Development of neurogenic detrusor overactivity (NDO) and overactive bladder are common consequences of spinal cord injury (SCI), severely affecting the patients’ quality of life. Reliable prognostic biomarkers are needed to measure SCI severity and effectiveness of therapeutic interventions. Bladder biopsies accurately reflect changes in the bladder wall, however, this is a highly invasive procedure not applicable in clinical practice. Urine is an attractive source of biomarkers as it can be collected non-invasively and changes in urinary protein abundance can reflect a person's health status. We hypothesized that urine from SCI patients treated with BoNTA contains specific proteins and metabolites that can reflect the severity of LUTD and BoNTA efficacy. To this end, we collected both urine and bladder dome biopsies from the SCI patients, receiving early BoNTA bladder injections and compared them with the placebo group.

Bladder base biopsies aiming to include detrusor muscle, and urine samples were collected from nine SCI patients. The subjects were mean 41.4 (range 20-62) years old at time of injury, with motor and sensory complete lesions (AIS classification A) spinal cord injury at level C7-Th11. Inclusion and urodynamic evaluation were performed, followed by treatment with BoNTA or placebo. The participants were randomized, assessed urodynamically and received first treatment (BoNTA or placebo) within 3 months after injury (Visit 1- V1). The second urodynamic evaluation, treatment and second set of biopsy and urine collection was three months later (Visit 2 – V2). Follow up with urodynamic evaluation, Quality of Life questionnaires and biopsy/urine collection were done 12 months after first visit (Visit 3 – V3). Total RNA was isolated from one set of biopsies for the bulk RNA sequencing and differential gene expression profiling.  Urinary proteomics was carried out using the SP3 protocol (Single‐Pot Solid‐Phase‐enhanced Sample Preparation) with paramagnetic beads washed with 50% ethanol.

Urodynamic Studies showed that none of the patients in the BoNTA group (n = 5) developed NDO with contractions above 40 cm H2O during the follow up period, whereas two patients in the placebo group (n = 4) developed NDO. Biopsy analysis showed a significant dysregulation of DNA repair pathways in the placebo group group, concomitant with NDO development. We describe a 28 gene DNA repair signature, altered in the placebo group after injury, and delineate RFC5 and RMI2 genes, encoding proteins required for DNA replication and homologous recombination-dependent DNA repair as NDO biomarkers. Urinary profiles of SCI patients (BoNTA and placebo groups) show dynamic changes in protein composition and abundance, correlated with dysregulation of bladder function and development of NDO in the placebo group. Specifically, we observed differences in the urinary levels of cytokines, proteins involved in DNA repair (DNAJC5, DDB1, and DNAJA), proteins indicative of oxidative stress and urothelial proteins.

Early BoNTA injections affected bladder contractility and immune cell-related signalling pathways, and prevented NDO. The changes in the bladder wall morphology and cell signalling, detected in the biopsies, included specific dysregulation of DNA repair processes, observed in placebo group only. These alterations were reflected in the urinary profiles, which were different between placebo and BoNTA with considerable longitudinal changes in both groups at 3 and 12 months after SCI.

Early BoNTA injections into the bladders of SCI patients preserved organ architecture, preventing DNA repair pathway dysregulation, indicative of NDO. Urine collection and analysis are valuable tools in the evaluation of bladder (dys)function and treatment efficacy.