Extra-cellular matrix modulation to improve bladder compliance in posterior urethral valves
Kevin X. Cao, MBBS, MRCS, MSc1, Maria Kolatsi-Joannou, PhD1, Cheuk Y. Man, MSc1, Navroop S. Johal, MB BCh FRCS PhD2, Peter M. Cuckow, MBBS FRCS PhD2, Christopher H. Fry, PhD3, Paul J. Winyard, BM BCh FRCPCH PhD1, David A. Long, PhD1.
1UCL Great Ormond Street Institute of Child Health, London, United Kingdom, 2Great Ormond Street Hospital for Children, London, United Kingdom, 3University of Bristol, Bristol, United Kingdom.
Posterior urethral valves (PUV) remains the condition singularly responsible for the greatest demand for renal replacement therapy in childhood. In research previously presented at SPU, we showed that severe PUV patients have higher extra-cellular matrix (ECM) content in their bladders - a possible aetiology for bladder dysfunction and consequent kidney scarring. Finding an anti-scarring therapy is thus a potential means to improve outcomes in PUV. We performed a series of pre-clinical experiments, evaluating an on-market family of drugs, the soluble guanylate cyclase (sGC) modulators, exploring their potential for fibrosis reduction in both an animal model of PUV and cell culture experiments.
8-week old male C57 mice (n=36) were used to create a PUV-analogue model, through partial-ligation of the urethra, around a temporary metallic stent, which were then orally-administered sGC modulators for two out of three weeks of the trial. Bladders were stained with picosirius red to allow evaluation of the smooth muscle to connective tissue ratio (SM:CTr), lower ratios indicating a greater content of ECM relative to muscle. Biomechanical assessments of the stress-strain relationship from these bladders were measured to derive elastic modulus, or stiffness from the steady-state tension (measured in kilopascals, kPa).
Primary cell cultures were established from children with PUV undergoing bladder surgery or kidney transplant and treated with the same drugs over a 7-day course to determine relative changes in both ECM proteins and changes to gene expression of ECM genes.
Our animal model generated a PUV-like morphology, reducing SM:CTr from 1.2 in sham surgery to 0.5 in the model, indicating a greater ECM content (p = 0.0019), as well as an increase in detrusor stiffness from 50kPa to 170kPa (p < 0.0001). These features highlight the relationship between ECM content and bladder compliance in vivo when a PUV-mimicking partial urethral obstruction is applied. Treatment with either sGC activator, cinaciguat or sGC stimulator, BAY 41-2272 (10mg/kg) kept both these variables in the same range as sham surgery (p < 0.0001), preventing the accumulation in ECM.
Culture of detrusor cells from patients with PUV in a pro-fibrotic 1% fetal bovine serum milieu resulted in a 20% increase in fibronectin protein and gene expression, compared to control milieu of 10% serum, relative to housekeeping gene/protein GAPDH. Treatment in 1% serum with BAY 41-2272 reduced expression of fibronectin protein by seven-fold and corresponding FN1 gene expression by 20%. No changes in alpha-smooth muscle actin gene or protein expression were seen with treatment, suggesting a mechanism for the function of these drugs may lie in inactivation rather than the suppression of myofibroblasts in the detrusor.
SGC modulators are newly-on-market drugs that demonstrate the ability to alter ECM-content and in parallel normalise bladder compliance in both animal and in vitro models of PUV. Repurposing of these drugs gives hope for altering the prognosis of what remains one of the most severe conditions in paediatric urology and through its continued study in the context of bladder fibrosis, shed light on the pathology of PUV.
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