Serum myoglobin modulates kidney injury via inducing ferroptosis after exertional heatstroke

Study design and participants

A retrospective cohort study was performed in the ICU of the General Hospital of Southern Theatre Command of Peoples Liberation Army from January 2008 to June 2019. The inclusion criterion was as follows: patients with “exertional heatstroke” caused by strenuous exercise performed in a high-temperature and high-humidity environment. The diagnostic criteria of exertional heatstroke were as follows: exposure to a high temperature or high humidity and a history of strenuous exercise; a clinical syndrome causing an excessively high body temperature (central temperature higher than 40℃); nervous system dysfunction (including delirium, cognitive impairment, coma, etc.); or systemic organ dysfunction. The exclusion criteria were as follows: 1) death or discharge within 24 h after admission; 2) incomplete data regarding key indicators; 3) incomplete outcome evaluation data obtained via telephone follow-up; and 4) a previous history of organ dysfunction, such as chronic kidney disease. AKI was defined as KDIGO standard.^[16] The primary outcome was the relationships between myoglobin levels and risk of AKI; the secondary outcome was composite outcome events with myoglobin levels and risk of AKI at discharge and death at 90 days.

The cause of heatstroke is strenuous outdoor training in southern China, and all patients are treated with cooling immediately on site and continue to cool till 39℃ (preferably 38.5℃ to 38.0℃) after admission. All patients received basic life support treatment according to their condition and were provided comprehensive treatment, including brain protection and mechanical ventilation. Moreover, appropriate volume management was performed for patients with AKI, and renal replacement therapy such as continuous renal replacement therapy (CRRT) was initiated, if necessary.

The study was approved by the Research Ethics Committee of the General Hospital of Southern Theatre Command of PLA (HE-2020-09). In view of the retrospective study design and depersonalization of data, the Ethics Committee agreed to waive the requirement for patient written informed consent but required that the patients be informed of the study details during a telephone follow-up.

Heat Stress Cell Model

HK-2 cells (National Collection of Authenticated Cell Cultures, CBP60447) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Lonza, Basel, Switzerland), and then HK-2 cells were treated with human myoglobin (882.35 nmol/L, cell assay concentration based on myoglobin renal threshold 1.5 mg/dL) (All from Abcam, Cambridge, MA, USA) at 43℃ for 2 h and rewarmed at 37℃ for different time points (0 h, 1 h, 3 h, 6 h, and 12 h). All cells are cultured at 37°C with 5% CO² (v/v). All media were supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/mL), streptomycin (100 U/mL), and 1× GlutaMAX Supplement (Thermo Fisher Scientific, Waltham, MA, USA).

p53 depletion

Small interference RNA (siRNA) sequence that target p53 was designed by Shanghai GenePharma Co. Ltd (Shanghai, China), as shown below: siP53 (5¢-CGAUGGUGUUACUUCCUGATT-3¢). siRNA transfection was performed according to the instruction of Lipofectamine 3000 Transfection Reagent Kit from Invitrogen (Thermo Fisher Scientific). HK-2 cells seeded on a 6-well plate were grown to 70%–90% confluence. Lipofectamine 3000 reagent and 5 μmol/L siRNA or control siRNA were mixed with Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA). The mixed solution was vortexed for 2 s to 3 s, and then incubated for 20 min at room temperature prior to incubation with cells for 6 h. Thereafter, the siRNA/Lipofectamine 3000 complex medium was replaced with the same volume of regular fetal bovine serum-supplemented culture medium. Then, 48 h after transfection, the depletion of p53 was confirmed by Western blotting, and cells were used in subsequent experiments.

CCK8

Human HK-2 cells were plated into 96-well flat bottom plates at 2×10⁵ cells/well, and incubated with myoglobin (882.35 nmol/ L) and heat stress treatment in medium containing 10% fetal calf serum (FCS) at 37 °C in 5% CO² in humidified air for 24 h. Thereafter, 10 μL CCK-8 solution (Dojindo, Nagasaki, Japan) was added to each well, and the optical density of T-cell proliferative activity was measured by the use of a microplate reader.

Western blot

Western blotting was performed to determine the expression of inositol-requiring protein 1 (IRE1) p-IRE1/IRE1, eIF2, p-eIF2, PERK, p-PERK, transcription factor 4 (ATF-4), transcription factor 6 (ATF-6), C/EBP-homologous protein (CHOP), p53, cystine glutamate transporter (SLC7A11), and glutathione peroxidase (GPX4) in HK-2 cells. Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Grand Island, NY, USA). Proteins of HK-2 cells were separated by SDS PAGE and then transferred electrophoretically onto polyvinylidenedifluoride (PVDF) membranes. The membranes were probed with anti-p-IRE1/IRE1 antibody, anti-ATF-4/6 antibody anti-CHOP antibody, anti-P53 antibody, anti-SLC7A11 antibody, anti-GPX4 antibody (Abcam, Cambridge, MA, USA), and polyclonal anti-actin antibodies (Santa Cruz Biotechnology, Dallas, USA). Protein bands were detected using an Odyssey System from LI-COR Biosciences, USA.

