- Email: [email protected]
Urinary Exosomes as a Source of Kidney Dysfunction Biomarker in Renal Transplantation
Urinary Exosomes as a Source of Kidney Dysfunction Biomarker in Renal Transplantation S. Alvarez, C. Suazo, A. Boltansky, M. Ursu, D. Carvajal, G. Innocenti, A. Vukusich, M. Hurtado, S. Villanueva, J.E. Carreño, A. Rogelio, and C.E. Irarrazabal ABSTRACT End-stage renal disease (ESRD) requires for its treatment permanent dialysis or kidney transplantation (KT). KT is the best clinical treatment, however, the early function of the allograft varies depending on multiple factors associated with cold ischemia time (CIT) and the allograft rejection process. It is known that serum creatinine is an insensitive and late marker for predicting graft recovery after KT, mainly in patients with delayed graft function (DGF). Neutrophil gelatinase-associated lipocalin (NGAL) is produced in the distal nephron and it is one of the most promising novel biomarkers for acute kidney injury (AKI) and chronic kidney disease (CKD). NGAL has been proposed to be a predictor of organ recovery from DGF after KT from donors after cardiac death. Because nonrenal diseases can also induce NGAL, more information is necessary to validate the sensitivity and specificity of urine and plasma NGAL in clinical samples. The exosomes are vesicles released into the urine from the kidney epithelium and they have been proposed as better source to explore as biomarker of renal dysfunction. The molecular composition of the urinary exosomes could be representative of the physiological or physiopathologic condition of the urinary system. We propose that determination of NGAL in urinary exosomes is a better predictor of kidney dysfunction after KT than other urinary fractions. We analyzed 15 kidney allograft recipients, with a mean age of 36 years (range, 16e60 years) and 75% were male: 11 living donors (LD) and 4 deceased donors (DD). The average length of CIT was 14 hours in DD and less than 1 hour in LD. Three patient developed DGF. Using Western blot analysis, NGAL was detectable in the cellular and exosomal fraction of the urine. The exosomes expressed higher levels of NGAL than the cellular fraction. The expression of NGAL was observed from the first day after transplantation. In the cellular fraction of the urine, no significant differences of NGAL were observed between the patients. However, the median of NGAL expression in the exosomes fraction was significantly higher in DD patient, from the first day after KT (P < .05). Moreover, we noticed that NGAL expression in exosomes remained elevated in the patients with DGF compared with non-DGF patients (P < .05). Considering the highest abundance of NGAL in the urinary exosomes and its correlation with DGF patients, we suggest the exosomal fraction as a more sensitive substrate to evaluate early biomarkers of DGF after KT.
K
IDNEY transplantation (KT) is the treatment of choice for end-stage renal disease (ESRD) because it prolongs patient survival, improves quality of life, and is less costly than dialysis.1e4 The use of newer immunosuppressive agents has decreased considerably the acute rejection.5,6 Unfortunately, a substantial proportion of grafts still fail within a decade.7 The allograft failure due to chronic allograft dysfuntion still remains as one of the leading causes of organ loss and death.8 It is necessary to explore new tools
From the Transplantation Unit, Davila Clinic (S.A., A.B., M.U., D.C., G.I., A.V., M.H.), and Laboratory of Molecular Physiology, University of Los Andes (C.S., S.V., J.E.C, A.R., C.E.I.), Santiago, Chile. Supported by FAI-MED-00309/University of Los Andes-Chile and FONDECYT-1100885. Address reprint requests to Carlos E. Irarrazabal, PhD, Molecular Physiology Laboratory, Universidad de los Andes, S. Carlos Apoquindo 2200-Las Condes, Santiago, Chile E-mail: cirarrazabal@ uandes.cl
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.08.079
Transplantation Proceedings, 45, 3719e3723 (2013)
3719
3720
ALVAREZ, SUAZO, BOLTANSKY ET AL Table 1. Demographic Information
Patient
Age
Gender
Ischemia (min)
Donor
DGF
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
42 41 44 60 24 57 40 18 40 42 38 34 16 36 22
M M M M F M F M M M F M M M M
67 35 32 1182 80 27 60 ND 92 720 60 120 300 1080 420
L L L D L L L L L D L L L D D
Y N N Y N N N N N N N N N Y N
Abbreviations: M, male; F, Female; L, living; D, deceased; DGF, delayed graft function; N, No; Y, Yes.
