Nitric Oxide Is an Important Mediator of Renal Tubular Epithelial Cell Death in Vitro and in Murine Experimental Hydronephrosis
2006; Elsevier BV; Volume: 169; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2006.050964
ISSN1525-2191
AutoresTiina Kipari, Jean‐François Cailhier, David A. Ferenbach, Simon J. Watson, Kris Houlberg, David Walbaum, Spike Clay, John Savill, Jeremy Hughes,
Tópico(s)Dialysis and Renal Disease Management
ResumoMacrophages play a pivotal role in tissue injury and fibrosis during renal inflammation. Although macrophages may induce apoptosis of renal tubular epithelial cells, the mechanisms involved are unclear. We used a microscopically quantifiable co-culture assay to dissect the cytotoxic interaction between murine bone marrow-derived macrophages and Madin-Darby canine kidney cells and primary murine renal tubular epithelial cells. The induction of tubular cell apoptosis by cytokine-activated macrophages was reduced by inhibitors of nitric oxide synthase whereas tubular cell proliferation was unaffected. Furthermore, cytokine-activated macrophages derived from mice targeted for the deletion of inducible nitric oxide synthase were noncytotoxic. We then examined the role of nitric oxide in vivo by inhibiting inducible nitric oxide synthase in the model of murine experimental hydronephrosis. l-N6-(1-iminoethyl)-lysine was administered in the drinking water between days 5 and 7 after ureteric obstruction. Macrophage infiltration was comparable between groups, but treatment significantly inhibited tubular cell apoptosis at day 7. Tubular cell proliferation was unaffected. Inducible nitric oxide synthase blockade also reduced interstitial cell apoptosis and increased collagen III deposition. These data indicate that nitric oxide is a key mediator of macrophage-directed tubular cell apoptosis in vitro and in vivo and also modulates tubulointerstitial fibrosis. Macrophages play a pivotal role in tissue injury and fibrosis during renal inflammation. Although macrophages may induce apoptosis of renal tubular epithelial cells, the mechanisms involved are unclear. We used a microscopically quantifiable co-culture assay to dissect the cytotoxic interaction between murine bone marrow-derived macrophages and Madin-Darby canine kidney cells and primary murine renal tubular epithelial cells. The induction of tubular cell apoptosis by cytokine-activated macrophages was reduced by inhibitors of nitric oxide synthase whereas tubular cell proliferation was unaffected. Furthermore, cytokine-activated macrophages derived from mice targeted for the deletion of inducible nitric oxide synthase were noncytotoxic. We then examined the role of nitric oxide in vivo by inhibiting inducible nitric oxide synthase in the model of murine experimental hydronephrosis. l-N6-(1-iminoethyl)-lysine was administered in the drinking water between days 5 and 7 after ureteric obstruction. Macrophage infiltration was comparable between groups, but treatment significantly inhibited tubular cell apoptosis at day 7. Tubular cell proliferation was unaffected. Inducible nitric oxide synthase blockade also reduced interstitial cell apoptosis and increased collagen III deposition. These data indicate that nitric oxide is a key mediator of macrophage-directed tubular cell apoptosis in vitro and in vivo and also modulates tubulointerstitial fibrosis. Macrophages are remarkably versatile cells that play a major role in many key biological processes including host defense, wound healing, development, tissue remodeling, acute inflammation, and the clearance of apoptotic cells.1Gordon S Macrophage-restricted molecules: role in differentiation and activation.Immunol Lett. 1999; 65: 5-8Crossref PubMed Scopus (74) Google Scholar, 2Leibovich SJ Ross R The role of the macrophage in wound repair. 