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Journal of Investigative Medicine:
doi: 10.2310/6650.2007.00015
EB Symposium

Biomarkers of Acute Kidney Injury: Early Diagnosis, Pathogenesis, and Recovery

Parikh, Chirag R.; Edelstein, Charles L.; Devarajan, Prasad; Cantley, Lloyd

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Author Information

From the Section of Nephrology (C.R.P., L.C.), Department of Medicine, Yale University School of Medicine, New Haven, CT; Clinical Epidemiology Research Center (C.R.P.), Veterans Affairs Medical Center, West Haven, CT; Division of Renal Diseases (C.L.E.), University of Colorado Health Sciences Center, Denver, CO; Cincinnati Children's Hospital Medical Center (P.D.), University of Cincinnati, Cincinnati, OH.

All authors contributed equally to this article.

This symposium was supported by the Society of American Federation of Medical Research, the NIH, and Abbott Diagnostics. This publication was made possible by Grant Number 1 R13 RR023236‐01 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH.

Address correspondence to: Dr. Chirag R. Parikh, Clinical Epidemiology Research Center, 950 Campbell Avenue, Mail Box 151B, Bldg 35A, Room 219, West Haven, CT 06492; e‐mail: chirag.parikh@yale.edu.

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Abstract

This review article represents the proceedings from the translational research symposium presented at the Federation of American Societies for Experimental Biology/American Federation for Medical Research (FASEB/AFMR) meeting in May 2007 in Washington, DC. This research symposium consisted of four talks, which are individually summarized below. The first talk gave a general overview of the biomarkers in the setting of acute kidney injury (AKI), along with some data from clinical biomarker studies. The second talk discussed the preclinical studies of the emerging biomarkers in the area of AKI. The third talk discussed the biomarkers of AKI after deceased kidney transplantation, which is a unique model of kidney ischemia‐reperfusion injury. The final talk discussed the role of circulating stem cells in renal tubule regeneration after ischemic injury and if they have potential for biomarkers of recovery or therapeutic intervention.

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Talk 1: Role of Biomarkers in Diagnosis and Prognosis of Acute Kidney Injury

Speaker: Chirag R. Parikh, MD, PhD

The incidence of acute kidney injury (AKI) in hospitalized patients is approximately 7%.1 The incidence of AKI in the intensive care unit ranges between 5 and 25%2,3 and carries an overall mortality rate of 50 to 80%.3‐5 Tremendous progress has been made in our understanding of the molecular mechanisms of AKI. However, a translation of these findings to diagnostics and therapeutics used in clinical practice of AKI remains challenging. The mortality and morbidity rates associated with AKI remain dismally high and have not appreciably improved during the last four decades. The lack of significant progress in the prevention and management of AKI has been attributed, in part, to the failure to identify suitable physiologic surrogate end points for use in research studies testing the efficacy of new interventions.6 For example, the standardized use of serum cardiac enzyme concentrations and electrocardiographic criteria has facilitated rapid progress in the management of coronary insufficiency, markedly decreasing the morbidity and mortality of acute myocardial infarction. By contrast, AKI prevention and therapy studies using nonspecific variables of AKI such as urine output and serum and urine chemistries have not yielded interventions proven to decrease morbidity (requirement for dialysis) and mortality. In fact, few AKI studies have demonstrated a beneficial effect on the most commonly used physiologic surrogate end points, serum urea nitrogen and creatinine concentrations.

The diagnosis of AKI is usually based on either the elevation of serum creatinine or the detection of oliguria. Serum creatinine, however, is a poor marker of early renal function because the serum concentration is greatly influenced by changes in muscle mass and tubular secretion. Hence, the normal reference interval is relatively wide, and the use of serum creatinine alone to follow disease progression is fraught with imprecision. There are numerous nonrenal factors influencing the serum creatinine concentration, such as body weight, race, age, gender, total body volume, drugs, muscle metabolism, and protein intake. In AKI, serum creatinine is an even poorer reflection of kidney function because the patients are not in steady state; hence, serum creatinine lags far behind renal injury. Furthermore, significant renal disease (eg, fibrosis) can exist with minimal or no change in creatinine because of renal reserve, enhanced tubular secretion of creatinine, or other factors. Possibly, the interventions would have been successful if they could be initiated at the onset of AKI rather than waiting several days for creatinine to rise. The issues discussed above increase the risk of failure in drug development. These issues also increase the variability in the outcomes and magnify the size and cost of clinical studies.

