Spinal cord injury (SCI) is one of the leading causes of bladder dysfunction, with overflow incontinence representing a hallmark clinical manifestation (Burns et al., 2001; Almajid et al., 2017). Neurogenic bladder secondary to SCI not only compromises the quality of life but also predisposes patients to recurrent urinary tract infections and progressive renal impairment (Almajid et al., 2002). However, most studies on post-SCI bladder dysfunction have focused on neural reflex circuits and detrusor electrophysiology, whereas alterations in local epithelial–stromal signaling networks remain largely unexplored (Ryu et al., 2018).

The activin–follistatin–inhibin (AFI) axis, a branch of the transforming growth factor-β (TGF-β) superfamily, plays pivotal roles in embryonic development, tissue repair, inflammation, fibrosis, and tumorigenesis (Hedger and de Kretser, 2013). The AFI axis comprises three functional components: (i) a ligand family (Inha, Inhba, Inhbb, Inhbc, and Inhbe) encoding distinct activin and inhibin isoforms that modulate cellular proliferation and differentiation; inhibins are heterodimers composed of inhibin α (INHA) and a β subunit (INHBB, INHBC, or INHBE); (ii) a receptor family (Acvr1, Acvr1b, Acvr1c, Acvr2a, and Acvr2b) that mediates ligand-induced small mother against decapentaplegic (SMAD) signaling; and (iii) the binding protein follistatin (Fst), which antagonizes activin signaling by competitive sequestration, thereby maintaining signaling homeostasis. Together, these elements form an integrated regulatory network of the AFI axis (De Jong, 1988; Barrero et al., 2009; Tsuchida et al., 2009). Previous studies have revealed a distinctive spatial organization of the AFI axis in the normal bladder: Activin A receptor type 1B (ACVR1B) and Activin A receptor type 2B (ACVR2B) are localized to the basolateral membranes of urothelial cells, activin-positive cells are predominantly distributed within the stromal compartment, and FST forms a thin, basement membrane–like barrier separating the two. This spatial segregation suggests that the AFI axis contributes to bladder homeostasis through fine-tuned paracrine regulation (Mao et al., 2025). Chronic bladder distension, inflammation, and tissue remodeling following SCI may disrupt this epithelial-stromal barrier, facilitating aberrant activin-receptor interactions and pathological activation of downstream signaling cascades. The lower urinary tract dysfunction resulting from SCI is frequently accompanied by progressive deterioration of the upper urinary tract, including kidney injury. While bladder dysfunction in neurogenic bladder has been extensively studied, there is increasing recognition that kidney dysfunction, especially secondary to high intravesical pressure and urinary retention, plays a critical role in the long-term morbidity of SCI patients (Wang et al., 2024). The AFI axis has been implicated in various pathophysiological processes, including inflammation and fibrosis, in multiple tissues. Given the evidence linking AFI axis components to kidney injury and fibrosis (Tsai et al., 2024; Wang et al., 2025), we aimed to profile the AFI axis in both the bladder and kidney to assess whether similar molecular alterations occur in the upper urinary tract.

Here, we established a standardized mouse SCI model and characterized SCI-associated voiding behavior using the voiding spot assay, together with gross bladder morphology and organ weight measurements. We subsequently profiled mRNA and protein expression of key AFI-axis components in the bladder and kidney, coupled with immunofluorescence-based spatial mapping. These analyses aimed to delineate how SCI remodels the AFI axis within the urinary tract, thereby providing molecular insight into the pathogenesis of neurogenic bladder and providing a foundation for future mechanistic and interventional validation.

This study aimed to elucidate how spinal cord injury reshapes the expression of the activin-follistatin-inhibin axis in the bladder and kidney, thereby advancing our understanding of the molecular underpinnings of neurogenic bladder dysfunction. Our findings demonstrate that SCI induces marked alterations in key AFI-axis components, most notably a robust upregulation of FST in the bladder and a significant reduction of INHA at both the mRNA and protein levels. For INHBE, although its mRNA expression in the bladder was decreased after SCI, no specific band could be detected by Western blot under our experimental conditions, precluding reliable assessment of overall protein abundance. By contrast, immunofluorescence revealed enhanced INHBE staining in the stromal compartment of SCI bladders. In the kidney, the AFI axis exhibited a distinct remodeling pattern: Inhbb expression was increased, whereas Inha and Inhbe were reduced at both the transcript and protein levels, with INHBE immunofluorescent staining showing no overt difference between sham and SCI groups. Together, these observations provide new molecular clues to how SCI perturbs bladder function and impacts the upper urinary tract, and they identify AFI-axis remodeling as a candidate molecular signature and highlight AFI-axis components as mechanistic nodes and/or biomarkers worthy of functional validation in neurogenic bladder.

