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.
In order for the bladder to store urine it must be compliant (pliable). It is imperative that it can hold urine at low pressures. Failure of this results in elevated bladder pressure, transmitting urine to the kidney resulting in glomerular injury, renal parenchymal fibrosis and failure. Deposition of extracellular matrix (ECM) within the bladder wall is the main reason for loss of bladder wall pliability. What leads to excess ECM deposition and resultant fibrosis remains unclear, but accumulated evidence suggests that inflammatory cascades play a significant role. In response to inflammatory insult, bladder fibrosis occurs as part of a wound healing process and the accumulation of ECM proteins (collagen types I and III) [
We have previously published a novel murine model of inflammatory bladder disease using the human cathelicidin LL-37 peptide to induce physiologic bladder inflammation [
To our knowledge, the RAGE pathway has not been described or implicated in inflammatory diseases of the bladder. RAGE is primarily involved in homeostasis and inflammation and is one of the primary receptors for high mobility group box 1 (HMGB1) [
In the lower urinary tract, HMGB1 is ubiquitously expressed in urothelial cells and extracellularly liberated into urine [
In this study we propose that LL-37 induced bladder inflammation involves RAGE, a common RAGE ligand HMGB1, and ultimately converges on NF-κB signaling. We investigated whether activation of these pathways in the bladder could be responsible for the sustained, proinflammatory phenotype.
Experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the University of Utah. LL-37 induced bladder inflammation was performed as previously described [8,40]. Briefly, 8 to 12-week old female C57BL/6 mice were catheterized, bladders emptied, then washed with 150 µL of 0.9% sodium chloride and emptied. After washing, 320 µM of LL-37 or saline (controls) were infused slowly and left indwelling for 1 hour. Tissues were harvested at either 12 or 24 hours and the bladders were split longitudinally. One section was fixed in 4% paraformaldehyde for histology and the other was either snap frozen in liquid nitrogen then stored at −80˚C for protein extraction, or placed in RNAlater (QIAGEN, Germantown, MD, USA) overnight at 4˚C then proceeded to RNA isolation or stored at −80˚C for future use.
5 µm sections were deparaffinized then rehydrated through xylene and graded alcohols. Endogenous peroxidase activity was blocked with 1% Hydrogen peroxide in TBST for 20’ and washed 3X in TBST for 3’. Antigen retrieval was performed (Vector laboratories, Burlingame, CA., Lot# V0421). To minimize non-specific antibody binding, sections were incubated for 60’ in 5% FBS in TBS with 0.3% Triton X-100. Sections were incubated overnight at 4˚C with primary antibody (Goat-Anti Mouse RAGE 1:200 in blocking solution, R&D, Minneapolis, MN, Lot# AF1179; or Rabbit-Anti Mouse HMGB1, 1:1000 in blocking solution, Abcam, Cambridge, MA, Lot# ab18256). Following O/N incubation, slides were washed 3X in TBST for 3’. Sections were then incubated for 60’ with biotinylated secondary antibody 1:2000, followed by Vectastatin Elite ABC Reagent (Vector Laboratories, Lot# PK-6100) diluted in TBST for 30’. Between incubations, sections were washed 3X for 3’ in TBST. For visualization of immunoreactivity sections were incubated in DAB peroxidase substrate for 20 - 40 sec. Sections were washed in ddH2O, counterstained, and dehydrated. Negative controls included incubation with TBST in place of the primary antibody and no immunoreactivity was observed.
Bladder tissues stored at −80˚C were homogenized in ice-cold RIPA lysis buffer (Thermo Scientific, Rockford, IL) with the addition of protease inhibitor (Sigma Aldrich) and phosphatase inhibitor (Thermo Scientific, Rockford, IL) using a Mini-Bead Beater homogenizer (BioSpec Products, Inc. Bartlesville, OK, USA). Isolated total protein was quantified using the BCA method (PIERCE, Rockford, IL). Equal amounts of protein were electrophoresed on a 4% - 12% SDS-polyacrylamide gel (InVitrogen) and subsequently transferred to a nitrocellulose membrane (0.45 m, InVitrogen). The membrane was blocked in washing solution (0.1% Tween 20 in PBS) containing 5% nonfat dry milk. The same primary antibody utilized for RAGE IHC was utilized for the Western Blot experiments. Secondary antibodies were incubated at room temperature for 1 hour in blocking solution. Proteins were visualized via ECLplus chemiluminescence reagents (GE healthcare life sciences, Pittsburgh, PA).
