Hexarelin alleviates apoptosis on ischemic acute kidney injury via MDM2/p53 pathway

Abstract

Introduction: Hexarelin exhibits significant protection against organ injury in models of ischemia/reperfusion (I/R)-induced injury (IRI). Nevertheless, the impact of Hexarelin on acute kidney injury (AKI) and its underlying mechanism remains unclear. In this study, we investigated the therapeutic potential of Hexarelin in I/R-induced AKI and elucidated its molecular mechanisms.

Methods: We assessed the protective effects of Hexarelin through both in vivo and in vitro experiments. In the I/R-induced AKI model, rats were pretreated with Hexarelin at 100 μg/kg/d for 7 days before being sacrificed 24 h post-IRI. Subsequently, kidney function, histology, and apoptosis were assessed. In vitro, hypoxia/reoxygenation (H/R)-induced HK-2 cell model was used to investigate the impact of Hexarelin on apoptosis in HK-2 cells. Then, we employed molecular docking using a pharmmapper server and autodock software to identify potential target proteins of Hexarelin.

Results: In this study, rats subjected to I/R developed severe kidney injury characterized by tubular necrosis, tubular dilatation, increased serum creatinine levels, and cell apoptosis. However, pretreatment with Hexarelin exhibited a protective effect by mitigating post-ischemic kidney pathological changes, improving renal function, and inhibiting apoptosis. This was achieved through the downregulation of conventional apoptosis-related genes, such as Caspase-3, Bax and Bad, and the upregulation of the anti-apoptotic protein Bcl-2. Consistent with the in vivo results, Hexarelin also reduced cell apoptosis in post-H/R HK-2 cells. Furthermore, our analysis using GSEA confirmed the essential role of the apoptosis pathway in I/R-induced AKI. Molecular docking revealed a strong binding affinity between Hexarelin and MDM2, suggesting the potential mechanism of Hexarelin's anti-apoptosis effect at least partially through its interaction with MDM2, a well-known negative regulator of apoptosis-related protein that of p53. To validate these findings, we evaluated the relative expression of MDM2 and p53 in I/R-induced AKI with or without Hexarelin pre-administration and observed a significant suppression of MDM2 and p53 by Hexarelin in both in vivo and in vitro experiments.

Conclusion: Collectively, Hexarelin was identified as a promising medication in protecting apoptosis against I/R-induced AKI.

Keywords: Acute kidney injury; Apoptosis; Hexarelin; Kidney ischemia–reperfusion injury; MDM2/p53.

Fig. 1

Hexarelin Attenuated Renal Function in I/R-induced AKI. A I/R-induced AKI model was established in SD rats; B serum creatine, BUN levels and relative expression of KIM-1 of different groups (C) H&E staining and (D) PAS staining of (I) control, (II) sham, (III) AKI, (IV) Hexarelin pretreatment before AKI and (V) Saline pretreatment before AKI groups; (VI) tubular injury score of different in vivo groups. Light microscopic images showing renal tubular epithelial cells shedding, tubular dilatation [yellow (III and V)], renal tubular epithelial cell necrosis, intratubular cast and basement membrane exposed [blue (III and V)], enlargement of bowman space [grey (III and V)], renal interstitial edema [red (III and V)], renal interstitial inflammatory cell infiltration [green (III and V)], severe degeneration and vacuolar of tubular epithelial cells [black (III and V)] in AKI and saline pretreatment before AKI groups. All data are shown as mean ± SD. *P < 0.05, **P < 0.01 versus control; ^#P < 0.05, ^##P < 0.01 versus AKI

Fig. 2

Hexarelin alleviated apoptosis after I/R-induced AKI. A TUNEL staining of (I) control, (II) sham, (III) AKI, (IV) Hexarelin-AKI and (V) Saline-AKI groups; (VI) quantitative analysis of TUNEL positive cell; B–E mRNA level of Caspase-3, Bax, Bad and Bcl-2; F–J Protein level of Caspase-3, Bax, Bad, Bcl-2 relative to β-actin detected by Western blotting. *P < 0.05, **P < 0.01 versus control; ^#P < 0.05, ^##P < 0.01 versus AKI

Fig. 3

Hexarelin mitigates cell damage after H/R exposure. A In vitro experiments were conducted using HK-2 cells administrated with different concentrations of Hexarelin before hypoxia for 9 h followed by Reoxygenation for 3 h; B qRT-PCR used to measure the relative expression of Hif-1α; C cell viability was detected using CCK-8 assay; D mRNA level of KIM-1 after Hexarelin administration followed by H/R treatment. *P < 0.05, **P < 0.01 versus control; ^#P < 0.05, ^##P < 0.01 versus AKI

Fig. 4

Hexarelin inhibited apoptosis in H/R-induced HK-2 cell. A Cell apoptosis of (I) H/R, pretreated with Hexarelin at a dose of (II)10^–6, (III) 10^–5 (IV) 10^–4 were measured via PI staining; B relative fluorescent intensity of control, H/R, Hexarelin pretreatment before H/R and PBS pretreatment before H/R groups; C–F mRNA level of Caspase-3, Bax, Bad and Bcl-2 measured by qRT-PCR; G–K protein level of Caspase-3, Bax, Bad and Bcl-2 detected by Western blot analysis. *P < 0.05, **P < 0.01 versus control; ^#P < 0.05, ^##P < 0.01 versus AKI

Fig. 5

Hexarelin protected apoptosis against I/R-induced AKI associated with MDM2 and p53. A GSEA analysis of GEO dataset of GSE98622 samples; B Volcano plot DEGs of IR-AKI samples and (C) Heatmap of genes of apoptosis signaling pathway; D 5 core transcription factors were obtained using MCC algorithm after transcription factors enrichment prediction analysis; E Molecular docking was performed to confirm the combination of Hexarelin and MDM2; F Detailed combination information of Hexarelin and MDM2, the two formed hydrogen bonds at the position of GLU-25 and THR-26 from MDM2 on a sizeable hydrophobic pocket in the N-terminal domain of MDM2

Fig. 6

The expression of MDM2 and p53 after I/R-induced AKI and H/R exposure. mRNA levels of (A) p53 and (B) MDM2 detected by qRT-PCR; protein levels C–G Level of p53, MDM2 tested by western blot analysis; H–I Immunohistochemistry was used to detect the expression of p53, MDM2 in (I) control, (II) sham, (III) AKI, (IV) Hexarelin-AKI, and (V) Saline-AKI groups. *P < 0.05, **P < 0.01 versus control; ^#P < 0.05, ^##P < 0.01 versus AKI

Fig. 7

The potential mechanisms of Hexarelin protect against I/R-induced AKI. Hexarelin targeted MDM2 subsequently decreased p53, inhibiting apoptotic cell transcription promoter to protect cell apoptosis against I/R-induced AKI