In vivo and ex vivo assessment of bladder hyper-permeability and using molecular targeted magnetic resonance imaging to detect claudin-2 in a mouse model for interstitial cystitis

Abstract

Objectives To determine if the URO-MCP-1 mouse model for bladder IC/BPS is associated with in vivo bladder hyper-permeability, as measured by contrast-enhanced MRI (CE-MRI), and assess whether molecular-targeted MRI (mt-MRI) can visualize in vivo claudin-2 expression as a result of bladder hyper-permeability. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects primarily women. It is known that permeability plays a substantial role in IC/BPS. Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. Claudin-2 is a molecular marker that is associated with increased hyperpermeability in the urothelium. Materials and methods CE-MRI was used to measure bladder hyper-permeability in the URO-MCP-1 mice. A claudin-2-specific mt-MRI probe was used to assess in vivo levels of claudin-2. The mt-MRI probe consists of an antibody against claudin-2 conjugated to albumin that had Gd-DTPA (gadolinium diethylenetriamine pentaacetate) and biotin attached. Verification of the presence of the mt-MRI probe was done by targeting the biotin moiety for the probe with streptavidin-horse radish peroxidase (SA-HRP). Trans-epithelial electrical resistance (TEER) was also used to assess bladder permeability. Results The URO-MCP-1 mouse model for IC/BPS was found to have a significant increase in bladder permeability, following liposaccharide (LPS) exposure, compared to saline-treated controls. mt-MRI- and histologically-detectable levels of the claudin-2 probe were found to increase with LPS -induced bladder urothelial hyper-permeability in the URO-MCP-1 IC mouse model. Levels of protein expression for claudin-2 were confirmed with immunohistochemistry and immunofluorescence imaging. Claudin-2 was also found to highly co-localize with zonula occlidens-1 (ZO-1), a tight junction protein. Conclusion The combination of CE-MRI and TEER approaches were able to demonstrate hyper-permeability, a known feature associated with some IC/BPS patients, in the LPS-exposed URO-MCP-1 mouse model. This MRI approach could be clinically translated to establish which IC/BPS patients have bladder hyper-permeability and help determine therapeutic options. In addition, the in vivo molecular-targeted imaging approach can provide invaluable information to enhance our understanding associated with bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing further therapeutic strategies.

SuperGAG biopolymers for treatment of excessive bladder permeability

“…Such studies could be performed using MRI and a Gd-labeled SuperGAG. 47,48 First in human clinical trials will then assess optimal dosing frequency in humans using well validated patient-reported outcomes measures (e.g., O'Leary-Sant questionnaires) 49 and bladder MRI permeability measures that can determine for how long SuperGAG provides protection.…”

“…Planned future research to take this concept to the clinic will include further optimization of SuperGAG biopolymer compositions to enhance binding to the urothelium to enhance duration of action, additional animal studies to assess improved efficacy and optimize dosing frequency to maintain impermeability in animals. Such studies could be performed using MRI and a Gd‐labeled SuperGAG 47,48 . First in human clinical trials will then assess optimal dosing frequency in humans using well validated patient‐reported outcomes measures (e.g., O’Leary‐Sant questionnaires) 49 and bladder MRI permeability measures that can determine for how long SuperGAG provides protection.…”

MRI as a Tool to Assess Interstitial Cystitis Associated Bladder and Brain Pathologies

“…CE-MRI of BWH could therefore also be applied to assess colon–bladder crosstalk in inflammatory bowel disease (IBD) patients, as well as other inflammatory colon diseases. Additionally, we were able to demonstrate an increase in MRI SIs in the regions surrounding the dome of the bladder wall in a transgenic URO-MCP-1 mouse model for IC/BPS following intravesical lipopolysaccharide (LPS) exposure (see Figure 1 D–F where there was a significant increase in the % difference in MRI SI in the LPS group ( p < 0.001) due to leakage of Gd-DTPA) [ 39 ]. A similar CE-MRI approach to assess urothelial regeneration in rat bladders, which were augmented with permeable porcine small intestinal submucosa, was also used [ 40 ], indicating that this methodology can be expanded to other bladder conditions.…”

“…Following the preclinical rat PS-induced BWH study, we then conducted a human pilot study comparing IC/BPS patients with normal individuals and clearly demonstrated the clinical feasibility of detecting BWH in IC/BPS [ 41 ]. Due to the increased thickness of the human bladder wall, BWH was detected within the bladder wall (see Figure 1 G–I where there was a significant increase in % difference in MRI SI for the IC bladders ( p < 0.001) compared to controls) [ 41 ] rather than the outside regions as seen in the preclinical models [ 28 , 38 , 39 ]. Another group used a mixture of both Gd-DTPA (Gadavist) and an iron oxide contrast agent, ferumoxytol, to further enhance bladder wall contrast in rats with induced cystitis (PS-induced) [ 42 ] and more recently in patients with IC [ 43 ].…”

“…PA-biotin) were used by our group to assess BWH biomarkers, such as decorin, vascular endothelial growth factor receptor 1 (VEGFR1) claudin-2 in a rat PS-induced BWH model (see Figure 3 A–C where the claudin expression is significantly increased in the PS group ( p < 0.05)) [ 46 ], and claudin-2 in the transgenic URO-MCP-1 mouse model for IC/BPS (see Figure 3 D–F where the claudin expression is significantly increased in the LPS group ( p < 0.05)) [ 39 ]. Contrast difference images indicate either the increased levels of claudin-2 [ 39 , 46 ] or a decrease in the levels of either decorin or VEGFR1 [ 46 ]. Decreased protein expression for decorin was previously found in feline interstitial cystitis bladders [ 18 ].…”