Augmenter of liver regeneration promotes mitochondrial biogenesis in renal ischemia–reperfusion injury


Mitochondria are the center of energy metabolism in the cell and the preferential target of various toxicants and ischemic injury. Renal ischemia–reperfusion (I/R) injury triggers proximal tubule injury and the mitochondria are believed to be the primary subcellular target of I/R injury. The promotion of mitochondrial biogenesis (MB) is critical for the prevention I/R injury. The results of our previous study showed that augmenter of liver regeneration (ALR) has anti-apoptotic and anti-oxidant functions. However, the modulatory mechanism of ALR remains unclear and warrants further investigation. To gain further insight into the role of ALR in MB, human kidney (HK)-2 cells were treated with lentiviruses carrying ALR short interfering RNA (siRNA) and a model of hypoxia reoxygenation (H/R) injury in vitro was created. We observed that knockdown of ALR promoted apoptosis of renal tubular cells and aggravated mitochondrial injury, as evidenced by the decrease in the mitochondrial respiratory proteins adenosine triphosphate (ATP) synthase subunit β, cytochrome c oxidase subunit 1, and nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) beta subcomplex 8. Meanwhile, the production of reactive oxygen species was increased and ATP levels were decreased significantly in HK-2 cells, as compared with the siRNA/control group (p < 0.05). In addition, the mitochondrial DNA copy number and membrane potential were markedly decreased. Furthermore, critical transcriptional regulators of MB (i.e., peroxisome proliferator-activated receptor-gamma coactivator 1 alpha, mitochondrial transcription factor A, sirtuin-1, and nuclear respiratory factor-1) were depleted in the siRNA/ALR group. Taken together, these findings unveil essential roles of ALR in the inhibition of renal tubular cell apoptosis and attenuation of mitochondrial dysfunction by promoting MB in AKI.


Acute kidney injury (AKI) is a severe clinical complication that is associated with a high mortality rate and socioeconomic loss due to the lack of effective therapeutic strategies to cure or delay the progression of the disease [1, 2]. In addition, AKI is the primary risk factor for chronic kidney disease (CKD), increasing the risk of stage 4 or 5 CKD by as much as 28-fold [3]. Renal ischemia–reperfusion (I/R) injury is a common cause of AKI with the renal proximal tubular cells being the particular target of I/R-induced AKI [4, 5]. The kidney is one of the most energy-demanding organs and, thus, has an abundant mitochondrial content, second to the heart, especially the cells of the proximal tubules [6,7,8]. Compelling evidence in nephrology research has established that mitochondrial dysfunction is central to the pathogenesis of AKI and triggers the dysfunction of renal tubular epithelial cells and eventual death. Epithelial cell injury in AKI is characterized by decreased production of adenosine triphosphate (ATP), increased formation of reactive oxygen species (ROS), and increased release of cytochrome c [9, 10]. Importantly, several studies have shown that mitochondrial dysfunction is a typical hallmark of I/R-induced kidney injury and occurs earlier than renal dysfunction or morphological injury [11]. Notably, mitochondria are dynamic organelles that maintain homeostasis and a variety of cellular functions, such as the main source of ROS production and cytosolic calcium levels. Most importantly, mitochondria are the main sites of ATP production for energy homeostasis in cells. Once mitochondrial dysfunction occurs, ATP depletion and ROS accumulation result in oxidative stress that leads to eventual cell death. In response, mitochondrial biogenesis (MB) occurs to maintain cellular homeostasis against oxidative stress and injury by forming new mitochondria [12]. Taken together, the ability of maintaining mitochondrial homeostasis by MB is critical to the proper function of mitochondria.

Augment of liver regeneration (ALR), also known as hepatic stimulator substance, specifically stimulates the regeneration of hepatocytes [13]. ALR is highly homologous to the yeast Erv family protein and is frequently expressed in human liver and kidney cells [14]. Several studies have found that ALR is located in the mitochondrial intermembrane space where it participates in the import of mitochondrial proteins [15, 16]. Other than its profound proliferative effect, ALR also protects steatotic hepatocytes from I/R injury by attenuating oxidative stress and mitochondrial dysfunction [17]. Alternatively, another investigation elucidated that liver-specific ALR knock-out (ALR/) mice had reduced hepatocyte mitochondrial respiratory function and increased oxidative stress [18].

