Microenergy Acoustic Pulse Therapy Reprograms the Multicellular Urethral Microenvironment After Simulated Childbirth Injury: A Single-Cell Transcriptomic Study

1. Introduction

Stress urinary incontinence (SUI) is defined as involuntary urine leakage during physical activity, exertion, coughing, or sneezing [1]. Vaginal delivery and aging are major risk factors for SUI, and the combination of childbirth-related tissue injury, estrogen deprivation, and age-related loss of regenerative capacity may contribute to persistent urethral dysfunction [2,3,4]. Current treatment is centered on pelvic floor muscle training, urethral bulking, and surgery; however, therapies that directly restore the damaged urethral microenvironment remain limited [5,6].Urethral continence depends on coordinated interactions among smooth muscle, the external striated urethral sphincter, connective tissue, vasculature, peripheral nerves, immune cells, and the urothelial barrier [7,8,9]. Vaginal distension models show that simulated childbirth injury can disrupt sphincter structure and extracellular matrix integrity, whereas ovariectomy and inhibition of collagen crosslinking can further aggravate tissue damage [10,11]. Our previous single-cell study further suggested that aging redirects urethral repair from coordinated regeneration toward muscle loss, denervation-related transcriptional changes, fibroblast activation, inflammation, extracellular matrix deposition, and urothelial senescence.

Microenergy acoustic pulse (MAP) is a low-intensity mechanical stimulation therapy designed to activate endogenous tissue repair without thermal ablation. In animal models of SUI, MAP improves leak point pressure and restores pelvic floor and urethral muscle structure, accompanied by activation of resident progenitor cells and enhancement of repair-related signaling [12]. However, the effects of MAP on individual urethral cell populations and multicellular communication networks remain unclear.

Single-cell RNA sequencing (scRNA-seq) can resolve rare cell states in complex tissues and infer ligand-receptor communication within the urethral microenvironment [13,14,15,16]. This exploratory study integrated single-cell data from eight animals representing aging, injury, fibrosis-prone injury, and MAP treatment states. We conceptualized the urethra as a dynamic ecosystem maintained by epithelial, vascular, immune, stromal, neural, and muscle cells and hypothesized that MAP promotes repair not by acting on muscle alone, but through three interrelated levels: reshaping cellular community structure, reconnecting intercellular communication networks, and improving intrinsic regenerative, senescence, and stress states.

2. Results

2.1. The Single-Cell Atlas Resolves a Complex Multicellular Urethral Ecosystem

The integrated dataset comprised 51,690 quality-controlled urethral cells from eight animals. UMAP visualization showed broad integration across individual samples, treatment groups, and ages while preserving biologically distinct cell populations. Unsupervised analysis identified 20 transcriptionally distinct clusters spanning ciliated, luminal, secretory, glandular, squamous, basal, tuft/chemosensory, neuroendocrine, and epithelial/neural-like states, together with endothelial, lymphatic endothelial, fibroblast, stromal/vascular-associated, immune, smooth muscle, striated muscle, and high-mitochondrial/stressed populations. These findings establish the urethra as a multicellular tissue ecosystem rather than an isolated muscular conduit (Figure 1A-D).

2.2. Injury and Aging Are Associated with Multicellular Repair Failure

Comparison of age and injury conditions indicated a shift from coordinated homeostasis toward a maladaptive repair state. Muscle populations showed reduced regenerative and neuron-projection-associated programs, consistent with impaired neuromuscular integrity. Fibroblast and stromal populations were enriched for extracellular-structure organization, growth-factor responses, and wound-remodeling programs, whereas immune populations showed inflammatory activation. Differentiated urothelial features were reduced and senescence-associated activity increased in selected epithelial states. Thus, childbirth-like injury, estrogen deprivation, fibrosis-prone remodeling, and aging were associated with coordinated disturbances involving muscle, matrix, vasculature, immunity, and epithelial barrier function (Figure 1D).

