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• W2064909102 abstract "Angiotensin-converting enzyme (ACE) inhibition induces glomerular repair in the Munich Wistar Frömter (MWF) rat, a model of spontaneous glomerular injury. In this study, we investigated whether this effect is related to changes in glomerular cell number, particularly of podocytes, which are progressively lost with age. MWF rats with advanced nephropathy were studied at both 40 weeks and after 20 weeks of observation either with or without treatment with the ACE inhibitor lisinopril. Forty-week-old Wistar rats were used as controls. In untreated MWF rats, proteinuria, hypertension, glomerulosclerosis, and renal function worsened, while lisinopril induced regression of both functional and structural changes. Despite glomerular hypercellularity in untreated MWF rats, the number of endothelial cells per glomerulus did not change, and podocyte number even decreased. ACE inhibition halted the progressive increase in glomerular cell number and enhanced endothelial cell volume density. Surprisingly, lisinopril not only halted age-related podocyte loss but also increased the number of glomerular podocytes above baseline, which was associated with an increased number of proliferating Wilms tumor 1-positive cells, loss of cyclin-dependent kinase inhibitor p27 expression, and increased number of parietal podocytes. These data indicate that ACE inhibition restructures glomerular capillary, primarily by restoring the podocyte population in this model of glomerular injury. Increased parietal podocyte number in lisinopril-treated MWF rats suggests that the remodeling of Bowman's capsule epithelial cells contributes to this effect. Angiotensin-converting enzyme (ACE) inhibition induces glomerular repair in the Munich Wistar Frömter (MWF) rat, a model of spontaneous glomerular injury. In this study, we investigated whether this effect is related to changes in glomerular cell number, particularly of podocytes, which are progressively lost with age. MWF rats with advanced nephropathy were studied at both 40 weeks and after 20 weeks of observation either with or without treatment with the ACE inhibitor lisinopril. Forty-week-old Wistar rats were used as controls. In untreated MWF rats, proteinuria, hypertension, glomerulosclerosis, and renal function worsened, while lisinopril induced regression of both functional and structural changes. Despite glomerular hypercellularity in untreated MWF rats, the number of endothelial cells per glomerulus did not change, and podocyte number even decreased. ACE inhibition halted the progressive increase in glomerular cell number and enhanced endothelial cell volume density. Surprisingly, lisinopril not only halted age-related podocyte loss but also increased the number of glomerular podocytes above baseline, which was associated with an increased number of proliferating Wilms tumor 1-positive cells, loss of cyclin-dependent kinase inhibitor p27 expression, and increased number of parietal podocytes. These data indicate that ACE inhibition restructures glomerular capillary, primarily by restoring the podocyte population in this model of glomerular injury. Increased parietal podocyte number in lisinopril-treated MWF rats suggests that the remodeling of Bowman's capsule epithelial cells contributes to this effect. Clinical studies have documented that single or multidrug antiproteinuric treatments based on angiotensin II blockade can stabilize, or even reverse, renal disease progression in both patients with diabetic and non-diabetic nephropathies even in advanced stages of the disease.1Ruggenenti P Schieppati A Remuzzi G Progression, remission, regression of chronic renal diseases.Lancet. 2001; 357: 1601-1608Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 2Barnett AH Bain SC Bouter P Karlberg B Madsbad S Jervell J Mustonen J Angiotensin-receptor blockade versus converting-enzyme inhibition in type 2 diabetes and nephropathy.N Engl J Med. 2004; 351: 1952-1961Crossref PubMed Scopus (772) Google Scholar, 3Wilmer WA Hebert LA Lewis EJ Rohde RD Whittier F Cattran D Levey AS Lewis JB Spitalewitz S Blumenthal S Bain RP Remission of nephrotic syndrome in type 1 diabetes: long-term follow-up of patients in the Captopril Study.Am J Kidney Dis. 1999; 34: 308-314Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 4Ruggenenti P Perna A Benini R Bertani T Zoccali C Maggiore Q Salvadori M Remuzzi G Investigators of the GISEN Group Gruppo Italiano Studi Epidemiologici in Nefrologia In chronic nephropathies prolonged ACE inhibition can induce remission: dynamics of time-dependent changes in GFR.J Am Soc Nephrol. 