Rhabdomyolysis induced AKI via the regulation of endoplasmic reticulum stress and oxidative stress in PTECs

Abstract

Although rhabdomyolysis-associated acute kidney injury (AKI) is a life-threatening clinical condition that accounts for about 10–40% of AKI cases, the mechanism underlying rhabdomyolysis-associated AKI has not been fully understood. In an in vivo study on the use of antioxidant N-acetyl-L-cysteine (NAC) to block endoplasmic reticulum (ER) stress and oxidative stress, functional and histologic results showed more progressive renal damages and increased expressions of caspase-12, cleaved caspase-3, BAX, GRP78 and CHOP among the glycerol-treated littermates (50%, 10 mL kg⁻¹, IM), compared with the control group. Serum levels of SOD and MDA levels decreased in the glycerol group, while the NAC pretreatment group exhibited partially reversed functional and histological changes. Moreover, in an in vitro study, cells apoptosis, levels of GRP78, CHOP, caspase-4, cleaved caspase-3, BAX and reactive oxygen species (ROS) expressions were found to increase in response to the myoglobin treatment (200 mM), highlighting the involvement of apoptosis of proximal tubule epithelial cells (PTECs) in the activation of ER stress and oxidative stress. Taken altogether, the results indicate that rhabdomyolysis-induced AKI may be due to activated ER stress and oxidative stress in PTECs.

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Introduction

Rhabdomyolysis is a clinical condition initiated by acute destruction of skeletal muscle. Such destruction, often irreversible, is caused by metabolism disorders, a crush injury, drugs (e.g. statin,² psychotropic drugs^3,4), toxins (e.g. alcohol,⁵ biotoxins,^6,7 methamphetamine⁸), infection, inflammation, etc.⁹ When skeletal muscle is damaged, intracellular proteins, in particular myoglobin, often leak into circulatory systems, resulting in AKI characterized by myoglobinuria and hyperkalemia.¹⁰ A recent study has reported that up to 15% of rhabdomyolysis patients developed AKI,¹¹ and the mortality due to crush injury-induced AKI could rise up to more than 40% without timely treatment.¹²

The experimental model of rhabdomyolysis-associated AKI is induced in vivo by glycerol injection and in vitro by myoglobin treatment of proximal tubule epithelial cells (PTECs). Some studies suggested that endoplasmic reticulum (ER) stress and oxidative stress are involved in PTECs apoptosis,^1,13–15 while other studies have shown that the administration of NAC has a protective effect on relieving rhabdomyolysis-associated AKI.^1,16,17

ER is a delicate organelle with a pivotal role in secretory protein folding, intracellular Ca²⁺ storage, and synthesis of unsaturated fatty acids, sterols and phospholipids.¹⁸ Work overload due to stressful conditions, including hypoxia, hypoglycaemia and calcium imbalance, can be detected by ER transmembrane receptors such as PERK (protein kinase RNA-like kinase),¹⁹ ATF6 (activating transcription factor 6) and IRE1, which are combined with the ER chaperone glucose-regulated protein 78 (GRP78).¹⁸ Activation of the unfolded protein response (UPR), which is initiated by these three receptors, releases GRP78 to restore normal ER function.²⁰ If the stress is prolonged, excessive ER stress induces cell apoptosis.^21–23 CCAAT/enhancer-binding protein–homologous protein (CHOP) is the objective gene with pro-apoptotic functions in both PERK and ATF6 mechanisms of ER stress.^23–25 Caspase-12, which is only expressed in rodents, is a major mediator of ER stress-induced apoptosis. ER stress occurs in the downstream receptors, such as IRE1 and ATF6, which activate caspase-12 and induce caspase-3 cleavage to initiate apoptosis.^17,26,27

On the other hand, oxidative stress is closely related to ER stress in the progression of DN, Parkinson disease, and rotavirus infection.^16,28,29 Previous studies highlighted that myoglobin, induced by rhabdomyolysis, can promote oxidative stress as a pro-oxidant enzyme.³⁰ Oxidative stress is caused by the imbalance between the generation of reactive oxygen species (ROS) and the defensive capability of antioxidants.³¹ It can cause cellular apoptosis via mitochondria-dependent and mitochondria-independent pathways and is involved in toxin-induced organ pathophysiology.³² Malondialdehyde (MDA) is a final metabolite product of lipid peroxidation, which is generated when overproduction of ROS alters the oxidant balance and disrupts the membrane lipid composition through lipid peroxidation. Superoxide dismutase (SOD), one of the most important antioxidant enzymes that act as the original protective system against ROS, is generated during oxidative stress. Thus, MDA and SOD are often used as biomarkers that measure the level of oxidative stress.^33–35

In our study, we hypothesize that the progressive kidney dysfunction of rhabdomyolysis-associated AKI is due to activated ER stress and oxidative stress, and the NAC pretreatment improves renal functions of rhabdomyolysis-associated AKI by blocking these two signaling pathways.

