The human proximal tubular epithelial cell line HK-2 was obtained from the American Type Culture Collection (ATCC), ATCC Cat# CRL-2190, RRID:CVCL_0302. The sources of the antibodies used in this study were as follow: anti-LC3B (Cell Signaling Technology, 2775), anti-LC3B (Novus, NB100-2220), anti-SQSTM1 (Abcam, ab109012), anti-Cleaved Caspase 3 (Sigma-Aldrich, AB3623), anti-GAPDH (Cell Signaling Technology, 2118), anti-PINK1 (Novus, BC100-494), anti-PARK2/Parkin (Cell Signaling Technology, 4211), anti-Tom20 (Santa Cruz Biotechnology, sc-17764), HRP-labeled Goat Anti-Rabbit IgG (Beyotime, A0208), HRP-labeled Goat Anti-mouse IgG (Beyotime, A0216), Alexa Fluor 488 (Thermo Fisher Scientific, A-21222). Human serum albumin (HAS/ALB) was purchased from Sigma Aldrich (St. Louis, MO, USA). Small interfering RNA (siRNA) targeting human Parkin, negative control siRNA, Parkin plasmids, and control empty plasmid vector were purchased from GenePharma (Shanghai, China). The sequences of siRNA oligonucleotides were sa follows: Parkin siRNA, 5${}^{\prime }$-CCUUCUGCCGGGAAUGUAATT; control siRNA, 5${}^{\prime }$-UUCUCCGAACGUGUCACGUTT.

Human proximal tubular epithelial HK-2 cells were grown in Dulbecco’s modified Eagle’s/F12 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (all from Gibco, Gaithersburg, MD, USA), and human recombinant epidermal growth factor (10 ng/mL, Thermo Fisher Scientific) at 37 °C under 5% CO2. Human serum albumin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Based on the results of our previous study, albumin overload experiments were performed by culturing HK-2 cells with 8 mg/mL albumin for 8 h [18]. Small interfering RNA (siRNA) targeting human Parkin, negative control siRNA, Parkin plasmids, and the control empty plasmid vector were purchased from GenePharma (Shanghai, China). Transfections were performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions.

Total RNA was extracted from cultured cells using the TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions after 48 h of transfection. RNA concentrations were estimated by Nanodrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Reverse transcription and qRT-PCR were performed using the Custom gene qRT-PCR Quantitation Kit (GenePharma, Shanghai, China) with specific primers. The sequences of the primers were: Parkin, forward: 5${}^{\prime }$-GGAGTGCAGTGCCGTATTTG-3${}^{\prime }$ and reverse: 5${}^{\prime }$-AGGGCTTGGTGGTTTTCTTGA-3${}^{\prime }$; GAPDH, forward: 5${}^{\prime }$-GGTGGTCTCCTCTGACTTCAA-3${}^{\prime }$ and reverse: 5${}^{\prime }$-GTTGCTGTAGCCAAATTCGTTGT-3${}^{\prime }$. The cycle threshold (Ct value) was detected by a CFX ConnectTM RT-PCR system (Bio-Rad). The relative quantity of mRNA transcripts was calculated using the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{ct}}$ method, and GAPDH was used as the endogenous control for normalization.

Total proteins were extracted from HK-2 cells with a protein extraction kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Protein concentrations were examined using a BCA assay kit (Thermo, Waltham, MA) according to the manufacturer’s instructions. The proteins were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Next, the proteins were transferred onto 0.22 $\mu$m polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA) and blocked with 5% non-fat dry milk in Tris-buffered saline and Tween 20 for 2 h at room temperature. The membranes were then incubated with primary antibodies overnight at 4 °C and with the appropriate horseradish peroxidase (HRP)-conjugated secondary IgG for 1 h at room temperature. Finally, chemiluminescence was imaged in a Chemi DocTM XRS + WB molecular imager using Image LabTM software (Bio-Rad, Herculaes, CA, USA), and band intensities were quantified with ImageJ 1.8.0 software (National Institutes of health, Bethesda, MD, USA).

Mitophagy was determined by the co-localization of autophagosomes and mitochondria. HK-2 cells cultured on coverslips were incubated with red-fluorescing MitoTracker Red (50 nM, C1035, Beyotime, Shanghai, China) for 15 min at 37 °C. After twice washing with PBS, these cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then they were incubated with 1% Triton X-100 for 10 min and blocked with 3% BSA buffer for 30 min. Subsequently, cells were incubated with an LC3 (1:100) antibody at 4 °C overnight and incubated with green fluorescence-labeled secondary antibody for 1 h at room temperature. Finally, 4, 6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA) was used as a counterstain. Images were visualized using an OLYMPUS confocal microscope (Tokyo, Japan). For quantification, over 100 cells in each group were examined for co-localization of autophagosomes (green) with mitochondria (red) to estimate the percentage of cells with mitophagosome formation.

