Progression of Murine Lupus Nephritis Is Linked to Acquired Renal Dnase1 Deficiency and Not to Up-Regulated Apoptosis
2009; Elsevier BV; Volume: 175; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2009.080943
ISSN1525-2191
AutoresNatalya Seredkina, Svetlana N. Zykova, Ole Petter Rekvig,
Tópico(s)interferon and immune responses
ResumoThe accumulation of apoptotic cells has been suggested as a possible mechanism of nucleosome conversion into self-antigens that may both initiate autoimmune responses and participate in immune complex deposition in lupus nephritis. In this study, we analyzed both the rate of transcription of apoptosis-related genes and the presence of activated apoptotic factors within kidneys of lupus-prone (NZB×NZW) F1 mice during disease progression. The results of this study demonstrated no activation of apoptotic pathways in kidneys of these lupus-prone mice at the time of appearance of anti-double standard DNA antibodies in serum, as well as the formation of mesangial immune deposits in glomeruli. In contrast, the transition of mesangial into membranoproliferative lupus nephritis coincided with an accumulation of activated caspase 3-positive cells in kidneys, in addition to a dramatic decrease in Dnase1 gene transcription. Highly reduced expression levels of the Dnase1 gene may be responsible for the accumulation of large chromatin-containing immune complexes in glomerular capillary membranes. Thus, the initiation of lupus nephritis is not linked to increased apoptotic activity in kidneys. The combined down-regulation of Dnase1 and the increased number of apoptotic cells, which is possibly due to their reduced clearance in affected kidneys, may together be responsible for the transformation of mild mesangial lupus nephritis into severe membranoproliferative, end-stage organ disease. The accumulation of apoptotic cells has been suggested as a possible mechanism of nucleosome conversion into self-antigens that may both initiate autoimmune responses and participate in immune complex deposition in lupus nephritis. In this study, we analyzed both the rate of transcription of apoptosis-related genes and the presence of activated apoptotic factors within kidneys of lupus-prone (NZB×NZW) F1 mice during disease progression. The results of this study demonstrated no activation of apoptotic pathways in kidneys of these lupus-prone mice at the time of appearance of anti-double standard DNA antibodies in serum, as well as the formation of mesangial immune deposits in glomeruli. In contrast, the transition of mesangial into membranoproliferative lupus nephritis coincided with an accumulation of activated caspase 3-positive cells in kidneys, in addition to a dramatic decrease in Dnase1 gene transcription. Highly reduced expression levels of the Dnase1 gene may be responsible for the accumulation of large chromatin-containing immune complexes in glomerular capillary membranes. Thus, the initiation of lupus nephritis is not linked to increased apoptotic activity in kidneys. The combined down-regulation of Dnase1 and the increased number of apoptotic cells, which is possibly due to their reduced clearance in affected kidneys, may together be responsible for the transformation of mild mesangial lupus nephritis into severe membranoproliferative, end-stage organ disease. Affection of kidneys is a major complication in systemic lupus erythematosus and lupus nephritis is associated with high rate of morbidity and mortality. According to the International Society of Nephrology/Renal Pathology Society classification criteria it is separated into six different classes from subclinical (class I) to end-stage disease (class VI).1Weening JJ D'Agati VD Schwartz MM Seshan SV Alpers CE Appel GB Balow JE Bruijn JA Cook T Ferrario F Fogo AB Ginzler EM Hebert L Hill G Hill P Jennette JC Kong NC Lesavre P Lockshin M Looi LM Makino H Moura LA Nagata M The classification of glomerulonephritis in systemic lupus erythematosus revisited.J Am Soc Nephrol. 