1. ACUTE KIDNEY INJURY Role of Novel Biomarkers R Bhimma Department of Paediatrics and Child Health Nelson R Mandela School of Medicine University of KwaZulu-Natal

2. Diagnosis Increase serum creatinine and/or blood urea nitrogen levels and/or decrease urine output Sudden impairment of kidney function occurring over a period of hours to days. Definition of AKI

3. Why is it important to detect AKI early?  A rise in serum creatinine of just 0.3mg/dl (26.5 mmol/l) has a four fold higher multivariable – adjusted risk of death.  Children with AKI may be at risk for long-term chronic kidney disease.

4. Acute Kidney Injury Network (AKIN) classification of AKI Serum creatinine (SCr) criteria Urine output (UO) criteria Stage 1≥0.3 mg/dl (26.5 μmol/l) rise or rise to 1.5–1.99 X baseline UO <0.5 ml/kg/h for 6 h Stage 2Rise to ≥2–2.99 X baselineUO <0.5 ml/kg/h for 12 h Stage 3Rise to ≥3 X baseline or ≥4 mg/dl (354 μmol/l) rise with an acute rise of at least 0.5 mg/dl (44 μmol/l) UO<0.3 ml/kg/h for 24 h or anuria for 12 h Ped Nephrol,10 July 2010, Zubaida Al-Ismaili

5. Modified pediatric Risk, Injury, Failure, Loss, End-stage kidney disease (pRIFLE) criteria Estimated creatinine clearances (eCCl) Urine output (UO) criteria RiskeCCl decreased by 25%UO<0.5 ml/kg/h for 8 h InjuryeCCl decreased by 50%UO<0.5 ml/kg/h for 16 h FailureeCCl decreased by 75% or eCCl <35 ml/min/1.73 m2 UO<0.3 ml/kg/h for 24 h or anuria for 12 h LossᵃPersistent failure >4 weeks End-stage kidney diseaseᵃPersistent failure >3 months a The pRIFLE stages “loss” and “end-stage kidney disease” are not AKI stages per se; they describe chronic outcomes of AKI

6. Limitations of serum creatinine and BUN Change extracellular volume depletion decreased kidney blood flow Influenced by other factors, which are not directly related to kidney damage, such as:  age  sex  body mass  nutritional status

7. Limitations of Serum creatinine and BUN cont…  Elevated serum creatinine concentrations are not specific for AKI and require differentiation from other pre-renal or extra-renal causes.  Serum creatinine concentrations are not specific for renal tubular lesions, pathogenetically related to AKI development.  Reflect the loss of glomerular filtration function, accompanying the development of AKI.

8. Limitations of serum creatinine and BUN cont…  Increases in serum creatinine are detected later than the actual GFR changes as creatinine accumulates over time.  Serum creatinine is a poor marker of kidney dysfunction as changes in its concentrations are neither sensitive nor specific in response to slight GFR alterations.  Changes in serum creatinine become apparent only when the kidneys have lost ≥50% of their functional capacity.

9. What is a biomarker? “ a characteristics that is objectively measured and evaluated as an indicator of normal biological process, pathogenic process, or pharmacologic response to a therapeutic intervention”. TestBiomarker HeightGrowth Urinary dipsticks for nitritesUTI ProteinuriaDisease severity in IgA nephropathy. Anti-GBM AbGood pastures syndrome

10. Ideal Biomarker AKI  Non – invasive  Easily obtainable  Measurable using standardized assays  Fast results  Incur reasonable cost to perform

11. Possible roles for novel kidney injury biomarkers Ismaili Z Al et al Ped Nephrol, 2010

12. Pathophysiological mechanics of AKI and repair

13. Proposed mechanisms for increased biomarker levels in plasma and urine (3) Glomerular filtration Increased biomarker levels in urine (1) Increased synthesis in extrarenal tissues (2) Release from circulating immune cells Increased biomarker levels in plasma (4) Impaired reabsorption in the proximal tubule (5) Increased synthesis in tubular cells (6) Release from infiltrating immune cells Martensson J et al BJA, 2012

