Intracellular Cyclic Nucleotide Analogues Inhibit in vitro Mitogenesis and Activation of Fibroblasts Derived from Obstructed Rat Kidneys

Abstract

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 µM 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 α-smooth muscle actin; α-SMA) and expression of CTGF (Northern blotting), a TGF-β₁-driven immediate early response gene. Serum-stimulated mitogenesis was decreased 27 ± 4% by 100 µM 8br-cAMP (p < 0.01), 49 ± 6% by 1,000 µM 8br-cAMP (p < 0.001) and 43 ± 7% by 1,000 µM 8br-cGMP (p < 0.01). 1,000 µM 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 α-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.

Long-term continuous sildenafil treatment ameliorates corporal veno-occlusive dysfunction (CVOD) induced by cavernosal nerve resection in rats

“…12,13,[24][25][26][27][28][29]32,33,37,38,50 This may occur via cGMP produced by the nitric oxide from iNOS, and/or the nitric oxide can directly reduce collagen synthesis, myofibroblast formation in the interstitial connective tissue, and reactive oxygen species. 26,[30][31][32] In contrast to our study with vardenafil in the BCNR rat, the slight stimulation of iNOS induction by sildenafil in the BCNR animals was not significant.…”

“…[24][25][26][27][28][29] The nitric oxide produced by iNOS, besides inhibiting collagen synthesis and the TGFb1 pathway, also quenches reactive oxygen species, and in some cases, the differentiation of fibroblasts to myofibroblasts, the cells that produce collagen in many fibrotic conditions. [30][31][32] In a recent study, we have shown that the PDE5 inhibitor, vardenafil, given for 45 days in the drinking water to rats subjected to bilateral cavernosal nerve resection (BCNR) [33][34][35] prevented the development of CVOD and the underlying SMC loss and fibrosis in the corpora cavernosa. 36 An antifibrotic effect by vardenafil and sildenafil was observed in the penile tunica albuginea in the rat model of Peyronie's disease, and in the case of sildenafil in the aged corpora smooth muscle.…”

Aldosterone Induces Kidney Fibroblast Proliferation via Activation of Growth Factor Receptors and PI3K/MAPK Signalling

“…These fibroblasts have stronger proliferative activity than those obtained from normal kidneys and more closely represent fibroblasts in diseased kidneys [14]. Dissected kidneys were incubated in collagen-coated wells in 10% FCS/DMEM/penicillin/streptomycin.…”

Anti-fibrotic effects of soluble guanylate cyclase stimulators and activators: A review of the preclinical evidence

“…The sGC stimulator BAY 41-2272 caused significant, dosedependent reductions in collagen I and fibronectin in TGF-b-stimulated human lung fibroblasts [45]. Exposure to the cGMP analog 8bromo-cGMP (1 mmol/L) caused a significant decrease in basal collagen synthesis as measured by hydroxyproline incorporation in rat renal interstitial fibroblasts [46]. As such, the sGC modulators as a class may hold potential to inhibit fibrosis by attenuating collagen production via suppression of TGF-b stimulation.…”