Knocking‑down Annexin A3 Attenuates Sepsis‑Induced Acute Kidney Injury: In‑Depth Insights from HK2 Cell Studies

Introduction

Acute kidney injury in sepsis is characterized by multiple pathogenic features. Among these, excessive inflammation, increased reactive oxygen species (ROS) production, ER stress, and apoptotic cell death are prominent. The interplay among these factors ultimately disrupts cellular function and kidney integrity. The HK2 cell model—a line of human proximal tubular epithelial cells—has been widely employed in preclinical studies to simulate renal epithelial injury in sepsis. Researchers have identified that upon exposure to endotoxins such as lipopolysaccharide (LPS), HK2 cells display heightened expression of annexin A3, a member of the annexin family known for its phospholipid‑binding properties in the presence of calcium ions.

Recent experimental evidence indicates that the knockdown of annexin A3 using RNA interference (siRNA) methods in LPS‑treated HK2 cells markedly reduces inflammatory cytokine expression, lowers oxidative stress markers, diminishes ER stress proteins, and results in decreased apoptosis. This suggests that annexin A3 is a key mediator in driving the deleterious cascade in sepsis‑induced AKI.

Background on Annexin A3

Annexin A3 is one of multiple annexin proteins that participate in numerous cellular processes such as membrane trafficking, signal transduction, and cell differentiation. Its expression is regulated in response to cellular stress, and its overexpression has been reported in several pathological conditions, including various cancers and inflammatory diseases. In the context of sepsis‑induced AKI, the elevation of annexin A3 contributes to the amplification of pro‑inflammatory signals and the generation of ROS. These reactive compounds, when produced in excess, can trigger a cascade of events leading to the activation of ER stress sensors and apoptotic pathways. The study under review demonstrates that by specifically targeting annexin A3, it is possible to attenuate the renal epithelial cell damage induced by septic processes.

The HK2 cell line represents a useful in vitro model to study kidney proximal tubule cell function because these cells share many physical and biochemical properties with human renal epithelium. When HK2 cells are challenged with LPS, mimicking bacterial infection, they undergo inflammatory activation, increased ROS production, and ER stress. As such, mitigating the impact of annexin A3 in these cells provides insights into the underlying mechanisms of renal injury and opens up new avenues for therapeutic interventions.

Experimental Methodology Overview

The experimental design to evaluate the role of annexin A3 in sepsis‑induced AKI in HK2 cells involves several key steps:

1. Establishing the In Vitro AKI Model:
   HK2 cells were exposed to various concentrations of LPS to mimic the inflammatory conditions of sepsis. Optimization of LPS concentration and treatment duration ensured a robust model characterized by increased cellular injury signals.

2. Knocking‑down Annexin A3 Expression:
   Specific small interfering RNAs (siRNAs) targeting annexin A3 were transfected into HK2 cells. Subsequent quantitative real‑time polymerase chain reaction (qRT‑PCR) and Western blot analyses confirmed a significant reduction in annexin A3 mRNA and protein levels.

3. Assessment of Cellular Viability and Proliferation:
   The proliferative capacity of HK2 cells under LPS‑induced stress was evaluated using the cell counting kit‑8 (CCK‑8) assay, colony formation assays, and 5‑ethynyl‑2′‑deoxyuridine (EdU) incorporation. Knocking‑down annexin A3 resulted in improved cell viability and colony growth, indicating a protective effect against LPS‑induced cytotoxicity.

4. Measurement of Inflammation, Oxidative Stress, and ER Stress:
   Enzyme‑linked immunosorbent assay (ELISA) kits were used to quantify inflammatory cytokines such as tumor necrosis factor‑alpha (TNF‑α), interleukin‑6 (IL‑6), and interleukin‑1β (IL‑1β). In parallel, antioxidant parameters such as glutathione (GSH) and superoxide dismutase (SOD) were measured, along with malondialdehyde (MDA) and ROS levels as indices of oxidative damage. Western blot analysis assessed the levels of ER stress markers including GRP78, CHOP, IRE1α, and ATF6.

5. Analysis of Apoptosis:
   Flow cytometry, accompanied by Annexin V‑fluorescein isothiocyanate (FITC) staining, was employed to quantify apoptotic cell populations. A reduction in apoptosis was observed in LPS‑treated cells following annexin A3 knockdown.

The combined methodologies allowed for comprehensive characterization of cellular responses and provided a strong mechanistic link between annexin A3 expression and sepsis‑induced cellular injury.

Key Experimental Results

The results from these combined experiments revealed that LPS treatment of HK2 cells significantly increased annexin A3 expression and triggered widespread inflammatory, oxidative, ER stress responses, and apoptotic cell death. However, when annexin A3 was knocked down via siRNA, these harmful effects were markedly reduced. The key findings include:

• Cell Viability and Proliferation:
  LPS‑treated HK2 cells exhibited a decrease in cell viability. Knockdown of annexin A3 improved cell survival and increased colony formation. EdU incorporation assays confirmed higher rates of DNA replication in the annexin A3 knockdown group compared to LPS‑only exposure.

• Inflammatory Markers:
  ELISA analyses demonstrated that the levels of TNF‑α, IL‑6, and IL‑1β were significantly elevated in LPS‑exposed cells. The suppression of annexin A3 expression resulted in a significant decrease in these cytokines, indicating reduced inflammation.

