Hypothesis / aims of study
Overactive bladder (OAB) is a common disease in childhood that severely affects children's physical and mental health. Its etiology remains unclear. This study aimed to investigate the preliminary mechanism of fructose-induced OAB via the liver-bladder axis using cross-organ multi-omics analysis, providing a foundation for novel diagnostic and therapeutic strategies for pediatric OAB.
Study design, materials and methods
Three-week-old male C57/B6 mice were fed a 60% fructose diet for 1 week to establish an OAB model, confirmed by urodynamic analysis. Histological changes in bladder tissues were evaluated by HE staining, mitoROS staining, and transmission electron microscopy. For in vitro mechanistic validation, primary rat bladder smooth muscle cells were cultured with fructose. LC/MS metabolomics, 13C metabolic flux tracing, and OCR mitochondrial function assays were performed to assess fructose uptake and metabolic utilization. Hepatic changes were analyzed via oil red O and Sirius red staining, transcriptomics, and metabolomics. Bladder smooth muscle contractility, RLC phosphorylation, and single-cell/bulk RNA sequencing were systematically evaluated.
Results
HE and mitoROS staining revealed significant pathological remodeling and ROS accumulation in the bladder of fructose-fed mice. TEM showed severe mitochondrial damage in BSMCs. LC/MS analysis demonstrated marked metabolic reprogramming in bladder tissues without fructose accumulation. 13C tracing and OCR experiments indicated minimal fructose contribution to glycolysis or the TCA cycle. Fructose was partially metabolized into lactate within 24 hours, with no significant pyruvate or malate production, suggesting passive cellular entry without active energy metabolism . Hepatic staining showed no significant pathological changes . RNA-seq revealed significant alterations in bile acid metabolism, cholesterol metabolism, and PPAR signaling pathways in the liver. Single-cell and bulk RNA-seq of bladder tissues showed activation of PI3K-AKT, NF-κB, and neurotrophin signaling in BSMCs. Fructose-treated BSMCs exhibited increased contractility and elevated RLC phosphorylation. Mechanistically, hepatic dysregulation of bile acid metabolism may lead to systemic overflow of secondary bile acids, activating TGR5 receptors in BSMCs and amplifying cholinergic GPCR signaling .
Interpretation of results
1. Bladder Local Changes:
Mitochondria are key organelles for energy metabolism and redox homeostasis in BSMCs; their structural damage impairs energy production, causes redox imbalance, and aggravates ROS-mediated injury. Bladder pathological remodeling combined with mitochondrial damage provides the structural basis for smooth muscle hypercontractility and OAB-like phenotype, consistent with clinical features of OAB.
2. Key Metabolic Finding:
Metabolomic analysis showed significant metabolic reprogramming but no fructose accumulation in the bladder. ¹³C tracing and OCR assays confirmed that fructose barely participates in bladder glycolysis or TCA cycle, only marginally metabolized into lactate within 24 hours. These findings challenge the conventional view, indicating fructose induces bladder dysfunction indirectly via systemic metabolic disorders, not local active metabolism.
3. Liver:
Liver histological staining showed no obvious MASLD or NASH, but RNA-seq revealed abnormalities in bile acid metabolism, cholesterol metabolism and PPAR signaling. High fructose triggers early hepatic molecular metabolic disorders prior to organic lesions, suggesting bladder dysfunction may precede hepatic metabolic diseases; PPAR dysregulation disrupts bile acid homeostasis, initiating systemic metabolic disorders.
4. Bladder Cellular Function:
RLC phosphorylation mediates BSMC hypercontractility, while NF-κB, PI3K-AKT and NGF/BDNF synergistically drive OAB phenotype via inflammation, contractile function and neural regulation.
5. Core Mechanism:
Dysregulated hepatic bile acid metabolism causes systemic overflow of secondary bile acids, which activate BSMC TGR5 receptors and amplify cholinergic GPCR signaling. TGR5 links hepatic metabolism to bladder smooth muscle function, and its coupling with cholinergic GPCR signaling explains fructose-induced bladder overactivity.