2004; 15: 241-250Crossref PubMed Scopus (1435) Google ScholarNucleosomes play a central role as potential inducers of autoantibody production and in formation of immune complexes.2Berden JH Licht R van Bruggen MC Tax WJ Role of nucleosomes for induction and glomerular binding of autoantibodies in lupus nephritis.Curr Opin Nephrol Hypertens. 1999; 8: 299-306Crossref PubMed Scopus (135) Google Scholar, 3Mjelle JE Rekvig OP Fenton KA Nucleosomes possess a high affinity for glomerular laminin and collagen IV and bind nephritogenic antibodies in murine lupus-like nephritis.Ann Rheum Dis. 2007; 66: 1661-1668Crossref PubMed Scopus (60) Google Scholar, 4Mohan C Adams S Stanik V Datta SK Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus.J Exp Med. 1993; 177: 1367-1381Crossref PubMed Scopus (628) Google Scholar, 5Kalaaji M Mortensen E Jorgensen L Olsen R Rekvig OP Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.Am J Pathol. 2006; 168: 1779-1792Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 6Kalaaji M Fenton KA Mortensen ES Olsen R Sturfelt G Alm P Rekvig OP Glomerular apoptotic nucleosomes are central target structures for nephritogenic antibodies in human SLE nephritis.Kidney Int. 2007; 71: 664-672Crossref PubMed Scopus (149) Google Scholar, 7Tax WJ Kramers C van Bruggen MC Berden JH Apoptosis, nucleosomes, and nephritis in systemic lupus erythematosus.Kidney Int. 1995; 48: 666-673Crossref PubMed Scopus (116) Google Scholar They are normal products of apoptosis, but it is still not clear how intracellular self-antigens like nucleosomes become immunogens capable of triggering and maintaining a strong and prolonged autoantibody production.8Kaplan MJ Apoptosis in systemic lupus erythematosus.Clin Immunol. 2004; 112: 210-218Crossref PubMed Scopus (76) Google Scholar, 9Mortensen ES Fenton KA Rekvig OP Lupus nephritis: the central role of nucleosomes revealed.Am J Pathol. 2008; 172: 275-283Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar One hypothesis indicates that a dysregulation of apoptosis might be responsible for transformation of apoptotic into secondary necrotic chromatin. Such necrotic chromatin may potentially induce cellular and humoral autoimmunity and particularly antibody production to double-stranded DNA (dsDNA) and nucleosomes.10Casciola-Rosen LA Anhalt G Rosen A Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes.J Exp Med. 1994; 179: 1317-1330Crossref PubMed Scopus (1527) Google Scholar, 11Dieker JW van der Vlag J Berden JH Deranged removal of apoptotic cells: its role in the genesis of lupus.Nephrol Dial Transplant. 2004; 19: 282-285Crossref PubMed Scopus (52) Google Scholar, 12Berden JH Grootscholten C Jurgen WC van der Vlag J Lupus nephritis: a nucleosome waste disposal defect?.J Nephrol. 2002; 15: S1-S10PubMed Google Scholar, 13Gaipl US Munoz LE Grossmayer G Lauber K Franz S Sarter K Voll RE Winkler T Kuhn A Kalden J Kern P Herrmann M Clearance deficiency and systemic lupus erythematosus (SLE).J Autoimmun. 2007; 28: 114-121Crossref PubMed Scopus (233) Google Scholar Most discussions concentrate on increased apoptotic activity and accumulation of apoptotic, secondary necrotic cells due to reduced clearance of the dead cells as central events in the evolution of lupus nephritis. Increased apoptotic activity among peripheral blood cells from systemic lupus erythematosus patients14Courtney PA Williamson K Crockard AD Bell AL Apoptotic lymphocytes in SLE.Lupus. 1998; 7: 498Crossref PubMed Scopus (4) Google Scholar, 15Emlen W Niebur J Kadera R Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus.J Immunol. 1994; 152: 3685-3692PubMed Google Scholar, 16Ren Y Tang J Mok MY Chan AW Wu A Lau CS Increased apoptotic neutrophils and macrophages and impaired macrophage phagocytic clearance of apoptotic neutrophils in systemic lupus erythematosus.Arthritis Rheum. 2003; 48: 2888-2897Crossref PubMed Scopus (281) Google Scholar and its positive correlation with autoantibody production and disease activity has been reported.17Courtney PA Crockard AD Williamson K Irvine AE Kennedy RJ Bell AL Increased apoptotic peripheral blood neutrophils in systemic lupus erythematosus: relations with disease activity, antibodies to double stranded DNA, and neutropenia.Ann Rheum Dis. 1999; 58: 309-314Crossref PubMed Scopus (198) Google Scholar Recent studies suggest the same for glomerular cell apoptosis in human and murine lupus nephritis.5Kalaaji M Mortensen E Jorgensen L Olsen R Rekvig OP Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.Am J Pathol. 