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• W2009672980 abstract "The small GTPase RhoA is activated by the angiotensin II (AngII) type 1 receptor (AT1R), which is part of the local renin-angiotensin system that is involved in podocyte injury preceding glomerular crescent formation. We demonstrated previously that inhibition of AT1R protects against crescentic glomerular injury in Fc receptor-deficient mice (γ−/−) with anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN). Here, we hypothesized that the RhoA kinase inhibitor, fasudil, attenuates AT1R-dependent crescentic GN. We examined anti-GBM GN in γ−/− mice with or without fasudil treatment, and further investigated the underlying mechanisms in cultured differentiated podocytes and leukocytes. Fasudil markedly attenuated crescentic GN with a significant decrease in proteinuria and hematuria, infiltration of T cells and monocytes/macrophages as well as their local proliferation, and preservation of podocyte-specific proteins, including WT-1 and nephrin, in glomeruli. In vitro studies showed that AngII induced the down-regulation of both nephrin and WT-1 expression in podocytes, which was reversed by fasudil in a dose-dependent manner. Additionally, fasudil blocked the AngII-induced migration of both macrophages and T cells. Furthermore, we also examined lipopolysaccharide-induced nephrotic syndrome in severe combined immunodeficiency disease mice and found that fasudil failed to block the development of proteinuria because of a B7-1-dependent podocyte injury. In conclusion, fasudil treatment prevents crescent formation and disease progression in anti-GBM GN by preventing AngII-induced podocyte injury and leukocyte migration. The small GTPase RhoA is activated by the angiotensin II (AngII) type 1 receptor (AT1R), which is part of the local renin-angiotensin system that is involved in podocyte injury preceding glomerular crescent formation. We demonstrated previously that inhibition of AT1R protects against crescentic glomerular injury in Fc receptor-deficient mice (γ−/−) with anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN). Here, we hypothesized that the RhoA kinase inhibitor, fasudil, attenuates AT1R-dependent crescentic GN. We examined anti-GBM GN in γ−/− mice with or without fasudil treatment, and further investigated the underlying mechanisms in cultured differentiated podocytes and leukocytes. Fasudil markedly attenuated crescentic GN with a significant decrease in proteinuria and hematuria, infiltration of T cells and monocytes/macrophages as well as their local proliferation, and preservation of podocyte-specific proteins, including WT-1 and nephrin, in glomeruli. In vitro studies showed that AngII induced the down-regulation of both nephrin and WT-1 expression in podocytes, which was reversed by fasudil in a dose-dependent manner. Additionally, fasudil blocked the AngII-induced migration of both macrophages and T cells. Furthermore, we also examined lipopolysaccharide-induced nephrotic syndrome in severe combined immunodeficiency disease mice and found that fasudil failed to block the development of proteinuria because of a B7-1-dependent podocyte injury. In conclusion, fasudil treatment prevents crescent formation and disease progression in anti-GBM GN by preventing AngII-induced podocyte injury and leukocyte migration. Crescentic glomerulonephritis (GN) is a manifestation of severe glomerular injury with a poor clinical outcome.1Tipping PG Holdsworth SR T cells in crescentic glomerulonephritis.J Am Soc Nephrol. 2006; 17: 1253-1263Crossref PubMed Scopus (152) Google Scholar It is observed in a variety of GN of immune origin, in particular anti-glomerular basement membrane (anti-GBM) disease2Pusey CD Anti-glomerular basement membrane disease.Kidney Int. 2003; 64: 1535-1550Crossref PubMed Scopus (177) Google Scholar and class IV lupus nephritis.3Weening 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 (1497) Google Scholar However, the pathogenesis of glomerular crescents remains unknown. It is generally considered that epithelial cells predominated in crescents of patients during the early phase of disease, whereas late phases were characterized by rupture of the basement membrane of Bowman's capsule and subsequent infiltration of cellular crescents, predominantly by macrophages.4Morita T Suzuki Y Churg J Structure and development of the glomerular crescent.Am J Pathol. 