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The resultant diminished tubulointerstitial macrophage infiltrate was associated with a reduction in tubular epithelial cell apoptosis. This finding was reinforced by our recent work that used conditional macrophage ablation in progressive nephrotoxic glomerulonephritis because macrophage ablation significantly reduced the level of tubular epithelial cell apoptosis.14Duffield JS Tipping PG Kipari T Cailhier JF Clay S Lang R Bonventre JV Hughes J Conditional ablation of macrophages halts progression of crescentic glomerulonephritis.Am J Pathol. 2005; 165: 1207-1219Abstract Full Text Full Text PDF Scopus (205) Google Scholar The role of macrophages in the nonimmunological model of experimental hydronephrosis induced by unilateral ureteric obstruction has also been studied.12Lenda DM Kikawada E Stanley ER Kelley VR Reduced macrophage recruitment, proliferation, and activation in colony-stimulating factor-1-deficient mice results in decreased tubular apoptosis during renal inflammation.J Immunol. 2003; 170: 3254-3262PubMed Google Scholar, 13Lange-Sperandio B Cachat F Thornhill BA Chevalier RL Selectins mediate macrophage infiltration in obstructive nephropathy in newborn mice.Kidney Int. 2002; 61: 516-524Crossref PubMed Scopus (86) Google Scholar Lenda and colleagues12Lenda DM Kikawada E Stanley ER Kelley VR Reduced macrophage recruitment, proliferation, and activation in colony-stimulating factor-1-deficient mice results in decreased tubular apoptosis during renal inflammation.J Immunol. 2003; 170: 3254-3262PubMed Google Scholar obstructed the kidneys of colony-stimulating-factor-1 (CSF-1)-deficient mice and demonstrated a reduced interstitial macrophage infiltrate associated with a reduced level of tubular epithelial cell death. Lange-Sperandio and colleagues13Lange-Sperandio B Cachat F Thornhill BA Chevalier RL Selectins mediate macrophage infiltration in obstructive nephropathy in newborn mice.Kidney Int. 2002; 61: 516-524Crossref PubMed Scopus (86) Google Scholar obstructed the kidneys of triple E-, P-, and L-selectin knockout mice or wild-type control mice within the first 48 hours after birth. Triple selectin knockout mice exhibited diminished tubulointerstitial macrophage infiltration and reduced levels of tubular epithelial cell death compared to control mice. Although tubular cell apoptosis is invariably present during renal injury, excessive levels of tubular cell death are highly undesirable and result in renal tubular atrophy, hypocellular scarring and eventual organ failure.15Gobe GC Axelsen RA Genesis of renal tubular atrophy in experimental hydronephrosis in the rat. Role of apoptosis.Lab Invest. 1987; 56: 273-281PubMed Google Scholar Indeed, the tubulointerstitium of the kidney plays an important role in all renal diseases irrespective of the nature of the original injury16Bohle A Wehrmann M Mackensen-Haen S Gise H Mickeler E Xiao TC Muller C Muller GA Pathogenesis of chronic renal failure in primary glomerulopathies.Nephrol Dial Transplant. 1994; 9: 4-12PubMed Google Scholar because there is a striking correlation between the severity of the tubulointerstitial changes in human biopsies and the subsequent development and progression of chronic renal failure to end-stage renal failure requiring dialysis.17Bohle A Muller GA Wehrmann M Mackensen-Haen S Xiao JC Pathogenesis of chronic renal failure in the primary glomerulopathies, renal vasculopathies, and chronic interstitial nephritides.Kidney Int Suppl. 1996; 54: S2-S9PubMed Google Scholar Currently, despite the documented correlation between the severity of macrophage infiltration and the level of tubular epithelial cell apoptosis,12Lenda DM Kikawada E Stanley ER Kelley VR Reduced macrophage recruitment, proliferation, and activation in colony-stimulating factor-1-deficient mice results in decreased tubular apoptosis during renal inflammation.J Immunol. 