兽研Background/Aim
Proteinuria is a key indicator of canine renal disease. While bacterial cystitis causes post-renal proteinuria, the extent to which sporadic bacterial cystitis increases the urine protein-to-creatinine ratio (UPC) is not well defined. This study aimed to quantitatively evaluate the effect of sporadic bacterial cystitis on the canine UPC and determine whether the UPC can distinguish sporadic infection from renal proteinuria.
Materials and Methods
A retrospective study was conducted on dogs evaluated between 2016 and 2025. The study included healthy dogs (n=37), dogs with sporadic bacterial cystitis (n=13), bacterial cystitis with comorbidities (n=65), chronic kidney disease (CKD; n=26), and hyperadrenocorticism (HAC; n=15).
Results
The median UPC in the sporadic bacterial cystitis group (0.20) showed no significant difference compared to healthy dogs (0.20; p=0.392). Conversely, dogs with bacterial cystitis with comorbidities (0.62) and those with CKD or HAC (0.73) exhibited significantly higher UPCs than the sporadic infection group (p<0.001). Distinguishing sporadic infection from renal proteinuria yielded an area under the curve of 0.9373, with an optimal cut-off of 0.425 (100% specificity, 73.2% sensitivity).
Conclusion
Sporadic bacterial cystitis alone does not cause clinically significant increases in the UPC in dogs. A UPC exceeding 0.32 in dogs with cystitis should prompt further investigation for underlying diseases, and a UPC greater than 0.425 suggests renal proteinuria. This approach prevents the misattribution of proteinuria and supports the timely diagnosis of renal diseases.
Introduction
Sporadic bacterial cystitis is a localized, acute lower urinary tract infection that occurs in otherwise healthy dogs and is characterized by transient bladder inflammation caused by opportunistic commensal bacteria ascending the urethra (1). Bacteria responsible for canine urinary tract infections typically originate from the intestinal tract or the skin (2); Escherichia coli (E. coli) is the most frequent species found, with females more commonly affected than males (3).
Proteinuria is a common clinical finding in dogs and is categorized as pre-renal, renal, or post-renal based on the site of origin (4). Pre-renal proteinuria occurs when excessive low-molecular-weight proteins, such as hemoglobin, myoglobin, or immunoglobulin light chains, circulate in the bloodstream (5). Renal proteinuria develops in association with glomerular barrier dysfunction or impaired tubular reabsorption (6), whereas post-renal proteinuria results from the entry of protein into the urine distal to the renal pelvis due to inflammatory, infectious, hemorrhagic, or neoplastic processes, or urolithiasis (5). Renal proteinuria represents both a diagnostic and prognostic marker in dogs and cats, with higher magnitudes correlating with greater risks of renal disease progression and mortality (7, 8). Accurate identification of renal proteinuria is essential, as persistent renal proteinuria itself can contribute to progressive kidney injury in dogs (4).
When assessing proteinuria in dogs, post-renal extra-urinary causes should first be excluded by obtaining urine via cystocentesis (1), because urine contamination during voiding from the distal part of the urethra, the vagina, or the prepuce can occur (3). Pre-renal causes can be excluded by evaluating plasma protein concentrations (4). Assessment of renal proteinuria is recommended after pre-renal and post-renal causes have been excluded (4). Although it is well recognized that post-renal disorders like bacterial cystitis induce proteinuria, the extent to which sporadic bacterial cystitis alone in dogs increases the urine protein-to-creatinine ratio (UPC) remains poorly defined.
Previous studies have shown that bacteriuria is a poor predictor of proteinuria, with positive urine cultures being neither associated with increased UPC nor showing meaningful reductions in UPC after resolution (9, 10). Even marked bacteriuria did not correlate with UPC magnitude (9), indicating no causal relationship between bacteriuria and UPC. Although an experimental model demonstrated substantial UPC increases after chemically induced cystitis, the effects of mucosal injury could not be distinguished from those of infection (11). Consequently, the degree to which naturally occurring sporadic bacterial cystitis contributes to elevations in UPC remains unknown.
This study aimed to quantitatively evaluate the effect of sporadic bacterial cystitis on UPC in dogs; to compare UPC values among dogs with sporadic bacterial cystitis, bacterial cystitis with comorbidities, and healthy dogs; and to determine the extent to which UPC elevations in dogs may distinguish sporadic bacterial cystitis from chronic kidney disease (CKD) and hyperadrenocorticism (HAC), which are well-established causes of renal proteinuria (4, 6-8).
Materials and Methods
Animals. This retrospective study reviewed the medical records of dogs evaluated at the Veterinary Teaching Hospital of Chungbuk National University between June 2016 and August 2025. For the comparison of dogs with sporadic bacterial cystitis, healthy dogs, and dogs with bacterial cystitis with comorbidities (Study 1), the study population included 37 healthy dogs, 13 dogs with sporadic bacterial cystitis, and 65 dogs with bacterial cystitis with comorbidities. For the comparison of dogs with sporadic bacterial cystitis, CKD, and HAC, which are major causes of renal proteinuria (Study 2), only dogs with UPC ≥0.2 were included to evaluate whether the magnitude of UPC elevation in dogs classified as having borderline proteinuria or proteinuria could help distinguish renal proteinuria from proteinuria attributed to cystitis.
The diagnosis of sporadic bacterial cystitis was established according to the 2019 International Society for Companion Animal Infectious Diseases guidelines (1), which required the presence of lower urinary tract signs such as pollakiuria, dysuria, stranguria, and hematuria, a positive aerobic urine culture, and urinalysis performed on urine collected by cystocentesis unless contraindicated. Sporadic bacterial cystitis was defined as an infection in otherwise healthy dogs without known urinary tract anatomical or functional abnormalities or relevant comorbidities such as systemic hypertension (systolic blood pressure>160 mmHg) (12), and occurring fewer than three times within the preceding 12 months.
CKD was diagnosed based on the International Renal Interest Society CKD guidelines (12), using clinical history, physical examination, urinalysis, blood analysis, and imaging findings.
The diagnosis of HAC was made according to previously established diagnostic criteria (13-15). HAC was suspected based on the patient’s history, physical examination, and urinalysis and blood analysis at admission. HAC was diagnosed by either a low-dose dexamethasone suppression test or an adrenocorticotropic hormone stimulation test.
Healthy dogs were defined as those with no clinical, physical, laboratory, or imaging abnormalities and without any diseases or conditions that could be considered pathological. Dogs with bacterial cystitis with comorbidities were defined as those exhibiting lower urinary tract signs and a positive aerobic urine culture, with urine collected by cystocentesis unless contraindicated, and accompanied by urinary tract anatomical or functional abnormalities or relevant comorbidities such as endocrinopathies. Dogs were excluded from the sporadic bacterial cystitis group if they had any conditions that could induce pre-renal, renal, or post-renal proteinuria. Exclusion criteria included systemic hypertension (systolic blood pressure>160 mmHg), systemic inflammation, neoplasia, immune-mediated disease, cardiac disease, potential underlying endocrine disease, recent treatment with antibiotics, anti-inflammatory drugs, or immunosuppressive medications within the preceding month, polypoid cystitis, pyelonephritis, or diabetes mellitus (4, 5).
This study was approved by the Institutional Animal Care and Use Committee of Chungbuk National University (approved number CBNUA-25-0156-02). The overall flowchart of dog inclusion and exclusion criteria, as well as group allocation for Study 1 and Study 2, is presented in Figure 1.
[图：Study flowchart illustrating dog selection and grouping for Study 1 and Study 2. Out of 207 dogs evaluated between 2016 ]
Review of medical records. Clinical findings, including physical examination, complete blood counts, serum biochemistry profiles, electrolyte measurements, urinalysis results, radiographic imaging, and abdominal ultrasonographic evaluations were reviewed comprehensively to determine eligibility for inclusion within each diagnostic group. The following data were extracted from the medical records of dogs: age, breed, sex, systolic blood pressure, urine dipstick results, urine sediment findings, and UPC.
Urinalysis. Urine samples were collected by cystocentesis in all dogs included in this study. Complete urinalysis was performed using a dipstick (IDEXX UA Strips, IDEXX Laboratories, Inc., Westbrook, ME, USA), and urine sediment examination was conducted with an automated analyzer (IDEXX SediVue Dx, IDEXX Laboratories, Inc.). For measurement of the UPC, urine samples were centrifuged at 1,500 rpm for 5 minutes, and only the supernatant was analyzed. The UPC was automatically determined using a biochemical analyzer (IDEXX Catalyst One; IDEXX Laboratories, Inc.).