1973; 72: 349-368PubMed Google Scholar, 5Min KW Gyorkey F Gyorkey P Yium JJ Eknoyan G The morphogenesis of glomerular crescents in rapidly progressive glomerulonephritis.Kidney Int. 1974; 5: 47-56Crossref PubMed Scopus (63) Google Scholar, 6Jennette JC Hipp CG The epithelial antigen phenotype of glomerular crescent cells.Am J Clin Pathol. 1986; 86: 274-280PubMed Google Scholar, 7Boucher A Droz D Adafer E Noel LH Relationship between the integrity of Bowman's capsule and the composition of cellular crescents in human crescentic glomerulonephritis.Lab Invest. 1987; 56: 526-533PubMed Google Scholar, 8Nagata M Horita S Shu Y Shibata S Hattori M Ito K Watanabe T Phenotypic characteristics and cyclin-dependent kinase inhibitors repression in hyperplastic epithelial pathology in idiopathic focal segmental glomerulosclerosis.Lab Invest. 2000; 80: 869-880Crossref PubMed Scopus (62) Google Scholar This picture is also confirmed in animal models with anti-GBM glomerulonephritis (anti-GBM GN).9Lan HY Nikolic-Paterson DJ Mu W Atkins RC Local macrophage proliferation in the pathogenesis of glomerular crescent formation in rat anti-glomerular basement membrane (GBM) glomerulonephritis.Clin Exp Immunol. 1997; 110: 233-240Crossref PubMed Scopus (41) Google Scholar, 10Le Hir M Keller C Eschmann V Hahnel B Hosser H Kriz W Podocyte bridges between the tuft and Bowman's capsule: an early event in experimental crescentic glomerulonephritis.J Am Soc Nephrol. 2001; 12: 2060-2071PubMed Google Scholar However, origin of the crescentic epithelial cells is still controversial. Moeller and colleagues11Moeller MJ Soofi A Hartmann I Le Hir M Wiggins R Kriz W Holzman LB Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.J Am Soc Nephrol. 2004; 15: 61-67Crossref PubMed Scopus (161) Google Scholar recently demonstrated by podocyte-specific 2.5P-Cre mouse with anti-GBM GN that visceral glomerular epithelial cells, podocytes, adhered to the parietal basement membrane and populated glomerular crescents during the early phase of cellular crescents. On the other hand, Tipping and Holdsworth1Tipping PG Holdsworth SR T cells in crescentic glomerulonephritis.J Am Soc Nephrol. 2006; 17: 1253-1263Crossref PubMed Scopus (152) Google Scholar demonstrated critical roles of macrophages and T cells in the crescent formation in this disease by their series of elegant studies. Therefore, podocytes and macrophages/T cells may be important players in the progression of crescent formation, and should be the targets for the treatments of crescentic GN. The podocyte is a highly differentiated cell of the kidney glomerulus that forms multiple interdigitating foot processes.12Arakawa M Tokunaga J A scanning electron microscope study of the glomerulus. Further consideration of the mechanism of the fusion of podocyte terminal processes in nephrotic rats.Lab Invest. 1972; 27: 366-371PubMed Google Scholar The neighboring foot processes derived from different podocyte plasma membranes are connected by a continuous membrane-like structure called a slit diaphragm (SD)13Rodewald R Karnovsky MJ Porous substructure of the glomerular slit diaphragm in the rat and mouse.J Cell Biol. 1974; 60: 423-433Crossref PubMed Scopus (439) Google Scholar or slit membrane.14Yamada E The fine structure of the renal glomerulus of the mouse.J Biophys Biochem Cytol. 1955; 1: 551-566Crossref PubMed Scopus (119) Google Scholar Several SD proteins, whose interactions are regulated with or without the actin cytoskeleton, have recently been identified.15Reiser J Kriz W Kretzler M Mundel P The glomerular slit diaphragm is a modified adherens junction.J Am Soc Nephrol. 2000; 11: 1-8Crossref PubMed Google Scholar It is widely accepted that podocyte dysfunction is involved in the development of proteinuria in certain kidney diseases such as minimal change nephrotic syndrome, focal segmental glomerulosclerosis, and membranous nephropathy. In addition, cytoskeletal changes of podocytes are critically involved in the pathogenesis of GN.16Morigi M Buelli S Angioletti S Zanchi C Longaretti L Zoja C Galbusera M Gastoldi S Mundel P Remuzzi G Benigni A In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: implication for permselective dysfunction of chronic nephropathies.Am J Pathol. 