Basic fibroblast growth factor expression is increased in human renal fibrogenesis and may mediate autocrine fibroblast proliferation.
BACKGROUND
Interstitial fibroblasts play a critical role in renal fibrogenesis, and autocrine proliferation of these cells may account for continuous matrix synthesis. Basic fibroblast growth factor (FGF-2) is mitogenic for most cells and exerts intracrine, autocrine, and paracrine effects on epithelial and mesenchymal cells. The aims of the present studies were to localize and quantitate the expression of FGF-2 in normal and pathologic human kidneys and to study the in vitro effects of FGF-2 on proliferation, differentiation, and matrix production of isolated cortical kidney fibroblasts.


METHODS
FGF-2 protein expression was localized by immunofluoresence double labelings in normal and fibrotic human kidneys. Subsequently, interstitial FGF-2 labeling was determined semiquantitatively in 8 normal kidneys and 39 kidneys with variable degrees of interstitial fibrosis and was correlated with the morphometrically determined interstitial cortical volume. In addition, FGF-2 expression was quantitated by immunoblot analysis in three normal and six fibrotic kidneys. FGF-2 mRNA was localized by in situ hybridizations. Seven primary cortical fibroblast lines were established, and expression of FGF-2 and FGF receptor-1 (FGFR-1) were examined. The effects of FGF-2 on cell proliferation were determined by bromodeoxyuridine incorporation and cell counts, those on differentiation into myofibroblasts by staining for alpha-smooth muscle actin, and those on matrix synthesis by enzyme-linked immunosorbent assay for collagen type I and fibronectin. Finally, proliferative activity in vivo was evaluated by expression of MIB-1 (Ki-67 antigen).


RESULTS
In normal kidneys, FGF-2 expression was confined to glomerular, vascular, and a few tubular as well as interstitial fibroblast-like cells. The expression of FGF-2 protein was increased in human kidneys, with tubulointerstitial scarring correlating with the degree of interstitial fibrosis (r = 0.84, P < 0.01). Immunoblot analyses confirmed a significant increase in FGF-2 protein expression in kidneys with interstitial scarring. In situ hybridization studies demonstrated low-level detection of FGF-2 mRNA in normal kidneys. However, FGF-2 mRNA expression was robustly up-regulated in interstitial and tubular cells in end-stage kidneys, indicating that these cells are the source of excess FGF-2 protein. Primary cortical fibroblasts express FGF-2 and FGFR-1 in vitro. FGF-2 induced a robust growth response in these cells that could be blocked specifically by a neutralizing FGF-2 antibody. Interestingly, the addition of the neutralizing antibody alone did reduce basal proliferation up to 31.5%. In addition, FGF-2 induced expression of alpha-smooth muscle actin up to 1.6-fold, but no significant effect was observed on the synthesis of collagen type I and fibronectin. Finally, staining for MIB-1 revealed a good correlation of interstitial FGF-2 positivity with interstitial and tubular proliferative activity (r = 0.71, P < 0.01 for interstitial proliferation, N = 30).


CONCLUSIONS
Interstitial FGF-2 protein and mRNA expression correlate with interstitial scarring. FGF-2 is a strong mitogen for cortical kidney fibroblasts and may promote autocrine fibroblast growth. Expression of FGF-2 correlates with interstitial and tubular proliferation in vivo.
Strutz
F
F
Department of Nephrology and Rheumatology, Georg-August-University Göttingen, Germany. fstrutz@gwdg.de
Zeisberg
M
M
Hemmerlein
B
B
Sattler
B
B
Hummel
K
K
Becker
V
V
Müller
G A
GA
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D019898:Autocrine Communication; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000473:pathology*; D005355:Fibrosis / Q000378:metabolism; D006801:Humans; D019394:Ki-67 Antigen / Q000378:metabolism; D007668:Kidney / Q000378:metabolism* / Q000473:pathology*; D007672:Kidney Cortex / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008934:Mitogens / Q000494:pharmacology; D009130:Muscle, Smooth / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism
D019394:Ki-67 Antigen; D008934:Mitogens; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; C496353:FGFR2 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2