2.3. MAP Remodels Cellular Composition and Restores Urethral Muscle Content

Cell-type composition differed across sham, VBDO, BAPN, and MAP tissues. VBDO and BAPN altered the relative abundance of epithelial, endothelial, lymphatic endothelial, fibroblast, immune, stromal/vascular-associated, smooth-muscle, and striated-muscle states. MAP did not simply reproduce the sham distribution; instead, it generated a distinct cellular configuration consistent with active multicellular remodeling (Figure 2A,B). Histologic examination supported this transcriptomic pattern. Pha/α-SMA staining showed marked disruption and loss of urethral muscle after VBDO and BAPN, whereas MAP-treated tissues displayed improved circumferential organization and greater pha-positive striated muscle and α-SMA-positive smooth muscle content. Quantification confirmed a significant recovery of muscle content with MAP relative to the injured groups, although restoration was incomplete compared with sham (Figure 2C,D).

2.4. MAP Alters Signaling Information Flow and Biological Repair Programs

CellChat-based information-flow comparisons demonstrated that MAP generated a signaling profile distinct from sham, VBDO, and BAPN (Figure 3A). MAP-associated increases involved extracellular-matrix and basement-membrane pathways, cell adhesion and junctional signaling, vascular remodeling, developmental regulation, and neural communication. Representative pathways included COLLAGEN, LAMININ, FN1, ANGPT, VEGF, PECAM1, CDH5, CLDN, NECTIN, JAM, WNT/ncWNT, TGFβ, BMP, RELN, SLIT, NCAM, NRXN, MPZ, and semaphorin signaling. In the MAP-versus-BAPN comparison, LAMININ- and FN1-associated information flow was particularly prominent, suggesting enhanced cell-matrix and basement-membrane communication (Figure 3B). Gene Ontology analysis showed that MAP-upregulated genes were enriched in cytoplasmic translation, oxidative energy metabolism, cellular respiration, Golgi-mediated transport, reactive oxygen species handling, postsynaptic organization, synapse regulation, and apoptotic-signal regulation. Conversely, genes relatively reduced after MAP were enriched in RNA processing, steroid-hormone responses, mesenchymal development, developmental growth, and persistent wound-remodeling programs. Together, these changes are consistent with a transition from prolonged injury signaling toward metabolically competent, stress-adapted, and structurally organized repair (Figure 3C).

2.5. MAP Reorganizes Epithelial-Stromal-Vascular-Muscle Communication

Differential CellChat networks comparing MAP with BAPN revealed broad changes in both the number and aggregate strength of predicted ligand-receptor interactions (Figure 4A). The largest network differences involved endothelial and lymphatic endothelial cells, fibroblasts, immune cells, luminal and secretory epithelial populations, and smooth and striated muscle. Incoming-signaling heatmaps showed that MAP changed the receptor-associated signals received by multiple cell populations, including muscle compartments, whereas outgoing-signaling heatmaps showed parallel changes in ligand-associated signals transmitted to surrounding vascular, immune, epithelial, and matrix-associated cells (Figure 4B,C). The distributed nature of these changes indicates that MAP did not act through a single dominant sender, receiver, or pathway but instead reorganized bidirectional communication across the urethral microenvironment.

2.6. MAP Partially Restores Stem/Progenitor-Associated Cellular States

Stem/progenitor-associated markers displayed lineage-restricted expression across the urethral atlas (Figure 5A). Epithelial progenitor-like populations expressed markers including Krt15 and Sox9, muscle-associated cells expressed Itga7, and vascular/stromal repair populations expressed Cd34 and Met. The combined stem/progenitor-like cell fraction was lower after VBDO than in sham tissues. BAPN showed limited recovery, whereas MAP was associated with a modest increase in this fraction (Figure 5B). Treatment-level marker analysis further showed preservation or re-expression of selected repair-associated genes, particularly Krt15 and Met, in MAP-treated tissues (Figure 5C). These observations support partial maintenance or reactivation of endogenous regenerative states but do not establish lineage expansion or complete regeneration.

