Intracellular signalling pathways relay information from the extracellular environment to the nucleus. These pathways are important determinants of cellular responses, especially during renal fibrosis where fibrogenic stresses activate the pathways and disparately affect survival, proliferation and differentiation of intrinsic and inflammatory renal cell populations, and the synthesis of extracellular matrix (ECM) proteins. The biomolecular pathways involved in renal fibrosis are not fully understood. The present thesis tested the hypothesis that fibrogenic stresses activate renal cell specific survival, death and differentiation responses through activation of the mitogen activated protein kinase (MAPK) pathways, in particular the extracellular signal regulated kinase (ERK) and c-jun amino (N) terminal kinase (JNK) pathways, or altemative signalling pathways. In vitro and in vivo models of renal fibrosis were
used to test this hypothesis.
In vitro: The relative outcomes (apoptosis, cell proliferation, differentiation, production of ECM) were analysed in three disparate renal cell types (tubular epithelial, vascular endothelial and fibroblast) after fibrogenic challenge with tumour necrosis factor-α (TNFα) (50ng/ml), transforming growth factor-β (TGFβ1) (l0ng/ml) or oxidative stress (ImM hydrogen peroxide). Cellular outcome was compared with activation or inhibition of ERK or JNK TNFα induction of apoptosis was limited to endothelial cells with no corresponding activation of ERK or JNK. TGFβ1 did not induce any significant changes in cell death or proliferation for any of the cell types, nor did it alter ERK or JNK activation. Apoptosis was the predominant biological outcome after oxidative stress, which was associated with a marked increase in ERK activation in all cell
populations (p<0.05). Specific inhibition of ERK by PD98059 indicated a pro-apoptotic role for ERK in oxidative stress-induced fibroblast apoptosis, however ERK was important for maintaining epithelial and endothelial cell survival. Transient JNK activation was evident only in epithelial and endothelial cells within the first five hours after oxidative stress. Specific inhibition of JNK by SP600125 supported a role for sustained JNK activation in the epithelial cell death pathway.
In vivo: Unilateral ureteral obstruction (UUO) induction of renal fibrosis in rats was used to examine the relationship between ERK, apoptosis, proliferation and differentiation in renal cells, in comparison with the in vitro results. UUO induction (6 hrs, 1-7 days) caused the progressive development of fibrosis in terms of myofibroblast activation, ECM accumulation, oxidative stress and tubular atrophy. Tubular
epithelial and interstitial mitosis and apoptosis were significantly increased after 2-7 days of UUO (p<0.01), which was accompanied by increases in ERK activation at 1-7 days of UUO (3-5 fold, p<0.05). Activated ERK localized to the tubular epithelium and, occasionally, to cells of the interstitium. A significant spatiotemporal correlation was found between activated ERK and tubular epithelial proliferation rather than apoptosis. Apoptotic interstitial cells were found to express ERK, however viable interstitial cells of the myofibroblast phenotype, identified by expression of a-smooth muscle actin, exhibited no patterns of activated ERK in UUO animals.
Alternative signalling pathways: Microarray technology was used to identify two genes with altered expression after oxidative stress. JunD was upregulated in renal epithelial cells but not fibroblasts. Janus kinase-3 (JAK3) was downregulated in both
epithelial cells and fibroblasts. There were some discrepancies between transcriptional and translational methods of expression analysis. Immunohistochemical analysis of JAK3 in the in vivo UUO model indicated that its distribution altered with progressive fibrosis and it localised with proliferating tubular epithelial cells. Double staining for activated ERK and JAK3 excluded the possibility of co-stimulatory pathways promoting tubular epithelial proliferation during tubulo-interstitial fibrosis.
Summary: There are renal cell specific differences in activation of the MAPK signal transduction pathways following fibrotic insult. Both in vitro and in vivo models suggest that ERK activation initiates the apoptotic death pathway in fibroblasts. The absence of this activation in vivo may allow their differentiation to myofibroblasts and their survival
during fibrosis. In epithelial and endothelial cells, ERK transduces either proliferative or survival signals. The ERK pathway may act as a dual regulator of survival and death in a renal cell-type dependent manner in the tubulo-interstitium during fibrosis, and it may prove to be a target for limiting the myofibroblast population without affecting detrimentally the survival patterns of intrinsic tubular epithelial and endothelial cells. Alternative signalling molecules such as JAK3 may be useful MAPK-independent targets for protecting renal tubular epithelium from atrophy associated with renal fibrosis.