Antibodies specific for Bcl-2-associated X protein (Bax; 2772), microtubule-associated protein light chain 3 (LC3; 12741T), cleaved caspase-3 (9661 for immunohistochemical staining), and β-tubulin (2128T) were procured from Cell Signaling Technology. A mouse monoclonal antibody targeting PHB2 (sc-133094) was sourced from Santa Cruz Biotechnology. Antibodies against B-cell lymphoma 2 (Bcl-2; ab182858), cleaved caspase-3 (ab32351 for Western blot), autophagy-related gene 7 (ATG7; ab133528), and P62 (ab56416) were procured from Abcam. Antibodies against β-actin (A20120A0702) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; A20120A0701) were obtained from BioTNT. All horseradish peroxidase (HRP)-conjugated secondary antibodies used for Western blot analysis were sourced from Jackson ImmunoResearch Laboratories. MitoSOX (M36008) was purchased from Thermo Fisher Scientific. A mitochondrial membrane potential assay kit containing JC-1 (C2003S) and ROS assay kit (S0033S) were obtained from Beyotime Biotechnology. A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) apoptosis detection kit (G1501) was sourced from Servicebio. Cisplatin (P4394) was acquired from Sigma-Aldrich.

HK-2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 (SH30023; Hyclone), supplemented with 10% fetal bovine serum (04-001-1A; BioInd) at 37 °C in a humidified incubator under 5% CO₂. PHB2-specific small interfering RNA (siRNA) and PHB2-overexpression (OE) plasmids were obtained from Genomeditech. After a 24-h incubation period in six-well plates, HK-2 cells were transfected with either 100 nM siRNA or 2 ng/mL plasmid using Lipo8000 (C0533; Beyotime Biotechnology) for an additional 48 h, according to the manufacturer’s protocol. Negative control groups were subjected to parallel transfection with scrambled siRNA or empty plasmid. The following siRNA sequences were used: 5′-CAUCACAGAAUCGUAUCUA-3′ and 5′-UAGAUACGAUUCUGUGAUG-3′. Subsequently, the HK-2 cells were exposed to 20 µM cisplatin for 24 h to induce cellular stress and assess the effects of PHB2 modulation on cisplatin-induced cellular responses.

All animal experiments were performed in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Shanghai Ninth People’s Hospital. Eight-week-old male C57BL/6 mice were acquired from Shanghai Jihui Laboratory Animal Center and housed in standard facilities maintained at constant humidity and temperature, with ad libitum access to water and chow. The animals were randomly assigned to four groups (n = 6 per group). PHB2 overexpression was achieved through adeno-associated virus (AAV) serotype 9 (AAV9)-mediated gene transfer. AAV9 vectors encoding mouse PHB2 were provided by Taitool Bioscience. In brief, the C57BL/6 mice were anesthetized via inhalation of 2% isoflurane. They were then injected with 4.4 × 10¹² viral genomes per milliliter of AAV2/9-CAG-PHB2-flag-WPRE-pA or AAV2/9-CAG-3×flag-WPRE-pA (control group) at five sites (10 μL per site) on the renal cortex by using a glass micropipette (1705RN; Hamilton). Renal injury was induced via a single intraperitoneal injection of cisplatin at a dose of 20 mg/kg body weight. Mice were euthanized at 28 days after AAV infection and 72 h after cisplatin injection via intraperitoneal administration of 150 mg/kg 3% pentobarbital sodium for further analysis.

Harvested kidneys were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 3-μm-thick slices. These sections were hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) staining. Tubular injury was scored according to the percentage of damaged tubules: 0, normal (no damaged tubule); 1, <25% damaged areas; 2, 25%–49% damaged areas; 3, 50%–74% damaged areas; and 4, ≥75% damaged areas. For immunohistochemical analyses, mouse kidneys were fixed in 4% paraformaldehyde for at least 24 h, embedded in paraffin, and sectioned. These sections were deparaffinized, hydrated in graded ethanol series, and stained using the peroxidase–antiperoxidase method. The sections were then incubated with the anti-PHB2 (1:100) and anti-cleaved caspase-3 (1:400) at 4 °C overnight and then with the corresponding secondary antibodies.

Fresh kidney tissues were harvested and sectioned into 1-mm³ cubes. These cubes were prefixed in 2% glutaraldehyde, according to standard procedures. Next, the tissues were washed thoroughly, subjected to a dehydration process, and embedded in resin. The embedded tissues were sliced into ultrathin (70-nm-thick) sections by using standard methods. These sections were stained with lead citrate for enhanced contrast and visibility under the microscope. Finally, the stained, embedded sections were examined and detected on the transmission electron microscope FEI Talos L120C.

