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

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

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

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

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

[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

[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

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

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

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].

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

“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.”

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: 0.1038/kisup.2012.1; MacLeod A. NCEPOD report on acute kidney injury- must do better. Lancet 2009; 374: 1405-1406