2006; 168: 1779-1792Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 18Makino H Sugiyama H Yamasaki Y Maeshima Y Wada J Kashihara N Glomerular cell apoptosis in human lupus nephritis.Virchows Arch. 2003; 443: 67-77Crossref PubMed Scopus (38) Google Scholar Such results are based on detection of apoptotic cells, whereas expression of apoptotic triggers and executioners has not been subjected to detailed investigations so far in lupus nephritis. Many studies have demonstrated impaired clearance of apoptotic cells in systemic lupus erythematosus.19Munoz LE Gaipl US Franz S Sheriff A Voll RE Kalden JR Herrmann M SLE–a disease of clearance deficiency?.Rheumatology (Oxford). 2005; 44: 1101-1107Crossref PubMed Scopus (177) Google Scholar, 20Gaipl US Franz S Voll RE Sheriff A Kalden JR Herrmann M Defects in the disposal of dying cells lead to autoimmunity.Curr Rheumatol Rep. 2004; 6: 401-407Crossref PubMed Scopus (32) Google Scholar, 21Kuenkele S Beyer TD Voll RE Kalden JR Herrmann M Impaired clearance of apoptotic cells in systemic lupus erythematosus: challenge of T and B cell tolerance.Curr Rheumatol Rep. 2003; 5: 175-177Crossref PubMed Scopus (23) Google Scholar This may result in accumulation of apoptotic cells without prior rise in the rate of apoptosis. Therefore, to determine whether there is an increase in the apoptotic activity in systemic lupus erythematosus, or whether accumulation of apoptotic or secondary necrotic debris may be due to reduced clearance, the apoptotic pathways need to be investigated.The extrinsic apoptotic pathway is initiated through the ligation of specific death receptors on the cell surface, which is followed by a cascade of enzymatic activations and identifiable morphological changes in cells and particularly in nuclei. Signaling is provided through the extrinsic pathway from receptors (Fas, tumor necrosis factor receptor superfamily, member 1a) toward activation of caspases through involvement of adaptor proteins Fas (TNFRSF6)-associated via death domain, TNFRSF1A-associated via death domain) that form bridges between downstream regulators and effectors.22Nagata S Apoptosis by death factor.Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4536) Google Scholar Anti-apoptotic (Bcl2l2) and pro-apoptotic (BCL2-associated X protein) members of the Bcl-2 protein family play a key role in controlling execution of the intrinsic apoptotic pathway.23Hengartner MO The biochemistry of apoptosis.Nature. 2000; 407: 770-776Crossref PubMed Scopus (6211) Google Scholar Thus, investigation of apoptotic processes needs an integrated assessment of apoptotic triggers, executioners and effectors.In this study, we analyzed whether there is an up-regulated apoptotic activity in kidneys of lupus-prone BW mice during nephritis progression since accumulation of apoptotic cells is an obligate observation during development of lupus nephritis.5Kalaaji M Mortensen E Jorgensen L Olsen R Rekvig OP Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.Am J Pathol. 2006; 168: 1779-1792Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 18Makino H Sugiyama H Yamasaki Y Maeshima Y Wada J Kashihara N Glomerular cell apoptosis in human lupus nephritis.Virchows Arch. 2003; 443: 67-77Crossref PubMed Scopus (38) Google Scholar We also analyzed whether accumulation of chromatin fragments in glomerular capillary membranes and mesangial matrix relates to reduced fragmentation of apoptotic chromatin by diminished transcription of the renal Dnase1 gene, and secondary to this, decreased clearance of large chromatin fragments.Materials and MethodsAnimalsFemale (NZBxNZW)F1 (BW) and BALB/c mice were purchased from Harlan (Blackthorn, UK), while MRL-lpr/lpr mice were purchased from Jackson Laboratory (Bar Harbor, Maine). Treatment and care of animals were in accordance with the guidelines of The Norwegian Ethical and Welfare Board for Research Animals, and the study was approved