2. Biomarkers of Acute Kidney Injury Dr Sameena Ghayur Shifa College of Medicine /Shifa International Hospital Sameena.ghayur@yahoo.com

3. AKI: A Common, Serious Problem  In 5% of all hospitalized patients, 50% patients in ICUs  The incidence is increasing –globally  Mortality rate 50 - 80% in dialyzed ICU patients– 4 Million die each year of AKI  AKI requiring dialysis is one of the most important independent predictors of death in ICU patients  25% of ICU dialysis survivors progress to ESRD within 3 years

4. Pathogenesis

5. Pathophysiology

6. AKI – A Systemic Condition Functional and structural extra-renal organ injury occurs in AKI Potential mediators uraemic toxins cytokines leukocytes

7. Definitions - KDIGO  Serum creatinine rises by ≥ 26µmol/L within 48 hours or  Serum creatinine rises ≥ 1.5X the reference value which is known or presumed to have occurred within one week or  Urine output is 6 consecutive hours

8. RIFLE Criteria Acute dialysis quality initiative(ADQI) group

9. Diagnosis of AKI is Often Delayed  Clinicians have used SCr to diagnose AKI for decades.  Acknowledged as inadequate gold standard:  Poor specificity in some settings that are not associated with kidney injury  Poor sensitivity in setting of adequate renal reserve  Relatively slow kinetics after injury  Varies widely with age, gender, diet, muscle mass, muscle metabolism, medications, hydration status  In AKI, serum creatinine can take several days to reach a new steady state

10. Diagnosis of AKI is Often Delayed  Considerable interest in identifying better biomarkers of tubular injury: potentially more accurate and earlier diagnosis

11. How to evaluate new biomarkers? Ideal Biomarker  Highly organ specific  Allow recognition of etiology of AKI  Correlate with histological findings  Correlate with degree of tubular damage  Noninvasive  Test be simple, quick, accurate, reliable, inexpensive and commonly available

12. Serum Biomarkers

13. Neutophil Gelatinase Associated Lipocalin (NGAL)  Growth and differentiation of Renal tubular epithelial cells  Bacteriostatic effect in distal urogenital tract by interfering with bacterial siderophore mediated iron aquisition J Am Soc Nephrol 2006: 17:1503-1520

14. Neutophil Gelatinase Associated Lipocalin (NGAL)

15. Neutophil Gelatinase associated Lipocalin (NGAL) Biomark Med. 2010April : 4 (2):265-280

16. Urine NGAL Platform  Abbott Diagnostics  ARCHITECT: Standardized clinical platform

17. Plasma NGAL Kit * In development. Currently not for sale in US

18. Cystatin C  Serum cystatin C -a non- glycosylated, 13.3-kDa protein belonging to cystatin protease inhibitors.  After glomerular filtration, it is fully catabolized in the proximal renal tubule and is not returned to blood.  When GFR decreases, cystatin C level begins to rise proportionately

19. Cystatin C  Endogenous, detected earlier than serum creatinine to diagnose and identify progress  Independent of age, sex, race, body mass and hydration  Nephlometry  Not diagnostically specific for AKI  Early marker of impaired glomerular function rather than tubular lesion Curr Med Chem 2007; 2007: 14 2314-2317 Blood Purif 2006; 24: 67-70

20. Uric Acid  Acute urate nephropathy  Marker of Imminent onset of AKI  Diagnostic marker  Active indicator of intra-renal injury to microvasculature  Potent regulator of endothelial NO levels  Inhibitor of proliferation and migration of epithelial cells Nucleosides Nucleosides Nucleotides Nucleic acid 2008; 27 (8): 967-78

21. Uric Acid

22. Urine Biomarkers

23. Urine as Clinical material for AKI  Urinary enzymes of renal origin  Urinary low molecular weight proteins  Gene products - AKI markers specially produced in the Kidney

24. Urinary Enzymes of Renal Origin  Site specific  Alkaline phosphatase, G glutamyl transpeptidase, Alanine aminotranpeptidase:  Reflect damage of brush border membrane, loss of micro villi  Glutatione transferase: proximal and distal tubules  Critically ill patients  N acetyl β Dglucosaminidase (NAG): lysosomes of proximal tubular cells Shock 2006;26:245-53 Nephrol DialTransplant 2003;18:543-51 J Am Soc Nephrol 2007;18:904-12

25. Urinary Enzymes of Renal origin  Very sensitive marker directly correlated with serum creatinine and reduced GFR  As early as first day of kidney injury  Predictive value low  Do not identify the cause or reversibility of process  May identify patients at Risk  Prognosis  Rapid inactivation of enzymes –collection and storage

26. Urinay low molecular weight proteins  α 1 microglobulin:Liver, bound to IgA, free form excreted in urine  β 2 microglobulin:Nephrotoxic agents, hypoxia  Instability of protein at pH <6 and alkalination of urine  RBP:Stability at low pH  Cystacin C:  Tubular proteinuria better predictor of AKI than enzymuria  ELISA

27. AKI markers specially produced in in Kidney  Protein products of genes specifically related to AKI  Urinary cytokines and chemokines  Structural and functional proteins of renal tubules

28. Protein Products of Genes Specifically related to AKI  CYR61:H eparin binding protein, member of family of EGF, signaling molecule, protective role in process of repair and neovascularization, earlier marker  KIM -1: Marker of ischemia and and toxic injury, transmenbrane and extracellular ectodomains, sensitive, specific, not affected by urine characteristics  NGAL Am J physiol Renal Physiol 2006;291:456-64 J Am Soc Nephrol 2007;18:2704-2714

29. Urinary Cytokines and Chemokines  Immune response-Role in pathogenesis  Non specific parameters  Gro α : 3 h after ischemia, after transplant  IL-18: Chemotactic, ischemic tissue damage, sensitivity and specificity of >90% Am J physiol Renal Physiol 2006;29:1187-1193 J Clin Inves 2001;107:1145-1152

30. Structural and Functional proteins of Renal tubules  F-Actin: Apical membrane of proximal tubular cells, pH change causes depolymerization, actin in the microvilli, 30 min after ischemia  NHE3: Most abundant sodium transporter in renal tubules (60-70% reabsorption), observed drop in sodium reabsoption, urinary excretion of NHE3 may be regarded as a marker of AKI Am J physiol Renal Physiol 1999;276:544-551 Am J Kidney Dis 2003; 42: 599-600

31. Summary Urinary Markers  Mainly used in experimental studies  Medical laboratories-sensitive, specific and relatively costly immunological methods  Require active validation  Guidelines need to be developed for urine collection, storage and centrifugation

32. Conclusion  AKI is a continuing problem in clinical practice associated with high mortality and morbidity  Standard lab diagnosis of AKI is based on determination of serum creatinine which is imperfect  Despite intense research no single ideal biomarker has yet been found  Proteins in urine and plasma are a step forward in the development of clinical practice with potential impact on treatment outcomes  They require validation and trials in large patient populations