Biomarkers and surrogate end‐point markers have many uses in laboratory and clinical investigations and in drug discovery. Biomarkers are useful for diagnosing, classifying, or grading the severity of disease in both laboratory and clinical settings. They may be able to provide efficacy, toxicity, and mechanistic information for the preclinical and clinical phases of drug discovery. Because biomarkers and surrogate end‐point markers can accelerate the speed and decrease the risk of drug discovery, they are highly sought after. A troponin‐like biomarker of AKI that is easily measured, unaffected by other biologic variables, and capable of both early detection and risk stratification would be a tremendous advance for clinical medicine (Figure 1).

Figure 1
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In the setting of AKI, biomarkers may assist with early diagnosis of kidney injury compared with creatinine, differential diagnosis of AKI, or prognosis of AKI. Clinical studies of two such biomarkers, interleukin‐18 (IL‐18) and neutrophil gelatinase‐associated lipocalin (NGAL), in AKI are discussed below.

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Interleukin‐18

IL‐18 was found, in human subjects, to be markedly elevated in established acute tubular necrosis (ATN).7 Importantly, this was in contrast to normal healthy subject controls and patients with chronic kidney disease, prerenal azotemia, urinary tract infection, and nephrotic syndrome. The area under the receiver operating characteristic (ROC) curve was 0.95, suggesting that IL‐18 is very accurate for diagnosis of ATN. This study supports the use of urinary IL‐18 in the differential diagnosis of AKI.

A subsequent study in acute respiratory distress syndrome (ARDS) patients in 2005 first investigated the potential of urinary IL‐18 as an early marker of AKI.8 On multivariate analysis, urine IL‐18 levels > 100 pg/mg predicted the development of AKI 24 hours before the serum creatinine, with an adjusted odds ratio of 6.5 and ROC of 73%. Importantly, urine IL‐18 on the day of initiation of mechanical ventilation was also predictive of mortality in ARDS patients independent of severity of illness scores, serum creatinine, and urine output.

Urinary IL‐18 has also demonstrated promise as an early marker of AKI following cardiac surgery.9 Using standard measures, specifically serum creatinine, AKI was first detected only 48 to 72 hours after cardiopulmonary bypass (CPB) in pediatric patients. We found that urine IL‐18 increased 4 to 6 hours after CPB, peaked at 12 hours, and remained elevated up to 48 hours. In this study, IL‐18 levels were also correlated with severity of AKI based on the duration (number of days) of AKI.

Urine IL‐18 is quite promising as a candidate biomarker for the early detection of AKI in ARDS and following CPB. IL‐18 also shows great promise as a biomarker in differential diagnosis and prognosis. Prospective studies in larger patient population are under way and will be a critical step in validating the role of this biomarker.

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Neutrophil Gelatinase‐Associated Lipocalin

The first human study of NGAL (serum and urine) was published in pediatric patients undergoing CPB in which 28% of all children developed AKI, not detectable by serum creatinine until 1 to 3 days after CPB. In contrast, urine NGAL measurements were elevated as early as 2 hours post‐CPB with a specificity of 1 and a sensitivity of 0.98, for a urine NGAL cutoff value of 50 μg/L. Although this study of pediatric patients at a single center represents a quite homogeneous population with no associated comorbidities, this was a pivotal trial exploring the potential of NGAL as a novel biomarker.

A similar study in adult cardiac surgery patients, in which 20% developed AKI following CPB, again demonstrated the potential of NGAL as an early biomarker.10 However, the performance characteristics of NGAL were significantly lower than in the pediatric study.

In a study of adult patients undergoing percutaneous coronary intervention, NGAL levels in the urine and the blood were found to be elevated as early as 2 hours postprocedure; however, the utility as a biomarker in this setting is limited as none of the patients went on to develop contrast nephropathy or AKI.11

The power and clinical utility of biomarkers in AKI will most likely emerge through a combination of biomarkers. The most interesting results were identified in the pediatric cardiac surgery study in which both NGAL and IL‐18 were found to increase, in tandem, well before the serum creatinine.9 When considered together, these two biomarkers have an added advantage in predicting the severity and number of days of AKI. The combined strength of these two biomarkers supports the development of a clinically useful biomarker panel for early prediction, as well as prognosis or risk stratification.

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References

1. Nash K, Hafeez A, Hou S. Hospital-acquired renal insufficiency Am J Kidney Dis 2002;39:930-6.

2. de Mendonca A, Vincent JL, Suter PM, et al. Acute renal failure in the ICU: risk factors and outcome evaluated by the SOFA score Intensive Care Med 2000;26:915-21.

3. Liano F, Junco E, Pascual J, et al. The spectrum of acute renal failure in the intensive care unit compared with that seen in other settings. The Madrid Acute Renal Failure Study Group Kidney Int Suppl 1998;66:S16-24.

4. Thadhani R, Pascual M, Bonventre JV. Acute renal failure N Engl J Med 1996;334:1448-60.

5. Schrier RW, Wang W, Poole B, Mitra A. Acute renal failure: definitions, diagnosis, pathogenesis, and therapy J Clin Invest 2004;114:5-14.

6. American Society of Nephrology Renal Research Report. J Am Soc Nephrol 2005;16:1886-903.

7. Parikh CR, Jani A, Melnikov VY, et al. Urinary interleukin-18 is a marker of human acute tubular necrosis Am J Kidney Dis 2004;43:405-14.

8. Parikh CR, Abraham E, Ancukiewicz M, Edelstein CL. Urine IL-18 is an early diagnostic marker for acute kidney injury and predicts mortality in the intensive care unit J Am Soc Nephrol 2005;16:3046-52.

9. Parikh CR, Mishra J, Thiessen-Philbrook H, et al. Urinary IL-18 is an early predictive biomarker of acute kidney injury after cardiac surgery Kidney Int 2006;70:199-203.

10. Wagener G, Jan M, Kim M, et al. Association between increases in urinary neutrophil gelatinase-associated lipocalin and acute renal dysfunction after adult cardiac surgery Anesthesiology 2006;105:485-91.

11. Bachorzewska-Gajewska H, Malyszko J, Sitniewska E, et al. Neutrophil-gelatinase-associated lipocalin and renal function after percutaneous coronary interventions Am J Nephrol 2006;26:287-92.

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Talk 2: Preclinical Studies of Biomarkers of Acute Kidney Injury

Speaker: Charles L. Edelstein MD, PhD

Preclinical studies of the pathophysiology of acute kidney injury (AKI) have contributed immensely to the development of biomarkers of AKI. The pathophysiology of AKI involves tubular, inflammatory, and vascular factors and has been reviewed in detail.1,2 Our preclinical studies of the pathophysiology of AKI involving caspase‐1 led to the discovery of interleukin (IL)‐18 as a biomarker of AKI.

Caspases are intracellular cysteine proteases. Caspase‐3 is the major mediator of apoptotic cell death. Caspase‐1 (previously known as IL‐1β‐converting enzyme) plays a major role in the activation of the proinflammatory cytokines IL‐1β and IL‐18.3 IL‐18 is involved in diverse functions, including inflammation (innate immunity), ischemic tissue injury, and T cell‐mediated immunity. IL‐18 is a mediator of ischemic tissue injury in the heart4 and brain.5 IL‐18 plays an important role in the activation of macrophages and natural killer cells.6 Thus, we determined whether caspase‐1‐mediated production of IL‐18 played a role in ischemic AKI.

Caspase‐1‐deficient mice developed less ischemic AKI as judged by renal function and renal histology.7 Lack of the active form of IL‐18 was investigated as a possible mechanism of this protection. Kidney IL‐18 was more than 100% increased in wild‐type AKI compared with sham‐operated controls. On immunoblot analysis, there was a conversion of the precursor to the active form of IL‐18 in AKI wild‐type mice but not in the caspase‐1‐deficient AKI mice and sham‐operated controls. To further analyze the role of IL‐18, wild‐type mice were injected with rabbit antimurine IL‐18 neutralizing antiserum prior to the ischemic insult. These mice were protected against AKI to a degree similar to that of caspase‐1‐deficient mice. The conclusion of this study was that IL‐18 plays a deleterious role in ischemic AKI.

Caspase‐deficient mice have provided extensive information about the role of individual caspases in disease processes. The study of caspase inhibitors is an important initial step toward the possible therapeutic effects of caspase inhibition. Thus, we determined the effect of the pancaspase inhibitor OPH‐001 on caspase‐1, IL‐18, neutrophil infiltration, and renal function in ischemic AKI.8 Mice with ischemic AKI treated with OPH‐001 had a marked (100%) reduction in blood urea nitrogen and serum creatinine and a highly significant reduction in the morphologic acute tubular necrosis (ATN) score than did vehicle‐treated mice. OPH‐001 significantly reduced the increase in caspase‐1 activity and IL‐18 and prevented neutrophil infiltration in the kidney during ischemic AKI. To further investigate whether this lack of neutrophil infiltration was contributing to the protection against ischemic AKI, a model of neutrophil depletion was developed. Neutrophil‐depleted mice had a small (18%) reduction in serum creatinine during ischemic AKI but no reduction in the ATN score despite a lack of neutrophil infiltration in the kidney. Remarkably, caspase‐1 activity and IL‐18 were still significantly increased in the kidney in neutrophil‐depleted mice with AKI. IL‐18 antiserum‐treated neutrophil‐depleted mice with ischemic AKI had a significant (75%) reduction in serum creatinine and a significant reduction in the ATN score compared with vehicle‐treated neutrophil‐depleted mice. These results suggest a novel neutrophil‐independent mechanism of IL‐18‐mediated ischemic AKI.

Studies in mice demonstrated that IL‐18 is a mediator of ischemic AKI.7,8 Immunohistochemistry of both mouse and human kidneys demonstrated an increase in IL‐18 protein in injured tubular epithelial cells in AKI kidneys compared with normal controls. Thus, the question arose as to whether IL‐18 could be released from the injured tubular epithelial cells into the urine and serve as a urinary biomarker of AKI. Urine IL‐18 was massively increased in mice with AKI compared with unmeasurable levels in control mice.7 Subsequent studies in humans demonstrated that urine IL‐18 is an early predictive biomarker of AKI.9‐12

Neutrophil gelatinase‐associated lipocalin (NGAL) is a 21 kDa protein of the lipocalin superfamily. NGAL is a critical component of innate immunity to bacterial infection and is expressed by immune cells, hepatocytes, and renal tubular cells in various disease states.13 NGAL protein increases massively in the kidney and urine in early ischemic AKI in mice.14

Kidney injury molecule 1 (KIM‐1) is a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain. KIM‐1 messenger ribonucleic acid and protein are expressed at a low level in normal kidney but are increased dramatically in postischemic kidney.15 Urinary KIM‐1 is a biomarker for the early detection of both cisplatin‐induced AKI and ischemic AKI in mice.16

In summary, in preclinical studies, the proinflammatory cytokine IL‐18 is both a mediator and a biomarker of AKI, as evidenced by the following: (1) IL‐18 expression increases in the kidney in AKI, (2) inhibition of IL‐18 is protective against AKI, and (3) IL‐18 increases in the urine in both mice and humans with AKI. Also, in preclinical studies, urinary NGAL and urinary KIM‐1 are early biomarkers of AKI in mice (Table 1).

Table 1
Table 1
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References
1. Lameire N, Van Biesen W, Vanholder R. The changing epidemiology of acute renal failure Nat Clin Pract Nephrol 2006;2:364-77.

2. Edelstein CL, Schrier RW. Pathophysiology of ischemic acute renal injury. In: Schrier RW, editor. Diseases of the kidney and urinary tract. Vol 2, 8th ed. Philadelphia: Lippincott, Williams and Wilkins; 2007. p. 930-61.

3. Dinarello CA. Biologic basis for interleukin-1 in disease Blood 1996;87:2095-147.

4. Pomerantz BJ, Reznikov LL, Harken AH, Dinarello CA. Inhibition of caspase 1 reduces human myocardial ischemic dysfunction via inhibition of IL-18 and IL-1beta Proc Natl Acad Sci U S A 2001;98:2871-6.

5. Hedtjarn M, Leverin AL, Eriksson K, et al. Interleukin-18 involvement in hypoxic-ischemic brain injury J Neurosci 2002;22:5910-9.

6. Lochner M, Forster I. Anti-interleukin-18 therapy in murine models of inflammatory bowel disease Pathobiology 2002;70:164-9.