Measurement of ROS and Fe2+

The level of intracellular ROS was determined by the oxidation-sensitive fluorescent probe DCFH-DA with flow cytometry. The level of Fe²⁺ was detected by an iron assay kit purchased from Nanjing Jiancheng Bioengineering Institute and tested at a wavelength of 520 nm.

Statistical analysis

The continuous variables conforming to a normal distribution are expressed as Mean ± SD. Count data and ordinal data are represented as medians and interquartile ranges (IQRs). Count data were compared using multiple independent samples nonparametric Kruskal–Wallis H tests, and measurement data for intergroup comparisons were analyzed using nonparametric Mann–Whitney U tests. Data sets were examined by one-way ANOVA. Statistical analyses were performed using SPSS Windows version 23.0 (SPSS Inc., Chicago, IL, USA), Empower (R) (http://www.empowerstats.com, X&Y solutions, Inc., Boston, MA, USA), and R (http://www.R-project.org) software. P values (two-tailed) less than 0.05 were considered statistically significant.

Outcomes and baseline characteristics of serum biomarkers of RM in patients who underwent EHS stratified according to the incidence of AKI

A total of 187 patients were included, and all of them were males. Of these, 82 (43.9%) patients had AKI at admission, and stage 1, stage 2, and stage 3 were 62/82 (75.6%), 11/82 (13.4%), and 9/82 (11.0%), respectively. 21 (12.4%) patients had AKI at discharge, and 10/21 (47.6%), 6/21 (28.6%), and 5/21 (23.8%) patients had stage 1, stage 2, and stage 3 AKI, respectively. The 90-day mortality was higher in AKI than in non-AKI (26.8% vs. 1.0%, P < 0.001). The levels of myoglobin (Mb) and creatine kinase (CK) at admission, 24 h, 48 h, and discharge were higher in patients with AKI than in non-AKI patients (Table 1).

Association of myoglobin and AKI and 90-day mortality following EHS

The characteristics of the patients, stratified according to quartiles of myoglobin level, are shown in Figure 1. For rhabdomyolysis after heatstroke, the incidence of AKI was 73.08% in the highest myoglobin quartile (≥ 1000 ng/mL) and 12.82% in the lowest quartile (< 127 ng/mL), which yielded an adjusted odds ratio of AKI that was 18.95 (95% confidence interval [CI], 6.00–59.83) times as high in the highest quartile as in the lowest quartile (Figure 1). The myoglobin level was also strongly associated with the combined outcome of acute kidney injury and death at 90 d (adjusted odds ratio, 7.9; 95% CI, 1.61–38.89) (Figure 2).

Myoglobin is associated with HK-2 cell ferroptosis in a heat stress model

To determine the role of myoglobin in the mechanism of human kidney proximal tubular (HK-2) cell function, HK-2 cells were treated with myoglobin at concentrations of 882.35nmol/L at 43℃ for 2 h and rewarmed at 37℃ at different time points (0 h, 1 h, 3 h, 6 h, and 12 h). First, we examined the effect of heat shock and myoglobin on the survival of HK-2 cells, and the cell survival rate began to decline after 0 h of rewarming (Figure 3A, P < 0.01). Simultaneously, with the extension of rewarming time, the cell survival rate decreased significantly; after 12 h of rewarming, the cell survival rate decreased to less than 50%. We further determined whether ferroptosis was involved in severe heatstroke complicated with AKI. Both of SLC7A11 and GPX4 expression in HK-2 cells were detected by Western blot. We found that compared with the control group at 37℃, the expression of p53 was significantly increased in response to myoglobin after heat stress for 0 h. With the extension of rewarming time, the expression increased and then decreased and reached its highest level at 3 h of rewarming (Figure 3B, P < 0.01). As shown in Figure 1C, the expression levels of SLC7A11 and GPX4 were significantly decreased after heat stress and myoglobin treatment for 0 h, and the expression continuously decreased with the extension of rewarming time. Furthermore, the change in intracellular iron content is a very important indicator of ferroptosis. We evaluated HK-2 cell ferroptosis by detecting the intracellular iron content and found that intracellular Fe²⁺ was markedly increased at 3 h of rewarming after heat stress and myoglobin treatment (Figure 3C, P < 0.01). Additionally, the level of ROS was significantly increased at 3 h of rewarming in the heat stress- and myoglobin-treated cell model, suggesting that heat shock and myoglobin could induce the production of ROS and activate oxidative stress in HK-2 cells (Figure 3D, P < 0.01). Then, silencing of the p53 gene was used via a siRNA approach. SLC7A11 and GPX4 expression in HK-2 cells was significantly decreased at 3 h of rewarming in response to myoglobin under heat stress, and these effects were prevented by p53 gene silencing in HK-2 cells (Figure 3E, P < 0.01). After p53 gene knockdown, intracellular iron content and ROS levels were significantly decreased compared to that of control-siRNA group under heat stress (Figure 3F and G, P < 0.01). Further, the additional groups for myoglobin (at concentrations of 882.35 nmol/L at 37℃ for 3 h) alone were provided in Figure 3H. Taken together, our data show that myoglobin appears to be related to human HK-2 cell ferroptosis under heat stress by affecting SLC7A11 and GPX4 expression, intracellular iron content, and the production of ROS.

