We have demonstrated that microarray data can be leveraged to identify gene expression patterns common in SSAKI. Although mRNA levels do not necessarily correlate with protein expression, analysis of MMP-8 and elastase-2 protein levels in the serum compartment indicates that our gene expression data can identify novel serum protein biomarkers for SSAKI.
Although sepsis is the most important predictor of AKI in critically ill patients [37
], few clinical studies are dedicated to identifying the early phenotype of patients with SSAKI. Interrogation of several large databases of critically ill adult patients has yielded variables that may be predictive of SSAKI but are not consistently reliable (Table ). The conclusions of these studies simply state that elevated proinflammatory markers portend adverse outcomes in acute kidney disease and death in patients with septic shock [39
Variables predictive of SSAKI in selected major adult trials
NGAL has received considerable attention as a potential biomarker for AKI. NGAL derivation and validation studies were primarily performed in ischemic or nephrotoxic AKI with great efficacy [13
], while investigations of NGAL biomarker utility in SSAKI have demonstrated variable results. In the general ICU population, serum NGAL levels have not been reliably demonstrated to be specific for SSAKI (Table ). We previously published that while day 1 serum NGAL levels were elevated, with acceptable sensitivity, in children who develop SSAKI, the specificity was quite poor (39%) [41
]. A recent prospective cohort study of 632 adult ICU patients demonstrated that the likelihood ratio for AKI (RIFLE Failure) was 1.71 for NGAL, and that elevated NGAL alone conferred an ROC area under curve of 0.77 for RIFLE Failure but when included in a 'most efficient clinical model' NGAL improved the ROC area under curve for RIFLE Failure to 0.96 [42
]. OK In this study, specificity was 50% for a plasma NGAL cut-off value of 168 ng/ml. Emergency room serum NGAL levels >150 ng/ml were highly sensitive for AKI within 72 hours (96%), but specificity was only 51% [43
]. NGAL is known to be released by activated neutrophils and appears to be intrinsically elevated in sepsis, which may explain its limitation as a biomarker specific for SSAKI.
A variable that may complicate delineation of biomarkers for SSAKI is that the pathophysiology of SSAKI may be different from that of ischemic or nephrotoxic AKI. Although acute tubular necrosis has traditionally been considered the etiology of AKI in sepsis, no conclusive pathologic evidence has demonstrated that this is true [18
]. Animal models of SSAKI demonstrate increased, rather than decreased, renal blood flow and a state of hyperdynamic AKI [44
]. The mechanisms mediating disease progression are multifactorial, and AKI during sepsis may be a result of acute tubular apoptosis
rather than acute tubular necrosis
]. Nonhemodynamic-dependent mechanisms of injury during sepsis, inflammatory and immunologic, may trigger this apoptotic response. This pathophysiology has probably decreased the utility of traditional tests based on the functionality of proximal tubular filtration [45
Our study demonstrates that microarray-derived gene expression data can provide a tool for discovery of candidate biomarkers for SSAKI. Outside the neonatal population, we are the first group to attempt to characterize biomarkers specific to SSAKI (and not all-cause AKI) in children. Our data represent the first 24 hours of presentation to medical care, a timeframe that can be considered a therapeutic window for acute intervention. It is important to note that our patients were restricted to having septic shock, an exclusion criterion not previously used in pediatric studies investigating AKI biomarkers. Additionally, our definition of SSAKI identifies patients with severe, persistent kidney injury up to day 7. These candidate biomarkers thus identify patients with resuscitation unresponsive AKI (that is, patients having high serum creatinine levels at admission who subsequently did not normalize with standard resuscitation). The expression patterns of the 21 upregulated gene probes demonstrate excellent sensitivity and good specificity for SSAKI (Figure ). The negative predictive value of nearly 100% carries obvious import to the bedside practitioner, but should be tempered by the fact that the prevalence of AKI in our cohort was relatively low (31/179, 17%). Individual performance calculations of two of these gene products, MMP-8 and elastase-2, demonstrate reasonable sensitivity in the derivation cohort (Table ), but even higher sensitivity in the validation cohort (Table ). Additionally, in the validation cohort, the specificity and negative predictive values of MMP-8 and elastase-2 increased further. Although MMP-8 and elastase-2 will require further prospective validation studies for confirmation, the results are a relevant demonstration that our gene expression methodology can successfully be leveraged against the tremendous heterogeneity in patients with SSAKI.
The biological links between MMP-8, elastase-2, and SSAKI are unclear at the present time. MMP-8 is a collagen-cleaving endopeptidase, secreted by neutrophils, involved in extracellular matrix remodeling. MMP-8 is implicated in the pathogenesis of numerous rheumatologic diseases and has been demonstrated as a biomarker for disease processes ranging from odontogenic inflammation, to left ventricular remodeling after myocardial infarction, to preterm labor [46
]. We have recently identified that MMP-8 may be important in the pathogenesis of septic shock [49
], and Hu and colleagues demonstrated that the use of an MMP-8 inhibitor protects mice against endotoxic shock [50
]. There are no previous reports of MMP-8 involvement in the progression of kidney injury. Neutrophil elastase (elastase-2) is a serine protease secreted by neutrophils in response to bacterial infection, is a marker of inflammation, and has been documented as a biomarker of neonatal sepsis [51
]. Interestingly, inhibition of neutrophil elastase reduced progressive renal injury seen in experimental direct endotoxin-induced AKI [52
Our study has several limitations. Since in many cases neither a baseline creatinine nor a height was available, the definition of SSAKI in our population was based on a referenced median-for-age creatinine [34
]. Importantly, the initial pediatric RIFLE study also identified AKI in patients based on assumed
estimated creatinine clearance when baseline creatinine levels were unavailable [53
]. Second, some of the performance characteristics (negative and positive predictive values) are influenced by the relatively low prevalence of AKI in our cohort. This is probably secondary to the stringent a priori
definition used for SSAKI. The intentional use of such a definition allowed us to identify the most severely affected patients with fluid-unresponsive AKI [22
]. Third, the data for MMP-8 and elastase-2 continue to demonstrate the relatively low specificity that many candidate biomarkers carry for SSAKI. Still, when all 21 upregulated gene probes are analyzed using the leave-one-out procedure, the specificity increases to 80%, a level not yet seen for SSAKI biomarkers. Fourth, neither MMP-8 nor elastase-2 has been studied formally in AKI disease pathogenesis, from sepsis or other causes. Since MMP-8 and elastase-2 are neutrophil-derived proteases, it is tempting to speculate that they contribute to neutrophil tissue invasion and end-organ damage. In support of such speculation, our microarray data indicate that septic shock is associated with increased expression of several basement membrane-related genes (for example, laminin-family genes) that contribute to neutrophil diapedesis. Finally, our approach relies on extrapolation of mRNA expression data for the derivation of serum protein-based biomarkers. This can be problematic in that mRNA expression does not always correlate with protein expression. What will be required in the future is exploration into the feasibility of measuring the other candidate biomarkers, such as IFNα-inducible protein 27, which demonstrated a more than twofold mRNA expression level between patients with and without SSAKI.