7. Melnikov VY, Ecder T, Fantuzzi G, et al. Impaired IL-18 processing protects caspase-1-deficient mice from ischemic acute renal failure J Clin Invest 2001;107:1145-52.

8. Melnikov VY, Faubel SG, Siegmund B, et al. Neutrophil-independent mechanisms of caspase-1- and IL-18-mediated ischemic acute tubular necrosis in mice J Clin Invest 2002;110:1083-91.

9. Parikh CR, Jani A, Melnikov VY, et al. Urinary interleukin-18 is a marker of human acute tubular necrosis Am J Kidney Dis 2004;43:405-14.

10. Parikh CR, Abraham E, Ancukiewicz M, Edelstein CL. Urine IL-18 is an early diagnostic marker for acute kidney injury and predicts mortality in the ICU J Am Soc Nephrol 2005;16:3046-52.

11. Parikh CR, Mishra J, Thiessen-Philbrook H, et al. Urinary IL-18 is an early predictive biomarker of acute kidney injury after cardiac surgery Kidney Int 2006;70:199-203.

12. Parikh CR, Jani A, Mishra J, et al. Urine NGAL and IL-18 are predictive biomarkers for delayed graft function following kidney transplantation Am J Transplant 2006;6:1639-45.

13. Schmidt-Ott KM, Mori K, Li JY, et al. Dual action of neutrophil gelatinase-associated lipocalin J Am Soc Nephrol 2007;18:407-13.

14. Mishra J, Ma Q, Prada A, et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury J Am Soc Nephrol 2003;14:2534-43.

15. Ichimura T, Bonventre JV, Bailly V, et al. Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury J Biol Chem 1998;273:4135-42.

16. Vaidya VS, Ramirez V, Ichimura T, et al. Urinary kidney injury molecule-1: a sensitive quantitative biomarker for early detection of kidney tubular injury Am J Physiol Renal Physiol 2006;290:F517-29.

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Talk 3: Biomarkers of Acute Kidney Injury after Deceased Kidney Transplantation

Speaker: Prasad Devarajan, MD

Acute kidney injury (AKI), also referred to as acute renal failure, represents a common and devastating problem in clinical medicine, with a persistently high mortality and morbidity.1,2 AKI owing to ischemia‐reperfusion occurs to some extent almost invariably in kidney transplants. The ensuing degree of renal dysfunction is proportionate to the duration of cold ischemia, rendering kidney transplantation a temporally defined and predictable model for the study of early events in AKI. Delayed graft function (DGF), most commonly defined as dialysis requirement within 7 days of surgery, is a serious consequence of AKI in the transplant setting and occurs in up to 10% of live donor and 50% of deceased donor kidney transplants.3 In addition to the well‐known clinical complications of acute renal failure and dialysis, DGF predisposes the graft to both acute and chronic rejection, is an independent risk factor for suboptimal graft function at 1 year post‐transplant, and increases the risk of chronic allograft nephropathy and graft loss. Clinical definitions of DGF employ urine output, creatinine reduction ratios, or dialysis requirement, but these variables typically identify DGF only several days after kidney transplantation. Consequently, early therapeutic interventions that have ameliorated DGF in animal models have been ineffective in human studies, at least in part owing to the paucity of early biomarkers for AKI.

In general, biomarker discovery proceeds in three phases of translation from the bench to the bedside. The initial discovery phase involves identification of candidate biomarkers using basic science technologies. The second translation phase requires the development of robust clinical assays for the candidate biomarkers and testing in defined populations. The final validation phase entails testing the assays in a large clinical trial. For the discovery phase, we used a genome‐wide interrogation strategy to identify kidney genes that are induced very early after ischemia‐reperfusion injury in animal models. We identified neutrophil gelatinase‐associated lipocalin (NGAL) as one of the most dramatically upregulated genes in the kidney after ischemia.4‐6 Importantly, NGAL protein was markedly induced and easily detected in the kidney tubules, urine, and plasma, very early after ischemia‐reperfusion injury in animals. For the translation phase, we designed a sensitive and specific NGAL enzyme‐linked immunosorbent assay (ELISA), with excellent inter‐ and intra‐assay coefficients of variation. Using this assay, NGAL was found to be a highly predictive urinary and plasma biomarker of AKI in patients undergoing cardiac surgery.7 We therefore tested the hypothesis that kidney and urine NGAL represent novel early biomarkers of AKI in another well‐defined human population, namely kidney transplantation. Since our previous studies have shown that urine interleukin‐18 (IL‐18) levels are increased in animal models and humans with established DGF,8 we also assessed the utility of IL‐18 measurements (using a commercially available ELISA kit) for the prediction of DGF.

