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 establish an in vitro renal injury model of amikacin(AKN)and investigate the protective effect and mechanism of vaccarin(VA)in the AKN-induced in vitro renal injury model.Methods Human renal tubular epithelial cells(HK-2)were cultured in vitro and incubated with different drugs of AKN or/and VA to de-termine the optimal drug concentration based on cell viability tested by MTT.The changes in intracellular oxidative stress were assessed using the dihydroethidium(DHE)probe and malondialdehyde(MDA)/glutathione(GSH)assay kits at different time points.Total RNA was extracted,and RT-qPCR was performed to detect the changes in the gene expression of kidney injury molecule-1(KIM-1)and neutropil gelatinase-associated lipocalin(NGAL).Western blot analysis was performed to detect the levels of ferroptosis-related markers solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4)in HK-2 cell lysis.Results High concentrations of AKN significantly decreased the viability of HK-2 cells in vitro,with a half maximal inhibitory concentration(IC50)of(5.74±0.47)mmol/L.VA at concentrations of 25-100 μmol/L increased the viability of AKN-stimulated HK-2 cells(P<0.05).After treatment with AKN(4 mmol/L),the mRNA expression levels of KIM-1 and NGAL were significantly higher than those of the negative control(NC)group(P<0.001).VA(50 μmol/L)signifi-cantly reduced the mRNA expression levels of KIM-1(P<0.01)and NGAL(P<0.05).The intensity of DHE staining increased after 3 hours of AKN treatment,but the difference was not statistically significant.However,the intensity of DHE staining was significantly higher in the 6-24 hours group compared to the 0-hour group(P<0.01).Furthermore,MDA levels significantly increased,while GSH levels significantly decreased after 6-24 hours of AKN treatment,with statistically significant differences(P<0.05).After 6-24 hours of AKN stimula-tion,the ferroptosis-related proteins SLC7A11 and GPX4 both significantly decreased(P<0.001).Co-incubation with VA for 24 hours effectively reversed the changes in DHE staining,MDA and GSH levels,as well as the chan-ges of SLC7A11 and GPX4 protein levels(P<0.001).Conclusion In this study,an in vitro renal injury model was established by stimulating HK-2 cells with high concentrations of AKN,and it was found that VA might allevi-ate the damage to renal tubular cells caused by AKN via inhibiting oxidative stress related ferroptosis.