Inflammatory macrophages and renal tubular epthelial cell apoptosis

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 evidentI 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.