(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).

(a) Albuminuria (b) blood urea nitrogen concentration and (c) proportion of crescentic glomeruli in Pod-Tet on-Cre Egfr^wt/wt and Pod-Tet on-Cre Egfr^loxP/loxP mice, 8 days after NTS-induced RPGN (P<0.05 for all comparisons). (d) Survival curve of challenged Pod-Tet on-Cre Egfr^loxP/loxP and littermate control mice in a severe model of RPGN. (* P<0.01). (e) Ultrastructural analysis of podocytes by transmission electron microscopy in NTS-treated Pod-Tet on-Cre Egfr^wt/wt and Pod-Tet on-Cre Egfr^loxP/loxP mice. More severe foot process effacement and irregular thickening of the GBM is visible in Pod-Tet on-Cre Egfr^wt/wt animals. Scale bars: upper panels 2μm, lower panels 1μm.

Representative images of immunostaining for HB-EGF using monoclonal sc-74526 antibody in sections of kidney biopsies from 8 random subjects diagnosed with non crescentic glomerulopathies (upper panels), including diabetic nephropathy, amyloidosis, minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), IgA nephropathy (IgAN), and membranous nephropathy (MN). Similarly, lower panels show immunostaining for HB-EGF in renal biopsies from 8 random subjects RPGN of various etiologies, including lupus nephritis, microscopic polyangiitis (MP), endocarditis (End), Goodpasture disease (Gp), and Wegener disease (Wg). Scale bar 50 μm.

(a) Representative image of in situ hybridization study in NTS-injected wild-type (Hbegf (+/+)) animals, showing proHB-EGF expression in glomeruli (G), especially in parietal glomerular epithelial cells (Pec), in podocytes (day 4) and in crescents (Cr) (day 8). White arrow indicates abundant proHB-EGF mRNA expression in areas where tuft-capsular podocyte bridges were present. Scale bar (orange), 50 μm. (b) Quantification by real-time RT-PCR of proHB-EGF mRNA in freshly isolated podocytes on day 6 after nephrotoxic serum injection (NTS) and in podocytes from non-injected control mice (CT) (n=3 per group). * P<0.05 versus controls. (c) Western blot analysis of phosphorylated EGFR and total EGFR in the renal cortex from non-challenged controls (CT), wild-type (Hbegf (+/+)) mice infused with NTS ((+/+)NTS), HB-EGF deficient mice infused with NTS ((−/−)NTS), and from Hbegf (+/+) animals that were given intraperitoneal injections of the EGFR tyrosine kinase inhibitor AG1478 ((+/+)NTS+AG1478). Values reported are means ± sem. (n=6–8 per group). (d) Immunofluorescence staining for phosphoEGFR in renal cortex from controls (CT), NTS-injected Hbegf (+/+) mice ((+/+)NTS), HB-EGF deficient mice infused with NTS ((−/−)NTS), and from NTS-injected Hbegf (+/+) animals treated by AG1478 ((+/+)NTS+AG1478), on day 8 after NTS administration. * P<0.05 versus controls at baseline, ** P<0.01 versus controls at baseline. ## P<0.01 versus mice treated with vehicle only. Pec: parietal glomerular epithelial cells; G: glomerulus; Cr: crescent.

(a) Survival curve for challenged Hbegf (+/+) and Hbegf (−/−) mice. In all cases, death was associated with severe renal damage with macroscopic hematuria (blood leakage into the urine) and animals died from renal failure (with 100% crescentic and necrotizing lesions at autopsy). (b) Masson Trichrome staining of kidneys and proportion of crescentic glomeruli in control mice and in NTS-injected Hbegf (+/+) and Hbegf (−/−) mice (day 8 post NTS) (Cr: crescents, G: glomeruli, Tc: tubules with proteinaceous casts) Scale bar, 50μm. (c) Ascites score as an index of albumin plasma loss and water and sodium retention, (d) albuminuria and (e) blood urea nitrogen concentrations in NTS-challenged Hbegf (+/+) and Hbegf (−/−) animals on day 8 post NTS, and in unchallenged controls (CT). Values reported are means ± sem. (n=9–12 per group). * P<0.05 versus controls at baseline, ** P<0.01 versus baseline, *** P<0.001 versus baseline. # P<0.05 versus NTS-treated (+/+), ## P<0.01 versus NTS-treated (+/+).

(a) Quantification by western blot analysis of phosphorylated EGFR and total EGFR in the renal cortex from non-challenged controls (CT), NTS-injected mice treated with vehicle alone, and NTS-injected mice treated with erlotinib either started twelve hours before administration of NTS (days 0–14) or in a curative protocol, started four days later (d4–14). Mice were euthanised after 14 days of RPGN. (b) Blood urea nitrogen concentration and (c) proportion of crescentic glomeruli in CT in the different groups of mice as in (a). Data are means ± sem, (n=9 mice per group). ** P<0.01 versus controls at baseline (CT), *** P<0.001 versus CT, ## P<0.01 versus vehicle, ## P<0.001 versus vehicle.
(d) Ultrastructural analysis of podocytes by transmission electron microscopy in erlotinib-treated and vehicle-treated mice, five days after injection of NTS. Scale bar 2 μm.
(e) Masson trichrome staining of renal cortex from a mouse treated with erlotinib (day 4–14) (left panel) and a vehicle-treated mouse (right panel) on day 14. Ne: necrotic glomerular lesions, Cr: cellular crescents, Tc: tubular proteinaceous casts, Infilt: diffuse CD3-positive cell infiltrates (Infilt) seen in vehicle-treated mice. Scale bar 100 μm.