1. Development of an Immunoassay for the Kidney-Specific Protein myo-Inositol Oxygenase, a Potential Biomarker of Acute Kidney Injury J.P. Gaut, D.L. Crimmins, M.F. Ohlendorf, C.M. Lockwood, T. A. Griest, N.A. Brada, M. Hoshi, B. Sato, R.S. Hotchkiss, S. Jain, and J.H. Ladenson May 2014 www.clinchem.org/content/60/5/747.full © Copyright 2014 by the American Association for Clinical Chemistry

2. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  Acute kidney injury (AKI) is a common and significant problem  Affects 45% of critically ill and 20% of hospitalized patients, resulting in increased morbidity and mortality  Even mild AKI increases risk of chronic kidney disease  The gold standard for diagnosis, creatinine and urine output, is nonspecific and insensitive  Depends of muscle mass, diet, and liver function  May not change until 50% of renal function is lost

3. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  Early and reliable AKI biomarkers are needed Since structural changes precede functional loss in most AKI cases, structural proteins may be most sensitive to damage  Structural biomarkers upregulated after injury  Kidney injury molecule-1, neutrophil gelatinase associated lipocalin, interleukin-18, tissue inhibitor of metalloproteinase-2, and insulin-like growth factor-binding protein-7  Intrinsic structural biomarkers of AKI  Alkaline phosphatase,  -glutathione-S-transferase,  -glutamyltranspeptidase, and N-acetyl-  -glucosaminidase

4. © Copyright 2009 by the American Association for Clinical Chemistry Question  What is the ideal acute kidney injury biomarker?

5. © Copyright 2009 by the American Association for Clinical Chemistry Materials & Methods  Identification of renal specific genes  Genechip array (Affymetrix) analyis was performed on brain, liver, spleen, kidney, skeletal muscle, lung, pancreas, heart, and small intestine dissected from three C57Bl/6 mice.  These data were mined for genes with mean difference values >10,000 in the kidney, expressed at >10-fold amounts in kidney relative to other tissues, and expressed in the proximal tubule.  Anti-myo-inositol oxygenase (Anti-MIOX) antibodies  Rabbit polyclonal and mouse monoclonal antibodies were generated to recombinant human GST-MIOX.  Recombinant MIOX was confirmed using N-terminal Edman sequencing.

6. © Copyright 2009 by the American Association for Clinical Chemistry Materials & Methods  MIOX Immunoassay  A sandwich immunoassay was developed for MIOX using two distinct mouse monoclonal antibodies and detected using an electrochemiluminescent detection system.  Recombinant human GST-MIOX was used to generate standard curves.  Mouse Model  8-12 week old C57Bl/6 mice were subjected to bilateral renal ischemia for 30 min.  One week prior to surgery, serum was collected and frozen at -80°C.  Two sham-operated animals underwent an identical procedure without vascular pedicle clamping.  24 hr following surgery, serum and kidneys were collected.

7. © Copyright 2009 by the American Association for Clinical Chemistry Materials & Methods  Human Patients  Critically ill adult patients were screened for increases in plasma creatinine occurring over 24-72 hr.  Patients were also screened for stable serum creatinine over 72 hr.  Patients with chronic kidney disease or who did not have Foley catheters were not included.  Remnant plasma samples were obtained before increases in plasma creatinine (time 0) and at the time of the creatinine increase (time 54).  One representative sample was retrieved for stable creatinine patients.  Patient medical records were reviewed for demographic information, urine output, and diagnosis.  Oliguria was defined as a urine output <0.5 mL/kg/h for at least 6 hr.

8. © Copyright 2009 by the American Association for Clinical Chemistry Question  What are the advantages and disadvantages of studying a biomarker using remnant samples?

9. © Copyright 2009 by the American Association for Clinical Chemistry Figure 1. (A) Miox mRNA expression in mouse tissues. (B) MIOX expression in human tissue homogenates (50  g protein/lane) with a rabbit polyclonal anti-MIOX antibody. The observed protein migrates between carbonic anhydrase (CA, 29 kDa) and ovalbumin (OVA, 45 kDa). SBTI – soybean trypsin inhibitor (20 kDa). No staining was observed in the absence of primary antibody. Renal Specificity of MIOX

10. © Copyright 2009 by the American Association for Clinical Chemistry Figure 2. (A) Western blot of normal human kidney homogenate (kidney) using the rabbit polyclonal (pAb R9544) and the mouse monoclonal anti-MIOX antibodies 12H06 and 1D10. GST-MIOX was cut with thrombin, run on the same gel, transferred to PVDF and protein stained (Protein Stain). The protein stained bands were subjected to Edman sequencing and confirmed to represent MIOX and GST. The only band that reacted with all three antibodies corresponded to recombinant MIOX (~33 kDa). Anti-MIOX Antibodies

11. © Copyright 2009 by the American Association for Clinical Chemistry Figure 4. (A) Miox was measured in mouse serum at baseline and 24 hours following AKI (n=5). No Miox was detected at baseline. Serum Miox was increased 24 hours post-injury (2.8 ± 0.7 ng/mL; mean ± SEM). *P<0.02 (paired t-test). (B) Representative section of renal cortex 24 hours post-injury. Extensive tubular necrosis is evident (arrows, H&E, 400x). No tubular necrosis was observed sham- operated animals. AKI Mouse Model

12. © Copyright 2009 by the American Association for Clinical Chemistry Figure 5. MIOX in critically ill and hospitalized patients. (A) Serum creatinine (SCr) peaked 54.3±3.8 hours (Time 54 h) relative to the preceding SCr measurement (Time 0 h), and was increased at Time 54 h in patients with AKI (**P<0.005). (B) Patients with AKI showed higher plasma MIOX concentrations at Time 0 h and Time 54 h compared to patients without AKI (*P=0.002). MIOX in Human Patients

13. © Copyright 2009 by the American Association for Clinical Chemistry Figure 6. (A) Serum creatinine is similar amongst all groups at Time 0 h. (B) Plasma MIOX increased at Time 0 h in patients with oliguric and dialysis-requiring AKI. * P <0.05. MIOX in Human Patients

14. © Copyright 2009 by the American Association for Clinical Chemistry Question  What are the advantages and disadvantages of MIOX as an acute kidney injury biomarker?  What additional questions about the biology of MIOX need to be answered?

15. © Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at www.clinchem.org Download the free Clinical Chemistry app on iTunes for additional content! Follow us