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Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis.

Bollée G, Flamant M, Schordan S, Fligny C, Rumpel E, Milon M, Schordan E, Sabaa N, Vandermeersch S, Galaup A, Rodenas A, Casal I, Sunnarborg SW, Salant DJ, Kopp JB, Threadgill DW, Quaggin SE, Dussaule JC, Germain S, Mesnard L, Endlich K, Boucheix C, Belenfant X, Callard P, Endlich N, Tharaux PL - Nat. Med. (2011)

Bottom Line: Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro.Likewise, pharmacological blockade of EGFR also improves the course of RPGN, even when started 4 d after the induction of experimental RPGN.This suggests that targeting the HB-EGF-EGFR pathway could also be beneficial in treatment of human RPGN.

View Article: PubMed Central - PubMed

Affiliation: Unité Mixte de Recherche (UMR) 970, Paris Cardiovascular Research Centre, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France.

ABSTRACT
Rapidly progressive glomerulonephritis (RPGN) is a life-threatening clinical syndrome and a morphological manifestation of severe glomerular injury that is marked by a proliferative histological pattern ('crescents') with accumulation of T cells and macrophages and proliferation of intrinsic glomerular cells. We show de novo induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in intrinsic glomerular epithelial cells (podocytes) from both mice and humans with RPGN. HB-EGF induction increases phosphorylation of the epidermal growth factor receptor (EGFR, also known as ErbB1) in mice with RPGN. In HB-EGF-deficient mice, EGFR activation in glomeruli is absent and the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro. Conditional deletion of the Egfr gene from podocytes of mice alleviates the severity of RPGN. Likewise, pharmacological blockade of EGFR also improves the course of RPGN, even when started 4 d after the induction of experimental RPGN. This suggests that targeting the HB-EGF-EGFR pathway could also be beneficial in treatment of human RPGN.

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HB-EGF induces a migratory phenotype in podocytes in vitro(a) Podocyte outgrowth over 6 days from decapsulated glomeruli of Hbegf (+/+) or Hbegf (−/−) mice (arrow). Cells are stained for WT-1 expression. (b) Outgrowth area from glomeruli of Hbegf (−/−) mice in the absence (light blue bar) or presence of the EGFR inhibitor AG1478 (500 nM) (AG, dark blue bar) and from glomeruli. Sparse outgrowth from glomeruli of Hbegf (−/−) mice in the absence (light grey bar) was rescued by addition of 30 nM HB-EGF (black bar). (c) Schematic drawing of podocyte outgrowth from isolated glomeruli, which was used as a combined migration/proliferation assay to assess the ability of crescent formation in vitro. Podocytes are in a stationary state (blue color) on the surface of capillary loops (grey circle), when glomeruli are plated. Subsequently, podocytes assume a migratory phenotye (orange color), characterized by apical protrusions, by attachment and by migration on the substratum. Later stages of outgrowth also involve proliferation. (d) Representative image of F-actin reorganisation and formation of ring-like actin structures (RiLiS) induced by HB-EGF (30 nM for 7 min) in differentiated podocytes, in the absence or presence of AG1478 (500 nM). The effect of HB-EGF to induce apical protrusions is abrogated in the presence of AG1478 (500 nM). (e) Quantitative analysis of RiLiS formation in differentiated podocytes. HB-EGF (30 nM) was added in the absence (Ctl) or presence of inhibitors: AG - AG1478 (500 nM), Wo - Wortmannin (100 nM), LY - LY294002 (30 μM), PD - PD98059 (25 μM), SB - SB203580 (25 μM). (f) BrdU incorporation in differentiated podocytes over 48 h. (g) Distance of migration of differentiated podocytes within 8 h in the wound assay. Data are means ± SEM (n=3–4 experiments). * P<0.05 vs. untreated Hbegf (+/+) glomeruli in (b) and vs. HB-EGF alone in (e–g). Scale bar : 300 μm in (a) and 30 μm in (d).
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Figure 2: HB-EGF induces a migratory phenotype in podocytes in vitro(a) Podocyte outgrowth over 6 days from decapsulated glomeruli of Hbegf (+/+) or Hbegf (−/−) mice (arrow). Cells are stained for WT-1 expression. (b) Outgrowth area from glomeruli of Hbegf (−/−) mice in the absence (light blue bar) or presence of the EGFR inhibitor AG1478 (500 nM) (AG, dark blue bar) and from glomeruli. Sparse outgrowth from glomeruli of Hbegf (−/−) mice in the absence (light grey bar) was rescued by addition of 30 nM HB-EGF (black bar). (c) Schematic drawing of podocyte outgrowth from isolated glomeruli, which was used as a combined migration/proliferation assay to assess the ability of crescent formation in vitro. Podocytes are in a stationary state (blue color) on the surface of capillary loops (grey circle), when glomeruli are plated. Subsequently, podocytes assume a migratory phenotye (orange color), characterized by apical protrusions, by attachment and by migration on the substratum. Later stages of outgrowth also involve proliferation. (d) Representative image of F-actin reorganisation and formation of ring-like actin structures (RiLiS) induced by HB-EGF (30 nM for 7 min) in differentiated podocytes, in the absence or presence of AG1478 (500 nM). The effect of HB-EGF to induce apical protrusions is abrogated in the presence of AG1478 (500 nM). (e) Quantitative analysis of RiLiS formation in differentiated podocytes. HB-EGF (30 nM) was added in the absence (Ctl) or presence of inhibitors: AG - AG1478 (500 nM), Wo - Wortmannin (100 nM), LY - LY294002 (30 μM), PD - PD98059 (25 μM), SB - SB203580 (25 μM). (f) BrdU incorporation in differentiated podocytes over 48 h. (g) Distance of migration of differentiated podocytes within 8 h in the wound assay. Data are means ± SEM (n=3–4 experiments). * P<0.05 vs. untreated Hbegf (+/+) glomeruli in (b) and vs. HB-EGF alone in (e–g). Scale bar : 300 μm in (a) and 30 μm in (d).

