Sepsis is associated with mitochondrial DNA damage and a reduced mitochondrial mass in the kidney of patients with sepsis-AKI

Sepsis is defined as a dysregulated host response to infection, which can lead to loss of organ homeostasis, multiple organ failure, and ultimately, death in patients [1]. Mortality and morbidity associated with sepsis and septic shock are high. In-hospital mortality rate amounts to 20–30% for sepsis and 40–60% for septic shock [1, 2]. Despite extensive research, the pathophysiology of sepsis remains incompletely understood. As a result, therapeutic options directed at the molecular cause of organ dysfunction are lacking, and the current therapy is limited to source control (i.e., antibiotics, drainage) and organ support [1‐4]. Up to 60% of patients with sepsis develop acute kidney injury (AKI) and sepsis is the most common cause of AKI in the intensive care unit (ICU) [5, 6]. The occurrence of AKI in sepsis is associated with failure of other organs and increased mortality [5, 6]. Thus, sepsis-AKI constitutes a serious medical health problem.

Mitochondria have emerged as key players in the pathogenesis of sepsis-AKI [7‐9]. Healthy mitochondria are essential for maintaining renal homeostasis and are important in supporting the metabolic challenge during sepsis [2, 7, 8]. Mitochondrial failure during sepsis results in renal ATP-depletion and increased levels of reactive oxygen species (ROS), which progresses into loss of cellular homeostasis and organ dysfunction [10]. Sepsis lowers ATP levels, increases biomarkers for mitochondrial dysfunction and reduces antioxidant defense, which are associated with poor survival, as demonstrated in muscle biopsies in 16 critically ill patients compared to 10 healthy, age-matched patients undergoing elective hip surgery [11]. It is known that accumulating levels of ROS during sepsis can cause damage to mitochondrial proteins and DNA in liver and heart from rats [12, 13], further impairing mitochondrial function and leading to a vicious circle in which ROS production continues to increase [12‐14]. Conversely, biogenesis of mitochondria is associated with an increased survival of septic shock [11], and experimental models of sepsis-AKI confirm the importance of maintaining healthy mitochondria for renal recovery and survival [15‐19].

To date, the precise molecular mechanisms of mitochondria in the pathogenesis of sepsis-AKI are incompletely understood, which hampers the development of a molecular targeted therapy to prevent or treat sepsis-AKI and improve outcome. The high metabolic burden during sepsis leads to increased ROS production by mitochondria. We hypothesized that damage to mitochondrial DNA during sepsis causes mitochondrial dysfunction with relevance to both short-term outcome and potentially also for long-term outcome in sepsis-AKI. We therefore explored the mRNA expression of mitochondrial quality mechanism pathways and studied mitochondrial DNA (mtDNA) oxidation and integrity in renal biopsies from sepsis-AKI patients and control subjects. Next, we validated these results in vitro, by inducing human umbilical vein endothelial cells (HUVECs) with lipopolysaccharide (LPS) for 48 h to mimic sepsis.

Sepsis leads to increased levels of ROS, which is in part due to the overwhelming immune response to facilitate bacterial killing [1, 27, 29]. In turn, ROS can cause collateral damage by oxidizing proteins, lipids and DNA [12, 13]. Subsequent damage to mtDNA can impair mitochondrial function and further increase ROS generation [27, 29]. Whether this process underlies the pathophysiology in sepsis-AKI was not yet known. Here, we demonstrate that sepsis-AKI patients have an upregulated mRNA expression of markers important in the renal oxidative stress response, leading to higher levels of mtDNA oxidation, mtDNA damage and a reduced number of mitochondria in the kidney. Despite the mitochondrial damage, we did not find evidence of compensatory upregulation of mitochondrial quality control mechanisms, which further substantiates mitochondrial dysfunction in sepsis-AKI. Together, these data demonstrate key mechanisms leading to mitochondrial failure in the pathophysiology of sepsis-AKI.

