DMEM/F-12 medium (cat. no. 12400-024), fetal bovine serum (FBS; cat. no. 16140-071), penicillin and streptomycin antibiotic mixture (cat. no. 15140122) and cell culture supplies were obtained from Gibco (Thermo Fisher Scientific, Inc.). D-glucose (cat. no. G8270), D-mannitol (cat. no. M4125), 2′,7′-dichlorofluorescin diacetate (DCFH-DA; cat. no. D6883), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; cat. no. D8417), ferrostatin-1 (cat. no. SML0583), bardoxolone methyl (CDDO Methyl Ester; cat. no. SMB00376), erastin (cat. no. E7781) and buthionine sulfoximine (cat. no. B2515) were purchased from Sigma-Aldrich; Merck KGaA. RSL3 (cat. no. S8155) and FIN56 (cat. no. S8254) were obtained from Selleck Chemicals. Antibody against salusin-β (cat. no. PAC026Hu01) and enzyme linked immunosorbent assay (ELISA) kits for salusin-β (cat. no. CEC026Hu) measurement were obtained Wuhan USCN Business Co., Ltd. Click-iT Plus EdU (5-ethynyl-2′-deoxyuridine) Alexa Fluor 594 imaging kits were procured from Thermo Fisher Scientific, Inc. (cat. no. C10639). Goat Anti-Rabbit IgG H&L (Alexa Fluor 488; cat. no. ab150077) was purchased from Abcam. The specific primers used in the present study were synthesized by Santa Cruz Biotechnology, Inc. Control short interfering (si)RNA (cat. no. sc-44239) and nuclear factor-erythroid-2-related factor 2 (Nrf-2) siRNA cat. no. (sc-37030) were bought from Santa Cruz Biotechnology, Inc. Lentivirus particles carrying salusin-β short hairpin (sh)RNA or negative control shRNA were produced by Jiman Biotechnology (Shanghai) Co., Ltd., as previously described (35,37). Lentivirus empty vectors or vectors expressing salusin-β were constructed by Jiman Biotechnology (Shanghai) Co., Ltd as previously described (33,35,38). Antibodies against β-actin (cat. no. 66009-1-Ig or cat. no. 20536-1-AP) and HRP-conjugated secondary antibodies (cat. nos. SA00001-1 and SA00001-2) were obtained from ProteinTech Group, Inc. Antibodies against glutathione peroxidase 4 (GPX4; cat. no. 52455), solute carrier family 7 (cationic amino acid transporter, y+ system) member 11 (SLC7A11; cat. no. 12691), ferritin heavy polypeptide 1 (FTH-1; cat. no. 4393), transferrin receptor 1 (TFR-1; cat. no. 13113) and Nrf-2 (cat. no. 12721) were purchased from Cell Signaling Technology, Inc. Cell Counting Kit-8 (CCK-8; cat. no. G021-1-1), lactate dehydrogenase (LDH) release kits (cat. no. A020-2-2), malondialdehyde (MDA) assay kit (cat. no. A003-1-2) and reduced glutathione (GSH) assay kits (cat. no. A006-2-1) were purchased from Nanjing Jiancheng Bioengineering Institute. An iron assay kit (cat. no. ab83366) was from Abcam. The selected concentrations of chemicals were in accord with previously published papers (19,39,40).

Human proximal tubular (HK-2) cells were procured from the American Type Culture Collection (cat. no. CRL-2190). HK-2 cells were cultured in DMEM/F-12 medium contained 10% FBS, streptomycin (100 mg/ml) and penicillin (100 U/ml) under a humidified atmosphere of 5% CO₂ at 37°C. When HK-2 cells reached ~70-80% confluence, these cells were challenged by 30 mM glucose (Sigma-Aldrich; Merck KGaA) at different time points. D-mannitol was used as a hyperosmolar control in the current study (5.5 mM D-glucose plus 24.5 mM D-mannitol) (41). In some experiments, HK-2 cells were pretreated with bardoxolone methyl (100 nM), or Ferrostatin-1 (1 µM) for 30 min before high glucose (HG) stimulation.

