To determine the efficacy and mechanism of a single-chain peptide derivative (β7-33) of human relaxin-2 (hRLX2) in reversing radiation-induced bladder fibrosis and lower urinary tract dysfunction (LUTD).  Radiation cystitis is a consequence of radiotherapy for pelvic malignancies.  Acutely, irradiation leads to reactive oxygen/nitrogen species in urothelial cells, apoptosis, barrier disruption, and inflammation.  Chronically, this results in collagen deposition, bladder fibrosis, and attenuated storage and voiding functions.  In severe cases, cystectomies are performed as current therapies do not reverse fibrosis.

We performed assays for the RXFP1-dependent formation of cyclic adenosine monophosphate (cAMP) and compared the effects of β7-33 to the native ligand hRLX2.  HEK-293T cells were seeded at a density of 26,000 cells per square centimeter and then transfected with  2.5*10^-13 g DNA per cell per plasmid using Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer instructions.  Cells were transfected with pCDNA3.1-RXFP1-GFP and pGloSensor-22f (Promega), with three replicates per condition.  24-32 hours after transfection, cells were equilibrated in 2% GloSensor cAMP Reagent (Promega) in 10% FBS in CO2-Independent Media for two hours before kinetic luminescence measurements were taken for 30 minutes on a SpectraFluor plate reader (Tecan).  Cells were treated with forskolin as a positive control.  Cells transfected with only pGloSensor-22f showed no response to hRLX2 or β7-33 (data not shown).
We previously developed a mouse model for selective bladder irradiation (10 Gray; 1 Gy = 100 rads) that results in chronic fibrosis within 6 weeks, with decreased bladder compliance, contractility, and overflow incontinence.  Seven weeks post-irradiation, female C57Bl/6 mice were continuously infused with β7-33 (400 μg/kg/day/14 days) or vehicle (saline) via subcutaneous osmotic pumps.  Mice were evaluated in vivo using urine spot analysis, cystometrograms and external urethral sphincter electromyograms; and in vitro using length-tension measurements and histology.
Results:  In HEK-293T cells transfected with RXFP1, β7-33 induced formation of cAMP in a manner comparable to hRLX2, though the peptide required concentrations roughly 100-fold higher for similar effects.  In irradiated mouse bladders, β7-33 lowered basal bladder pressure, increased voiding intercontractile interval (ICI), improved voiding efficiency, and increased bladder compliance and contractility.

In HEK-293T cells transfected with RXFP1, β7-33 induced formation of cAMP in a manner comparable to hRLX2, though the peptide required concentrations roughly 100-fold higher for similar effects.  In irradiated mouse bladders, β7-33 lowered basal bladder pressure, increased voiding intercontractile interval (ICI), improved voiding efficiency, and increased bladder compliance and contractility.

Treatment outcomes were likely caused by the activation of the Relaxin Family Peptide Receptors 1 and 2 (RXFP1 and RXFP2).  In the urinary bladder, these receptors are expressed on the detrusor.  hRLX2 is known to activate both RXFP1 and to a lesser extent RXFP2.  While the irradiated, untreated mice show a short ICI with non-voiding contractions, the treated mice show much longer ICI and markedly reduced evidence of non-voiding contractions.  The much lower baseline bladder pressure seen in treated mice is strong evidence of a reduction in fibrosis, as is the marked decrease in passive tension in the length-tension measurements.  Treatment with β7-33 did not increase active tension as treatment with full-length hRLX2 hormone is known to do [1], which in combination with the 100-fold lower response in the cAMP assay produced by β7-33 supports previous findings that β7-33 does not promote angiogenesis, which is downstream of elevated cAMP levels in cells with active RXFP1.

β7-33 may be a new therapeutic option for rescuing bladders with chronic radiation cystitis.  β7-33 offers a notable advantage over hRLX2 in that β7-33 does not induce angiogenesis as does hRLX2, making it safer to use in patients undergoing radiotherapy for pelvic cancers. 
As hRLX2 can activate both RXFP1 and RXFP2, future investigations will determine the mechanism of action using mice lacking expression of either RXFP1 or RXFP2.  Further mechanistic investigation will address which signal transduction pathways are required for rescue of bladder function. We will also confirm the effects of β7-33 on collagen content and bladder wall architecture. 
Another notable potential impact of therapy with either hRLX2 or β7-33 is the known anti-inflammatory effects of hRLX2 signaling.  Inflammation plays a causal role in numerous bladder pathologies including fibrosis.  hRLX2 has been shown to inhibit vascular inflammation, to reduce macrophage pro-inflammatory cytokine secretion, and to reduce inflammation in hearts damaged by either myocardial infarction or aging.  We plan to initiate a careful inspection of the effects on inflammation in radiation cystitis produced by both hRLX2 and β7-33.

Ikeda, Y.; Zabbarova, I. V.; Birder, L. A.; Wipf, P.; Getchell, S. E.; Tyagi, P.; Fry, C. H.; Drake, M. J.; Kanai, A. J. Relaxin-2 Therapy Reverses Radiation-Induced Fibrosis and Restores Bladder Function in Mice. Neurourol. Urodyn. 2018 Nov; 37(8): 2441-2451.