Results

Glycerol induced acute kidney injury

After the glycerol injection, serum creatinine (sCr) markedly increased in a time-dependent manner and the peak was observed (74.72 ± 5.34 μmol L⁻¹) at 48 h (Fig. 1A). Creatine kinase (CK) significantly increased in the glycerol-injected group, suggesting structural damages to muscle tissues (Fig. 1B). Histologic evidence of kidney tissues exhibited the development and progression of proximal tubule dilation, epithelial simplification, infiltration of inflammatory cells and cast formation in the glycerol-injected group, as opposed to the control group (Fig. 1C).

Fig. 1 Glycerol induced acute kidney injury. Serum creatinine elevated in glycerol injection treatment group on a time-related pattern (A). CK levels increased in response to glycerol injection (B). Histologic changes were observed after glycerol injection at different time points (C, black arrow: proximal tubule dilation; white arrow: epithelial simplification; yellow arrow: cast formation; red arrow: infiltration of inflammatory cells). Values are mean ± standard error of mean (SEM) (n = 5–7 for each group). ***P < 0.001; **P < 0.01; *P < 0.05.

Myoglobin induced the apoptosis of PTECs

Morphologic changes of PTECs by electron microscope showed that myoglobin treatment induced mitochondrial swelling, expansion of ER and nucleoplasm accumulation, reflecting early phase of apoptosis (Fig. 2B). Results of TdT-mediated dUTP Nick-End Labelling (TUNEL) staining of kidney tissues indicated that the injection of glycerol induced PTECs apoptosis, but the number of TUNEL-positive cell/HPF decreased at 48 h compared with that of 24 h (Fig. 2A and C). In an in vitro study, flow cytometry, which measured the apoptosis rate, was consistent with that of TUNEL staining in each group. The highest apoptosis rate happened at 24 h after myoglobin treatment (Fig. 2D and E).

Fig. 2 Myoglobin induced the apoptosis of PTECs. Kidney tissues were subject to TUNEL staining (A), and the positive staining cells were counted in each field. Original magnification, ×200. HPF, high-powered field (C). At different time points after treatment, morphologic changes of renal tubular epithelial cell were observed under the electron microscope (B, white arrow: expansion of endoplasmic reticulum; black arrow: swelling mitochondrial; yellow arrow: nucleoplasm accumulation; blue arrow: infiltration of inflammatory cells). Flow cytometry revealed apoptosis in HK-2 cells (D and E). Values are mean ± SEM (n = 5–7 for each group). ***P < 0.001; **P < 0.01; *P < 0.05.

Myoglobin up-regulates caspase-related apoptosis

In tissues immunohistochemistry (IHC) analysis, we observed that the apoptosis-related protein expression of cleaved caspase-3, Bcl-2 associated X protein (BAX) and caspase-12 time-dependently increased in the glycerol treatment group, compared with the control group (Fig. 3A). Western blot analysis demonstrated similar changes in the apoptosis-relevant proteins levels (Fig. 3B). The mRNA levels of cleaved caspase-3, caspase-4, BAX and Bcl-2 were measured by qPCR in the in vitro study. The results indicated that the mRNA levels of cleaved caspase-3 and caspase-4 were up-regulated in response to myoglobin treatment, but there were no significant differences in BAX or Bcl-2 mRNA levels (Fig. 3D). However, the protein levels of cleaved caspase-3, BAX and caspase-4 were elevated in a time-dependent manner in an in vitro study, which was consistent with that of the in vivo study (Fig. 3C). IHC staining of HK-2 cells, a human renal proximal tubule epithelial cell line, exhibited similar changes in cleaved caspase-3 and BAX proteins levels, compared with that of the in vivo study (Fig. 3E).

