Intracellular cyclic nucleotide analogues inhibit in vitro mitogenesis and activation of fibroblasts derived from obstructed rat kidneys.
As several studies indirectly suggest that inhibiting the intracellular breakdown of cyclic nucleotides may inhibit fibrogenesis, this study used membrane permeable cyclic nucleotide analogues to examine the role of cAMP and cGMP signaling pathways in the regulation of renal fibroblast function. Fibroblasts were isolated by explant outgrowth culture of rat kidneys post unilateral ureteric obstruction. Subcultured cells were exposed to 10- 1,000 microM of the cyclic nucleotide analogues 8-bromo-cAMP (8br-cAMP) and 8-bromo-cGMP (8br-cGMP). Functional parameters examined included mitogenesis (thymidine incorporation), collagen synthesis (proline incorporation), myofibroblast differentiation (Western blotting for alpha-smooth muscle actin; alpha-SMA) and expression of CTGF (Northern blotting), a TGF-beta(1)-driven immediate early response gene. Serum-stimulated mitogenesis was decreased 27 +/- 4% by 100 microM 8br-cAMP (p < 0.01), 49 +/- 6% by 1,000 microM 8br-cAMP (p < 0.001) and 43 +/- 7% by 1,000 microM 8br-cGMP (p < 0.01). 1,000 microM 8br-cAMP and 8br-cGMP reduced basal collagen synthesis by 80 +/- 5 and 60 +/- 21% respectively (both p < 0.05). Maximum dose of 8br-cAMP but not 8br-cGMP inhibited basal expression of the differentiation marker alpha-SMA by 43 +/- 33 (p < 0.05), resulted in a more rounded cell morphology and reduced expression of CTGF by 39 +/- 24% (p < 0.05). Measurement of mitochondrial activity confirmed that effects were independent of cell toxicity. In conclusion, cyclic nucleotides inhibit fibrogenesis in vitro. Strategies which elevate intracellular cyclic nucleotide concentrations may therefore be therapeutically valuable in preventing the proliferation and activation of fibroblasts in progressive renal disease.
Hewitson
T D
TD
Department of Nephrology, The Royal Melbourne Hospital, Melbourne, Vic., Australia. Tim.Hewitson@mh.org.au
Martic
M
M
Darby
I A
IA
Kelynack
K J
KJ
Bisucci
T
T
Tait
M G
MG
Becker
G J
GJ
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology / Q000633:toxicity; D000199:Actins / Q000096:biosynthesis; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D055513:Connective Tissue Growth Factor; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005355:Fibrosis; D017874:Immediate-Early Proteins / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D007668:Kidney / Q000166:cytology* / Q000473:pathology*; D009712:Nucleotides, Cyclic / Q000494:pharmacology*; D051381:Rats; D014517:Ureteral Obstruction / Q000473:pathology
D000199:Actins; C526316:CCN2 protein, rat; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D009712:Nucleotides, Cyclic; D055513:Connective Tissue Growth Factor; D015124:8-Bromo Cyclic Adenosine Monophosphate; C016276:8-bromocyclic GMP; D003094:Collagen; D004247:DNA; D000242:Cyclic AMP; D006152:Cyclic GMP