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. 2026 Sep 24;48(1):2728429. doi: 10.1080/0886022X.2026.2728429

Sigma-1 receptor protects against renal ischemia-reperfusion injury by enhancing mitophagy via Rac1 regulation

Siyuan Gong 1,*, Yonghong Xiong 1,*, Wenyuan Li 1, Yan Leng 1, Xinqi Deng 1, Bihan Wang 1, Baichuan Yang 1, Yuhang Yang 1, Wei Li 1,✉
PMCID: PMC13618200  PMID: 42785975

Abstract

Renal ischemia-reperfusion injury (IRI) is a leading cause of acute kidney injury and is associated with mitochondrial dysfunction, excessive reactive oxygen species (ROS) production, and tubular cell apoptosis. Sigma-1 receptor (Sigma1R), an intracellular chaperone, helps maintain mitochondrial homeostasis and cell survival. Here, Sigma1R expression was significantly downregulated in renal IRI. Fluvoxamine treatment ameliorated renal dysfunction and reduced apoptosis in vivo, whereas Sigma1R overexpression preserved mitochondrial membrane potential and attenuated ROS accumulation in HK-2 cells subjected to hypoxia/reoxygenation. The findings support the functional involvement of Rac1 in Sigma1R-mediated enhancement of PINK1/Parkin-mediated mitophagy, which may facilitate the clearance of damaged mitochondria and restore mitochondrial quality control. Overall, Rac1 is involved in Sigma1R-mediated mitophagy and mitochondrial protection, highlighting Sigma1R as a potential therapeutic target for renal IRI.

Keywords: Ischemia-reperfusion injury, mitophagy, sigma-1 receptor, Rac1

Introduction

Renal IRI is a common cause of perioperative renal injury, frequently occurring during kidney transplantation [1]. Renal IRI causes significant tissue damage and remains a challenge without effective treatments. Renal IRI triggers a cascade of deleterious events including mitochondrial damage, accumulation of ROS, and activation of pro-apoptotic signaling pathways, all of which contribute to acute kidney injury and subsequent chronic renal dysfunction [2,3]. Despite advances in perioperative management, IRI continues to be associated with high morbidity and mortality, and no effective pharmacological therapy is currently available.

Mitochondria play a pivotal role in IRI, with excessive production of mitochondrial ROS recognized as a critical factor in its pathogenesis [4]. Mitophagy is a central quality control process by which damaged mitochondria are selectively removed, thereby preventing the spread of injury signals, preserving bioenergetics, and modulating cell survival [5,6]. Proper activation of mitophagy helps maintain mitochondrial quality control, limit ROS accumulation, and attenuate cell death [4,7].

Initially characterized as an intracellular chaperone, Sigma1R stabilizes key proteins at the endoplasmic reticulum (ER)-mitochondrial interface, ensuring proper calcium transfer that is critical for mitochondrial ATP production and cell survival [8,9]. Studies have shown that Sigma1R activation confers renoprotective effects in experimental models of IRI by mitigating oxidative stress, reducing inflammatory cytokine release, and improving mitochondrial dynamics [9,10]. A growing body of evidence demonstrates that Sigma1R activation can restore impaired autophagy, thereby promoting the clearance of damaged cellular components including defective mitochondria [11,12]. In renal IRI, efficient mitophagy is critical because damaged mitochondria contribute to further cell injury by releasing pro-apoptotic factors and excessive ROS [13].

In parallel, Rac1, a member of the Rho family of small GTPases, regulates mitochondrial dynamics [14,15]. Rac1 modulates NADPH oxidase activity and thereby promotes stress-induced ROS production; excessive Rac1 activity has also been linked to cytoskeletal disruption and mitochondrial fragmentation [16]. Downregulation of Rac1 can reduce excessive ROS, potentially creating conditions that favor the clearance of dysfunctional mitochondria by mitophagy [17,18].

Rac1 is required for normal cytoskeletal function and cell motility, whereas its overactivation under stress can drive excessive ROS production through NADPH oxidase [19]. Previous work has reported an interaction between Sigma1R and Rac1 in mitochondrial compartments [12]. This reported association suggests that Sigma1R may influence Rac1-related signaling and downstream ROS production, thereby providing a potential mechanistic link to mitophagy in renal IRI.

In this study, we investigated whether Sigma1R confers renoprotection through a mechanism involving Rac1 and mitophagy, with the aim of clarifying the molecular basis of renal IRI and identifying potential therapeutic targets.

