Pirfenidone reduces in vitro rat renal fibroblast activation and mitogenesis.
BACKGROUND
Fibroblasts have been universally recognised in tubulointerstitial injury, where their presence has been shown to be a marker of disease progression. Recently, pirfenidone (PF) has been shown to both ameliorate progressive fibrosis and reduce established scarring after ureteric obstruction (UUO) in the rat, suggesting that it is a novel anti-fibrotic agent. The objective of this study was therefore to determine if these effects include down-regulation of fibroblast function.


METHODS
Cortical fibroblasts were obtained from outgrowth cultures of renal tissue isolated from kidneys 3 days after UUO and constituted 100% of cells studied. Functional studies examined the effects of 20 and 200 microg/ml PF on basal serum stimulated activity. Activation was examined by western blotting for alpha smooth muscle actin (alphaSMA) and connective tissue growth factor (CTGF). Cell proliferation, collagenase activity and collagen production were determined from kinetic studies, zymography for MMP2 and [3H] proline incorporation in collagenous proteins respectively.


RESULTS
Proliferation, as measured by [3H] thymidine incorporation, was reduced in dose dependent manner by 20 and 200 microg/ml PF (p<0.05; 200 vs 0 microg/ml). Likewise, 200 microg/ml PF reduced cell population growth over 5 days of culture (p<0.05 vs 0 microg/ml). PF (200 microg/ml) decreased alphaSMA and CTGF protein expression to 66+/-13 and 37+/-26% of basal levels respectively (both p<0.05 vs 0 microg/ml). Synthesis of collagen was unaffected by PF. Maximal dose of PF produced a modest reduction in MMP2 lytic activity (p=0.05). Effects of PF were independent of cell toxicity.


CONCLUSIONS
Down-regulation of renal fibroblast activation and proliferation are specific actions of PF.
Hewitson
T D
TD
Department of Nephrology, The Royal Melbourne Hospital, Australia. tim.hewitson@mh.org.au
Kelynack
K J
KJ
Tait
M G
MG
Martic
M
M
Jones
C L
CL
Margolin
S B
SB
Becker
G J
GJ
D000199:Actins / Q000037:antagonists & inhibitors; D000818:Animals; D015415:Biomarkers; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D055513:Connective Tissue Growth Factor; D005347:Fibroblasts / Q000187:drug effects* / Q000502:physiology*; D006133:Growth Substances; D017874:Immediate-Early Proteins / Q000037:antagonists & inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D007668:Kidney / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D008297:Male; D061965:Matrix Metalloproteinase Inhibitors; D008938:Mitosis / Q000187:drug effects*; D009130:Muscle, Smooth / Q000378:metabolism; D011728:Pyridones / Q000494:pharmacology* / Q000506:poisoning; D051381:Rats; D017207:Rats, Sprague-Dawley
D000199:Actins; D015415:Biomarkers; C526316:CCN2 protein, rat; D006133:Growth Substances; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D061965:Matrix Metalloproteinase Inhibitors; D011728:Pyridones; D055513:Connective Tissue Growth Factor; D003094:Collagen; C093844:pirfenidone