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NGAL - Acute kidney injury biomarker


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NGAL - Acute kidney injury biomarker

  1. 1. Acute Kidney InjuryAcute Kidney Injury Current status & emerging scopeCurrent status & emerging scope
  2. 2. Introduction to AKIIntroduction to AKI Delay in diagnosis and initiation of therapy that may prevent nephron loss AKI secondary to ischemic injury Common & potentially devastating problem A persistently high rate of mortality despite significant advances in supportive care Outcome Why?
  3. 3. AKI – diagnostic criteriaAKI – diagnostic criteria Mehta et al. Critical Care 2007 11:R31
  4. 4. AKI – Clinical stagingAKI – Clinical staging Mehta et al. Critical Care 2007 11:R31
  5. 5. current simple test for renal status AKI - MarkersAKI - Markers Contemporary markersEffective tests but inadequate for early inference Emerging Biomarkers Albumin Urea Creatinine Cystatin C KIM-1 IL-18 NGAL eGFR
  6. 6. What is the current simpleWhat is the current simple test?test?  In individuals with diabetes:In individuals with diabetes: – ““Spot” urine albumin to creatinine ratioSpot” urine albumin to creatinine ratio  In others at risk:In others at risk: – ““Spot” urine albumin to creatinine ratio ORSpot” urine albumin to creatinine ratio OR standard dipstickstandard dipstick (Bouleware, et al., 2003)(Bouleware, et al., 2003) – Estimate GFR from serum creatinine usingEstimate GFR from serum creatinine using the MDRD prediction equation.the MDRD prediction equation. *24 hour urine collections are NOT needed. Diabetics should be tested once a year. Others at risk testing less frequently as long as normal. back
  7. 7. AKI - CreatinineAKI - Creatinine  Does not rise until ~50% of renal function is lost, typically 48-72 hrs following acute injury. – Creatinine is correlated with muscle mass and nutrition – MDRD equation has not been validated for: – elderly (over 70 years of age) – pregnant women – serious comorbid conditions – extremes of body size, muscle mass, or nutritional status Nature Clinical Practice Nephrology, 27 November 2007
  8. 8. AKI - CreatinineAKI - Creatinine  Does not rise until ~50% of renal function is lost, typically 48-72 hrs following acute injury. – Creatinine based GFR equations are imprecise when GFR>60 – Acutely ill have greater variability in results – Inpatients have shown poorer agreement than outpatients between eGFR and measured GFR Nature Clinical Practice Nephrology, 27 November 2007
  9. 9. AKI - PossibilityAKI - Possibility  Possibility to identify biomarkers of AKI that can enable detect / impact high risk patients - 1. To diagnose AKI earlier, prior to overt azotemia development when majority of nephron loss may have already occurred. 2. To reduce risk for a disastrous outcome, & 3. To prevent progression with early intervention Nature Clinical Practice Nephrology, 27 November 2007
  10. 10. AKI - Reasons for focusAKI - Reasons for focus  AdvantagesAdvantages – Patients, physicians, industryPatients, physicians, industry  BeliefBelief – Direct relationship between duration and mortality – Early forms of AKI are often reversible and potentially effective preventive and therapeutic measures are frequently delayed due to lack of early diagnostic markers – Early diagnosis → timely institution of measures → prevention of progression [animal and human studies reveal a narrow “window of opportunity”] – A biomarker test that is simple, non-invasive and ofA biomarker test that is simple, non-invasive and of early diagnostic and prognostic value.early diagnostic and prognostic value.