Fig. 3 Myoglobin up-regulated caspase-related apoptosis signaling pathway. At different time points after glycerol injection treatment, mouse renal cortical tissues were subject to IHC (A) and western blot analysis with indicated antibodies (B). HK-2 cells were harvested after indicated treatment and subject to western blot analysis (C) and qPCR (D). HK-2 cells were subject to IHC with indicated antibodies (E). ***/^###P < 0.001; **P < 0.01; */^#P < 0.05.

Myoglobin aggravated expressions of ER stress markers

CHOP and GRP78, of which expressions were induced during ER stress, significantly increased in response to the glycerol injection (Fig. 4A and B). The mRNA levels of CHOP and GRP78 increased at 24 h and 48 h in myoglobin-treated HK-2 cells (Fig. 4D). As shown in Fig. 4C, the GRP78 protein level was up-regulated at 2 h, while the significant rise of CHOP levels was observed at 8 h in the in vitro study. The results highlighted that rhabdomyolysis-associated myoglobin may activate ER stress in HK-2 cells by regulating CHOP and GRP78.

Fig. 4 Myoglobin aggravated ER stress featured proteins expressions. Mouse renal cortical tissues were subject to IHC (A) at different time points after glycerol injection treatment and western blot analysis with indicated antibodies (B). HK-2 cells were harvested after indicated treatment and subject to western blot analysis (C) and qPCR (D). ***/^###P < 0.001; **P < 0.01; */^#P < 0.05.

Myoglobin activated oxidative stress

The serum levels of MDA and SOD were examined to evaluate the degree of oxidative stress in mice with rhabdomyolysis-associated AKI. As for the SOD levels, there were no significant changes until 2 h after the glycerol injection. But the SOD level was significantly reduced afterwards (Fig. 5A), while the MDA increased in response to the treatment (Fig. 5B). The 2′,7′-fluorescence probe was used to test the activation of ROS. We detected strong fluorescence after 2 h and 4 h in myoglobin-treated HK-2 cells (Fig. 5C). The results of other groups were not shown because of overwhelmingly strong fluorescence to be collected under the same parameters.

Fig. 5 Myoglobin activated oxidative stress and induced ROS accumulation. Serum levels of SOD (A) and MDA (B) were tested using commercial ELISA kits. ROS activation was marked with DCFH-DA and observed by confocal laser scanning microscope (C). *P < 0.05.

NAC improved renal function by the regulation of apoptosis, ER stress and oxidative stress in glycerol-induced AKI

NAC was an antioxidant that partially blocks the oxidative stress and ER stress. The NAC pretreatment reduced sCr levels in the NAC-block group, compared with the treatment group (Fig. 6B), whereas no significant differences in CK levels were observed between the NAC-block and treatment groups (Fig. 6C). Since the highest TUNEL score was detected at 24 h after the glycerol injection, we harvested the kidney of NAC-treated models at 24 h for further investigation. TUNEL staining indicated that fewer tubular epithelial cells underwent apoptosis in the NAC group than in the treatment group (Fig. 6F). Caspase-12, BAX and cleaved caspase-3 proteins, which are related to apoptosis, were inhibited in the NAC group, through IHC staining (Fig. 6A) and western blot analysis (Fig. 3B). This implicates that NAC may protect kidney functions from glycerol induced AKI by blocking the caspase-related apoptosis signalling pathway. Moreover, both CHOP and GRP78 expressions decreased during ER stress in response to the NAC pretreatment, suggesting that NAC ameliorated ER stress and further improved kidney functions (Fig. 6A and 4B). The serum levels of MDA decreased while the SOD level increased in response to the NAC pretreatment, indicating the important role of suppression of oxidative stress in renal protection (Fig. 6D and E).

Fig. 6 NAC improved renal function by regulating apoptosis, ER stress and oxidative stress in glycerol-induced AKI models. NAC was administrated at 24 h, and mouse renal cortical lysates were subject to IHC with the indicated antibodies (A). Serum creatinine decreased in the NAC-block groups (B), but CK levels showed no differences between NAC-block and glycerol treatment groups (C). Kidney tissues were subject to TUNEL staining (F), and the positive staining cells were counted in each field. Original magnification, ×200. HPF. Serum levels of SOD (D) and MDA (E) were tested using commercial ELISA kits. *Compared with the control groups. ^#Compared with the treatment groups. ***P < 0.001; **P < 0.01; *P < 0.05; ^#P < 0.05.