1999; 10: 997-1006PubMed Google Scholar, 5Ruggenenti P Brenner BM Remuzzi G Remission achieved in chronic nephropathy by a multidrug approach targeted at urinary protein excretion.Nephron. 2001; 88: 254-259Crossref PubMed Scopus (35) Google Scholar Actually, regression of proteinuria and glomerulosclerosis by angiotensin converting enzyme (ACE) inhibition or angiotensin II type 1 receptor (AT1R) blockade has also been documented in experimental models of progressive nephropathies, such as puromycin aminonucleoside,6Marinides GN Groggel GC Cohen AH Border WA Enalapril and low protein reverse chronic puromycin aminonucleoside nephropathy.Kidney Int. 1990; 37: 749-757Crossref PubMed Scopus (68) Google Scholar chronic nitric oxide synthase inhibition,7Boffa JJ Lu Y Placier S Stefanski A Dussaule JC Chatziantoniou C Regression of renal vascular and glomerular fibrosis: role of angiotensin II receptor antagonism and matrix metalloproteinases.J Am Soc Nephrol. 2003; 14: 1132-1144Crossref PubMed Scopus (179) Google Scholar renal mass ablation,8Adamczak M Gross ML Krtil J Koch A Tyralla K Amann K Ritz E Reversal of glomerulosclerosis after high-dose enalapril treatment in subtotally nephrectomized rats.J Am Soc Nephrol. 2003; 14: 2833-2842Crossref PubMed Scopus (150) Google Scholar, 9Fujihara CK Velho M Malheiros DM Zatz R An extremely high dose of losartan affords superior renoprotection in the remnant model.Kidney Int. 2005; 67: 1913-1924Crossref PubMed Scopus (77) Google Scholar, 10Ma LJ Nakamura S Aldigier JC Rossini M Yang H Liang X Nakamura I Marcantoni C Fogo AB Regression of glomerulosclerosis with high-dose angiotensin inhibition is linked to decreased plasminogen activator inhibitor-1.J Am Soc Nephrol. 2005; 16: 966-976Crossref PubMed Scopus (130) Google Scholar aging,11Ma LJ Nakamura S Whitsitt JS Marcantoni C Davidson JM Fogo AB Regression of sclerosis in aging by an angiotensin inhibition-induced decrease in PAI-1.Kidney Int. 2000; 58: 2425-2436Crossref PubMed Scopus (128) Google Scholar and the Munich Wistar Frömter (MWF) rat.12Remuzzi A Fassi A Bertani T Perico N Remuzzi G ACE inhibition induces regression of proteinuria and halts progression of renal damage in a genetic model of progressive nephropathy.Am J Kidney Dis. 1999; 34: 626-632Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 13Remuzzi A Gagliardini E Donadoni C Fassi A Sangalli F Lepre MS Remuzzi G Benigni A Effect of angiotensin II antagonism on the regression of kidney disease in the rat.Kidney Int. 2002; 62: 885-894Crossref PubMed Scopus (80) Google Scholar, 14Remuzzi A Gagliardini E Sangalli F Bonomelli M Piccinelli M Benigni A Remuzzi G ACE inhibition reduces glomerulosclerosis and regenerates glomerular tissue in a model of progressive renal disease.Kidney Int. 2006; 69: 1124-1130Crossref PubMed Scopus (106) Google Scholar In the latter study, combined treatment with an ACE inhibitor and an AT1R blocker given from 25 to 40 weeks of age completely reversed proteinuria, and halted progressive glomerulosclerosis, particularly in glomeruli with mild sclerotic lesions.13Remuzzi A Gagliardini E Donadoni C Fassi A Sangalli F Lepre MS Remuzzi G Benigni A Effect of angiotensin II antagonism on the regression of kidney disease in the rat.Kidney Int. 2002; 62: 885-894Crossref PubMed Scopus (80) Google Scholar Recently three-dimensional reconstruction of the capillary tuft by serial section analysis allowed us to document the effects of administration of a high dose of an ACE inhibitor starting at 50 weeks of age, when rats had a more advanced nephropathy. This treatment not only remarkably reduced sclerosis volume in most glomeruli, but also increased the volume of the glomerular tuft occupied by intact capillary by up to 40%, indicating consistent glomerular tuft repair.14Remuzzi A Gagliardini E Sangalli F Bonomelli M Piccinelli M Benigni A Remuzzi G ACE inhibition reduces glomerulosclerosis and regenerates glomerular tissue in a model of progressive renal disease.Kidney Int. 2006; 69: 1124-1130Crossref PubMed Scopus (106) Google Scholar So far, the therapeutic effect of angiotensin II blockade has been mainly attributed to its capability to control extracellular matrix deposition. Inhibition of collagen synthesis,7Boffa JJ Lu Y Placier S Stefanski A Dussaule JC Chatziantoniou C Regression of renal vascular and glomerular fibrosis: role of angiotensin II receptor antagonism and matrix metalloproteinases.J Am Soc Nephrol. 