Complement C5b-9 Induces Receptor Tyrosine Kinase Transactivation in Glomerular Epithelial Cells
1999; Elsevier BV; Volume: 155; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65485-5
ISSN1525-2191
AutoresAndrey V. Cybulsky, Tomoko Takano, Joan Papillon, Alison J. McTavish,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoIn the passive Heymann nephritis (PHN) model of membranous nephropathy, C5b-9 induces glomerular epithelial cell (GEC. injury and proteinuria, which is partially mediated via production of eicosanoids. Using rat GEC in culture, we demonstrated that sublytic C5b-9 induced tyrosine phosphorylation of the epidermal growth factor receptor (EGF-R), Neu, fibroblast growth factor receptor-2, and hepatocyte growth factor receptor. In addition, C5b-9 stimulated increases in tyrosine204 phosphorylation of extracellular signal-regulated kinase-2 (ERK2), as well as free [3H]arachidonic acid (AA) and prostaglandin E2 (PGE2). Phosphorylated EGF-R bound the adaptor protein, Grb2, and the EGF-R-selective tyrphostin, AG1478, blocked the C5b-9-induced ERK2 phosphorylation, [3H]AA release, and PGE2 production by 45 to 65%, supporting a functional role for EGF-R kinase in mediating the activation of these pathways. Glomeruli isolated from rats with PHN demonstrated increases in ERK2 tyrosine204 phosphorylation and PGE2 production, as compared with glomeruli from control rats, and these increases were partially inhibited with AG1478. Thus, C5b-9 induces transactivation of receptor tyrosine kinases, in association with ERK2 activation, AA release, and PGE2 production in cultured GEC and glomerulonephritis in vivo. Transactivated tyrosine kinases may serve as scaffolds for assembly and/or activation of proteins, which then lead to activation of the ERK2 cascade and AA metabolism. In the passive Heymann nephritis (PHN) model of membranous nephropathy, C5b-9 induces glomerular epithelial cell (GEC. injury and proteinuria, which is partially mediated via production of eicosanoids. Using rat GEC in culture, we demonstrated that sublytic C5b-9 induced tyrosine phosphorylation of the epidermal growth factor receptor (EGF-R), Neu, fibroblast growth factor receptor-2, and hepatocyte growth factor receptor. In addition, C5b-9 stimulated increases in tyrosine204 phosphorylation of extracellular signal-regulated kinase-2 (ERK2), as well as free [3H]arachidonic acid (AA) and prostaglandin E2 (PGE2). Phosphorylated EGF-R bound the adaptor protein, Grb2, and the EGF-R-selective tyrphostin, AG1478, blocked the C5b-9-induced ERK2 phosphorylation, [3H]AA release, and PGE2 production by 45 to 65%, supporting a functional role for EGF-R kinase in mediating the activation of these pathways. Glomeruli isolated from rats with PHN demonstrated increases in ERK2 tyrosine204 phosphorylation and PGE2 production, as compared with glomeruli from control rats, and these increases were partially inhibited with AG1478. Thus, C5b-9 induces transactivation of receptor tyrosine kinases, in association with ERK2 activation, AA release, and PGE2 production in cultured GEC and glomerulonephritis in vivo. Transactivated tyrosine kinases may serve as scaffolds for assembly and/or activation of proteins, which then lead to activation of the ERK2 cascade and AA metabolism. The complement system plays an important role in mediating inflammation, cytolysis, and phagocytosis. Activation of the complement cascade near a cell surface leads to assembly of terminal components, exposure of hydrophobic domains, and insertion of the C5b-9 membrane attack complex into the lipid bilayer of the plasma membrane.1Morgan BP Effects of the membrane attack complex of complement on nucleated cells.Curr Top Microbiol Immunol. 