- Email: [email protected]
Urinary Biomarkers in Acute Kidney Injury: Ready for Prime Time?
Urinary Biomarkers in Acute Kidney Injury: Ready for Prime Time? Related Article, p. 632
A
cute kidney injury (AKI) is a serious and potentially devastating complication in hospitalized patients. In the setting of cardiac surgery with cardiopulmonary bypass (CPB), acute kidney failure requiring dialysis occurs in approximately 1%-2% of patients and is associated with a mortality in excess of 60%.1,2 Less severe stages of AKI, not requiring dialysis, can occur in up to 17% of patients3,4 and are independently associated with a 19-fold increase in shortterm mortality.3 Even mild kidney impairment, defined as a rise in serum creatinine of 25% over baseline, is associated with a doubling in longterm mortality up to 10 years after surgery.4 The negative effect is independent of other prognostic factors, and persists even if kidney function recovers to baseline.4 In order for potential therapeutic interventions to be successful, it is critical to identify biomarkers that are capable of detecting AKI early in the course of the disease. In this issue of the American Journal of Kidney Diseases, Devarajan et al utilize unbiased proteomic techniques to identify ␣1-microglobulin, ␣1-acid glycoprotein, and albumin as diagnostic and prognostic markers of AKI in children undergoing CPB.5 Ischemic AKI represents a sequence of events conceptually separated into initiation, extension, maintenance, and repair phases. The initiation and extension phases are characterized by alteration of microvascular hemodynamics and activation of inflammatory pathways in response to ischemic insult. Tubular epithelial injury resulting from these insults can induce apoptosis and, in severe injury, necrosis, luminal sloughing of epithelial cells, tubular obstruction, and back leak. Sublethally injured tubular cells can lose their cytoskeletal integrity, brush border membranes, and cell polarity. Mislocalization of adhesion molecules leads to loss of viable epithelial cells into the tubular lumen. In the repair phase, surviving epithelial cells undergo dedifferentiation, proliferation, and subsequently redifferentiation leading to restitution of normal tubular epithelium (reviewed in detail in6,7). Although this paradigm has provided a framework for possible therapeutic interventions in animal models of AKI, similar interventions in
humans repeatedly fail to alter the course of clinical AKI (such as insulin-like growth factor 8 or atrial natriuretic peptide9). There are several potential reasons for this. First, the current paradigm is derived from animal models, and so may not be completely applicable to human AKI. Second, even if the paradigm is broadly applicable, it is probably incomplete. The response of the kidney to an ischemic insult is complex and multifaceted, involving induction of multiple pathways, some of them protective (adaptive), others injurious (maladaptive), and many of them undiscovered. Finally, current, noninvasive serum markers of AKI (such as creatinine or cystatin C) are markers of filtration rather than injury and become abnormal well after AKI has been established, rendering early intervention impossible10 and giving no insight into the degree or nature of kidney injury or the pathophysiology of human AKI. Recent research efforts, therefore, have focused on identification of urinary biomarkers of kidney injury. This is an attractive strategy for several reasons. First, urine is easily accessible in human cohorts. Second, in the presence of tubular injury, the urine is likely to contain additional proteins (biomarkers) that are either filtered but not reclaimed by the injured tubule or released or secreted into the urine by the injured tubular cell or by infiltrating inflammatory cells. Third, discovery of novel proteins associated with AKI may uncover novel pathways of kidney injury or repair. This might provide the basis for both the early diagnosis and therapy of AKI. Recent advances in genomics, metabolomics, and proteomics provide an alternative and complementary approach to the process of biomarker discovery. These technologies allow a systematic interrogation of the genome, metabolome, or proteome, respectively, aimed at identifying genes or proteins associated with disease (or absence of disease). Once identified, the role Address correspondence to Martina Reslerova, MD, PhD, Nephrology Section, University of Manitoba, St. Boniface General Hospital, 409 Tache Ave, Winnipeg, MB R2H 2A6. E-mail: [email protected] © 2010 by the National Kidney Foundation, Inc. 0272-6386/10/5604-0002$36.00/0 doi:10.1053/j.ajkd.2010.08.004
American Journal of Kidney Diseases, Vol 56, No 4 (October), 2010: pp 609-611
609
610