Tissues were homogenized on ice with a hand homogenizor, then total RNA was purified according to manufacturer’s instructions (Qiagen RNeasy fibrous mini kit, Germantown, MD, USA). cDNA synthesis was performed following manufacturer instructions (ABI, Foster City, CA). Primer’s for all genes of interest (RAGE, TNFα, IL-6, IL-1β, GM-CSF, COX-2) were synthesized and purchased from Applied Biosystems (Foster City, CA). Gene expression was quantified using the Taqman Gene Expression Assay (ABI, Foster City, CA) on an Applied Biosystems 7900 HT instrument. The data sets were analyzed with normalization, variance stabilization, and log2 transformation. The 2−∆∆Ct method was utilized in the analysis for differences in relative gene expression [
To establish if baseline RAGE expression was present in normal bladders and to test for potential changes in RAGE expression after LL-37 challenge, both saline control and those exposed to LL-37 were subjected to RAGE IHC. Saline instilled control bladders harvested after 12 and 24 hours are demonstrated in Figures 1A and B (U—urothelium, SBM—submucosa, LP—lamina propria, SMC—smooth muscle cell layer).
LL-37 instilled bladders harvested after 12 and 24 hours are demonstrated in Figures 1C and D. In saline controls, strong RAGE immunoreactivity (brown stain)
was observed along the umbrella cells, with moderate intensity within the cytoplasmic regions in the urothelial (U) layer. No RAGE signal was observed in the SBM layer and LP, except for the arteriolar vessels had a moderate signal (rectangle). RAGE was detectable in the SMC layer. In LL-37 instilled bladders, similar RAGE expression patterns were observed compared to saline control tissues except for a qualitative loss of RAGE signal along superficial umbrella cells, but increased RAGE detection in the endothelium lining venules (
To help understand the impact of HMGB1 on LL-37 induced bladder inflammation, IHC was employed to determine HMGB1 expression patterns in both saline control versus LL-37 challenged bladders (
smooth muscle (Figures 2A and B). Analysis of the LL-37 instilled bladders showed similar ubiquitous HMGB1 immunoreactivity to saline control tissues, except for positive detection in all acute inflammatory cells (primarily PMNs) within the inflamed tissues, along with a qualitative increased staining pattern in the smooth muscle cell layer (Figures 2C and D).
In order to quantitate RAGE expression, both qRT-PCR and Western Blot methods were employed. In the qRTPCR experiments, total RNA was isolated from both saline control and LL-37 inflamed bladders. Tissue and RNA isolation was performed 24 hours after either saline or LL-37 exposure. LL-37 inflamed tissues demonstrated a near 10-fold reduction in RAGE RNA levels (
To quantitate the amount of RAGE protein in LL-37 challenged bladders, Western Blot analysis was performed (
qRT-PCR results were further validated with decreased RAGE protein expression from our Western Blot experiments.
HMGB1 signaling via RAGE can lead to the activation of NF-κB. Therefore, we wanted to investigate five common downstream genes activated by NF-κB [18-24]. We hypothesized that LL-37 challenged tissues would yield up-regulation of all five genes (TNFα, IL-6, IL-1β, GM-CSF, COX-2) when compared to saline controls. To test this hypothesis, we utilized qRT-PCR from mRNA isolated in 12 & 24 h tissues, for both LL-37 challenged bladders and saline controls.
Using our previously established model, we created bladder inflammation using the cathelicidin LL-37 [8,40], which has been found to be elevated in urine samples obtained from pediatric patients with either cystitis or pyelonephritis [
Unexpectedly, a reduction in RAGE expression was noted in inflamed tissues compared to controls. Although, IHC for the RAGE ligand HMGB1 demonstrated moderate differences between control and LL-37 inflamed tissues. We mainly observed increased expression patterns in the acute inflammatory cells and smooth muscle cells within the inflamed samples. Despite diminished
RAGE expression, the moderate qualitative increase in HMGB1 expression seen in the inflamed tissues would suggest alternative receptor pathways are involved in the profound activation of NF-κB dependent genes. Again, four of five common downstream genes activated by NF-κB were substantially up-regulated within our LL-37 challenged tissues.
It has been well described that Toll-like receptors (TLRs), specifically TLR2 and TLR4, are involved in HMGB1 signaling [43-45]. HMGB1 can act as a ligand and bind not only the RAGE receptor [
We have innovatively employed our reproducible LL-37 induced cystitis model to gain further insight towards a possible mechanistic pathway involved in bladder inflammation. This work opens the door for future studies involving the inflammatory ligand HMGB1, RAGE, and the corresponding receptor signaling pathways that implicate NF-κB.
Portions of this work have been supported in part by the following Grants: NIH SBIR 1R43DK093413 (SO, GDP, JZ) from the National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK); NIH K12 Career Development Award in Children’s Health Research UL1RR025764 (SO) from the University of Utah Center for Clinical and Translational Sciences (CCTS) and National Center for Advancing Translational Sciences (NCATS), American Urologic Association (AUA)/Pfizer Benign Urologic Diseases Award (SO), Primary Children’s Medical Center Foundation Integrated Science Award (SO, GDP), Primary Children’s Medical Center Foundation Early Career Development Award (SO).