2003; 170: 3254-3262PubMed Google Scholar, 13Lange-Sperandio B Cachat F Thornhill BA Chevalier RL Selectins mediate macrophage infiltration in obstructive nephropathy in newborn mice.Kidney Int. 2002; 61: 516-524Crossref PubMed Scopus (86) Google Scholar, 14Duffield JS Tipping PG Kipari T Cailhier JF Clay S Lang R Bonventre JV Hughes J Conditional ablation of macrophages halts progression of crescentic glomerulonephritis.Am J Pathol. 2005; 165: 1207-1219Abstract Full Text Full Text PDF Scopus (205) Google Scholar there is scant data regarding the mechanisms involved in macrophage-mediated tubular cell apoptosis. Inflammatory macrophages produce myriad proapoptotic mediators that may kill neighboring cells, including nitric oxide (NO), tumor necrosis factor-α (TNF-α), as well as Fas ligand (FasL).18Duffield JS Erwig L-P Wei X-Q Liew FY Rees AJ Savill JS Activated macrophages direct apoptosis and suppress mitosis of mesangial cells.J Immunol. 2000; 164: 2110-2119PubMed Google Scholar, 19Duffield JS Ware CF Ryffel B Savill J Suppression by apoptotic cells defines tumor necrosis factor-mediated induction of glomerular mesangial cell apoptosis by activated macrophages.Am J Pathol. 2001; 159: 1397-1404Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 20Brown SB Savill J Phagocytosis triggers macrophage release of Fas ligand and induces apoptosis of bystander leukocytes.J Immunol. 1999; 162: 480-485PubMed Google Scholar Previous in vitro studies addressing this issue used both macrophage and murine tubular cell lines21Lange-Sperandio B Fulda S Vandewalle A Chevalier RL Macrophages induce apoptosis in proximal tubule cells.Pediatr Nephrol. 2003; 18: 335-341PubMed Google Scholar with cytokine-activated J774 macrophages inducing apoptosis of murine PKSV-PR proximal tubular cells. This study and previous work by Tesch and colleagues,11Tesch GH Schwarting A Kinoshita K Lan HY Rollins BJ Kelley VR Monocyte chemoattractant protein-1 promotes macrophage-mediated tubular injury, but not glomerular injury, in nephrotoxic serum nephritis.J Clin Invest. 1999; 103: 73-80Crossref PubMed Scopus (237) Google Scholar however, did not determine the nature of the macrophage death effector although no role for TNF-α, FasL, or transforming growth factor-β was demonstrable.21Lange-Sperandio B Fulda S Vandewalle A Chevalier RL Macrophages induce apoptosis in proximal tubule cells.Pediatr Nephrol. 2003; 18: 335-341PubMed Google Scholar In this study we have used a well-established microscopically quantifiable co-culture assay18Duffield JS Erwig L-P Wei X-Q Liew FY Rees AJ Savill JS Activated macrophages direct apoptosis and suppress mitosis of mesangial cells.J Immunol. 2000; 164: 2110-2119PubMed Google Scholar and the model of experimental hydronephrosis12Lenda DM Kikawada E Stanley ER Kelley VR Reduced macrophage recruitment, proliferation, and activation in colony-stimulating factor-1-deficient mice results in decreased tubular apoptosis during renal inflammation.J Immunol. 2003; 170: 3254-3262PubMed Google Scholar, 13Lange-Sperandio B Cachat F Thornhill BA Chevalier RL Selectins mediate macrophage infiltration in obstructive nephropathy in newborn mice.Kidney Int. 2002; 61: 516-524Crossref PubMed Scopus (86) Google Scholar, 22Diamond JR Macrophages and progressive renal disease in experimental hydronephrosis.Am J Kidney Dis. 1995; 26: 133-140Abstract Full Text PDF PubMed Scopus (87) Google Scholar to examine the cytotoxic mechanism underlying macrophage-mediated tubular epithelial cell death in vitro and in vivo. We demonstrate an important role for macrophage-derived NO in the induction of tubular cell apoptosis in vitro and during