SPI1 exacerbates neurogenic bladder dysfunction by enhancing NRSF-mediated suppression of HCN1 and inducing endoplasmic reticulum stress.

Neurogenic bladder (NB) severely impacts patients' quality of life. This study was designed to explore the molecular mechanisms underlying the pathophysiology of NB-associated bladder dysfunction. A rat model of NB was established via spinal cord injury (SCI). Bladder function was evaluated by urodynamic testing and hematoxylin-eosin staining of tissue sections. The expression levels of SPI1, HCN1, neuronal restrictive silencing factor (NRSF), and endoplasmic reticulum (ER) stress-related proteins in bladder tissues or cells were examined using immunohistochemistry, Western blot, and reverse transcription quantitative polymerase chain reaction. Cell viability was measured with the Cell Counting Kit-8 assay. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry. Potential binding sites between SPI1 and the NRSF promoter were predicted using the JASPAR database and then validated by dual-luciferase reporter and chromatin immunoprecipitation assays. The role of ER stress in NB was investigated using the inhibitor 4-phenylbutyric acid or the inducer tunicamycin. Bladder tissues from NB rats exhibited increased apoptosis and a significant upregulation of ER stress-related proteins. Furthermore, expression of SPI1 and NRSF was elevated, whereas the expression of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) was reduced in bladder tissues from NB rats. Further analysis revealed that SPI1 binds to the NRSF promoter and upregulates its expression, thereby enhancing the repression of HCN1. Knockdown of SPI1 significantly alleviated bladder dysfunction, attenuated ER stress, and inhibited apoptosis. These effects were reversed by overexpression of NRSF. This study demonstrates that SPI1 contributes to bladder dysfunction by upregulating NRSF, which subsequently potentiates the inhibition of HCN1 and triggers ER stress. The findings identify promising molecular targets and signaling pathways for the intervention and treatment of NB.