# Temporal Proteomic and Phosphoproteomic Profiling Deciphers Molecular Dynamics of Acute-to-Chronic Kidney Disease After Ischemia-Reperfusion Injury, With Dock2 Emerging as a Key Regulator

## TL;DR

This study uses proteomic and phosphoproteomic profiling to uncover molecular changes in kidney injury and identifies Dock2 as a potential therapeutic target.

## Contribution

The study is the first to reveal the role and mechanism of Dock2 in the progression from acute to chronic kidney disease after injury.

## Key findings

- Sustained activation of NF-κB signaling and impaired fatty acid β-oxidation were observed during AKI-to-CKD transition.
- Dock2 was identified as a key mediator of proinflammatory responses in tubular epithelial cells via the IKKβ/NF-κB pathway.
- Pharmacological inhibition of Dock2 reduced renal injury, inflammation, and fibrosis in vivo.

## Abstract

Acute kidney injury (AKI), characterized by a rapid decline in renal function, has high mortality rates and frequently progresses to chronic kidney disease (CKD). A major contributor to AKI is ischemia-reperfusion injury (IRI). However, the global molecular changes underlying the AKI-to-CKD transition post-IRI remain to be fully elucidated. Using 4D label-free proteomic and phosphoproteomic analyses in a murine unilateral IRI model at 1 h, 1 day, 3 days, 7 days, and 28 days post injury, we systematically identified dysregulated proteins, phosphoproteins, and signaling pathways involved in the progression from AKI to CKD. Critically, these analyses consistently revealed the enrichment and sustained activation of NF-κB signaling, a key pathway driving inflammatory and fibrotic responses, across multiple time points. In addition, we identified significant impairment of fatty acid β-oxidation. Notably, our omics analysis specifically identified the dedicator of cytokinesis (Dock) protein family, with Dock2 emerging as a prime candidate due to its known immune regulatory functions. Dock2 expression showed significant upregulation post-IRI and was found predominantly localized to injured tubular epithelial cells. Functional validation demonstrated that Dock2 knockdown attenuated proinflammatory responses in tubular epithelial cells by inhibiting IKKβ-mediated NF-κB activation in vitro. Consistently, pharmacological inhibition of Dock2 by CPYPP ameliorated renal tubular injury, inflammation, and fibrosis in vivo. To our knowledge, this is the first study to reveal the role and mechanism of Dock2 in the AKI-to-CKD progression post-IRI. In conclusion, our findings delineate molecular mechanisms underpinning the transition from AKI to CKD and nominate Dock2 as a promising therapeutic target for mitigating this process.

•Temporal proteomic and phosphoproteomic analysis reveals molecular dynamics in renal IRI.•Sustained NF-κB activation and impaired FAO across AKI-to-CKD transition.•Dock2 mediates proinflammatory responses in TECs through the IKKβ/NF-κB pathway.•Targeting Dock2 mitigates AKI-to-CKD progression after IRI.

Temporal proteomic and phosphoproteomic analysis reveals molecular dynamics in renal IRI.

Sustained NF-κB activation and impaired FAO across AKI-to-CKD transition.

Dock2 mediates proinflammatory responses in TECs through the IKKβ/NF-κB pathway.

Targeting Dock2 mitigates AKI-to-CKD progression after IRI.

This 4D label-free proteomic and phosphoproteomic profiling systematically identified dysregulated proteins, phosphoproteins, and signaling pathways in AKI-to-CKD transition post-IRI. The enrichment and sustained activation of NF-κB signaling and impaired fatty acid β-oxidation (FAO) were revealed. Genetic and pharmacological targeting demonstrated Dock2 as a key mediator of proinflammatory responses in renal tubule cells via the IKKβ/NF-κB pathway. These findings nominate Dock2 as a promising therapeutic target to mitigate AKI-to-CKD progression post-IRI.