We first tested the hypothesis that expression of NGAL in the kidney is an early marker of AKI following transplantation. Sections from paraffin‐embedded protocol biopsy specimens obtained at approximately 1 hour of reperfusion after transplantation of 13 deceased and 12 living‐related renal allografts were examined in a double‐blind fashion for expression of NGAL by immunohistochemistry. Using a scoring system of 0 (no staining) to 3 (most intense staining), NGAL expression was significantly increased in deceased donor specimens (2.3 ± 0.8 versus 0.8 ± 0.7 in living donors, p < .001). NGAL staining was strongly correlated with peak postoperative serum creatinine, which occurred days later (r = .86, p < .001). Patients who developed DGF requiring dialysis during the first week post‐transplantation displayed the most intense NGAL staining. Thus, kidney NGAL represents a novel predictive biomarker of DGF following transplantation.9 However, the invasive nature of a kidney biopsy limits the clinical utility of this biomarker.

We then tested whether urinary NGAL and IL‐18 represent early, noninvasive biomarkers for DGF (defined as dialysis requirement within the first week after transplantation). In a prospective multicenter study, urine samples collected on day 0 from recipients of living donor kidneys (n = 23), deceased kidneys with prompt graft function (n = 20), and deceased kidneys with DGF (n = 10) were analyzed in a double‐blind fashion by ELISA for NGAL and IL‐18. In patients with DGF, peak postoperative serum creatinine requiring dialysis typically occurred 2 to 4 days after transplantation. Urine NGAL and IL‐18 values were significantly different in the three groups on day 0, with maximally elevated levels noted in the DGF group (p < .0001). The receiver operating characteristic curve for prediction of DGF based on urine NGAL or IL‐18 at day 0 revealed an area under the curve of 0.9 for both assays, indicative of excellent biomarkers. By multivariate analysis, both urine NGAL and IL‐18 on day 0 predicted the trend in serum creatinine in the post‐transplantation period after adjusting for the effects of age, gender, race, urine output, and cold ischemia time (p < .01). Thus, both urine NGAL and IL‐18 represent early, predictive biomarkers of DGF.10 Based on our previous results in subjects who developed AKI following cardiac surgery11 and in analogy with cardiac markers that have revolutionized the care of patients with chest pain, we propose that both urinary NGAL and IL‐18 may represent sequential biomarkers of DGF, as illustrated in Figure 2.

Figure 2
Figure 2
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However, there are limitations to this study that apply to the field of biomarkers of AKI in general. First, the majority of reports represent a relatively small number of patients, and the results will need to be prospectively validated in a larger population. Second, the possible confounding effects of comorbid conditions on NGAL and IL‐18 excretion are unknown. Third, it will be important to partner with industry to design point‐of‐care kits and platforms for biomarker panels that (1) are easy to perform at the bedside or in a standard clinical laboratory, using easily accessible samples such as blood or urine; (2) are highly sensitive to facilitate early detection and with a wide dynamic range and cutoff values that allow for risk stratification; (3) are highly specific for AKI and enable the identification of AKI subtypes, etiologies, and duration; and (4) exhibit strong biomarker properties on receiver operating characteristic curves. The availability of such tools will enable the design of rational interventional studies that are initiated early in the course of AKI.

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References
1. Lamiere N, Van Biesen W, Vanholder R. Acute renal failure Lancet 2005;365:417-30.

2. Devarajan P. Update on mechanisms of ischemic acute kidney injury J Am Soc Nephrol 2006;17:1503-20.

3. Perico N, Cattaneo D, Sayegh MH, Remuzzi G. Delayed graft function in kidney transplantation Lancet 2004;364:1814-27.

4. Supavekin S, Zhang W, Kucherlapati R, et al. Differential gene expression following early renal ischemia-reperfusion Kidney Int 2003;63:1714-24.

