1. ©2013 Astute Medical, Inc. PN 0138 Rev B 2013/03/19

2. A Rigorous Discovery-Validation Path Was Taken
Discovery Study
340 proteins analyzed
(including KIM-1, urine NGAL, plasma NGAL, Cystatin-C, IL-18, pi-GST, and L-FABP)
Validation Study
Primary Endpoint: moderate to severe AKI (KDIGO stage 2-3) within 12 hours of
sample collection
(based on serum creatinine and hourly urine output)
Kashani et al. Critical Care 2013, 17:R25

3. Revolutionary Biomarkers Were Identified
Biomarkers identified through hypotheses based on AKI pathophysiology
340 candidate biomarkers identified
Biomarkers ranked by ability to predict development of AKI RIFLE I or F within 12 to 36 hours
All possible combinations of two-four biomarkers (novel or previously described) were ranked
Top performing biomarkers identified
Tissue Inhibitor of Metalloproteinases-2 (TIMP-2)
Insulin-like Growth Factor Binding-Protein 7 (IGFBP7)
Kashani et al. Critical Care 2013, 17:R25

4. TIMP-2 and IGFBP7 Outperform Existing Biomarkers
FIGURE 2 from publication
[TIMP-2]•[IGFBP7] exhibited an AUC of 0.80 for development of AKI (stage 2 or 3) within 12 hours and alone IGFBP7 and TIMP-2 each exhibited an AUC of 0.76 and 0.79 respectively.
The AUC for urine [TIMP-2]•[IGFBP7] was significantly greater (p<0.002) than any of these existing biomarkers.
AUC for [TIMP-2]•[IGFBP7] was significantly greater than any existing biomarkers
Kashani et al. Critical Care 2013, 17:R25

5. TIMP-2 and IGFBP7 Work Well In Important Subgroups
Sepsis
Surgery
Kashani et al. Critical Care 2013, 17:R25

6. [TIMP-2]•[IGFBP7] Has a Compelling Specificity Profile
Urine NGAL (ng/mL)
[TIMP-2]•[IGFBP7] ((ng/mL)2 / 1000)
FIGURE 3 from publication
In terms of discrimination between AKI of different severities and various non-AKI conditions including chronic kidney disease, [TIMP-2]•[IGFBP7] showed clear separation between AKI and non-AKI conditions.
Kashani et al. Critical Care 2013, 17:R25

7. [TIMP-2]•[IGFBP7] Has a Compelling Specificity Profile
Urine KIM-1 (ng/mL)
[TIMP-2]•[IGFBP7] ((ng/mL)2 / 1000)
FIGURE 3 from publication
In terms of discrimination between AKI of different severities and various non-AKI conditions including chronic kidney disease, [TIMP-2]•[IGFBP7] showed clear separation between AKI and non-AKI conditions.
Kashani et al. Critical Care 2013, 17:R25

8. MAKE30 Analysis Shows TIMP-2 and IGFBP7 Are Clinically Meaningful Biomarkers
KDIGO 2-3 in 12h
MAKE30 (30 days)
Death
RRT
Persistently elevated sCr
(2x over baseline)
MAKE30
FIGURE 4 in publication
Risk of AKI (KDIGO stage 2-3 within 12 hours) and MAKE30 elevated sharply for [TIMP-2]•[IGFBP7] above 0.3 and almost quintupled and doubled, respectively, for [TIMP-2]•[IGFBP7] above 2.0.
Kashani et al. Critical Care 2013, 17:R25

9. TIMP-2 and IGFBP7 Have Compelling Mechanistic Origins in Early Cellular Injury
Proposed mechanism
IGFBP7 & TIMP-2 are markers of G1 cell cycle arrest during early cell injury
Renal tubular cells enter a short period of G1 cell-cycle arrest following injury
G1 cell-cycle arrest presumably prevents the cells from dividing when the DNA may be damaged
IGFBP7 & TIMP-2 may also signal in autocrine and paracrine fashions, spreading ‘alarm’ from the site of injury
Both markers may be involved in G1 cell cycle arrest and may signal that the renal epithelium has been stressed and has shut down function but may still be able to recover without permanent injury to the organ.
Importantly, TIMP-2 and IGFBP7 appear to be able to signal in autocrine and paracrine fashions, thus spreading the “alarm” from the site of injury.
TIMP-2 and IGFBP7 are known to be involed in the response to a wide variety of insults.
FIGURE 5 in publication
These processes and marker signals occur early enough in injury to take action
Kashani et al. Critical Care 2013, 17:R25

10. TIMP-2 and IGFBP7 Remain Highly Significant In Clinical Models With Risk Factors
Variable
P-value
(Cox PH Model)
(GEE Model)
[TIMP-2]•[IGFBP7]
<0.0001
Age
0.35
0.32
APACHE III Score
0.067
Hypertension
0.004
0.013
Nephrotoxic drugs
0.12
Liver Disease
0.069
0.057
Sepsis
0.64
Diabetes
0.29
Chronic Kidney Disease
0.27
Serum Creatinine
C-stat (AUC)
0.87
TABLE S4 & S5 IN ADDITIONAL FILE 1 of PUBLICATION.
[TIMP-2]•[IGFBP7] significantly improved risk prediction when added to a nine-parameter clinical model (including serum creatinine at matched time points with biomarkers) for the primary endpoint, using time to event, IDI, cfNRI and risk assessment plot analyses. All analyses showed significant enhancement by the addition of [TIMP-2]•[IGFBP7] with [TIMP-2]•[IGFBP7] remaining strongly associated with AKI in all models.
Endpoint was KDIGO 2-3 (RIFLE I/F) within 12 hours. All clinical risk factors with univariate p-value ≤ 0.1 for predicting the endpoint were included in the models. Net reclassification and integrated discrimination improvement analyses were also performed and showed significant enhancement of the models by [TIMP-2][IGFBP7].

11. Key Messages
Both IGFBP7 and TIMP-2 are inducers of G1 cell cycle arrest, a key mechanism implicated in AKI
IGFBP7 and TIMP-2 are new biomarkers for AKI and perform better than existing biomarkers for predicting the development of moderate or severe AKI (KDIGO stage 2 or 3) within 12 hours of sample collection
[TIMP-2]•[IGFBP7] significantly improved risk stratification when analyzed using Cox proportional hazards model, generalized estimating equation, integrated discrimination improvement or net reclassification improvement
Risk for major adverse kidney events within 30 days (MAKE30) elevated sharply above [TIMP-2]•[IGFBP7] values > 0.3 and doubled when [TIMP-2]•[IGFBP7] values were > 2.0
Kashani et al. Critical Care 2013, 17:R25

12. “An intriguing implication of this study is that, because both of these new markers can be upregulated in response to a wide range of noxious stimuli, they have the potential to be a nonspecific alarm raised by the renal tubules in response to stress.
Detecting this alarm will permit several things to happen, including appropriate
triage of patients, more intensive monitoring, and perhaps early involvement from specialists in nephrology and critical care who can promptly evaluate these patients while they are still in the golden hours of this disease prior to irreversible damage to the kidneys.”

13. KDIGO Management Options
….Why Risk Assessment Is Needed and What To Do For a Positive Test Result
KDIGO Consensus Guideline for AKI
Actions recommended to start when patients are at high risk…
…But NO available method to reliably identify high risk to aid clinical judgment
…often resulting in failure to initiate kidney-sparing management strategies
KDIGO: Kidney Disease Improving Global Outcomes; Kidney International Supplements (2012) 2, 1; doi: /kisup ; MacLeod A. NCEPOD report on acute kidney injury- must do better. Lancet 2009; 374: