Plasma Neutrophil Gelatinase-Associated Lipocalin (NGAL) Correlations With Cystatin C, Serum Creatinine, and Glomerular Filtration Rate in Patients After Heart and Lung Transplantation

Plasma Neutrophil Gelatinase-Associated Lipocalin (NGAL) Correlations With Cystatin C, Serum Creatinine, and Glomerular Filtration Rate in Patients After Heart and Lung Transplantation M. Szewczyk, T. Wielkoszyn´ski, M. Zakliczyn´ski, and M. Zembala ABSTRACT Objective. Kidney injury represents a major clinical problem in orthotopic heart transplant (OHT) patients which seriously increases the mortality rate. The aim of this work was to evaluate the utility of a new kidney damage marker—neutrophil gelatinaseassociated lipocalin (NGAL)—and its correlations with cystatin C, creatinine, and glomerular filtration rate (GFR) among patients after heart or lung transplantation. Materials and Methods. We examined serum samples from 71 patients after heart transplantation, 7 patients after lung transplantation, and 20 healthy controls to measure serum NGAL using an enzyme-linked immunosorbent assay (ELISA) method; cystatin C using latex immunonephelometry; and creatinine using the alkaline-picrate method. Results. Serum NGAL levels were significantly elevated among transplant patients, but did not significantly correlate with cystatin C in the transplant group (R ⫽ .13; P ⫽ .11) or in the control group (R ⫽ .26; P ⫽ .26), or GFR in the transplant group (R ⫽ ⫺.09; P ⫽ .25) or in the control cohort (R ⫽ .22; P ⫽ .36). In the transplant group, serum NGAL positively correlated with serum creatinine (R ⫽ .27; P ⫽ .001). Conclusions. Plasma NGAL was not a specific biomarker for monitoring chronic renal disorders. We did not exclude other pathologies that might contribute to increased serum NGAL levels. EUTROPHIL GELATINASE-associated lipocalin (NGAL) is a 21-kD protein of the secreted lipocalin superfamily of cytosolic proteins with barrel-like structures, carrying hydropholic ligands.1 The ligand of NGAL, a siderophore, was discovered by Goetz et al.2 Siderophores are a diverse group of small, nonpeptide, iron (Fe3⫹)binding chemicals produced in bacteria, fungi, and plants. Surprisingly, NGAL-bacterial siderophore-iron complex shows much stronger activity than NGAL-siderophore (without iron) or apo-NGAL (without siderophore).1 The biological activity of NGAL depends on the presence of iron.3 NGAL seems to display complex activities beyond its antimicrobial effects. Mori et al4 reported that NGAL protein accumulated in blood, urine, and renal proximal and distal tubules in acute renal failure. In cases associated with renal ischemia (sepsis, hypovolemia, and heart failure), nephrotoxicity (antibiotics, cisplatin, bisphosphonate, nonsteroidal anti-inflammatory drugs, radiocontrast, and hemoglobinuria), kidney parenchymal damage, hemolyticuremic syndrome, and posttransplant rejection,4 plasma

N

NGAL levels are elevated. NGAL expression is induced not only in kidney injury, but also in epithelial inflammation of the intestine,5 skin and airways,6 bacterial infections,7 and cancer.8 Moreover, for clinical usefulness, blood and urinary NGAL concentrations may be regarded as extremely early markers of acute kidney disease, namely, as early as 1 to 2 hours after the cardiopulmonary bypass surgery in children and adults.1 Increased NGAL levels are observed at 2 to 4 hours after percutanous coronary intervention (PCI), because contrast nephropathy is a frequent, potentially serious complication of PCI.9 The aim of this work was to evaluate the utility of serum NGAL concentrations to assess renal function as well as From the Silesian Center for Heart Diseases (M.S., M.Za., M.Ze.), Zabrze, Poland and Department of Chemistry (T.W.), Medical University of Silesia, Zabrze, Poland. Address reprint requests to Marta Szewczyk, Silesian Center for Heart Diseases, ul. Szpitalna 2, 41-800 Zabrze, Poland. E-mail: [email protected]

0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.08.018

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Transplantation Proceedings, 41, 3242–3243 (2009)

UTILITY OF NGAL AS KIDNEY DAMAGE MARKER

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correlate with cystatin C, creatinine, and glomerular filtration rate (GFR) among patients after heart or lung transplantation. MATERIALS AND METHODS We studied 78 recipients of cardiac (n ⫽ 71) or lung (n ⫽ 7) transplantations including 62 men and 16 women whose overall mean age was 46 ⫾ 12 years. Serum was separated and frozen from blood samples collected from patients at visits to examine immunosuppressive drug levels. The control group consisted of 20 healthy volunteers of overall mean age of 43 ⫾ 5 years. Serum NGAL was measured using an enzyme-linked immunosorbent assay (ELISA) method (Hycult Biotechnology); cystatin C using latex immunonephelometry (Siemens), and creatinine using an alkaline-picrate method (Cobas Integra, Roche). The statistical analysis was performed using the Mann-Whitney U test with correlations evaluated using the Spearman rank correlation test.