14. Proposed phase of biomarker development GoalsPhaseBiomarker aims Discovery phase Phase 1 Identify leads for AKI biomarker. Prioritize identified leads Translational phase Phase 2 Evaluate diagnostic accuracy Assess the ability to distinguish AKI from non-AKI Phase 3 Evaluate the capacity of the biomarker to detect preclinical AKI or other AKI characteristic (e.g. severity, prognosis) Define criteria for a positive screening test (e.g. cut-off) Validation phase Phase 4 Determine the operating characteristics of the biomarker-based screening test in a relevant population by determining the detection rate and the false referral rate (prospective study) Phase 5 To estimate the reduction in AKI morbidity and mortality afforded by the screening test (effects on population) Coca SG, et al (2008) Urinary biomarkers for acute kidney injury: perspectives on translation. Clin J Am Soc Nephrol 3:481–490, Pepe MS, et al. (2001) Phases of biomarker development for early detection of cancer. J Natl Cancer Inst 93:1054–1061

15. Summary of calculating diagnostic characteristics: sensitivity, specificity, positive predictive value, and negative predicative value. Disease presentDisease not present Biomarker test positiveTrue positive (TP)False positive (FP)TP+FP=total with positive test Biomarker test negativeFalse negative (FN)True negative (TN)FN+TN=total with negative test TP+FN=total with diseaseFP+TN=total with no disease How to calculate diagnostic characteristics SensitivityTP/(TP+FN): TP/total with disease SpecificityTN/(TN+FP): TN/total without disease Positive predictive value (PPV) TP/(TP+FP): TP/total with positive testProbability of actually having the disease when test is positive Negative predictive value (NPV) TN/(TN+FN): TN/total with negative testProbability of not having the disease when test is negative Ismaili Z Al et al Ped Nephrol,10 July 2010,

16. Hypothetical ROC: plot of the sensitivity of a test vs. 1-specificity for may different cut-off values of a biomarker

17. Kidney Biomarkers  > 20 protein biomarkers have been intensively studied.  Urinary biomarkers are regarded as more non invasive, easy to measure, easily obtainable and provide earlier detection of AKI.  Depending on the time of appearance after AKI, urinary biomarkers many be classified as those of structural injury or functional injury.

18. Biomarkers of structural injury Type of biomarkersSelective sites and associated types of injury Kidney injury molecule-1 (KIM-1)*Proximal tubule injury (Ischemic AKI, nephrotoxins, RCC) n-acetyl glucosaminadase (NAG)Proximal tubule injury Neutrophil gelatinase-associated lipocalin (NGAL)* Tubule and collecting duct injury (Ischemic AKI, nephrotoxins, delay allograft renal function) Interleukin (IL)-18*Tubule injury (AKI, delayed allograft renal function) ClusterinTubule injury

19. Biomarkers of functional injury Cystatin C*Glomerular injury; in urine indicates proximal tubule injury Total protein β2-microglobulin albuminGlomerular and tubular dysfunction Brush border antigens Adenosine deaminase binding protein Carbonic anhydraseProximal tubule injury Other tubular antigens Urinary enzymes N-acetyl-β-D-glucosaminidase Alanine aminopeptidase Cathepsin BProximal tubule injury γ-glutamyltransferase α-glutathione-S-transferase β-glucosidaseProximal tubule > distal tubule injury Alkaline phosphatase Lactate dehydrogenaseDistal tubule > proximal tubule injury

20. Others Type IV CollagenGlomerular injury Gamma-glutamyl transferase (γ-GT) Liver type fatty acid binding protein (L- FABP) Tubular epithelium injury Retinol binding protein 4 (RBP4) Sodium/hydrogen exchanger isoform Alpha-glutathione S-transferase (α- GST) Proximal tubule injury Exosomal fetuin-A Tamm-horsfall glycoproteinDistal tubule injury pi-glutathione S-transferase (π-GST)