UPC values were interpreted according to the manufacturer’s guidelines. When either urine protein or urine creatinine values exceeded the reportable analytical range, the UPC was calculated based on the medical relevance of proteinuria in the patient. In cases where both urine protein and urine creatinine exceeded the measurable range, samples were manually diluted at a 1:2 ratio prior to repeat analysis.
Aerobic urine cultures were performed for all dogs. Samples were inoculated onto blood agar plates, and culture results were considered positive when bacterial colonies were observed after incubation under aerobic conditions.
Each individual dog served as a unique biological replicate (n=37 for Healthy, n=13 for sporadic bacterial cystitis, and n=65 for bacterial cystitis with comorbidities in Study 1; n=7 for sporadic bacterial cystitis and n=41 for CKD or HAC in Study 2). Given the clinical nature of the sample collection from client-owned dogs, laboratory measurements were conducted once per sample in accordance with standardized clinical diagnostic protocols. Analytical precision was assured through daily quality control checks and regular calibration of all automated analyzers according to the manufacturers’ instructions.
Statistical analysis. All statistical analyses were performed using commercial software. Statistical tests were conducted with SPSS version 31 for Windows (IBM Corp., Armonk, NY, USA), and figures and receiver operating characteristic (ROC) curves were generated using Prism 10.0 (GraphPad Software Inc., La Jolla, CA, USA). The Shapiro-Wilk test was used to assess the normality of continuous variables; all data were determined to be non-normally distributed. Continuous variables were expressed as medians with interquartile ranges (IQR).
For Study 1, comparisons among the three groups (healthy, sporadic bacterial cystitis, and bacterial cystitis with comorbidities) were performed using the Kruskal-Wallis test, followed by Dunn’s post-hoc test. ROC curve analysis was used to evaluate the ability of UPC to differentiate sporadic bacterial cystitis from bacterial cystitis with comorbidities, and the area under the curve (AUC) was calculated. The optimal cut-off value was determined using the Youden index, and the corresponding sensitivity and specificity were obtained.
For Study 2, comparisons between two groups (sporadic bacterial cystitis vs. CKD + HAC) were performed using the Mann-Whitney U test. Comparisons among the three groups (sporadic bacterial cystitis, CKD, and HAC) were performed using the Kruskal-Wallis test with Dunn’s post-hoc test. ROC curve analyses were additionally conducted to assess the discriminatory ability of UPC for sporadic bacterial cystitis versus CKD + HAC, sporadic bacterial cystitis versus CKD, and sporadic bacterial cystitis versus HAC. The AUC, Youden index-derived optimal cut-off values, sensitivity, and specificity were calculated for each ROC curve.
A p-value <0.05 was considered statistically significant. For statistical analyses, censored UPC values (e.g., <0.20) were replaced with their respective boundary values (e.g., 0.20).
Results
Study 1 population. A total of 115 dogs were included in Study 1. Thirty-seven healthy dogs were included. Of the 129 dogs initially identified as having bacterial cystitis, 51 were excluded because a complete urinalysis had not been performed. A total of 78 dogs were diagnosed with bacterial cystitis; among them, 13 (17%) were classified as having sporadic bacterial cystitis, whereas 65 (83%) had concurrent underlying diseases or conditions that could influence cystitis and were therefore categorized as bacterial cystitis with comorbidities. Demographic data for the three groups are presented in Table I. The bacterial cystitis with comorbidities group represented an older population with a median age of 12 years, compared to 7 years for both the healthy and sporadic bacterial cystitis groups. Sex distribution varied by group; the sporadic bacterial cystitis group consisted mostly of females, while the healthy group included a larger proportion of intact males. Body weights were similar across all groups.