We previously reported that ALR protected human kidney (HK)-2 cells from oxidative injury via the mitochondrial respiratory chain [19]. Furthermore, injection of exogenous recombinant human augmenter of liver regeneration effectively reduced tubular injury and restored renal function in rats subjected to I/R injury [20]. Therefore, ALR may also protect the kidney from I/R injury [21], although the underlying mechanism remains unclear. Therefore, in this study, HK-2 cells were treated with short interfering RNA (siRNA) against ALR and a model of hypoxia-reoxygenation (H/R) injury was created to elucidate the role of ALR in I/R injury in vitro.


Preparation of the reagents

HK-2 cells were acquired from American Type Culture Collection (ATCC, USA) and maintained in Dulbecco’s minimum essential medium plus F12 (DMEM/F12, BI, Israel) supplemented with 10% Fetal bovine serum (FBS) and 1% penicillin/streptomycin. The siRNA/ALR lentivirus with puromycin and control shRNA lentiviral were designed by Genechem Co (Ltd, Shanghai, China). The JC-1 kit and Annexin V-PE Apoptosis Detection kit were purchased from Sigma-Aldrich (MO, USA). Dichlorofluorescin diacetate (DCFH-DA) obtained from Sigma-Aldrich Corporation. The cationic fluorescent dye tetramethylrhodamine methyl ester (TMRM) and 4,6-diamidino-2-phenylindole (DAPI) were purchased from AAT Bioquest (California, USA) and Keygen Biotech (KeyGen Biotech. Co. Ltd., Nanjing, China), respectively. The ALR primer, Reverse Transcription kit and its Real-time PCR kit were purchased from Takara Biotechnology (Shiga, Japan). Antibodies anti-COX1, anti-TFAM, anti-SIRT1, anti-NRF-1, anti-ATPS-β, anti-NDUFB 8, anti-PGC-1α antibodies used for Western blotting were purchased from Abcam (Cam bridge, UK). Cell immunofluorescence ALR antibody and DyLight 549 goat anti-mouse IgG were from Santa Cruz Biotechnology (CA, USA).

Cell culture and transfection

HK-2 cells were plated in DMEM/F12 supplement with 10% FBS and 1% penicillin/streptomycin in a 37 °C cell incubator (Thermo Fisher, MA, USA) with 5% CO2. The siRNA/ALR and siRNA/control were transfected at a multiplicity of Infection (MOI) of 6 for 72 h following the manufacturer’s instructions. Then all the cells were selected with 3 ug/ml of puromycin (Sigma–Aldrich Corporation) to perform for the next experiment.

Mode of I/R in HK-2

For induction of HR injury in vitro, the medium was displaced with serum-deprived media overnight to synchronize cell growth under the normal culture condition (37 °C, 95% air and 5% CO2). On the next day, HK-2 cells were washed with sterile phosphate-buffered saline (PBS) twice and then maintained with serum- deprived, d-glucose free media in a hypoxia chamber (Thermo Fisher, MA, USA) with 95% N2, 5% CO2 and 1% O2 at 37 °C for 6 h. Six hours later, HK-2 cells were removed to a general chamber (95% air and 5% CO2) and complete medium at 37 °C for indicated time point (3 h, 6 h, 12 h, 24 h). Finally, cells were collected at the indicated time point for biochemical analyses.

RNA isolation and real-time quantitative PCR

Total RNA was extracted from infected and normal HK-2 cells using the total RNA isolation kit. Each sample was synthesized and reverse transcribed according to the manufacturer’s protocol. The sequences of gene-specific primers were as follows: (ALR:5′-CAGAAGCGGGACACCAAGTTT-3′ and 5′-CACACTCCTCACAGGGGTAA-3′; GAPDH:5′-TGACTTCAACAGCGACACCCA-3′ and 5′-CACCCTGTTGCTGTAGCCAAA-3′; NDUFB 8: 5′-ACAGGAACCGTGTGGATACAT-3′ and 5′-CCCCACCCAGCACATGAAT-3′; COX1: 5′-ACAGCGATGGCTATTGAGGAGTAT-3′ and 5′-CACAGCACCAATCCTACCTCCAT-3′; ATPS-β: 5′-CGCATTTTGGTACTACACCACG-3′and 5′-TGCCTGTGATCTCTCTGACATAA-3′). All experiments were repeated at least in triplicate with three independent replicates for each group. To quantify the gene expression, a relative quantification method: \({2^{ - \Delta \Delta {{\text{C}}_{\text{t}}}}}\) was used.