Apoptosis in cells was detected through the TUNEL assay, according to the manufacturer’s instructions. In brief, fixed and permeabilized HK-2 cells and paraffin-embedded sections were incubated with 56 µL of the TUNEL reaction mixture at 37 °C for 1 h in the dark. The samples were subsequently stained with 4′,6-diamidino-2-phenylindole for 8 min. TUNEL-positive cells within a 0.25-mm² area were enumerated.

Intracellular and mitochondrial ROS levels in viable HK-2 cells were measured using ROS Assay Kit and MitoSOX, according to the manufacturer’s instructions. In brief, cells were incubated with 10 µL of 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37 °C for 20 min or with 10 µL of 5 µM MitoSOX at 37 °C for 30 min in the dark. Mitochondrial transmembrane potential was evaluated using a JC-1 assay kit, as described previously (Xu et al., 2019). After treatment with cisplatin for 24 h, HK-2 cells were labeled with JC-1 (300 nM) at 37 °C for 20 min.

Total RNA was isolated and reverse-transcribed by using a PrimeScript RT reagent kit (Takara), according to the manufacturer’s protocol. Real-time polymerase chain reaction (PCR) was performed on an ABI Prism 7,500 sequence detection system, and the relative expression levels of the target genes were normalized against the expression of GAPDH or 18s ribosomal RNA (rRNA) as an internal control. The specific primer sequences employed for the PCR reactions were as follows: mitochondrial ATP synthase 6 (mt-Atp6), 5′-AGGACGAACATGAACCCTAAT-3′ and 5′-CAGCTCATAGTGGAATGGCTA-3′ for mouse; mitochondrial cytochrome b (mt-Cytb), 5′-CCCAGACAACTACATACCAGC-3′ and 5′-GATTAAGGCTAGGACACCTCC-3′ for mouse; 18s rRNA, 5′-GTCTCAAAGATTAAGCCATGC-3′ and 5′-GACCAAAGGAACCATAACTGA-3′ for mouse; interleukin-6 (IL-6), 5′-AACAACCTGAACCTTCCAAAG-3′ and 5′-CAAACTCCAAAAGACCAGTGA-3′ for cell; IL-6, 5′-TAGTCCTTCCTACCCCAATTTCC-3′; 5′-TTGGTCCTTAGCCACTCCTTC-3′for mouse; tumor necrosis factor-α (TNF-α), 5′-CCAGGCAGTCAGATCATCTTC-3′ and 5′-GCTTGAGGGTTTGCTACAACA-3′ for cell; TNF-α, 5′-CCCTCACACTCAGATCATCTTCT-3′; 5′-GCTACGACGTGGGCTACAG-3′ for mouse; GAPDH, 5′-GACACTGAGCAAGAGAGGCCCTA-3′ and 5′-TGGATGAAATTGTGAGGA-3′ for mouse and GAPDH 5′-CCTCTGACTTCAACAGCGACA-3′ and 5′-ATGAGCTTGACAAAGTGGTCGT-3′ for cell.

Kidney cells were collected and lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology), according to the manufacturer’s protocol. All protein samples were separated through 10%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. After they were blocked with 5% nonfat milk for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight. The membranes were subsequently incubated with secondary antibodies at room temperature for 1 h. Immunoreactive band intensities were visualized based on enhanced chemiluminescence, and densitometric analyses for quantification were performed using Image J.

All data, presented as means ± standard deviations (SDs), were analyzed using one-way ANOVA for comparisons. A P-value of <0.05 was deemed to indicate statistical significance.

Our Western blot analysis revealed that after 24-h in vitro stimulation with cisplatin at varying concentrations, HK2 cells exhibited a gradual increase in PHB2 expression with an increase in cisplatin concentrations (Figures 1A,B). This finding was further corroborated by our in vivo observations: immunohistochemical staining of mouse renal tissues revealed PHB2 deposition in renal tubules after cisplatin treatment (Figures 1C,D). Similarly, intraperitoneal injection of cisplatin significantly increased PHB2 expression in mice (Figures 1E,F).

Western blot analysis of HK2 cells stimulated with cisplatin at different concentrations for 24 h revealed a significant increase in the expression of apoptosis-related proteins such as cleaved caspase-3 and Bax but a decrease in Bcl-2 expression along with a corresponding decline in the Bcl/Bax ratio (Figures 2A,B). These changes were consistent with the pathological manifestations of renal tubular necrosis observed in our cisplatin-treated mice (Figure 1C). Moreover, autophagy-related proteins exhibited dose-dependent changes in expression: as the cisplatin concentration increased, the LC3 II/LC3 I ratio and ATG7 level decreased and P62 levels increased (Figures 2C,D).

