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).
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.
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).
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