2003; 14: 1132-1144Crossref PubMed Scopus (179) Google Scholar transforming growth factor-β,14Remuzzi A Gagliardini E Sangalli F Bonomelli M Piccinelli M Benigni A Remuzzi G ACE inhibition reduces glomerulosclerosis and regenerates glomerular tissue in a model of progressive renal disease.Kidney Int. 2006; 69: 1124-1130Crossref PubMed Scopus (106) Google Scholar and plasminogen activator inhibitor-1 expression10Ma LJ Nakamura S Aldigier JC Rossini M Yang H Liang X Nakamura I Marcantoni C Fogo AB Regression of glomerulosclerosis with high-dose angiotensin inhibition is linked to decreased plasminogen activator inhibitor-1.J Am Soc Nephrol. 2005; 16: 966-976Crossref PubMed Scopus (130) Google Scholar, 11Ma LJ Nakamura S Whitsitt JS Marcantoni C Davidson JM Fogo AB Regression of sclerosis in aging by an angiotensin inhibition-induced decrease in PAI-1.Kidney Int. 2000; 58: 2425-2436Crossref PubMed Scopus (128) Google Scholar were indeed proposed as possible mechanisms responsible for sclerosis regression. However, the possibility that ACE inhibitors or AT1R antagonists can modulate glomerular cell survival and repair is intriguing and not well explored yet.Podocyte loss has been recognized as a causal factor for renal disease progression. A recent study performed in transgenic rats for human diphtheria toxin receptor has clearly documented a strict correlation between the extent of podocyte depletion, obtained by titrating the administration of the corresponding toxin, and defined stages of glomerular damage ranging from transient proteinuria to progressive decline of renal function.15Wharram BL Goyal M Wiggins JE Sanden SK Hussain S Filipiak WE Saunders TL Dysko RC Kohno K Holzman LB Wiggins RC Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene.J Am Soc Nephrol. 2005; 16: 2941-2952Crossref PubMed Scopus (572) Google Scholar We have recently reported in the male MWF rat that podocyte number is progressively reduced with age, and this may importantly contribute to glomerular permselective defect, proteinuria, and renal scarring on the long-term.16Macconi D Bonomelli M Benigni A Plati T Sangalli F Longaretti L Conti S Kawachi H Hill P Remuzzi G Remuzzi A Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury.Am J Pathol. 2006; 168: 42-54Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar This evidence prompted us to characterize changes in resident glomerular cells and infiltrating/inflammatory cells during the development of sclerotic lesions in progressive proteinuric nephropathy in male MWF rats, and to investigate which, among glomerular components, is the key player for glomerular capillary restructuring and repair induced by ACE inhibition therapy.Materials and MethodsStudy DesignTwenty-nine male MWF rats from our colony17Remuzzi A Puntorieri S Alfano M Macconi D Abbate M Bertani T Remuzzi G Pathophysiologic implications of proteinuria in a rat model of progressive glomerular injury.Lab Invest. 1992; 67: 572-579PubMed Google Scholar and six Wistar rats (Charles River S.p.A, Calco, Italy) were used in this study. MWF rats were divided into three groups. Group 1 (n = 10) consisting of untreated animals, was studied at 40 weeks of age, at which time we previously documented about 50% podocyte loss associated with massive proteinuria and glomerulosclerosis16Macconi D Bonomelli M Benigni A Plati T Sangalli F Longaretti L Conti S Kawachi H Hill P Remuzzi G Remuzzi A Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury.Am J Pathol. 