2.7. MAP Selectively Modulates SASP Activity Across Urethral Lineages

SASP module scores varied substantially by cell type and treatment (Figure 5D). Relatively high activity occurred in selected squamous/basal epithelial, fibroblast, epithelial/neural-like, neuroendocrine/chemosensory, and muscle-associated populations, whereas several endothelial, immune, and other epithelial states showed lower scores. MAP did not uniformly suppress SASP across all lineages. Instead, the pattern was consistent with selective reduction of detrimental stress- and inflammation-associated activity in vascular, immune, smooth-muscle, and striated-muscle compartments while preserving epithelial and stromal remodeling signals that may contribute to repair. The schematic model summarizes this lineage-specific balance between limiting chronic injury signaling and maintaining repair-associated communication (Figure 5E).

2.8. MAP is Associated with Reduced Ndrg1 Expression in Urethral Striated Muscle

Ndrg1 expression was distributed across selected urethral populations and was most evident in particular epithelial- and muscle-associated states (Figure 6A,B). Within the striated-muscle compartment, mean Ndrg1 expression was lower in MAP-treated tissue than in BAPN and sham tissue (Figure 6C). Because VBDO showed a different, non-monotonic pattern and Ndrg1 has context-dependent roles in hypoxia adaptation, differentiation, and tissue protection, this observation should not be interpreted as a simple dose-response relationship or as evidence that Ndrg1 is uniformly detrimental. Rather, the MAP-versus-BAPN reduction is consistent with attenuation of a persistent stress-associated state in the fibrosis-prone injured muscle compartment. The proposed model links this change to improved metabolic adaptation, synaptic repair, oxidative-stress handling, and reduced maladaptive muscle remodeling (Figure 6D).

3. Discussion

3.1. Restoring the Urethral Ecosystem

For decades, stress urinary incontinence (SUI) has often been viewed as a relatively simple mechanical “plumbing failure,” primarily attributed to lax urethral support or insufficient sphincter contraction, with treatment therefore focused on tightening, supporting, or repairing damaged tissues. Single-cell transcriptomics, however, reveals a far more complex picture. The urethra is not a passive conduit composed of a single muscular structure; rather, it is a dynamic tissue ecosystem maintained by epithelial, vascular, immune, stromal, neural, and muscle cells. SUI, particularly when associated with aging and childbirth injury, may therefore represent widespread dysfunction of this multicellular ecosystem rather than an isolated mechanical failure of muscle.

In this study, scRNA-seq captured active transcriptional programs in individual cells, and UMAP grouped cells with similar expression profiles into distinct clusters, enabling construction of a high-resolution cellular atlas of the rat urethra. The integrated analysis identified 20 transcriptionally distinct populations spanning epithelial, endothelial, lymphatic endothelial, immune, fibroblast, stromal/vascular-associated, smooth muscle, striated muscle, neuroendocrine, and stressed cell states. This atlas not only describes the cellular composition of the urethra but also provides a framework for tracking how the abundance, state, and communication of each cell type change after injury, aging, and treatment.

From an ecosystem perspective, MAP may promote tissue repair at four interrelated levels. First, MAP reshapes cellular community structure. Composition analysis showed redistribution of epithelial, stromal, vascular, immune, and muscle-associated populations in MAP relative to VBDO and BAPN. These changes did not represent a simple return of all populations to sham proportions; rather, they suggested formation of a distinct repair-associated cellular configuration, indicating that MAP may alter which cells are present in injured tissue and the relative balance among lineages. Second, MAP reconnects intercellular communication networks. CellChat analysis showed broad changes in incoming and outgoing signaling across epithelial, endothelial, lymphatic endothelial, immune, fibroblast, and muscle populations. Reorganized pathways involved ECM and basement membrane signaling, cell adhesion, angiogenesis, neural guidance, and neuromuscular communication. In other words, MAP not only changes cellular composition but may also alter the biological instructions that cells send and receive, enabling vascular, immune, matrix, epithelial, and muscle compartments to coordinate tissue repair in new ways. Third, MAP improves selected cell states. Injured cells may become trapped in persistent stress or senescence and release inflammatory and matrix-remodeling mediators through the SASP. Our findings suggest that MAP does not globally suppress all SASP activity; instead, it reduces senescence-associated stress in selected vascular and striated-muscle compartments while preserving signals in epithelial and stromal cells that may be required for repair. Ndrg1 is a critical gene for muscle regeneration[17]. Lower Ndrg1 expression in MAP than in BAPN striated muscle was also consistent with reduced persistent injury stress. By dampening maladaptive “alarm” signaling while preserving beneficial repair cues, MAP may create a microenvironment more permissive for re-entry into regenerative programs.

