheep and mouse immunoglobulin was seen in both cyclin I +/+ and –/– mice at the same time points (data not shown), confirming comparable initiating injury. Consistent with our in vitro data, glomerular cell apoptosis was increased 4-fold in the cyclin I –/– mice at day 7 of nephritis (0.07 + 0.03 versus 0.28 + 0.08 apoptotic cells per glomerular cross-section, p < 0.005), and these apoptotic cells were in a podocyte distribution (Fig. 4, A and B). To characterize the consequences of the early increased apoptosis, the number of podocytes was determined by counting WT-1-positive cells. There was no difference in podocyte number in unmanipulated mice (Fig. 5, A and B). However, by day 14 after disease induction podocyte number was significantly less in the cyclin I –/– versus wild-type mice (1.46 + 1.24 versus 3.19 + 0.90 cells per glomerular cross-section; p < 0.01) (Fig. 5, E and F).FIGURE 5Podocyte loss, matrix accumulation, and glomerulosclerosis are increased in cyclin I –/– nephritic mice. Podocytes were identified by immunostaining for WT-1. In unmanipulated animals, a similar complement of podocytes was seen in both the cyclin I +/+ and –/– mice (A and B). In contrast, at day 14 of nephritis there were significantly fewer podocytes in the cyclin I –/– mice (E and F). C and D, there was no difference in base-line immunostaining for fibronectin in the cyclin I +/+ and –/– mice. G and H, at day 14 of nephritis, there was greater accumulation of fibronectin in the cyclin I –/– mice. I, podocyte loss at day 14 was greater in the cyclin I –/– mice. J, glomerulosclerosis at day 7 was similar in the two groups but progressed to extensive involvement in the –/– mice at day 14. K, there was a strong correlation between the decline in WT-1-positive podocytes and glomerulosclerosis for both groups of mice (R2 = 0.616). **, p < 0.01; ***, p < 0.005. Original magnification ×400 (A–H).View Large Image Figure ViewerDownload Hi-res image Download (PPT) As a decline in podocyte number has been reported to underlie pathological extracellular matrix accumulation and progressive glomerulosclerosis in both experimental and human disease, these parameters were assessed
Referência(s)