21. Neutrophil gelatinase – associated lipocalin (NGAL)  25–kDa polypeptide covalently bound to gelatinase secreted from activated human neutrophils.  Most consistent biomarkers found during AKI.  Predominantly found in proliferating nuclear antigen– positive proximal tubule cells.  Predicts the occurrence of AKI in paediatric and adult patients after cardiac surgery. Cont…

22. Neutrophil gelatinase – associated lipocalin (NGAL)  AUC- ROC for NGAL is 0,92 at 2 hours and 1,00 at 4 hours CBP.  Also predicts mobility and mortality in children who undergo cardiac surgery.  Also predicts development of DGF.

23. Kidney Injury Molecule-1 (KIM-1)  Orphan trans-membrane receptor of unknown function.  Undetectable in normal kidney tissue or urine.  Markedly increased in ischaemic and nephrotoxic proximal tubular cell injury, and in renal cell carcinoma.  Higher urinary KIM-1 associated with worse outcome in established AKI. Cont… W. K. Han et al Advances in Clinical Chemistry, Vol. 49, 2009, pp. 73-97.

24. Kidney Injury Molecule-1 (KIM-1)  AUC – ROC 0,57 at 2 hours, 0,83 at 12 hours, and 0,78 at 24 hours after cardiac surgery.  Also predicts graft loss in renal transplant patients.  Useful in some studies to differentiate between AKI, CKD and normal patients. W. K. Han et al Advances in Clinical Chemistry, Vol. 49, 2009, pp. 73-97.

25. Interleukin (IL) -18  Pro-inflammatory cytokine  Mediates inflammatory process during ischaemic, sepsis and nephrotic AKI.  Recruits neutrophils during ischaemic injury.  Can predict AKI 1 day ahead of serum creatinine. Cont…

26. Interleukin (IL) -18  AUC – ROC 0.73 for development of AKI in patients with acute respiratory distress syndrome.  Independent predictor of mortality in the above group of patients.  Also elevated after CPB --- ROC- AUC 0.61 at 4 hours, 0.75 at 12 hours and 0.73 at 24 hours.

27. Cystatin C  13 kDa cysteine protease inhibitor.  Secreted by all nucleated cells into plasma.  Freely filtered at the glomerulus and not secreted in the tubules.  Less influenced by factors other than GFR (e.g. age, sex, race, or muscle mass).  Therefore change in serum and urinary cystatin C is more sensitive than a change in serum Cr in predicting change in GFR. Cont…

28. Cystatin C  Predicts the risk of AKI-associated CV morbidity.  Levels > 1.0mg/L also predict CV events and mortality in patients with a GFR >60mls/min/1.73m².  Increase urinary cystatin C and alpha 1 - microglobulin are predictors of an unfavourable outcome in ATN.  Has higher sensitivity and higher negative predictive value in determining GFR in CKD.

29. Proposed Biomarker strategies for Renal Replacement Therapy (RRT)- initiation in AKI.  Some studies have shown better renal outcomes in patients who start RRT while in RIFLE-risk or injury compared to failure.  However some patients may recover spontaneously.  Biomarkers like NGAL, cystatin C, NAG, KIM-1 and alpha 1 - microglobulin can help distinguish patients in whom RRT will be needed.

30. Proposed biomarkers – based strategy for RRT Consider initiating RRT BM indicates RRT necessary Severe AKI (AKIN stage 3) MILD/ MODERATE AKI (Akin stage 1-2) Conservative treatment BM Intermediate BM indicate RRT Unlikely Measure BMAKI present

31. Conclusion  Need a ‘panel’ of biomarkers making it possible for early detection, treatment, and hopefully, preventing AKI.  As cardiology moved form LDH to troponins for the diagnosis of MI, intensive care nephrology will hopefully evolve from serum Cr to tissue specific injury biomarkers.