2006; 168: 42-54Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar (MWF 40W). Group 2 (n = 10) was left untreated and followed from 40 to 60 weeks of age (MWF 60W), whereas rats of group 3 (n = 9) were treated with a high dose of ACE inhibitor lisinopril in drinking water from 40 to 60 weeks of age (MWF+LIS 60W). The dose of lisinopril was progressively increased from 80 to 100 mg/L from 50 to 60 weeks to provide a better control of systolic blood pressure. A group of Wistar rats was studied at 40 weeks of age and used as control (Wistar 40W). Systolic blood pressure, urinary protein excretion, and serum creatinine were periodically measured during the observation period by conventional methods.14Remuzzi A Gagliardini E Sangalli F Bonomelli M Piccinelli M Benigni A Remuzzi G ACE inhibition reduces glomerulosclerosis and regenerates glomerular tissue in a model of progressive renal disease.Kidney Int. 2006; 69: 1124-1130Crossref PubMed Scopus (106) Google Scholar, 16Macconi D Bonomelli M Benigni A Plati T Sangalli F Longaretti L Conti S Kawachi H Hill P Remuzzi G Remuzzi A Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury.Am J Pathol. 2006; 168: 42-54Abstract Full Text Full Text PDF PubMed Scopus (121) Google ScholarAll animals were maintained in a temperature-controlled room regulated with a 12-hour light/dark cycle, and they had free access to water and food (standard rat chow containing 18.5% protein by weight). Animal care and treatment were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policies (EEC Council Directive 86/609, OJL 358, 1987; DL n116, G.U., suppl. 40, 18/2/1992, Circolare No.8, G.U., 14/7/1994; Guide for the Care and Use of Laboratory Animals, National Research Council, 1996). At sacrifice, both kidneys were perfused under anesthesia with PBS and processed as previously described in detail.16Macconi D Bonomelli M Benigni A Plati T Sangalli F Longaretti L Conti S Kawachi H Hill P Remuzzi G Remuzzi A Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury.Am J Pathol. 2006; 168: 42-54Abstract Full Text Full Text PDF PubMed Scopus (121) Google ScholarIn additional experiments, two groups of untreated or lisinopril treated (from 40 to 60 weeks) MWF rats (n = 2 each) were injected with 5-bromo-2′-deoxyuridine (BrdU, Sigma Aldrich, St Louis, MO, i.p. 50 mg/kg dissolved in saline) for 5 days (the last administration 2 hours before the sacrifice). In addition the thymidine analog was also given in the drinking water for the same period of time to obtain a final dose of 100 mg/kg. Kidney specimens were fixed in paraformaldehyde 4% and processed for immunohistochemistry.ImmunohistochemistryThe identification and count of resident and infiltrating cells per glomerulus, proliferating cells, and parietal podocytes, and the evaluation of cyclin-dependent kinase inhibitor expression was performed on kidney sections by single or multiple immunostaining for the following antigens: Wilms's tumor 1 (WT1), a podocyte-specific nuclear antigen18Mundlos S Pelletier J Darveau A Bachmann M Winterpacht A Zabel B Nuclear localization of the protein encoded by the Wilms' tumor gene WT1 in embryonic and adult tissues.Development. 1993; 119: 1329-1341Crossref PubMed Google Scholar; RECA-1, a cell surface antigen expressed by all rat endothelial cells (ECs)19Duijvestijn AM van Goor H Klatter F Majoor GD van Bussel E van Breda Vriesman PJ Antibodies defining rat endothelial cells: rECA-1, a pan-endothelial cell-specific monoclonal antibody.Lab Invest. 1992; 66: 459-466PubMed Google Scholar; Thy1.1, a mesangial cell (MC) antigen20Ricono JM Xu YC Arar M Jin DC Barnes JL Abboud HE Morphological insights into the origin of glomerular endothelial and mesangial cells and their precursors.J Histochem Cytochem. 2003; 51: 141-150Crossref PubMed Scopus (46) Google Scholar; ED1, an antigen present in the rat monocytes and macrophages; Ki-67, a nuclear antigen expressed during all active phases of the cell cycle (G1 to M)21Scholzen T Gerdes J The Ki-67 protein: from the known and the unknown.J Cell Physiol. 2000; 182: 311-322Crossref PubMed Scopus (3434) Google Scholar; the protein gene product 9.5, an ubiquitin C-terminal hydrolase found to localize in parietal epithelial cells (PECs) of the Bowman's capsule in normal rat22Shirato I Asanuma K Takeda Y Hayashi K Tomino Y Protein gene product 9.5 is selectively localized in parietal epithelial cells of Bowman's capsule in the rat kidney.J Am Soc Nephrol. 