Kidney-Based Molecular Diagnostics of Freshwater Drowning: Comparative Evaluation of SP-A2 and AQP5 in an Animal...

Background/Objectives: Establishing drowning as the cause of death remains one of the most challenging tasks in forensic pathology, particularly when classical autopsy findings are absent, nonspecific, or compromised by decomposition. Molecular biomarkers may provide objective adjunctive evidence in such cases. This study investigated the diagnostic utility of Aquaporin-5 (AQP5) and Surfactant Protein A2 (SP-A2) measured in renal tissue as potential biomarkers of freshwater drowning and evaluated the influence of different freshwater environments on their expression. Methods: A controlled experimental study was performed using 29 adult Wistar rats allocated to freshwater drowning groups (Boračko Lake, n = 11; Jablaničko Lake, n = 11) and non-drowning control groups subjected to mechanical asphyxia followed by one hour of postmortem immersion (n = 7). Kidney tissues were collected within a maximum of 5 min after death and analyzed for AQP5 and SP-A2 concentrations using enzyme-linked immunosorbent assay (ELISA). Group differences were evaluated using Kruskal-Wallis and Dunn's post hoc tests, while effect sizes were estimated using Cliff's delta. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic performance of each biomarker in distinguishing drowning from non-drowning deaths. Results: AQP5 concentrations differed significantly among groups (χ² = 16.05, p = 0.00033), with large effect sizes observed in all pairwise comparisons. However, diagnostic performance was poor, as ROC analysis demonstrated no significant discrimination between drowning and non-drowning cases (AUC = 0.534, 95% CI: 0.314-0.754; p = 0.762). In contrast, SP-A2 showed significant intergroup differences (χ² = 6.69, p = 0.035) and moderate diagnostic accuracy for drowning identification (AUC = 0.752, 95% CI: 0.567-0.937; p = 0.008), with a sensitivity of 77.2% and specificity of 81.8% at the optimal threshold. Notably, AQP5 expression appeared suggestive of environmental modulation, whereas SP-A2 demonstrated greater relevance to the drowning process itself. Conclusions: Renal SP-A2 showed moderate diagnostic utility as a supportive biomarker for drowning, whereas AQP5 lacked sufficient discriminatory power for cause-of-death determination. The marked variability of AQP5 across freshwater environments suggests that it reflects environmental modulation rather than drowning-specific biological responses. Although neither marker is suitable as a standalone diagnostic tool, renal SP-A2 may represent a useful adjunct in forensic drowning investigations, particularly when pulmonary tissue is unavailable or unsuitable for analysis. These findings should be considered preliminary and applicable only to freshwater drowning conditions, as saltwater drowning was not investigated in the present study. Further validation in human forensic casework and larger experimental cohorts is warranted.