tubulointerstitial inflammation in vivo. In addition, our data reinforces a role for NO in modulating tubulointerstitial fibrosis and scarring. Tissue culture reagents were purchased from Life Technologies (Paisley, UK). Tissue culture plastics were obtained from Costar (Loughborough, Leicestershire, UK) and Falcon (Runcorn, Cheshire, UK). Cytokines were purchased from R&D Systems (Abingdon, Oxon, UK) and Peprotech EC Ltd. (London, UK). l-N6-(1-iminoethyl)-lysine (L-NIL) and the control inactive isomer d-N6-(1-iminoethyl)-lysine (D-NIL) were purchased from Fluorochem Ltd. (Old Glossop, Derbyshire, UK). All other reagents were from Sigma-Aldrich Co. Ltd. (Poole, UK) unless otherwise stated. Inducible nitric oxide synthase (iNOS) knockout23Wei XQ Charles IG Smith A Ure J Feng GJ Huang FP Xu D Muller W Moncada S Liew FY Altered immune responses in mice lacking inducible nitric oxide synthase.Nature. 1995; 375: 408-411Crossref PubMed Scopus (1159) Google Scholar and wild-type control mice were obtained from B and K Universal (Hull, UK). iNOS knockout and wild-type mice were on the 129/sv background. CB7BL/6 and FVB/N mice were bred at the University of Edinburgh. Bone marrow-derived macrophages were used in these studies and were prepared from FVB/N mice, C57BL/6, or iNOS knockout and wild-type mice as described previously.18Duffield JS Erwig L-P Wei X-Q Liew FY Rees AJ Savill JS Activated macrophages direct apoptosis and suppress mitosis of mesangial cells.J Immunol. 2000; 164: 2110-2119PubMed Google Scholar Briefly, bone marrow was isolated from femurs by standard sterile techniques and matured for 7 days in sterile Teflon wells in Dulbecco's modified Eagle's medium (DMEM)/F12 medium with 10% heat inactivated fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% L929 cell-conditioned medium as a source of M-CSF. Macrophages were greater than 98% positive for the macrophage marker F4/80 by flow cytometry. Madin-Darby canine kidney (MDCK) cells (a gift from Dr. J. Davie, University of Edinburgh) were cultured as described previously.24Yang YL Guh JY Yang ML Lai YH Tsai JH Hung WC Chang CC Chuang LY Interaction between high glucose and TGF-beta in cell cycle protein regulations in MDCK cells.J Am Soc Nephrol. 1998; 9: 182-193PubMed Google Scholar Briefly, cells were grown as a monolayer culture in 75-cm2 culture flasks and maintained with Eagle's minimum essential medium containing 1% nonessential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated FCS in a humidified atmosphere of 5% CO2 at 37°C. Murine primary tubular epithelial (PTE) cells were derived from the kidneys of C57BL/6 mice after microdissection and brief collagenase digestion.25Sato M Muragaki Y Saika S Roberts AB Ooshima A Targeted disruption of TGF-{beta}1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction.J Clin Invest. 2003; 112: 1486-1494Crossref PubMed Scopus (718) Google Scholar PTE cells were grown as a monolayer culture in 25-cm2 culture flasks and maintained with DMEM/F12 medium containing insulin (10 μg/ml), transferrin (5.5 μg/ml), selenium (5 ng/ml), epidermal growth factor (25 ng/ml), and dexamethasone (36 ng/ml). PTE cells were cytokeratin-positive and vimentin-negative by immunochemistry. MDCK or PTE cells were prelabeled with fluorescent CellTracker Green whereas in some experiments mature macrophages (7 to 10 days) were prelabeled with fluorescent CellTracker Orange (both CellTracker dyes obtained from Molecular Probes, Eugene, OR). Cells were washed with serum-free medium and incubated for 30 minutes (macrophage) or 1 hour (epithelial cells) in serum-free medium containing the respective CellTracker dye at a concentration of 5 ng/ml. Cells were washed in medium containing 10% FCS to remove unbound CellTracker dye. Epithelial cells were then trypsinized and