Materials and methods

Animals

Male C57BL/6 mice (8–10 weeks old, 22–25 g) were purchased from Shubeili (Wuhan, China). The animals were housed in a specific pathogen-free (SPF) facility under standard environmentally controlled conditions (temperature 22 ± 2 °C, relative humidity 50 ± 10%, and a 12-h light/dark cycle) with ad libitum access to standard laboratory chow and water. Mice were randomly allocated into the experimental groups. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Renmin Hospital, Wuhan University (Approval No. WDRM(F)20200604), and conducted in accordance with the ARRIVE guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Renal I/R injury model

To induce renal IRI, mice were anesthetized with pentobarbital sodium (50 mg/kg via intraperitoneal injection). A midline abdominal incision was made, and both renal pedicles were carefully exposed and clamped with non-traumatic microvascular clamps for 30 min to induce ischemia. Reperfusion was initiated by removing the clamps, and the kidneys were observed for color recovery. Sham-operated mice underwent the same procedure without clamping. Body temperature was maintained at 37 ± 0.5 °C using a thermostatically controlled heating pad throughout the surgery and during the recovery period. Postoperatively, animals received a subcutaneous injection of buprenorphine (0.1 mg/kg) for analgesia to minimize suffering. Mice were euthanized at 24 h after reperfusion via cervical dislocation under deep anesthesia. The deep anesthesia was induced by an intraperitoneal injection of sodium pentobarbital (150 mg/kg). All euthanasia and anesthesia procedures were performed in strict accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals.

Treatment

For pharmacological intervention, mice in the SHAM+FLU and IRI+FLU groups received FLU (MedChemExpress, Shanghai, China) at 20 mg/kg via intraperitoneal injection 30 min before the corresponding sham procedure or renal ischemia, respectively. Mice in the vehicle groups received an equivalent vehicle solution.

Cell culture and treatment

Human proximal tubular epithelial cells (HK-2) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Minimum Essential Medium (MEM; Hyclone, Beijing, China) supplemented with 10% fetal bovine serum (FBS; Bioexplorer, USA) at 37 °C in a humidified atmosphere containing 5% CO2. Cells were passaged every 2–3 days and used at 70–80% confluence.

For H/R injury, cells were incubated under hypoxic conditions (1% O2, 5% CO2, 94% N2) for 24 h, followed by reoxygenation in normoxic conditions (21% O2, 5% CO2) for 12 h.

Stable overexpression

Lentiviral vectors encoding human Sigma1R or Rac1 were purchased from Focusbiology (Shanghai, China). HK-2 cells were seeded at ∼40–50% confluence and transduced with lentivirus at a multiplicity of infection (MOI) of 20. After 24 h, the viral supernatant was removed and cells were cultured in fresh complete MEM. At 48 h post-transduction, cells were subjected to puromycin selection and maintained under selection for 3–5 days until untransduced control cells were eliminated. Surviving cells were expanded and used for subsequent experiments. Stable overexpression was confirmed by Western blotting. All viral work was performed under BSL-2 conditions in accordance with institutional biosafety regulations.

siRNA transfection

Small interfering RNAs (siRNAs) targeting human Rac1 and a non-targeting control siRNA were purchased from Focusbiology (Shanghai, China). HK-2 cells were seeded in 6-well plates and transfected with siRNA (final concentration: 50 nM) using Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 6 h, the medium was replaced with fresh complete MEM, and cells were cultured for an additional 48 h before subsequent analyses. The efficiency of Rac1 knockdown was confirmed by Western blotting.

Serum biochemical analysis

BUN and Scr levels were measured using commercial kits according to the manufacturer’s instructions (BioAssay Systems, Hayward, CA, USA). Serum KIM-1 and NGAL levels were measured using commercial mouse ELISA kits (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader, and serum concentrations were calculated from the corresponding standard curves.

Measurement of intracellular ATP

Intracellular ATP levels were measured using an ATP Assay Kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. After the indicated treatments, HK-2 cells were lysed with the provided lysis buffer on ice for 5 min and centrifuged at 12,000 × g for 5 min at 4 °C. The supernatant was collected, and ATP content was determined by mixing 100 µL of ATP detection working solution with 20 µL of sample. Luminescence was immediately measured using a microplate luminometer. ATP levels were normalized to protein concentration and expressed as µmol/g protein.

