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

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A Rigorous Discovery-Validation Path Was Taken

Discovery Study340 proteins analyzed

(including KIM-1, urine NGAL, plasma NGAL, Cystatin-C, IL-18,

pi-GST, and L-FABP)

Validation StudyPrimary Endpoint: moderate to

severe AKI (KDIGO stage 2-3) within 12 hours of sample collection

(based on serum creatinine and hourly urine output)

Sapphire Study35 sites

(20 North America, 15 Europe)Age > 21, Critically Ill3, no AKI (Stage 2 or 3)4

N = 744

Vienna CohortAge > 18,

in ICU + SepsisN = 134

Duke CohortAge > 18,

At least 1 risk factor1

N = 123

Mayo CohortAge > 18,

At least 1 risk factor2

N = 265

N = 7285

No AKIN = 416

AKI Stage 1N = 211

AKI Stage 2N = 83

AKI Stage 3N = 18

16 patients excluded (2 withdrew consent, 7

lost to follow-up, 7 with invalid or missing test

results)

Best Two Markers

Dis

cove

ryV

alid

atio

n

Within12 hrs

Kashani et al. Critical Care 2013, 17:R25

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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

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TIMP-2 and IGFBP7 Outperform Existing Biomarkers

AUC for [TIMP-2]•[IGFBP7] was significantly greater than any existing biomarkers

Kashani et al. Critical Care 2013, 17:R25

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TIMP-2 and IGFBP7 Work Well In Important Subgroups

Sepsis Surgery

Kashani et al. Critical Care 2013, 17:R25

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[TIMP-2]•[IGFBP7] Has a Compelling Specificity Profile

Urine NGAL (ng/mL) [TIMP-2]•[IGFBP7] ((ng/mL)2 / 1000)

Kashani et al. Critical Care 2013, 17:R25

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[TIMP-2]•[IGFBP7] Has a Compelling Specificity Profile

Urine KIM-1 (ng/mL) [TIMP-2]•[IGFBP7] ((ng/mL)2 / 1000)

Kashani et al. Critical Care 2013, 17:R25

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MAKE30 Analysis Shows TIMP-2 and IGFBP7 Are Clinically Meaningful Biomarkers

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Risk

for A

KI (K

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-3)

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[TIMP-2]•[IGFBP7] ((ng/mL)²/1000)

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KDIGO 2-3 in 12h

MAKE30MAKE30 (30 days)DeathRRTPersistently elevated sCr (2x over baseline)

Kashani et al. Critical Care 2013, 17:R25

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TIMP-2 and IGFBP7 Have Compelling Mechanistic Origins in Early Cellular Injury

Proposed mechanismIGFBP7 & 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

These processes and marker signals occur early enough in injury to take action

Kashani et al. Critical Care 2013, 17:R25

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TIMP-2 and IGFBP7 Remain Highly Significant In Clinical Models With Risk Factors

VariableP-value

(Cox PH Model)P-value

(GEE Model)[TIMP-2]•[IGFBP7] <0.0001 <0.0001

Age 0.35 0.32

APACHE III Score 0.35 0.067

Hypertension 0.004 0.013

Nephrotoxic drugs 0.12 0.013

Liver Disease 0.069 0.057

Sepsis 0.32 0.64

Diabetes 0.29 0.35

Chronic Kidney Disease 0.27 0.64

Serum Creatinine <0.0001 <0.0001

C-stat (AUC) 0.87 0.87

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].

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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

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“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 appropriatetriage 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.”

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KDIGO Consensus Guideline for AKI

KDIGO: Kidney Disease Improving Global Outcomes; Kidney International Supplements (2012) 2, 1; doi: 0.1038/kisup.2012.1; MacLeod A. NCEPOD report on acute kidney injury- must do better. Lancet 2009; 374: 1405-1406

Actions recommended to start when patients are at high risk…

…But NO available method to reliably identify high risk to aid clinicaljudgment …often resulting in

failure to initiate kidney-sparing management strategies

….Why Risk Assessment Is Needed and What To Do For a Positive Test Result

KDIGO Management Options