added to 48-well plates at a density to cover 60 to 70% of the well surface: 1 × 104 MDCK cells/well and 1.5 × 104 PTE cells/well. Wells were washed after 2 to 4 hours to remove nonadherent cells. Macrophages were added to epithelial cells at a ratio of two macrophages to one epithelial cell. MDCK experiments were conducted in DMEM/F12 medium containing 10% FCS, and PTE cell experiments were conducted in PTE cell medium containing 0.1% FCS. Selected co-cultures were activated with lipopolysaccharide (LPS) (1 μg/ml) and murine interferon-γ (IFN-γ) (100 U/ml), with nonactivated co-cultures being exposed to medium alone. After 24 hours of incubation, the undisturbed co-cultures underwent in situ fixation with formaldehyde (4% final concentration) to ensure retention of apoptotic cells.18Duffield JS Erwig L-P Wei X-Q Liew FY Rees AJ Savill JS Activated macrophages direct apoptosis and suppress mitosis of mesangial cells.J Immunol. 2000; 164: 2110-2119PubMed Google Scholar Fixed co-cultures were then stained with Hoechst 33342 at 1 μg/ml in phosphate-buffered saline (PBS) for 15 minutes. Using inverted fluorescent microscopy, nonoverlapping fields from each well were randomly and blindly chosen so that at least 200 epithelial cells were counted per well. Apoptotic epithelial cells were identified by their green condensed cytoplasm and pyknotic nuclei. Mitotic epithelial cells were discernible by their characteristic chromatin pattern. The number of apoptotic or mitotic epithelial cells was counted and expressed as apoptotic or mitotic cells per high-power field or as a percentage of the total number of epithelial cells (percent apoptosis). All experimental conditions were performed in triplicate and experiments performed on at least three separate occasions. Macrophage and primary PTE cell cultures were derived from at least three different animals. Experimental hydronephrosis was induced by performing unilateral ureteric obstruction.26Hughes J Brown P Shankland SJ Cyclin kinase inhibitor p21CIP1/WAF1 limits interstitial cell proliferation following ureteric obstruction.Am J Physiol. 1999; 277: F948-F956PubMed Google Scholar The left ureter of age-matched male FVB/N mice was ligated under inhalational anesthesia. Mice were administered either L-NIL or the control inactive isomer D-NIL in the drinking water (1 mg/ml) for 48 hours before sacrifice at day 7 (n = seven to eight per group).27Westenfeld R Gawlik A de Heer E Kitahara M Abou-Rebyeh F Floege J Ketteler M Selective inhibition of inducible nitric oxide synthase enhances intraglomerular coagulation in chronic anti-Thy 1 nephritis.Kidney Int. 2002; 61: 834-838Crossref PubMed Scopus (16) Google Scholar, 28Reilly CM Farrelly LW Viti D Redmond ST Hutchison F Ruiz P Manning P Connor J Gilkeson GS Modulation of renal disease in MRL/lpr mice by pharmacologic inhibition of inducible nitric oxide synthase.Kidney Int. 2002; 61: 839-846Crossref PubMed Scopus (60) Google Scholar The removed kidneys were cut longitudinally and fixed in either 10% buffered formalin or methyl Carnoy's solution (60% methanol, 30% chloroform, and 10% acetic acid) and embedded in paraffin. All experiments were performed in accordance with the UK Government Home Office regulations. To examine renal histology, 4-μm sections were stained with periodic acid-Schiff (PAS) reagent and counterstained with hematoxylin. Tubulointerstitial macrophage infiltration was quantified after immunostaining for the murine macrophage marker F4/80. Briefly, methyl Carnoy's fixed tissue sections were deparaffinized, rehydrated in ethanol, and incubated in 3% H2O2 in methanol to block endogenous peroxidase activity. Tissue sections were then incubated with rat monoclonal antibody (IgG2b) directed against mouse F4/80 (1/1000 dilution; Caltag