1992; 178: 115-140PubMed Google Scholar, 2Nicholson-Weller A Halperin JA Membrane signaling by complement C5b-9, the membrane attack complex.Immunol Res. 1993; 12: 244-257Crossref PubMed Scopus (131) Google Scholar Assembly of C5b-9 results in formation of transmembrane channels or rearrangement of membrane lipids with loss of membrane integrity. Nucleated cells require multiple C5b-9 lesions for lysis, but at lower doses, C5b-9 induces sublytic injury and various metabolic effects. These may include a rise in cytosolic Ca2+ concentration, alterations in membrane lipids and proteins, activation of phospholipases, protein kinases, and G-proteins, and induction of growth factors and proliferation.1Morgan BP Effects of the membrane attack complex of complement on nucleated cells.Curr Top Microbiol Immunol. 1992; 178: 115-140PubMed Google Scholar, 2Nicholson-Weller A Halperin JA Membrane signaling by complement C5b-9, the membrane attack complex.Immunol Res. 1993; 12: 244-257Crossref PubMed Scopus (131) Google Scholar, 3Cybulsky AV Salant DJ Quigg RJ Badalamenti J Bonventre JV Complement C5b-9 complex activates phospholipases in glomerular epithelial cells.Am J Physiol. 1989; 257: F826-F836PubMed Google Scholar, 4Cybulsky AV Bonventre JV Quigg RJ Lieberthal W Salant DJ Cytosolic calcium and protein kinase C reduce complement-mediated glomerular epithelial injury.Kidney Int. 1990; 38: 803-811Crossref PubMed Scopus (48) Google Scholar, 5Niculescu F Rus H Shin ML Receptor-independent activation of guanine nucleotide-binding regulatory proteins by terminal complement complexes.J Biol Chem. 1994; 269: 4417-4423Abstract Full Text PDF PubMed Google Scholar, 6Niculescu F Rus H van Biesen T Shin ML Activation of Ras and mitogen-activated protein kinase pathway by terminal complement complexes is G protein dependent.J Immunol. 1997; 158: 4405-4412PubMed Google Scholar, 7Halperin JA Taratuska A Nicholson-Weller A Terminal complement complex C5b-9 stimulates mitogenesis in 3T3 cells.J Clin Invest. 1993; 91: 1974-1978Crossref PubMed Scopus (106) Google ScholarThe C5b-9 membrane attack complex induces injury in diverse renal glomerular diseases. For example, in the rat passive Heymann nephritis (PHN) model of membranous nephropathy, impairment of glomerular capillary wall permselectivity (proteinuria) is mediated by antibody and C5b-9.8Cybulsky AV Rennke HG Feintzeig ID Salant DJ Complement-induced glomerular epithelial cell injury: role of the membrane attack complex in rat membranous nephropathy.J Clin Invest. 1986; 77: 1096-1107Crossref PubMed Google Scholar, 9Kerjaschki D Schulze M Binder S Kain R Ojha PP Susani M Horvat R Baker PJ Couser WG Transcellular transport and membrane insertion of the C5b-9 membrane attack complex of complement by glomerular epithelial cells in experimental membranous nephropathy.J Immunol. 1989; 143: 546-552PubMed Google Scholar The primary target of C5b-9 is the visceral glomerular epithelial cell (GEC), which suffers noncytolytic injury.8Cybulsky AV Rennke HG Feintzeig ID Salant DJ Complement-induced glomerular epithelial cell injury: role of the membrane attack complex in rat membranous nephropathy.J Clin Invest. 1986; 77: 1096-1107Crossref PubMed Google Scholar, 9Kerjaschki D Schulze M Binder S Kain R Ojha PP Susani M Horvat R Baker PJ Couser WG Transcellular transport and membrane insertion of the C5b-9 membrane attack complex of complement by glomerular epithelial cells in experimental membranous nephropathy.J Immunol. 1989; 143: 546-552PubMed Google Scholar C5b-9 also stimulates production of glomerular eicosanoids, including prostaglandin E2 (PGE2) and thromboxane A2,10Stahl RAK Adler S Baker PJ Chen YP Pritzl PM Couser WG Enhanced glomerular prostaglandin formation in experimental membranous nephropathy.Kidney Int. 1987; 31: 1126-1131Crossref PubMed Scopus (60) Google Scholar, 11Weise WJ Natori Y Levine JS O'Meara YM Minto AW Manning EC Goldstein DJ Abrahamson DR Salant DJ Fish oil has protective and therapeutic effects on proteinuria in passive Heymann nephritis.Kidney Int. 1993; 43: 359-368Crossref PubMed Scopus (36) Google Scholar, 12Nagao T Nagamatsu T Suzuki Y Effect of DP-1904, a thromboxane A2 synthase inhibitor, on passive Heymann nephritis in rats.Eur J Pharmacol. 