  11. 11. AKI -AKI - Ischaemic kidney responseIschaemic kidney response Multifaceted response of the kidney to ischaemia 1 Persistent vasoconstriction 2 Tubular obstruction 3 Cellular structural changes 4 Metabolic alterations 5Inflammatory response
  12. 12. In ischaemic kidney, the proximal tubule cells undergo a complex temporal sequence of events, including Ischaemic kidneyIschaemic kidney manifestationsmanifestations 2 Cell death due to apoptosis and Necrosis 3 Dedifferentiation & proliferation of viable cells 4 Reestablishment of the epithelial phenotype 1Loss of cell polarity Interventions for innovative and effective therapy must: 1. oppose tubule cell death and / or 2. enhance the recovery phase
  13. 13. Emerging biomarkersEmerging biomarkers in renal diseasein renal disease
  14. 14. Emerging biomarkersEmerging biomarkers  Multidisciplinary functional approach to - 1. identify the most relevant biomarker because  current markers rise after function is already lost and window of benefit from intervention is closed or closing. 1. detect renal tubular injury and / or dysfunction at an early stage  before a decline in GFR is detected by increased serum creatinine levels.  Some promising biomarkers include: KIM-1 IL-18 NGAL Cystatin-C
  15. 15. Emerging biomarkersEmerging biomarkers  Tubular enzymes  Neutrophil gelatinase-associated lipocalin  Kidney injury molecule 1  Low-molecular weight urinary proteins –alpha1-and beta2-microglobulin–retinol-binding protein– adenosine deaminase-binding protein–cystatinC  cysteine-rich protein 61  urinary interleukins/adhesion molecules,  management of the patient at risk for ARF.  glomerular filtration  proatrialnatriuretic peptide (1-98)  Cystatin C KIM-1 IL-18 NGAL Cystatin-C
  16. 16. Our understandingOur understanding Emerging biomarkersEmerging biomarkers
  17. 17. Cystatin C – what is it?Cystatin C – what is it?  Single chain basic protein, non-glycosylated, 13,360 kD that acts as an inhibitor of cysteine proteases  Synthesized by all nucleated cells  Constant production rate (gene of housekeeping type) – not influenced by acute phase reaction – not influenced by endogenous or analytical factors – not influenced by muscle mass, food intake, or body surface KIM-1 IL-18 NGAL Cystatin-C
  18. 18. Cystatin C - characteristicsCystatin C - characteristics  Cleared by free glomerular filtration, and not secreted by tubules or eliminated by any extra-renal route  A marker of kidney function that may alsoA marker of kidney function that may also be associated with inflammationbe associated with inflammation – Reflective of mildly impaired kidney function with increased inflammation.. KIM-1 IL-18 NGAL Cystatin-C
  19. 19. Cystatin C - StudyCystatin C - Study  85 patients at high risk of ARF – 44 patients developed ARF – 41 served as controls.  Serum creatinine & cystatin C were determined daily.  ↑ cystatin C significantly preceded that of creatinine.  At 1.5+0.6 days, cystatin C ↑ by 50%, earlier compared to creatinine. KIM-1 IL-18 NGAL Cystatin-C Kidney Int.2004 Sep;66(3):1115-22
  20. 20. Cystatin C - SummaryCystatin C - Summary  Serum cystatin C is a useful marker & may detect ARF 1-2 days earlier than creatinine.  Over 20 years, most studies show that cystatin C is a better predictor of GFR than creatinine – Meta-analysis - 49 studies, 4,492 individuals  Dharnidharka et al. AJKD 2002  Cystatin C’s advantage over creatinine as a GFR measure is probably greatest in the elderly and non-CKD persons KIM-1 IL-18 NGAL Cystatin-C
  21. 21. KIM-1 – what is it?KIM-1 – what is it?  Kidney Injury Molecule – 1 is a Type 1 transmembrane protein whose ectodomain is shed from cells upon injury.  Expression is markedly up-regulated in the proximal tubule in the post-ischemic rat kidney.  Early studies suggest that urinary KIM-1 levels may serve as a sensitive method for detecting early kidney injury. KIM-1 IL-18 NGAL Cystatin-C
