Discovery of Molecular Pathways Underlying NOX4-Derived Oxidative Stress–Mediated Hypertension in Dahl Salt-Sensitive Rat

Background: NOX4 knockout reduces salt-induced hypertension in Dahl salt-sensitive (SS) rats, and prior work has described associated transcriptomic and metabolic changes. However, because the knockout is germline, these differences may reflect developmental compensation or strain-specific adaptations rather than direct effects of NOX4-derived oxidative stress. To address this, we used salt-insensitive Sprague-Dawley (SD) rats as a reference and focused on genes whose salt responses were similar in SD and SSNOX4−/− rats but specifically altered in hypertensive SS rats, enriching for candidates more directly linked to NOX4-dependent oxidative stress in the kidney. Methods: RNA-seq and metabolomic dataset from the kidneys (cortex (Cx) and outer medulla (OM)) of SS, SD, and SSNOX4-/- rats studied under 0.4% low-salt (LS) and 7 days 4.0% high-salt (HS) conditions were obtained from our previously published studies but reanalyzed using the most recent reference genome sequence (GRCr8). To avoid misclassifying non-significant genes as similar, we applied a Two One-Sided Tests (TOST) procedure (equivalence margin ±0.3 log 2 fold change (FC) for RNA-seq, ±0.8 log 2 FC for metabolomics) to HS–LS salt responses in SD versus NOX4-KO rats, and defined as equivalent those genes or metabolites whose responses were statistically indistinguishable between these two salt-resistant backgrounds. Within this TOST-equivalent subsets, we then performed a directional contrast test using Linear Models for Microarray Data (limma) to compare HS–LS responses across the three strains and identified genes and metabolites in which only SS rats showed significantly divergent salt-induced changes (i.e. the direction of the HS-induced change in SS was opposite to that in both control strains). Results: Of the 43,360 detected genes, 2,556 in Cx and 112 in OM passed TOST. Among these, 456 in Cx and 24 in OM were significantly changed only in SS (raw p < 0.05). KEGG pathway analysis of these genes showed that many inflammation-related pathways previously reported in the salt responses of SS, SSNOX4−/−, and SD rats were not observed. Instead, pathways such as “autophagy”, “lysosome”, “Ubiquitin mediated proteolysis”, and “2-Oxocarboxylic acid metabolism” were significantly enriched in the cortex (Cx) (p < 0.05). No pathway was enriched in the outer medulla (OM). When we applied the same analytical approach to the metabolomics data, 33 of the 3240 metabolites in the Cx and 33 of 3082 in OM passed the pipeline and had Human Metabolome Database (HMDB) IDs. In the integrated gene–metabolite network analysis, shifts in adenine, 5-methylcytosine, and deoxycytidine were detected in the Cx, which may reflect alterations in DNA stability and nucleic-acid turnover. No pathways reached significance in the combined gene–metabolite network analysis in OM. Conclusion: The TOST-based analytical pipeline enabled more selective identification of pathways through which NOX-derived oxidative stress may contribute to hypertension and kidney injury in SS rats. This filtering reduced broad inflammatory signatures and instead highlighted protein turnover (autophagy, lysosome, ubiquitin–proteasome) and nucleotide/cofactor metabolic rewiring as pathways in the Cx that were uniquely altered in hypertensive SS rats, consistent with the region of higher NOX4 expression. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.