Augmenter of liver regeneration protects the kidney from ischaemia-reperfusion injury in ferroptosis

Abstract

Acute kidney injury (AKI) is a common and severe clinical condition with high morbidity and mortality. Ischaemia-reperfusion (I/R) injury remains the major cause of AKI in the clinic. Ferroptosis is a recently discovered form of programmed cell death (PCD) that is characterized by iron-dependent accumulation of reactive oxygen species (ROS). Compelling evidence has shown that renal tubular cell death involves ferroptosis, although the underlying mechanisms remain unclear. Augmenter of liver regeneration (ALR) is a widely distributed multifunctional protein that is expressed in many tissues. Our previous study demonstrated that ALR possesses an anti-oxidant function. However, the modulatory mechanism of ALR remains unclear and warrants further investigation. Here, to elucidate the role of ALR in ferroptosis, ALR expression was inhibited using short hairpin RNA lentivirals (shRNA) in vitro model of I/R-induced AKI. The results suggest that the level of ferroptosis is increased, particularly in the shRNA/ALR group, accompanied by increased ROS and mitochondrial damage. Furthermore, inhibition of system xc- with erastin aggravates ferroptosis, particularly silencing of the expression of ALR. Unexpectedly, we demonstrate a novel signalling pathway of ferroptosis. In summary, we show for the first time that silencing ALR aggravates ferroptosis in an in vitro model of I/R. Notably, we show that I/R induced kidney ferroptosis is mediated by ALR, which is linked to the glutathione-glutathione peroxidase (GSH-GPx) system.

Keywords: acute kidney injury (AKI); augmenter of liver regeneration (ALR); ferroptosis; ischaemia-reperfusion (I/R) injury; reactive oxygen species (ROS).

Figure 1

Ferroptosis was more apparent at H6R12 h, and augmenter of liver regeneration (ALR) was up‐regulated after I/R injury. A, Representative Western blot demonstrating the expression of acyl‐CoA synthetase long‐chain family member 4 (ACSL4), transferrin receptor (TFR), SLC7A11, glutathione peroxidase (GPX)₄ and ALR. B, The ratio of ACSL4, TFR, SLC7A11, GPX ₄ and ALR was quantified by densitometry based on immunoblot images and depicted as the radio of the indicated proteins normalized to β‐actin. *P < 0.05 and **P < 0.01 compared to the normal group

Figure 2

Reactive oxygen species (ROS) levels in Human kidney proximal tubular cells were evaluated using a dichloro‐dihydro‐fluorescein diacetate (DCFH‐DA) kit. A, C, ROS levels were measured by flow cytometry. B, D, The mean ROS fluorescence intensities in the short hairpin RNA lentivirals (shRNA)/augmenter of liver regeneration and shRNA/control groups in vitro. The mean ± SD represents three independent experiments (n = 3). *P < 0.05 and **P < 0.01 compared to the normal group. ^## P < 0.05 as compared to the hypoxia‐reoxygenation group at the same time‐point

Figure 3

Effect of short hairpin RNA lentivirals (shRNA)/augmenter of liver regeneration (ALR) on ALR expression. Human kidney proximal tubular cells were transduced with shRNA/ALR or shRNA/control for 72 h, puromycin selected, and tested for ALR expression. A, B, Representative Western blot demonstrating the expression of ALR normalized to β‐actin. C, To evaluate the knockdown of ALR mRNA expression, real‐time PCR was performed and normalized to β‐actin. **P < 0.01 versus the normal group

Figure 4

Cell counting kit (CCK8) assay of cell proliferation. Human kidney proximal tubular (HK‐2) cells transfected with short hairpin RNA lentivirals (shRNA)/control or shRNA/augmenter of liver regeneration (ALR) for the indicated times and analysed by using colorimetry at 450 nm. No significant difference in HK‐2 proliferation was observed between the shRNA/ALR group and shRNA/control group

Figure 5

Silencing of augmenter of liver regeneration (ALR) aggravates I/R induced ferroptosis in vitro. A, The expression of acyl‐CoA synthetase long‐chain family member 4 (ACSL4), SLC7A11, glutathione peroxidase‐4 (GPX ₄) and transferrin receptor (TFR) was analysed by Western blotting in human kidney proximal tubular cells. (**P < 0.01 and *P < 0.05). B, GPX ₄ expression and localization after silencing ALR and I/R injury. GPX ₄ expression was examined by confocal laser‐scanning microscopy, scale bar = 20 μm. C, Relative content of GPXs. Data are presented as the mean ± SD of three independent experiments. (*P < 0.05 and **P < 0.01)

Figure 6

Transmission electron microscopy images in the short hairpin RNA lentivirals (shRNA)/augmenter of liver regeneration (ALR) groups and shRNA/control groups subjected to H/R. Magnification is × 6,000 or × 15,000, scale bars = 2 μm or 1 μm, respectively

Figure 7

Ferroptosis inhibition and silencing of augmenter of liver regeneration (ALR) enhanced ferroptosis in human kidney proximal tubular cells at H6R12. SLC7A11, acyl‐CoA synthetase long‐chain family member 4 (ACSL4) and glutathione peroxidase‐4 (GPX ₄) expression was determined by immunoblotting. Data are shown as the mean ± SD.**P < 0.01 and *P < 0.05

Figure 8

Augmenter of liver regeneration (ALR) interacts with glutathione peroxidase‐4 (GPX ₄) and the signalling pathway for ferroptosis. A, Mapping ALR and colocalization of ALR and GPX ₄ using confocal laser‐scanning microscopy. ALR is located in the mitochondria. ALR and GPX ₄ colocalization in the cytoplasm, the bar scale represents 50 μmol/L. B, Endogenous GPX ₄ and endogenous ALR in intact human kidney proximal tubular cells by co‐immunoprecipitation approach. C, ALR mediates the signalling pathways of ferroptosis