The way cells deal with ROS under inflammatory circumstances is important for the distinction between survival, long-term complications, or mortality of patients with sepsis [30]. Induction of NGAL, mnSOD and HIF1α is indicative of renal oxidative stress and injury [31‐33]. One of the first markers of renal injury is upregulation of NGAL expression [34], as demonstrated in our biopsies derived from patients with sepsis-AKI. Increased NGAL expression is regulated by ROS to suppress bacterial growth and modulate the inflammatory response [31]. In turn, NGAL activates antioxidant defense mechanisms, leading to the upregulation of mnSOD, as demonstrated in vitro [31]. Accordingly, we found a positive correlation between NGAL expression and mnSOD expression in the renal biopsies. The importance of upregulated mnSOD in sepsis is confirmed in mouse models of sepsis (i.e., lipopolysaccharide [LPS] injection and cecal ligature and puncture [CLP]), where higher mnSOD expression prevented ATP depletion and subsequent mortality [33, 35, 36]. Similarly, we found an increased gene expression of mnSOD expression in patients with sepsis-AKI as compared to control subjects. Upregulation of HIF1α switches mitochondrial aerobic respiration to glycolysis metabolism, thereby bypassing the dysfunctional ROS-producing mitochondria and lowering oxidative stress [32]. Higher HIF1α expression levels were detected in whole blood cells from patients with septic shock [37], in line with the increased expression in the kidney as demonstrated here in sepsis-AKI. In contrast to NGAL, mnSOD and HIF1α, the expression of SIRT1, involved in inhibiting oxidative stress and the suppression of biogenesis and mitophagy [38, 39], was downregulated in sepsis-AKI patients. Taken together, sepsis-AKI is associated with upregulation of genes encoding molecules involved in inhibiting inflammation and antioxidant defense in the kidney.

Compared to control subjects, sepsis-AKI patients had upregulated mRNA expression of oxidative damage markers and high levels of mitochondrial DNA damage. Also 48 h of LPS induction in HUVECs caused an increase in mnSOD expression and mtDNA damage. Although several studies demonstrated increased biomarkers suggestive of mitochondrial dysfunction in sepsis [10, 11, 15], to our knowledge only one other study so far demonstrated the occurrence of mtDNA damage in patients with sepsis, as illustrated by the depletion of mtDNA defined by RT-qPCR in monocytes and lymphocytes [40]. Extending on the observations of these previous studies, we now directly demonstrate the presence of mtDNA damage in the septic kidney. Whereas mtDNA damage in monocytes and lymphocytes correlated with the APACHE score, we did not find such a correlation between mtDNA damage and APACHE score in sepsis-AKI patients, which might be due to the small sample size (n = 147 vs n = 12, respectively). The observed mtDNA damage in the kidney in patients with sepsis-AKI is in line with findings from experimental murine sepsis models, which showed profound damage to mtDNA in skeletal muscle and 50% mtDNA depletion in the liver [9, 41]. mtDNA damage was associated with increased DNA oxidation (i.e., 8-oxoG) in sepsis-AKI as compared to control subjects, while immunofluorescent staining showed co-localization of DNA oxidation with both nuclei and mitochondria. In line with this observation, sepsis in mice also leads to accumulation of 8-oxoG in mitochondria and mitochondrial dysfunction, as demonstrated in the brain [42]. Hence, mitochondrial dysfunction leading to increased oxidative stress, oxidation of mtDNA and damage that further impairs mitochondrial function might play a key role in the pathophysiology sepsis-AKI.