2000; 11: 2381-2386PubMed Google Scholar; p27, a cyclin-dependent kinase inhibitor. Cell nuclei were labeled by 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI) and the glomerular tuft by the wheat germ agglutinin (WGA). All of the immunofluorescence experiments were performed on sections (3 μm) from paraformaldehyde-lysine-periodate-fixed kidney specimens unless otherwise specified. Antibody incubations were performed at room temperature unless otherwise specified. After antigen unmasking (10 mmol/L citrate buffer, pH 6.0 for 20 minutes at 100°C) and blocking of non-specific sites (1% bovine serum albumin), sections were incubated with a mouse monoclonal antibody (moAb) to Ki-67 (dil 1:100 in PBS, Novacastra Laboratories Ltd, Newcastle, UK) overnight at 4°C, followed by Cy5-conjugated F(ab′)2 fragment donkey anti-mouse IgG (affinity purified 10 μg/ml in PBS, Jackson Immunoresearch Laboratories Inc., West Grove, PA) for 1 hour. After washing, sections were further incubated with a rabbit polyclonal antibody to WT1 (WT1 [C-19] 4 μg/ml in PBS, Santa Cruz Biotechnology, Santa Cruz, CA) for 3 hours, followed by Cy3-conjugated goat anti-rabbit IgG (12.5 μg/ml in PBS, Jackson Immunoresearch) for 1 hour, and finally with the fluorescein isothiocyanate (FITC)-conjugated WGA (12.5 μg/ml in PBS, Vector Laboratories Inc., Burlingame, CA) for 15 minutes at room temperature and with the nuclear stain DAPI (Sigma Aldrich, St Louis, MO) for 20 minutes at room temperature. At least 30 glomerular sections per animal were randomly acquired by confocal laser scanning microscope (LS510 Meta, Zeiss, Jena, Germany) and examined by an observer unaware of the identity of samples. For each glomerulus separate images for each marker were acquired and digitally merged. Glomerular cells were identified as DAPI-positive cells and counted, and the number of cells per glomerulus was estimated by morphometric analysis. By comparing superimposed images of WT1 and DAPI with those of Ki-67 plus DAPI, we identified and counted the number of cells positive for WT1, Ki-67, or both. Proliferating cells were identified as DAPI-positive cells expressing also Ki-67. Their percentage, in respect to the total cell number, was calculated to estimate the number of proliferating cells per glomerulus. Glomerular podocytes were identified as WT1-positive cells and counted as previously described.16Macconi D Bonomelli M Benigni A Plati T Sangalli F Longaretti L Conti S Kawachi H Hill P Remuzzi G Remuzzi A Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury.Am J Pathol. 2006; 168: 42-54Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar Finally, the percentage of WT1-positive cells also expressing Ki-67 was calculated, to estimate the number of proliferating WT1-positive cells per glomerulus.To validate the method for identification of proliferating WT1-positive glomerular cells, we verified whether WT1-positive cells expressing Ki-67 were also stained for BrdU. Paraformaldehyde-fixed kidney sections (5 μm) were pretreated for BrdU immunostaining by DNA denaturation (2M/L HCl for 15 minutes at 37°C) followed by 10 minutes in 0.1 M/L borate buffer (pH 8.5). After blocking, sections were incubated with WT1 (C-19) antibody overnight at 4°C followed by Cy3-conjugated goat anti-rabbit IgG (12.5 μg/ml in PBS) for 1 hour. Then sections were incubated with a moAb to Ki-67 (dil 1:100 in PBS) for 3 hours followed by Cy5-conjugated F(ab′)2 fragment donkey anti-mouse IgG (10 μg/ml in PBS) for 1 hour and finally with a FITC-conjugated moAb to BrdU (dil 1:4 in PBS, Roche Diagnostics Co, IN) for 1 hour and DAPI. For each glomerulus separate images of WT1, Ki-67, BrdU, and DAPI were obtained and in the merged image the simultaneous expression of Ki-67 and BrdU was evaluated in WT1-positive cells.To evaluate the expression of p27 in podocytes, kidney sections were incubated with goat polyclonal antibody to p27 (3 μg/ml in PBS, Santa Cruz) overnight at 4°C followed by FITC-conjugated donkey anti-goat IgG (25 μg/ml in PBS, Jackson) for 1 hour. Then sections were incubated with WT1 (C-19) antibody (4 μg/ml in PBS) for 3 hours followed by