# Time-resolved urinary proteomics reveals heme-associated oxidative stress responses in neonatal hypoxic-ischaemic encephalopathy

## TL;DR

This study uses urine proteomics to track oxidative stress and heme metabolism changes in newborns with brain injury due to lack of oxygen, identifying key proteins and pathways involved.

## Contribution

The study provides the first time-resolved urinary proteomic analysis in neonatal HIE, revealing dynamic molecular responses and key regulators.

## Key findings

- Early HIE urine showed elevated hemoglobin and heme scavenging proteins like haptoglobin and hemopexin.
- Pathways related to heme detoxification, lipoprotein remodeling, and PPAR signaling were activated in HIE.
- Six upstream regulators, including IL1A, TNF, and PPARD, were identified as coordinating inflammation and metabolic adaptation.

## Abstract

Neonatal hypoxic-ischemic encephalopathy (HIE) triggers systemic oxidative stress and redox imbalance, contributing to multi-organ injury. Urine is a noninvasive matrix for longitudinal profiling of molecular responses, yet time-resolved proteomic studies in HIE are limited.

We performed longitudinal SWATH-MS proteomic profiling of urine from term neonates with moderate-to-severe HIE treated with therapeutic hypothermia (n = 16) and non-asphyxiated controls (n = 19) at six time points during the first eight days of life. Proteins identified in ≥ 80% of samples were quantified, and differential abundance, temporal clustering, pathway enrichment, and upstream regulatory networks were analyzed.

Approximately 1,000 proteins were quantified per sample. A total of 438 proteins were differentially abundant, with most changes transient and ten persistent. Early HIE urine showed marked elevation of hemoglobin subunits and sequential induction of haptoglobin and hemopexin, indicating staged heme scavenging and oxidative stress responses. Pathway enrichment revealed inhibition of neutrophil-associated innate immunity and activation of heme detoxification, lipoprotein remodeling, and PPAR signaling. Temporal clustering demonstrated stage-specific proteomic transitions, with partial normalization toward controls by days 6-8. Ingenuity Pathway Analysis identified six upstream regulators: Interleukin-1 alpha (IL1A), Tumor Necrosis Factor (TNF), ETS Homologous Factor (EHF), Peroxisome Proliferator-Activated Receptor Delta (PPARD), Thioredoxin-Interacting Protein (TXNIP), and Solute Carrier Family 2 Member 3 (SLC2A3), coordinating inflammation, redox control, and metabolic adaptation.

We identified transient and sustained proteomic shifts that trace coordinated changes in oxidative stress, heme metabolism, and metabolic adaptation, alongside key regulators such as IL1A, TNF, EHF, PPARD, and TXNIP. The progression from early injury-related divergence to partial recovery by day 8 highlights the dynamic nature of post-insult remodeling. These findings support urinary proteomics as a robust, non-invasive tool for probing HIE pathophysiology and point to promising biomarker and pathway candidates for future studies.

The online version contains supplementary material available at 10.1186/s40348-026-00222-7.