• Oxidative Stress:
  LPS treatment led to a decrease in antioxidants (GSH and SOD) and an increase in oxidative damage markers (MDA and ROS). Knockdown of annexin A3 restored antioxidant levels and reduced oxidative markers.

• ER Stress and Apoptosis:
  Western blot analysis revealed that ER stress proteins (GRP78, CHOP, IRE1α, ATF6) were upregulated by LPS treatment. Annexin A3 silencing brought these protein levels closer to baseline. Correspondingly, the percentage of apoptotic cells, as determined by flow cytometry, was significantly lower in cells with reduced annexin A3 expression.

Table 1. Summary of Effects of Annexin A3 Knockdown in LPS‑Treated HK2 Cells

Note: Data in this table represent averaged experimental observations from multiple independent replicates.

Discussion and Implications

The upregulation of annexin A3 in HK2 cells under LPS-induced stress suggests that this protein plays a central role in mediating the deleterious cellular responses typical of sepsis-induced AKI. By facilitating inflammatory signaling, ROS production, and ER stress, annexin A3 contributes to tubular cell apoptosis and kidney dysfunction.

The finding that the targeted knockdown of annexin A3 can reverse these harmful effects is of substantial clinical interest. Suppression of annexin A3 not only restored cell viability and proliferative capacity but also normalized the levels of key mediators of inflammation and oxidative damage. The significant reductions in ER stress markers further underscore the role of annexin A3 in maintaining protein folding homeostasis under septic conditions.

These data imply that therapeutic strategies aimed at inhibiting annexin A3 expression or function may offer protective benefits for patients suffering from sepsis-induced AKI. Future research may explore the development of small molecules or biologics capable of modulating annexin A3 activity in vivo. Moreover, the HK2 cell model provides a valuable platform for screening potential therapeutics targeted against annexin A3 and for further elucidating the molecular pathways by which annexin A3 modulates renal cell injury.

Notably, the multifactorial nature of sepsis-induced AKI necessitates that any prospective treatment must address inflammation, oxidative stress, and ER stress concurrently. Annexin A3 modulation represents a promising avenue, as its inhibition appears to have a broad-spectrum impact across these different pathological processes. Despite these promising in vitro results, further validation using animal models and eventually clinical trials will be required to ascertain the safety and efficacy of annexin A3-targeted interventions.

Data Tables

In addition to Table 1, researchers may consider compiling supplementary data tables that detail experimental conditions, reagent concentrations, and time‑course studies. Below is an example of a supplementary table:

Table 2. Experimental Conditions and Observed Responses in LPS‑Induced HK2 Cells

Values are approximate and represent average effects from multiple experimental replicates.

Frequently Asked Questions (FAQ)

What role does annexin A3 play in sepsis-induced acute kidney injury?
Annexin A3 is implicated in the modulation of inflammatory responses, oxidative stress, and ER stress. During sepsis, its elevated expression contributes to kidney cell apoptosis and overall renal dysfunction. Knocking down annexin A3 has been shown to reduce these harmful effects, suggesting that it is a key mediator of sepsis-induced damage in renal epithelial cells.

How are HK2 cells used in this research?
HK2 cells are human proximal tubular epithelial cells that serve as an in vitro model for studying kidney function. In the context of sepsis-induced AKI, these cells are exposed to LPS to mimic the inflammatory environment seen in septic patients. Changes in cellular viability, inflammation, oxidative stress, and apoptosis are then measured to understand the impact of different treatments.

What experimental techniques were employed to assess the impact of annexin A3 knockdown?
Researchers used a combination of molecular and cellular biology techniques including:

• siRNA-mediated knockdown to reduce annexin A3 expression.
• Quantitative real‑time PCR and Western blotting to measure mRNA and protein levels.
• CCK‑8, colony formation, and EdU assays to evaluate cell proliferation and viability.
• ELISA for quantifying inflammatory cytokines.
• Flow cytometry to assess apoptosis.
• Assays for oxidative stress (measuring GSH, SOD, MDA, and ROS levels) and Western blotting for ER stress markers.

Can these findings be translated into clinical therapies?
While the in vitro results are promising, further research—beginning with animal model studies and eventually clinical trials—is necessary to establish the safety and therapeutic efficacy of targeting annexin A3 in patients with sepsis-induced AKI. The study provides a strong mechanistic basis suggesting that such therapies might help ameliorate renal injury in sepsis.

What are the broader clinical implications of this research?
Since sepsis-induced AKI is a severe complication leading to high rates of morbidity and mortality, identifying new molecular targets like annexin A3 opens up potential avenues for novel therapeutic interventions. By mitigating renal cell injury, such treatments could improve patient outcomes, reduce the progression to chronic kidney disease, and decrease overall health care burdens associated with sepsis.

References

1. Su, J., Wang, L., Guan, X., Li, N., & Sun, L. (2024). Knocking‑down annexin A3 suppresses inflammation, oxidative stress, apoptosis, and endoplasmic reticulum stress to attenuate sepsis‑induced acute kidney injury in HK2 cells. CytoJournal, 21, 75. https://doi.org/10.25259/Cytojournal_64_2024