Measurement of mitochondrial ROS

Mitochondrial reactive oxygen species (ROS) levels were detected using the MitoSOX™ Red mitochondrial superoxide indicator (MedChemExpress, Shanghai, China) according to the manufacturer’s protocol. After the indicated treatments, HK-2 cells were incubated with 5 µM MitoSOX™ reagent at 37 °C for 15 min in the dark. The cells were then washed three times with warm PBS and immediately observed under a fluorescence microscope (Olympus, Tokyo, Japan). The fluorescence intensity was quantified using ImageJ software, and relative mitochondrial ROS levels were normalized to those of the control group.

Measurement of mitochondrial membrane potential

Mitochondrial membrane potential (Δψm) was assessed using the JC-1 assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, HK-2 cells were incubated with JC-1 staining solution at 37 °C for 20 min in the dark. After washing twice with JC-1 buffer, the cells were immediately observed under a fluorescence microscope (Olympus, Tokyo, Japan). Red fluorescence (J-aggregates) and green fluorescence (monomers) were quantified using ImageJ software. The ratio of red to green fluorescence intensity was calculated to represent Δψm, with higher ratios indicating healthier mitochondrial membrane potential.

Mitochondria-lysosome colocalization assay

HK-2 cells were incubated with MitoTracker Green and LysoTracker Red (Beyotime, Shanghai, China) at 37 °C for 30 min in the dark. After washing with pre-warmed culture medium, fluorescence images were captured using an inverted fluorescence microscope (Leica, Germany). Colocalization between mitochondria and lysosomes was analyzed using ImageJ software to assess mitochondria-lysosome colocalization.

Histological analysis

After euthanasia, kidneys were excised, fixed in 4% paraformaldehyde for 24 h, dehydrated, and embedded in paraffin. Sections (4 µm) were cut and stained with hematoxylin and eosin (HE) according to standard protocols. The corticomedullary junction was selected for histological assessment, and tubular injury was evaluated by an experienced pathologist in a blinded manner using the Paller scoring criteria.

TUNEL staining of renal tissue

Paraffin-embedded kidney sections (5 µm) were deparaffinized, rehydrated, and treated with proteinase K (20 µg/mL) for 15 min at room temperature. After washing with PBS, the sections were incubated with a TUNEL kit (Roche, Germany) at 37 °C in the dark and then counterstained with DAPI. After three PBS washes, sections were mounted with antifade medium and imaged using an inverted fluorescence microscope. The apoptotic index was calculated as the percentage of TUNEL-positive nuclei among total DAPI-stained nuclei.

Western blotting

All reagents for PAGE and Western blotting were purchased from Servicebio (Wuhan, China). Kidney tissues or HK-2 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using a BCA assay (Beyotime, Shanghai, China). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against Sigma1R, LC3B, p62, Parkin, PINK1, TOM20, cleaved caspase-3, total caspase-3, Rac1, or β-actin (Cell Signaling Technology, USA). After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Bands were visualized using enhanced chemiluminescence and quantified using ImageJ.

Statistical analysis

All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.4.1 (GraphPad Software, USA). Prior to parametric testing, data normality and homogeneity of variances were verified using the Shapiro-Wilk test and Levene’s test, respectively. Comparisons between two groups were conducted using an unpaired Student’s t-test, while comparisons among three or more groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. A p-value < 0.05 was considered statistically significant.

Results

Sigma1R expression is downregulated in renal IRI

To evaluate the effect of renal IRI on Sigma1R expression, we first confirmed successful model establishment using multiple indicators. Compared with the sham group, the IRI group exhibited significantly elevated blood urea nitrogen (BUN), serum creatinine (Scr), serum kidney injury molecule-1 (KIM-1), and serum neutrophil gelatinase-associated lipocalin (NGAL) levels (Figure 1A–D), indicating impaired renal function and tubular injury. Histological examination of the corticomedullary junction by hematoxylin and eosin (HE) staining (Figure 1F) showed relatively preserved tubular architecture in the sham group, whereas IRI kidneys exhibited marked tubular epithelial injury, tubular dilation, vacuolar degeneration, and structural disruption. Tubular injury was further quantified using the Paller score (Figure 1E). Apoptosis was evaluated by Western blot analysis of cleaved and total caspase-3, with the cleaved caspase-3/total caspase-3 ratio significantly increased in IRI kidneys (Figure 1G–H). Consistently, the apoptotic index, calculated as the percentage of TUNEL-positive nuclei among total DAPI-stained nuclei, was significantly increased in the IRI group (Figure 1I), as confirmed by TUNEL staining (Figure 1J). Western blotting further showed that Sigma1R expression was significantly lower in IRI kidneys than in sham controls (Figure 1K and L). These findings confirm model establishment and indicate that Sigma1R is downregulated in renal IRI.