HK-2 cells were transfected with the mKeima-Red-Mito-7 (Addgene, 56018) plasmid using Lipofectamine 3000 for 40 h and then treated with 8 mg/mL albumin for another 8 h. Nuclei were counterstained with Hoechst 33342 (C1027, Beyotime, China). The cells were imaged using an OLYMPUS confocal microscope (Ex = 550 nm, Em = 620 nm for acidic red fluorescence, Tokyo, Japan). For quantification, more than 20 cells in each group were examined for autolysosome formation to estimate the autolysosome dots/cell.

$\mathrm{\Delta }$$\psi$m was detected using a JC-1 assay kit (C2003S, Beyotime, China). After the indicated treatments, HK-2 cells cultured in 6‑well plates were then washed with PBS and incubated with 1 ml JC-1 staining solution (5 $\mu$g/mL) at 37 °C for 20 min. Subsequently, HK-2 cells were washed twice with JC-1 buffer solution, and a fresh medium was added for detection. Images were captured captured using an AXIO Observer D1 fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and were analyzed with ImageJ software to obtain the mean densities of the regions of interest, which were normalized to those of the control group. In cells with high mt$\mathrm{\Delta }$$\mathrm{\Psi }$, JC-1 aggregates in the mitochondria and gives off a red fluorescence. In cells with low mt$\mathrm{\Delta }$$\mathrm{\Psi }$, the JC-1 fails to aggregate in the mitochondria and remains in the cytoplasm in its monomer form, which exhibits green fluorescence. The value of mt$\mathrm{\Delta }$$\mathrm{\Psi }$ was expressed as the ratio of red/green fluorescence intensity.

Intracellular ATP levels were detected using an ATP assay kit (S0026, Beyotime, China). After indicated treatments, HK-2 cells cultured in 6-well plates were washed twice with PBS and lysed with ATP Cell Lysis solution in an ice bath. Then, the cell lysates were centrifuged at 12,000 $\times$ g for 5 min at 4 °C, and supernatants were collected. In a 96-well culture plate, 20 $\mu$L of each supernatant was mixed with 100 $\mu$L of ATP detection working dilution. Relative luminescence was assessed using a Varioskan LUX microplate reader (Thermo Scientific, Waltham, MA, USA). The standard curve for the quantification was prepared each time. ATP content was normalized by protein.

HK-2 cells were incubated in 10 $\mu$M of the oxidation-sensitive fluorescent probe, 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) (S0033S, Beyotime) at 37 °C for 30 min, then washed with serum-free medium three times. Fluorescent images were photographed using a fluorescence microscope. The multifunctional microplate reader detected ROS levels at the $\sim$485/528 nm wavelength.

HK-2 cells were incubated in 5 $\mu$M MitoSOXTM Red (M36008, Invitrogen, Carlsbad, CA, USA) at 37 °C for 10 min, and then washed with warm buffer three times. Images were photographed using a fluorescence microscope. The multifunctional microplate reader detected ROS levels at the $\sim$510/580 nm wavelength.

Apoptosis was determined using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) apoptosis assay kit (C1088, Beyotime, China). HK-2 cells cultured on coverslips were washed with ice-cold PBS and subsequently fixed with 4% paraformaldehyde. Then, the slides were incubated with 1% Triton X-100 for 5 min and then incubated with 50 $\mu$L/slide TUNEL reaction mixture for 1 h at 37 °C. After incubation, slides were washed 3 times, and nuclei were stained with DAPI. The images were captured using a fluorescence microscope. To calculate the apoptosis percentage, five high-power fields ($\times$400) were evaluated for TUNEL-positive cells in each group.

All statistical tests were performed with SPSS 16.0 software (IBM, Chicago, IL, USA). Quantitative data were expressed as means $\pm$ SD of at least three independent experiments. Student’s t-test compared results between two groups. One-way analysis of variance (ANOVA) was employed for the comparisons among multiple groups followed by Tukey’s post hoc test. Significance was considered as p 0.05.