Caspase-related apoptosis, ER stress and ROS accumulation in myoglobin-treated HK-2 cells

At 24 h after myoglobin treatment, when apoptosis rate was supposed to peak, flow cytometry of HK-2 cells confirmed NAC ameliorated apoptosis (Fig. 7A). Expressions of BAX, cleaved caspase-3, caspase-4 and CHOP mRNAs significantly decreased in NAC-block group compared with treatment group. However, GRP78 and Bcl-2 mRNA levels did not exhibit significant differences between treatment and NAC-block groups (Fig. 7B). Furthermore, western blot revealed downregulation of levels of BAX, cleaved caspase-3, caspase-4, and CHOP and GRP78, in response to NAC treatment (Fig. 3C and 4C). ROS levels by the 2′,7′-fluorescence probe were markedly reduced in the NAC group, confirming that the NAC treatment suppresses the oxidative stress (Fig. 7C).

Fig. 7 NAC improved renal function by the regulation of apoptosis, ER stress and oxidative stress in glycerol-induced AKI models. NAC blockage was applied in vivo as mentioned in concise methods. Apoptosis rates of HK-2 cells were revealed by flow cytometry (A). HK-2 cells were subject to qPCR to examine the mRNA levels of relevant proteins (B). ROS significantly decreased in NAC-block group (C). Values are mean ± SEM (n = 5–7 for each group). ***P < 0.001; **P < 0.01; *P < 0.05.

Discussion

Through both in vivo and in vitro studies, we explored the underlying mechanisms of rhabdomyolysis-induced AKI. Our study confirmed our hypothesis that myoglobin, the key pathogenic protein of rhabdomyolysis, induced the AKI by activating the ER stress and oxidative stress, triggering caspase-3 activation and promoting the apoptosis of PTECs. The antioxidant NAC exhibited protective effects on rhabdomyolysis-associated AKI by the blockage of ER stress and oxidative stress in PTECs (Fig. 8).

Fig. 8 Mechanism for the role of ER stress and oxidative stress activation in the rhabdomyolysis-associated AKI and NAC ameliorates the injury by blocking ER stress and oxidative stress. NAC, N-acetylcysteine; ER, Endoplasmic Reticulum; AKI, acute kidney injury.

Rhabdomyolysis-associated AKI model was established by intramuscular injection of glycerol at bilateral back limbs. With the skeletal muscle degeneration and muscle enzyme leakage, myoglobin was released into the circulation with many other intercellular substances including CK.^10,36 The myoglobin filtered through the glomerular filtration barrier and become metabolized, resulting in kidney function damages.¹⁰ Similar CK levels in the glycerol injection group and the NAC pretreatment group supported that the glycerol injection induces muscle degeneration equally. Greater sCr levels were observed for the glycerol injection littermates, compared with the NAC pretreatment littermates. This hinted more reduction in kidney functions among the glycerol injection littermates. Histopathological examination results were consistent with the changes of kidney functions. TUNEL staining and flow cytometry results illustrated the key role of PETCs apoptosis in the progression of rhabdomyolysis-associated AKI.

ER stress was an important cellular response to systemic stress, which is necessary for resisting injury. However, when ER stress is beyond the capacity of the compensatory machinery, cells undergo apoptosis. Considerable studies indicated that ER stress in PTECs plays essential roles in the development of kidney diseases including AKI, primary glomerular disease, diabetic nephropathy (DN), cisplatin-induced nephrotoxicity, and renal ischemia–reperfusion injury.^4–7 In this study, we found that GRP78, CHOP and caspase-12 (or caspase-4 in human) were up-regulated at both the gene and protein levels in response to in vivo glycerol injection and in vitro myoglobin treatment. The results indicated that ER stress is involved in PETCs apoptosis in rhabdomyolysis-associated AKI. Furthermore, NAC pretreatment significantly decreased the PETCs apoptosis, and down-regulated GRP78, CHOP and caspase-12 (or caspase-4 in human), at both gene and protein levels. Thus, blocking ER stress could ameliorate PETCs apoptosis in rhabdomyolysis-associated AKI.