Figure 1.

Multi-panel figure comparing SHAM and IRI groups: bar graphs of serum markers, histology images, and Western blots of caspase-3 and sigma-1 receptor. The figure includes multiple panels (A-L) comparing biological measures between SHAM and IRI groups. Bar graphs (A-E, H, I) show significantly elevated blood urea nitrogen, serum creatinine, serum KIM-1, and NGAL levels in IRI. Panel F highlights HE-stained histology images, showing normal SHAM tissue and damaged IRI tissue. Western blots in Panel G reveal higher cleaved caspase-3 in IRI, while Panel H quantifies this with a higher ratio of cleaved to total caspase-3 in IRI. Panel J presents TUNEL staining, indicating increased apoptosis in IRI, and Panels K-L show reduced sigma-1 receptor levels in IRI.

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Sigma1R expression is downregulated in renal IRI. (A) Blood urea nitrogen (BUN), (B) serum creatinine (Scr), (C) serum kidney injury molecule-1 (KIM-1), and (D) serum neutrophil gelatinase-associated lipocalin (NGAL) levels in the sham and IRI groups (n = 6 animals per group). (E) Tubular injury quantified using the Paller score (n = 6 animals per group). (F) Representative HE-stained sections of the corticomedullary junction. (G-H) Representative Western blots of cleaved caspase-3 and total caspase-3 and quantification of the cleaved caspase-3/total caspase-3 ratio in renal tissue. (I) Apoptotic index calculated as the percentage of TUNEL-positive nuclei among total DAPI-stained nuclei. (J) Representative TUNEL/DAPI staining and merged images. (K-L) Representative Western blot and densitometric quantification of Sigma1R in renal tissue. Scale bars = 50 µm in F and 20 µm in J. Data are presented as means ± SD; n = 6 animals per group. ****p < 0.0001.

Sigma1R agonist fluvoxamine ameliorates renal injury in IRI

To evaluate whether FLU modulates Sigma1R expression, renal Sigma1R protein levels were assessed in the sham and sham + FLU groups. Western blot analysis showed that FLU treatment significantly increased Sigma1R protein expression compared with the sham group (Figure 2A and B). FLU treatment significantly reduced BUN, serum creatinine (Scr), serum KIM-1, and serum NGAL levels in the IRI+FLU group compared with the IRI group, indicating improved renal function and reduced kidney injury (Figure 2C–F). No significant differences in these parameters were observed between the sham and sham + FLU groups. Paller scoring further showed reduced tubular injury in the IRI+FLU group compared with the IRI group (Figure 2G). HE staining of the corticomedullary junction demonstrated marked tubular epithelial injury, tubular dilation, vacuolar degeneration, and structural disruption in the IRI group, whereas these pathological changes were attenuated by FLU treatment (Figure 2H). Apoptosis was evaluated by Western blot analysis of cleaved and total caspase-3. The cleaved caspase-3/total caspase-3 ratio was significantly increased in the IRI group and reduced following FLU treatment (Figure 2I and J). Consistently, TUNEL staining and apoptotic index analysis showed increased apoptosis in the IRI group, which was attenuated by FLU treatment (Figure 2K and L).

Figure 2.

Multi-panel figure showing Western blots, bar graphs of serum markers, and histological images comparing treatments in kidney studies. This complex multi-panel figure displays various experimental data across four treatment conditions: SHAM, SHAM+FLU, IRI, and IRI+FLU. Panel A presents Western blots for Sigma1R (27 kDa) and ß-actin (45 kDa). Panel B shows relative Sigma1R levels. Panels C-G depict bar graphs for blood urea nitrogen, serum creatinine, KIM-1, NGAL, and Paller scores, highlighting significant differences, especially in IRI. Panel H presents histological images of H&E stained kidney sections. Panel I includes Western blots for cleaved (14 kDa) and total caspase-3 (35 kDa). Panel J compares the ratio of cleaved to total caspase-3 across groups. Panel K shows TUNEL assay fluorescence images displaying apoptosis markers. Panel L quantifies the apoptosis index, emphasizing differences between treatment groups.