by the Institutional Review Board.Induction of Proteinuria in BALB/c Mice by Injecting Highly Pure Murine Anti-dsDNA Monoclonal Antibodies (mAbs)Healthy 10-week-old BALB/c mice (n = 3) where injected twice a week for 4 weeks with 200 μg purified murine anti-dsDNA mAbs (DNA6 mAb, kindly provided by T Marion, Memphis, TN) each time, similar to protocols used by others.24Ehrenstein MR Katz DR Griffiths MH Papadaki L Winkler TH Kalden JR Isenberg DA Human IgG anti-DNA antibodies deposit in kidneys and induce proteinuria in SCID mice.Kidney Int. 1995; 48: 705-711Crossref PubMed Scopus (178) Google Scholar, 25Kramers C Hylkema MN van Bruggen MC van de Lagemaat R Dijkman HB Assmann KJ Smeenk RJ Berden JH Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo.J Clin Invest. 1994; 94: 568-577Crossref PubMed Scopus (252) Google Scholar These were purified according to the protocol III of Kramers et al,25Kramers C Hylkema MN van Bruggen MC van de Lagemaat R Dijkman HB Assmann KJ Smeenk RJ Berden JH Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo.J Clin Invest. 1994; 94: 568-577Crossref PubMed Scopus (252) Google Scholar and were shown to contain only IgG heavy and light chains, and not DNA or histone proteins (data not shown). Granular deposits of chromatin-IgG complexes appeared in glomeruli at end-point in the injected mice (data not shown), and severe proteinuria developed progressively in these mice to levels comparable with those in BW and MRL-lpr/lpr mice with end-stage organ disease.Collection of Samples from MiceSerum samples from BW and sex-, age-matched BALB/c mice were collected from 4-, 8-, or 20-week-old (group 1, 2, and 3 respectively) and at the time of full-blown nephritis (group 4, mice ≥26 weeks old), while sera from MRL-lpr/lpr and BALB/c mice injected with anti-dsDNA mAbs were collected at end-point when mice suffered from severe proteinuria. Sera were stored at −20°C. Proteinuria was monitored weekly with sticks from Bayer Diagnostics (Bridgend, UK). At the age of 4, 8, 20 weeks, or after indication of severe nephritis (proteinuria ≥3g/L), animals were euthanized by CO2-suffocation. Each group contained 5 to 6 animals. Kidneys were extirpated, cut, and fixed in RNAlater (Qiagen Nordic, Norway) for further analysis of gene transcription, or fixed in 4% buffered depolymerized paraformaldehyde and embedded in paraffin for immunohistochemical analysis, or fixed in 8% buffered depolymerized paraformaldehyde for electron microscopy. The same samples were collected from sex- and age-matched BALB/c control mice. MRL-lpr/lpr mice and BALB/c mice injected with anti-dsDNA mAbs were used as proteinuric control mice to analyze if proteinuria per se affected renal gene transcription and apoptotic activities in BW mice. Samples from MRL-lpr/lpr mice were collected at age of 4 weeks and at severe nephritis. Injected BALB/c mice with proteinuria and age-matched non-treated BALB/c mice were euthanized at 14 weeks of age. Control groups included three animals in each. Organs from those animals were collected in the same way as from BW mice.Anti-dsDNA Enzyme-Linked Immunosorbent AssayCalf thymus dsDNA and Sigma fast orthophenylendiamine substrate were purchased from Sigma-Aldrich (St. Louis, MO). Murine serum antibodies against calf thymus dsDNA were detected and titrated by standard indirect enzyme-linked immunosorbent assay, as described in detail.26Rekvig OP Moens U Sundsfjord A Bredholt G Osei A Haaheim H Traavik T Arnesen E Haga HJ Experimental expression in mice and spontaneous expression in human SLE of polyomavirus T-antigen. A molecular basis for induction of antibodies to DNA and eukaryotic transcription factors.J Clin Invest. 1997; 99: 2045-2054Crossref PubMed Scopus (104) Google ScholarColocalization Immune Electron MicroscopyImmune electron microscopy was performed on murine kidney sections as previously described.5Kalaaji M Mortensen E Jorgensen L Olsen R Rekvig OP Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.Am J Pathol. 