2005; 166: 1309-1320Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar Blocking cytoskeleton rearrangement using a RhoA kinase inhibitor prevented the activation of nuclear factor (NF)-κB and Ap-1, suggesting a direct link between cytoskeleton and transcriptional regulation in podocytes.16Morigi M Buelli S Angioletti S Zanchi C Longaretti L Zoja C Galbusera M Gastoldi S Mundel P Remuzzi G Benigni A In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: implication for permselective dysfunction of chronic nephropathies.Am J Pathol. 2005; 166: 1309-1320Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar Small GTPases of the Rho family are key regulators of the cellular cytoskeleton. RhoA is involved in the regulation of stress fiber and focal adhesion formation, cell morphology, cell aggregation, cadherin-mediated cell-cell adhesion, cell motility, cytokinesis, membrane ruffling, neurite retraction, microvilli formation, and smooth muscle contraction.17Van Aelst L D'Souza-Schorey C Rho GTPases and signaling networks.Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2101) Google Scholar, 18Hall A Rho GTPases and the actin cytoskeleton.Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar, 19Katoh K Kano Y Amano M Onishi H Kaibuchi K Fujiwara K Rho-kinase-mediated contraction of isolated stress fibers.J Cell Biol. 2001; 153: 569-584Crossref PubMed Scopus (271) Google Scholar Fasudil and Y-27632 specifically inhibit RhoA kinase activity by competing for ATP binding, and are useful tools for evaluating the cellular function of RhoA kinase. RhoA functions in response to various heterotrimeric G protein-coupled receptor agonists.20Seasholtz TM Majumdar M Brown JH Rho as a mediator of G protein-coupled receptor signaling.Mol Pharmacol. 1999; 55: 949-956Crossref PubMed Scopus (206) Google Scholar Agonists such as lysophosphatidic acid, thrombin, and thromboxane A2 induce cytoskeletal alteration through Gα12 and/or Gα13 subunits in nonmuscle cells.21Gohla A Schultz G Offermanns S Role for G(12)/G(13) in agonist-induced vascular smooth muscle cell contraction.Circ Res. 2000; 87: 221-227Crossref PubMed Scopus (199) Google Scholar Receptors for the vasoconstrictive agents, angiotensin II (AngII), endothelin, and vasopressin were also recently linked to Gα12/13 and Gq activation.21Gohla A Schultz G Offermanns S Role for G(12)/G(13) in agonist-induced vascular smooth muscle cell contraction.Circ Res. 2000; 87: 221-227Crossref PubMed Scopus (199) Google Scholar Clinical and experimental studies have implicated AngII in the regulation of expression of adhesion molecules in many diseases.22Suzuki Y Ruiz-Ortega M Lorenzo O Ruperez M Esteban V Egido J Inflammation and angiotensin II.Int J Biochem Cell Biol. 2003; 35: 881-900Crossref PubMed Scopus (579) Google Scholar In addition, AngII enhances chemokine expression in various tissues and cell types.23Nahmod KA Vermeulen ME Raiden S Salamone G Gamberale R Fernandez-Calotti P Alvarez A Nahmod V Giordano M Geffner JR Control of dendritic cell differentiation by angiotensin II.FASEB J. 2003; 17: 491-493PubMed Google Scholar In particular, immunocompetent cells are equipped with components of the renin-angiotensin system (RAS) and contribute to AngII generation.22Suzuki Y Ruiz-Ortega M Lorenzo O Ruperez M Esteban V Egido J Inflammation and angiotensin II.Int J Biochem Cell Biol. 2003; 35: 881-900Crossref PubMed Scopus (579) Google Scholar, 23Nahmod KA Vermeulen ME Raiden S Salamone G Gamberale R Fernandez-Calotti P Alvarez A Nahmod V Giordano M Geffner JR Control of dendritic cell differentiation by angiotensin II.FASEB J. 2003; 17: 491-493PubMed Google Scholar, 24Rodríguez-Iturbe B Pons H Herrera-Acosta J Johnson RJ Role of immunocompetent cells in nonimmune renal diseases.Kidney Int. 2001; 59: 1626-1640Crossref PubMed Scopus (165) Google Scholar These findings suggest that RAS may influence the prognosis of many renal diseases in association with activation of the immune system. Furthermore, Th1-predominant immune responses promote crescent formation in the experimental models.1Tipping PG Holdsworth SR T cells in crescentic glomerulonephritis.J Am Soc Nephrol. 