Mentions: In vivo podocytes are terminally differentiated and stationary cells. During crescent formation in mice, however, podocytes assume a migratory phenotype, attach with their apical membrane onto the parietal basement membrane and start to proliferate12–14. Recent data confirm that podocytes contribute to crescents also in humans15,16. As an assay for podocyte crescent formation, we measured outgrowth of WT-1 positive cells from isolated decapsulated mouse glomeruli. The area of podocyte outgrowth strongly depended on the presence of a functional Hbegf allele (Fig. 2a,b). Of note, there was no gene-dose effect as similar results were obtained with Hbegf (+/−) and Hbegf (+/+) cells (not shown). Treatment with AG1478 suppressed podocyte outgrowth in mice carrying one or two functional Hbegf alleles, and addition of HB-EGF to the medium rescued podocyte outgrowth from Hbegf (−/−) glomeruli (Fig. 2a,b). Similar to the steps that characterize crescent formation, glomerular outgrowth of podocytes from decapsulated glomeruli involves formation of apical protrusions in podocytes, attachment and migration away from glomerular capillaries on the substrate as well as proliferation (Fig. 2c). We assessed podocyte outgrowth in vitro employing a podocyte cell line17 with endogenous expression of proHB-EGF mRNA (Supplementary Fig. 1a). Applying specific inhibitors, we examined the involvement of intracellular signalling pathways in podocyte outgrowth: the PI3 kinase pathway that is involved in actin dynamics; the classical MAP kinase pathway that is essential for proliferation; and the p38 MAP kinase pathway that may be relevant for directed migration. HB-EGF caused a complete reorganisation of the F-actin cytoskeleton, and massive formation of RiLiS (ring-like actin structures) and dorsal ruffles (Fig. 2d), indicating apically directed motility. Apical actin protrusions were completely abrogated by PI3 kinase inhibitors wortmannin and LY294002, but insensitive to a highly selective inhibitor of MEK1 (PD 98059) and to a p38 MAPK inhibitor (SB 203280) (Fig. 2e). Proliferation as determined by BrdU incorporation (Supplementary Fig. 1b) was reduced by PD 98059 in the presence of HB-EGF (Fig. 2f). The migration rate of podocytes was doubled by addition of recombinant HB-EGF (Fig. 2g and Supplementary Fig. 1c), which is known to involve a variety of cellular processes such as polarisation, protrusion dynamics, proliferation, cell-matrix and cell-cell adhesion18. Inhibition of PI3 kinase, MEK1 and p38 MAPK all significantly reduced the migration rate. The effects of HB-EGF on podocytes were mediated by the EGFR as they were blocked by AG1478 (Fig. 2d–g). Notably, EGFR tyrosine kinase inhibition mimicked the effect of HB-EGF deficiency on glomerular outgrowth (Fig. 2a,b) indicating that EGFR/ErbB1 is the prominent, if not only receptor, mediating podocyte proliferation and migration. Thus, HB-EGF triggers a phenotypic switch in podocytes that is required for crescent formation, and that involves multiple signalling pathways.


Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis.