1996; 316: 73-80Crossref PubMed Scopus (18) Google Scholar and the latter may enhance proteinuria in certain models of membranous nephropathy.11Weise WJ Natori Y Levine JS O'Meara YM Minto AW Manning EC Goldstein DJ Abrahamson DR Salant DJ Fish oil has protective and therapeutic effects on proteinuria in passive Heymann nephritis.Kidney Int. 1993; 43: 359-368Crossref PubMed Scopus (36) Google Scholar, 12Nagao T Nagamatsu T Suzuki Y Effect of DP-1904, a thromboxane A2 synthase inhibitor, on passive Heymann nephritis in rats.Eur J Pharmacol. 1996; 316: 73-80Crossref PubMed Scopus (18) Google Scholar, 13Cybulsky AV Lieberthal W Quigg RJ Rennke HJ Salant DJ A role for thromboxane in complement-mediated glomerular injury.Am J Pathol. 1987; 128: 45-51PubMed Google Scholar, 14Zoja C Benigni A Verroust P Ronco P Bertani T Remuzzi G Indomethacin reduces proteinuria in passive Heymann nephritis in rats.Kidney Int. 1987; 31: 1335-1343Crossref PubMed Scopus (45) Google Scholar This effect of thromboxane A2 may be through an increase in glomerular transcapillary pressure, which appears to be responsible for a portion of the enhanced urine protein excretion.15Gabbai FB Gushwa LC Wilson CB Blantz RC An evaluation of the development of experimental membranous nephropathy.Kidney Int. 1987; 31: 1267-1278Crossref PubMed Scopus (24) Google Scholar, 16Yoshioka T Rennke HG Salant DJ Deen WM Ichikawa I Role of abnormally high transmural pressure in the permselectivity defect of glomerular capillary wall: a study in early passive Heymann nephritis.Circ Res. 1987; 61: 531-538Crossref PubMed Scopus (120) Google ScholarWe have used well differentiated rat GEC in culture to define biochemical pathways that are activated by sublytic C5b-9. By analogy to GEC in vivo, in cultured GEC, sublytic C5b-9 injures plasma membranes.17Quigg RJ Cybulsky AV Jacobs JB Salant DJ Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells.Kidney Int. 1988; 34: 43-52Crossref PubMed Scopus (87) Google Scholar Sublytic C5b-9 also increases cytosolic Ca2+ concentration,3Cybulsky AV Salant DJ Quigg RJ Badalamenti J Bonventre JV Complement C5b-9 complex activates phospholipases in glomerular epithelial cells.Am J Physiol. 1989; 257: F826-F836PubMed Google Scholar, 18Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar stimulates phospholipases C, including phospholipase C-γ1, which leads to production of inositol phosphates and 1,2-diacylglycerol,3Cybulsky AV Salant DJ Quigg RJ Badalamenti J Bonventre JV Complement C5b-9 complex activates phospholipases in glomerular epithelial cells.Am J Physiol. 1989; 257: F826-F836PubMed Google Scholar, 19Cybulsky AV Cyr MD Phosphatidylcholine-directed phospholipase C: activation by complement C5b-9.Am J Physiol. 1993; 265: F551-F560PubMed Google Scholar, 20Cybulsky AV Papillon J McTavish AJ Complement activates phospholipases and protein kinases in glomerular epithelial cells.Kidney Int. 1998; 54: 360-372Crossref PubMed Scopus (48) Google Scholar stimulates activities of protein kinase C (PKC) and extracellular signal-regulated kinase-2 (ERK2),20Cybulsky AV Papillon J McTavish AJ Complement activates phospholipases and protein kinases in glomerular epithelial cells.Kidney Int. 1998; 54: 360-372Crossref PubMed Scopus (48) Google Scholar activates cytosolic phospholipase A2 (cPLA2), and releases arachidonic acid (AA) and eicosanoids.18Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar, 20Cybulsky AV Papillon J McTavish AJ Complement activates phospholipases and protein kinases in glomerular epithelial cells.Kidney Int. 1998; 54: 360-372Crossref PubMed Scopus (48) Google Scholar, 21Cybulsky AV Release of arachidonic acid by complement C5b-9 complex in glomerular epithelial cells.Am J Physiol. 