Serum amylase levels are commonly used to detect pancreatic injury. Amylase levels were slightly above normal 6 h after CLP in both Live-P and Die-P mice (Fig. 3). However, at 24 h, there was a significant increase in amylase levels for the Die-P mice. Because the levels were only 60% higher than normal, this probably indicates a statistically significant but biologically irrelevant pancreatic injury, with much higher levels being typically detected in the presence of severe pancreatitis.
Kidney injury has been traditionally detected by measurements of BUN and creatinine (27). At 6 h after CLP, the average levels of BUN for Live-P mice were slightly above the upper limit of normal (Fig. 4A). In Die-P at the same time point, they were significantly higher than the Live-P values and were almost 2-fold above normal. By 24 h, the BUN levels returned to normal in Live-P mice, but they continued to rise in Die-P mice to a level almost five times higher than Live-P at the same time point. These changes in BUN are similar to the alterations observed in plasma levels of IL-6 (25, 28). We compared BUN and IL-6 and demonstrated a close correlation between the two measurements (Fig. 4B).
Interleukin 6 levels predict whether mice will die before the end of day 5 after CLP (23), but the exact day of death is still uncertain. To measure the BUN levels closer to the time of death, mice underwent CLP and then had blood samples collected daily until death or the end of the experiment on day 5 after CLP. The BUN levels recorded within the last 24 h of life (last sample collected before death) for the animals that succumbed to sepsis (Dead by day 5) were above normal and significantly higher than the levels recorded at corresponding time points in mice that survived past day 5 (Fig. 5A).
The considerable difference between Live-P and Die-P murine BUN levels at 24 h after CLP observed in Figure 4 suggests that BUN may be able to predict sepsis survival. To test this idea, CLP was performed and animals were followed for survival until after day 5 and retrospectively separated into two groups: Dead by day 5 and Alive after day 5. Note that these groups were not the same as those predicted to live or die based on plasma IL-6. The BUN levels were measured in samples collected at 24 h after CLP from these mice (Fig. 5B). The mean BUN levels recorded were virtually identical to the BUN levels in mice separated based on IL-6 survival prediction in Figure 4 (Alive after day 5 versus Live-P and Dead by day 5 versus Die-P). A receiver-operator characteristic curve analysis was performed based on the data in Figure 5, and the area under the curve was 0.96, indicating excellent prediction capability (Fig. 5C). A cutoff level of 44 mg/dL predicted the outcome, with 92% sensitivity and 85% specificity. Mice with BUN levels higher than 44 mg/dL had a relative risk for mortality of 17.6 (95% confidence interval, 4.5–69.4) compared with the mice with lower levels. The data in Figures 4 and 5 indicate that kidney injury develops in the first 24 h of the evolution of sepsis in mice that will succumb to the disease.
A more specific marker of kidney function, cystatin C, was also measured at 24 h after CLP. Within 24 h, the cystatin C levels of both Live-P and Die-P mice were higher than those in normal mice (Fig. 6A). The difference between the Live-P and Die-P was small but statistically significant. Levels of this biomarker are considered to increase early in the development of kidney injury (27), and this small increase at 24 h could be exacerbated closer to death in Die-P mice if development of injury is a fast preterminal event.
To test if increased kidney injury develops closer to death in septic animals, mice were followed for survival until after day 5 after CLP and had their daily blood samples used to measure cystatin C (same mice as in Fig. 5A). The cystatin C levels were measured in samples collected within 24 h of death from mice that succumbed during the first 5 days after CLP and compared with levels measured at matched time points in mice that survived until after day 5. As seen in Figure 6B, the mean of the cystatin C levels of the survivors was similar to normal value. The nonsurvivors, however, had significantly higher levels than the survivors and normal mice. The mean for the levels in Dead by day 5 was 2.4 times higher than the mean of the normal mice. Taken together, the data presented in Figure 6 indicate that more significant kidney injury develops closer to death in septic mice, particularly in the last 24 h.
Liver, pancreas, spleen, and kidney injuries were evaluated in the murine CLP model of sepsis to determine if their presence could explain the pathophysiology of why some animals die while others survive. The histology of liver, pancreas, and kidney tissues was unremarkable 24 or 48 h into the evolution of sepsis for both mice predicted to die and survive. Other than changes such as lymphocyte apoptosis in the spleens of both groups, the lack of histological findings is consistent with the situation in human sepsis (26) and confirms CLP as a model for sepsis research that more closely reflects the human pathology. It should be noted that the current study compared the histology of septic mice predicted to die with that of septic mice predicted to live, whereas most previous studies compare septic mice with normal mice. The similarities between the CLP model of sepsis and human sepsis are shown in the Table, Supplemental Digital Content 2, at http://links.lww.com/SHK/A188. Our group has also recently published that there is no significant lung injury or dysfunction in the murine CLP model of sepsis (6). In the current study, some biomarker measurements of organ injury were elevated, particularly at 24 h after CLP in the mice predicted to die. Biologically significant injury, however, was only found for the kidneys, especially within the last 24 h of life for the animals that died after CLP. Although the association of sepsis and AKI is clearly established (8), our findings highlight the importance and timing of kidney injury in the murine CLP sepsis model.