Transfection of HK2 cells with PHB2-siRNA significantly reduced PHB2 levels (Figures 3A,B). After subsequent stimulation with 20 μmol/L cisplatin for 24 h, PHB2 siRNA transfection significantly exacerbated cisplatin-induced cell apoptosis, as indicated by increased expression of cleaved caspase-3 and Bax and decreased Bcl-2 expression, along with a decline in the Bcl/Bax ratio to the lowest level among all groups (Figures 3A,C). TUNEL assay also revealed increased tubular cell apoptosis in the PHB2-deficient group after cisplatin stimulation, consistent with our Western blot analysis results (Figures 3D,E).

Our DCFH-DA and MitoSOX staining results demonstrated that cisplatin stimulation increased both intracellular and mitochondrial ROS production, which was further elevated by PHB2-siRNA transfection; thus, PHB2 deficiency promotes ROS production (Figures 4A,B). JC-1 staining revealed a significant shift from red to green fluorescence in the group transfected with PHB2-siRNA and stimulated with cisplatin, indicating a severe loss of mitochondrial membrane potential (Figures 4C,D). Western blot analysis further revealed that PHB2-siRNA transfection led to a further reduction in the LC3 II/LC3 I ratio and ATG7 levels and an increase in P62 levels compared with the cisplatin-only group. Therefore, PHB2 deficiency exacerbated autophagy inhibition (Figures 4E,F). Moreover, the lack of PHB2 resulted in a further elevation of the inflammatory cytokines IL-6 and TNF-α upon cisplatin stimulation, suggesting a potential link between PHB2 and the regulation of inflammation (Figure 4G).

Transfection of HK2 cells with a PHB2-OE plasmid (Figures 5A,B) significantly reversed the increases in Bax expression and decreases in Bcl-2 expression induced by cisplatin (Figures 5C,D). Moreover, the TUNEL assay revealed that PHB2-OE plasmid transfection effectively reduced HK2 cell apoptosis caused by cisplatin, consistent with our Western blot analysis results (Figures 5E,F). Thus, PHB2 overexpression had a protective effect on renal tubules.

DCFH-DA and MitoSOX staining revealed that PHB2-OE plasmid transfection effectively reduced cisplatin-induced cellular and mitochondrial ROS accumulation (Figures 6A,B). Moreover, JC-1 staining directly demonstrated alterations in mitochondrial membrane potential, with increased red fluorescence intensity and decreased green fluorescence intensity in the group with PHB2 overexpression followed by cisplatin stimulation. Thus, PHB2 overexpression reversed the loss of mitochondrial membrane potential caused by cisplatin (Figures 6C,D). Western blot analysis further showed that PHB2 overexpression improved cisplatin-induced autophagy inhibition, as evidenced by increased LC3 II/LC3 I ratio and ATG7 expression (Figures 6E,F).

C57BL/6 mice were locally injected with AAV into one kidney and sacrificed 1 month after injection. Before euthanasia, the mice were administered 20 mg/kg cisplatin intraperitoneally. Western blot revealed a significant increase in PHB2 protein expression after local AAV injection (Figures 7A,B). Paraffin-embedded renal tissue sections were subjected to HE and PAS staining. The results demonstrated that AAV injection significantly mitigated cisplatin-induced loss of brush border, cast formation, lumen dilation of renal tubules, and tubular epithelial cell degeneration (Figure 7D), accompanied by a corresponding reduction in the tubular injury score (Figure 7E). AAV injection also attenuated the cisplatin-induced elevation in urea nitrogen levels (Figure 7C). Furthermore, TUNEL assay and immunohistochemical staining for cleaved caspase-3 indicated that PHB2 overexpression effectively reduced cellular apoptosis (Figures 7F–H). Taken together, these findings suggested that PHB2 overexpression effectively mitigates cellular apoptosis, preserves renal function, and alleviates cisplatin-induced AKI.

Transmission electron microscopy revealed mitochondrial swelling and cristae loss in renal tubular cells of mice treated with cisplatin. Notably, AAV-mediated PHB2 overexpression amended these mitochondrial abnormalities (Figure 8A). Furthermore, Western blot analysis and immunohistochemical staining revealed that PHB2 overexpression reversed cisplatin-induced autophagy inhibition, marked by an increased LC3 II/LC3 I ratio and downregulation of p62 (Figures 8A–D). Concurrently, cisplatin exposure significantly reduced the RNA levels of genes related to mitochondrial energy metabolism (Figure 8E). PHB2 overexpression ameliorated mitochondrial damage caused by cisplatin. Consistent with the in vitro findings, PHB2 overexpression in mouse kidneys also alleviated the cisplatin-induced inflammatory response (Figure 8F).