2006; 168: 1779-1792Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar Sections were incubated with rabbit anti-mouse IgG (RaM IgG, Cappel, ICN Pharmaceuticals, Inc.) and protein A conjugated with 5-nm gold particles (PAG-5 nm, University of Utrecht, The Netherlands) followed by fixation with glutaraldehyde and saturation of free aldehyde groups by glycine. In a next step, the same sections were incubated with the anti-dsDNA mAb DNA6 and protein A conjugated with 10-nm gold particles (PAG-10 nm) to determine whether in vivo-bound autoantibodies co-localized with DNA6 in extracellular chromatin. Micrographs were taken using a Jeol JEM-1010 Transmission Electron Microscope (Tokyo, Japan).Immunohistochemical AnalysisApoptotic factors in kidneys of the mice included in this study were detected by using Envision+Dual Link System-HRP (diaminobenzidine+) (Dako Norden A/S, Denmark). After deparaffinization of 4-μm paraffin-embedded kidneys sections, they were heated in citrate buffer, pH 6.0, for 20 minutes at 98°C, then blocked with peroxide for 5 minutes, followed by 60 minutes incubation with blocking solution (0.2% Triton X-100, 2% goat serum, lamb serum, fetal calf serum, and 2% bovine serum albumin) to prevent non-specific antibody binding. Subsequently, sections were incubated for 30 minutes with primary antibody to stain for activated caspase 3 (rabbit anti-mouse active caspase 3 antibody diluted 1:500, R&D Systems, Abinodon, UK), activated caspase 9 (rabbit anti-mouse activated caspase 9 antibody 1:2000, Nordic Biosite AB, Sweden), apoptotic peptidase activating factor 1 (rabbit anti-mouse APAF1 antibody 1:75, Acris Antibodies GmbH, Germany) or negative controls (normal rabbit IgG at the same concentration as primary antibodies, R&D Systems, Abinodon, UK). Secondary antibody and diaminobenzidine chromogen solution were used according to instruction from Dako. Sections were counterstained with hematoxylin and Scott's solution. To evaluate the number of positively stained cells we counted them in 10 view fields (magnification = original ×20) per kidney section.Gene Expression AnalysisTotal RNA was isolated with EZ-1 RNA tissue mini kit (Qiagen). The quality of RNA was analyzed by Agilent Bioanalyzer using RNA 6000 assay kit (Agilent Technologies, Inc., CA). cDNA was transcribed from RNA by using cDNA Archive kit (Applied Biosystems, CA). For real time PCR we used TaqMan Gene Expression Assays (Applied Biosystems, CA): tumor necrosis factor (TNF)-α Mm00443258_m1; FAS receptor Mm00433237_m1; TNFR I Mm00441875_m1; TNFR II Mm00441889_m1; TRAF2 Mm00801978_m1; FADD Mm00438861_ m1; TRADD Mm01251029_m1, Bcl2l2 Mm00432054_m1; BAX Mm00432050_m1; Cytochrome C somatic Mm01621044_g1; APAF1 Mm00437530_m1; caspase 9 Mm00516563_m1; caspase 3 Mm01195085_m1; Dnase 1 Mm01342389_g1; endogenous control – Mouse actin β 4352933E, and TATA binding protein Mm00446973_m1. The assays were performed on ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Expression levels were calculated using the ddCT method. Data are given as fold change compared with transcription in 4-week-old mice.Camptothecin-Induced Apoptosis in Isolated KidneysMice were euthanized by CO2 and kidneys were immediately extirpated and placed into 24 well plates (half a kidney per well) with apoptosis inducing solution–5 μmol/L camptothecin (Sigma-Aldrich, St Louis, MO) in RPMI-1640 with 10% fetal calf serum. The kidneys were incubated for 0, 2, 4, or 6 hours at 37°C in 5% CO2, then cut and fixed in 4% buffered depolymerized paraformaldehyde for morphological studies or in RNAlater (Qiagen) for further analyses of gene transcription. DNA fragmentation was determined by Agilent bioanalyzer as described before.27Zykova SN Seredkina N Benjaminsen J Rekvig OP Reduced fragmentation of apoptotic chromatin is associated with nephritis in lupus-prone (NZB x NZW)F(1) mice.Arthritis Rheum. 