Immunohistochemical Expression of Activated Caspase-3 as a Marker of Apoptosis in Glomeruli of Human Lupus Nephritis

Background: The role of apoptosis in lupus nephritis (LN) is still controversial. One of the key events in the process of apoptosis is activation of caspase-3. Studies of experimental models suggested that activated caspase-3 is a reliable indicator of apoptotic rate, with a favorable comparison against terminal transferase-mediated DNA nick-end labeling (TUNEL) assay. Our aim is to study apoptosis in various forms of LN and its relationship to histomorphological changes and selected laboratory findings by using activated caspase-3 as a novel marker of apoptosis. Methods: We investigated glomerular cell apoptosis in 51 biopsy specimens from patients with LN classified according to the International Society of Nephrology/Renal Pathology Society classification by using the TUNEL method and immunohistochemistry against activated caspase-3. In addition, activity and chronicity indices were calculated and anti–Ki-67 antibody was used to estimate proliferative activity. Results: Activated caspase-3–positive cells were present in glomeruli of 88.2% of cases, observed in the glomerular tuft and cellular and fibrocellular crescents. In the glomerular tuft, they were found mainly in segments with active inflammatory lesions. There was good correlation between apoptotic index assessed by using activated caspase-3 immunolabeling and the TUNEL method (r = 0.72; P < 0.01). We observed a significant positive correlation between apoptotic index and LN class (P < 0.001). Apoptotic index correlated significantly with activity index, proliferation index, and daily protein excretion (P < 0.001), but not chronicity index, creatinine concentration, or anti-DNA antibody-binding activity in serum. Conclusion: Apoptotic rate is greater in severe active glomerular lesions in human LN, suggesting that apoptosis may be involved in augmenting inflammation in human LN. Background: The role of apoptosis in lupus nephritis (LN) is still controversial. One of the key events in the process of apoptosis is activation of caspase-3. Studies of experimental models suggested that activated caspase-3 is a reliable indicator of apoptotic rate, with a favorable comparison against terminal transferase-mediated DNA nick-end labeling (TUNEL) assay. Our aim is to study apoptosis in various forms of LN and its relationship to histomorphological changes and selected laboratory findings by using activated caspase-3 as a novel marker of apoptosis. Methods: We investigated glomerular cell apoptosis in 51 biopsy specimens from patients with LN classified according to the International Society of Nephrology/Renal Pathology Society classification by using the TUNEL method and immunohistochemistry against activated caspase-3. In addition, activity and chronicity indices were calculated and anti–Ki-67 antibody was used to estimate proliferative activity. Results: Activated caspase-3–positive cells were present in glomeruli of 88.2% of cases, observed in the glomerular tuft and cellular and fibrocellular crescents. In the glomerular tuft, they were found mainly in segments with active inflammatory lesions. There was good correlation between apoptotic index assessed by using activated caspase-3 immunolabeling and the TUNEL method (r = 0.72; P < 0.01). We observed a significant positive correlation between apoptotic index and LN class (P < 0.001). Apoptotic index correlated significantly with activity index, proliferation index, and daily protein excretion (P < 0.001), but not chronicity index, creatinine concentration, or anti-DNA antibody-binding activity in serum. Conclusion: Apoptotic rate is greater in severe active glomerular lesions in human LN, suggesting that apoptosis may be involved in augmenting inflammation in human LN. APOPTOSIS IS A HIGHLY programmed form of cell death that has a significant role in such physiological processes as normal cell turnover, as well as many pathological conditions. It undoubtedly is involved in morphogenesis and a variety of pathophysiological conditions of the kidney, ranging from cyst formation to inflammation and glomerular or interstitial scarring.1Mene P. Amore A. Apoptosis Potential role in renal diseases.Nephrol Dial Transplant. 