Western blot analysis

Total proteins were isolated and protein concentrations from whole HK-2 cells lysates were quantified by BCA protein assay reagent (KeyGen Biotech. Co. Ltd., Nanjing, China). Briefly, 20 ug per lane were separated by 10–15% SDS-PAGE gels and proteins from samples were transferred to PVDF membranes, blocked, blotted with indicated antibodies and detected with Enhanced chemiluminescence (ECL) reagents according to standard procedures.

Apoptosis assay

Apoptotic cells were determined using Annexin V-APC detection Kit according to the manufacturer’s instruction. Briefly, cells were harvested using 0.2% trypsin free EDTA, then washed twice with PBS and resuspended in 50 µl lysis buffer. A total of 5 ul Annexin V-APC and DAPI were mixed and incubated for 10 min in the dark at 37°C and suspended in 300 µl PBS. Then all the samples were detected by flow cytometry within 1 h.

Colocalization of ALR and mitochondrial

Cells were plated in confocal dishes then further incubated overnight. After incubated with 100 nM TMRM for 20 min at 37 °C and then fixed with 4% paraformaldehyde for 15 min at room temperature. All cells were permeabilized with 0.3% Trion X-100 for 15 min and washes with PBS. Next, non-specific sites were blocked with 5% BSA and incubated overnight at 4 °C in primary antibody (1:50 in PBS). The dishes were washed with PBS for three times and then further incubated at room temperature with Goat anti-mouse IgG (FITC) for 60 min. After being washed, dishes were applied with 4,6-diamidino-2-phenylindole (DAPI, blue) for 5 min and then imaged by laser scanning confocal microscopy.

Determination of ROS levels

HK-2 cells were incubated with dichlorofluorescindiacetate (DCFH-DA), which enters cells and is converted to the fluorescent product DCF upon oxidation. So the intracellular ROS levels could be detected by DCFH-DA. Firstly, cells were seeded in 60 mm dishes at a density of 4 × 105 cells/plate and allowed to adhere for 24 h in advance. After with or without I/R treatment, HK-2 cells were exposed to 10 µM DCFH-DA and incubated in a 37 °C incubator for 15 min. At last, cells were washed with DMEM/F12 with serum-deprived media three times and analyzed by flow cytometry with 485 nm excitation wavelengths and 535 nm emission wavelengths.

Real-time PCR for mitochondrial DNA (mtDNA) copy number

The amount of mtDNA relative to Nuclear DNA (nDNA) was measured by q-PCR analysis. Total DNA were isolated from HK-2 cells by Genomic DNA Isolation Kit (Biovision, Wuhan, China) according to the standard protocols. After DNA was quantified and 20 ng of total DNA was used for q-PCR. Mitochondrial encoded NADH dehydrogenase 1(ND1) was used to measure mitochondrial copy number and was normalized to nuclear-encoded β-actin. Primers sequences for ND1 and β-actin were as follows: ND1 sense: 5′-TCTCACCATCGCTCTTCTACT-3′; DN1 antisense: 5′-AGGCTAGAGGTGGCTAGAATAA-3′; β-actin sense; 5′-CATGTACGTTGCTATCCAGGC-3′; β-actin antisense:5′- CTCCTTAATGTCACGCACGAT-3′. The SYBR Premix Ex Taq II Kit (Takara, Japan) was performed for real-time monitoring of amplification (20 ng of template, 40 cycles: 95 °C for 10 min, 95 °C for 15 s, 60 °C for 1 min). Amplification of β-actin was carried out to measure the relative mtDNA copy number, analysed by the radio of mtDNA copy number to β-actin copy number.