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Study 1 - Comparison of UPCs in three groups of dogs: healthy, sporadic bacterial cystitis, and bacterial cystitis with comorbidities. UPCs were measured in healthy dogs, dogs with sporadic bacterial cystitis, and dogs with bacterial cystitis with comorbidities. In healthy dogs (n=37), the median UPC was 0.2000 (IQR=0.0550-0.2000). Dogs with sporadic bacterial cystitis (n=13) had UPCs of 0.2000 (IQR=0.0900-0.2250) and dogs with bacterial cystitis with comorbidities (n=65) had UPCs of 0.6200 (IQR=0.3050-2.0800). UPCs in dogs with bacterial cystitis with comorbidities were significantly higher than those in dogs with sporadic bacterial cystitis (p<0.001) and healthy dogs (p<0.0001). However, there was no significant difference between healthy dogs and dogs with sporadic bacterial cystitis (p=0.392) (Figure 2).
[图：Comparison of UPC values among healthy dogs, dogs with SBC, and dogs with BCC (Study 1). Significant differences in UPC ]
Study 1 - ROC curve analysis for differentiating sporadic bacterial cystitis from bacterial cystitis with comorbidities. The ROC curve analysis was performed to evaluate the performance of UPC in distinguishing sporadic bacterial cystitis from bacterial cystitis with comorbidities. The AUC was 0.8834 [95% confidence interval (CI)=0.8061-0.9608; p<0.0001]. Based on this curve, the optimal UPC cut-off value was 0.3200, which yielded a specificity of 92.31% and a sensitivity of 73.85% (Figure 3).
[图：ROC curve analysis of UPC for differentiating SBC from BCC in dogs (Study 1). The AUC was 0.8834 (95% CI=0.8061-0.9608).]
Study 2 population. A total of 48 dogs were included in Study 2, consisting of 7 dogs with sporadic bacterial cystitis, 26 dogs with CKD, and 15 dogs with HAC. Demographic data for the dogs included in Study 2 are summarized in Table II. The CKD and HAC groups represented older populations with median ages of 13 and 11 years, respectively, compared to 7 years for the sporadic bacterial cystitis group. Regarding body weight, the CKD group (median, 3.9 kg) was notably lighter than the sporadic bacterial cystitis and HAC groups. Sex distribution also varied; the sporadic bacterial cystitis group was exclusively female, whereas the CKD group comprised a higher proportion of males compared to the predominantly female HAC group.
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Study 2 - Comparison of UPCs in three groups of dogs: sporadic bacterial cystitis, CKD and HAC. UPCs were measured in dogs with sporadic bacterial cystitis, CKD, and HAC, and dogs with UPC≥0.2 were included. When analyzing the combined CKD and HAC groups, dogs with sporadic bacterial cystitis (n=7) had a significantly lower median UPC (0.2000; IQR=0.2000-0.3100) than those in the CKD + HAC group (n=41; 0.7300; IQR=0.3750-1.5550; p<0.0001) (Figure 4A). When evaluating the CKD and HAC groups independently, both demonstrated significantly higher UPCs compared to the sporadic bacterial cystitis group. The median UPC was 0.6650 for dogs with CKD (n=26; IQR=0.3400-1.1175; p<0.01) and 1.1900 for dogs with HAC (n=15; IQR=0.5100-2.0800; p<0.001). No significant difference was observed between the CKD and HAC groups (p=0.167) (Figure 4B).
[图：Comparison of UPC values among dogs with SBC, CKD, and HAC (Study 2). (A) Significant differences in UPC were observed b]
Study 2 - ROC curve analysis for distinguishing sporadic bacterial cystitis from CKD and HAC. ROC curve analysis demonstrated the diagnostic performance of UPC in distinguishing sporadic bacterial cystitis from CKD and HAC. The AUCs and optimal cut-off values were as follows:
- Sporadic vs. Combined (CKD+HAC): AUC 0.9373 (95% CI=0.8572-1.000); optimal cut-off 0.4250 (73.17% sensitivity, 100% specificity) (Figure 5A).
[图：ROC curve analysis of UPC for differentiating SBC from CKD and HAC in dogs (Study 2). (A) For the comparison between SBC]
- Sporadic vs. CKD: AUC 0.9231 (95% CI=0.8195-1.000); optimal cut-off 0.2600 (96.15% sensitivity, 71.43% specificity) (Figure 5B).
- Sporadic vs. HAC: AUC 0.9619 (95% CI=0.8897-1.000); optimal cut-off 0.4400 (86.67% sensitivity, 100% specificity) (Figure 5C).