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Sigma1R agonist fluvoxamine ameliorates renal injury in IRI. (A-B) Representative Western blot and densitometric analysis of Sigma1R protein expression in the sham and sham + FLU groups. (C) Blood urea nitrogen (BUN), (D) serum creatinine (Scr), (E) serum kidney injury molecule-1 (KIM-1), and (F) serum neutrophil gelatinase-associated lipocalin (NGAL) levels in each group. (G) Quantification of renal tubular injury using the Paller score. (H) Representative HE-stained sections of the corticomedullary junction showing histopathological changes. (I-J) Representative Western blots of cleaved caspase-3 and total caspase-3 and quantification of the cleaved caspase-3/total caspase-3 ratio in renal tissues. (K) Representative TUNEL staining, DAPI staining, and merged images. (L) Apoptotic index calculated as the percentage of TUNEL-positive nuclei among total DAPI-stained nuclei. Scale bars = 50 µm in H and 20 µm in K. Data are presented as means ± SD; n = 6 animals per group. ns, not significant; ****p < 0.0001.

Sigma1R overexpression attenuates mitochondrial dysfunction in HK-2 cells

To assess Sigma1R-associated mitochondrial effects in vitro, HK-2 cells were transduced with a Sigma1R-overexpressing lentivirus. Western blotting confirmed Sigma1R overexpression relative to the vector control (Figure 3A and B). H/R reduced the JC-1 red/green fluorescence ratio relative to the NC group, whereas Sigma1R overexpression increased the ratio relative to the HR+Vector group (Figure 3D and E). H/R also increased mitochondrial ROS, whereas Sigma1R overexpression reduced ROS accumulation relative to the HR+Vector group (Figure 3F and G). Consistent with these findings, H/R reduced intracellular ATP content, whereas Sigma1R overexpression increased ATP content relative to the HR+Vector group (Figure 3C). Together, these results indicate that Sigma1R overexpression preserves mitochondrial membrane potential, reduces oxidative stress, and maintains bioenergetic capacity during H/R.

Figure 3.

Multi-panel figure with Western blot and bar graphs of Sigma1R, ATP content, JC-1 ratios, and ROS levels; fluorescence images display monomers and aggregates. The figure includes multiple panels (A-G). Panel A shows a Western blot for Sigma1R (27 kDa) and ß-actin (45 kDa). Panel B presents a bar graph illustrating relative Sigma1R levels, with significant expression in the SIG-OE group. Panel C depicts ATP content among NC, HR, HR + Vector, and HR + SIG-OE groups. Panel D features fluorescence microscopy images distinguishing monomers (green) and aggregates (red) across all groups. Panel E shows the ratio of JC-1 fluorescence, highlighting differences in mitochondrial membrane potential. Panel F includes images of MitoSox (red) and MitoTracker (green) with merged views. Finally, Panel G displays ROS levels, indicating significant differences across treatments.

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Sigma1R overexpression attenuates mitochondrial dysfunction in HK-2 cells. (A-B) Western blotting and quantification confirming Sigma1R overexpression in HK-2 cells transduced with a Sigma1R-expressing lentivirus. (C) Intracellular ATP content. (D-E) Representative JC-1 images and quantification of the red/green fluorescence ratio. (F-G) Representative MitoSOX images and quantification of mitochondrial ROS. Scale bars = 50 µm in D and F. Data are presented as mean ± SD; n = 3 independent experiments. ns, not significant; ***p < 0.001, ****p < 0.0001.

Sigma1R overexpression enhances mitophagy in HK-2 cells

To investigate whether Sigma1R overexpression promotes mitophagy during H/R injury, we examined key mitophagy-related proteins and Rac1 by Western blotting. Compared with the HR+Vector group, the HR+Sig-OE group showed significantly lower Rac1, p62, and TOM20 protein levels, together with increased LC3B-II, PINK1, and Parkin levels (Figure 4A–G). These changes were consistent with enhanced mitophagy and indicated that Sigma1R overexpression attenuated H/R-induced Rac1 upregulation. We then assessed mitochondria–lysosome colocalization by fluorescence microscopy. H/R increased mitochondria–lysosome colocalization compared with the NC group, and Sigma1R overexpression further increased this measure relative to the H/R group (Figure 4H and I). Together, these results indicate that Sigma1R overexpression enhances mitophagy in HK-2 cells during H/R injury and is associated with reduced Rac1 protein expression.

Figure 4.

Multi-panel image showing Western blot results for various proteins and bar graphs quantifying their levels across conditions. The figure consists of several panels: Panel A shows a Western blot with protein bands for LC3B I/II, Rac1, P62, Parkin, PINK1, TOM20 across conditions NC, HR, HR+Vector, and HR+Sig-OE, with molecular weights indicated. Panels B-G display bar graphs quantifying relative protein levels, with significant changes highlighted. Panel H features fluorescent images displaying lysosome (red) and mitochondria (green) staining for each condition. Panel I provides a bar graph quantifying cells with Mito-Lyso contacts, showing significant differences between experimental groups.