Previous studies have demonstrated that stress is a critical pathogenic factor in the initiation, development, and progression of most renal disease models. Recent studies have shown that ROS contributes to the cancer cell apoptosis.³⁷ In this study, we revealed glycerol injection increased serum MDA level and reduced SOD level, suggesting that oxidative stress evolves during the rhabdomyolysis-associated AKI. However, the serum SOD levels did not decrease at the early stage of AKI. This was partly due to the compensatory mechanism that generated extra SOD in response to oxidative stress. But when the generation of SOD could not keep pace with the excessive oxidative stress, the SOD level would decline. In the in vitro study, we observed excessive ROS accumulation in response to myoglobin treatment. Furthermore, the NAC pretreatment restored MDA, SOD and ROS levels, along with the PETCs apoptosis, indicating that blockage of oxidative stress could ameliorate PETCs apoptosis.

In summary, rhabdomyolysis leads to the release of myoglobin and aggravates AKI by activating ER stress and oxidative stress and by causing the apoptosis of PTECs. Hence, blockage of ER stress and oxidative stress in PTECs might alleviate AKI through the antioxidant NAC.

Experimental

Animal

This study adheres to the “Principles of Laboratory Animal Care” (National Institutes of Health publication 85-23, revised 1985) that seeks to minimize both the number of animals used and any suffering that they might experience, and conducted according to the Guidelines for the Care and Use of Laboratory Animals of West China Medical School. The animal protocol was approved by the Animal Care and Use Committee of Sichuan University (IACUC no. 20100318). Healthy 8 week male C57BL/6 mice were housed in a controlled environment (constant temperature at 20 ± 2 °C and humidity at 50–60% with a 12 h light and 12 h dark cycle) and had free access to food and water. After 1 week of adaptation to the housing conditions, the mice were randomly divided into three groups (n = 7 per group): the control group, the treatment group (glycerol injection); and the NAC-block group. The mice were sacrificed at 2 h, 4 h, 8 h, 12 h, 24 h and 48 h after the exposure. Blood sample was collected and stored at −80 °C. The upper half of the left kidney was quickly removed and frozen in liquid nitrogen, then stored at −80 °C until processed for western blot analysis. The lower half of the left kidney was fixed in 2.5% glutaraldehyde for 2 hours at 4 °C and processed for electron microscope. The right kidney was quickly removed and fixed in 10% phosphate buffered formalin for HE staining, IHC and TUNEL assay.

Myoglobin-induced AKI model

The mice were injected with 50% glycerol dissolved in 0.9% normal saline (10 μL g⁻¹) at bilateral back limbs to induce rhabdomyolysis and myoglobin-induced AKI model. The AKI model was considered established when the level of serum creatinine of the treatment group rose up to 2 times of their control littermates.

NAC blockage

NAC (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was dissolved in DMSO and diluted in 0.9% normal saline to be administered at a dose of 400 μg g⁻¹, and was intraperitoneally injected 30 minutes before the glycerol injection of myoglobin-induced AKI model.

Serum analysis

Serum creatinine levels and CK levels were evaluated by high performance liquid chromatography (HPLC) conducted by the Institute of Drug Clinical Trial and the GCP center of West China Hospital. Serum MDA (BioVision, Inc., San Francisco, CA, USA), and SOD (Cayman Chemical, Ann Arbor, MI, USA) activities were measured using the respective commercially available kits according to the manufacturers' protocols.

Histologic examination

The right kidney, fixed in 10% phosphate buffered formalin, was dehydrated in a graded series of alcohol concentrations and embedded in paraffin. Kidney blocks were cut into 2 μm sections and then subject to HE staining for morphologic analysis and TUNEL staining for cell apoptosis and IHC. HE-stained tissue sections were viewed by light microscopy at magnifications of ×200 or ×400. For semi-quantitative analysis of morphological changes, high-magnification (×200) fields of the cortex and outer stripe of the outer medulla were randomly selected. All quantifications were performed blindedly. Histopathological changes were evaluated by the percentage of injured/damaged renal tubules, as indicated by tubular lysis, dilation, disruption, and cast formation. Tissue damages were scored on a scale of 0–4, with 0, 1, 2, 3, 4 corresponding to 0%, <25%, 26–50%, 51–75%, ≥76% of injured/damaged renal tubules, respectively. For the TdT-mediated dUTP Nick-End Labelling (TUNEL) staining, the sections were stained using an in situ cell death detection kit, fluorescein (Roche, Basel, Switzerland), according to the manufacturer's protocol for paraffin-embedded sections. Positive cells were counted at magnification of ×200, and at least 10 fields per section for each sample were examined (n = 5–7).