Discussion
In this study, we compared UPCs between dogs with sporadic bacterial cystitis – after excluding all other potential causes of proteinuria, and those with other diseases to quantitatively assess the extent to which sporadic bacterial cystitis influences UPC in dogs. First, no significant difference in UPC was observed between dogs with sporadic bacterial cystitis and healthy controls, whereas dogs with proteinuria associated with CKD, HAC, or bacterial cystitis with comorbidities showed significantly higher UPC values than those with sporadic bacterial cystitis. The optimal cut-off values derived from the ROC curves allowed clear discrimination between the groups based on UPC. The UPC serves as a pivotal indicator not only for differentiating sporadic bacterial cystitis from bacterial cystitis with comorbidities but also for distinguishing sporadic infections from proteinuria secondary to HAC or CKD.
In human medicine, the relationship between urinary tract infection (UTI) and proteinuria or microalbuminuria has been thoroughly studied (16-27). Studies in children show no association between asymptomatic bacteriuria and proteinuria (28-31). Rather, patients with diabetic nephropathy, nephrotic syndrome, or other renal disorders who already have proteinuria appear more susceptible to developing bacteriuria (20, 25, 26). In symptomatic UTIs, tubular proteinuria is a well-recognized feature of febrile upper urinary tract infections (pyelonephritis) (16, 17, 19, 24). However, there is limited research investigating the characteristics of proteinuria in patients with cystitis, and existing findings indicate that while albuminuria may occur physiologically, it is not a consistent feature (19, 32). Taken together, while upper urinary tract infections (pyelonephritis) can lead to tubular proteinuria, there is insufficient evidence that bacterial cystitis alone produces significant proteinuria.
Non-renal proteinuria in dogs is traditionally attributed to lower urinary tract inflammation or hemorrhage, as inflammation increases vascular permeability and facilitates the escape of proteins into surrounding tissues (8, 33, 34). However, evidence from multiple studies suggests that hematuria, pyuria, and bacteriuria do not meaningfully contribute to significant proteinuria. For instance, the presence of hematuria or pyuria in dogs has shown no clear correlation with UPC values (11, 35). Furthermore, only 49.8% of samples with active sediment were found to be proteinuric, and neither the resolution of active sediment nor a positive culture was associated with a significant reduction in UPC (10). Similarly, bacteriuria has proven to be a poor predictor of proteinuria (9, 10). Even in dogs with marked bacterial growth, UPC values remained consistently below 0.5, with negligible agreement between culture status and abnormal UPC levels (9). Collectively, these findings indicate that a causal relationship between bacterial cystitis and significant proteinuria is unlikely. While previous research relied primarily on culture results, the present study further strengthens this conclusion by strictly excluding dogs with concurrent diseases and comorbidities, thereby isolating the specific impact of bacterial cystitis.
An experimental study investigated the effect of bacterial cystitis on UPC by inducing the condition in five dogs through intravesical instillation of sulfosalicylic acid, followed by inoculation with E. coli (11). Marked increases in UPC were observed, ranging from 1.5 to 40.8. Although the results of the present study were not consistent with those of the experimental study, they aligned with previous human studies on cystitis-related proteinuria and canine studies about the relationship between bacteriuria and UPC, indicating that there is insufficient evidence that bacterial cystitis alone produces clinically meaningful or persistent proteinuria. The experimental study results were derived from surgically induced cystitis, suggesting that the increase in UPC might have been the effect of chemical mucosal irritation, not from the effect of cystitis.
In this study, the optimal UPC cut-off to differentiate sporadic bacterial cystitis from bacterial cystitis with comorbidities was 0.32. Beyond identifying these comorbidities, ROC curve analysis demonstrated excellent diagnostic performance for distinguishing proteinuria induced by sporadic bacterial cystitis from that associated with CKD and HAC, with AUC values exceeding 0.90 in all comparisons (36). Specifically, among dogs with UPC ≥0.2 (the threshold for borderline proteinuria), an optimal cut-off of 0.425 was identified to effectively differentiate sporadic bacterial cystitis from renal-origin proteinuria (CKD and HAC). Although the small sample size of the sporadic bacterial cystitis group necessitates caution when interpreting the diagnostic accuracy of these cut-off values, our results indicate that clinically significant proteinuria should not be solely attributed to sporadic bacterial cystitis. Misclassification may obscure clinically meaningful renal proteinuria, the early and accurate recognition of which is essential for determining prognosis and guiding appropriate diagnostic evaluation and management (4, 7, 8).
