Poly(ADP-ribosyl)ation (PARylation) is a specific form of post-translational modification (PTM) predominantly triggered by the activation of poly-ADP-ribose polymerase 1 (PARP1). However, the role and mechanism of PARylation in the advancement of acute kidney injury (AKI) remain undetermined. Here, we demonstrated the significant upregulation of PARP1 and its associated PARylation in murine models of AKI, consistent with renal biopsy findings in patients with AKI. This elevation in PARP1 expression might be attributed to trimethylation of histone H3 lysine 4 (H3K4me3). Furthermore, a reduction in PARylation levels mitigated renal dysfunction in the AKI mouse models. Mechanistically, liquid chromatography-mass spectrometry indicated that PARylation mainly occurred in receptor for activated C kinase 1 (RACK1), thereby facilitating its subsequent phosphorylation. Moreover, the phosphorylation of RACK1 enhanced its dimerization and accelerated the ubiquitination-mediated hypoxia inducible factor-1α (HIF-1α) degradation, thereby exacerbating kidney injury. Additionally, we identified a PARP1 proteolysis-targeting chimera (PROTAC), A19, as a PARP1 degrader that demonstrated superior protective effects against renal injury compared with PJ34, a previously identified PARP1 inhibitor. Collectively, both genetic and drug-based inhibition of PARylation mitigated kidney injury, indicating that the PARylated RACK1/HIF-1α axis could be a promising therapeutic target for AKI treatment.

Objective:To investigate the regulatory effects and possible mechanisms of recombinant adenovirus-mediated hypoxia inducible factor-1 alpha(HIF-1α)transfection on HIF-1α in post-hypoxic rat cortical neurons.Methods:(1)Preparation of rats' cerebral cortical neurons in primary culture and hypoxia model.Recombinant adenovirus-mediated HIF-1α(AdHIF-1α)transfection and recombinant Ad transduction in normal and hypoxic cells.Then they will be divided into normal control group, hypoxia group, the recombinant adenovirus hypoxia-inducible factor-1α(AdHIF-1α)gene transfection group and recombinant adenovirus empty vector(Ad)transfection group.(2)To observe the transfection of AdHIF-1α/ Ad under fluorescence microscopy.The expression of hypoxia-inducible factor-1 alpha(HIF-1α)were observed by Western blot analysis in each group of neurons at 12 h、24 h、48 h and 72 h.Results:Hypoxic neurons transfected with Ad/AdHIF-lα showed the obvious expression 48 h under fluorescence microscopy.At each time of the AdHIF-1α gene transfection group, the expression of HIF-1α is significantly increased, the positive expression emerges at 12 h, the expression increased at 24 h, and it reaches the peak at 48 h, until 72 h declines, and it has statistical significance compared with the hypoxia group( P<0.01, P<0.05), but the Ad group has no statistically significant differences compared with the hypoxia group. Conclusions:The transfection of recombinant adenovirus-mediated HIF-1α gene can significantly increase the expression levels of HIF-1α in hypoxic neurons, and it may play a protective role in the neurons after hypoxia.

Objective:To investigate the transfection of recombinant adenovirus vectors containing the hypoxia inducible factor-1α gene (AdHIF-1α)in rat brain microvascular endothelial cells(BMECs) and to provide a theoretical basis for the treatment of hypoxic BMECs by AdHIF-1α.Methods:Rat BMECs were isolated, identified, and cultured in a maintenance medium containing 100 μmol/L cobalt dichloride (CoCl 2), establishing a hypoxia model of BMECs; then AdHIF-1α was transfected into hypoxic BMECs.The transfection of fluorescent protein was observed under a fluorescence microscope. Results:Transfection of AdHIF-1α into BMECs was monitored under a fluorescence microscope at 12 h, 24 h, 48 h and 72 h, respectively.Minor fluorescence began to appear at 12 h (0.13±0.01), and the fluorescence expression increased at 24 h (0.46±0.03, q=25.88, P<0.01), was most obvious at 48 h (0.97±0.05, q=40.00, P<0.01), and decreased at 72 h (0.38±0.02, q=46.28, P<0.01). Conclusions:Recombinant adenovirus vectors containing AdHIF-1α can be transfected into hypoxic BMECs in vitro.