Aquaporins (AQPs) are a family of transmembrane proteins that facilitate the rapid and selective transport of water across cell membranes. Among them, aquaporin-2 (AQP2) is best characterized in the renal collecting ducts, where its expression and apical membrane insertion are tightly regulated by vasopressin via the V2 receptor pathway [1, 2]. The importance of AQP2 in maintaining water balance in the kidney has been well established, especially in the context of urine concentration mechanisms. However, the distribution of AQP2 is not limited to renal tissues. Recent studies have demonstrated its expression in extrarenal locations in humans, including the urinary bladder urothelium, suggesting that AQP2 may also participate in modulating water permeability in the lower urinary tract [3]. Given the bladder’s role as both a storage organ and a sensory structure, precise regulation of water handling within the urothelium could influence not only hydration balance but also urothelial signaling, sensation, and barrier function.

Vasopressin is a multifunctional neurohormone that regulates water homeostasis, cardiovascular tone, and stress responses through 3 G protein-coupled receptors: V1a, V1b, and V2. While V2 receptors primarily mediate renal water reabsorption via AQP2 trafficking in the collecting ducts, V1a receptors are widely expressed in extrarenal tissues, where they modulate vascular tone, intracellular calcium signaling, and epithelial function, including in the urinary bladder [4]. Importantly, vasopressin V1a receptor (AVP-V1a) activation can trigger intracellular signaling cascades that may influence the subcellular localization of AQP2, thereby linking neurohormonal and osmotic regulation pathways.

The urinary bladder is densely innervated and highly responsive to neural stimuli, which modulate not only detrusor contractility but also urothelial biology, including water channel expression. In our previous work, conducted by our group, we investigated how aquaporins respond under conditions of detrusor overactivity induced by bladder outlet obstruction in rats. That study demonstrated significant upregulation of AQP2, AQP3, endothelial nitric oxide synthase, and neuronal nitric oxide synthase in the bladder urothelium, suggesting that mechanical or neural stimuli may influence epithelial water and nitric oxide handling [5]. Building on this, Langdale et al. [6] reported that electrical stimulation of the pelvic nerve significantly increased bladder capacity in a prostaglandin E₂ (PGE₂)-induced overactive bladder (OAB) model in rats, indicating that pelvic nerve activity modulates bladder function via afferent mechanisms and potentially through epithelial regulatory pathways as well.

Building upon this emerging evidence, the present study hypothesized that electrical stimulation of the pelvic nerve would induce translocation of AQP2 and AVP-V1a from the cytoplasm to the membrane in the bladder urothelium of rats. By combining immunohistochemical and biochemical analyses, we aimed to elucidate a possible neurophysiological mechanism through which bladder water transport is regulated. These findings may contribute to our understanding of how sensory input and autonomic signaling modulate urothelial function and fluid homeostasis in both health and disease.

The present study demonstrates that electrical stimulation of the pelvic nerve may induce translocation of AQP2 and AVPV1a from the cytosol to the plasma membrane in rat urinary bladder urothelial cells. This observation might suggest a novel neurophysiological mechanism regulating urothelial water transport and expands our understanding of bladder sensory and secretory modulation under autonomic control. Notably, Langdale et al. [6] demonstrated that electrical stimulation of the pelvic nerve significantly increased bladder capacity in a PGE₂-induced OAB model in rats, suggesting that pelvic nerve activity may modulate afferent signaling and detrusor excitability. Although their focus was primarily on functional bladder capacity rather than epithelial mechanisms, their findings support the broader concept that pelvic nerve stimulation can influence multiple aspects of bladder physiology. In this context, our observation of AQP2 and AVP-V1a translocation in the urothelium following pelvic nerve stimulation may represent a complementary epithelial response that parallels neuromodulatory effects on bladder storage function.

AQP2 is traditionally recognized for its role in renal water reabsorption, where vasopressin binding to V2 receptors initiates a signaling cascade leading to AQP2 phosphorylation and its insertion into the apical membrane of collecting duct cells [1, 2]. Although this mechanism has been extensively studied in the kidney, AQP2 has also been identified in extrarenal sites such as the bladder urothelium [3, 5]. In these tissues, its precise function remains less defined, yet its presence indicates a potential epithelial adaptation for regulating water permeability in response to physiological demands. Our data show that neuromodulation via pelvic nerve stimulation facilitates the translocation of AQP2 to the urothelial membrane, echoing the renal paradigm where cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling regulates AQP2 trafficking [1]. Beyond classical pathways, recent research highlights additional regulatory mechanisms for AQP2 movement. Yanagawa et al. [7] reported that the vesicle trafficking protein LRBA (lipopolysaccharide-responsive and beige-like anchor) directs AQP2 to the plasma membrane following vasopressin stimulation, demonstrating that multiple intracellular systems coordinate AQP2 localization. These findings support the idea that AQP2 translocation in the bladder maybe governed by a complex network of signaling proteins, not solely cAMP/PKA activity.

