To elucidate pathways in bladder inflammation, we
employed our physiologically relevant LL-37 induced cystitis model. Based on
inflammatory studies involving other organ systems implicating the receptor
for advanced glycation end-products (RAGE), we first hypothesized that RAGE is
critically involved in LL-37 induced cystitis. We further hypothesized that a common
RAGE ligand high mobility group box 1 (HMGB1) is up-regulated in bladders
challenged with LL-37. Finally, we hypothesized that NF-κB dependent inflammatory
genes are activated in LL-37 induced cystitis. Testing our first hypothesis,
C57Bl/6 mice were challenged with either saline (control) or 320 μM of LL-37
intravesically for 1 hr. After 12 or 24 hours, tissues were examined with
immunohistochemistry (IHC) for RAGE, and both mRNA and protein isolation for
respective qRT-PCR and Western Blot analysis. Our second hypothesis was tested
by employing HMGB1 IHC. Testing our final hypothesis, qRT-PCR was performed investigating
five genes: TNFα, IL-6, IL-1β, GM-CSF, COX-2. In control and LL-37 challenged
tissues, IHC for RAGE revealed similar qualitative expression. Evaluation with
qRT-PCR and Western Blot for RAGE revealed diminished expression at the mRNA
and protein level within LL-37 challenged bladders. IHC for HMGB1 revealed a
moderate qualitative increase within LL-37 challenged tissues. Finally, with
the exception of TNFα, all NF-κB dependent inflammatory genes yielded
substantial up-regulation. We have employed our LL-37 induced cystitis model to
gain insight to wards a possible mechanistic pathway involved in bladder
inflammation. This work provides data for future studies involving the
inflammatory ligand HMGB1, RAGE, and receptor pathways that activate NF-κB.