EGF-related growth factors in the pathogenesis of murine ARPKD.
BACKGROUND
Epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and their receptor, EGFR, play key roles in polycystic kidney disease (PKD) pathogenesis. Renal expression of two related growth factors, amphiregulin and heparin-binding EGF, has not been examined previously in PKD. The aims of this study of murine autosomal-recessive polycystic kidney disease (ARPKD) were (1) to characterize amphiregulin and heparin-binding EGF expression in cystic versus normal kidneys and cells; and (2) to identify the functional effects of abnormal EGF-related growth factor expression.


METHODS
Amphiregulin and heparin-binding-EGF expression were examined by immunohistology and Western blot of kidneys and conditionally-immortalized collecting tubule cells obtained from cystic bpk mice (a murine model of ARPKD) and normal littermates. EGF, TGF-alpha, amphiregulin, and heparin-binding EGF in vitro effects on cystic and control collecting tubule cells were assessed by cell proliferation, cyst fluid mitogenicity, and EGFR activation.


RESULTS
By immunohistology, amphiregulin and heparin-binding EGF localized to apical and basolateral surfaces of proximal tubule cysts > normal proximal tubules. In cystic collecting tubules, heparin-binding EGF (but not amphiregulin) localized to both apical and basolateral surfaces; whereas in normal collecting tubules, amphiregulin and heparin-binding EGF localized to the basolateral surface only. Increased amphiregulin and heparin-binding EGF expression by Western blot was seen in cystic vs. normal kidneys and increased heparin-binding EGF (but not amphiregulin) expression was present in cystic collecting tubule cell lines vs. controls. EGF, TGF-alpha, amphiregulin, and heparin-binding EGF were all mitogenic to cystic > control collecting tubule cells. Immunoprecipitation of EGF and TGF-alpha reduced cyst fluid mitogenicity by almost 80%, whereas heparin-binding EGF and amphiregulin immunoprecipitations had minimal effects. Differential receptor activation was also seen: Heparin-binding EGF markedly activated EGFR (>EGF = TGF-alpha > amphiregulin), with a greater effect seen in cystic vs. control collecting tubule cells.


CONCLUSIONS
Multiple EGF-related growth factors are abnormally expressed in murine ARPKD and may have differential roles in disease pathogenesis. In particular, newly identified abnormalities in heparin-binding EGF expression in cystic kidneys and cells may have important implications for disease pathogenesis.
MacRae Dell
Katherine
K
Rainbow Center for Childhood PKD, Department of Pediatrics, Division of Pediatric Nephrology, Rainbow Babies and Children's Hospital and Case Western Reserve University, Cleveland, Ohio 44106, USA. kmd8@po.cwru.edu
Nemo
Raghad
R
Sweeney
William E
WE
Avner
Ellis D
ED
D066258:Amphiregulin; D000818:Animals; D002455:Cell Division; D020420:Cyst Fluid / Q000378:metabolism; D004195:Disease Models, Animal; D066255:EGF Family of Proteins; D004815:Epidermal Growth Factor / Q000502:physiology*; D066246:ErbB Receptors / Q000502:physiology; D006023:Glycoproteins / Q000502:physiology; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins / Q000502:physiology; D007685:Kidney Tubules, Collecting / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D008817:Mice, Mutant Strains; D008934:Mitogens / Q000378:metabolism; D017044:Polycystic Kidney, Autosomal Recessive / Q000209:etiology* / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D016211:Transforming Growth Factor alpha / Q000502:physiology
D066258:Amphiregulin; C587849:Areg protein, mouse; D066255:EGF Family of Proteins; D006023:Glycoproteins; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008934:Mitogens; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D066246:ErbB Receptors