and function of these genes or proteins can be further explored in experimental models. This approach thus inverts the traditional “discovery paradigm,” since an understanding of mechanism follows, rather than precedes, discovery of a biomarker. Devarajan et al have successfully utilized this approach in the discovery and validation of neutrophil gelatinase-associated lipocalin (NGAL) as a novel biomarker of AKI.11 There are multiple different proteomics approaches that can be utilized in biomarker discovery. One approach is low throughput techniques combining a separation step (eg, reversed-phase high-performance liquid chromatography [RP-HPLC] or gel) followed by tandem mass spectrometry (MS-MS). This provides an in-depth, high content analysis of the urinary proteome with the benefits of specific protein identification, the ability to detect low abundance proteins, and the ability to determine differential expression of proteins. However, this does not provide absolute quantification, so candidate proteins must have specific high-throughput assays developed (eg, enzyme-linked immunosorbent assay) to measure test performance in larger validation sets of patients. The report by Devarajan et al utilized archived urine samples in a test set of 30 children undergoing CPB surgery to analyze the differences in the AKI versus non-AKI proteome. This analysis was performed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) which is an alternative high-throughput, low-content proteomic approach. The limitations of SELDI-TOF MS are that it detects only protein peak patterns and does not provide candidate protein identification or quantification. It is also limited in the ability to detect low abundance proteins. Devarajan et al were able to identify 3 protein peaks (28.5, 43, and 66 kDa) in the AKI group 2 hours after surgery. Using in-gel digestion and MS-MS, they subsequently identified ␣1-microglobulin, ␣1acid glycoprotein, and albumin as the respective protein peaks. The observed differences between AKI and non-AKI at 2 hours after surgery were then confirmed quantitatively for each of the proteins and the findings carefully validated in a separate group of 365 children. The diagnostic test statistics and area under the receiver-operator charac-
Ho et al
teristic curve for each of these markers show that they add prognostic information related to increase in serum creatinine, length of hospital stay, and duration of AKI. This is clinically relevant and potentially useful as the additional prognostic information of these markers was measured early in the course of development (2 hours postoperatively). They have also demonstrated that these markers are independent predictors of AKI on multivariate analysis, in addition to CPB time. The current study by Devarajan et al illustrates the applicability of a SELDI-TOF MS approach to biomarker discovery and validation. The validation of relatively nonspecific markers of tubular injury as predictors of AKI is somewhat paradoxical in that these novel technologies take us back to the basics, confirming albuminuria as one of the prognostic factors for development of AKI. This observation is promising as this simple measurement is already widely available and could be easily implemented by clinicians at the bedside. These findings thus confirm the relevance of proteomic techniques in both experimental and clinical nephrology. Should these proteins now be routinely measured postoperatively in children undergoing CPB? In our view, there are still several limitations to the present study that preclude such a broad recommendation. First, although levels of the biomarkers do predict risk, the positive and negative predictive values are not sufficient to rule in or rule out AKI in an individual patient. Second, there are no specific validated interventions that could be applied based on this prognostic information. Nevertheless, these findings are useful in study settings to identify patients at risk of AKI who might be included in interventional trials. Finally, the question remains of whether these findings are generalizable to adult populations of AKI in the setting of CPB or other types of AKI. In conclusion, proteomic techniques are capable of not only identifying novel biomarkers (and thereby novel pathways) of AKI but also of validating the role of previously known proteins in the diagnosis and/or prognosis of AKI, as in the current study. With further improvement in our prognostic and diagnostic abilities in AKI, nephrologists may soon find themselves apply-
Editorial
611
ing specific therapeutic interventions and changing the course of AKI. Julie Ho, MD Martina Reslerova, MD, PhD Claudio Rigatto, MD, MSc University of Manitoba Winnipeg, Canada
ACKNOWLEDGEMENTS Financial Disclosure: The authors declare that they have no relevant financial interests.