2006; 17: 1253-1263Crossref PubMed Scopus (152) Google Scholar, 25Suzuki Y Gomez-Guerrero C Shirato I Lopez-Franco O Hernandez-Vargas P Sanjuan G Ruiz-Ortega M Sugaya T Okumura K Tomino Y Ra C Egido J Susceptibility to T cell-mediated injury in immune complex disease is linked to local activation of renin-angiotensin system: the role of NF-AT pathway.J Immunol. 2002; 169: 4136-4146Crossref PubMed Scopus (39) Google Scholar We demonstrated previously the marked protective action of an AngII type I receptor (AT1R) antagonist against crescentic glomerular injury in FcR-deficient mice (γ−/− mice) with anti-GBM GN.26Suzuki Y Shirato I Okumura K Ravetch JV Takai T Tomino Y Ra C Distinct contribution of Fc receptors and angiotensin II-dependent pathways in anti-GBM glomerulonephritis.Kidney Int. 1998; 54: 1166-1174Crossref PubMed Scopus (145) Google Scholar Indeed, anti-GBM GN was completely attenuated in bone marrow chimeras of γ−/− and AT1R−/− mice.25Suzuki Y Gomez-Guerrero C Shirato I Lopez-Franco O Hernandez-Vargas P Sanjuan G Ruiz-Ortega M Sugaya T Okumura K Tomino Y Ra C Egido J Susceptibility to T cell-mediated injury in immune complex disease is linked to local activation of renin-angiotensin system: the role of NF-AT pathway.J Immunol. 2002; 169: 4136-4146Crossref PubMed Scopus (39) Google Scholar These protective outcomes were linked to attenuated infiltration of macrophages and T cells into glomeruli.26Suzuki Y Shirato I Okumura K Ravetch JV Takai T Tomino Y Ra C Distinct contribution of Fc receptors and angiotensin II-dependent pathways in anti-GBM glomerulonephritis.Kidney Int. 1998; 54: 1166-1174Crossref PubMed Scopus (145) Google Scholar, 27Gómez-Guerrero C Lopez-Franco O Suzuki Y Sanjuan G Hernandez-Vargas P Blanco J Egido J Nitric oxide production in renal cells by immune complexes: role of kinases and nuclear factor-kappaB.Kidney Int. 2002; 62: 2022-2034Crossref PubMed Scopus (36) Google Scholar Based on this, the purpose of the present study is to examine whether a RhoA kinase inhibitor ameliorates the AngII-dependent crescentic glomerular injury via podocyte protection or prevention of leukocyte recruitment. The γ−/− mice were generated by homologous recombination, as described previously,26Suzuki Y Shirato I Okumura K Ravetch JV Takai T Tomino Y Ra C Distinct contribution of Fc receptors and angiotensin II-dependent pathways in anti-GBM glomerulonephritis.Kidney Int. 1998; 54: 1166-1174Crossref PubMed Scopus (145) Google Scholar, 28Poole A Gibbins JM Turner M van Vugt MJ van de Winkel JG Saito T Tybulewicz VL Watson SP The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen.EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (398) Google Scholar and were fed regular chow. All animal procedures were conducted in accordance with the guidelines for the care and use of laboratory animals approved by Juntendo University School of Medicine. All experiments used age-matched 8- to 10-week-old female animals. Mice were divided into three groups, anti-GBM GN without treatment (untreated group; no Tx group), fasudil-treated anti-GBM GN (fasudil-treated group; Tx group), and delayed fasudil-treated anti-GBM GN (delayed fasudil-treated group; delayed Tx group) groups. Blood pressure was measured by the tail-cuff method using an automatic sphygmomanometer (Softron, Tokyo, Japan) before injection with nephrotoxic serum (NTS) (Kyowa Hakko Kogyo Co., Tokyo, Japan) and before sacrifice (day 14). Before each measurement, the mice were placed in a Plexiglas cage at 37°C for 5 minutes. The method used for preparation of NTS has been described previously.26Suzuki Y Shirato I Okumura K Ravetch JV Takai T Tomino Y Ra C Distinct contribution of Fc receptors and angiotensin II-dependent pathways in anti-GBM glomerulonephritis.Kidney Int. 1998; 54: 1166-1174Crossref PubMed Scopus (145) Google Scholar Anti-GBM GN was induced by a single intravenous injection of NTS through the tail vein of mice preimmunized with rabbit IgG and complete Freund adjuvant 5 days earlier. We administered NTS at a dose of 200 μl/20 