We started our investigations by examining mitochondrial function during albumin overload treatment of HK-2 cells. As we know, a series of redox reactions creates an electrochemical gradient through the mitochondrial electron transport chain, which drives the synthesis of ATP and generates the mitochondrial membrane potential ($\mathrm{\Delta }$$\psi$m). Therefore, we firstly measured the $\mathrm{\Delta }$$\psi$m and ATP generation to evaluated mitochondrial function. The results revealed a clear decrease of $\mathrm{\Delta }$$\psi$m (Fig. 1A,B) and ATP generation (Fig. 1C) in albumin overload-treated HK-2 cells, indicating mitochondrial dysfunction after albumin overload. Mitochondria, as the source of intracellular ROS, when confronting with oxidative stress, is the primary target attacked by ROS, and produces excessive amounts of ROS due to the damage to enzymes in the electron transport chain. Given this, we assessed the intracellular ROS by DCFH-DA and mitochondrial ROS by MitoSOX Red staining respectively. The results showed that both intracellular ROS and mitochondrial ROS were increased by albumin overload (Fig. 1D,E).

Fig. 1.

Albumin overload induced mitochondrial dysfunction, ROS generation and mitophagy activation in HK-2 cells. (A) Representative images of JC-1 staining showing red fluorescence of JC-1 aggregate and green signal of monomer. Scale bar: 20 $\mu$m. (B) Quantification of the ratio of JC-1 aggregate to JC-1 monomer. (C) ATP contents. (D) Representative images of DCFH-DA staining for intracellular ROS and MitoSOX staining for mitochondrial ROS. Scale bar: 20 $\mu$m. (E) Quantification of DCFH-DA fluorescence intensity and MitoSOX fluorescence intensity. (F) Representative blots. (G) Densitometry of protein signals. (H) Representative images of mitophagy. Mitochondria were labeled with MitoTracker Red and autophagosomes were labeled with LC3 antibody. Scale bar: 10 $\mu$m. (I) Quantitative analysis of mitophagosome formation. (J) Representative images of autolysosomes formation. HK-2 cells were transfected with the mKeima-Red-Mito-7 plasmid, followed by treatment with 8 mg/mL albumin. The fluorescence was detected by confocal microscope. Scale bar = 10 $\mu$m. (K) Quantitative analysis of autolysosomes formation. Data are depicted as the mean $\pm$ SD from three independent experiments. *p 0.05; **p 0.01; ***p 0.001. Ctrl, control; ALB, albumin.

We then examined the occurrence of mitophagy during albumin overload in HK-2 cells. Immunoblot analyses revealed that albumin overload induced the conversion of LC3-I to LC3-II and decreased the level of SQSTM1 (Fig. 1F,G), indicating autophagy activation. Immunoblot analyses confirmed that albumin overload induced a marked reduction in translocase of outer mitochondrial membrane 20 homolog (Tom20) (Fig. 1F,G), suggesting mitochondrial clearance by mitophagy. We then performed some direct observations of mitophagy by assessing mitophagosome formation following the colocalization of autophagosomes and mitochondria. These evaluations were performed by labeling mitochondrial with MitoTracker Red and autophagosome staining with a fluorescently labeled LC3 antibody (Fig. 1H). These evaluations revealed very few LC3 green-fluorescent puncta within the control cells, indicating a low level of autophagy. In contrast, albumin overload significantly increased the expression of LC3, indicating increased autophagosome formation. Notably, some LC3 green-fluorescent puncta co-localized with the Mito-Tracker signals in cells following albumin overload (Fig. 1H,I), indicating mitophagosome formation. To further confirm the occurrence of mitophagy, mito-Keima, a pH-sensitive fluorescent protein, was transfected into HK-2 cells to determine mitochondrial movement from the cytoplasm to the lysosome. As shown in Fig. 1J,K, red spots appeared in the cytoplasm upon albumin overload, indicating that mitochondria tended to form autolysosomes. These results confirmed the activation of autophagy and mitophagy during albumin overload in HK-2 cells.

HK-2 cells exposed to albumin overload showed increased expression of PINK1, Parkin, and phospho-Parkin (Fig. 2A,B), indicating activation of the PINK1/Parkin-dependent mitophagy pathway. Given this, we determined the role of this pathway in these cells via the controlled inhibition of Parkin expression using Parkin siRNA transfection (Fig. 2C–E). There was a significant decrease in albumin overload-induced mitophagy and autolysosomes in Parkin siRNA-treated cells (Fig. 2F–I), which was accompanied by the partial restoration of Tom20 expression in these cells (Fig. 2A,B). Conversely, the overexpression of Parkin (Fig. 3A–E) further increased albumin overload-induced mitophagy and autolysosomes (Fig. 3F–I), as well as Tom20 degradation (Fig. 3D,E). Taken together, these results indicate that the PINK1/Parkin pathway critically contributes to tubular cell mitophagy in response to albumin overload.

