Hypothesis / aims of study
Painful Bladder Syndrome / Interstitial Cystitis (PBS/IC) is a disease of the urinary bladder that causes increased urgency to void and pelvic pain. It is thought that increased bladder sensations in PBS/IC are due to sensitization of bladder afferent nerves by inflammatory mediators. Current therapies aimed at known targets in the inflammatory or nociceptive pathways have, to date, been ineffective at reducing the symptoms in PBS/IC patients. To find potential new treatments for PBS/IC, we have attempted to express a non-native chloride channel (named EG3RF) in bladder afferent neurons. The EG3RF channel was created by fusing the transmembrane domain of the human glycine α subunit with the extracellular domain of the prokaryotic ELIC channel. Pharmacologically, this channel can be activated by either decreased extracellular pH caused by inflammation or by application of biogenic amines (e.g., cysteamine or propylamine) but has no activity under normal conditions. The aim of our study was to show that EG3RF, expressed in bladder afferent nerves through intravesical transfection, would be activated by inflammation in an animal model of PBS/IC (intraperitoneal cyclophosphamide), preventing their sensitization and thus eliminating the bladder overactivity normally exhibited in the model. During the course of the study, it also became apparent that EG3RF transfection directly reduced the inflammation induced by cyclophosphamide treatment. Our study is the first to demonstrate a potential gene therapy treatment for PBS/IC that reverses both bladder overactivity and bladder inflammation.
Study design, materials and methods
Prior to in vivo transfection, a plasmid encoding the EG3RF sequence was encapsulated in cationic liposomes at a ratio of 10:1 (lipid to plasmid), with a final concentration of plasmid of 200nM. For transfection, female Sprague Dawley rats (~200-250g) were anesthetized using isoflurane (2-4% in oxygen) and catheterized transurethrally using a 20g angiocatheter. The bladder was then instilled with 0.5 ml of a 0.1% solution of n-Dodecyl-β-D-maltoside in a Krebs buffer solution for 1 minute to disrupt the urothelial barrier. After washing twice with Krebs solution, 0.5 ml of the liposome/plasmid solution was instilled into the bladder and left for 1 hour. The animals were then allowed to recover from anesthesia and returned to their cage for 1 week to allow the channel to be expressed. At the end of the week, bladder inflammation was induced using an intraperitoneal injection of cyclophosphamide (CYP, 150mg/kg), followed by 24 hours of metabolic cage recordings. Urethane anesthetized bladder cystometry was performed after the metabolic cage studies. Successful transfection of tissue was confirmed by collection of the bladder smooth muscle, urothelium and L6-S1 dorsal root ganglia at the end of cystometry for extraction of mRNA for PCR. RT-PCR was performed using primers designed to amplify the ELIC portion of EG3RF’s sequence to prevent false positives from any homology of the glycine portion to the rat ortholog.
To quantify bladder edema in response to CYP treatment in non-transfected and EG3RF transfected animals, a subset of the animals above had a catheter (PE 50 intramedic tubing) implanted into the jugular vein during preparations for cystometry. At the end of cystometry, Evans Blue dye (50mg/kg) was injected through the catheter. After 15 minutes, rats were sacrificed by decapitation, exsanguinated and the bladder removed. After weighing, the bladder was placed in 3 ml formamide for 72 hours. The dye present in the formamide solution was quantified by measuring optical density and then the concentration was estimated using a standard curve. All readings were normalized by bladder weight.
The cell-based portion of our study used immortalized normal human urothelial cells (TRT-HU1) at passage number 25-35. Cells were plated on either cell culture treated 96-well plates for ATP measurements or collagen coated glass coverslips for calcium imaging. Cells were used for experiments after 2-3 days when they had reached 50-70% confluency. To transfect cultured cells, the gene encoding EG3RF was incorporated into a BacMam viral delivery system and incubated with the cells 24 hours after plating. To measure extracellular ATP concentrations, a luciferin/luciferase assay was performed. Lysosomal ATP release was stimulated in transfected and non-transfected cells with chloroquine (100µM). Lysosomal calcium release was examined using Fura-2 based calcium imaging. Lysosomal calcium release was stimulated using the TRPML1 agonist ML-SA1 (100µM). Activation of EG3RF was accomplished using cysteamine (1mM).
Results
PCR amplification confirmed that intravesical transfection of the EG3RF plasmid resulted in receptor mRNA expression in the urothelium, detrusor and L6-S1 DRGs one week after transfection. Metabolic cage experiments indicated no differences in voiding function between EG3RF transfected and control animals in the absence of CYP treatment. CYP treatment decreased intercontraction interval during cystometry in anesthetized rats, which was significantly attenuated in EG3RF transfected animals (See Figure 1A). Cysteamine or propylamine treatment decreased bladder activity in both metabolic cage and cystometry experiments (31mg/kg ip or iv, respectively) in CYP treated rats but was ineffective in normal rats.
During the course of the cystometry experiments, it was noted that EG3RF transfected rats also exhibited much less bladder edema in response to CYP than non-transfected rats. Plasma extravasation of Evans Blue dye was greatly increased after CYP treatment in non-transected rats; however, plasma extravasation was significantly reduced in EG3RF transfected animals (Figure 1B). To attempt to elucidate the cellular mechanism of this decreased inflammation, we examined EG3RF’s ability to inhibit lysosomal ATP release, which has previously been implicated in bladder inflammation [1]. Stimulation of EG3RF transfected cells with cysteamine significantly attenuated chloroquine-mediated ATP release from the human TRT-HU1 urothelial cell line. Additionally, Fura-2 based calcium imaging demonstrated that EG3RF activation in urothelial cells inhibits the calcium transient elicited by stimulation of the lysosomal calcium channel TRPML1.
Interpretation of results
Transfection of EG3RF into the rat bladder successfully attenuated the increased frequency of voiding induced by CYP. This improvement occurred even in the absence of exogenous agonists, most likely because the receptor can be activated by the acidic extracellular environment induced by inflammation. Activation of the receptor with biogenic amines such as cysteamine or propylamine further reduced the excitation of bladder reflexes. Transfection of EG3RF had no effect in normal animals, indicating that the treatment would spare normal bladder function. While we have hypothesized that the beneficial effects of the treatment are due to a reduction in afferent nerve excitability, the observed reduction in inflammation suggests a secondary site of action; possibly the urothelium. Based on EG3RF’s ability to reduce lysosomal calcium transients and lysosomal ATP release, it is also possible that EG3RF’s anti-inflammatory and analgesic effects are mediated through reduced lysosomal ATP release driving inflammation and afferent excitability.