Figure 1

The distributions of AKI and AKI stage at different time. AKI: acute kidney injury.

Figure 2

Primary outcome and secondary outcome. (A) Primary outcome: myoglobin levels and risk of AKI. This model was adjusted for age. Quartile 1 was the reference (R) group. Quartile1<127, Quartile2: 127–468.9, Quartile3: 468.9–1000, Quartile4≥1000 Quartile1 5/39 (12.82%), Quartile2 12/40 (30.0%), Quartile3 14/28 (50.0%), Quartile4 38/52 (73.08%). (B) Secondary outcome: myoglobin levels and risk of AKI at discharge and death at 90 d. Quartile1 0 (0.0%), Quartile2 3/40 (7.5%), Quartile3 3/29 (10.3%), Quartile4 18/52 (34.6%). AKI: acute kidney injury.

Myoglobin modulates HK-2 cell ferroptosis involving ERS after heat stress

We further analyzed the expression of ERS-related proteins by Western blot, mainly detected the three classical pathways of ERS, and analyzed the expression of ATF-4, CHOP, ATF-6, IRE1, and p-IRE1. HK-2 cells treated with myoglobin and heat stress showed elevated expression of phosphorylation of PKR-like ER kinase (PERK) p-IRE1/IRE1, ATF-4, ATF-6 and CHOP, and with the extension of rewarming time, their expression first increased and then decreased (Figure 4A, P < 0.01). To confirm this, we used an ERS inhibitor (4-BPA) to treat HK-2 cells. There was a significant increase in the expression of p-PERR/PERK, p-eIF2, p-IRE1/IRE1, ATF-4, ATF-6, and CHOP at 3 h of rewarming in response to myoglobin under heat stress; thus, pretreatment with 4-BPA markedly decreased the expression of p-PERR/PERK, p-eIF2, p-IRE1/IRE1, ATF-4, ATF-6, and CHOP in the heat stress and myoglobin groups (Figure 4B, P < 0.01). Together, these data reveal the important role of myoglobin in aggravating the ERS response in HK-2 cells during heatstroke. Further, we found that the cell survival rate was significantly decreased after heat stress and myoglobin treatment rewarming for 3 h, and the cell survival rate was markedly increased in the 4-BPA + myoglobin+ heat stress group (Figure 4C, P < 0.01). Western blot analysis showed that inhibition of ERS using 4-BPA decreased p53 activation and enhanced SLC7A11 and GPX4 expression in HK-2 cells after heat stress and myoglobin treatment (Figure 4D, P < 0.01). Notably, intracellular Fe²⁺ and ROS were significantly decreased in response to heat stress and myoglobin combined with 4-BPA treatment rewarming for 3 h (Figure 4 E and F, P < 0.01), indicating that inhibiting ERS could suppress the accumulation of iron and ROS production induced by myoglobin under heat stress.

Baicalein protects HK-2 cell ferroptosis induced by myoglobin under heat stress by inhibiting ERS

Recent studies have reported that ERS and ferroptosis are involved in AKI induced by heat stress combined with myoglobin, and ERS inhibitors can effectively inhibit ferroptosis and alleviate heat stress and myoglobin-mediated kidney injury. Therefore, we considered whether there are clinical drugs that can inhibit ferroptosis by regulating ERS to alleviate rhabdomyolysis-induced AKI after EHS. In the present study, as expected, treatment with baicalein increased the cell survival rate after heat stress and myoglobin treatment rewarming for 3 h (Figure 5A, P < 0.01), suggesting the protective role of baicalein in AKI under heat stress. Furthermore, baicalein inhibited the activation of ERS signaling, as demonstrated by decreased expression of p-PERK/PERK, p-eIF2, p-IRE1/IRE1, ATF-4, ATF-6, and CHOP in the heat stress and myoglobin treatment rewarming for 3 h group (Figure 5B, P < 0.01). Simultaneously, baicalein alleviated ferroptosis in HK-2 cells, as shown by decreased p53 activation and elevated SLC7A11 and GPX4 expression (Figure 5C, P < 0.01). Notably, intracellular Fe²⁺ and ROS were markedly decreased in response to heat stress and myoglobin combined with baicalein treatment rewarming for 3 h (Figure 5 D and E, P < 0.01), indicating that baicalein could inhibit the accumulation of iron and ROS production induced by myoglobin under heat stress.