  22. 22. KIM-1 studyKIM-1 study  The most widely cited study in the field is oneThe most widely cited study in the field is one by Han et al, studying KIM-1 for elevation ofby Han et al, studying KIM-1 for elevation of KIM-1 in the urine, as a biomarker for ARF.KIM-1 in the urine, as a biomarker for ARF. – 23 patients with ARF,23 patients with ARF, – 9 with chronic renal failure (CRF), and9 with chronic renal failure (CRF), and – 9 normal subjects9 normal subjects  This KIM-1 study used azotemia to identify ARFThis KIM-1 study used azotemia to identify ARF patients and the results showed the following:patients and the results showed the following: – Urinary KIM-1 was elevated in patients with ARFUrinary KIM-1 was elevated in patients with ARF secondary to ischemic ATN,secondary to ischemic ATN, – No elevations seen in CRF patients & patients withNo elevations seen in CRF patients & patients with ARF due to other causes, as well as normal subjectsARF due to other causes, as well as normal subjects – No sensitivity, specificity, or ROC data was provided.No sensitivity, specificity, or ROC data was provided. KIM-1 IL-18 NGAL Cystatin-C
  23. 23. IL-18 - studyIL-18 - study  Biomarker of human acute tubular necrosis.  In the most popular IL-18 study, Parikh et al examined urinary levels of IL-18 in – 11 healthy controls, – 14 patients with ATN, – 8 with pre-renal azotemia, – 5 with UTI, and – 12 with chronic renal failure.  Again, as noted for the KIM-1 study, study subjects were diagnosed using azotemia as a criterion. KIM-1 IL-18 NGAL Cystatin-C
  24. 24. IL-18 - summaryIL-18 - summary  The results showed urinary IL-18 to be a good marker for renal failure secondary to ATN, as compared to all other causes in the study,  ROC analysis showed a sensitivity of 95%, and a specificity of 82%. KIM-1 IL-18 NGAL Cystatin-C
  25. 25. FocusFocus NGALNGAL
  26. 26. NGAL – what is it?NGAL – what is it?  Neutrophil gelatinase–associated lipocalin (NGAL) is a 25-kDa epithelial protein that is covalently bound to gelatinase from human neutrophils.  178 aa disulfide-bridged polypeptide chain  Calculated molecular mass: 22 kDa  Apparent molecular mass: 25 kDa (glycosylation)  Forms complex with 92-kDa matrix metalloproteinase-9 (MMP-9; gelatinase B) - capable of protecting from degradation by interacting with this protein. KIM-1 IL-18 NGAL Cystatin-C
  27. 27. NGAL – when is it found?NGAL – when is it found?  NGAL has also been linked to apoptosis in reproductive tissues.  Epithelial cells of the involuting mammary gland and uterus express high levels of NGAL, temporally coinciding with a period of maximal apoptosis.  Thus, it is likely that a subset of epithelial cells may utilize this mechanism to regulate their own demise KIM-1 IL-18 NGAL Cystatin-C
  28. 28. NGAL – why is it found?NGAL – why is it found?  Induced in response to apoptotic stressors and may be a compensatory response that involves cell defence pathways and thus play a role in cell survival.  Most up-regulated gene in the kidney after AKI in animal models and especially high levels after renal ischaemia  Levels increase within minutes of injury  Spills into blood and urine and released in inflammation from several epithelia. KIM-1 IL-18 NGAL Cystatin-C
  29. 29. NGAL – where is it found?NGAL – where is it found?  Expression is markedly induced in stimulated epithelia and expressed in:xpressed in: – Secondary granules of neutrophilsSecondary granules of neutrophils – Certain epithelial cells (gut, lung, uterus,Certain epithelial cells (gut, lung, uterus, mammary gland, kidney)mammary gland, kidney)  Elevated in the serum of patients with acute bacterial infections, in the sputum of patients with asthma or COPD, and the bronchial fluid from the emphysematous lung.  NGAL counters the morphologic response of renal tubular epithelial cells to ischemia. KIM-1 IL-18 NGAL Cystatin-C
  30. 30. NGAL - AvailabilityNGAL - Availability NGAL may be expressed by the damaged tubule to induce re-epithelialization and proliferation NGAL is expressed by the penetrating ureteric bud and triggers nephrogenesis by stimulating the conversion of mesenchymal cells into kidney epithelia Renal research findings suggest During kidney development