Cy5-conjugated goat anti-rabbit IgG (7.5 μg/ml in PBS, Jackson) for 1 hour and finally with rhodamine WGA (Vector) and DAPI. At least 30 glomeruli per animal were acquired and the percentage of WT1-positive cells expressing p27 was calculated.To evaluate glomerular expression of the glial cell line-derived neutrophic factor (GDNF), kidney sections were incubated with a moAb raised against amino acids 78 to 134, representing the mature GDNF of human origin (4 μg/ml in PBS, Santa Cruz) overnight at 4°C, followed by Cy3-conjugated F(ab′)2 fragment donkey anti-mouse IgG (6.5 μg/ml in PBS, Jackson) for 1 hour and finally with FITC-WGA (Vector) and DAPI. From 40 to 60 glomeruli per section were randomly chosen and blindly examined.To estimate the presence of parietal podocytes, kidney sections were incubated overnight at 4°C with a moAb to WT1 (WT1 [F-6] 4 μg/ml in PBS, Santa Cruz) followed by Cy3-conjugated F(ab′)2 fragment donkey anti-mouse IgG (6.5 μg/ml in PBS, Jackson) for 1 hour and then with rabbit anti-human protein gene product 9.5 antibody (1:200, AbD Serotec, Kidlington, Oxford, UK) for 3 hours, followed by Cy5-conjugated goat anti-rabbit IgG (7.5 μg/ml in PBS) for 1 hour. Finally FITC-WGA and DAPI were added. PECs in Bowman's capsule were identified by co-staining with DAPI and protein gene product, and by morphology (WGA staining of Bowman's capsule). Parietal podocytes were identified as PEC expressing WT1. The number of PECs and parietal podocytes was determined using nuclear count in at least 20 glomeruli for each animal, and the length of Bowman's capsule was also measured. Then the parietal podocyte/PEC number ratio and PEC number/Bowman's capsule length ratio were computed.To investigate the presence of inflammatory cells infiltrating the glomerular tuft, kidney sections were incubated with a moAb to ED1 (dil 1:100, Chemicon, Temecula, CA) for 1 hour, followed by Cy3-conjugated F(ab′)2 fragment donkey anti-mouse IgG (6.5 μg/ml in PBS) for 1 hour and then with FITC-WGA. At least 30 glomeruli for each animal were acquired and ED1-positive cells counted.To estimate glomerular ECs, sections were incubated with a mouse anti-rat RECA-1 moAb (10 μg/ml Serotec) for 1 hour, followed by Cy5-conjugated F(ab′)2 fragment donkey anti-mouse IgG (10 μg/ml in PBS) for 1 hour, and then with FITC-WGA and DAPI. Glomerular cells were identified by counting DAPI-positive nuclei surrounded by RECA-1 staining in at least 30 glomeruli per animal. Another series of sections was used to estimate the volume density (Vv) of glomerular ECs using 15 glomerular sections per animal.To estimate the number of glomerular MCs, sections were incubated with a mouse anti-rat Thy1.1 moAb (dil 1:100, AbD Serotec) for 1 hour, followed by Cy3-conjugated F(ab′)2 fragment donkey anti-mouse IgG (6.5 μg/ml in PBS, Jackson) for 1 hour, and then with FITC-WGA and DAPI. Glomerular MCs were identified by counting DAPI-positive nuclei surrounded by Thy1.1 staining in at least 30 glomeruli per animal.Morphometric AnalysisThe extent of glomerular sclerotic lesions was estimated on a single kidney cortical section (3.5 μm in thickness) stained with periodic acid-Schiff examined by light microscopy (Zeiss, Jena, Germany) using computer-based morphometric analysis. For each kidney section, about 50 glomeruli were systematically digitized (glomeruli were consecutively encountered moving the microscope stage with an S-shape path) using a ×40 objective, and digital images were acquired and processed with interactive tools (Image J, http://rsb.info.nih.gov/ij) to outline separately the outer polygon of the glomerular capillary tuft and, if present, the sclerotic region of the glomerular tuft. The percentage of glomerular volume occupied by sclerotic changes was then calculated as the percent ratio of the sclerotic surface area over the total area of capillary tuft, based on the principle that area density is equivalent to volume density.23Weibel ER Stereological methods.in: Practical methods for biological morphometry. Academic Press Inc., London1979: 40-116Google ScholarEstimation of mean glomerular volume (VG) was performed on digital images of cortical tissue either stained with periodic acid-Schiff or labeled with fluorescein-WGA as previously described.16Macconi D Bonomelli M Benigni A Plati T Sangalli F Longaretti L Conti S Kawachi H Hill P Remuzzi G Remuzzi A Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury.Am J Pathol. 