Bollée G, Flamant M, Schordan S, Fligny C, Rumpel E, Milon M, Schordan E, Sabaa N, Vandermeersch S, Galaup A, Rodenas A, Casal I, Sunnarborg SW, Salant DJ, Kopp JB, Threadgill DW, Quaggin SE, Dussaule JC, Germain S, Mesnard L, Endlich K, Boucheix C, Belenfant X, Callard P, Endlich N, Tharaux PL - Nat. Med. (2011)

HB-EGF induces a migratory phenotype in podocytes in vitro(a) Podocyte outgrowth over 6 days from decapsulated glomeruli of Hbegf (+/+) or Hbegf (−/−) mice (arrow). Cells are stained for WT-1 expression. (b) Outgrowth area from glomeruli of Hbegf (−/−) mice in the absence (light blue bar) or presence of the EGFR inhibitor AG1478 (500 nM) (AG, dark blue bar) and from glomeruli. Sparse outgrowth from glomeruli of Hbegf (−/−) mice in the absence (light grey bar) was rescued by addition of 30 nM HB-EGF (black bar). (c) Schematic drawing of podocyte outgrowth from isolated glomeruli, which was used as a combined migration/proliferation assay to assess the ability of crescent formation in vitro. Podocytes are in a stationary state (blue color) on the surface of capillary loops (grey circle), when glomeruli are plated. Subsequently, podocytes assume a migratory phenotye (orange color), characterized by apical protrusions, by attachment and by migration on the substratum. Later stages of outgrowth also involve proliferation. (d) Representative image of F-actin reorganisation and formation of ring-like actin structures (RiLiS) induced by HB-EGF (30 nM for 7 min) in differentiated podocytes, in the absence or presence of AG1478 (500 nM). The effect of HB-EGF to induce apical protrusions is abrogated in the presence of AG1478 (500 nM). (e) Quantitative analysis of RiLiS formation in differentiated podocytes. HB-EGF (30 nM) was added in the absence (Ctl) or presence of inhibitors: AG - AG1478 (500 nM), Wo - Wortmannin (100 nM), LY - LY294002 (30 μM), PD - PD98059 (25 μM), SB - SB203580 (25 μM). (f) BrdU incorporation in differentiated podocytes over 48 h. (g) Distance of migration of differentiated podocytes within 8 h in the wound assay. Data are means ± SEM (n=3–4 experiments). * P<0.05 vs. untreated Hbegf (+/+) glomeruli in (b) and vs. HB-EGF alone in (e–g). Scale bar : 300 μm in (a) and 30 μm in (d).
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Figure 2: HB-EGF induces a migratory phenotype in podocytes in vitro(a) Podocyte outgrowth over 6 days from decapsulated glomeruli of Hbegf (+/+) or Hbegf (−/−) mice (arrow). Cells are stained for WT-1 expression. (b) Outgrowth area from glomeruli of Hbegf (−/−) mice in the absence (light blue bar) or presence of the EGFR inhibitor AG1478 (500 nM) (AG, dark blue bar) and from glomeruli. Sparse outgrowth from glomeruli of Hbegf (−/−) mice in the absence (light grey bar) was rescued by addition of 30 nM HB-EGF (black bar). (c) Schematic drawing of podocyte outgrowth from isolated glomeruli, which was used as a combined migration/proliferation assay to assess the ability of crescent formation in vitro. Podocytes are in a stationary state (blue color) on the surface of capillary loops (grey circle), when glomeruli are plated. Subsequently, podocytes assume a migratory phenotye (orange color), characterized by apical protrusions, by attachment and by migration on the substratum. Later stages of outgrowth also involve proliferation. (d) Representative image of F-actin reorganisation and formation of ring-like actin structures (RiLiS) induced by HB-EGF (30 nM for 7 min) in differentiated podocytes, in the absence or presence of AG1478 (500 nM). The effect of HB-EGF to induce apical protrusions is abrogated in the presence of AG1478 (500 nM). (e) Quantitative analysis of RiLiS formation in differentiated podocytes. HB-EGF (30 nM) was added in the absence (Ctl) or presence of inhibitors: AG - AG1478 (500 nM), Wo - Wortmannin (100 nM), LY - LY294002 (30 μM), PD - PD98059 (25 μM), SB - SB203580 (25 μM). (f) BrdU incorporation in differentiated podocytes over 48 h. (g) Distance of migration of differentiated podocytes within 8 h in the wound assay. Data are means ± SEM (n=3–4 experiments). * P<0.05 vs. untreated Hbegf (+/+) glomeruli in (b) and vs. HB-EGF alone in (e–g). Scale bar : 300 μm in (a) and 30 μm in (d).
Mentions: In vivo podocytes are terminally differentiated and stationary cells. During crescent formation in mice, however, podocytes assume a migratory phenotype, attach with their apical membrane onto the parietal basement membrane and start to proliferate12–14. Recent data confirm that podocytes contribute to crescents also in humans15,16. As an assay for podocyte crescent formation, we measured outgrowth of WT-1 positive cells from isolated decapsulated mouse glomeruli. The area of podocyte outgrowth strongly depended on the presence of a functional Hbegf allele (Fig. 2a,b). Of note, there was no gene-dose effect as similar results were obtained with Hbegf (+/−) and Hbegf (+/+) cells (not shown). Treatment with AG1478 suppressed podocyte outgrowth in mice carrying one or two functional Hbegf alleles, and addition of HB-EGF to the medium rescued podocyte outgrowth from Hbegf (−/−) glomeruli (Fig. 2a,b). Similar to the steps that characterize crescent formation, glomerular outgrowth of podocytes from decapsulated glomeruli involves formation of apical protrusions in podocytes, attachment and migration away from glomerular capillaries on the substrate as well as proliferation (Fig. 2c). We assessed podocyte outgrowth in vitro employing a podocyte cell line17 with endogenous expression of proHB-EGF mRNA (Supplementary Fig. 1a). Applying specific inhibitors, we examined the involvement of intracellular signalling pathways in podocyte outgrowth: the PI3 kinase pathway that is involved in actin dynamics; the classical MAP kinase pathway that is essential for proliferation; and the p38 MAP kinase pathway that may be relevant for directed migration. HB-EGF caused a complete reorganisation of the F-actin cytoskeleton, and massive formation of RiLiS (ring-like actin structures) and dorsal ruffles (Fig. 2d), indicating apically directed motility. Apical actin protrusions were completely abrogated by PI3 kinase inhibitors wortmannin and LY294002, but insensitive to a highly selective inhibitor of MEK1 (PD 98059) and to a p38 MAPK inhibitor (SB 203280) (Fig. 2e). Proliferation as determined by BrdU incorporation (Supplementary Fig. 1b) was reduced by PD 98059 in the presence of HB-EGF (Fig. 2f). The migration rate of podocytes was doubled by addition of recombinant HB-EGF (Fig. 2g and Supplementary Fig. 1c), which is known to involve a variety of cellular processes such as polarisation, protrusion dynamics, proliferation, cell-matrix and cell-cell adhesion18. Inhibition of PI3 kinase, MEK1 and p38 MAPK all significantly reduced the migration rate. The effects of HB-EGF on podocytes were mediated by the EGFR as they were blocked by AG1478 (Fig. 2d–g). Notably, EGFR tyrosine kinase inhibition mimicked the effect of HB-EGF deficiency on glomerular outgrowth (Fig. 2a,b) indicating that EGFR/ErbB1 is the prominent, if not only receptor, mediating podocyte proliferation and migration. Thus, HB-EGF triggers a phenotypic switch in podocytes that is required for crescent formation, and that involves multiple signalling pathways.

