Abstract
Macrophages (Mφ) play a key role in renal inflammation and may be
cytotoxic to resident cells within tissues. I began this thesis by examining the effect
of Mφ upon the level of apoptosis and proliferation in tubular epithelial cells in vitro.
I used a microscopically quantifiable co-culture assay to determine whether
inflammatory Mφ modulated apoptosis and proliferation of (i) Madine-Darby canine
kidney (MDCK) cells and (ii) primary murine tubular epithelial cells. Bone marrow derived Mφ and target tubular cells were differentially labeled with fluorochromes
and interacted for 24 hrs. Apoptosis and proliferation was quantified by fluorescent
microscopy after fixation and Hoechst staining. Quiescent non-activated Mφ did not
induce significant apoptosis of tubular cells whilst cytokine activated (LPS/IFN-γ)
Mφ induced marked apoptosis of both MDKC and primary tubular cells.
Pharmacological inhibition of nitric oxide (NO) production with L-NAME or
inclusion of cytokine activated Mcj) derived from inducible nitric oxide synthase
(iNOS) knockout (KO) mice reduced apoptosis in both MDKC and primary tubular
cells. MDCK cell proliferation was significantly suppressed by both non-activated
and activated Mφ from iNOS WT and iNOS KO mice indicating that it was an NOindependent effect. In contrast, no effect upon primary tubular cell proliferation was
evident
I then went on to examine the role of NO in vivo in the murine model of
unilateral ureteric obstruction (UUO) characterised by tubular cell apoptosis and
interstitial fibrosis. Initially UUO was performed in iNOS KO and wild-type mice
but iNOS KO mice exhibited significantly increased Mφ infiltration. Therefore, the
specific iNOS inhibitor L-NIL (control D-NIL) was administered between days 5 to
7 following UUO. Mice were sacrificed at day 7 and the obstructed kidney removed
for histological analysis. L-NIL treatment did not affect Mφ infiltration but did
reduce both tubular and interstitial cell apoptosis. Proliferation of tubular cells and
interstitial cells was unaffected. Although myofibroblast accumulation was
unaffected, interstitial fibrosis was significantly increased by L-NIL treatment.
I then went on to examine the role of NO in vivo in the murine model of
unilateral ureteric obstruction (UUO) characterised by tubular cell apoptosis and
interstitial fibrosis. Initially UUO was performed in iNOS KO and wild-type mice
but iNOS KO mice exhibited significantly increased Mφ infiltration. Therefore, the
specific iNOS inhibitor L-NIL (control D-NIL) was administered between days 5 to
7 following UUO. Mice were sacrificed at day 7 and the obstructed kidney removed
for histological analysis. L-NIL treatment did not affect Mφ infiltration but did
reduce both tubular and interstitial cell apoptosis. Proliferation of tubular cells and
interstitial cells was unaffected. Although myofibroblast accumulation was
unaffected, interstitial fibrosis was significantly increased by L-NIL treatment.