REFERENCES 1. Chertow GM, Lazarus JM, Christiansen CL, et al. Preoperative renal risk stratification. Circulation. 1997;95(4): 878-884. 2. Thakar CV, Arrigain S, Worley S, Yared JP, Paganini EP. A clinical score to predict acute renal failure after cardiac surgery. J Am Soc Nephrol. 2005;16(1):162-168. 3. Mangano CM, Diamondstone LS, Ramsay JG, Aggarwal A, Herskowitz A, Mangano DT. Renal dysfunction after myocardial revascularization: risk factors, adverse outcomes, and hospital resource utilization. Ann Intern Med. 1998;128(3):194-203.
4. Loef BG, Epema AH, Smilde TD, et al. Immediate postoperative renal function deterioration in cardiac surgical patients predicts in-hospital mortality and long-term survival. J Am Soc Nephrol. 2005;16(1):195-200. 5. Devarajan P, Krawczeski CD, Nguyen MT, Kathman T, Wang Z, Parikh CR. Proteomic identification of early biomarkers of acute kidney injury after cardiac surgery in children. Am J Kidney Dis. 2010;56(4):632-642. 6. Bonventre JV, Weinberg JM. Recent advances in the pathophysiology of ischemic acute renal failure. J Am Soc Nephrol. 2003;14(8):2199-2210. 7. Sutton TA, Fisher CJ, Molitoris BA. Microvascular endothelial injury and dysfunction during ischemic acute renal failure. Kidney Int. 2002;62(5):1539-1549. 8. Hirschberg R, Kopple J, Lipsett P, et al. Multicenter clinical trial of recombinant human insulin-like growth factor I in patients with acute renal failure. 1999;55(6):24232432. 9. Lewis J, Salem MM, Chertow GM, et al. Atrial natriuretic factor in oliguric acute renal failure. Am J Kidney Dis. 2000;36(4):767-774. 10. Bellomo R, Kellum JA, Ronco C. Defining acute renal failure: physiological principles. Intensive Care Med. 2004;30(1):33-37. 11. Ronco C. N-GAL: diagnosing AKI as soon as possible. Crit Care. 2007;11(6):173.
A
cute kidney injury (AKI) is a serious and potentially devastating complication in hospitalized patients. In the setting of cardiac surgery with cardiopulmonary bypass (CPB), acute kidney failure requiring dialysis occurs in approximately 1%-2% of patients and is associated with a mortality in excess of 60%.1,2 Less severe stages of AKI, not requiring dialysis, can occur in up to 17% of patients3,4 and are independently associated with a 19-fold increase in shortterm mortality.3 Even mild kidney impairment, defined as a rise in serum creatinine of 25% over baseline, is associated with a doubling in longterm mortality up to 10 years after surgery.4 The negative effect is independent of other prognostic factors, and persists even if kidney function recovers to baseline.4 In order for potential therapeutic interventions to be successful, it is critical to identify biomarkers that are capable of detecting AKI early in the course of the disease. In this issue of the American Journal of Kidney Diseases, Devarajan et al utilize unbiased proteomic techniques to identify ␣1-microglobulin, ␣1-acid glycoprotein, and albumin as diagnostic and prognostic markers of AKI in children undergoing CPB.5 Ischemic AKI represents a sequence of events conceptually separated into initiation, extension, maintenance, and repair phases. The initiation and extension phases are characterized by alteration of microvascular hemodynamics and activation of inflammatory pathways in response to ischemic insult. Tubular epithelial injury resulting from these insults can induce apoptosis and, in severe injury, necrosis, luminal sloughing of epithelial cells, tubular obstruction, and back leak. Sublethally injured tubular cells can lose their cytoskeletal integrity, brush border membranes, and cell polarity. Mislocalization of adhesion molecules leads to loss of viable epithelial cells into the tubular lumen. In the repair phase, surviving epithelial cells undergo dedifferentiation, proliferation, and subsequently redifferentiation leading to restitution of normal tubular epithelium (reviewed in detail in6,7). Although this paradigm has provided a framework for possible therapeutic interventions in animal models of AKI, similar interventions in