As cellular composition, muscle structure, communication networks, and cell states are remodeled simultaneously, endogenous repair potential may also recover. The MAP group showed a modest increase in the stem/progenitor-like fraction and increased expression of repair-associated markers including Krt15 and Met. Although these changes are insufficient to demonstrate lineage expansion or complete tissue regeneration, they support a biologically coherent model in which MAP shifts the damaged urethra from a state dominated by persistent inflammation, fibrosis, and stress toward one that is more favorable for progenitor survival, differentiation, and tissue integration.

The central implication of this study is therefore not that MAP simply “strengthens muscle,” [18] but that it offers a broader therapeutic framework: treating the urethra as a living, interdependent cellular ecosystem and promoting global repair by simultaneously modulating cellular community structure, intercellular communication, and intrinsic cell states. It is important to emphasize that the present findings are derived mainly from exploratory single-cell data and do not establish complete biological recovery after MAP. Nevertheless, they provide clear, testable directions for future functional, histologic, and mechanistic validation.

3.2. MAP-Mediated Multicellular Repair Mechanisms and Translational Implications

This exploratory single-cell study extends SUI from a disorder of the sphincter alone to a failure of multicellular tissue repair[16]. By integrating four treatment states and two ages, we found that MAP was associated with broad reorganization of the injured urethral microenvironment. MAP altered cellular composition, restored urethral muscle content, and enhanced communication pathways related to ECM and basement membrane organization, vascular support, cell adhesion, neural guidance, and synaptic structure, and partially restored progenitor-like states. These findings provide a potential mechanistic explanation for the functional and structural improvements previously observed after MAP treatment in animal models of SUI [12].

Urethral sphincter function depends on both muscle integrity and coordinated neuromuscular activation. Age-associated reduction in neuron-projection programs and neuromuscular junctions may contribute to persistent SUI. In the present study, MAP enriched RELN, SLIT, semaphorin, NCAM, NRXN, MPZ, glutamate-related signaling, and postsynaptic organization programs. These pathways are involved in axon guidance, synaptic adhesion, Schwann cell/myelin-associated communication, and neuromuscular patterning. MAP may therefore support the neural component of sphincter repair in addition to activating muscle progenitors. This hypothesis requires validation through nerve tracing, electrophysiology, and quantitative NMJ analysis in independent MAP-treated cohorts.

MAP also markedly altered ECM and cell-adhesion signaling. Increased collagen and laminin information flow should not be interpreted simply as promotion of fibrosis. During tissue healing, these ligand-receptor systems may reflect basement membrane repair, matrix-guided cell migration, vascular stabilization, and muscle attachment. Concurrent enhancement of FN1, cadherin, nectin, claudin, JAM, and PECAM1/CDH5 pathways further suggests coordinated structural remodeling. Notably, GO analysis showed reduced persistent wound-healing and mesenchymal-development programs together with enhanced oxidative metabolism and stress adaptation, a combination more consistent with maturation of repair than with uncontrolled fibrosis. Nevertheless, collagen organization and mechanical properties require histologic and biochemical validation.

Vascular signaling may represent another important mechanism. ANGPT, VEGF, PECAM1, and CDH5 pathways were enriched in selected MAP comparisons, and endothelial and lymphatic endothelial cells occupied prominent positions in differential communication networks. Adequate perfusion is essential for progenitor activation, muscle metabolism, immune resolution, and matrix turnover. Mechanical stimulation may therefore act in part by improving the vascular niche that supports urethral regeneration.

