Myo-inositol oxygenase expression profile modulates pathogenic ferroptosis in the renal proximal tubule

Abstract

Overexpression of myo-inositol oxygenase (MIOX), a proximal tubular enzyme, exacerbates cellular redox injury in acute kidney injury (AKI). Ferroptosis, a newly coined term associated with lipid hydroperoxidation, plays a critical role in the pathogenesis of AKI. Whether or not MIOX exacerbates tubular damage by accelerating ferroptosis in cisplatin-induced AKI remains elusive. Cisplatin-treated HK-2 cells exhibited notable cell death, which was reduced by ferroptosis inhibitors. Also, alterations in various ferroptosis metabolic sensors, including lipid hydroperoxidation, glutathione peroxidase 4 (GPX4) activity, NADPH and reduced glutathione (GSH) levels, and ferritinophagy, were observed. These perturbations were accentuated by MIOX overexpression, while ameliorated by MIOX knockdown. Likewise, cisplatin-treated CD1 mice exhibited tubular damage and derangement of renal physiological parameters, which were alleviated by ferrostatin-1, a ferroptosis inhibitor. To investigate the relevance of MIOX to ferroptosis, WT mice, MIOX-overexpressing transgenic (MIOX-Tg) mice, and MIOX-KO mice were subjected to cisplatin treatment. In comparison with cisplatin-treated WT mice, cisplatin-treated MIOX-Tg mice had more severe renal pathological changes and perturbations in ferroptosis metabolic sensors, which were minimal in cisplatin-treated MIOX-KO mice. In conclusion, these findings indicate that ferroptosis, an integral process in the pathogenesis of cisplatin-induced AKI, is modulated by the expression profile of MIOX.

Keywords: Apoptosis; Nephrology.

Figure 1. Expression of MIOX and ferritinophagy markers in kidneys of patients with ATN, as assessed by immunohistochemistry.

Scanning-magnification photomicrographs revealed that MIOX was expressed in the proximal tubular cells, and its expression was increased notably in patients with ATN, as readily seen in high-magnification photomicrographs (C and D vs. A and B). No MIOX expression was seen in the glomerulus. The ferritinophagy biomarkers NCOA4 (cargo receptor of ferritin) and FTH1 (heavy chain of ferritin) were seen localized in the renal tubular epithelia, and their expression was notably decreased in patients with ATN (G and H vs. E and F, and K and L vs. I and J). Like MIOX, no significant expression of ferritinophagy markers was noted in the glomerular compartment. Scale bars: 50 μm. Panels G and K are from consecutive sections from the same patient sample but stained with different antibodies.

Figure 2. Ferroptosis is an essential part of cisplatin-induced HK-2 cell death.

Normally, the HK-2 cells exhibited a flat epithelial morphology, demonstrated by phase-contrast microscopy and direct visualization of H&E staining (A and D, red arrow and arrowhead). Cisplatin (CP) treatment caused shrinkage of HK-2 cells (B and E, yellow arrows and arrowheads), while cotreatment of cisplatin and Fer-1 led to a reversion of their morphology (C and F, blue arrow and arrowhead). TUNEL staining revealed notable DNA damage in cisplatin-treated HK-2 cells, and cotreatment with Fer-1 reduced the cell death (G–I). MTT assay revealed reduced cell viability following cisplatin exposure, which was rescued by Fer-1 treatment (J) (n = 6; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). Deferoxamine (DFO) and Z-VAD(OMe)-FMK (VAD) treatment also led to an increased survival of cells, while Nec-1 was ineffective (J and K) (n = 6; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). To investigate the intracellular dynamics of iron in HK-2 cells following cisplatin treatment, distribution of ferrum and heavy chain of ferritin (FTH1) was assessed. FTH1 was found distributed diffusely in the cytoplasm (green) and minimally in the lysosomes (red) (L–N). Interestingly, FTH1 was seen heavily colocalized in the lysosomes following cisplatin treatment (O–Q). The ferrum was also found to be marginally codistributed within the lysosomes, and the codistribution markedly increased following cisplatin treatment (R–W). Scale bars: 30 μm.

Figure 3. Ferroptosis inhibition attenuates cisplatin-induced AKI.

Cisplatin treatment led to a disruption of tubular epithelia, loss of brush borders, and cast formation, which were alleviated by the administration of Fer-1 (A–C). In addition, PAS staining revealed sloughing off of the epithelia and shedding of PAS-positive material in the tubular lumina following cisplatin treatment (E vs. D). These changes were attenuated by the prior treatment of Fer-1 (F vs. E). As assessed by SDS-PAGE, cisplatin treatment increased urinary albumin excretion, but not in mice pretreated with Fer-1 (G). Similarly, Fer-1 treatment attenuated cisplatin-induced elevation of serum creatinine levels (H) (n = 6; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). Besides, the increase in tubular damage score and mRNA levels of KIM-1 and NGAL induced by cisplatin was also alleviated by the administration of Fer-1 (I–K) (n = 4; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). Scale bars: 50 μm.