After treatment, HK-2 cells were washed with ice-cold PBS and lysed in RIPA lysis buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 1 mM EDTA and 2% Triton X-100) supplemented with protease inhibitor cocktail (Roche Diagnostics) and the protein contents were determined by using BCA colorimetric protein kit (P0012; Beyotime Institute of Biotechnology). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer was added into cell lysates and boiled for 5 min. Samples and pre-stained markers were added as required. Equal amount of cell lysates (30 µg) was separated onto SDS-PAGE gels (8–12%) and transferred onto polyvinylidene fluoride membranes. Membranes were sealed with 5% skimmed milk powder for 1 h at room temperature, followed by incubation with primary antibodies at 4°C overnight. The following primary antibodies were used: Salusin-β (1:500; cat. no. PAC026Hu01; Wuhan USCN Business Co., Ltd), β-actin (1:1,000; cat. no. 66009-1-Ig; ProteinTech Group, Inc.), GPX4 (1:1,000; cat. no. 52455; Cell Signaling Technology, Inc.), SLC7A11 (1:1,000; cat. no. 12691; Cell Signaling Technology, Inc.), FTH-1 (1:1,000; cat. no. 4393; Cell Signaling Technology, Inc.), TFR-1 (1:1,000; cat. no. 13113; Cell Signaling Technology, Inc.) and Nrf-2 (1:1,000; cat. no. 12721; Cell Signaling Technology, Inc.). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. nos. SA00001-1 and SA00001-2; ProteinTech Group, Inc.) for 1 h at room temperature. Immunodetection was conducted using an ECL system (Fusion FX7 imaging system; Vilber China). Protein expression was semi-quantified using Image Lab software (version 5.2.1; Bio-Rad Laboratories, Inc.).

HK-2 cells were fixed with 4% paraformaldehyde for 20 min at room temperature. The fixed cells were then permeabilized with 0.1% Triton X-100 for 10 min at room temperature and 5% goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) for 1 h. The expressions of salusin-β in HK-2 cells were detected by incubation of primary antibody against salusin-β at 4°C overnight. Afterwards, these cells were incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) for 1 h at 37°C and the nucleus was stained by DAPI for 10 min at room temperature. The positive immunofluorescence images were captured by a fluorescence microscopy (Olympus BX6 with DP72 camera; Olympus Corporation; magnification, ×200). A total of six visual fields in each group were randomly selected in each group.

Total RNA was isolated from cells (2×10⁵) by TRIzol^® reagent (cat. no. 9108; Thermo Fisher Scientific, Inc.). All steps were performed according to the manufacturer's protocols. The purity and concentration of RNA were detected using NanoDrop (Thermo Fisher Scientific, Inc.). The first cDNA synthesis was conducted using GoScript Reverse Transcription System (Promega Corporation) in the presence of 0.5 g of total RNA in each sample. Following the manufacturer's instructions, RT-qPCR was carried out using the qPCR SYBR-Green Master Mix (cat. no. 11119ES60; Shanghai Yeasen Biotechnology Co., Ltd.) under the Applied Biosystems 7500 Real Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 120 sec, followed by 40 amplification cycles at 95°C for 15 sec, and annealing and extension at 60°C for 20 sec. The relative expression levels were calculated based on the 2^−ΔΔCq method (42). These experiments were repeated three independent times. The primer sequences used for RT-qPCR are listed in Table SI.

The levels of salusin-β in cellular lysate were analyzed through the commercial ELISA kits according to the instructions as previously described (43,44). Briefly, the samples and diluted standards (50 µl) were added into the appropriate wells, respectively. 50 µl of Detection Reagent A was added into each well and incubated for 1 h at 37°C. After washing three times with 1X Wash Solution, 100 µl of Detection Reagent B working solution was added into each well and incubated for 30 min at 37°C. Substrate Solution and Stop Solution were subsequently added into each well and the liquid turned yellow. The measurement of optical density was performed using a microplate reader (SYNERGY H4; BioTek Instruments, Inc.) at 450 nm immediately. The levels of salusin-β were expressed as pg per mg protein by normalizing to the protein contents in each sample.

Cell viability was evaluated using the CCK-8 according to the manufacturer's instructions. In brief, HK-2 cells were seeded onto 96-well plates (1×10⁴ cells/well) and treated with normal glucose (NG) and high glucose (HG), respectively. The culture medium was replaced with 100 µl fresh medium supplemented with 10 µl of the CCK-8 solution in each well. After incubating for 2 h at 37°C, measurement of the absorbance at 450 nm was carried out by using a microplate reader (SYNERGY H4; BioTek Instruments, Inc.). In addition, Click-iT Plus EdU (5-ethynyl-2′-deoxyuridine) Alexa Fluor™ 594 imaging kits were used for measurement of HK-2 cell proliferation according to the manufacturer's protocols. The red and blue fluorescence signals were imaged by a fluorescence microscopy (Olympus BX6 with DP72 camera; Olympus Corporation; magnification, ×200). A total of 6 visual fields were randomly selected in each group and the average number of positive cells was calculated.