Mitochondrial membrane potential (MMP) assessment

MMP was measured by a fluorescent probe JC-1 in cells after I/R treatment. Shortly, JC-1 reagent B (200×), a novel cationic carbocyanine dye that accumulates in mitochondria, ultrapure water, and JC-1 buffer (5×) were mixed at a ratio of 1:160:40. HK-2 cells were collected and 500 µl JC-1 staining fluid and incubated with JC-1 working solution for 15 min at 37 °C. HK-2 cells were washed three times with DMEM/F12 and measured using FACS with 485 nm excitation and 528 nm emission wavelengths. We also used the cationic fluorescent dye TMRM to assess changes in MMP in HK-2 cells. TMRM is readily sequestered by healthy mitochondria, but its fluorescence is rapidly lost when MMP is dissipated. Cells were seeded in confocal dishes before treated with or without I/R. HK-2 cell were washed twice with PBS and incubated for 20 min at 37 °C with 100 nM TMRM for staining mitochondria and then washed the cells twice with PBS carefully. Finally, the cell colonies for each well were analyzed by a confocal microplate imager with scale bar represents 10 µm, 549 nm laser excitation and 573 nm emission.

The intracellular ATP level in HK-2 cells

The intracellular ATP content was measured by using an enhanced ATP assay kit, (Beyotime Biotechnology, Shanghai, China). All cells including siRNA/ALR group and siRNA/control group were harvested by centrifugation (12,000×g, 4 °C, 5 min) and washed with sterile PBS. Then all the cells were lyzed by lysis buffer according to the standard protocols. The concentration of ATP was calculated according to a standard curve and then read with an ELISA reader (BioTek, Winooski, VT, USA).

Transmission electronic microscope

After specified treatment, cells from each sample were subsequently collected and the morphologic alterations are assessed by transmission electron microscopy (Hitachi, Tokyo, Japan).

Cell immunofluorescence

Cells were plated on confocal dish and treated with I/R. After incubated with TMRM (100 µM) for 20 min, HK-2 cells were perfused with 4% paraformaldehyde in 0.1M phosphate buffer for 20 min, and then washed with PBS, permeabilized with 0.5% Triton-X100 in PBS for 10 min. After three washes with PBS carefully, cells were blocked with 5% BSA and incubated overnight at 4 °C with anti-ALR antibody (1:50, PBS). The dishes were then washed three times with PBS and incubated for 1 h at room temperature with a 1:50 dilution of biotinylated goat anti-mouse IgG in PBS. Finally, cell nuclei were counterstained with DAPI (blue) for 5 min and viewed by laser scanning confocal microscopy. All colocalization studies were blinded.

Statistical analysis

Parametric data in multiple groups were performed with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for comparisons of multiple groups. All data presented as mean ± standard deviation (SD). Single asterisk and double asterisk indicate statistically significant at P < 0.05 and P < 0.01, respectively.


I/R triggers mitochondrial damage and ALR was upregulated in vitro

Western blot analyses demonstrated that ALR expression was significantly upregulated, then followed by a gradual decrease. Meanwhile, the expression levels of adenosine triphosphate (ATP) synthase subunit β (ATPS-β), cytochrome c oxidase subunit 1 (COX1), and nicotinamide adenine dinucleotide: ubiquinone oxidoreductase subunit B8 (NDUFB8) gradually decreased at 3, 6, and 12 h after reperfusion, then increased at 24 h (Fig. 1). Mitochondrial injury was more notable at ischemia 6 h and reperfusion 12 h (I6R12). So, I6R12 was selected as an observation point to perform the next experiments.

Fig. 1

The expression levels of ALR, ATPS-β, COX1, and NDUFB8 in HK-2 cells were determined by western blot analysis. ALR was upregulated and ATPS-β, COX1, and NDUFB8 were downregulated following I/R injury. Parametric data were performed with ANOVA followed by Tukey’s post hoc test. Bar graphs represent the mean ± standard deviation (SD). *p < 0.05 and **p < 0.01 versus the normal control group

The effect of ALR silencing on colocalization of ALR and mitochondria

ALR is a cytochrome c reductase that is localized to the mitochondrial intermembrane space, where it is involved with mitochondrial transcripts, as confirmed by confocal laser-scanning microscopy (Fig. 2a). Additionally, after silencing of ALR in HK-2 cells, the expression levels of ALR, as quantified by western blot analysis, were significantly lower in the siRNA/ALR group (p < 0.01), as compared with the normal and shRNA/control groups (Fig. 2b). Similar results were obtained by real-time PCR (Fig. 2b). These results demonstrate the transfection efficiency of HK-2 cells.