The modest recovery of stem/progenitor-like populations provides a cellular link to previous observations that MAP activates resident muscle progenitors [7,12]. Krt15, Met, Sox9, Itga7, and Cd34 marked distinct epithelial, muscle, and stromal/vascular repair populations. MAP did not fully restore the overall progenitor fraction to the sham level, suggesting partial reactivation rather than complete reversal of injury. Tissue repair depends not only on progenitor abundance but also on microenvironmental signals that regulate survival, differentiation, and integration. Changes in WNT, NOTCH, growth-factor, ECM, and vascular communication may therefore be as important as changes in stem-like cell proportions.

SASP signaling was also strongly lineage dependent. Global suppression of SASP may not necessarily be beneficial because transient inflammatory and matrix-remodeling signals are required for tissue repair. MAP reduced stress-related activity in selected vascular and striated-muscle populations while preserving repair-associated remodeling programs in epithelial and stromal compartments [16]. This pattern supports selective reprogramming rather than global suppression. Lower Ndrg1 expression in MAP versus BAPN striated muscle was consistent with reduced persistent stress; however, Ndrg1 has context-dependent roles in differentiation, hypoxia adaptation, and tissue protection and should not be regarded as a stand-alone marker of fibrosis.

The translational appeal of MAP lies in its noninvasive nature and its potential to act on several pathologic compartments simultaneously. Older patients with SUI may have muscle atrophy, denervation, vascular insufficiency, fibrosis, immune dysregulation, and urothelial dysfunction. A treatment directed only at muscle contraction may fail to correct these interacting defects. The present findings suggest that mechanical stimulation may promote a more coordinated regenerative niche, although efficacy, dose-response relationships, durability, and safety require evaluation in adequately powered studies.

This study has important limitations. First, the transcriptomic cohort included only eight animals, with one animal in each age-by-treatment condition. The large number of cells obtained from each animal does not constitute independent biological replication; therefore, formal treatment efficacy or age-by-treatment interactions cannot be inferred. Second, CellChat infers communication from transcript abundance and curated ligand-receptor databases and does not demonstrate protein-level signaling or physical interaction. Third, stem/progenitor and SASP classifications depend on gene-set definitions and require lineage tracing and orthogonal validation. Fourth, the MAP group lacked independent functional and histologic replication within the present dataset. Finally, balanced downsampling may improve comparability but reduce detection of rare populations. These results should therefore be considered hypothesis-generating evidence to guide targeted validation.

4. Materials and Methods

4.1. Animals and Experimental Design

All animal procedures were conducted in accordance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco. This scRNA-seq study included eight female Sprague-Dawley rats. Animals were assigned to four groups: sham control, vaginal balloon dilation plus ovariectomy (VBDO), VBDO plus beta-aminopropionitrile (BAPN), and VBDO plus MAP treatment. Each group contained one 24-week-old and one 48-week-old animal, yielding a 4-treatment × 2-age experimental design (Table 1).

Sham animals underwent anesthesia, vaginal catheter insertion, and surgical preparation without balloon dilation or ovariectomy. Simulated childbirth injury was induced using an 18-Fr Foley balloon filled with 4 mL of water and maintained under a 130-g load for 4 h; bilateral ovariectomy was performed 1 week later[11,12]. BAPN-treated animals received intraperitoneal BAPN at 300 mg/kg twice weekly for 4 weeks, followed by a 1-week washout. MAP was applied to the pelvic floor/urethral region using previously established parameters: an energy flux density of 0.033 mJ/mm², 3 Hz, 500 pulses per session, twice weekly for 4 weeks [12]. Tissues were collected after completion of treatment.