g body weight to both groups. The selection of this dose was based on results of preliminary studies, which showed that it was sufficient to induce proteinuria and severe renal damage in γ−/− mice. None of the mice developed anaphylactic symptoms after injection of NTS. Urinary protein was determined by the Knight's method as described previously.26Suzuki Y Shirato I Okumura K Ravetch JV Takai T Tomino Y Ra C Distinct contribution of Fc receptors and angiotensin II-dependent pathways in anti-GBM glomerulonephritis.Kidney Int. 1998; 54: 1166-1174Crossref PubMed Scopus (145) Google Scholar Blood samples were taken from the orbital venous plexus before sacrifice. Kidneys were perfused with cold saline and removed under general anesthesia, and then either frozen in liquid nitrogen or fixed in 4% paraformaldehyde. Fasudil (Asahi Kasei Pharma Co., Tokyo, Japan) was selected to inhibit RhoA kinase because it is the only such inhibitor practically available for long-term in vivo use.29Satoh S Utsunomiya T Tsurui K Kobayashi T Ikegaki I Sasaki Y Asano T Pharmacological profile of hydroxy fasudil as a selective rho kinase inhibitor on ischemic brain damage.Life Sci. 2001; 69: 1441-1453Crossref PubMed Scopus (147) Google Scholar, 30Higashi M Shimokawa H Hattori T Hiroki J Mukai Y Morikawa K Ichiki T Takahashi S Takeshita A Long-term inhibition of Rho-kinase suppresses angiotensin II-induced cardiovascular hypertrophy in rats in vivo: effect on endothelial NAD(P)H oxidase system.Circ Res. 2003; 93: 767-775Crossref PubMed Scopus (390) Google Scholar In the treatment group, fasudil (10 mg/kg body weight/day) was intraperitoneally administered daily throughout the experiment from 4 days before (n = 6) (fasudil-treated group) or 7days after (n = 5) (delayed fasudil-treated group) NTS injection. To determine the number of proliferating cells, mice were injected intraperitoneally with BrdU (5-bromo-2′-deoxyuridine; Calbiochem, La Jolla, CA) at 2 mg/20 g body weight at 16, 8, and 4 hours before sacrifice.31López-Franco O Suzuki Y Sanjuan G Blanco J Hernandez-Vargas P Yo Y Kopp J Egido J Gomez-Guerrero C Nuclear factor-kappa B inhibitors as potential novel anti-inflammatory agents for the treatment of immune glomerulonephritis.Am J Pathol. 2002; 161: 1497-1505Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar An immortalized murine podocyte clone was kindly provided by Dr. Peter Mundel (Mount Sinai School of Medicine, New York, NY). The preparation and characterization of these cells have been described in detail previously.32Saleem MA Ni L Witherden I Tryggvason K Ruotsalainen V Mundel P Mathieson PW Co-localization of nephrin, podocin, and the actin cytoskeleton: evidence for a role in podocyte foot process formation.Am J Pathol. 2002; 161: 1459-1466Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar Podocytes were maintained in RPMI 1640 medium (Sigma, Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Inc., Gaithersburg, MD), 100 U/ml penicillin G, and 100 μg/ml streptomycin in a humidified atmosphere with 5% CO2. During propagation, the culture medium was supplemented with 10 U/ml recombinant mouse interferon-γ (PeproTech EC, London, UK) to enhance the expression of T-antigen, and the cells were cultured at 33°C (permissive conditions). Podocytes were cultured on type I collagen-coated dishes (Asahi Techno Glass Co., Tokyo, Japan) at 37°C (nonpermissive conditions) to induce differentiation without interferon-γ. At least 10 days were required to induce differentiation. Podocytes cultured for 10 to 20 passages were used in all experiments. For starvation conditions, podocytes were cultured in fetal calf serum-free medium containing 5% bovine serum albumin for 12 hours after differentiation. Jurkat T cells (ATCC TIB-152; Sumisho Pharma, Tokyo, Japan) and human immature monocyte/macrophage U-937 cells (ATCC U-937, Sumisho Pharma) were cultured and then maintained in RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Inc.), 100 U/ml penicillin G, and 100 μg/ml streptomycin in a humidified atmosphere with 5% CO2 at 37°C. The chemotactic activity induced by AngII was evaluated in 24-well Transwell chemotaxis chambers (Costar Corning, Rochester, NY) as described previously.33Falk W Goodwin Jr, RH Leonard EJ A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration.J Immunol Methods. 