HK-2 cells in logarithmic growth were transduced with lentivirus particles carrying salusin-β shRNA or negative control shRNA (10⁹ titer/ml; MOI=50, 50 µl lentivirus particles per 1×10⁶ cells) for 48 h at 37°C and then challenged by HG stimulation for another 48 h at 37°C. For overexpression of salusin-β in HK-2 cells, HK-2 cells were transduced with lentivirus expressing salusin-β vectors or empty vectors (10¹⁰ titer/ml; MOI=50, 50 µl lentivirus particles per 1×10⁶ cells) for 48 h at 37°C before administration of NG or HG for 48 h. After that, cells were harvested for biochemical testing at the desired time points. For Nrf-2 knockdown, HK-2 cells were plated the day before siRNA transfection. A non-targeting control siRNA (50 nM) and Nrf-2 siRNA (50 nM) were transduced to HK-2 cells for 48 h at 37°C by using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. The medium was changed after 4–6 h of transfection. Subsequent experiments were performed at 48 h post-transfection.

Total iron levels in cell lysates were quantified by an iron assay kit (ab83366, Abcam). Briefly, the collected HK-2 cells were lysed in the iron assay buffer and the supernatants were obtained by centrifugation at 16,000 × g for 10 min at 4°C. A total of 5 ml of iron reducing agent was mixed into cell samples (50 ml) for total iron analysis. Next, the iron probe solution (100 ml) was added and incubated for 1 h at 25°C under dark conditions. Later, the absorbance at the wavelength of 593 nm was detected by spectrophotometry.

The collected cells were homogenized by the MDA lysis buffer and the supernatants were collected after centrifugation at 13,800 × g for 10 min at 4°C. The MDA concentrations in cell lysates were assessed using a lipid peroxidation assay kit according to the manufacturer's instructions. In short, 200 µl of the supernatant from each homogenized sample was placed into a tube, followed by incubation of the thiobarbituric acid solution (200 µl) for 40 min at 95°C. These samples were cooled to room temperature and centrifuged (1,500 × g for 10 min) to remove insoluble sediment. The supernatants were collected and the absorbance was measured at 532 nm using a microplate reader (SYNERGY H4; BioTek Instruments, Inc.).

HK-2 cells were washed with ice-cold PBS and homogenized by vibrating homogenizer with cold PBS and the supernatants were collected after centrifugation at 13,800 × g for 10 min at 4°C. GSH assay mixture (125 µl) was added into each well for 5 min at room temperature and the optical density was determined at the wavelength of 405 nm. The concentrations of GSH were then quantified by comparing the optical density of the samples to the standard curve, respectively.

Intracellular ROS levels in HK-2 cells were evaluated by dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining as previously described (45,46). HK-2 cells were initially cultured in 6-well plates for 48 h and DCFH- DA fluorescent dye (10 µM) was added and incubated for 20 min at 37°C in the dark and then were washed with PBS. The fluorescence intensity was measured by a fluorescence microscope (Olympus BX6 with DP72 camera; Olympus Corporation) with an excitation wavelength at 488 nm and an emission wavelength at 525 nm. The images were observed and captured in 10 randomly selected fields of view from 4–6 repeated experiments in 1–2 randomly selected fields of view (magnification, ×200).

LDH contents were measured using LDH cytotoxicity assay kits as previously reported (47). The standards and culture supernatants were obtained and added into the appropriate wells (20 µl), matrix buffer (25 µl) and coenzyme I (5 µl) were sequentially added. The liquid system was incubated for 15 min at 37°C. Later, 2,4,-Dinitrophenylhydrazine (25 µl) was added and incubated for 15 min at 37°C. Sodium hydroxide solution (0.4 M, 250 µl) were then added and incubated for 5 min at room temperature. The reactions were read at 450 nm by using a microtiter plate reader (SYNERGY H4; BioTek Instruments, Inc.). Mean LDH release of control cells was indexed to 100%, the value used for normalization.