Fig. 2.

Silencing of Parkin inhibited albumin overload-induced mitophagy in HK-2 cells. HK-2 cells were transfected with Parkin siRNA or control siRNA for 48 h followed by treatment with albumin (8 mg/mL) for 8 h. (A) Representative blots. (B) Densitometry of protein signals. *p 0.05 vs. Ctrl; ${}^{\mathrm{\#}}$p 0.05 vs. si-control + ALB; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. si-control + ALB. (C) Quantification of Parkin mRNA expression. * p 0.05. (D) Representative blots. (E) Densitometry of protein signals. ***p 0.001. (F) Representative images of mitophagy. Scale bar: 10 $\mu$m. (G) Quantitative analysis of mitophagosome formation. (H) Representative images of autolysosomes formation. Scale bar = 10 $\mu$m. (I) Quantitative analysis of autolysosomes formation. *p 0.05, **p 0.01. Data are depicted as the mean $\pm$ SD from three independent experiments. Ctrl, control; ALB, albumin; si-control, control siRNA; si-Parkin, Parkin siRNA.

Fig. 3.

Overexpression of Parkin enhanced albumin overload-induced mitophagy in HK-2 cells. HK-2 cells were transfected with Parkin overexpression plasmids or empty vector for 48 h followed by treatment with albumin (8 mg/mL) for 8 h. (A) Quantification of Parkin mRNA expression. ***p 0.001. (B) Representative blots. (C) Densitometry of protein signals. *p 0.05. (D) Representative blots. (E) Densitometry of protein signals. *p 0.05 vs. Ctrl; **p 0.01 vs. Ctrl; ${}^{\mathrm{\#}}$p 0.05 vs. empty vector + ALB. (F) Representative images of mitophagy. Scale bar: 10 $\mu$m. (G) Quantitative analysis of mitophagosome formation. (H) Representative images of autolysosomes formation. Scale bar = 10 $\mu$m. (I) Quantitative analysis of autolysosomes formation. *p 0.05. Data are depicted as the mean $\pm$ SD from three independent experiments. Ctrl, control; ALB, albumin; empty vector, control empty plasmid vector; Parkin OE, Parkin overexpression plasmids.

Mitophagy plays an essential role in clearing damaged mitochondria. Therefore, mitophagy deficiency may allow the accumulation of damaged mitochondria, thereby impacting mitochondrial function. To determine the effect of PINK1/Parkin-mediated mitophagy on mitochondrial function, we first assessed the effect of Parkin silencing on $\mathrm{\Delta }$$\psi$m, ATP generation, and ROS production during albumin overload in HK-2 cells. Under control conditions, $\mathrm{\Delta }$$\psi$m loss and ROS production were at low levels in both control siRNA-transfected HK-2 cells and Parkin siRNA-transfected HK-2 cells (Supplementary Fig. 1). Silencing of Parkin further accelerated albumin overload-induced loss of $\mathrm{\Delta }$$\psi$m (Fig. 4A,B). Consistently, silencing of Parkin aggravated the inhibitory effect of albumin overload on ATP production (Fig. 4C). Also, silencing of Parkin enhanced albumin overload-induced intracellular and mitochondrial ROS levels (Fig. 4D–G).

Fig. 4.

Silencing of Parkin aggravated albumin overload-induced mitochondrial dysfunction and ROS generation. (A) Representative images of JC-1 staining showing red fluorescence of JC-1 aggregate and green signal of monomer. Scale bar: 20 $\mu$m. (B) Quantification of the ratio of JC-1 aggregate to JC-1 monomer. (C) ATP contents. (D) Representative images of DCFH-DA staining for intracellular ROS. Scale bar: 20 $\mu$m. (E) Quantification of DCFH-DA fluorescence intensity. (F) Representative images of MitoSOX staining for mitochondrial ROS. Scale bar: 20 $\mu$m. (G) Quantification of MitoSOX fluorescence intensity. Data are depicted as the mean $\pm$ SD from three independent experiments. *p 0.05; ***p 0.001. ALB, albumin; si-control, control siRNA; si-Parkin, Parkin siRNA.