1998; 13: 1936-1943Crossref PubMed Scopus (26) Google Scholar In glomerular diseases, apoptosis appears to have a dual role. Efficient deletion by apoptosis of excessive, damaged, or nonfunctioning renal cells and infiltrating inflammatory cells is beneficial for the resolution of glomerulonephritis.2Baker A.J. Mooney A. Huges J. Lombardi D. Johnson R.J. Savill J. Mesangial cell apoptosis The major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis.J Clin Invest. 1994; 94: 2105-2116Crossref PubMed Scopus (399) Google Scholar, 3Shimizu A. Kitamura H. Masuda Y. Ishizaki M. Sugisaki Y. Yamanaka N. Apoptosis in the repair process of experimental proliferative glomerulonephritis.Kidney Int. 1995; 47: 114-121Crossref PubMed Scopus (176) Google Scholar Conversely, inappropriate activation of apoptosis may contribute to the deletion of glomerular and tubular cells, thus participating in the pathogenesis of glomerulosclerosis and tubular atrophy.4Shimizu A. Masuda Y. Kitamura H. Ishizaki M. Sugisaki Y. Yamanaka N. Apoptosis in progressive crescentic glomerulonephritis.Lab Invest. 1996; 74: 941-951PubMed Google Scholar, 5Sugiyama H. Kashihara N. Makino H. Yamasaki Y. Ota A. Apoptosis in glomerular sclerosis.Kidney Int. 1996; 49: 103-111Crossref PubMed Scopus (260) Google Scholar, 6Savill J. Mooney A. Hughes J. Apoptosis and renal scarring.Kidney Int Suppl. 1996; 54: S14-S17PubMed Google Scholar Lupus nephritis (LN) is one of the most severe forms of organ involvement in patients with systemic lupus erythematosus (SLE), with a variegated histomorphological picture and unpredictable course. The significance of apoptosis in patients with LN is not clear. Some investigators reported decreased apoptosis in renal biopsy specimens from patients with LN compared with those with other proliferative glomerulonephritides and controls,7Soto H. Mosquera J. Rodriguez-Iturbe B. Henriquez La Roche C. Pinto A. Apoptosis in proliferative glomerulonephritis Decreased apoptosis expression in lupus nephritis.Nephrol Dial Transplant. 1997; 12: 273-280Crossref PubMed Scopus (58) Google Scholar, 8Lu F.M. Shen W. Zhang X.R. Guo M.Y. Lin S.Y. Dysregulation of apoptosis A possible mechanism leading to chronic progressive renal histological changes in lupus nephritis.Chin Med J. 2000; 113: 1082-1086PubMed Google Scholar, 9Wargowska-Danilewicz M. Danilewicz M. Apoptosis in lupus SLE-N IV and non-lupus mesangiocapillary glomerulonephritis type I MCGN I. A comparative study.J Nephrol. 1998; 11: 44-49PubMed Google Scholar whereas others found an increased number of apoptotic cells not only in active lesions, but also in chronic and sclerotic lesions of patients with LN.5Sugiyama H. Kashihara N. Makino H. Yamasaki Y. Ota A. Apoptosis in glomerular sclerosis.Kidney Int. 1996; 49: 103-111Crossref PubMed Scopus (260) Google Scholar, 10Makino 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 An important controversial issue is the identification of apoptotic cells. Apoptotic cells can be recognized by characteristic morphological features, such as chromatin condensation, cell shrinkage, nuclear fragmentation, and final engulfment of apoptotic bodies by macrophages.11Geske F.J. Gerschenson L.E. The biology of apoptosis.Hum Pathol. 2001; 32: 1029-1038Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar However, these features are difficult to observe in a damaged and inflamed glomerulus with proliferative and necrotic lesions. Furthermore, morphology alone does not enable the recognition of cells early in the apoptotic pathway.12Walker J.A. Quirke P. Viewing apoptosis through a “TUNEL.”.J Pathol. 