Results were expressed as mean ± standard deviation. All results were analyzed by GraphPad Prism 6.0 (GraphPad Software, Inc.). All data were tested for normality and equal variance. If the data passed these tests, comparisons between two groups were made by unpaired Student's t tests. One way ANOVA was used for multiple-group comparisons. When ANOVA was significant, post hoc testing of differences between groups was conducted by using Bonferroni tests. The independent experiments were repeated at least four times. P<0.05 was considered to indicate a statistically significant difference.

To test whether HG altered salusin-β expression in HK-2 cells, HK-2 cells were treated with increasing doses of glucose at various time points. Upon HG treatment (25 mM), the protein expression of salusin-β was upregulated in HK-2 cells and this was further increased with increasing glucose concentrations, reaching its maximal effect at 30 mM (Fig. 1A and B). Thus, the dose of glucose was selected as 30 mM for the following experiments. Time-course experiments showed that the protein expression of salusin-β was increased slightly after HG treatment for 24 h (Fig. 1C and D). However, the protein expression of salusin-β was markedly augmented in HK-2 cells exposed to HG for 48 and 72 h (Fig. 1C and D). Accordingly, HG treatment for 48 h was chosen as a model of cell damage during the subsequent experiments. Furthermore, HG-induced upregulation of salusin-β in HK-2 cells was confirmed by immunofluorescence (Fig. 1E), RT-qPCR (Fig. 1F) and ELISA results (Fig. 1G), respectively. To study the effects of osmolality on the expression of salusin-β, D-mannitol was used as a hyperosmolar control (5.5 mM D-glucose plus 24.5 mM D-mannitol) (41,48). Notably, the protein and mRNA expressions of salusin-β were markedly incremented in HG-incubated HK-2 cells, but not in hyperosmolar D-mannitol-treated cells (Fig. S1A-C). These results suggested that aberrant expressions of salusin-β in HK-2 cells were an effect of glucotoxicity, rather than from hyperglycemia-induced hyperosmolarity.

Previous studies have highlighted the importance of ferroptosis in the development of DN as inhibition of ferroptosis can effectively delay the progression of DN (18,19). It is reported that salusin-β serves as an inducer of oxidative stress in HK-2 cells through increased ROS levels and decreased GSH activities (35), key characteristics of ferroptosis (15,49). On this basis it was hypothesized that salusin-β might be involved in HG-induced ferroptosis in HK-2 cells. To test this hypothesis, the protein and mRNA expressions of ferroptosis-related genes, such as GPX4, SLC7A11, FTH-1 and TFR-1, in salusin-β-deficient HK-2 cells under HG conditions were examined. Similar to a previous report (19), the protein expressions of GPX4, SLC7A11 and FTH-1 were downregulated, while TFR-1 protein expression was upregulated in HK-2 cells exposed to HG (Fig. 2A and B). However, deletion of salusin-β (Fig. S2A) reversed abnormal expressions of these proteins induced by HG (Fig. 2A and B). In line with this, RT-qPCR results showed a parallel change in mRNA expression levels of GPX4, SLC7A11, FTH-1 and TFR-1 (Fig. 2C). Meanwhile, incubation of HK-2 cells with HG promoted the concentration of iron and induced the end products of lipid peroxidation, MDA, accompanied by a decrease in GSH contents (Fig. 2D-F). Notably, the ferroptosis-related changes in HG-induced HK-2 cells were rescued when salusin-β was absent (Fig. 2D-F). In addition, knockdown of salusin-β attenuated HG-elicited massive generation of ROS, as evidenced by DCFH-DA staining (Fig. 2G and H). Importantly, knockdown of salusin-β significantly inhibited HG-triggered cell viability decline and LDH release, suggesting the protective effects of salusin-β deficiency against HG-induced cytotoxicity in HK-2 cells (Fig. 2I-K). Furthermore, induction of ferroptosis and decreased cell viability were not observed in D-mannitol-incubated HK-2 cells (Fig. S1D-G). These preliminary results suggested that the antagonistic effects of salusin-β knockdown on ferroptosis may be dependent on the expressions of antioxidant genes (GPX4 and SLC7A11) and iron metabolism-related genes (FTH-1 and TFR-1).