1991; 261: F427-F436PubMed Google Scholar, 22Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar Our studies suggest that the functional consequences of cPLA2 activation include production of eicosanoids and exacerbation of complement-induced GEC injury.22Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar The functional role of ERK2 activation remains to be determined.Although we and others have characterized multiple signaling pathways that are activated by C5b-9, less is known about plasma membrane-associated events triggered by C5b-9 assembly, or the activation of very early steps in these pathways. There is no known transmembrane receptor for C5b-9, however, in B cell lines, the C5b-9 complex is reported to couple to heterotrimeric G-proteins, which can activate downstream effectors.5Niculescu F Rus H Shin ML Receptor-independent activation of guanine nucleotide-binding regulatory proteins by terminal complement complexes.J Biol Chem. 1994; 269: 4417-4423Abstract Full Text PDF PubMed Google Scholar The present study addresses another mechanism by which assembly of C5b-9 may lead to the activation of protein kinases and phospholipases, ie, the transactivation of receptor tyrosine kinases (RTKs). Generally, RTKs function as transmembrane receptors for peptide ligands such as growth factors and contain an intrinsic tyrosine kinase as a part of their cytoplasmic domains.23Carpenter G Receptors for epidermal growth factor and other polypeptide mitogens.Ann Rev Biochem. 1987; 56: 881-914Crossref PubMed Scopus (1042) Google Scholar, 24Schlessinger J Ullrich A Growth factor signaling by receptor tyrosine kinases.Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1287) Google Scholar, 25Malarkey K Belham CM Paul A Graham A McLees A Scott PH Plevin R The regulation of tyrosine kinase signalling pathways by growth factor and G-protein-coupled receptors.Biochem J. 1995; 309: 361-375Crossref PubMed Scopus (264) Google Scholar It is believed that binding of a peptide ligand to the extracellular domain of the corresponding RTK leads to an increase in tyrosine kinase activity, in association with phosphorylation of the receptor on multiple cytoplasmic tyrosine residues, and phosphorylation of substrate proteins.23Carpenter G Receptors for epidermal growth factor and other polypeptide mitogens.Ann Rev Biochem. 1987; 56: 881-914Crossref PubMed Scopus (1042) Google Scholar, 24Schlessinger J Ullrich A Growth factor signaling by receptor tyrosine kinases.Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1287) Google Scholar, 25Malarkey K Belham CM Paul A Graham A McLees A Scott PH Plevin R The regulation of tyrosine kinase signalling pathways by growth factor and G-protein-coupled receptors.Biochem J. 1995; 309: 361-375Crossref PubMed Scopus (264) Google Scholar The signal may then be transmitted to nuclear or cytoplasmic effectors through a series of adaptor molecules and serine/threonine protein kinases known collectively as the mitogen-activated protein kinase pathway.24Schlessinger J Ullrich A Growth factor signaling by receptor tyrosine kinases.Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1287) Google Scholar, 25Malarkey K Belham CM Paul A Graham A McLees A Scott PH Plevin R The regulation of tyrosine kinase signalling pathways by growth factor and G-protein-coupled receptors.Biochem J. 1995; 309: 361-375Crossref PubMed Scopus (264) Google Scholar Alternatively, enzymes, including phospholipase C-γ1, can associate with the phosphorylated RTK and undergo activation.24Schlessinger J Ullrich A Growth factor signaling by receptor tyrosine kinases.Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1287) Google Scholar, 25Malarkey K Belham CM Paul A Graham A McLees A Scott PH Plevin R The regulation of tyrosine kinase signalling pathways by growth factor and G-protein-coupled receptors.Biochem J. 1995; 309: 361-375Crossref PubMed Scopus (264) Google Scholar Recently, it has been reported that the epidermal growth factor receptor (EGF-R) tyrosine kinase can be activated not only by EGF, but also, in the absence of natural ligand, via transactivation by G-protein-coupled receptors.26Daub H Weiss FU Wallasch C Ullrich A Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors.Nature. 