2000; 195: 275-276Crossref Scopus (56) Google Scholar Several techniques for the detection of apoptotic cells were developed; the most widely used is the terminal transferase-mediated DNA nick-end labeling (TUNEL) assay, which recognizes cells containing DNA strand breaks. However, DNA strand breaks are not a unique feature of apoptosis, but also occur in necrosis, during repair of reversibly damaged DNA, and postmortem autolysis.13Grasl-Kraupp B. Ruttkay-Nedecky B. Koudelka H. Bukowska K. Bursch W. Schulte-Hermann R. In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death A cautionary note.Hepatology. 1995; 21: 1465-1468PubMed Google Scholar, 14Gown A.M. Willingham M.C. Improved detection of apoptotic cells in archival paraffin sections Immunohistochemistry using antibodies to cleaved caspase 3.J Histochem Cytochem. 2002; 50: 449-454Crossref PubMed Scopus (285) Google Scholar The TUNEL technique also can label nonapoptotic nuclei showing signs of active gene transcription.15Kockx M.M. Muhring J. Knaapen M.W. de Meyer G.R. RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis.Am J Pathol. 1998; 152: 885-888PubMed Google Scholar The validity of the TUNEL method to detect apoptosis therefore is controversial. Recently, antibodies that recognize activated (cleaved) caspase-3 became available commercially. Studies of various experimental models of apoptosis suggested that this is a reliable indicator of apoptotic rate, with a favorable comparison against TUNEL assay.12Walker J.A. Quirke P. Viewing apoptosis through a “TUNEL.”.J Pathol. 2000; 195: 275-276Crossref Scopus (56) Google Scholar, 14Gown A.M. Willingham M.C. Improved detection of apoptotic cells in archival paraffin sections Immunohistochemistry using antibodies to cleaved caspase 3.J Histochem Cytochem. 2002; 50: 449-454Crossref PubMed Scopus (285) Google Scholar, 16Duan W.R. Garner D.S. Williams S.D. Funckes-Shippy C.L. Spath I.S. Blomme E.A. Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts.J Pathol. 2003; 199: 221-228Crossref PubMed Scopus (288) Google Scholar Only a few studies of apoptosis in renal biopsy specimens of patients with lupus were reported; these were based on a limited number of patients and used electron microscopy or TUNEL assay to detect apoptotic cells.5Sugiyama H. Kashihara N. Makino H. Yamasaki Y. Ota A. Apoptosis in glomerular sclerosis.Kidney Int. 1996; 49: 103-111Crossref PubMed Scopus (260) Google Scholar, 7Soto H. Mosquera J. Rodriguez-Iturbe B. Henriquez La Roche C. Pinto A. Apoptosis in proliferative glomerulonephritis Decreased apoptosis expression in lupus nephritis.Nephrol Dial Transplant. 1997; 12: 273-280Crossref PubMed Scopus (58) Google Scholar, 8Lu F.M. Shen W. Zhang X.R. Guo M.Y. Lin S.Y. Dysregulation of apoptosis A possible mechanism leading to chronic progressive renal histological changes in lupus nephritis.Chin Med J. 2000; 113: 1082-1086PubMed Google Scholar, 9Wargowska-Danilewicz M. Danilewicz M. Apoptosis in lupus SLE-N IV and non-lupus mesangiocapillary glomerulonephritis type I MCGN I. A comparative study.J Nephrol. 1998; 11: 44-49PubMed Google Scholar, 10Makino 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, 17Wang J.S. Tseng H.H. Shih D.F. Ger L.P. Expression of inducible nitric oxide synthase and apoptosis in human lupus nephritis.Nephron. 1997; 97: 404-411Crossref Scopus (46) Google Scholar The aim of our study is to analyze the immunohistochemical expression of activated (cleaved) caspase-3 in glomeruli of patients with LN and evaluate the apoptotic rate and relationship between apoptosis and histopathologic and clinical parameters. We included in the study 51 archival renal biopsy specimens of 43 patients (39 women, 4 men) aged 10 to 70 years (28.3 ± 12.6 years) fulfilling revised criteria for the classification of SLE.18Hochberg M.C. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus.Arthritis Rheum. 1997; 40 (1725-1725)Crossref Scopus (8624) Google Scholar Four patients underwent 1 repeated biopsy and 3 patients underwent 2 repeated biopsies of the kidney. Renal tissue was examined by means of traditional light, immunofluorescence, and electron microscopy according to standardized techniques. LN was classified according to the International Society of Nephrology/Renal Pathology Society 2003 proposal,19Weening J.J. D’Agati V.D. Schwartz M.M. et al.The classification of glomerulonephritis in systemic lupus erythematosus revisited.J Am Soc Nephrol. 