One of the principal findings of this work is the pronounced upregulation of FST in the bladder following SCI, accompanied by preservation of its characteristic suburothelial and stromal localization. As a high-affinity activin-binding protein, follistatin constrains the activity of TGF-β family ligands within the local microenvironment by limiting ligand availability to their receptors (Hedger and de Kretser, 2013). In the present study, FST was increased at the mRNA, protein, and immunofluorescence levels in SCI bladders, without an apparent reconfiguration of its spatial distribution. This pattern supports a model in which FST responds to chronic distension, inflammation, and matrix remodeling primarily through quantitative amplification rather than positional redistribution. Importantly, fibrosis- and inflammation-related tissue readouts (e.g., ECM deposition or inflammatory cytokine markers) were not directly quantified in this study, and thus these interpretations remain inferential. Such upregulation may represent a protective attempt to buffer excessive activin activity, yet under certain conditions, excessive sequestration of activin could also impair appropriate repair responses or epithelial differentiation and thereby contribute to maladaptive remodeling.

Within the bladder, the concordant decrease of INHA at both transcript and protein levels adds a second layer of imbalance to the AFI axis. INHA encodes the α subunit of inhibin, which antagonizes activin signaling by competitively engaging activin receptors (De Jong, 1988). Thus, the overall reduction of INHA suggests a shift of the activin–inhibin equilibrium toward diminished inhibin-mediated antagonism. Immunofluorescence refines this picture further: in sham bladders, INHA is predominantly localized to the urothelium and suburothelial region, whereas in SCI bladders, staining intensity in these layers remains largely unchanged but shows a modest increase in the stroma. This combination of “global reduction with relative stromal enrichment” indicates that SCI not only attenuates the overall inhibitory capacity of inhibin toward activin, but also remodels the AFI-axis architecture at the epithelial-stromal interface. Such remodeling may alter how activin signals are integrated among urothelial cells, suburothelial myofibroblasts, and resident immune cells, with potential consequences for barrier integrity, matrix turnover, and the chronic inflammatory milieu characteristic of neurogenic bladder.

The behavior of INHBE in the bladder further underscores the complexity of AFI-axis regulation across the gene-protein-tissue continuum. At the transcript level, Inhbe was significantly downregulated after SCI; however, under our experimental conditions, immunoblotting did not yield a resolvable INHBE-specific band, consistent with very low abundance in bladder homogenates and/or limited antibody performance for quantitative Western blotting. To obtain spatially resolved information, we therefore performed immunofluorescence, which revealed increased INHBE signal after SCI in both the urothelium and the stroma, with a more prominent enhancement in the stromal compartment. This divergence between a homogenate-level mRNA reduction and compartment-enriched fluorescence gains is best interpreted as platform- and compartment-dependent, rather than as a single-layer contradiction. qRT-PCR reports an average transcript signal pooled from heterogeneous tissue fractions, whereas immunofluorescence captures antigen signals within defined microanatomical compartments; thus, SCI-associated shifts in tissue composition/cellular sources, together with post-transcriptional control, differential protein stability across microenvironments, and methodological differences between bulk extraction and in situ detection, could jointly contribute to the observed pattern. Importantly, these observations do not negate the consistent transcript-level decrease, but instead highlight that spatial redistribution and local enrichment can occur despite an overall reduction in transcript abundance, and that conventional bulk assays may under-represent such compartment-specific modulation. Future compartment-resolved approaches (e.g., urothelium-stroma separation, laser-capture microdissection, or spatial transcriptomics) will be valuable for reconciling transcript- and tissue-level readouts.

In the kidney, the AFI axis exhibited a remodeling pattern distinct from that observed in the bladder. Quantitative RT–PCR revealed upregulation of Acvr1b and Inhbb, alongside downregulation of Inha, Inhbc, and Inhbe. Consistent with these findings, Western blot analysis confirmed increased INHBB protein levels and decreased INHA and INHBE, while total INHBC protein remained largely unchanged. Immunofluorescence demonstrated that INHA, INHBB, and INHBE were broadly distributed throughout the renal parenchyma in both sham and SCI groups. Following SCI, the overall staining pattern was largely preserved, with only a reduction in INHA signal intensity, a modest enhancement of INHBB, and essentially stable INHBE staining. These observations suggest that, in contrast to the more pronounced spatial remodeling seen in the bladder, the renal response to SCI is mediated primarily through fine-tuning of AFI-axis expression levels within a pre-existing anatomical framework. The combination of INHBB upregulation with INHA and INHBE downregulation may shift activin family signaling toward a more pro-inflammatory or pro-fibrotic profile, in line with previous reports linking activin family members to renal injury and fibrosis (Wijayarathna and de Kretser, 2016; Bian et al., 2025; Nagayama et al., 2025).