Laboratories, Northampton, UK) at 4°C overnight, followed by a mouse-adsorbed biotinylated rabbit anti-rat IgG (1/1000 dilution; Vector Laboratories, Peterborough, UK) at room temperature for 30 minutes. For iNOS immunostaining, tissue sections were incubated with a polyclonal rabbit antibody (1/50 dilution; Abcam Laboratories, Cambridge, UK) at 4°C overnight, followed by a biotinylated goat anti-rabbit IgG (1/300 dilution; DakoCytomation, Glostrup, Denmark) at room temperature for 30 minutes. The tubulointerstitial myofibroblast population was quantified after immunostaining for the myofibroblast marker α-smooth muscle actin. Tissue sections were incubated with monoclonal mouse anti-human α-smooth muscle actin that cross-reacts with murine α-smooth muscle actin [clone 1A4 (IgG2a), 1/1000 dilution; Sigma-Aldrich Co. Ltd., Poole, UK] at 4°C overnight, followed by a biotinylated rat-anti-mouse IgG2a (1/100 dilution; Zymed Laboratories, San Francisco, CA) at room temperature for 30 minutes. Tubulointerstitial fibrosis was quantified after immunostaining for collagen III. Tissue sections were incubated with goat-anti-human type III collagen antibody, which also detects murine type III collagen (1/50 dilution; Cambridge BioScience Ltd., Cambridge, UK) at 4°C overnight, followed by a biotinylated rabbit-anti-goat IgG (1/100 dilution; Vector Laboratories) at room temperature for 30 minutes. After washing in PBS, sections were incubated in horseradish peroxidase-conjugated avidin D (1/2000 dilution; Vector Laboratories) at room temperature for 20 minutes. Color was developed using diaminobenzidine as the chromogen and counterstained with methyl green or hematoxylin. An irrelevant isotype control primary antibody served as negative control. Positive control tissue included sections from diseased mice that were known to express F4/80-positive macrophages or exhibit significant renal scarring. Interstitial macrophage infiltration was quantified in a blinded manner by analyzing 10 sequentially selected nonoverlapping fields of renal cortex of F4/80-stained sections at ×100 magnification using computer-assisted image analysis (ImageJ 1.30h; National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/Java1.3.1_03).14Duffield JS Tipping PG Kipari T Cailhier JF Clay S Lang R Bonventre JV Hughes J Conditional ablation of macrophages halts progression of crescentic glomerulonephritis.Am J Pathol. 2005; 165: 1207-1219Abstract Full Text Full Text PDF Scopus (205) Google Scholar, 29Hunter MG Hurwitz S Bellamy CO Duffield JS Quantitative morphometry of lupus nephritis: the significance of collagen, tubular space, and inflammatory infiltrate.Kidney Int. 2005; 67: 94-102Crossref PubMed Scopus (32) Google Scholar, 30Duffield JS Forbes SJ Constandinou CM Clay S Partolina M Vuthoori S Wu S Lang R Iredale JP Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.J Clin Invest. 2005; 115: 56-65Crossref PubMed Scopus (1328) Google Scholar Macrophage infiltration was expressed as the percentage of tissue surface area positive for F4/80 staining.14Duffield JS Tipping PG Kipari T Cailhier JF Clay S Lang R Bonventre JV Hughes J Conditional ablation of macrophages halts progression of crescentic glomerulonephritis.Am J Pathol. 2005; 165: 1207-1219Abstract Full Text Full Text PDF Scopus (205) Google Scholar, 31Ophascharoensuk V Giachelli CM Gordon K Hughes J Pichler R Brown P Liaw L Schmidt R Shankland SJ Alpers CE Couser WG Johnson RJ Obstructive uropathy in the mouse: role of osteopontin in interstitial fibrosis and apoptosis.Kidney Int. 1999; 56: 571-580Crossref PubMed Scopus (256) Google Scholar Tubulointerstitial collagen III deposition was quantified in a similar manner while myofibroblast accumulation was expressed as the percentage of tissue surface area positive for α-smooth muscle actin