1980; 33: 239-247Crossref PubMed Scopus (251) Google Scholar The lower wells were loaded in triplicate with 600 μl of RPMI 1640 medium containing various concentrations of fasudil and AngII. The upper compartments were loaded with 100 μl of cell suspension containing 5 × 105 Jurkat or U-937 cells, which were starved for 12 hours beforehand. The chambers were incubated with or without fasudil at 37°C for 3 hours, and then AngII was added to each chamber at the same temperature for 6 hours to assess chemotaxis of the T cells and macrophages. Migrating cells in the lower compartment were counted by flow cytometry.27Gómez-Guerrero C Lopez-Franco O Suzuki Y Sanjuan G Hernandez-Vargas P Blanco J Egido J Nitric oxide production in renal cells by immune complexes: role of kinases and nuclear factor-kappaB.Kidney Int. 2002; 62: 2022-2034Crossref PubMed Scopus (36) Google Scholar Chemotaxis data were expressed relative to the number of migrating cells in the control (no AngII or fasudil) for macrophages and in the cells treated with AngII at a concentration of 10−8 mol/L for T cells. Cell proliferation was evaluated in 24-well chambers (Asahi Techno Glass Co.) loaded in triplicate with 100 μl of cell suspension containing 2 × 105 Jurkat or U-937 cells, which were starved for 12 hours beforehand, followed by 600 μl of RPMI 1640. The chambers were incubated with or without fasudil treatment at 37°C for 3 hours, and then AngII was added to each chamber at the same temperature for 24 hours to assess proliferative activities. Cell count was determined by flow cytometry.25Suzuki Y Gomez-Guerrero C Shirato I Lopez-Franco O Hernandez-Vargas P Sanjuan G Ruiz-Ortega M Sugaya T Okumura K Tomino Y Ra C Egido J Susceptibility to T cell-mediated injury in immune complex disease is linked to local activation of renin-angiotensin system: the role of NF-AT pathway.J Immunol. 2002; 169: 4136-4146Crossref PubMed Scopus (39) Google Scholar Some of the cultured cells were also used for immunohistochemistry to examine cytoskeletal change. Proliferation data were expressed relative to the average activity in control cells. Wound healing assays were conducted exactly as previously described34Asanuma K Yanagida-Asanuma E Faul C Tomino Y Kim K Mundel P Synaptopodin orchestrates actin organization and cell motility via regulation of RhoA signalling.Nat Cell Biol. 2006; 8: 485-491Crossref PubMed Scopus (329) Google Scholar, 35Yanagida-Asanuma E Asanuma K Kim K Donnelly M Young Choi H Hyung Chang J Suetsugu S Tomino Y Takenawa T Faul C Mundel P Synaptopodin protects against proteinuria by disrupting Cdc42:IRSp53:Mena signaling complexes in kidney podocytes.Am J Pathol. 2007; 171: 415-427Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar with some modification. Differentiated wild-type podocytes (each 5 × 105) were seeded on type I collagen-coated six-well plates and wounded with a sterile 200-μl pipette tip. Wounded monolayers were washed with phosphate-buffered saline (PBS) and incubated in RPMI 1640 medium. Time-lapse images were taken with a ×10 phase-contrast objective on a microscope (Nikon, Tokyo, Japan) at 0 and 24 hours. At the indicated time points, the monolayers were photographed using the grid as a marker, and the wound width (μm) was measured at each time point using Leica FW4000 software (Leica, Wetzlar, Germany). Migratory rates were calculated as (A − B)/A × 100%, with A and B reflecting the width of the wound at 0 or 24 hours, respectively. The data represent the mean ± SEM of six independent experiments. Kidney sections (3-μm thickness) were fixed in 4% paraformaldehyde and stained with periodic acid-Schiff reagent or Masson Trichrome to assess histological changes by light microscopy. Immunohistochemistry was performed on 3-μm-thick formalin-fixed paraffin sections of the kidney. Sections were first deparaffinized and rehydrated before an overnight incubation with mouse anti-human WT-1 (1:100; DAKO, Tokyo, Japan) or polyclonal guinea pig anti-mouse nephrin (1:100; Progen, Heidelberg, Germany). For WT-1, the sections were then incubated with horseradish peroxidase-labeled