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Sigma1R overexpression enhances mitophagy and reduces Rac1 protein expression in HK-2 cells. (A) Representative Western blots of LC3B, Rac1, p62, Parkin, PINK1, TOM20, and β-actin. (B-G) Densitometric quantification of LC3B-II, Rac1, p62, Parkin, PINK1, and TOM20, respectively. Sigma1R overexpression decreased Rac1, p62, and TOM20 protein levels while increasing LC3B-II, PINK1, and Parkin levels relative to the HR+Vector group. (H) Representative LysoTracker, MitoTracker, merged, and enlarged images. (I) Quantification of mitochondria-lysosome colocalization. Scale bars = 50 µm in the original images and 20 µm in the enlarged images. Data are presented as mean ± SD; n = 3 independent experiments. ns, not significant; ***p < 0.001, ****p < 0.0001.

Rac1 knockdown promotes mitophagy in HK-2 cells

Rac1, a small GTPase of the Rho family, regulates mitochondrial dynamics, ROS generation, and autophagy pathways. To examine the relationship between Rac1 and mitophagy, Rac1 was silenced using siRNA. Western blot analysis confirmed a significant reduction in Rac1 protein expression following Rac1 knockdown (Figure 5A and B). Rac1 silencing significantly increased LC3B-II, Parkin, and PINK1 protein levels while decreasing p62 and TOM20 levels compared with the HR+siNC group (Figure 5C–H), consistent with enhanced mitophagy. Mitochondria-lysosome colocalization was also increased following Rac1 knockdown (Figure 5I and J). Together, these findings indicate that Rac1 knockdown enhances mitophagy in HK-2 cells during H/R injury. The similar phenotypes observed following Sigma1R overexpression and Rac1 knockdown support the functional involvement of Rac1 in Sigma1R-associated mitophagy during H/R injury.

Figure 5.

Multi-panel figure with Western blots for Rac1 and ß-actin, bar graphs of LC3B, P62, Parkin, PINK1, TOM20, and fluorescence microscopy images. The figure consists of ten panels (A-J). Panel A shows a Western blot for Rac1 (21 kDa) and ß-actin (45 kDa) across NC, siNC, and Rac1-siRNA. Panel B presents bar graphs for relative Rac1 protein levels, highlighting a significant decrease in Rac1-siRNA. Panel C contains a Western blot for LC3B I (16 kDa), LC3B II (14 kDa), P62 (62 kDa), Parkin (50 kDa), PINK1 (65 kDa), TOM20 (15 kDa), and ß-actin. Panels D-H show bar graphs quantifying the relative protein levels of LC3B, P62, Parkin, PINK1, and TOM20, indicating significant differences among conditions. Panel I features fluorescence microscopy images (LysoTracker and Mitotracker) demonstrating cellular contacts, with merged and zoomed-in views. Panel J illustrates the percentage of cells with mito-lyso contacts, highlighting significant differences across treatments.

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Rac1 knockdown promotes mitophagy in HK-2 cells. (A-B) Representative Western blot and densitometric analysis confirming reduced Rac1 protein expression following Rac1 knockdown. (C-H) Representative Western blots and quantitative analyses of mitophagy-related proteins, including LC3B, p62, Parkin, PINK1, and TOM20. Rac1 knockdown increased LC3B-II, PINK1, and Parkin levels while decreasing p62 and TOM20 levels compared with the HR+siNC group. (I-J) Representative images and quantitative analysis of mitochondria-lysosome colocalization. Scale bars = 50 µm in the original images and 20 µm in the enlarged images. Data are presented as means ± SD; n = 3 independent experiments. ns, not significant; **p < 0.01, ***p < 0.001, ****p < 0.0001.

Rac1 overexpression partially attenuates Sigma1R-associated mitophagy and mitochondrial protection in HK-2 cells

To further examine the functional relationship between Sigma1R and Rac1, Rac1 overexpression efficiency was first confirmed by Western blotting (Figure 6A and B). HK-2 cells exposed to H/R were then assigned to the HR, HR+Rac1-OE, HR+Sig-OE, and HR+Sig-OE+Rac1-OE groups. Compared with the HR group, Sigma1R overexpression increased LC3B-II, Parkin, and PINK1 levels while decreasing p62 and TOM20 levels. In contrast, Rac1 overexpression produced the opposite pattern. Importantly, simultaneous Rac1 overexpression partially attenuated the mitophagy-associated protein changes induced by Sigma1R overexpression (Figure 6C–H).

