Myofibroblast loss during renal remodelling
Vernon, Madeleine Anne
Renal fibrosis, the final endpoint of renal disease of any cause, is characterised by myofibroblast deposition of extracellular matrix (ECM) and commonly studied using the unilateral ureteric obstruction (UUO) model. Macrophages are multifunctional cells and involved in renal injury, repair and scarring. Work in other organs has shown that fibrosis is not necessarily irreversible and we established and characterised the murine model of reversible unilateral ureteric obstruction (R-UUO) to investigate the potential reversibility of fibrosis and the underlying mechanism with a particular focus upon the role of macrophages. Reversal of UUO was performed at day 7 and R-UUO kidneys exhibited rapid and profound loss of α-smooth muscle actin (αSMA) positive myofibroblasts over the subsequent 7 days. Loss of αSMA+ myofibroblasts was accompanied by limited and variable degradation of ECM components including collagens I and III. αSMA/TUNEL double staining suggested that some myofibroblasts underwent apoptosis. Infiltrating macrophages were abundant at D7 UUO and persisted at all time points following reversal, however, there was a reduction in the F4/80+ population at D7 Reversal by flow cytometry. Of the F4/80+ population two distinct subpopulations could be identified, F4/80Hi and F4/80Lo cells. The relative contribution of these macrophage populations to the total renal macrophage pool did not alter in obstruction or reversal. The F4/80Hi population was characterised by increased expression of CD11c and decreased expression of CD11b and Ly6C (F4/80HiCD11cHiCD11bLoLy6CLo), whereas the F4/80Lo population was characterised by increased expression of CD11b and Ly6C and decreased expression of CD11c (F4/80LoCD11cLoCD11bHiLy6CHi). CD11b was decreased in both F4/80+ populations during reversal, with altered Ly6C profiles, indicating that the phenotype of the macrophages in each population is different and may change during reversal. Indeed, macrophages isolated by flow cytometry utilising anti-F4/80-APC conjugated antibodies had altered mRNA profiles with D7 reversal associated with decreased mRNA expression of mannose receptor and TGFβ. Previous work in the liver indicates that macrophages may promote or inhibit tissue scarring. In order to ascertain whether macrophages were involved in the loss of αSMA+ myofibroblasts we depleted macrophages after reversal by either administering diphtheria toxin to transgenic CD11b-DTR mice or antagonising colony stimulating factor-1 (CSF-1), a key macrophage mitogen and growth factor, by administering antibodies to the CSF-1 receptor. Both approaches significantly depleted macrophage infiltration but did not retard the loss of αSMA+ myofibroblasts indicating that myofibroblast loss was macrophage independent. Lastly, we investigated the potential role of tissue stiffness in myofibroblast loss following UUO reversal. Primary renal myofibroblasts were cultured from obstructed kidneys, carefully phenotyped and cultured on matrices of differing stiffness. The susceptibility of myofibroblasts to apoptosis increased as the matrix stiffness fell. These data suggest that the altered mechanical microenvironment of the decompressed kidney may be a key stimulus for the macrophage independent loss of myofibroblasts that follows the reversal of UUO.