  31. 31. NGAL - RoleNGAL - Role NGAL [Neutrophil Gelatinase Associated Lipocalin 5 Tilts the overall balance of proximal tubule cell fate toward cell survival after ischemic injury 1 Upregulated in the post- ischemic kidney, in tubular epithelial cells that are undergoing proliferation 2 Can enhance the epithelial phenotype, based at least in part on its ability to ameliorate tubule cell apoptosis and enhance tubule cell proliferation 3 Functions as an iron- transporting protein during nephrogenesis 4 Plays a renoprotective role in ischemic ARF
  32. 32. NGAL – Iron transportNGAL – Iron transport NGAL Based iron mechanisms operative in the post- ischemic kidney 5 Delivery of iron into cells is crucial for cell growth and renal regeneration after nephrotoxic injury 1 Because NGAL can be endocytosed by the proximal tubule, the protein could potentially recycle iron into viable cells 2 May serve as a reservoir for iron that is released from tubule cells that are damaged by nephrotoxic injury 3 Removes iron, a reactive molecule, from the site of tissue injury, thereby limiting iron-mediated cytotoxicity 4 May also facilitate the removal of excess intracellular iron, thereby limiting oxidant mediated apoptosis
  33. 33. NGAL in kidney - explanationNGAL in kidney - explanation KIM-1 IL-18 NGAL Cystatin-C A B The forest fire therapy for kidney function: these figures represent 2 patients with both having 60% viable nephrons as green trees and 40% damaged nephrons due to ischaemia. However, patient B has much stronger response than patient A. It is proposed that sreum creatinine or GFR is indicative of the viable nephron while renal NGA indicates the extent of active lesion. Viable nephron Damaged nephron Active lesion
  34. 34. NGAL studyNGAL study  The most promising study to date is the NGAL study by Mishra et al. who followed a population of children coming off cardiopulmonary bypass for the development of ARF and correlation to rises in urine and serum NGAL.  Of the 71 study subjects, 20 developed ARF (diagnosed by rise in serum creatinine).  Serum and urine NGAL were shown to rise within 2 hours off cardiopulmonary bypass, a full 24-36 hours before the development of azotemia. KIM-1 IL-18 NGAL Cystatin-C
  35. 35. NGAL - ConclusionsNGAL - Conclusions  Using a cut-off of 25 ug/ml, urine NGAL had a sensitivity of 100% and specificity of 98%, whereas serum NGAL had a sensitivity of 70% and specificity of 94%.  Furthermore, ROC analysis showed urine NGAL to have and area under the curve of 0.998, and serum NGAL had a corresponding value of 0.906, showing excellent characteristics as markers for ARF in this setting. KIM-1 IL-18 NGAL Cystatin-C
  36. 36. NGAL – Another studyNGAL – Another study  In an established murine model of renal ischemia-reperfusion injury, IV NGAL given before, during, or after ischemia resulted in marked amelioration of the morphologic and functional consequences, as evidenced by - – A significant decrease in the histopathologic damage to tubules – A significant decrease in serum creatinine measurements. – A reduction in the number of apoptotic tubule cells, and – An increase in proliferating PCT after ischemic injury. Amelioration of Ischemic Acute Renal Injury by Neutrophil Gelatinase–Associated Lipocalin by JAYA MISHRA et al, J Am Soc Nephrol 15: 3073–3082, 2004.
  37. 37. NGAL response to renalNGAL response to renal ischaemia - serumischaemia - serum KIM-1 IL-18 NGAL Cystatin-C
  38. 38. NGAL response to renalNGAL response to renal ischaemia - urineischaemia - urine KIM-1 IL-18 NGAL Cystatin-C
  39. 39. NGA- Scope aheadNGA- Scope ahead
  40. 40. The futureThe future AKI biomarkersAKI biomarkers
  41. 41. Cardiac biomarkersCardiac biomarkers KIM-1 IL-18 NGAL Cystatin-C
  42. 42. AKI Panel markers - perception KIM-1 IL-18 NGAL Cystatin-C Cystatin C IL-18 KIM-1 NGAL ????
  43. 43. Questions needing clarityQuestions needing clarity  When is AKI being diagnosed currently?When is AKI being diagnosed currently?  How sensitive is NGAL relatively?How sensitive is NGAL relatively?  How specific is NGAL relatively?How specific is NGAL relatively?  When does NGAL secretion begin?When does NGAL secretion begin?  What is the normal level of NGAL?What is the normal level of NGAL?  Is NGAL diagnostic or prognostic?Is NGAL diagnostic or prognostic?  How does NGAL compare with IL-18?How does NGAL compare with IL-18?  How does NGAL compare with cystatin CHow does NGAL compare with cystatin C or KIM-1?or KIM-1?