2004; 15: 241-250Crossref PubMed Scopus (1418) Google Scholar based on the modified World Health Organization classification from 1995.20Churg J. Bernstein J. Glassock R.J. Renal Disease. Igaku-Shoin, New York, NY1995Google Scholar Biopsy specimens also were quantitatively and semiquantitatively assessed for the presence of active and chronic lesions. Based on these features, activity and chronicity indices were calculated.21Austin H.A. Muenz L.R. Joyce K.M. Antonovych T.A. Balow J.E. Diffuse proliferative lupus nephritis Identification of specific pathologic features affecting renal outcome.Kidney Int. 1984; 25: 689-695Crossref PubMed Scopus (579) Google Scholar Five renal biopsy specimens with no morphological abnormalities under light and immunofluorescence microscopy from patients with hematuria from extrarenal causes served as controls. For immunohistochemistry, 5-μm thick sections from archival formalin-fixed paraffin-embedded tissues were placed on poly-l-lysine–coated slides, deparaffinized, and subjected to heat-induced epitope retrieval (pressure cooking in EDTA buffer at pH 8.0). For analysis of activated (cleaved) caspase-3 expression, we used monoclonal mouse antibody against human cleaved caspase-3 at a dilution of 1:30 (Cell Signaling, Danvers, MA). According to the manufacturer, anticleaved caspase-3 antibody specifically recognizes the large fragment (17 kd) of activated, but not full-length, caspase-3. Anti–Ki-67 (MIB-1) antibody (Dako, Glostrup, Denmark) at 1:20 dilution was used to detect proliferative activity of glomerular cells. Bound antibodies were detected by using a sensitive streptavidin-biotin-peroxidase technique (iVIEW DAB Detection Kit; Ventana Medical Systems, Tucson, AZ) and NexES IHC automated slide stainer (Ventana Medical Systems) according to the manufacturer’s instructions. An endogenous biotin blocking kit (Endogenous Biotin Blocking Kit; Ventana Medical Systems) was included in the procedure. Thirty-six renal biopsy specimens were available for TUNEL assay. DNA fragmentation associated with apoptosis was detected by the addition of fluorescein-labeled nucleotides to free 3′ hydroxyl groups in DNA using an In Situ Cell Death Detection Kit, POD (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions on 1 of the serial sections. After pretreatment by using microwave irradiation for 5 minutes in 0.1 mol/L of citrate buffer, pH 6.0, at 97°C, slides were cooled rapidly and incubated with TUNEL reaction mixture for 60 minutes at room temperature. This was followed by incubation with antifluorescein antibody Fab fragments conjugated with horseradish peroxidase at a 1:10 dilution for 20 minutes at room temperature and subsequent detection with substrate 3,3′-diaminobenzidine (both Dako) for 5 minutes. Slides were counterstained with hematoxylin. Sections treated without primary antibodies or without terminal deoxynucleotidyl transferase served as negative controls. For positive controls, adenoma of the large intestine was processed in the same way as renal tissue. Caspase-3–, TUNEL-, and Ki-67–positive cells were counted in all glomeruli (5 to 36 glomeruli/specimen; average, 14.7 glomeruli) in each biopsy specimen. Mean numbers of caspase-3–, TUNEL-, and Ki-67–positive stained cells per glomerulus were determined as the glomerular apoptotic index, TUNEL index, and proliferation index, respectively. Apoptotic and TUNEL indices were compared with proliferation index, histological indices, and laboratory parameters. Laboratory findings at the time of the biopsy, including anti–double-stranded DNA autoantibody, serum creatinine concentration, and daily urinary protein excretion, were obtained from patient records. Statistical analysis of quantitative data was performed by using SPSS, version 12.0.1, for Windows (SPSS Inc, Chicago, IL). Differences between groups were evaluated by means of Mann-Whitney