Increased levels of AST, amylase, BUN, and cystatin C indicate a level of multiple organ injury. Two alternate hypotheses exist to explain the findings. Multiple small insults to different organ systems could act in combination/synergy to produce lethality in murine sepsis. However, the levels measured would not be expected to indicate substantial alterations in organ function. More likely, intra-abdominal sepsis causes a mild degree of multiple organ damage but not overt failure of the liver, pancreas, or kidney at this time point. In models of acetaminophen-induced acute liver injury, AST levels of 15,000 U/L were reported at 24 h, representing more than a 50-fold increase over those seen in our septic mice (29). Amylase in lethal acute pancreatitis measured more than 12,000 U/L, higher than an 8-fold elevation compared with our CLP mice (30). In our current study, BUN levels of 90 mg/dL approached those seen in a lethal model of renal ischemia-reperfusion injury of about 98 mg/dL at 24 h (31). There may be progressive organ injury closer to death, and the measurements for cystatin C indicated that more significant kidney injury did develop in 24 h before death in nonsurviving mice. Obtaining histological evidence for this increased kidney injury is not feasible with current methods. The prediction based on plasma IL-6 levels indicates whether the animal will die in the first 5 days after CLP but not the exact moment.
The pancreatic blood supply seems to be affected earlier than that of other organs in sepsis (32), raising the possibility of increased injury. A small elevation in amylase levels was detected for Die-P mice at 24 h, indicative of only limited injury at this time point in the evolution of sepsis (33). The levels reached are consistent with previously described findings in sepsis studies using the murine CLP model (34). Similarly, the AST levels suggest only mild liver injury at 24 h and only slightly worse in mice predicted to die. A recent study does show an increasing trend in AST levels until 48 h after CLP in nonsurvivors, but this was in the setting of a double-hit model (sepsis after trauma/hemorrhage) (35). Even though closer to death, the level of kidney injury seems significant, because 24 h before death, there was a substantial increase compared with those of mice that survived, as indicated by cystatin C levels in this study. Examining these organs together, it is apparent that a small degree of multiple organ injury manifests at 24 h into the progression of CLP sepsis, particularly in the animals that were going to die. Because limited blood volume can be collected in our nonlethal serial sampling technique, daily monitoring of AST and amylase levels was not possible with our techniques. Consequently, we could not test if the progression to overt kidney injury in dying mice is also associated with severe liver and pancreas injury throughout the acute phase.
Historically, serum creatinine has been the most frequently used endogenous marker for estimating the glomerular filtration rate (GFR). However, it is not considered ideal because the levels are influenced by factors, such as diet, muscle mass, sex, race, and age; it is actively secreted by the proximal tubules that can lead to a 10% to 40% overestimation of the GFR; and it does not work well for detecting smaller decreases, that is, less than 40% of GFR (36). Cystatin C, a 13-kd endogenous cystein proteinase inhibitor, has recently been proposed to be a better marker for GFR, and two meta-analyses that looked at more than 100 studies comparing it with serum creatinine have confirmed this (37, 38). Cystatin C has previously been used to diagnose AKI in the CLP model (39). It is constantly produced by a housekeeping-type gene in all nucleated cells, without being affected by muscle mass, sex, or age. It is freely filtered by the glomeruli and not secreted by tubular cells, but it is reabsorbed and fully catabolized by proximal renal tubular cells. Serum cystatin C has been shown to be the best for early detection of AKI or for diagnosis of already established AKI (27). Cystatin C has also been shown to be a more sensitive and earlier marker for renal dysfunction than BUN and creatinine in mouse models of AKI (40).
Some of the formulas based on cystatin C indicate a linear relation between GFR and the reciprocal of serum cystatin C (41). This indicates that the increase in cystatin C levels seen in this study for septic mice within 24 h of death would correspond to a decrease of more than 50% in GFR, indicative of injury by the RIFLE classification criteria (42). Although this study did not attempt to uncover mechanisms of kidney injury in sepsis, a better understanding of the moment when it happens after CLP should help future mechanistic studies. A 2010 report of cystatin C levels in CLP sepsis found no difference between sham- and CLP-operated rats, but the values obtained were about 10 times lower than in our study (43). This could be an interspecies difference because our levels are more in line with previous reports of kidney injury in mice (39, 40) or caused by a difference in illness severity modulated by the lethality of the septic insult.
As a marker of kidney function, BUN has been considered to be even less specific than creatinine because its levels are affected, apart from glomerular filtration, by tubular reabsorption and also by the rate of its production, which in turn is influenced by protein intake, tissue protein catabolism, gastrointestinal bleeding, and corticosteroid therapy. As such, urea nitrogen is a reflection of many simultaneous pathological processes. The timing of some of these processes in CLP sepsis could explain why the BUN values measured in the last 24 h of life decrease when compared with those measured at 24 h after CLP, while the more specific marker of kidney function, cystatin C, increased in the same time span. Blood urea nitrogen levels higher than 40 mg/dL on admission to the intensive care unit were associated with increased mortality in critically ill patients, particularly at 30 days but even up to a year (44). The same BUN levels were also associated with a higher risk of positive blood cultures. From the data obtained in our study, a level of 44 mg/dL at 24 h after CLP was highly predictive of 5-day mortality, indicating that BUN could be useful as a biomarker for sepsis mortality. In addition, there was a close correlation of BUN and IL-6 (a well-established biomarker for sepsis mortality).
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