In the context of bladder physiology, vasopressin’s effects are not restricted to V2 receptor activation. Cafarchio et al. [8] demonstrated that both V1a and V2 receptor pathways contribute to increased intravesical pressure in anesthetized rats, suggesting cooperative roles for multiple receptor subtypes in regulating smooth muscle and epithelial function. Furthermore, Zhang et al. [9] found that V1a receptor activation in vascular smooth muscle cells triggers downstream proliferative signaling through the GRK2-EGFR-ERK1/2 axis, while other studies have linked AVP receptors to hormonal regulation in pancreatic and adrenal tissues [10]. These observations imply that pelvic nerve stimulation may engage overlapping AVP pathways, potentially explaining the observed co-translocation of AQP2 and AVP-V1a in our model.

Although both AQP2 and AVP-V1a were observed to translocate in response to the same neural input, it remains uncertain whether this reflects direct interaction or parallel responses to shared upstream signals. Vasopressin is a key hormone in fluid homeostasis and has been shown to modulate detrusor contractility through V1a receptors in the bladder. Yoshimura and Chancellor [11] previously described that autonomic inputs regulate both afferent sensory pathways and smooth muscle tone in the lower urinary tract, offering a mechanistic basis for neural modulation of bladder epithelium. Birder et al. [12] further demonstrated age-related upregulation of vasopressin receptors in both kidney and bladder, indicating that AVP signaling may have age-related implications in bladder regulation as well. From a clinical perspective, disturbances in vasopressin signaling have been implicated in conditions such as nocturia and overactive bladder, as recognized in pathophysiologic frameworks established by the International Continence Society [13]. Our findings contribute to this understanding by suggesting that the epithelial translocation of AQP2 and AVP-V1a is part of a coordinated response to neural input, reflecting a regulatory mechanism for bladder water permeability under autonomic control.

Previous animal models have revealed that AQP expression in the bladder can be influenced by diverse non-neuronal stimuli. In our prior study, caveolin-1 deficiency in mice was associated with altered AQP1 expression and bladder dysfunction, suggesting that structural membrane proteins may modulate aquaporin function [14]. Additionally, we have shown that estrogen deficiency following ovariectomy alters AQP2 and AQP3 expression, implicating hormonal regulation in the control of urothelial permeability [15]. These data, along with our current findings, underscore the multifactorial nature of AQP regulation in the bladder, shaped by neural, hormonal, and structural inputs. Importantly, the mechanism identified in this study is distinct in that it is driven by direct neural stimulation, rather than mechanical injury, hormonal status, or urine volume overload. Our demonstration of partial membrane relocalization of AQP2 and AVP-V1a following pelvic nerve stimulation suggests an autonomic regulatory axis that could serve as a therapeutic target. This mechanism may be especially relevant in disorders characterized by disrupted fluid homeostasis or impaired bladder sensation, such as neurogenic bladder or nocturnal polyuria.

Collectively, these findings reinforce the view that the bladder urothelium is a dynamic and neuroresponsive tissue interface. These results also provoke broader questions about how the bladder urothelium senses and adapts to its environment. Given its established role in neurotransmitter release and afferent signal modulation, vasopressin-mediated changes in epithelial permeability may influence not only bladder compliance but also sensory perception and voiding behavior. While our study was performed in an animal model, these findings may offer translational value in understanding human bladder disorders characterized by dysregulated fluid handling or autonomic dysfunction. From a therapeutic standpoint, interventions that influence aquaporin trafficking or AVP receptor signaling could represent novel approaches to treating lower urinary tract symptoms. Precise targeting of urothelial water channels in response to autonomic input may allow for symptom control with fewer side effects than systemic pharmacologic agents.

Nevertheless, several limitations should be acknowledged. First, we employed an acute stimulation model that may not fully reflect the repetitive or sustained neural activity observed during physiological bladder cycles. Future studies should incorporate chronic or repeated stimulation protocols to assess long-term regulatory effects. Second, although we confirmed translocation of AQP2 and AVP-V1a, we did not evaluate functional outcomes such as water transport, epithelial conductance, or urodynamic changes. These endpoints are critical to establish the physiological significance of our findings. Third, while immunofluorescence supported protein relocalization, we could not precisely differentiate between cytosolic and membrane compartments, as whole-bladder tissues were used. Fourth, the signaling mechanisms driving AVP-V1a translocation remain unclear. Potential involvement of pathways such as cAMP/PKA or GRK2–ERK1/2 should be investigated in future studies using pharmacological or genetic tools. Lastly, the lack of standardized bladder volume prior to tissue harvest may have introduced variability in hydrostatic pressure, potentially influencing molecular responses. Addressing these limitations through mechanistic and functional studies will help define the physiological role and therapeutic potential of neural control over urothelial water transport.

In conclusion, this study demonstrates that pelvic nerve stimulation induces the membrane translocation of AQP2 and AVP-V1a in the bladder urothelium, independent of mechanical or hormonal triggers. These findings reveal a novel autonomic regulatory mechanism for epithelial water handling, suggesting a possible role in regulating bladder water permeability and highlighting the bladder urothelium as an active target of neural modulation. This mechanism may have therapeutic relevance in conditions such as neurogenic bladder or nocturnal polyuria, where impaired sensory or fluid regulation plays a central role.