Figure 3

Myoglobin is associated with HK-2 cell ferroptosis in a heat stress model. (A) Human HK-2 cells were treated with myoglobin at concentrations of 882.35nmol/L at 43℃ for 2 h and rewarmed at 37℃ at different time points (0 h, 1 h, 3 h, 6 h, and 12 h). The effect of heat stress and myoglobin on the survival of HK-2 cells and the cell survival rate were examined. (B) Western blot and quantitative analyses (B1) were performed to evaluate the expression of p53, SLC7A11, and GPX4. (C and D) Intracellular Fe²⁺ and ROS in HK-2 cells were detected. (E, E1) HK-2 cells were transfected with control (ctrl) siRNA or siP53 for 48 h, treated with myoglobin at a concentration of 882.35nmol/L at 43℃ for 2 h, and rewarmed at 37℃ for 3 h. (F and G) HK-2 cells were transfected with control (ctrl) siRNA or siP53 for 48 h, treated with myoglobin at a concentration of 882.35 nmol/L at 43℃ for 2 h, and rewarmed at 37℃ for 3 h. Intracellular Fe²⁺ and ROS in HK-2 cells were detected. (H and H1) Western blot and quantitative analyses were performed evaluate the expression of p53, SLC7A11, and GPX4. Western blot and quantitative analyses were performed evaluate the expression of p53, SLC7A11, and GPX4; The values are the mean ± SD from triplicate independent experiments with statistical significance: *P < 0.05 vs. the control group; ^#P < 0.05 vs. the scrambled+rewarmed group.

Figure 4

Myoglobin modulates HK-2 cell ferroptosis involving endoplasmic reticulum stress after heat stress. (A, A1) Human HK-2 cells were treated with myoglobin at concentrations of 882.35nmol/L at 43℃ for 2 h and rewarmed at 37℃ at different time points (0 h, 1 h, 3 h, 6 h, and 12 h). The expression levels of p-IRE1/IRE1, ATF-4, ATF-6, and CHOP were measured by Western blot. (B, B1, B2, B3, B4) Human HK-2 cells were pretreated with 4-BPA, treated with myoglobin at concentrations of 882.35nmol/L at 43℃ for 2 h, and rewarmed at 37℃ for 3 h. The expression levels of p-PERR/PERK, p-eIF2, p-IRE1/IRE1, ATF-4, ATF-6, and CHOP were measured by Western blot. (C) Human HK-2 cells were pretreated with 4-BPA, treated with myoglobin at concentrations of 882.35nmol/L at 43℃ for 2 h, and rewarmed at 37℃ for 3 h. The cell survival rate of HK-2 cells was examined by CCK8. (D) Western blot and quantitative analyses (D1) were performed to evaluate the expression of P53, SLC7A11, and GPX4. (E and F) Intracellular Fe²⁺ and ROS in HK-2 cells were detected. The values are the mean ± SD from triplicate independent experiments with statistical significance: *P < 0.05 the control group; ^#P < 0.05 vs. the Mb group.

Figure 5

Baicalein protects HK-2 cell ferroptosis induced by myoglobin under heat stress by inhibiting endoplasmic reticulum stress. (A) Human HK-2 cells were treated with myoglobin at a concentration of 882.35nmol/L and baicalein (Selleck Chemicals, USA) at a concentration of 20 μmol/L at 43℃ for 2 h and rewarmed at 37℃ for 3 h. HK-2 cells were treated with DMSO as control. The cell survival rate of HK-2 cells was examined by CCK8. (B, B1, B2, B3, B4) Total protein was stained, and p-PERR/PERK, p-eIF2, p-IRE1/IRE1, ATF-4, ATF-6, and CHOP were analyzed by Western blotting. (C) Western blot and quantitative analyses (C1) were performed to evaluate the expression of p53, SLC7A11, and GPX4. (D and E) Intracellular Fe²⁺ and ROS in HK-2 cells were detected. The values are the mean ± SD from triplicate independent experiments with statistical significance: *P < 0.05 the DMSO group; ^#P < 0.05 vs. the Mb group.