Fig. 2

Localization of ALR and the effect of ALR gene silencing on HK-2 cells. a Colocalization of ALR and mitochondria was determined by confocal microscopy (scale bar 50 µm). b HK-2 cells were treated with ALR shRNA lentivirus and lentiviral vectors, respectively. ALR expression was verified by western blot analysis and normalized to that of β-actin. ALR mRNA expression was evaluated by real-time PCR analysis. Parametric data were performed with ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD. ** P < 0.01, versus the siRNA/control group

Changes of ROS levels after I/R in stable ALR siRNA cells

ALR acts as a growth factor with antioxidative functions. To determine whether ALR protects against mitochondrial damage, intracellular ROS levels were measured by flow cytometry. As shown in Fig. 3, ROS levels were markedly increased in the shRNA/ALR group after I/R treatment for 12 h (mean fluorescence intensity, 9967.33 ± 733.67 versus 1947.67 ± 272.67 AU, respectively, p < 0.01).

Fig. 3

Knockdown of ALR increased intracellular ROS levels in HK-2 cells. ROS accumulation was measured by DCFH-DA kit. a ROS levels were measured by flow cytometry. b The relative levels of fluorescence intensity are expressed in a bar graph. Parametric data were performed with ANOVA followed by Tukey’s post hoc test. Values are presented as the mean ± SD (**p < 0.01)

Silencing of ALR exacerbate mitochondrial activity and morphologic injury

Mitochondria provide cellular energy in the form of ATP utilizing substrates from tricarboxylic acid. Upon mitochondrial injury, oxidative phosphorylation is impaired with subsequent insufficient intercellular ATP production. Hence, protecting mitochondrial function is critical to alleviate I/R injury. To further assess the effect of silencing of ALR in HK-2 cells, indicators of mitochondrial activity, such as mitochondrial membrane potential (MMP), ATP levels, and mitochondrial morphology, were determined. After 12 h of I/R, the MMP of the siRNA/ALR group decreased significantly, as compared with the siRNA/control and normal groups (Fig. 4a–c). In addition, the levels of mitochondrial ATP are an indicator of cell death. As shown in Fig. 4d, ATP levels were decreased significantly after I/R injury in the siRNA/ALR group, as compared with the siRNA/control group. Mitochondrial dysfunction can also have structural repercussions reflected by morphological alterations. Electron microscopy showed that mitochondria appeared on average smaller and the membranes were stained darker with distinct disorder of the mitochondrial crests (Fig. 4e).

Fig. 4

The effect of siRNA/ALR on mitochondrial function and morphology. a Effects of ALR gene silencing on the MMP of HK-2 cells. Cells were stained with tetramethyl rhodamine methyl ester (100 µM) for 20 min after I/R treatment and detected by confocal laser-scanning microscopy (scale bar 10 µm). b MMP was evaluated using JC-1 dye and quantified by flow cytometry. c A bar graph of MMP. d ATP levels were measured using an ATP assay kit. e Electron microscopic analysis of mitochondria in HK-2 cells subjected to I/R. The black arrows indicate morphologic alterations of the mitochondria in all groups. Parametric data were performed with ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD of three experiments, *p < 0.05, **p < 0.01, as compared with the indicated sample

Silencing of ALR induced HK-2 cells apoptosis

Interestingly, damaged mitochondria are known to alter the mitochondrial membrane and open the mitochondrial transition pores, which release cytochrome c into the cytosol, ultimately leading to apoptotic cell death. Therefore, apoptosis was assessed after I/R treatment in vitro (Fig. 5). The apoptotic rate of HK-2 cells was evaluated with a flow cytometer. Based on the status of staining for allophycocyanin (APC) and proliferation dye eFluor 450 (PB450-A), the apoptotic population was composed as follows: viable cells (APC/PB450-A), early apoptotic cells (APC/PB450-A+), late apoptotic cells (APC+/PB450-A+), and necrotic cells (APC+/PB450-A-). The proportion of apoptotic cells was greater in the siRNA/ALR group than the siRNA/control group (31.26 ± 1.38% vs. 16.93 ± 0.87%, respectively, p < 0.01).