2006; 168: 42-54Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar The latter was used for the calculation of the mean number of podocytes per glomerulus. VG was calculated using the formulaVG=(β/k)(Am)3/2where Am is the mean glomerular cross-sectional area, k = 1.01 is a size distribution coefficient, and β = 1.38 is the shape coefficient for glomeruli that are assumed to be spherical.23Weibel ER Stereological methods.in: Practical methods for biological morphometry. Academic Press Inc., London1979: 40-116Google ScholarThe mean number of resident and infiltrating cells per glomerular tuft was calculated as the product of cell volume density (Nv) and (VG),16Macconi D Bonomelli M Benigni A Plati T Sangalli F Longaretti L Conti S Kawachi H Hill P Remuzzi G Remuzzi A Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury.Am J Pathol. 2006; 168: 42-54Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar as previously described in details. Briefly, Nv was calculated as NV = NA / , where NA, the cell nuclear profile area density, is the ratio between the number of cell nuclear profiles and the measured glomerular capillary tuft area. is the average diameter of cell nuclei that we estimated from major and minor axis of nuclear sections, measured on digital images assuming ellipsoidal shape of cell nuclei. This technique was used to estimate average number of infiltrating cells per glomerulus, total number of resident cells, podocytes, ECs, and MCs. To further verify the precision of podocyte estimation, we used serial sections of an entire glomerular capillary and calculated the actual number of podocytes using high-resolution digitized images. Briefly, kidney tissue was obtained from a normal Wistar rat (40 weeks of age). Serial sections (1 μm thick) of a whole glomerular tuft were cut from an epon resin embedded sample (more than 200 sections in total) and sequentially collected. Digital micrographs of toluidine blue stained sections were acquired, from the beginning to the end of the Bowman's capsule (141 sections), by optical microscopy (at 100x). Images were digitally rotated and translated using image analysis software developed in house to generate a stack of aligned images containing the entire capillary tuft. In each image of the stack the nuclei of podocytes were identified and marked with a colored circle on all images, as reported in supplemental Figure S1 (see http://ajp.amjpathol.org) for a representative section. The image stack was then digitally processed for automatic segmentation of colored volumes and counting, each volume corresponding to a podocyte nucleus. There is a complete agreement of the estimation of the podocyte number with the two techniques (supplemental Figure S1, see http://ajp.amjpathol.org).The volume of capillary tuft occupied by ECs was determined on the basis of area density. To this purpose, digital images of capillary tuft outline were thresholded on green color for WGA staining to identify the area occupied by capillary cells and matrix and on red color to identify RECA-1 staining. Then the ratio between red and green pixels was calculated as the percentage of tuft cellular volume occupied by ECs.Transmission Electron Microscopy of Visceral Podocytes and PECsFor transmission electron microscopy analysis, ultrathin sections (60 to 100 nm) were cut, from cortical kidney tissue samples embedded in epon resin, by an ultramicrotome (LKB, Bromme, Sweden), collected on copper grids, and stained with uranyl acetate and lead citrate. Ultrastructural analysis was performed by transmission electron microscopy (Morgagni 268D, Philips, Eindhoven, The Netherlands).Statistical Analys" @default.
• W2064909102 created "2016-06-24" @default.
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• W2064909102 date "2009-03-01" @default.
• W2064909102 modified "2023-10-17" @default.
• W2064909102 title "Podocyte Repopulation Contributes to Regression of Glomerular Injury Induced by Ace Inhibition" @default.
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