4.2. Single-Cell RNA Sequencing and Data Preprocessing

Fresh urethral tissue was enzymatically dissociated to generate single-cell suspensions. Libraries were constructed using the 10x Genomics Chromium Single Cell 3′ platform and sequenced on an Illumina NovaSeq instrument. Raw reads were aligned to the Rattus norvegicus reference genome using Cell Ranger. Cells with 500-9,000 detected genes, fewer than 25,000 unique molecular identifiers, and less than 20% mitochondrial transcripts were retained. Doublets, low-quality droplets, and cells without reliable annotation were removed. Seurat was used for normalization, variable-feature selection, integration, principal-component analysis, neighborhood construction, clustering, and UMAP visualization. The complete quality-controlled dataset contained 51,690 cells. This approach reduced domination by samples with higher cell recovery but did not increase the number of biological replicates.

4.3. Cell-Type Annotation and Differential Expression Analysis

Clusters were annotated using canonical markers and differentially expressed marker genes. Reanalysis resolved 20 transcriptionally distinct clusters, including ciliated epithelial, luminal urothelial, secretory epithelial, secretory/glandular epithelial, squamous epithelial, squamous/basal epithelial, tuft/chemosensory epithelial, neuroendocrine/chemosensory, epithelial/neural-like, neural/epithelial-like, endothelial, endothelial subtype, lymphatic endothelial, fibroblast, stromal/vascular-associated, immune, smooth muscle, striated muscle, and high-mitochondrial/stressed populations.Differentially expressed genes were identified using the Wilcoxon rank-sum test in Seurat. GO enrichment analysis was performed for genes increased or decreased in MAP relative to BAPN. Because each age-by-treatment condition was represented by one animal, enrichment results were interpreted as exploratory.

4.4. Stem/Progenitor and SASP Analyses

Stem/progenitor-like states were assessed using a prespecified marker panel spanning epithelial, stromal, and muscle progenitor programs. Representative genes included Krt15, Met, Sox9, Itga7, Cd34, Lgr5, and Ccnc1. Cells meeting the predefined combined marker-expression criterion were classified as stem/progenitor-like, and their proportions were summarized by treatment group.A core SASP module score was calculated for each cell using Seurat module scoring and summarized by cell type and treatment group. Because SASP programs differ among lineages and may contain both maladaptive inflammatory signals and repair-associated remodeling signals, changes were interpreted in a cell type-specific manner rather than as a uniform global effect.

4.5. Cell-Cell Communication Analysis

CellChat was used to infer ligand-receptor communication among major urethral cell populations and to compare MAP with sham, VBDO, and BAPN. Outcomes included the number and aggregate strength of predicted interactions, incoming and outgoing signaling patterns, and relative information flow across signaling families. Pathways of particular interest included ECM and basement membrane signaling (COLLAGEN, LAMININ, FN1), angiogenic signaling (ANGPT, VEGF), adhesion and junctional signaling (CDH, JAM, NECTIN, CLDN, PECAM1), developmental and regenerative signaling (WNT, ncWNT, NOTCH, EGF, FGF, TGFβ), and neural/neuromuscular communication (AGRN, NCAM, NRXN, RELN, SLIT, semaphorins, Netrin, MPZ, and glutamate-related signaling).

4.6. Statistical Considerations

The animal was considered the biological unit. This was an exploratory single-cell study with one animal in each age-by-treatment condition. Cell-level tests were used to prioritize candidate genes and pathways rather than to infer population-level treatment efficacy. Accordingly, individual cells were not treated as independent biological replicates, and formal age-by-treatment interaction testing was not performed. Descriptive values, adjusted P values from transcriptomic analyses, and sample-level data points are reported where available. Conclusions are expressed using association-based language and require validation in larger independent cohorts.

5. Conclusions

MAP therapy was associated with coordinated remodeling of the urethral ecosystem after simulated childbirth injury. The updated data support four interrelated effects: redistribution of epithelial, vascular, immune, stromal, and muscle communities; recovery of urethral muscle content; reconnection of matrix, adhesion, angiogenic, neural, and neuromuscular communication networks; and selective improvement of stem/progenitor-associated, SASP, and injury-stress states. These exploratory findings support viewing SUI as a multicellular tissue-ecosystem imbalance and suggest that MAP may shift the urethra from maladaptive remodeling toward a more regenerative state. Independent biological replication, functional testing, histologic validation, and targeted mechanistic studies remain necessary.