humans repeatedly fail to alter the course of clinical AKI (such as insulin-like growth factor 8 or atrial natriuretic peptide9). There are several potential reasons for this. First, the current paradigm is derived from animal models, and so may not be completely applicable to human AKI. Second, even if the paradigm is broadly applicable, it is probably incomplete. The response of the kidney to an ischemic insult is complex and multifaceted, involving induction of multiple pathways, some of them protective (adaptive), others injurious (maladaptive), and many of them undiscovered. Finally, current, noninvasive serum markers of AKI (such as creatinine or cystatin C) are markers of filtration rather than injury and become abnormal well after AKI has been established, rendering early intervention impossible10 and giving no insight into the degree or nature of kidney injury or the pathophysiology of human AKI. Recent research efforts, therefore, have focused on identification of urinary biomarkers of kidney injury. This is an attractive strategy for several reasons. First, urine is easily accessible in human cohorts. Second, in the presence of tubular injury, the urine is likely to contain additional proteins (biomarkers) that are either filtered but not reclaimed by the injured tubule or released or secreted into the urine by the injured tubular cell or by infiltrating inflammatory cells. Third, discovery of novel proteins associated with AKI may uncover novel pathways of kidney injury or repair. This might provide the basis for both the early diagnosis and therapy of AKI. Recent advances in genomics, metabolomics, and proteomics provide an alternative and complementary approach to the process of biomarker discovery. These technologies allow a systematic interrogation of the genome, metabolome, or proteome, respectively, aimed at identifying genes or proteins associated with disease (or absence of disease). Once identified, the role Address correspondence to Martina Reslerova, MD, PhD, Nephrology Section, University of Manitoba, St. Boniface General Hospital, 409 Tache Ave, Winnipeg, MB R2H 2A6. E-mail: [email protected] © 2010 by the National Kidney Foundation, Inc. 0272-6386/10/5604-0002$36.00/0 doi:10.1053/j.ajkd.2010.08.004
American Journal of Kidney Diseases, Vol 56, No 4 (October), 2010: pp 609-611
609
610
and function of these genes or proteins can be further explored in experimental models. This approach thus inverts the traditional “discovery paradigm,” since an understanding of mechanism follows, rather than precedes, discovery of a biomarker. Devarajan et al have successfully utilized this approach in the discovery and validation of neutrophil gelatinase-associated lipocalin (NGAL) as a novel biomarker of AKI.11 There are multiple different proteomics approaches that can be utilized in biomarker discovery. One approach is low throughput techniques combining a separation step (eg, reversed-phase high-performance liquid chromatography [RP-HPLC] or gel) followed by tandem mass spectrometry (MS-MS). This provides an in-depth, high content analysis of the urinary proteome with the benefits of specific protein identification, the ability to detect low abundance proteins, and the ability to determine differential expression of proteins. However, this does not provide absolute quantification, so candidate proteins must have specific high-throughput assays developed (eg, enzyme-linked immunosorbent assay) to measure test performance in larger validation sets of patients. The report by Devarajan et al utilized archived urine samples in a test set of 30 children undergoing CPB surgery to analyze the differences in the AKI versus non-AKI proteome. This analysis was performed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) which is an alternative high-throughput, low-content proteomic approach. The limitations of SELDI-TOF MS are that it detects only protein peak patterns and does not provide candidate protein identification or quantification. It is also limited in the ability to detect low abundance proteins. Devarajan et al were able to identify 3 protein peaks (28.5, 43, and 66 kDa) in the AKI group 2 hours after surgery. Using in-gel digestion and MS-MS, they subsequently identified ␣1-microglobulin, ␣1acid glycoprotein, and albumin as the respective protein peaks. The observed differences between AKI and non-AKI at 2 hours after surgery were then confirmed quantitatively for each of the proteins and the findings carefully validated in a separate group of 365 children. The diagnostic test statistics and area under the receiver-operator charac-
Ho et al