U test; multiple comparisons, by means of Kruskal-Wallis test; and Spearman rank correlation test was used for correlation analysis. P less than 0.05 is accepted as statistically significant. Numerical variables are expressed as mean ± SD. Of 51 renal biopsy specimens, 8 were classified as class II (mesangial proliferative); 15, as class III (focal proliferative); and 28, as class IV (diffuse proliferative) LN. In class IV, there were 10 cases of diffuse segmental LN and 18 cases of diffuse global LN. Eleven patients had renal insufficiency, which was severe (serum creatinine level > 2.26 mg/dL [>200 μmol/L]) in 2 cases. Daily urinary protein excretion ranged from 0.02 to 14.9 g/d (mean, 2.8 ± 3.5 g/d). Anti–double-stranded DNA autoantibodies were positive in 42 (82.5%) cases, and binding was high (>0.80; Farr assay) in 34 cases. The remaining 9 patients (17.5%) had negative anti-DNA binding test results. Caspase-3–positive cells were present in glomeruli of 45 of 51 renal biopsy specimens (88.2%). The reaction was moderate to strong and detected in cytoplasm of apoptotic cells. Caspase-3–positive cells frequently showed morphological characteristics of apoptotic cells (condensed and fragmented chromatin, shrunken cytoplasm), although some cells with normal cellular morphological characteristics also were immunolabeled. Apoptotic index ranged from 0 to 1.5 (mean, 0.38 ± 0.35). TUNEL-positive cells were identified in glomeruli of 33 of 36 biopsy specimens (91.66%). The reaction was moderate to strong and was detected in nuclei and perinuclear regions of cells with morphological characteristics consistent with apoptosis. In approximately one third of cases, there was moderate background staining of all nuclei in the specimen, which sometimes was more intensive at the specimen margins, making distinction of apoptotic cells from viable cells very difficult. The number of cells detected with the TUNEL method was larger than the number of activated caspase-3–positive apoptotic cells. TUNEL index ranged from 0 to 2.09 (mean, 0.68 ± 0.62). Nevertheless, there was good correlation between the TUNEL index and apoptotic index, assessed by using activated caspase-3 immunolabeling (r = 0.72; P < 0.01). Caspase-3–positive cells were present in either the glomerular tuft (Fig 1A) or cellular and fibrocellular crescents (Fig 1C). In the glomerular tuft, we found positive cells mainly in segments with intensive inflammatory changes. Localization of TUNEL-positive cells corresponded well with activated caspase-3 staining, although TUNEL-positive cells also were observed frequently in areas of segmental necrosis (Fig 2C).Fig 2TUNEL-positive cells in glomeruli of diffuse LN. Apoptotic cell in glomerular tuft with proliferative changes and in tubular epithelium (arrows): (A) TUNEL method and (B) hematoxylin and eosin stain in a subsequent serial section. Groups of TUNEL-positive cells in necrotic lesions of diffuse global LN with necrotizing arteriolitis: (C) TUNEL method and (D) hematoxylin and eosin stain in a subsequent serial section.View Large Image Figure ViewerDownload (PPT) We did not observe caspase-3–positive cells in glomeruli of the healthy control group and only exceptional positive cells were detected with the TUNEL method. Caspase-3–positive cells were detected in 3 of 8 patients (37.5%) with class II LN, 14 of 15 patients (93.9%) with class III, and all patients with class IV. There was a significant correlation between apoptotic index and LN classification (P < 0.001; Fig 3). Apoptotic index was significantly higher in those with class III compared with class II LN (0.29 ± 0.215 versus 0.02 ± 0.028; P < 0.001) and class IV compared with class III (0.52 ± 0.370 versus 0.29 ± 0.215; P < 0.05). There were more caspase-3–positive cells in specimens of global compared with segmental class IV LN, but the difference was not statistically significant. Activity index ranged from 0 to 18 (mean, 8.1 ± 4.6), and chronicity index ranged from 0 to 9 (mean, 2.2 ± 2.5). Proliferation index in patients with LN ranged from 