2008; 58: 813-825Crossref PubMed Scopus (26) Google ScholarStatistical AnalysisTwo-way analysis of variance was used to compare the Ct values between the age groups and the strains in the gene expression study. Differences were regarded significant at P < 0.05 provided at least twofold changes of mRNA levels. One-way analysis of variance with Dunett post hoc test was used to assess the differences in the number of active caspase-3-positive cells on the sections of kidneys with various degrees of morphological changes.ResultsAnalyses of Serum Antibodies against dsDNA and Renal Morphology in BALB/c and BW MiceThe four age groups of BW mice were analyzed for appearance of circulatory anti-dsDNA antibodies and for morphological changes in glomeruli. Figure 1A demonstrate serum levels of anti-dsDNA antibodies in each group of BW mice (results given as mean OD490 ± SEM for each group). Sex and age matched BALB/c mice were negative for anti-dsDNA antibodies (data not shown).Four- and 8-week-old BALB/c or BW mice had normal kidney morphology and no serum anti-dsDNA antibodies. Immune electron microscopy analysis did not show any immune complex deposits, nor electron dense structures in glomeruli of groups 1–3 BW mice (data not shown). Group 3 mice had low levels of anti-dsDNA autoantibodies (Figure 1A).The nephritic group 4 mice (age ≥26 weeks old, n = 6) with high levels of anti-dsDNA antibodies (Figure 1A) was divided into two subgroups due to distinctly different morphological patterns observed by immune electron microscopy, three animals in each. Group 4A had granular deposits of IgG in the mesangial matrix only (Figure 1B), while group 4B demonstrated massive deposition of IgG associated with EDS in glomerular capillary membranes (Figure 1C). Glomerular in vivo-bound IgG were strictly associated with EDS in group 4A and group 4B mice as demonstrated by immune electron microscopy. Furthermore, these in vivo-bound IgG molecules (traced by 5-nm gold particles) co-localized with anti-dsDNA mAb DNA6 added to the sections in vitro (traced by 10-nm gold particles), indicating presence of immune complexes consisting of IgG and chromatin fragments (see Figure 1, B and C, for details).Immunohistochemical Detection of Apoptotic Cells in KidneysTo determine the amount of apoptotic cells in kidneys we stained tissue sections with antibodies against several apoptotic proteins. Activated caspase 3 and 9 and APAF1 were chosen as reliable indicators for presence of apoptotic cells compared with the use of for example, the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay.28Jeruc J Vizjak A Rozman B Ferluga D Immunohistochemical expression of activated caspase-3 as a marker of apoptosis in glomeruli of human lupus nephritis.Am J Kidney Dis. 2006; 48: 410-418Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 29Baima B Sticherling M How specific is the TUNEL reaction? An account of a histochemical study on human skin.Am J Dermatopathol. 2002; 24: 130-134Crossref PubMed Scopus (26) Google Scholar These factors reflect activation of both the intrinsic and extrinsic apoptotic pathways.Immunohistochemical analysis showed no difference between staining of activated caspase 3 and 9 or APAF 1 in kidneys from groups 1 (Figure 2, A–C, respectively), 2 and 3 (data not shown) of the BW mice compared with age-matched BALB/c mice.Figure 2Detection of apoptotic factors in kidneys of young (A–C) and nephritic (D–F) BW mice. Data are demonstrated for activated caspase 3 (A, D), APAF1 (B, E) and activated caspase 9 (C, F) in kidneys from 4-week-old (upper panels) and nephritic (lower panels) BW mice. There is increased numbers of activated caspase 3-positive cells in tubular cells from proteinuric BW mice (D) and also activated caspase 3-positive structures in tubular lumina (inset in D), whereas no staining was observed in kidney sections from young BW mice (A). Activated caspase 9 and APAF 1 were absent in kidneys from young BW mice (B, C, for APAF1 and activated caspase 9, respectively), but gave strong staining in tubular lumina in the proteinuric kidneys (E, F, respectively). There was no staining in negative controls using non-specific rabbit IgG (inset in F). Magnification = original ×40. Arrows in D point at activated caspase 3-positive cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Slightly increased number of active caspase 3-positive cells was found in kidneys of nephritic mice (Figure 2D), mostly located in tubuli and interstitial area (Figure 2D, arrows), and few apoptotic cells were observed in