In contrast to the above results, overexpression of salusin-β (Fig. S2B) aggravated the effects of HG on the protein (Fig. 3A-B) and mRNA (Fig. 3C) expressions of GPX4, SLC7A11, FTH-1 and TFR-1 in HK-2 cells. Notably, salusin-β overexpression itself also changed the expressions of GPX4, SLC7A11, FTH-1 and TFR-1 in HK-2 cells at both protein and mRNA levels (Fig. 3A-C), indicating the critical implication of salusin-β in the process of ferroptosis. In addition, ectopic overexpression of salusin-β further deteriorated HG-induced ferroptosis in HK-2 cells, as manifested by more iron accumulation, MDA contents and lower GSH contents (Fig. 3D-F). DCFH-DA staining results showed that overexpression of salusin-β worsened the effects of HG on ROS generation in HK-2 cells (Fig. 3G and H). HG-induced decrease in cell viability and increase in LDH release were further exacerbated by ectopic overexpression of salusin-β (Fig. 3I and K). Additionally, salusin-β overexpression led to spontaneous cytotoxicity on HK-2 cells, as indicated by measurement of cell viability and LDH release (Fig. 3I and K).

As a master antioxidant regulatory transcription factor, Nrf-2 is found to prevent ferroptosis-related cell death via upregulating antioxidant enzymes, including heme oxygenase-1 (HO-1), GPX4 and SLC7A11 (50). In addition, Nrf-2 serves a vital role in the regulation of ferroptosis-related genes, such as genes for thiol-dependent antioxidant system, ion metabolism, enzymatic detoxification of reactive carbonyl species and carbonyls, nicotinamide adenine dinucleotide phosphate system and ROS generation from mitochondria or extra-mitochondria (51). Activation of Nrf-2 signaling is anticipated to open up a novel way to treat ferroptosis-associated diseases, including DN (19). As expected, Bardoxolone methyl, an inducer of Nrf-2 (Fig. 4A and B), upregulated the mRNA expression levels of GPX4 and SLC7A11 and reversed HG-induced inhibition of GPX4 and SLC7A11 mRNA levels (Fig. 4C and D). The contents of iron and MDA were enhanced (Fig. 4E and G), whereas GSH levels and cell viability were diminished in HK-2 cells challenged by HG (Fig. 4F and 4H) and these effects were noticeably dampened by application of Bardoxolone methyl. The present study next investigated whether Nrf-2 signaling was involved in salusin-β-mediated ferroptosis in HG-stimulated HK-2 cells. As shown in Fig. 4I, the protein expression of Nrf-2 was significantly increased in HG-incubated cells being treated with salusin-β shRNA compared with the control shRNA group. By contrast, the decreased expression of Nrf-2 was further exacerbated by overexpression of salusin-β in HG-stimulated HK-2 cells (Fig. 4J). The data clearly demonstrated that salusin-β might be responsible for HG-induced ferroptosis via inhibiting the Nrf-2 signaling pathway.

To further verify the implication of the Nrf-2 signaling cascade in salusin-β-mediated ferroptosis in HK-2 cells, Nrf-2 siRNA and Nrf-2 inducer Bardoxolone methyl were used in the present study. In the absence of Nrf-2 (Fig. S3), the suppressive effects of salusin-β shRNA on HG-induced cell damage, including iron deposition, GSH deletion, MDA accumulation and cell viability decline, were abolished (Fig. 5A and D). In parallel to this, the stimulatory effects of salusin-β on HG-induced ferroptosis occurrence were clearly compromised by Bardoxolone methyl pretreatment (Fig. 5E-H). Collectively, the molecular mechanism by which salusin-β contributed to ferroptosis appeared to involve Nrf-2-mediated regulation of antioxidant system and ion metabolism.

The above results demonstrated that salusin-β participated in HG-induced HK-2 cell ferroptosis in a Nrf-2-dependent manner, it may be interesting to know whether salusin-β was changed in the process of ferroptosis. Western blotting results showed that the protein expression of salusin-β was unexpectedly upregulated by several ferroptosis activators, including erastin (X_c⁻ inhibitors), GPX4 inhibitors (RSL3 and FIN56) and GSH synthase inhibitor (buthionine sulfoximine) (Fig. 6A). Conversely, pretreatment of ferrostatin-1 (a ferroptosis inhibitor) downregulated the increased expression of salusin-β in HG-treated HK-2 cells (Fig. 6B). These results suggested that interaction of salusin-β with ferroptosis formed a positive feedback, thereby contributing to HG-induced HK-2 cell injury and DN.