Electron microscopy

After being fixed in cold 2.5% glutaraldehyde for 2 h at 4 °C, kidney tissues were washed with phosphate-buffered saline (PBS) (0.2 mol L⁻¹, pH 7.4) for 2 h, fixed with 1% osmic acid for 2 h, and then washed six times with PBS for 10 min per wash. The samples were dehydrated with ethanol and cleaned with epoxypropane. They were embedded in EPON 812 overnight at room temperature. Ultrathin sections (40–60 nm) were cut (EM UC61rt, Leica) and stained with uranyl acetate/lead citrate. These sections were subsequently visualized using a transmission electron microscope (H-7650, Hitachi). The figures shown were at magnification of ×10 000.

Immunohistochemistry

Mice were euthanized at various time points after injury, and kidneys were harvested, followed by overnight fixation in 10% phosphate buffered formalin. The fixed kidneys were dehydrated through a graded series of ethanol, embedded in paraffin, sectioned (5 mm), and mounted on glass slides.

HK-2 cells were cultured in 24-well plates on glass coverslips embedded with polylysine. After indicated treatments, the cells were washed thrice with PBS and fixed in 4% paraformaldehyde at 4 °C. Paraformaldehyde was removed 20 minutes later. The coverslips were washed thrice with PBS, followed by incubation with 3% H₂O₂ at room temperature for 10 minutes. The coverslips were washed thrice with PBS and mounted on glass slides.

The slides were blocked with 2.5% normal goat serum and incubated with primary antibodies at 4 °C. The slides were washed thrice in PBS, and VECTASTAIN ABC Kit (Vector, Burlingame, CA, USA) was used for staining following the manufacturer's instruction. The sections were counterstained with hematoxylin. Images were captured using an AxioCam HRc digital camera (Carl Zeiss), and the positive-staining cells were counted in each of 3400 fields, with 10 fields randomly chosen for each section. Mean values of data were reported as mean ± standard error of the mean (SEM) (n = 5–7 for each group).

Western blot analysis

Mouse kidney cortexes were dissected and homogenized in radio immune precipitation (RIPA) lysis buffer (P0013B, Beyotime Biotechnology, China). HK-2 cells were harvested in RIPA lysis buffer after the indicated treatment. After centrifugation at 13 000 rpm for 15 minutes at 4 °C, equal amounts of protein lysate were loaded directly on 12.5% SDS-PAGE, transferred onto Immun-Blot PVDF (polyvinylidene fluoride) membrane for protein blotting (0.2 μm, Bio-Rad Laboratories, Inc) by wet electroblotting. The membranes were blocked for 1 hour at room temperature with 5% skim milk dissolved in TBS and washed thrice with TBS containing 0.05% Tween 20 (TBS-T). Next, the membranes were probed with the indicated primary antibodies at 4 °C overnight. The membranes were washed thrice with TBS-T and incubated with the indicated secondary antibody for 2 hours at room temperature. The membranes were washed thrice with TBS-T, and immunoblots were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA) with Bio-Rad ChemiDoc MP. The signals were quantified with the Image J program (NIH, Bethesda, MD, USA). The ratios for the proteins examined were normalized to β-actin and were expressed as the mean ± SEM.

Antibodies

In in vivo study, antibodies against caspase-12, BAX, CHOP, and GRP78 were purchased from Abcam (Cambridge, MA, USA), while cleaved caspase-3 antibody was from Cell Signalling Technology (CST, Beverly, MA, USA). In the in vitro study, antibodies against caspase-4, cleaved caspase-3, BAX and CHOP were purchased from CST, and GRP78 antibody was from Abcam. β-Actin antibody and all secondary antibodies were purchased from R&D Systems (MI, USA). Details of antibodies are provided in ESI (Table S1†).

Ferrous myoglobin media

Ferrous myoglobin is a type of cytotoxic that induces AKI.³⁸ Myoglobin from equine skeletal muscle (M5696, Sigma Aldrich Corporation, St. Louis, MO, USA) and ascorbic acid (Sigma Aldrich Corporation, St. Louis, MO, USA) were dissolved in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 media (DMEM/F12, HyClone, GE Healthcare Life Sciences, UT, USA) containing 10% fetal calf serum (FBS, HyClone, GE Healthcare Life Sciences, UT, USA) to prepare ferrous myoglobin immediately before experiment. The final concentration of myoglobin was 200 mM, while that of ascorbic acid was 2 mM. According to the existing literature^39,40 and our pilot study, we applied ferrous myoglobin at a concentration of 200 mM. The ferrous myoglobin media was filtered through 0.2 μm sterile filter before finally applied to cells.

