Involvement of Phosphatase and Tensin Homolog-Induced Putative Kinase 1–Parkin-Mediated Mitophagy in Septic Acute Kidney Injury
Sepsis-associated acute kidney injury (AKI) is a critical condition that significantly contributes to morbidity and mortality in intensive care units, accounting for approximately 50% of all AKI cases. Despite advances in medical science, the pathophysiology of septic AKI remains poorly understood, and current therapeutic strategies are limited. This study investigates the role of mitophagy, a selective form of autophagy that targets damaged mitochondria, in the pathogenesis of septic AKI. Specifically, the research focuses on the involvement of the phosphatase and tensin homolog-induced putative kinase 1 (PINK1)-Parkin pathway in regulating mitophagy and its potential protective effects against renal injury during sepsis.
Background and Rationale
Sepsis is a life-threatening condition characterized by a dysregulated host response to infection, leading to organ dysfunction. AKI is a common complication of sepsis, and its development is associated with prolonged hospitalization and increased mortality. Unlike non-septic AKI, the pathogenesis of septic AKI is not solely driven by hemodynamic changes but also involves cellular stress responses, including oxidative injury and mitochondrial dysfunction. Autophagy, a cellular process that degrades and recycles damaged organelles and macromolecules, has been implicated in the protection against kidney injury in various experimental models. However, the specific role of mitophagy, particularly the PINK1-Parkin pathway, in septic AKI remains unclear.
Objectives and Hypotheses
This study aimed to determine whether the PINK1-Parkin mitophagy pathway is activated in renal tubular epithelial cells (RTECs) during septic AKI and to explore its effects on cell apoptosis and renal function. The researchers hypothesized that PINK1-Parkin-mediated mitophagy exerts a protective role against sepsis-induced renal injury by mitigating mitochondrial dysfunction and reducing apoptosis.
Methods
Experimental Models
The study employed both in vitro and in vivo models to investigate the role of mitophagy in septic AKI. In vitro experiments were conducted using the human RTEC cell line HK-2, which was stimulated with bacterial lipopolysaccharide (LPS) to mimic septic conditions. In vivo experiments utilized a rat model of septic AKI induced by cecal ligation and perforation (CLP).
Mitophagy and Apoptosis Assessment
Mitophagy-related gene expression was assessed using Western blot analysis to measure the levels of key proteins, including microtubule-associated protein 1 light chain 3 alpha (LC3-II), Beclin-1 (BECN-1), and p62. Electron microscopy was used to observe autophagy-related ultrastructural features in rat RTECs. Gain- and loss-of-function approaches were employed to investigate the role of the PINK1-Parkin pathway in HK-2 cell mitophagy. Specifically, PINK1 was either knocked down using short interfering RNA (siRNA) or overexpressed using adenoviral vectors.
Apoptosis was assessed by measuring the expression of proapoptotic proteins, including cleaved caspase-3 and BAX, using Western blot analysis. Flow cytometry was used to quantify apoptotic rates in HK-2 cells treated with autophagy activators (rapamycin and brefeldin A) or inhibitors (3-methyladenine and chloroquine).
Renal Function Assessment
In vivo, the effects of mitophagy modulation on renal function were evaluated by measuring blood urea nitrogen (BUN) and serum creatinine levels in rats subjected to CLP and treated with autophagy activators or inhibitors.
Results
Septic Insults Induce Mitophagy in RTECs
Both in vitro and in vivo experiments demonstrated that septic insults significantly induce mitophagy in RTECs. In HK-2 cells, LPS stimulation led to a marked increase in LC3-II and BECN-1 protein expression, peaking at 4 hours post-stimulation. Similarly, in rat RTECs, LC3-II, BECN-1, and p62 protein levels were significantly elevated, with peak expression observed at 2 hours post-CLP. Electron microscopy revealed mitochondrial deformation and the formation of mitolysosomes in RTECs from septic rats, indicating active mitophagy.
The PINK1-Parkin Pathway is Essential for Mitophagy
Loss- and gain-of-function experiments in HK-2 cells demonstrated that the PINK1-Parkin pathway plays a crucial role in mitophagy regulation. Knockdown of PINK1 significantly attenuated LPS-induced LC3-II expression, while PINK1 overexpression enhanced LC3-II levels. These findings suggest that the PINK1-Parkin pathway is essential for mitophagy activation in RTECs exposed to septic conditions.
Mitophagy Protects RTECs from Apoptosis
LPS stimulation induced apoptosis in HK-2 cells, as evidenced by increased expression of cleaved caspase-3 and BAX. However, autophagy activators (rapamycin and brefeldin A) prevented further apoptosis, whereas autophagy inhibitors (3-methyladenine and chloroquine) exacerbated apoptosis. Similarly, PINK1 overexpression significantly reduced cleaved caspase-3 levels in LPS-stimulated HK-2 cells, while PINK1 depletion enhanced apoptosis. These results indicate that mitophagy activation protects RTECs from sepsis-induced apoptosis.
Mitophagy Improves Renal Function in Septic AKI
In vivo experiments showed that autophagy activators (rapamycin and brefeldin A) significantly reduced BUN and serum creatinine levels in rats with CLP-induced septic AKI, indicating improved renal function. Conversely, autophagy inhibitors (3-methyladenine and chloroquine) further increased BUN levels, although serum creatinine levels remained unchanged. These findings suggest that mitophagy activation has a protective effect on renal function during sepsis.
Discussion
This study provides compelling evidence that mitophagy, particularly the PINK1-Parkin pathway, plays a protective role in septic AKI. The findings indicate that septic insults rapidly induce mitophagy in RTECs, leading to the clearance of damaged mitochondria and the prevention of apoptosis. The PINK1-Parkin pathway is essential for this process, as PINK1 knockdown attenuates mitophagy, while PINK1 overexpression enhances it.
The protective effects of mitophagy are further supported by the observation that autophagy activators reduce apoptosis and improve renal function in septic AKI models. These results align with previous studies showing that mitophagy activation protects against renal injury in other forms of AKI, such as ischemia-reperfusion injury and cisplatin-induced nephrotoxicity.
The study also highlights the dual role of autophagy in cell survival and death. While moderate activation of mitophagy protects RTECs from apoptosis, excessive autophagy may lead to mitochondrial damage and exacerbate cell death. Therefore, the therapeutic modulation of mitophagy must be carefully balanced to achieve optimal protection against septic AKI.
Conclusion
In conclusion, this study demonstrates that PINK1-Parkin-mediated mitophagy is activated in RTECs during septic AKI and exerts a protective role by mitigating mitochondrial dysfunction and reducing apoptosis. The findings suggest that targeting the PINK1-Parkin pathway may offer a novel therapeutic strategy for the prevention and treatment of septic AKI. Further research is needed to explore the precise mechanisms by which PINK1 regulates Parkin and to develop targeted therapies that enhance mitophagy in the context of sepsis.
doi.org/10.1097/CM9.0000000000000448
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