PROTAC-Mediated HDAC7 Degradation Attenuates AKI Injury by Alleviating Deacetylation of the Mitophagy-Related Protein VCP

Acute kidney injury (AKI) is characterized by an abrupt loss of kidney function with high morbidity and mortality, yet no effective drug for AKI is available. Post-protein modifications play important roles in the occurrence and development of many diseases. Histone deacetylase 7 (HDAC7) belongs to the class IIa family of histone deacetylases and plays a role in the expression of pro-inflammatory genes in macrophages during inflammation. However, there has been insufficient investigation into the expression, function, and molecular mechanism of this enzyme in clinical patients, animals, and cellular models of acute kidney injury.

Materials and Methods

Male C57BL/6 mice (6–8 weeks, 20–22 g, Experimental Animal Centre of Anhui Medical University) were subjected to cecal ligation-puncture (CLP) or renal ischemia-reperfusion (I/R) to model sepsis- or I/R-induced AKI. An immortalized murine tubular epithelial cell line (mTECs, authenticated STR and mycoplasma-free) was maintained in DMEM/5 % FBS and challenged with LPS (20 µg ml⁻¹, 24 h) or 1 % O₂ hypoxia (12 h) plus 3 h re-oxygenation (H/R) to mimic septic or ischemic stress. HDAC7 was stably knocked down with shRNA-lentivirus (GV115) or over-expressed/mutated (K8Q/R, K251Q/R) using Hanbio-built plasmids; efficacy was verified by Western blot for HDAC7, KIM-1, VCP, DRP1, PINK1 and BNIP3. Protein interactions were captured by anti-HDAC7 co-immunoprecipitation and probed for VCP, HA and pan-acetyl-lysine.For histology, kidneys fixed in 4 % PFA were paraffin-sectioned (4 µm) and stained with H&E or PAS; injury was scored (0–4) over ≥ 10 random fields. Immunohistochemistry for HDAC7, KIM-1 and F4/80 and immunofluorescence for acetylation and mitochondrial markers were imaged on Olympus IX83 or Zeiss LSM880 confocal and quantified with ImageJ. RNA extracted with TRIzol was reverse-transcribed and analysed by SYBR-Green qRT-PCR (Bio-Rad CFX) with β-actin as reference.Mitochondrial phenotype was interrogated with Seahorse XFp analyser (basal and maximal OCR, ECAR), Cox8-EGFP-mCherry mitophagy reporter and JC-1 membrane-potential assay. Surface ultrastructure was visualized by scanning electron microscopy after glutaraldehyde fixation, graded ethanol dehydration and gold sputter-coating (Gemini SEM 300). Global acetylome profiling was performed by LC-MS/MS (Jingjie PTM Biolabs). All data are mean ± SD; comparisons used two-tailed t-test or one-way ANOVA with Tukey–Kramer post-test (JMP Pro 15.2 / EZR 1.55), significance set at P < 0.05.

1. HDAC7 was significantly upregulated in Humans and Mice with AKI

We investigated Zn²⁺-dependent HDACs in AKI using snRNA-seq and identified HDAC7 as specifically upregulated in proximal tubules. This was validated in human AKI biopsies, I/R and CLP mouse models, and in vitro under H/R or LPS stress, confirming HDAC7’s central role in tubular injury.

2.HDAC7 cKO Protects Against I/R- CLP-Induced Renal Dysfunction, Injury, and Inflammation in Mice.

To investigate the role of HDAC7 in AKI, we established a renal tubule–specific HDAC7 conditional knockout (cKO) mouse model(HDAC7flox/flox/ggt-Cre).Tubule-specific HDAC7 knockout in mice reduced renal injury after ischemia/reperfusion, as shown by decreased BUN, creatinine, KIM-1, NGAL levels, and improved histology, indicating a key role for HDAC7 in AKI progression. HDAC7 silencing in HK2 cells attenuated H/R- and LPS-induced inflammatory responses and KIM-1 expression, while HDAC7 overexpression exacerbated cellular injury and inflammation, indicating a pro-inflammatory role for HDAC7 in renal tubular damage.

3. HDAC7-Mediated Acetylation of the Mitophagy-Associated Protein VCP Contributes to LPS-Induced Injury in mTECs

To investigate how HDAC7 regulates renal injury, we performed global acetylome profiling in mTECs and identified 423 significantly altered acetylation sites after LPS treatment, with notable enrichment in mitochondrial and autophagy-related pathways. Valosin-containing protein (VCP), a key ATPase involved in mitophagy, showed increased acetylation at K8 and K251 sites under LPS stimulation. Co-IP and subcellular fractionation confirmed that HDAC7 interacts with VCP primarily on mitochondria. Although VCP total protein levels were unchanged, its acetylation was enhanced by LPS, and VCP knockdown reduced inflammation and injury marker KIM-1. These results indicate that HDAC7-mediated acetylation of VCP impairs mitochondrial function and promotes tubular cell damage during LPS-induced injury.

4.The Acetylation of VCP at K8 Rather Than K251 Contributes to Regulating Mitophagy and mTECs injury

By generating site-specific acetylation mutants of VCP, we found that acetylation at K8, but not K251, plays a critical role in regulating mitophagy and mitochondrial function in mTECs. The acetyl-mimetic mutant VCP-K8Q impaired mitochondrial-lysosomal colocalization, suppressed mitophagy, reduced OCR and ATP production, and promoted inflammatory injury. In contrast, the deacetylation-mimetic mutant VCP-K8R enhanced mitochondrial function and mitigated damage. These results identify K8 acetylation as a key regulatory mechanism in VCP-mediated tubular injury.

5.HDAC7 PROTAC B22 confers renal protection bothin vitro and in vivo

We developed B22, a highly specific HDAC7-targeting PROTAC degrader, which effectively reduces HDAC7 protein via the ubiquitin-proteasome system without significant cytotoxicity. In LPS- or H/R-stimulated mTECs, B22 pretreatment downregulated Kim-1, inflammatory cytokines, and pyroptosis markers, while enhancing mitophagy and preserving mitochondrial morphology. The protective effects were accompanied by reduced VCP expression and supported by structural modeling confirming ternary complex formation. These results demonstrate that B22 confers potent anti-inflammatory and cytoprotective benefits in vitro by selectively degrading HDAC7.

In summary, this study identifies HDAC7 as a key epigenetic regulator promoting inflammation and mitochondrial dysfunction in acute kidney injury. Through HDAC7-mediated acetylation of VCP at lysine 8, mitophagy is suppressed, exacerbating tubular cell damage. We further developed B22, a novel HDAC7-targeting PROTAC degrader, which effectively attenuates renal injury and inflammation both in vitro and in vivo by facilitating HDAC7 degradation and restoring VCP function. These findings not only elucidate the HDAC7–VCP axis as a central mechanism in AKI but also highlight the therapeutic potential of PROTAC-based HDAC7 targeting as a promising strategy for the treatment of kidney injury.