In interpreting the renal findings, we emphasize that kidney alterations observed after SCI may predominantly reflect secondary consequences of lower urinary tract dysfunction—such as sustained urinary retention and elevated intravesical pressure with potential upper-tract reflux—rather than a direct effect of the thoracic lesion per se. Accordingly, the renal AFI-axis changes detected here are discussed in the context of post-SCI voiding dysfunction imposing pressure/retention-related stress on the upper urinary tract. Our data therefore support an association between SCI-induced bladder dysfunction and renal AFI-axis remodeling, while the direct versus secondary nature of these renal alterations remains to be resolved.

Several methodological considerations must be taken into account when interpreting these renal findings. First, although Acvr1b mRNA levels were increased after SCI, its basal expression was low, and the lack of a highly specific and sensitive primary antibody precluded reliable quantification of ACVR1B protein by Western blot. As a result, conclusions regarding activin receptor remodeling rest predominantly on transcriptional data and are not supported by direct protein-level validation. Second, while INHBC could be readily detected and quantified by Western blot in both bladder and kidney, we were unable to obtain antibodies that yielded specific, interpretable immunofluorescent signals in tissue sections, preventing assessment of its spatial redistribution during SCI. These technical limitations do not negate the mRNA and bulk protein trends we observed, but they do constrain inferences about AFI-axis reorganization at the microanatomical level and warrant caution when extrapolating these molecular changes to specific pathophysiological processes.

Our study provides a novel molecular perspective on the pathophysiology of neurogenic bladder following SCI. Previous work has focused predominantly on neural circuit reorganization, detrusor overactivity, and growth factor-related pathways, such as the proNGF/p75 axis in the SCI bladder (de Groat, 1998; Ryu et al., 2018; Peyronnet et al., 2019; Ma et al., 2023),whereas the AFI axis has been studied mainly in the context of reproductive endocrinology, inflammation, and pregnancy-related disorders (Perakakis et al., 2018; Bouzoni et al., 2020; Bouzoni et al., 2022). Building on prior mapping of AFI-axis distribution in the normal bladder,the present study is, to our knowledge, the first to demonstrate at a systems level that SCI-induced neurogenic bladder is accompanied not only by local remodeling of AFI-axis components within the bladder wall, but also by parallel alterations in upper urinary tract structures such as the kidney (Mao et al., 2025). By integrating data from voiding spot assays, organ weight measurements, gross morphology, qRT-PCR, Western blotting, and spatially resolved immunofluorescence, we delineate a cross-organ, multi-node pattern of AFI-axis reprogramming that would be difficult to capture using any single methodological approach.

These findings have several important implications. Neurogenic bladder after SCI is fundamentally initiated by disruption of afferent and efferent neural pathways that govern bladder sensation and detrusor–sphincter coordination. Within this well-established neurogenic framework, progressive bladder-wall remodeling—particularly fibrosis and epithelial-stromal reprogramming—can accumulate and contribute to persistent dysfunction and heightened upper-tract vulnerability. Our data therefore position AFI-axis remodeling as a molecular correlate of bladder and renal tissue remodeling that accompanies SCI-induced voiding dysfunction, rather than as an alternative explanation that diminishes the central role of neural injury. Although the present study is descriptive and does not directly interrogate cell-specific mechanisms, the observed, compartment-resolved shifts in AFI-axis members are consistent with prior evidence linking activin/inhibin/follistatin signaling to repair and fibrotic remodeling, and motivate future functional studies to define how AFI-axis perturbation interfaces with post-SCI fibrosis and long-term bladder deterioration. In line with this broader framework, activin signaling—via the TGF-β superfamily and SMAD2/3-related programs—has been functionally implicated in fibrotic remodeling in multiple organ systems. In the bladder, partial inhibition of the activin type I receptor ALK4 (ACVR1B) has been reported to attenuate fibrosis in experimental obstruction models, supporting a profibrotic ALK4–SMAD2/3 axis (Wang et al., 2021). In the kidney, activin A has been linked to inflammatory and profibrotic injury responses, and antagonizing activin A signaling via follistatin has been shown to reduce renal fibrosis and inflammation in disease models (Bian et al., 2025). However, because we did not directly manipulate AFI-axis components or quantify fibrosis/inflammation markers in the current dataset, these mechanistic links remain hypothesis-generating and require targeted validation in future studies. From a translational perspective, AFI-axis constituents may serve as candidate molecular markers for disease stratification, progression monitoring, and therapeutic response. The emerging evidence that targeting FST or activin-receptor interactions can be beneficial in other fibrotic and inflammatory conditions suggests that judicious modulation of this axis in neurogenic bladder could complement existing neuromodulatory or surgical interventions (Tsuchida et al., 2009).