Figure 4. Cisplatin leads to excessive mitochondrial ROS generation and MIOX overexpression, which in turn accentuates cisplatin-induced lipid hydroperoxidation in HK-2 cells.

Cisplatin treatment increased DHE staining, indicative of mitochondrial ROS, in HK-2 cells, and it was partially reduced by MitoQ treatment (A–D) (n = 6; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). Immunoblotting studies revealed increased expression of MIOX following 4 hours of cisplatin treatment (F, left). No obvious MIOX upregulation was observed after 20 hours of cisplatin treatment (F, right). Besides, the status of MIOX overexpression and gene disruption in HK-2 cells was confirmed by immunoblotting studies (F, right; third, fourth, and sixth lanes). Fluorescence microscopy revealed that cisplatin treatment for 20 hours led to increased 4-HNE staining, indicative of lipid hydroperoxidation, in HK-2 cells (E, G, and H) (n = 6; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). The hydroperoxidation was accentuated by the overexpression of MIOX while attenuated by MIOX gene disruption (E and I–L). Similar changes in 4-HNE levels in vitro were observed by immunoblotting analyses (F, right). Scale bars: 50 μm.

Figure 5. Overexpression of MIOX exacerbates cell death, while its gene disruption inhibits it, in cisplatin-treated HK-2 cells.

TUNEL staining revealed severe DNA damage in cisplatin-treated HK-2 cells, which was further promoted by MIOX overexpression, and alleviated by MIOX gene disruption (A–F). Cisplatin treatment led to shrinkage of HK-2 cells after 48 hours, and the maximal contraction was seen in MIOX-overexpressing cells, as compared with the controls (G–J). MIOX siRNA treatment partially reversed these changes (L vs. K). As indicated by MTT assay, cisplatin-induced cell death was alleviated by MIOX siRNA treatment; however, it was accentuated by MIOX overexpression (M and N) (n = 6; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP + empty vector group, 1-way ANOVA with Dunn’s multiple comparisons for M and 2-tailed Student’s t test for N). MTT experiments also revealed that RSL3 (ferroptosis inducer) caused massive ferroptosis-specific cell death in HK-2 cells, which was exacerbated by overexpression of MIOX and attenuated by its gene disruption (O and P) (n = 6; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the RSL3 + empty vector group, 1-way ANOVA with Dunn’s multiple comparisons for O and 2-tailed Student’s t test for P). Scale bars: 30 μm.

Figure 6. Knockdown of MIOX inhibits, while its overexpression accelerates, ferritinophagy in cisplatin-treated HK-2 cells.

Immunofluorescence microscopy revealed a decreased fluorescence related to the expression of NCOA4 in cisplatin-treated HK-2 cells (B vs. A). The immunofluorescence intensity was maximally reduced in cisplatin-treated MIOX-overexpressing cells, and it was partially restored following MIOX siRNA treatment (C–F). Similarly, there was a substantial downregulation of FTH1 in cisplatin-treated HK-2 cells, and a further decrease in FTH1-related immunofluorescence was observed in cisplatin-treated MIOX-overexpressing cells, whereas MIOX siRNA treatment prevented its downregulation (G–L). The changes in NCOA4 and FTH1 expression levels were confirmed by immunoblotting studies (M–O) (n = 4; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). Scale bars: 50 μm.

Figure 7. Overexpression of MIOX accentuates, whereas its gene disruption attenuates, ferritin uptake by the lysosomes and accumulation of free iron after cisplatin treatment.

Normally, ferritin (FTH1, green) was seen localized primarily in the cytoplasm and in small amounts in the lysosome (LAMP1, red) in untreated HK-2 cells (A and B). Immunofluorescence microscopy revealed considerable translocation of FTH1 into the lysosomal compartment in cisplatin-treated HK-2 cells (C, D, and M) (n = 4; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). The translocation was tremendously enhanced in cisplatin-treated MIOX-overexpressing cells, while remarkably disrupted by the transfection of MIOX siRNA (E–M). To measure intracellular free iron levels, labile iron pool (LIP) assays were performed. The results indicated a marked increase of intracellular free iron concentration in HK-2 cells after cisplatin treatment (N) (n = 4; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). The concentration of free iron was seen further increased in cisplatin-treated MIOX-overexpressing cells, and it was attenuated by MIOX gene disruption (N). Scale bars: 50 μm.

Figure 8. MIOX overexpression promotes lysosomal permeability and decreases GSH concentration, GPX4 activity, and NADPH levels in cisplatin-treated HK-2 cells.