staining excluding blood vessels. Methyl Carnoy's fixed tissue sections were deparaffinized, rehydrated, and incubated in 3% H2O2 in methanol to block endogenous peroxidase activity. Nonspecific binding was blocked by incubation in 2% goat serum, 1% bovine serum albumin, 0.1% Triton X-100, and 0.05% Tween 20 for 30 minutes at room temperature. Tissue sections were then sequentially incubated in the following antibodies: 1) polyclonal rabbit anti-iNOS (1/50 dilution; BD Biosciences Pharmingen, Oxford, UK); 2) AlexaFluor-488-conjugated goat anti-rabbit IgG (1/300 dilution; Molecular Probes); 3) rat anti-mouse F4/80 (IgG2b, 1/1000 dilution; Caltag Laboratories, Northampton, UK); 4) mouse-adsorbed biotinylated rabbit anti-rat IgG (1/1000 dilution, Vector Laboratories); and 5) Alexa Fluor-568-conjugated streptavidin (1/300 dilution, Molecular Probes). Primary antibodies were incubated overnight at 4°C, with remaining incubations being performed at room temperature for 30 minutes. After each incubation step tissue sections were washed three times in Tris-buffered saline (pH 7.6) for 5 minutes. To assess the specificity of the immunostaining, tissue sections were incubated with nonimmune rabbit or rat IgG in place of the primary anti-iNOS or anti-F4/80 antibodies and then processed under identical conditions. Tissue sections were mounted with anti-fade mounting medium (Vector Laboratories), and double-immunofluorescent staining was analyzed by inverted fluorescent microscopy. Apoptotic cells were detected by the terminal dUTP nick-end labeling (TUNEL) assay as previously described.32Baker AJ Mooney A Hughes J Lombardi D Johnson RJ 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 (403) Google Scholar Briefly, 4-μm formalin-fixed tissue sections were deparaffinized and rehydrated in ethanol followed by an antigen retrieval step comprising of boiling in 0.01 mol/L sodium citrate buffer for 2 minutes. Sections were then incubated with proteinase K (6.2 μg/ml), followed by TdT (300 enzyme U/ml; Amersham Pharmacia Biotech) and Bio-14-dATP (0.94 nmol/L; Gibco BRL, Life Technologies, Paisley, Scotland). Biotinylated ATP was detected using the RTU Vectastain Elite ABC Reagent (Vector Laboratories) and slides were counterstained with methyl green and eosin. As a positive control, slides were pretreated with DNase I (20 Kunitz U/ml; Roche Molecular Biochemicals, Lewes, UK). Cells were regarded as TUNEL-positive if they exhibited stained nuclei with an apoptotic morphology. Tubular and interstitial cell apoptosis was quantified in a blinded manner by counting the number of TUNEL-positive tubular and interstitial cells in 20 to 25 sequentially selected nonoverlapping fields of renal cortex at ×400 magnification. Data were expressed as the mean number ± SEM per high-power field. PAS-stained tissue sections were used to quantify proximal and distal tubular cell proliferation because proximal tubular cells exhibit a characteristic PAS-positive luminal brush border.33Hughes J Johnson RJ Role of Fas (CD95) in tubulointerstitial disease induced by unilateral ureteric ligation (UUO).Am J Physiol. 1999; 277: F26-F32PubMed Google Scholar Mitotic cells were readily identifiable, and proximal and distal cell proliferation were calculated in a blinded manner by counting the number of mitotic proximal and distal tubular epithelial cells in 20 to 25 sequentially selected nonoverlapping fields of renal cortex at ×400 magnification and expressed as the mean number ± SEM per high-power field. All results are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism 3.02/Instat 1.1 (GraphPad Software, San Diego, CA). The Student's t-test was used for comparisons involving two groups, and statistical differences among multiple group
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