5. Mishra J, Ma Q, Prada A, et al. Identification of NGAL as a novel urinary biomarker for ischemic injury J Am Soc Nephrol 2003;4:2534-43.

6. Devarajan P, Mishra J, Supavekin S, et al. Gene expression in early ischemic renal injury: clues towards pathogenesis, biomarker discovery and novel therapeutics Mol Genet Metab 2003;80:365-76.

7. Mishra J, Dent C, Tarabishi R, et al. Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury following cardiac surgery Lancet 2005;365:1231-8.

8. Parikh CR, Jani A, Meinikov VY, et al. Urinary interleukin-18 is a marker of human acute tubular necrosis Am J Kidney Dis 2004;43:405-14.

9. Mishra J, Ma Q, Kelly C, et al. Kidney NGAL is a novel early marker of acute injury following transplantation Pediatr Nephrol 2006;21:856-63.

10. Parikh CR, Jani A, Mishra J, et al. Urine NGAL and IL-18 are predictive biomarkers for delayed graft function following kidney transplantation Am J Transplant 2006;6:1639-45.

11. Parikh CR, Mishra J, Thiessen-Philbrook H, et al. Urinary IL-18 is an early predictive biomarker of acute kidney injury after cardiac surgery Kidney Int 2006;70:199-203.

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Talk 4: Stem Cells in Acute Kidney Injury: Therapeutic Potential or Biomarkers?

Speaker: Lloyd Cantley, MD

The epithelial cells of the renal tubule are uniquely susceptible to injury owing to their relatively poor oxygen supply compared to energy demand and their role in transporting large amounts of solute during the process of reclamation of the glomerular filtrate. This susceptibility to injury leads to a fairly high incidence of acute kidney injury (AKI) under conditions of hemodynamic stress and the need for pathways of epithelial protection and/or repair if the organism is to survive.

Recently, biopsy studies of female kidneys transplanted into human male recipients have suggested that Y chromosome‐containing cells can be found in the tubular epithelium.1,2 Similar results have been obtained in mice in which male bone marrow was transplanted into female recipients, leading to the hypothesis that bone marrow may contain stem cells that are mobilized into the circulation and directly contribute to tubular repair following injury.3,4

Analysis of circulating nucleated cells in uninjured mice reveals that the vast majority, 99.5%, express surface markers, identifying them as lymphocytes, monocytes, or polymorphonuclear leukocytes. The remaining 0.5% are lineage negative and will attach to a substrate and proliferate under standard tissue culture conditions. Continuous culture of peripheral blood from the mouse for > 14 days reveals that these lineage‐negative cells lack expression of the hematopoietic stem cell (HSC) marker c‐Kit or of the endothelial progenitor cell marker Flk1. Instead, the expression profile of these cells is most similar to that of marrow stromal cells (MSCs; also called mesenchymal stem cells).

Following transient ischemia and reperfusion, the kidney has been found to upregulate the expression of several factors that can mobilize bone marrow‐derived cells into the circulation, including interleukin‐6, stromal derived factor‐1 (SDF‐1), and Groα (also called keratinocyte‐derived chemokine [KC]). Consistent with this, circulating lineage‐negative cells increased following ischemic renal injury and represented approximately 2 to 2.5% of nucleated cells at 24 to 48 hours following reperfusion. Of note, there was a wide variation in cell mobilization, with approximately 30% of mice exhibiting no detectable mobilization of lineage‐negative cells. However, if mice are subjected to bone marrow ablation prior to the ischemia‐reperfusion, thus eliminating these bone marrow‐derived cells, initial renal injury was more severe than in control mice, and there was no evidence of recovery during a 7‐day follow‐up. This negative effect of bone marrow ablation was prevented by intravenous administration of lineage‐negative (ie, HSC + MSC) bone marrow cells after the renal injury.4