Lysosomal permeability was investigated by AO staining. AO-associated green fluorescence in the cytoplasm increased, while red fluorescence in the lysosome decreased, suggesting increased lysosomal permeability of HK-2 cells following cisplatin treatment (B vs. A). The changes in fluorescence were accentuated in cisplatin-treated MIOX-overexpressing cells but attenuated in cisplatin-treated cells transfected with MIOX siRNA (C–F). The expression of GPX4, a key enzyme for ferroptosis inhibition, decreased after cisplatin treatment (M). A substantial decline in GPX4 activity was also observed in both HK-2 cells and MIOX-overexpressing cells, and this decrease was negated by the concomitant transfection with MIOX siRNA (N) (n = 6; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). The status of intracellular GSH levels was assessed by monobromobimane (MBB) staining. Blue fluorescence was decreased after cisplatin treatment, and further decreased in cisplatin-treated MIOX-overexpressing cells, while partially restored by MIOX siRNA transfection (G–L and O) (n = 6; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). The NADPH levels were also found to be low in MIOX-overexpressing cells and cisplatin-treated cells (P). There was further depletion of NADPH in cisplatin-treated MIOX-overexpressing cells, which was blocked by MIOX gene disruption (P) (n = 6; *P < 0.05 compared with the control group, ^#P < 0.05 compared with the CP group, 1-way ANOVA with Dunn’s multiple comparisons). Scale bars: 30 μm.

Figure 9. Overexpression of MIOX exacerbates, while its gene disruption alleviates, renal tubular injury, lipid hydroperoxidation, and decline in GPX4 activity and NADPH levels in cisplatin-induced AKI.

The expression profile of MIOX in WT, MIOX-Tg, and MIOX-KO mice was demonstrated by immunoblotting studies (M). Cisplatin treatment led to severe renal tubular injury in WT mice, which was accentuated in MIOX-Tg mice but attenuated in MIOX-KO mice (A–F). Similarly, NGAL mRNA levels increased in cisplatin-treated WT mice and MIOX-Tg mice, and a minimal increase was observed in cisplatin-treated MIOX-KO mice (P) (n = 4; *P < 0.05 compared with the WT control group, ^#P < 0.05 compared with the WT CP group, 1-way ANOVA with Dunn’s multiple comparisons). 4-HNE expression levels were used to assess the status of lipid hydroperoxidation. Immunofluorescence and immunoblotting studies revealed an increase in 4-HNE levels in cisplatin-treated WT mice, which was maximal in cisplatin-treated MIOX-Tg mice but minimal in cisplatin-treated MIOX-KO mice (G–M). Besides, a decline in GPX4 activity was observed in cisplatin-treated WT mice and MIOX-Tg mice but not in cisplatin-treated MIOX-KO mice (N) (n = 6; *P < 0.05 compared with the WT control group, ^#P < 0.05 compared with the WT CP group, 1-way ANOVA with Dunn’s multiple comparisons), although GPX4 expression remained stable after cisplatin treatment (M). The NADPH levels in untreated MIOX-Tg mice were much lower than those in WT mice (O) (n = 6; *P < 0.05 compared with the WT control group, ^#P < 0.05 compared with the WT CP group, 1-way ANOVA with Dunn’s multiple comparisons). Both WT and MIOX-Tg mice had markedly decreased NADPH levels following cisplatin treatment, while a moderate reduction was seen in cisplatin-treated MIOX-KO mice (O). Scale bars: 30 μm.

Figure 10. MIOX overexpression promotes ferritinophagy in cisplatin-induced AKI.

Immunofluorescence microscopy showed that both ferritin and NCOA4 were mainly expressed in renal tubular epithelia (A–L). The expression of NCOA4 decreased in cisplatin-treated WT mice; however, MIOX-KO mice were unaffected, as indicated by immunofluorescence and immunoblotting studies (B vs. A, F vs. E, and M). The maximal decrease in the NCOA4 expression was observed in cisplatin-treated MIOX-Tg mice (D vs. B and C, and M). Intriguingly, FTH1 expression increased markedly in cisplatin-treated WT mice (H vs. G, and M). This upregulation may be due to the feedback mechanism (21), which was substantiated by reverse transcriptase PCR analyses in the present study. The analyses revealed increased FTH1 and decreased transferrin mRNA levels in cisplatin-treated kidneys (N and O) (n = 6; *P < 0.05 compared with the control group, 2-tailed Student’s t test). FTH1 expression in cisplatin-treated MIOX-Tg mice was maximally increased, while no obvious changes were noted in cisplatin-treated MIOX-KO mice (J vs. H and I, L vs. K, and M). Scale bars: 30 μm.