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.

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A variety of phenotypes of P. aeruginosa can be isolated from the sputum of cystic fibrosis (CF) patients colonised by this organism. Previous studies have described an association between some of these phenotypes and suggested that their emergence was due to conversion or selection by temperate phage. As a result I carried out, (a) a comparative survey of phenotypes in 200 CF and 100 non-CF cultures of P. aeruginosa and a statistical analysis of their association, (b) a study of the rates of mutation for the predominant phenotypes and their genetic linkage, (c) a study of the incidence and properties of temperate phages in CF and non-CF cultures and their association with strain phenotype. A wide range of phenotypes were found in CF and the most frequent were mucoid colony type (50%), atypical serotype (86%), serum sensitivity (74%) and extreme sensitivity to antibiotics and antimicrobial agents, especially to carbenicillin (20%), Irgasan (25%), nalidixic acid (20%), EDTA (20%), cetrimide (10%) and trimethoprim (20%). In contrast, less than 1% of non-CF isolates exhibited these characteristics. Statistical analysis showed that with the exception of antibiotic hypersensitivity, the phenotypes of CF cultures were generally unrelated. Mutation rates determined for the predominant CF phenotypes showed that they were strain dependent and varied between each distinct phenotype. Although some evidence was found for linkage between some of the phenotypes, it was also strain related and the majority of traits varied independently of each other. The incidence of temperate phage was similar in CF and non-CF cultures. Furthermore, the lysogeny state did not influence strain phenotype. P. aeruginosa temperate phages isolated from both collections were similar in host range and serological properties, but were distinct from the typing phages. The selection and examination of the features of phage resistant variants by the use of virulent and temperate phages showed that some cultures exhibited characteristics similar to those isolated from CF. No evidence was found for the role of phage in promoting phenotype change in CF.

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