  44. 44. NGAL compared to IL-18NGAL compared to IL-18 KIM-1 IL-18 NGAL Cystatin-C NGAL and IL-18: Biomarkers in tandem Parikh et al, Kidney Int70:199- 203, 2006
  45. 45. NGAL compared to Cystatin CNGAL compared to Cystatin C KIM-1 IL-18 NGAL Cystatin-C Vandevoorde and Devarajan, J Am Soc Nephrol 2006, 17:404A
  46. 46. Postmortem of the studiesPostmortem of the studies  The KIM-1 and IL-18 studies are both plagued by the fact that urine levels of these markers were determined after the diagnosis of ARF was certain (by azotemia), and therapy initiated at such a time may not result in improved clinical outcomes as compared to current standards of care. KIM-1 IL-18 NGAL Cystatin-C
  47. 47. Postmortem of the studiesPostmortem of the studies  These tests may be of clinical utility to identify patients with ARF secondary to ischemic ATN (in the case of KIM-1), or ATN in general (in the case of IL-18), for potential targeted therapy of these disease processes in the years ahead. KIM-1 IL-18 NGAL Cystatin-C
  48. 48. Postmortem of the studiesPostmortem of the studies  However the NGAL study succeeds in this respect since NGAL measurements were made during the development of disease, showing a robust rise in serum and urine NGAL 24-36 hours before a rise in serum creatinine, when the initiation of therapy may avoid or diminish overt renal failure. KIM-1 IL-18 NGAL Cystatin-C
  49. 49. Who should be tested &Who should be tested & treated?treated?  With diabetes:With diabetes: – With urine albumin/creatinine ratios more than 30mgWith urine albumin/creatinine ratios more than 30mg albumin/1 gram creatininealbumin/1 gram creatinine  Without diabetes:Without diabetes: – With urine albumin/creatinine ratios more than 300mgWith urine albumin/creatinine ratios more than 300mg albumin/1 gram creatinine corresponding to about 1+albumin/1 gram creatinine corresponding to about 1+ on standard dipstickon standard dipstick  Any patient:Any patient: – With estimated GFR less than 60 mL/min/1.73 m2With estimated GFR less than 60 mL/min/1.73 m2
  50. 50. Therapy to prevent progressionTherapy to prevent progression  Intensive glycemic control lessens progressionIntensive glycemic control lessens progression from microalbuminuria in type 1 diabetesfrom microalbuminuria in type 1 diabetes – DCCT, 1993DCCT, 1993  Antihypertensive therapy with ACE InhibitorsAntihypertensive therapy with ACE Inhibitors lessens proteinuria and progressionlessens proteinuria and progression – Giatras, et al., 1997Giatras, et al., 1997 – Psait, et al., 2000Psait, et al., 2000 – Jafar, et al., 2001Jafar, et al., 2001  Low protein diets lessen progressionLow protein diets lessen progression – Fouque, et al., 1992Fouque, et al., 1992 – Pedrini, et al., 1996Pedrini, et al., 1996 – Kasiske, et al., 1998Kasiske, et al., 1998 Meta-Analyses Meta-Analyses
  51. 51. Update on the ImmunocalinsUpdate on the Immunocalins LipocalinsLipocalins
  52. 52. ImmunocalinsImmunocalins  γγ-microglobulin-microglobulin  ββ-microglobulin-microglobulin  αα22-microglobulin-microglobulin  αα11-microglobulin-microglobulin  Ig L-chainsIg L-chains  ββ22-microglobulin-microglobulin  RBPRBP [RETINOL BINDING PROTEIN][RETINOL BINDING PROTEIN]  αα11-microglobulin,-microglobulin, Protein HCProtein HC αα11-microglobulin-microglobulin  Recognition in 1985:Recognition in 1985: αα11-m + RBP + others =-m + RBP + others = new protein superfamily = Lipocalinsnew protein superfamily = Lipocalins New LMW-proteins isolated by BerggNew LMW-proteins isolated by Berggåård et al.rd et al.
  53. 53. The ImmunocalinsThe Immunocalins
  54. 54. The ImmunocalinsThe Immunocalins
  55. 55. ConclusionConclusion  Biomarkers detected in urine or serum shortly after AKI, have been suggested to contribute to prediction of ARF.  However, it needs to be borne in mind that excretion of these biomarkers may also increase after reversible and mild renal dysfunction and may not necessarily be associated with persistent or irreversible damage. Shock 2006; Sep 26(3):245-53