teristic curve for each of these markers show that they add prognostic information related to increase in serum creatinine, length of hospital stay, and duration of AKI. This is clinically relevant and potentially useful as the additional prognostic information of these markers was measured early in the course of development (2 hours postoperatively). They have also demonstrated that these markers are independent predictors of AKI on multivariate analysis, in addition to CPB time. The current study by Devarajan et al illustrates the applicability of a SELDI-TOF MS approach to biomarker discovery and validation. The validation of relatively nonspecific markers of tubular injury as predictors of AKI is somewhat paradoxical in that these novel technologies take us back to the basics, confirming albuminuria as one of the prognostic factors for development of AKI. This observation is promising as this simple measurement is already widely available and could be easily implemented by clinicians at the bedside. These findings thus confirm the relevance of proteomic techniques in both experimental and clinical nephrology. Should these proteins now be routinely measured postoperatively in children undergoing CPB? In our view, there are still several limitations to the present study that preclude such a broad recommendation. First, although levels of the biomarkers do predict risk, the positive and negative predictive values are not sufficient to rule in or rule out AKI in an individual patient. Second, there are no specific validated interventions that could be applied based on this prognostic information. Nevertheless, these findings are useful in study settings to identify patients at risk of AKI who might be included in interventional trials. Finally, the question remains of whether these findings are generalizable to adult populations of AKI in the setting of CPB or other types of AKI. In conclusion, proteomic techniques are capable of not only identifying novel biomarkers (and thereby novel pathways) of AKI but also of validating the role of previously known proteins in the diagnosis and/or prognosis of AKI, as in the current study. With further improvement in our prognostic and diagnostic abilities in AKI, nephrologists may soon find themselves apply-
Editorial
611
ing specific therapeutic interventions and changing the course of AKI. Julie Ho, MD Martina Reslerova, MD, PhD Claudio Rigatto, MD, MSc University of Manitoba Winnipeg, Canada
ACKNOWLEDGEMENTS Financial Disclosure: The authors declare that they have no relevant financial interests.
REFERENCES 1. Chertow GM, Lazarus JM, Christiansen CL, et al. Preoperative renal risk stratification. Circulation. 1997;95(4): 878-884. 2. Thakar CV, Arrigain S, Worley S, Yared JP, Paganini EP. A clinical score to predict acute renal failure after cardiac surgery. J Am Soc Nephrol. 2005;16(1):162-168. 3. Mangano CM, Diamondstone LS, Ramsay JG, Aggarwal A, Herskowitz A, Mangano DT. Renal dysfunction after myocardial revascularization: risk factors, adverse outcomes, and hospital resource utilization. Ann Intern Med. 1998;128(3):194-203.
4. Loef BG, Epema AH, Smilde TD, et al. Immediate postoperative renal function deterioration in cardiac surgical patients predicts in-hospital mortality and long-term survival. J Am Soc Nephrol. 2005;16(1):195-200. 5. Devarajan P, Krawczeski CD, Nguyen MT, Kathman T, Wang Z, Parikh CR. Proteomic identification of early biomarkers of acute kidney injury after cardiac surgery in children. Am J Kidney Dis. 2010;56(4):632-642. 6. Bonventre JV, Weinberg JM. Recent advances in the pathophysiology of ischemic acute renal failure. J Am Soc Nephrol. 2003;14(8):2199-2210. 7. Sutton TA, Fisher CJ, Molitoris BA. Microvascular endothelial injury and dysfunction during ischemic acute renal failure. Kidney Int. 2002;62(5):1539-1549. 8. Hirschberg R, Kopple J, Lipsett P, et al. Multicenter clinical trial of recombinant human insulin-like growth factor I in patients with acute renal failure. 1999;55(6):24232432. 9. Lewis J, Salem MM, Chertow GM, et al. Atrial natriuretic factor in oliguric acute renal failure. Am J Kidney Dis. 2000;36(4):767-774. 10. Bellomo R, Kellum JA, Ronco C. Defining acute renal failure: physiological principles. Intensive Care Med. 2004;30(1):33-37. 11. Ronco C. N-GAL: diagnosing AKI as soon as possible. Crit Care. 2007;11(6):173.