Figure 6.

Multi-panel figure displaying Western blots, bar graphs, and microscopy images of Rac1, LC3B, P62, ATP, and ROS in various conditions. The figure features multiple panels (A-O) showcasing Western blots, bar graphs, and fluorescence microscopy to analyze Rac1 expression, protein levels (LC3B, P62, etc.), ATP content, mitochondrial-lysosomal interactions, membrane potential, and ROS levels across treatments (HR, Sigma1R, Rac1). Panel A shows Rac1 and ß-actin bands, while Panel B compares Rac1 levels, revealing significant overexpression in Rac1-OE cells. Panels C-O further contrast protein expressions and cellular conditions, including microscopy images that highlight differences in mitochondrial dynamics and ROS levels, with statistical significance noted throughout.

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Rac1 overexpression partially attenuates Sigma1R-associated mitophagy and mitochondrial protection in HK-2 cells. (A-B) Representative Western blot and densitometric analysis confirming Rac1 overexpression in HK-2 cells. (C) Representative Western blots of LC3B, p62, Parkin, PINK1, TOM20, and β-actin in the HR, HR+Rac1-OE, HR+Sig-OE, and HR+Sig-OE+Rac1-OE groups. (D-H) Densitometric quantification of LC3B-II, p62, Parkin, PINK1, and TOM20, respectively. (I) Intracellular ATP content. (J-K) Representative LysoTracker, MitoTracker, merged, and enlarged images and quantification of mitochondria-lysosome colocalization. (L-M) Representative JC-1 images and quantification of the red/green fluorescence ratio. (N-O) Representative MitoSOX images and quantification of mitochondrial ROS levels. Scale bars = 50 µm in the original images and 20 µm in the enlarged images in J; scale bars = 50 µm in L and N. Data are presented as means ± SD; n = 3 independent experiments. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Sigma1R overexpression also increased intracellular ATP content, whereas Rac1 overexpression reduced ATP levels. Simultaneous Rac1 overexpression partially attenuated the Sigma1R-associated increase in ATP content (Figure 6I). Fluorescence analysis further showed that Sigma1R overexpression increased mitochondria-lysosome colocalization, whereas Rac1 overexpression reduced colocalization, and dual overexpression produced an intermediate phenotype (Figure 6J and K). JC-1 analysis showed that Sigma1R overexpression preserved mitochondrial membrane potential, whereas Rac1 overexpression attenuated this effect (Figure 6L and M). Consistently, Sigma1R overexpression reduced mitochondrial ROS accumulation, while simultaneous Rac1 overexpression partially reversed this reduction (Figure 6N and O). Together, these findings support the functional involvement of Rac1 in Sigma1R-associated mitophagy and mitochondrial protection during H/R injury.

Discussion

Renal IRI causes tissue damage through a burst of ROS and inflammatory responses [20]. Mitochondrial dysfunction is central to this pathology, and the controlled elimination of dysfunctional mitochondria by mitophagy is crucial for cellular recovery and survival [5]. Sigma1R is increasingly recognized as a chaperone that modulates cellular stress responses and mitochondrial function [21]. Although Sigma1R has been studied predominantly in neuronal tissues, accumulating evidence indicates that it is also present in renal cells, where it influences mitochondrial dynamics and cell survival [22,23]. Sigma1R activation can attenuate the release of inflammatory cytokines, including interleukin-1β, interleukin-6, and tumor necrosis factor-α [11,24]. However, the precise protective mechanism of Sigma1R in renal IRI remains unclear.

This study shows that Sigma1R expression is downregulated in renal IRI. FLU treatment improved renal function and reduced apoptosis in vivo, whereas Sigma1R overexpression attenuated H/R-induced mitochondrial dysfunction in H/R-treated HK-2 cells. Rac1 was functionally involved in Sigma1R-associated enhancement of PINK1/Parkin-mediated mitophagy. These findings support a mechanistic link among Sigma1R, Rac1, and mitochondrial quality control in renal IRI.