0 to 5.44 (mean, 1.56 ± 1.38) and correlated with LN classification (P < 0.001). In normal control kidneys, proliferation index ranged from 0 to 0.17 (mean, 0.10 ± 0.102). Apoptotic index correlated significantly with activity and proliferation indices (r = 0.63 and 0.63; P < 0.001; Fig 4A, B). There was weak correlation between apoptotic and chronicity indices (r = 0.42; P < 0.05) that disappeared when class II cases were excluded from the calculation. Apoptotic index correlated significantly with daily protein excretion (r = 0.68; P < 0.001; Fig 4C). There was weak correlation between apoptotic index and renal function, assessed by using serum creatinine concentration (r = 0.29; P = 0.039), that was lost when class II cases were excluded from the calculation. There was no correlation between apoptotic index and immunoserological activity of lupus assessed by using anti–double-stranded DNA antibodies (P = 0.89). In various glomerulonephritides, apoptosis was linked with many processes, such as cell proliferation, inflammatory cell infiltration, sclerosis, or the repair process in glomeruli.2Baker A.J. Mooney A. Huges J. Lombardi D. Johnson R.J. Savill J. Mesangial cell apoptosis The major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis.J Clin Invest. 1994; 94: 2105-2116Crossref PubMed Scopus (399) Google Scholar, 3Shimizu A. Kitamura H. Masuda Y. Ishizaki M. Sugisaki Y. Yamanaka N. Apoptosis in the repair process of experimental proliferative glomerulonephritis.Kidney Int. 1995; 47: 114-121Crossref PubMed Scopus (176) Google Scholar, 4Shimizu A. Masuda Y. Kitamura H. Ishizaki M. Sugisaki Y. Yamanaka N. Apoptosis in progressive crescentic glomerulonephritis.Lab Invest. 1996; 74: 941-951PubMed Google Scholar, 5Sugiyama H. Kashihara N. Makino H. Yamasaki Y. Ota A. Apoptosis in glomerular sclerosis.Kidney Int. 1996; 49: 103-111Crossref PubMed Scopus (260) Google Scholar Only a few studies examined apoptotic rate in patients with LN, and only in a limited number of patients and with controversial results. Some investigators suggested that reduced apoptosis is a characteristic of proliferative LN associated with chronic histopathologic changes,7Soto H. Mosquera J. Rodriguez-Iturbe B. Henriquez La Roche C. Pinto A. Apoptosis in proliferative glomerulonephritis Decreased apoptosis expression in lupus nephritis.Nephrol Dial Transplant. 1997; 12: 273-280Crossref PubMed Scopus (58) Google Scholar, 8Lu F.M. Shen W. Zhang X.R. Guo M.Y. Lin S.Y. Dysregulation of apoptosis A possible mechanism leading to chronic progressive renal histological changes in lupus nephritis.Chin Med J. 2000; 113: 1082-1086PubMed Google Scholar whereas others found an increased number of apoptotic cells in both active and sclerotic lesions of patients with LN.5Sugiyama H. Kashihara N. Makino H. Yamasaki Y. Ota A. Apoptosis in glomerular sclerosis.Kidney Int. 1996; 49: 103-111Crossref PubMed Scopus (260) Google Scholar, 10Makino 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 The apoptotic process is extremely rapid, and a cell may undergo apoptosis and disappear within about 1 or 2 hours, making quantification of the phenomenon in tissue sections challenging.22Amore A. Coppo R. Role of apoptosis in pathogenesis and progression of renal disease.Nephron. 2000; 86: 99-104Crossref PubMed Scopus (30) Google Scholar Previous studies used electron microscopy and/or the TUNEL method to detect apoptotic cells.5Sugiyama H. Kashihara N. Makino H. Yamasaki Y. Ota A. Apoptosis in glomerular sclerosis.Kidney Int. 1996; 49: 103-111Crossref PubMed Scopus (260) Google Scholar, 7Soto H. Mosquera J. Rodriguez-Iturbe B. Henriquez La Roche C. Pinto A. Apoptosis in proliferative glomerulonephritis Decreased apoptosis expression in lupus nephritis.Nephrol Dial Transplant. 1997; 12: 273-280Crossref PubMed Scopus (58) Google Scholar, 8Lu F.M. Shen W. Zhang X.R. Guo M.Y. Lin S.Y. Dysregulation of apoptosis A possible mechanism leading to chronic progressive renal histological changes in lupus nephritis.Chin Med J. 2000; 113: 1082-1086PubMed Google Scholar, 9Wargowska-Danilewicz M. Danilewicz M. Apoptosis in lupus SLE-N IV and non-lupus mesangiocapillary glomerulonephritis type I MCGN I. A comparative study.J Nephrol. 