RESULTS

Serum NGAL, cystatin C, and creatinine concentrations as well as GFR values are presented in Table 1. All examined parameters (except for creatinine) were significantly elevated among transplant patients. We did not observe a significant correlation between serum NGAL and cystatin C levels in the transplant group (R ⫽ .1315; P ⫽ .1162) or in the control group (R ⫽ .2627; P ⫽ .2629) similarly, GFR was not correlated with NGAL in the transplant group (R ⫽ .0964; P ⫽ .2505) in the controls (R ⫽ .2176; P ⫽ .3567). Serum NGAL levels significantly and positively correlated with serum creatinine (R ⫽ .2670; P ⫽ .0013) among the transplant group. DISCUSSION

NGAL expression is an extensively studieds biomarker for renal diseases, but many other pathologies are associated with elevated levels of urinary or humoral NGAL. However, measurement of NGAL levels in other disorders, eg, infection, must be treated with caution, since bystander neoplastic conditions or renal disorders themselves increase Table 1. Concentrations (Means ⴞ SD) of Serum NGAL, Cystatin C, and Creatinine as Well as GFR Values in Transplant Recipients and Controls

NGAL (ng/mL) Cystatin C (mg/mL) GFR (mL/min) Creatinine (␮mol/L)

Transplant Group

Control Group

P

168.5 ⫾ 73.1 1.46 ⫾ 0.75 59.8 ⫾ 29.7 111.4 ⫾ 59.1

64.1 ⫾ 31.4 0.60 ⫾ 0.08 154.8 ⫾ 25.3 88.2 ⫾ 25.4

.05 .05 .01 NS

NGAL levels. These conditions therefore distort the measurements and interpretations of the results. The results reported herein have confirmed that NGAL is potentially important biomarker in clinical nephrology, extending to chronic kidney disease as an index of its pathophysiologic role in tubular adaptations to renal damage.10 Sustained production of NGAL in chronic kidney disease is probably a compensatory defensive mechanism, similar to that during acute kidney injury.10 We did not observe significant correlations between serum NGAL levels and renal function parameters of cystatin C and GFR. We could not exclude that other pathologies may contribute to increased serum NGAL levels: hypertension, endothelial dysfunction, atherosclerosis, and vascular damage. Since NGAL was measured in blood serum, this concentration expresses overall body NGAL production (systemic NGAL balance), not only the local, renal NGAL production. Also, immunosuppressive drugs and other conditions may cause significantly higher NGAL levels and cystatin C concentrations and GFR values among the transplant group compared with the controls. REFERENCES 1. Mori K, Nakao K: Neutrophil gelatinase-associated lipocalin as the real-time indicator of active kidney damage. Kidney Int 71:967, 2007 2. Goetz DH, Holmes MA, Borregaard N, et al: The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell 10:1033, 2002 3. Barasch J, Mori K: Cell biology: iron thievery. Nature 432: 811, 2004 4. Mori K, Lee HT, Rapoport D, et al: Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest 115:610, 2005 5. Playford RJ, Belo A, Poulsom R, et al: Effects of mouse and human lipocalin homologues 24p3/lcn 2 and neutrophil gelatinaseassociated lipocalin on gastrointestinal mucosal integrity and repair. Gastroenterology 131:809, 2006 6. Cowland JB, Sorensen OE, Sehested M, et al: Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, but not TNF-alpha. J Immunol 171:6630, 2003 7. Flo TH, Smith KD, Sato S, et al: Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432:917, 2004 8. Hanal J, Mammoto T, Seth P, et al: Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cell. J Biol Chem 280:13641, 2005 9. Bachorzewska-Gajewska H, Malyszko J, Sitniewska E, et al: Neutrophil-gelatinase-associated lipocalin and renal function after percutaneous coronary interventions. Am J Nephrol 26:287, 2006 10. Thomas LN, Barasch J, Devarajan P: Biomarkers in acute and chronic kidney disease. Curr Opin Nephrol Hypertens 17:127, 2008