1996; 379: 557-560Crossref PubMed Scopus (1314) Google Scholar, 27Daub H Wallasch C Lankenau A Herrlich A Ullrich A Signal characteristics of G protein-transactivated EGF receptor.EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (586) Google Scholar, 28Eguchi S Numaguchi K Iwasaki H Matsumoto T Yamakawa T Utsonomiya H Motley ED Kawakatsu H Owada KM Hirata Y Marumo F Inagami T Calcium-dependent epidermal growth factor receptor transactivation mediates angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells.J Biol Chem. 1998; 273: 8890-8896Crossref PubMed Scopus (503) Google Scholar Using rat GEC in culture, we demonstrated that early signals elicited by C5b-9 assembly in the plasma membrane include tyrosine phosphorylation of various RTKs, including EGF-R. Inhibition of the EGF-R tyrosine kinase, in part, abolishes C5b-9-induced activation of ERK2 and cPLA2 and production of PGE2. Furthermore, we extended these results in cultured GEC to C5b-9-mediated injury in vivo by demonstrating that ERK2 activity and PGE2 synthesis are increased in glomeruli of rats with PHN and that these increases are partially blocked after inhibition of the EGF-R tyrosine kinase.Materials and MethodsMaterialsTissue culture reagents were obtained from Life Technologies (Burlington, ON). C8-deficient serum and purified C8 were purchased from Quidel (San Diego, CA). Lipids, phorbol myristate acetate (PMA), digitonin, AG1478, PGE2, and anti-PGE2 antiserum were purchased from Sigma Chemical Co. (St. Louis, MO). EGF, basic fibroblast growth factor (bFGF), and hepatocyte growth factor were from Collaborative Research (Bedford, MA). PP1 was from Biomol Research Laboratories (Plymouth Meeting, PA), and RG50864 (AG213) was from Rhone-Poulenc Rorer (Collegeville, PA). Anti-phosphotyrosine monoclonal antibody, PY20, was from Transduction Laboratories (Lexington, KY). Rabbit antibodies to Neu, FGF-R2, Met, and ERK2 and agarose-conjugated glutathione S-transferase (GST)-Grb2-(1–217) fusion protein were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-phosphoERK antibody was purchased from New England Biolabs (Mississauga, ON). Rabbit anti-EGF-R antibody, RK2, and sheep anti-rat Fx1A were described previously.29Cybulsky AV McTavish AJ Extracellular matrix is required for MAP kinase activation and proliferation of rat glomerular epithelial cells.Biochem Biophys Res Comm. 1997; 231: 160-166Crossref PubMed Scopus (19) Google Scholar, 30Salant DJ Cybulsky AV Experimental glomerulonephritis.in: DiSabato G Methods in Enzymology. Vol 162. Academic Press, New York1988: 421-461Google Scholar [3H]AA (100 Ci/mmol), and [3H]PGE2 (200 Ci/mmol) were from New England Nuclear (Boston, MA). Electrophoresis and immunoblotting reagents were from Bio-Rad Laboratories (Mississauga, ON). Male Sprague-Dawley rats were purchased from Charles River Canada (St. Constant, PQ).Cell Culture and TransfectionRat GEC culture and characterization have been published previously.17Quigg RJ Cybulsky AV Jacobs JB Salant DJ Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells.Kidney Int. 1988; 34: 43-52Crossref PubMed Scopus (87) Google Scholar GEC were cultured in K1 medium on plastic substratum. Experiments measuring free [3H]AA used GEC that stably overexpress cPLA2. Parental GEC or GEC that express the neomycin-resistance gene (neo) were generally used in other experiments. Production and characterization of these cell lines were described previously.18Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar, 22Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google ScholarIncubation of GEC with ComplementThe standard protocol involved incubation of GEC in monolayer culture with rabbit anti-GEC antiserum (5–10% v/v) in modified Krebs-Henseleit buffer containing 145 mmol/L NaCl, 5 mmol/L KCl, 0.5 mmol/L MgSO4, 1 mmol/L Na2HPO4, 0.5 mmol/L CaCl2, 5 mmol/L glucose, and 20 mmol/L Hepes, pH 7.4, for 40 minutes at 22°C.3Cybulsky AV Salant DJ Quigg RJ Badalamenti J Bonventre JV Complement C5b-9 complex activates phospholipases in glomerular epithelial cells.Am J Physiol. 