Mitochondrial quality control mechanisms, consisting of biogenesis (making new mitochondria), fission/fusion of mitochondria and mitophagy (removal of damaged mitochondria), can counteract mitochondrial damage and subsequent dysfunction [7, 8, 43]. Sepsis-AKI patients had lower mRNA expression of TFAM (marker for biogenesis) and PINK and PARKIN (markers for mitophagy), but no change in mRNA expression of MFN2 and DRP1 (markers for fusion and fission). HUVECs induced with LPS for 48 h showed a similar pattern, where TFAM was decreased, but no change in PINK, MFN2 or DRP1. Similar to our findings, sepsis is associated with increased mRNA expression and protein levels of TFAM in muscle, suggestive of biogenesis and a lowered mitochondrial mass [11]. Adequate compensation of mitochondrial damage seems to play an important role in determining the outcome of sepsis, as mRNA expression of Peroxisome Proliferator-activated Receptor Gamma Coactivator 1-alpha (PGC1α) (marker of biogenesis upstream of TFAM) is associated with survival from sepsis [11]. Also, LPS-induced mice with AKI suffer from decreased mRNA expression of PGC1α in the renal cortex, whereas overexpression of PGC1α shows recovery from LPS-induced AKI [15]. The protective role of mitophagy is illustrated by PARK2-deficient mice, which exhibited degradation of mitochondrial functions and impaired recovery of cardiac contractility in sepsis [44]. Additionally, inhibition of mitophagy in mice increases the sensitivity of multiple organ failure and death from sepsis [44, 45]. Here, we found a decreased expression of PINK1 and PARKIN, the main mitophagy regulators, in the septic kidney, which suggests that removal of damaged mitochondria by mitophagy is impaired. Lastly, we found no changes in mRNA expression DRP1 or MFN2 (markers for fission/fusion) in renal biopsies or LPS treated HUVECs. Accordingly, fission and fusion did not differ between septic patients and controls in human PBMCs [46]. However, since PINK1 and PARKIN mediate degradation of MFN2 and activation of DRP1 to prevent fusion while promoting fission [47, 48], we cannot conclude that protein levels or activity of PINK1 and PARKIN and hence fusion/fission are unaltered in sepsis. Together, sepsis is associated with mtDNA damage in sepsis without upregulation of genes encoding for mitochondrial quality control processes to safeguard the mitochondria.

A strength of our study is the use of fresh direct postmortem kidney biopsies from sepsis-AKI patients and control subjects, which allowed us to directly investigate the effect of sepsis on mitochondria in the kidney in association with immunohistochemical analysis, albeit in a small cohort of patients, while other studies estimate kidney and mitochondrial damage indirect using surrogate markers in urine or blood. However, we could only analyze non-survivors, which represent the most severe critically ill patients, as it would be ethically unacceptable to obtain renal biopsies from living patients with sepsis-AKI. Postmortem changes in mRNA expression should be taken into consideration when interpreting our data, but are unlikely to have been of major relevance, as samples were collected as quickly as possible after death (24–150-min postmortem), and the kidney is among the less susceptible organs to changes in RNA integrity and gene expression as demonstrated up to 24-h postmortem [49]. Lastly, there is uncertainty as to whether renal cell carcinoma might have affected mitochondrial mass or DNA levels in surrounding healthy tissue. However, mtDNA copy number, DNA content and activities of mitochondrial enzymes are shown to be reduced within RCC tissue [50, 51]. Furthermore, impairment of mitochondrial DNA levels and mass depends on RCC aggressiveness, a trend that is not found in healthy tissue within the same kidney [52, 53]. Thus, in contrast to effects of cancer on mitochondrial mass and DNA within the tumor, this does not seem to be the case for surrounding tissue. However, even if mitochondrial mass and DNA in surrounding tissue would have been affected by the tumor, this would have led to an underestimation of the effect of sepsis and thus not compromise our findings.

Our findings shed new light on the contribution of mitochondria in the pathophysiology of sepsis-AKI. We reveal that sepsis induces oxidation of nuclear and mitochondrial DNA and mtDNA damage, without signs of upregulation of mitochondrial quality control mechanisms, resulting in a reduced mitochondrial mass in the septic kidney. These findings are of clinical relevance, as sepsis is the major cause of AKI and death in critically ill patients. Dissecting the molecular mechanisms leading to mitochondrial dysfunction in sepsis-AKI is crucial for the development of novel targeted therapies to prevent or treat sepsis-AKI and potentially improve the survival. Since it is not known whether mtDNA damage is repaired after survival from sepsis, our findings might also be of relevance for long-term outcomes of sepsis. Given the role of mitochondria in the pathophysiology of sepsis-AKI, pharmacologic strategies directed at maintaining mitochondrial function, limiting oxidative stress and mtDNA damage, or enhancing mitochondrial quality control to ameliorate mitochondrial damage, might successfully prevent or halt AKI in sepsis.