To test the hypothesis that renal injury mobilizes bone marrow stem cells into the circulation so that they can enter the kidney and mediate tubular regeneration, we studied the effects of infusion of either lineage‐negative bone marrow cells (containing both HSCs and MSCs) or MSCs alone in mice subjected to renal ischemia‐reperfusion or cisplatin nephrotoxicity. To track the injected cells, male donor cells were injected into female recipient mice, with fluorescence in situ hybridization performed on kidney sections at 1 to 7 days after renal injury to identify Y chromosome‐containing cells. In animals that had received transplantation with 2 to 5 × 105 lineage‐negative bone marrow cells (containing both the HSC and MSC cell populations), multiple Y chromosome‐containing cells were detected in the renal interstitium, primarily in areas surrounding the injured tubules. The majority (≈70%) of these interstitial cells were found to be macrophages. In contrast to this large number of interstitial cells, only rare examples of Y chromosome cells residing within the tubules were seen (< 0.01% of all tubular cells). Furthermore, using BrdU uptake, PCNA staining and Ki‐67 staining, only one example of a bone marrow‐derived cell entering the tubule and undergoing proliferation was detected. The vast majority of cells undergoing proliferation in response to the injury were instead judged to be endogenous tubular cells.

Since bone marrow cells can enter the kidney following injury but fail to become tubular cells in significant numbers under these conditions, we examined the alternative possibility that these cells exhibited paracrine or endocrine effects that positively influence the response of endogenous tubular cells to renal injury. Studies by several groups have demonstrated that the MSC fraction of bone marrow can exert renoprotective effects when injected intravenously.5,6 Our experiments confirmed these observations, showing that blood urea nitrogen (BUN) values were lower in mice that received 2 × 105 MSCs after treatment with cisplatin compared with mice that received vehicle control. Histologic examination of the kidneys from these experiments revealed no examples of Y chromosome‐containing cells in the renal tubules and only rare examples of Y chromosome‐containing cells in the renal interstitium. Instead, TUNEL staining demonstrated that the rate of apoptosis of endogenous tubular cells was significantly less in MSC‐treated mice, whereas PCNA staining and BrdU uptake revealed that the rates of proliferation were higher. From these results, we conclude that MSCs protect the kidney from acute injury via an endocrine effect.

To further examine the possibility that MSCs secrete factors that have endocrine effects on renal epithelia, we used coculture of renal epithelia and MSCs. Primary cultures of cells isolated from microdissected renal tubule segments, or immortalized mouse renal proximal tubule cells, were found to proliferate in response to coculture with MSCs or with media conditioned by previous exposure to MSCs (MSC‐CM). Furthermore, cisplatin‐mediated death of mouse proximal tubule cells was partially inhibited by culture in MSC‐CM but not by culture in conditioned media from mouse proximal tubule cells themselves. Finally, daily intraperitoneal injection of MSC‐CM diminished the rates of apoptosis, tubule necrosis, and rise in BUN seen following cisplatin administration to the mouse.

Cumulatively, these studies argue that bone marrow‐derived cells are mobilized into the circulation in small numbers following AKI, although the large variation in this mobilization makes it unlikely that quantitation of these cells will be a useful biomarker for AKI. The majority of these cells appear to be HSC derivatives that can enter the renal interstitium and differentiate into inflammatory cells such as macrophages or, in rare cases, differentiate into or fuse with tubular cells. However, a second population of mobilized bone marrow cells are the MSCs that do not enter the kidney parenchyma in appreciable numbers but instead appear to secrete factors that are renoprotective (Figure 3). The importance of mobilization and/or activation of these cells for the induction of the renoprotective effect remains to be determined.

Figure 3
Figure 3
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References
1. Poulsom R, Forbes SJ, Hodivala-Dilke K, et al. Bone marrow contributes to renal parenchymal turnover and regeneration J Pathol 2001;195:229-35.

2. Gupta S, Verfaillie C, Chmielewski D, et al. A role for extrarenal cells in the regeneration following acute renal failure Kidney Int 2002;62:1285-90.

3. Lin F, Cordes K, Li L, et al. Hematopoietic stem cells contribute to the regeneration of renal tubules after renal ischemia-reperfusion injury in mice J Am Soc Nephrol 2003;14:1188-99.

4. Kale S, Karihaloo A, Clark PR, et al. Bone marrow stem cells contribute to repair of the ischemically injured renal tubule J Clin Invest 2003;112:42-9. [Epub 2003 Jun 2016].

5. Morigi M, Imberti B, Zoja C, et al. Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure J Am Soc Nephrol 2004;15:1794-804.

6. Togel F, Hu Z, Weiss K, et al. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms Am J Physiol Renal Physiol 2005;15:F31-42.

Key words:: renal failure; translational research; stem cells; delayed graft function

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