1989; 257: F826-F836PubMed Google Scholar, 4Cybulsky AV Bonventre JV Quigg RJ Lieberthal W Salant DJ Cytosolic calcium and protein kinase C reduce complement-mediated glomerular epithelial injury.Kidney Int. 1990; 38: 803-811Crossref PubMed Scopus (48) Google Scholar, 19Cybulsky AV Cyr MD Phosphatidylcholine-directed phospholipase C: activation by complement C5b-9.Am J Physiol. 1993; 265: F551-F560PubMed Google Scholar, 20Cybulsky AV Papillon J McTavish AJ Complement activates phospholipases and protein kinases in glomerular epithelial cells.Kidney Int. 1998; 54: 360-372Crossref PubMed Scopus (48) Google Scholar, 21Cybulsky AV Release of arachidonic acid by complement C5b-9 complex in glomerular epithelial cells.Am J Physiol. 1991; 261: F427-F436PubMed Google Scholar, 22Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar GEC were then incubated at 37°C with normal human serum (2.5% v/v in Krebs-Henseleit buffer), or with heat-inactivated (decomplemented. human serum (56°C) in controls. In some experiments, antibody-sensitized GEC were incubated with 2.5% C8-deficient human serum (C8DS) supplemented with purified C8 (80 μg/ml undiluted serum), or with 2.5% C8DS alone in controls. We have generally used heterologous complement to facilitate studies with complement-deficient sera and to minimize possible signaling via complement-regulatory proteins; however, in previous studies, results of several experiments were confirmed with homologous (rat) complement.18Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar Sublytic concentrations of complement (<5% normal human serum) were established previously.22Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar In GEC, complement is not activated in the absence of antibody.17Quigg RJ Cybulsky AV Jacobs JB Salant DJ Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells.Kidney Int. 1988; 34: 43-52Crossref PubMed Scopus (87) Google ScholarSublytic complement cytotoxicity was monitored by measuring release of biscarboxyethyl carboxyfluorescein (BCECF) as detailed previously.17Quigg RJ Cybulsky AV Jacobs JB Salant DJ Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells.Kidney Int. 1988; 34: 43-52Crossref PubMed Scopus (87) Google Scholar, 22Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar Complement lysis was determined by measuring release of lactate dehydrogenase, similarly to the method described previously.17Quigg RJ Cybulsky AV Jacobs JB Salant DJ Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells.Kidney Int. 1988; 34: 43-52Crossref PubMed Scopus (87) Google ScholarInduction of PHNPHN was induced by a single intravenous injection of 0.4 ml of sheep anti-rat Fx1A antiserum.30Salant DJ Cybulsky AV Experimental glomerulonephritis.in: DiSabato G Methods in Enzymology. Vol 162. Academic Press, New York1988: 421-461Google Scholar Urine was collected overnight at day 13–14 postinjection. Glomeruli were isolated by differential sieving on day 1430Salant DJ Cybulsky AV Experimental glomerulonephritis.in: DiSabato G Methods in Enzymology. Vol 162. Academic Press, New York1988: 421-461Google Scholar and were suspended in modified Krebs-Henseleit buffer for experiments.Tyrosine PhosphorylationAfter incubation with antibody and complement, ∼6 × 106 GEC were lysed, and proteins were immunoprecipitated with primary rabbit antisera directed to specific RTKs, as described previously.25Malarkey K Belham CM Paul A Graham A McLees A Scott PH Plevin R The regulation of tyrosine kinase signalling pathways by growth factor and G-protein-coupled receptors.Biochem J. 1995; 309: 361-375Crossref PubMed Scopus (264) Google Scholar After immunoprecipitation, immune complexes were incubated with agarose-coupled protein A. For analysis of GST-Grb2-associated proteins, ∼2 × 107 GEC were lysed and incubated with agarose-conjugated GST-Grb2 fusion protein (4 μg) for 3 hours at 4°C. Complexes were boiled in Laemmli sample buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Proteins were then electrophoretically transferred onto nitrocellulose paper, blocked with 3% bovine serum