Our results showed that Sigma1R expression was downregulated in renal IRI. This observation is consistent with previous reports describing the cytoprotective effects of Sigma1R in several organ systems. During renal IRI, ischemia halts ATP production and promotes metabolic-waste accumulation, whereas reperfusion triggers excessive ROS production that damages mitochondrial membranes and proteins [12,20]. H/R similarly injures mitochondria in HK-2 cells [25]. In this model, Sigma1R overexpression reduced ROS accumulation and preserved mitochondrial membrane potential. Sigma1R overexpression also increased the LC3B-II/LC3B-I ratio and Parkin levels while decreasing TOM20 and p62 levels. These changes are consistent with enhanced mitophagy and may contribute to mitochondrial protection.

Mitophagy preserves cellular function by selectively sequestering and degrading damaged mitochondria [5]. Failure to clear these organelles sustains ROS production and activates proapoptotic pathways [26]. Efficient mitophagy is therefore important for limiting mitochondrial injury during IRI. Previous work has shown that Sigma1R agonists such as FLU reduce renal apoptosis and improve kidney function [27]. Consistent with these reports, our data support a protective role for Sigma1R in renal IRI. In HK-2 cells, Sigma1R overexpression alleviated H/R-induced mitochondrial dysfunction and increased PINK1, Parkin, and LC3B-II levels. Because PINK1/Parkin-dependent mitophagy is a major pathway for damaged-mitochondria clearance, these findings suggest that Sigma1R may protect mitochondrial quality control by enhancing mitophagy.

Sigma1R interacts with regulatory proteins and signaling molecules that contribute to mitochondrial homeostasis [28]. Previous work has reported that Sigma1R can interact with Rac1 in mitochondrial membranes [29]. Rac1 regulates cytoskeletal rearrangement, cell proliferation, and ROS production through NADPH oxidase. Its role in ROS generation provides a plausible functional connection to mitochondrial damage and mitophagy. Sigma1R-Rac1 interactions have also been implicated in macrophage efferocytosis after ischemic injury [12]. Because these interactions depend on ligand and cellular context, Sigma1R may influence Rac1-associated signaling under specific conditions. In our H/R model, Rac1 knockdown produced phenotypes similar to Sigma1R overexpression, including enhanced PINK1/Parkin-associated mitophagy.

These findings suggest that Rac1 is functionally involved in Sigma1R-mediated mitochondrial protection and mitophagy during H/R injury. Because Rac1 activity was not measured directly, the data do not establish that Sigma1R suppresses Rac1 activity or interrupts NADPH oxidase signaling. Rac1 may nevertheless influence mitophagy through several pathways. As a regulator of NADPH oxidase, Rac1 can increase ROS generation, mitochondrial depolarization, and subsequent PINK1/Parkin activation. Rac1 can also interact with Bcl-2 and potentially modulate Beclin-1-dependent autophagy initiation. In addition, simvastatin-mediated inhibition of Rac1-mTOR signaling enhances autophagy in coronary arterial myocytes, suggesting that Rac1-associated signaling can affect autophagic processes, including mitophagy [30].

To further examine the functional relationship between Sigma1R and Rac1, we performed a co-overexpression experiment in HK-2 cells. Sigma1R overexpression enhanced mitophagy and attenuated mitochondrial dysfunction, whereas simultaneous Rac1 overexpression partially attenuated these effects. Rac1 co-overexpression reduced the Sigma1R-associated increases in PINK1 and Parkin and attenuated TOM20 degradation. It also partially reversed the reductions in mitochondrial ROS and membrane-potential loss, while decreasing ATP content relative to Sigma1R overexpression alone. These findings support the functional involvement of Rac1 in Sigma1R-mediated cytoprotection.

In summary, our findings support a protective role for Sigma1R in renal ischemia-reperfusion injury through maintenance of mitochondrial integrity and enhancement of PINK1/Parkin-mediated mitophagy. Rac1 was functionally involved in Sigma1R-mediated mitochondrial protection and mitophagy; however, because Rac1 activity was not measured directly, the data do not demonstrate direct inhibition of Rac1 by Sigma1R. The Sigma1R-Rac1 relationship may therefore contribute to mitochondrial quality control in renal IRI. This study has several limitations: Rac1 activity was not measured, mitophagy was assessed using static markers rather than dynamic flux assays, and mitochondrial respiration was not evaluated by oxygen-consumption analysis. Despite these limitations, the findings support Sigma1R as a potential therapeutic target for mitigating mitochondrial injury after ischemic stress.

Funding Statement

This work was supported by the National Natural Science Foundation of China [grant number 82272232].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are publicly available in the Harvard Dataverse repository at https://doi.org/10.7910/DVN/UNEHB8.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are publicly available in the Harvard Dataverse repository at https://doi.org/10.7910/DVN/UNEHB8.

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