Discovery and validation of cell cycle arrest biomarkers in human acute kidney injury

Abstract

Introduction: Acute kidney injury (AKI) can evolve quickly and clinical measures of function often fail to detect AKI at a time when interventions are likely to provide benefit. Identifying early markers of kidney damage has been difficult due to the complex nature of human AKI, in which multiple etiologies exist. The objective of this study was to identify and validate novel biomarkers of AKI.

Methods: We performed two multicenter observational studies in critically ill patients at risk for AKI - discovery and validation. The top two markers from discovery were validated in a second study (Sapphire) and compared to a number of previously described biomarkers. In the discovery phase, we enrolled 522 adults in three distinct cohorts including patients with sepsis, shock, major surgery, and trauma and examined over 300 markers. In the Sapphire validation study, we enrolled 744 adult subjects with critical illness and without evidence of AKI at enrollment; the final analysis cohort was a heterogeneous sample of 728 critically ill patients. The primary endpoint was moderate to severe AKI (KDIGO stage 2 to 3) within 12 hours of sample collection.

Results: Moderate to severe AKI occurred in 14% of Sapphire subjects. The two top biomarkers from discovery were validated. Urine insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2), both inducers of G1 cell cycle arrest, a key mechanism implicated in AKI, together demonstrated an AUC of 0.80 (0.76 and 0.79 alone). Urine [TIMP-2]·[IGFBP7] was significantly superior to all previously described markers of AKI (P <0.002), none of which achieved an AUC >0.72. Furthermore, [TIMP-2]·[IGFBP7] significantly improved risk stratification when added to a nine-variable clinical model when analyzed using Cox proportional hazards model, generalized estimating equation, integrated discrimination improvement or net reclassification improvement. Finally, in sensitivity analyses [TIMP-2]·[IGFBP7] remained significant and superior to all other markers regardless of changes in reference creatinine method.

Conclusions: Two novel markers for AKI have been identified and validated in independent multicenter cohorts. Both markers are superior to existing markers, provide additional information over clinical variables and add mechanistic insight into AKI.

Trial registration: ClinicalTrials.gov number NCT01209169.

Figure 1

Study design and number of patients in cohorts. ¹Risk factors included sepsis, hypotension, major trauma, hemorrhage, radiocontrast exposure, or major surgery or requirement for ICU admission. All enrolled patients were in the ICU. ²Risk factors included hypotension, sepsis, IV antibiotics, radiocontrast exposure, increased intra-abdominal pressure with acute decompensated heart failure, or severe trauma as the primary reason for ICU admission and likely to be in the ICU for 48 hours. ³Critical illness was defined as admission to an ICU and sepsis-related organ failure assessment (SOFA) score [32] ≥2 for respiratory or ≥1 for cardiovascular. ⁴Initially patients with acute kidney injury (AKI) stage 1 were also excluded but this was changed at the first protocol amendment. ⁵A total of 728 patients had test results for urinary biomarkers. A total of 726 patients had test results for plasma biomarkers.

Figure 2

Area under the receiver-operating characteristics curve (AUC) for novel urinary biomarkers and existing biomarkers of acute kidney injury for the primary Sapphire study endpoint (KDIGO stage 2 or 3 within 12 hours of sample collection). Samples were collected within 18 hours of enrollment. The AUC for urinary [TIMP-2]·[IGFBP7] is larger than for the existing biomarkers (P value <0.002). IGFBP7, insulin-like growth factor-binding protein 7; IL-18, interleukin-18; KIM-1, kidney injury marker-1; L-FABP, liver fatty acid-binding protein; NGAL, neutrophil gelatinase-associated lipocalin; pi-GST, pi-Glutathione S-transferase; TIMP-2, tissue inhibitor of metalloproteinases-2.

Figure 3

Discrimination between non-AKI conditions and AKI of different severities for (A) urine [TIMP-2]·[IGFBP7], (B) urine NGAL, and (C) urine KIM-1. Open boxes represent Sapphire subjects who did not have AKI (of any stage) within seven days. Shaded boxes represent Sapphire subjects stratified by maximum AKI stage within 12 hours of sample collection. Boxes and whiskers show interquartile ranges and total observed ranges (censored by 1.5 times the box range), respectively. Samples were collected within 18 hours of enrollment. AKI, acute kidney injury; IGFBP7, insulin-like growth factor-binding protein 7; KIM-1, kidney injury marker-1; NGAL, neutrophil gelatinase-associated lipocalin; TIMP-2, tissue inhibitor of metalloproteinases-2.

Figure 4

Risk for KDIGO stage 2 to 3 AKI (A) and MAKE₃₀ (B) as a function of urine [TIMP-2]·[IGFBP7]. Risk at each [TIMP-2]·[IGFBP7] value along the abscissa was calculated as follows: the number of samples positive for the endpoint that had [TIMP-2]·[IGFBP7] above the abscissa value divided by the total number of samples that had [TIMP-2]·[IGFBP7] above the abscissa value. Slightly more than 50% of the samples had a [TIMP-2]·[IGFBP7] value above 0.3 where risk began to elevate sharply and about 10% of the samples had a [TIMP-2]·[IGFBP7] value above 2.0 where risk almost doubled and quintupled for MAKE₃₀ and AKI, respectively. AKI, acute kidney injury; IGFBP7, insulin-like growth factor-binding protein 7; TIMP-2, tissue inhibitor of metalloproteinases-2.

Figure 5

Proposed mechanistic involvement of the novel biomarkers in AKI: initial tubular cells sustain injury by various insults. In response to DNA and possibly other forms of damage, IGFBP7 and TIMP-2 are expressed in the tubular cells. IGFBP7 directly increases the expression of p53 and p21 and TIMP-2 stimulates p27 expression. These effects are conducted in an autocrine and paracrine manner via IGFBP7 and TIMP-2 receptors. The p proteins in turn, block the effect of the cyclin-dependent protein kinase complexes (CyclD-CDK4 and CyclE-CDK2) on the cell cycle promotion, thereby resulting in G₁ cell cycle arrest for short periods of time presumably to avoid cells with possible damage from dividing. AKI, acute kidney injury; IGFBP7, insulin-like growth factor-binding protein 7; TIMP-2, tissue inhibitor of metalloproteinases-2.