Given these results we went on to evaluate the effects of upregulating mitophagy via Parkin overexpression on albumin overload-induced mitochondrial dysfunction in HK-2 cells. Under control conditions, $\mathrm{\Delta }$$\psi$m loss and ROS production were at low levels in both control plasmid empty vector-transfected HK-2 cells and Parkin overexpression plasmids-transfected HK-2 cells (Supplementary Fig. 1). Parkin overexpression alleviated the albumin overload-induced loss of $\mathrm{\Delta }$$\mathrm{\Psi }$m (Fig. 5A,B) and reduction in ATP levels (Fig. 5C). Consistently, Parkin overexpression inhibited albumin overload-induced intracellular and mitochondrial ROS generation in HK-2 cells (Fig. 5D–G). Thus, these results indicate the critical role of PINK1/Parkin-mediated mitophagy in the clearance of damaged mitochondria and protection of mitochondrial function during albumin overload in HK-2 cells.

Fig. 5.

Overexpression of Parkin attenuated albumin overload-induced mitochondrial dysfunction and ROS generation. (A) Representative images of JC-1 staining showing red fluorescence of JC-1 aggregate and green signal of monomer. Scale bar: 20 $\mu$m. (B) Quantification of the ratio of JC-1 aggregate to JC-1 monomer. (C) ATP contents. (D) Representative images of DCFH-DA staining for intracellular ROS. Scale bar: 20 $\mu$m. (E) Quantification of DCFH-DA fluorescence intensity. (F) Representative images of MitoSOX staining for mitochondrial ROS. Scale bar: 20 $\mu$m. (G) Quantification of MitoSOX fluorescence intensity. Data are depicted as the mean $\pm$ SD from three independent experiments. *p 0.05; **p 0.01; ***p 0.001. ALB, albumin; empty vector, control empty plasmid vector; Parkin OE, Parkin overexpression plasmids.

We then examined the effect of inhibiting mitophagy on apoptosis following albumin overload by silencing Parkin expression. Under control conditions, apoptosis rate and the expression of cleaved caspase 3 were at low levels in both control siRNA-transfected HK-2 cells and Parkin siRNA-transfected HK-2 cells (Supplementary Fig. 2). Upon albumin overload, many cells presented with nuclear morphology typical of apoptosis and were TUNEL-positive (Fig. 6A). Cell counting revealed that albumin overload induced apoptosis in ~17% of the control siRNA-transfected HK-2 cells, but this increased to ~25% in Parkin siRNA-transfected HK-2 cells (Fig. 6B). These results were confirmed by an immunoblot analysis that revealed a markedly increased expression of cleaved caspase 3 in Parkin siRNA-transfected cells compared to that in the control cells (Fig. 6C,D).

Fig. 6.

Silencing of Parkin aggravated albumin overload-induced apoptosis in HK-2 cells. (A) Representative images of TUNEL staining of HK-2 cells. Scale bar: 20 $\mu$m. (B) Apoptosis percentage. (C) Whole-cell lysates were collected for western blotting analysis of cleaved caspase 3 and GAPDH (loading control). (D) Densitometry of cleaved caspase 3. Data are depicted as the mean $\pm$ SD from three independent experiments. *p 0.05; **p 0.01. ALB, albumin; si-control, control siRNA; si-Parkin, Parkin siRNA.

In contrast to mitophagy deficiency, the effect of the upregulation of mitophagy via Parkin overexpression on albumin overload-induced apoptosis was investigated. Under control conditions, apoptosis rate and the expression of cleaved caspase 3 were at low levels in both control plasmid empty vector-transfected HK-2 cells and Parkin overexpression plasmids-transfected HK-2 cells (Supplementary Fig. 2). In response to albumin overload, HK-2 cells with Parkin overexpression plasmids showed fewer TUNEL-positive cells (Fig. 7A,B) and lower levels of Cleaved caspase 3 than control plasmid empty vector cells (Fig. 7C,D). Collectively, these findings provide compelling evidence for the pro-survival role of PINK1/Parkin mediated mitophagy in albumin overload-induced HK-2 cell injury.

Fig. 7.

Overexpression of Parkin attenuated albumin overload-induced apoptosis in HK-2 cells. (A) Representative images of TUNEL staining of HK-2 cells. Scale bar: 20 $\mu$m. (B) Apoptosis percentage. (C) Whole-cell lysates were collected for western blotting analysis of Cleaved caspase 3 and GAPDH (loading control). (D) Densitometry of Cleaved caspase 3. Data are depicted as the mean $\pm$ SD from three independent experiments. *p 0.05; ***p 0.001. ALB, albumin; empty vector, control empty plasmid vector; Parkin OE, Parkin overexpression plasmids.