antibody as secondary antibody (polymer-horseradish peroxidase-labeled anti-mouse antibody; Dako Cytomation, Carpinteria, CA). For nephrin, the sections were reacted with horseradish peroxidase-labeled rabbit anti-guinea pig antibody as secondary antibody (Zymed, San Francisco, CA) at room temperature for 1 hour. Immunoreactivity was detected using an enhanced DAB kit (Dako Cytomation). Nephrin-positive areas in each glomerulus were quantified with KS-400 image analysis software (Carl Zeiss MicroImaging Japan, Tokyo, Japan). Double staining of F4/80 and BrdU was performed with phycoerythrin-labeled anti-mouse F4/80 (1:50; Cedarlane, Ontario, Canada) and fluorescein isothiocyanate-labeled anti-mouse BrdU antibodies (1:100; Santa Cruz Biotechnology, Santa Cruz, CA). Sclerotic change was defined by an increase in fibrillar material and hyalinosis containing acellular, structureless material composed of glycoproteins and lipids.36Tisher CC Brenner BM Renal Pathology with Clinical and Functional Correlations. J.B. Lippincott Company, Philadelphia1994: 355Google Scholar We identified that the sclerotic glomeruli include sclerotic changes of more than 50% within a glomerulus. We checked at least 200 glomeruli in each animal by Masson Trichrome staining and assessed the percentage of pathological glomeruli. The frozen kidney sections were prepared using a cryostat and stained with the following antibodies after acetone fixation: monoclonal rat anti-mouse CD4 (L3T4) antibody (BD PharMingen, San Jose, CA) or monoclonal rat anti-mouse CD8a (Ly-2) antibody (BD PharMingen) for 1 hour, and then horseradish peroxidase-labeled goat anti-rat IgG antibody (Simplestain MAX-PO, rat; Nichirei, Tokyo, Japan) as a secondary antibody for 1 hour. Bound antibodies were detected using an enhanced DAB kit (Dako Cytomation) as mentioned above. Cytoskeletal changes with or without fasudil treatment were analyzed by immunofluorescence. For in vitro cellular staining, Jurkat or U-937 cells were incubated on the sterile cover glasses (IWAKI brand, Scitech Division, Asahi Techno Glass). These cover glasses were directly fixed with 2% paraformaldehyde and 4% sucrose, permeabilized with 0.03% Triton X-100 for 5 minutes, and then blocked with 2% bovine serum albumin, 2% fetal calf serum, and 0.2% fish gelatin in PBS. The cells were incubated with rhodamine phalloidin (Molecular Probes, Tokyo, Japan) for 1 hour and then mounted in Prolong antifade mounting media (Thermo Shandon, Pittsburgh, PA). Fluorescence images were recorded using a confocal microscope (FV-1000; Olympus, Tokyo, Japan) with excitation at 488 nm and detection at 500 to 600 nm for fluorescein isothiocyanate, and with excitation at 543 nm and long-pass detection at 555 to 655 nm for tetramethyl-rhodamine isothiocyanate. Total RNA was isolated from the frozen kidney tissues or cultured podocytes (3 × 106 cells) on 15-cm dishes37Shih NY Li J Karpitskii V Nguyen A Dustin ML Kanagawa O Miner JH Shaw AS Congenital nephrotic syndrome in mice lacking CD2-associated protein.Science. 1999; 286: 312-315Crossref PubMed Scopus (700) Google Scholar using TRIZOL reagent (Life Technologies Inc., Carlsbad, CA) according to the instructions provided by the manufacturer. One-μg aliquots of total RNA were reverse-transcribed using oligo (dT) primers (Life Technologies) and reverse transcriptase, Superscript II (Life Technologies). The product was analyzed by real-time PCR using SYBR Green (Applied Biosystems, Warrington, UK) technology on a PRISM7500 instrument (Applied Biosystems, Foster City, CA). For most of the transcripts, forward and reverse primers were identified according to the primer information available from UniSTS at the National Center for Biotechnology Information. Primer sequences are provided below. The reaction mixture consisted of 1 μl of diluted template, 12.5 μl of SYBR Green PCR Master" @default.
• W2009672980 created "2016-06-24" @default.
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• W2009672980 title "Amelioration of Crescentic Glomerulonephritis by RhoA Kinase Inhibitor, Fasudil, through Podocyte Protection and Prevention of Leukocyte Migration" @default.
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