Bottom Line: Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro.Likewise, pharmacological blockade of EGFR also improves the course of RPGN, even when started 4 d after the induction of experimental RPGN.This suggests that targeting the HB-EGF-EGFR pathway could also be beneficial in treatment of human RPGN.

View Article: PubMed Central - PubMed

Affiliation: Unité Mixte de Recherche (UMR) 970, Paris Cardiovascular Research Centre, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France.

ABSTRACT
Rapidly progressive glomerulonephritis (RPGN) is a life-threatening clinical syndrome and a morphological manifestation of severe glomerular injury that is marked by a proliferative histological pattern ('crescents') with accumulation of T cells and macrophages and proliferation of intrinsic glomerular cells. We show de novo induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in intrinsic glomerular epithelial cells (podocytes) from both mice and humans with RPGN. HB-EGF induction increases phosphorylation of the epidermal growth factor receptor (EGFR, also known as ErbB1) in mice with RPGN. In HB-EGF-deficient mice, EGFR activation in glomeruli is absent and the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro. Conditional deletion of the Egfr gene from podocytes of mice alleviates the severity of RPGN. Likewise, pharmacological blockade of EGFR also improves the course of RPGN, even when started 4 d after the induction of experimental RPGN. This suggests that targeting the HB-EGF-EGFR pathway could also be beneficial in treatment of human RPGN.

Show MeSH
Related in: MedlinePlus