1998; 11: 44-49PubMed Google Scholar, 10Makino 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, 17Wang J.S. Tseng H.H. Shih D.F. Ger L.P. Expression of inducible nitric oxide synthase and apoptosis in human lupus nephritis.Nephron. 1997; 97: 404-411Crossref Scopus (46) Google Scholar, 23Tashiro K. Kodera S. Takahashi Y. Horikoshi S. Shirato I. Tomino Y. Detection of apoptotic cells in glomeruli of patients with IgA nephropathy.Nephron. 1998; 79: 21-27Crossref PubMed Scopus (25) Google Scholar Although ultrastructural morphology is a reliable tool for detecting apoptotic cells, usually only a few glomeruli are available for electron microscopic studies, making this technique unsuitable for quantification of apoptotic cells in disease settings in which apoptosis is a rare event. The TUNEL assay is associated with a number of technical problems and also labels necrotic, autolytic, and transcriptionally active cells, in addition to apoptotic cells.12Walker J.A. Quirke P. Viewing apoptosis through a “TUNEL.”.J Pathol. 2000; 195: 275-276Crossref Scopus (56) Google Scholar, 13Grasl-Kraupp B. Ruttkay-Nedecky B. Koudelka H. Bukowska K. Bursch W. Schulte-Hermann R. In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death A cautionary note.Hepatology. 1995; 21: 1465-1468PubMed Google Scholar, 14Gown A.M. Willingham M.C. Improved detection of apoptotic cells in archival paraffin sections Immunohistochemistry using antibodies to cleaved caspase 3.J Histochem Cytochem. 2002; 50: 449-454Crossref PubMed Scopus (285) Google Scholar, 15Kockx M.M. Muhring J. Knaapen M.W. de Meyer G.R. RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis.Am J Pathol. 1998; 152: 885-888PubMed Google Scholar, 16Duan W.R. Garner D.S. Williams S.D. Funckes-Shippy C.L. Spath I.S. Blomme E.A. Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts.J Pathol. 2003; 199: 221-228Crossref PubMed Scopus (288) Google Scholar Activation of caspases (cleavage of procaspase to activated caspase) is one of the key events in the process of apoptosis. Caspase-3 is a central effector caspase in many cells, leading to proteolytic cleavage of a large number of proteins, and is responsible for apoptosis-associated chromatin margination, DNA fragmentation, and nuclear collapse.24Slee E.A. Adrain C. Martin S.J. Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis.J Biol Chem. 2001; 276: 7320-7326Crossref PubMed Scopus (807) Google Scholar When cleaved through activation of the apoptotic cascade, the peptide end of this activated caspase represents a novel epitope not present in normal cells. Detection of this novel epitope is believed to be a unique and sensitive indicator of apoptosis. Studies of various human tissues and cells showed that immunohistochemical detection of activated caspase-3 is a useful tool for identifying apoptotic cells in archival material, even before all morphological features of apoptosis occur.14Gown A.M. Willingham M.C. Improved detection of apoptotic cells in archival paraffin sections Immunohistochemistry using antibodies to cleaved caspase 3.J Histochem Cytochem. 2002; 50: 449-454Crossref PubMed Scopus (285) Google Scholar, 16Duan W.R. Garner D.S. Williams S.D. Funckes-Shippy C.L. Spath I.S. Blomme E.A. Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts.J Pathol. 2003; 199: 221-228Crossref PubMed Scopus (288) Google Scholar Previous studies showed increased caspase-3 activity in various animal models of renal disease, including ischemia-reperfusion injury,25Daemen M.A.R.C. van’t Veer C. Danecker G. et al.Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation.J Clin Invest. 1999; 104: 541-549Crossref PubMed Scopus (484) Google Scholar cyclosporine A–induced nephrotoxicity,26Shihab F.S. Andoh T.F. Tanner A.M. Yi H. Bennet W.M. Expression of apoptosis in regulatory genes in chronic cyclosporin nephrotoxicity favours apoptosis.Kidney Int. 1999; 5