Fig. 5

Silencing of ALR resulted in apparent apoptosis at 12 h. a Apoptotic HK-2 cells were detected by Annexin V-APC/DAPI staining. b The mean percentage of positively stained cells. Parametric data were performed with ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD (**p < 0.01)

siRNA/ALR suppressed MB and mtDNA copy number

The expression levels of MB-related genes (i.e., ATPS-β, NDUFB8, and COX1) in HK-2 cells are shown in Fig. 6a. After I/R treatment for 12 h, total protein lysates were collected and subjected to western blot analysis. The results showed that the expression levels of ATPS-β, NDUFB8, and COX1 were significantly decreased after I/R injury, as compared with the normal and siRNA/control groups (p < 0.05). The results of real-time PCR analyses were similar (Fig. 6b). Moreover, the mtDNA content was decreased in the siRNA/ALR group after I/R exposure (Fig. 6c). Taken together, these data revealed that ALR played a crucial role in both renal I/R injury and repression of MB.

Fig. 6

Effects of silencing of ALR on MMP and mtDNA copy number in HK-2 cells. a The expression levels of ATPS-β, COX1, and NDUFB8 were measured by western blot analysis after I/R for 12 h. The ratios of ATPS-β, COX1, and NDUFB8 were quantified by densitometry based on immunoblot images. b Real-time quantitative PCR analysis showing mRNA expression levels of ATPS-β, COX1, and NDUFB8 in HK-2 cells following I/R for 12 h. The expression levels were normalized to that of glyceraldehyde 3-phosphate dehydrogenase. c Relative mtDNA content was analyzed by qRT-PCR. Parametric data were performed with ANOVA followed by Tukey’s post hoc test. Bar graphs represent the mean ± SD. *p < 0.05, **p < 0.01 as compared with the indicated sample

SiRNA/ALR depressed critical transcriptional regulators of MB

MB regulatory factors were further investigated. Briefly, HK-2 cells were subjected to I/R injury for 12 h and protein expression levels were quantified by western blot analysis. Densitometric analysis of sirtuin-1 (SIRT-1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α), and NRF-1 were normalized to β-actin expression, respectively. As shown in Fig. 7, the expression levels of SIRT-1, PGC-1α, and NRF-1 were decreased in the siRNA/ALR group following I/R injury.

Fig. 7

Effects of ALR silencing on MB regulatory factors. The expression levels of ALR, SIRT-1, PGC-1α, and NRF-1 were quantified by western blot analysis following I/R for 12 h. Quantification of the relative protein expression levels of SIRT-1, PGC-1α, NRF-1, and ALR. Parametric data were performed with ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD (*p < 0.05), as compared with the indicated sample


I/R remains the most common cause of AKI in hospitalized patients, especially among postsurgical patients. Unfortunately, the care of patients with AKI is at present largely symptomatic and supportive treatment [22]. The pathophysiology of renal I/R injury can be briefly summarized as a primary acute energy deficit during ischemic AKI, followed by the release of cellular adenosine, which modulates tissue inflammation, finally resulting cytoskeletal changes in endothelial cells and the death of renal tubular cells because of ATP depletion [23,24,25]. Unfortunately, ATP deficiency is prolonged after the reperfusion stage due to abnormal mitochondrial morphology and function [9]. Additionally, the production of cellular ROS caused by lipid peroxidation is the primary factor that promotes the inflammatory response. In our previous study, ALR increased I/R-induced AKI and accelerated kidney recovery in rats. Similarly, ALR played a protective role in the mitochondrial response to H2O2-induced oxidative stress [19]. This evidence indicates that ALR could effectively protect the kidneys by modulation of cellular ROS production, apoptotic gene expression [19], and the nuclear factor-κB pathway [26]. As a cytokine, ALR is mainly located in the membrane gap of mitochondria [27], consistent with the confocal microscopy results (Fig. 2a). Furthermore, ALR is considered a mitochondrial intermembrane space protein that is involved in the mitochondrial localization of mitochondrial intermembrane space import and assembly protein 40 homolog and transfer of electrons to cytochrome c and oxygen [28]. Thus, ALR could reduce the production of oxygen free radicals and detoxify ROS. Accordingly, with knockdown of ALR, intracellular ROS increased significantly, as compared with the siRNA/control group (Fig. 3), suggesting that ALR participates in ROS detoxification as a sensor of transferred oxygen. However, the effect of PGC-1α under conditions of oxidative stress and cardiac I/R remains controversial [29, 30]. Transient induction of PGC-1α in mouse cardiac-derived H9c2 cells increased cell death after ischemia-reoxygenation injury [30]. Similarly, PGC-1α was found to promote the proliferation of human breast cancer cells [31].