At the same time, several limitations of this study warrant cautious interpretation. First, the present dataset was generated in a single mouse strain and sex with modest group sizes (n = 3 per group) and at a single post-injury endpoint, which limits statistical power and may not capture sex-dependent effects or temporal dynamics of AFI-axis remodeling. Although the post-injury interval and endpoint are specified in the Methods, we emphasize here that the current study represents a focused snapshot rather than a longitudinal trajectory. In this context, we used a male cohort to provide an initial, internally consistent molecular snapshot of AFI-axis remodeling after SCI and to align with the male predominance reported in traumatic SCI at the population level (World Health Organization, 2024); nevertheless, murine lower urinary tract function exhibits sex-dependent voiding–sphincter coordination, and recent work using cystometry combined with EUS-EMG has demonstrated distinct sex-specific patterns in spinal-intact animals and after SCI with corresponding differences in voiding efficiency (Hashimoto et al., 2024). Accordingly, the functional interpretation and AFI-axis remodeling reported here should be considered male-specific and not assumed to generalize across sexes. Future studies with larger cohorts, inclusion of female mice, and multiple post-injury time points will be essential to define robustness, inter-individual variability, and the evolution of AFI-axis changes across the acute-to-chronic transition. Furthermore, our work is primarily descriptive and correlative: we did not directly manipulate AFI-axis components—for example, via gene knockdown or pharmacological blockade—to establish causal links between AFI reprogramming and functional changes in the bladder or kidney. In addition, because fibrosis/inflammation markers in bladder and kidney (e.g., collagen/fibronectin/α-SMA or inflammatory cytokine and immune-cell infiltration readouts) were not assessed, the functional relevance of AFI-axis remodeling to these downstream pathophysiological processes should be interpreted with caution. Because vesicoureteral reflux/hydronephrosis, urodynamic parameters, and renal functional indices were not directly assessed, we cannot discriminate whether the renal changes are direct consequences of SCI or secondary to bladder dysfunction. And the kidney assessment was limited to gross inspection, and dedicated renal pathology readouts (e.g., histology for subtle hydronephrosis, tubular injury, or fibrosis) were not performed; therefore, microscopic renal changes cannot be excluded. In addition, technical constraints prevented detection of INHBE protein in the bladder by Western blot, reliable assessment of ACVR1B protein in the kidney, and immunofluorescent validation of INHBC localization in either organ. Functional evaluation of bladder performance was largely based on voiding spot assays and organ weights, and although the voiding spot assay provides a practical, noninvasive readout of spontaneous voiding behavior, it does not provide pressure–flow information and cannot distinguish specific urodynamic mechanisms underlying neurogenic lower urinary tract dysfunction (e.g., detrusor overactivity, detrusor–sphincter dyssynergia, or impaired contractility with retention). We therefore describe an overflow incontinence–like voiding pattern as an interpretation based on convergent VSA features (leakage/tailing and altered spot morphology) together with gross evidence of bladder distension and increased bladder weight, while acknowledging that definitive classification would require cystometry (± EUS electromyography) and objective post-void residual quantification. For clarity, this terminology is used as an operational, descriptive analogy to convey an incomplete-emptying–type behavior (overdistended bladder with dribbling/leakage), rather than a urodynamically confirmed diagnosis. Moreover, because pressure–flow parameters and sphincter activity were not measured in the present study, we cannot determine whether detrusor overactivity, reflex/high-pressure voiding, or detrusor–sphincter dyssynergia contributed to the observed VSA patterns. Future studies incorporating longitudinal urodynamics, single-cell or spatial transcriptomics, and targeted interventions against specific AFI-axis members will be essential to clarify how this signaling axis contributes to the onset and progression of neurogenic bladder.

In summary, this study demonstrates that SCI-related overflow incontinence is accompanied by structured remodeling of the AFI axis in both the bladder and kidney, characterized by tissue- and compartment-specific upregulation of FST and INHBB and downregulation of INHA and INHBE. AFI-axis reprogramming thus emerges as a key molecular feature of neurogenic bladder and its associated upper tract adaptations. Components of the AFI axis, including FST and distinct inhibin/activin subunits, represent candidate mechanistic nodes and potential biomarkers, and their therapeutic relevance should be evaluated in future interventional studies with objective functional endpoints.