Cell culture

HK-2 cell was obtained from American Type Culture Collection (ATCC). Cells were cultured in DMEM/F-12 containing 10% FBS, and incubated at 37 °C in a humidified atmosphere of air/CO₂ (95 : 5). We divided the cells in exponential growth state into 3 groups: for treatment group, cells were incubated with 200 mM ferrous myoglobin for indicated times. For control groups, cells were incubated for indicated times with complete medium alone. For the NAC-block group, cells were incubated with NAC at 2.5 mM 30 min prior to myoglobin treatment.

Flow cytometry

The cells were harvested and the samples were prepared using the BD Annexin V-FITC/PI apoptosis detection kit according to manufacturer's instruction. The signals were detected using a CytoFLEX by Beckman & Coulter.

RNA isolation and quantitative real-time PCR

Total RNA was extracted from HK-2 cells with an RNeasy kit according to the manufacturer's instructions (Takara, Japan). The RNA concentration and purity were confirmed with Nanodrop 2000. The RNA quality was tested by agarose gel electrophoresis followed by cDNA synthesis. Real-time PCR was performed with the CFX96TM Real-Time System (Bio-Rad, Hercules, USA) and SYBR Premix Ex TaqTM II (Tli RNase H Plus) (Takara). The following primers were used for quantitative PCR: glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-forward (5′-AGA AGG CTG GGG CTC ATT TG-3′), reverse (5′-AGG GGC CAT CCA CAG TCT TC-3′); caspase-4-forward (5′-GAG GGC ATT TGC TAC CAG AC-3′), reverse (5′-ATG CAC AGT TCC GCA GAT T-3′); caspase-3-forward (5′-AGA TGG TTT GAG CCT GAG CA-3′), reverse (5′-CAG TGC GTA TGG AGA AAT GG-3′); BAX-forward (5′-CCG ATT CAT CTA CCC TGC TG-3′), reverse (5′-TGA GCA ATT CCA GAG GCA GT-3′); Bcl-2-forward (5′-CAA CAC AGA CCC ACC CAGA-3′), reverse (5′-TGG CTT CAT ACC ACA GGT TTC-3′); CHOP-forward (5′-CTT CTC TGG CTT GGC TGA CT-3′), reverse (5′-TCC CTT GGT CTT CCT CCT CT-3′); gene expression was analysed using the 2^−ΔΔC_t method by normalizing with GAPDH gene expression in all experiments.

Measurement of intercellular ROS level

DCFH-DA was used as an ROS probe. HK-2 cells were incubated in 6-well plates, exposed to the indicated treatment, and then incubated with 10 μM DCFH-DA (D6883, Sigma Aldrich Corporation, St. Louis, MO, USA) for 30 min. After removing the DCFH-DA from the wells, the cells were washed thrice with PBS and the signals were detected by Nikon A1Si confocal laser scanning microscope (Nikon, Japan).

Statistical analysis

Descriptive data were presented as mean ± SEM. Two sample t-tests were used for statistical analysis. For multiple group comparisons, ANOVA and Bonferroni-corrected t-tests were used; a two-sided P value < 0.05 was considered statistically significant.

Conclusion

The in vivo and in vitro studies confirmed that myoglobin, a key pathogenic protein of rhabdomyolysis, induces AKI by activating ER stress and oxidative stress, leading to caspase-3 activation, and finally causing the apoptosis of PTECs. Moreover, by blocking ER stress and oxidative stress in PTECs, the antioxidant NAC alleviates kidney damages caused by rhabdomyolysis-associated AKI.

Contributions

Research design and conducted experiments: Feng YY, Ma L., Liu LF, Zhang XM, Shi M., Zhang L. and Fu P. Performed data analysis: Zhang XM, Guo F., Huang RS, and Zhang L. Contributed to the writing of the manuscript and English revision: Feng YY, Ma L., Hong HG, Li Y.

Acknowledgements

This study was supported by Natural Science Foundation of China (No. 81270818, 81570668, 11528102).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra18865f

‡ These authors contributed equally to this work.

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