Accumulating evidence suggests protective effects of ALR in I/R-induced AKI, as ALR expression was upregulated after I/R injury, peaking at 12 h (Fig. 1). After I/R treatment, the levels of intracellular ROS were significantly increased (Fig. 3), which resulted in mitochondrial shrinkage (Fig. 4e) and a significant increase in the proportion of apoptotic cells (Fig. 5a). Mitochondrial proteins, such as PGC-1α, were depleted early after I/R injury, which was in agreement with the findings of earlier studies [11, 32], demonstrating that PGC-1α protects human renal tubule cells under conditions of oxidative stress.

In renal I/R models, the mitochondrial proteins COX1, NDUFB8, and ATPS-β were associated with mitochondrial damage and MB [11]. To observe the role of ALR in mitochondria, ALR was knocked down in HK-2 cells. After I/R treatment in vitro, there was a remarkable increase in the abundance of the mitochondrial proteins NDUFB8, ATPS-β, and COX1, suggesting that knockdown of ALR exaggerated mitochondrial injury, consistent with the depletion of ATP (Fig. 4d) and MMP (Fig. 4b). Also, persistent hypoxia and knockdown of ALR could trigger changes in mitochondrial morphology in HK-2 cells (Fig. 4e). Early studies recognized the importance of mitochondria as the hub of energy metabolism in eukaryotic cells [33]. MB is a series of complex and sophisticated processes that are regulated by a series of nuclear transcription factors and coactivators. PGC-1α, a transcriptional coactivator, was identified as a primary regulator of MB and a key regulator of energy metabolism, which plays an important role in mitochondrial oxidative phosphorylation, the tricarboxylic acid cycle, fatty acid metabolism in hepatocytes, and mitochondrial function [34, 35]. Furthermore, the deacetylation of PGC-1α by the nuclear protein SIRT1 has been extensively implicated in MB. PGC-1α co-activates nuclear respiratory factor 1, which then activates transcription factor A (mitochondrial), rather than directly triggering the transcription of mtDNA-related genes [36]. Finally, the mitochondrial content increases and mitochondrial dysfunction is repaired, but within narrow limits. Taken together, the direct effect of MB is to increase the expression levels of mitochondrial respiratory chain genes (i.e., NDUFB8, COX1, and ATPS-β), to a certain extent, enhance oxidative phosphorylation, and increase the levels of ATP for the repair of cellular damage.

The results of this study showed that knockdown of ALR in HK-2 cells increased the levels of cellular ROS and damaged mitochondrial morphology and function, which may be associated with inhibition of MB and further aggravation of mitochondrial injury in I/R-induced AKI.


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This work was supported by grants from the National Natural Science Foundation of China (81873604, 30971364), the Natural Science Foundation Project of CQ CSTC (cstc2015jcyjA10069), the Medical Scientific Research Projects of the Chongqing Health and Family Planning Commission (20142031), and the Fund for Fostering Talent in Scientific Research of Chongqing Medical University (201404).

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Correspondence to Xiao-hui Liao.

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Huang, L., Long, R., Jiang, G. et al. Augmenter of liver regeneration promotes mitochondrial biogenesis in renal ischemia–reperfusion injury. Apoptosis 23, 695–706 (2018).

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  • Augmenter of liver regeneration
  • Mitochondrial biogenesis
  • Ischemia–reperfusion injury
  • Reactive oxygen species