In this study, only 13 dogs (17%) met the criteria for pure sporadic bacterial cystitis, reflecting the rarity of truly isolated infections in clinical practice. This is consistent with previous reports indicating that most dogs with bacteriuria have concurrent renal or systemic diseases, as multiple host defense mechanisms normally prevent bacterial colonization of the lower urinary tract (37). These defenses include adequate urine volume, frequent and complete voiding, and maintenance of urinary continence, as well as anatomic protective structures such as urethral high-pressure zones and the surface characteristics of the urothelium. Mucosal defense barriers – particularly antibody production, the glycosaminoglycan layer, and intrinsic antimicrobial properties of the urothelium – further limit infection, while the chemical properties of urine (e.g., extreme pH ranges, hyperosmolality, and high urea concentration) exert additional antimicrobial effects. Finally, systemic immune competence provides an essential layer of protection. Together, these mechanisms help explain why sporadic bacterial cystitis in dogs is relatively uncommon and why it does not typically produce sufficient mucosal inflammation to result in clinically significant proteinuria.
Study limitations. First, the number of dogs with strictly defined sporadic bacterial cystitis was small, which may limit the statistical power to detect subtle differences between groups and increase the potential risk of a Type II error. While strict inclusion criteria were essential to isolate pure sporadic bacterial cystitis without confounding comorbidities, this small sample size warrants caution when interpreting non-significant findings and may restrict the generalizability of our results. Second, day-to-day biological variation in UPC was not assessed, and fluctuations in urine protein excretion could have influenced individual measurements. Third, urine sediment activity was not incorporated into the analysis, preventing evaluation of potential associations between sediment findings and proteinuria. Fourth, comparisons were not stratified by CKD stage, which may influence the magnitude of renal proteinuria. Finally, among dogs with bacterial cystitis accompanied by comorbidities, proteinuria was not analyzed according to the specific type of underlying disorder, limiting interpretation of how individual diseases contribute to UPC variability. More accurate analysis of urinary proteins in dogs with sporadic bacterial cystitis, using SDS-PAGE or identifying microalbuminuria, could benefit future studies by distinguishing patterns of proteinuria among different diseases (21, 22). In addition to quantitative parameters such as UPC, non-invasive biomarker discovery using urine samples has rapidly expanded in canine urology, as demonstrated by the potential of urinary exosomal NMP-22 for diagnosing canine urothelial carcinoma (38). Furthermore, evaluating local tissue inflammation and cellular/microvascular responses remains critical across various disease models (39, 40). Integrating advanced urinary profiling with UPC evaluation may further refine the differentiation of sporadic bacterial cystitis from other lower urinary tract pathologies and systemic comorbidities.
Conclusion
This study indicates that sporadic bacterial cystitis alone in dogs does not result in clinically meaningful increases in UPC, whereas the presence of comorbid conditions is associated with substantially higher values. Therefore, a UPC exceeding 0.32 in dogs with bacterial cystitis should prompt further investigation for underlying renal or systemic diseases rather than being attributed solely to cystitis. Moreover, when the cause of proteinuria remains unclear at or above the borderline threshold (UPC ≥0.2), a UPC greater than 0.425 is more suggestive of renal proteinuria, such as CKD or HAC, than of sporadic bacterial cystitis. This threshold-based approach may help prevent the misinterpretation of proteinuria, support the timely diagnosis of renal disease, and ultimately improve clinical outcomes.
Conflicts of Interest
The Authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Authors’ Contributions
JL: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. YC: Investigation, Methodology, Resources, Writing – review & editing. YK: Investigation, Methodology, Writing – review & editing. HK: Formal analysis, Software, Writing – review & editing. B-TK: Supervision, Validation, Writing – review & editing. TY: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.
Funding
This research was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agriculture and Food Convergence Technologies Program for Research Manpower development funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number: RS-2024-00398561).
Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, a large language model (ChatGPT, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.