glomeruli. Interestingly, antibodies against activated caspase 3 and 9 and APAF 1 gave strong staining of structures in tubular lumina in nephritic kidneys (Figure 2, D (inset), E, and F, for activated caspase 3, caspase 9, and APAF 1, respectively). No staining was found in tubular lumina in nephritic kidneys using nonspecific rabbit IgG (insert in Figure 2F). Other proteinuric kidneys, like those from severely proteinuric BALB/c mice injected with anti-dsDNA antibodies, did not contain caspase 3 and 9 and APAF 1 positive structures in their lumina (data not shown). These latter data argue against the idea that debris in tubular lumina is a non-specific result of proteinuria. When analyzing kidneys from severely nephritic MRL-lpr/lpr mice, which have a distinctly different genetic background for development of lupus-like nephritis, smaller and fewer apoptotic structures could be observed in tubular lumina compared with what was observed in the nephritic BW kidneys (data not shown).The number of activated caspase 3-positive cells was determined in kidney sections of nephritic BW mice in comparison with younger BW and age matched BALB/c mice (Figure 3 right axis) and plotted against the absolute number of renal cells in the same slides (Figure 3 left axis). There was no significant difference in the number of activated caspase 3-positive cells between BALB/c kidneys from different age groups, while in group 4B of BW mice the number of activated caspase 3-positive cells was significantly increased, as compared with young BW group 1 mice (P < 0.05). This rise did not correspond to elevation of absolute number of renal cells in the nephritic group. There was no significant difference in the number of activated caspase 3-positive cells in kidneys from nephritic BW mice compared with age-matched BALB/c mice. Subgroups 4A and 4B BALB/c mice were divided in compliance with age of group 4A and 4B BW mice. In control experiments, we observed that sections of kidneys from BALB/c mice with severe proteinuria after injection of anti-dsDNA mAbs had no more activated caspase 3-positive cells than young or age-matched non-treated BALB/c mice (Figure 4A). Similarly, in MRL-lpr/lpr mice with severe nephritis, kidneys contained only slightly more activated caspase 3-positive cells than young animals of the same strain. The difference was, however, not statistically significant (Figure 4A), thus indicating that proteinuria did not cause increased apoptosis in these proteinuric mice.Figure 3Quantitative analysis of activated caspase 3-positive cells in kidneys from BALB/c and BW mice. Activated caspase 3-positive cells were counted on the tissue sections (right axis) and plotted against the absolute number of renal cells in the same slides (left axis). Number of cells is given as average of 10 view fields per kidney section from all mice (n = 5 for groups 1 and 3, and n = 3 for groups 4A and 4B) of the different age groups. In BALB/c kidneys, the number of caspase 3-positive cells remained the same in all of the age groups, whereas in BW kidneys, there was a significant increase in group 4B, as compared with group 1. Asterisk in group 4B indicates a significant increase in activated caspase 3-positive cells compared with group 1 (P < 0.05). BALB/c groups 4A and 4B corresponds to age of Group 4A (mesangial matrix deposits) and 4B (capillary membrane deposits) nephritic BW mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Number of activated caspase 3 positive cells and changes in Dnase I gene expression in kidneys with proteinuria based on different etiologies. An averaged number of activated caspase 3-positive cells obtained by counting 10 view fields per kidney section from different groups of mice is presented. The bars represent mean of the groups (± SEM; n = 5 for group 1 of BW and n = 3 for the rest of the groups) A: Data from proteinuric kidneys are presented in comparison with results from young animals of the same strain. For BALB/c mice data are given also from non-treated age-matched animals. Significant increase in the number of activated caspase 3-positive cells was found only in g
Referência(s)