albumin/2% ovalbumin, and incubated with antibody to phosphotyrosine. Blots were developed using alkaline phosphatase-conjugated secondary antibody and colorimetric detection.31Kee N McTavish AJ Papillon J Cybulsky AV Receptor protein tyrosine kinases in perinatal developing rat kidney.Kidney Int. 1997; 52 (erratum Kidney Int 1997, 52:1165–1167): 309-317Crossref PubMed Scopus (21) Google Scholar To assess ERK2 activation, ∼1 × 106 GEC were lysed and 25 μg of lysate were subjected to SDS-PAGE. Activation of ERK2 was monitored by immunoblotting with anti-phosphoERK antibody, ie, antibody that reacts with ERK phosphotyrosine204. RTK tyrosine phosphorylation and ERK2 tyrosine204 phosphorylation were quantitated by densitometry, as described previously.29Cybulsky AV McTavish AJ Extracellular matrix is required for MAP kinase activation and proliferation of rat glomerular epithelial cells.Biochem Biophys Res Comm. 1997; 231: 160-166Crossref PubMed Scopus (19) Google Scholar Tyrosine phosphorylation of glomerular proteins was monitored in an analogous manner. Immunoblotting for RTK or ERK2 proteins was carried out as described above, except that blots were developed with RTK- or ERK2-specific antibodies.Measurement of Free [3H]AAGEC phospholipids were labeled to isotopic equilibrium with [3H]AA for 48 to 72 hours, as detailed previously.3Cybulsky AV Salant DJ Quigg RJ Badalamenti J Bonventre JV Complement C5b-9 complex activates phospholipases in glomerular epithelial cells.Am J Physiol. 1989; 257: F826-F836PubMed Google Scholar, 18Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar, 19Cybulsky AV Cyr MD Phosphatidylcholine-directed phospholipase C: activation by complement C5b-9.Am J Physiol. 1993; 265: F551-F560PubMed Google Scholar, 20Cybulsky AV Papillon J McTavish AJ Complement activates phospholipases and protein kinases in glomerular epithelial cells.Kidney Int. 1998; 54: 360-372Crossref PubMed Scopus (48) Google Scholar, 21Cybulsky AV Release of arachidonic acid by complement C5b-9 complex in glomerular epithelial cells.Am J Physiol. 1991; 261: F427-F436PubMed Google Scholar, 22Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar Lipids were extracted from ∼1 × 106 cells and cell supernatants. Methods for extracting and separating radiolabeled lipids by thin layer chromatography are published.3Cybulsky AV Salant DJ Quigg RJ Badalamenti J Bonventre JV Complement C5b-9 complex activates phospholipases in glomerular epithelial cells.Am J Physiol. 1989; 257: F826-F836PubMed Google Scholar, 18Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar, 19Cybulsky AV Cyr MD Phosphatidylcholine-directed phospholipase C: activation by complement C5b-9.Am J Physiol. 1993; 265: F551-F560PubMed Google Scholar, 20Cybulsky AV Papillon J McTavish AJ Complement activates phospholipases and protein kinases in glomerular epithelial cells.Kidney Int. 1998; 54: 360-372Crossref PubMed Scopus (48) Google Scholar, 21Cybulsky AV Release of arachidonic acid by complement C5b-9 complex in glomerular epithelial cells.Am J Physiol. 1991; 261: F427-F436PubMed Google Scholar, 22Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google ScholarMeasurement of PGE2PGE2 was measured by radioimmunoassay in lipid extracts of GEC plus culture supernatants, or in glomerular supernatants, using anti-PGE2 antiserum and [3H]PGE2 tracer, as described previously,7Halperin JA Taratuska A Nicholson-Weller A Terminal complement complex C5b-9 stimulates mitogenesis in 3T3 cells.J Clin Invest. 1993; 91: 1974-1978Crossref PubMed Scopus (106) Google Scholar and according to the manufacturer's protocol (Sigma). For GEC, [3H]PGE2 (1000 cpm) was added just before extraction to correct for extraction efficiency. Briefly, samples were incubated with [3H]PGE2 and anti-PGE2 antibody for 1 hour at 4°C, after which unbound PGE2 was removed by the addition of activated charcoal. The radioactivity of the supernatant was counted in a β-scintillation counter, a
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