for early detection of renal injury in the post-transplantation period to decide the right therapeutic strategy. Neutrophil gelatinase-associated lipocalin (NGAL) is emerging as a novel biomarker of acute kidney injury (AKI) from several etiologies, such as cardiac surgery, contrast nephropathy, KT, and sepsis.9 This protein is produced in several human tissues and particularly in the distal nephron. It has siderophore-chelating property and acts as an irontransporting shuttle.9,10 NGAL increases in both serum and urine 48 hours before the increase of creatinine, and shows a strong correlation with changes in creatinine concentrations.11 NGAL has been found to work as an early, predictive biomarker of delayed graft function (DGF) following KT.12e16 However, urinary NGAL along with other urine metalloproteinases are also promising biomarkers for the presence of brain tumor,17 inflammatory bowel diseases,18 breast cancer,19 and urinary tract infection.20 Additional studies are necessary to validate the sensitivity and specificity of urine NGAL in kidney injury in patients with other comorbidities. We hypothesize that vesicles present in the urine could be a better source to explore as kidney injury biomarkers. The exosomes are nanovesicles (40e100 nm of diameter), released from different types of cells and have been found in fluids such as blood,21 amniotic fluid,22,23 breast milk,24 platelets,25 synovial fluid,26 bronchoalveolar lavage fluid,27 malignant ascites,28,29 and urine.30 The exosomas are produced by the endocytic and exocytic activity in physiological and physiopathologic conditions.31 The study of the exosome composition has demonstrated that it contain
proteins from different cell compartments, such us plasmatic membrane (NKCC2 and CD64),30 cytoplasmatic glyceraldehyde-3-phosphate dehydrogenase (GAPDH),30 and nuclear (AFT3 and WT-1)32 compartments. The protein composition of the exosomal fluid constitutes a potential new method to evaluate biomarkers of kidney disease. The aim of the present study was to evaluate the NGAL abundance in the urine exosome and to study its relationship with renal injury after KT. MATERIALS AND METHODS Patients We analyzed 15 patients during 2009 from the Transplant Unit of the Davila Clinic: 11 living donors (LD) and 4 deceased donors (DD). The mean age was 37 years old (range, 16e60 years) and 75% were male. The average cold ischemia time (CIT) was 14 hours to DD (n ¼ 4) and 1.3 hours to LD (n ¼ 11; Table 1). The immunosuppression was standard based on Thymoglobulin and Simulect for induction, and tacrolimus, mycophenolate mofetil, and steroids for long-term therapy. All patients signed an informed consent form. DGF was considered whenever hemodialysis was required in the post-KT period.
Urine Samples We collected urine samples from patients undergoing renal transplantation at 24, 48, and 72 hours post-transplantation. The samples were stored at -80 C. To 10 mL of urine a protease inhibitor mix was added (Complex Mini, Roche) and processed to the purification of urinary fractions. The cell fraction was obtained after 17,000g centrifugation for 15 minutes at 4 C. The overfluid was further centrifuged at 200,000g for 1 hour at 4 C to obtain the fraction rich in urinary exosomes. Both fractions were treated with lysis buffer (100 mmol/L Tris-HCl, pH 6.8, 500 mmol/L NaCl, 10% Tween 20), plus protease inhibitor (Complete Mini, Roche Applied Science, Indianapolis, Ind, United States). The protein concentration was determined in each sample using the commercial kit BCA Protein Assay Reagent (Pierce) and the lysates were stored at -80 C.
Western Blot The soluble proteins were separated on 7.5 or 10% Tris-Glycine gels and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, Calif, United States). Western blot analysis was performed according to standard conditions. In brief, nonspecific binding was blocked with 5% nonfat milk for 45 minutes at room temperature. Then membranes were incubated with mouse antiLipocain-2/NGAL (R&D Systems, Inc) overnight at 4 C. After washing with 0.1% Tween-20 in PBS, blots were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at room temperature. Proteins were detected
Table 2. Evolution of SCr and FENA in the POD Total (n ¼ 15)
LD (n ¼ 11)
DD (n ¼ 4)
POD
SCr mg/dL
FENA
SCr mg/dL
FENa
SCr mg/dL
1 2 3
6.2 3.6 4.3 4.4 3.0 2.9
30.6 þ 84.54 21.2 þ 73.45 22.3 þ 75.27
5.2 2.6 3.2 2.7 2.2 1.8
7.6 þ 5.0 2.3 þ 1.3 2.6 þ 2.0
8.2 5.4 7.6 7.2 5.6 4.3
Note: POD, Postoperative days; SCr, Serum Creatinine; FENa, Fractional Excretion of Sodium; LD, Living donor; DD, deceased donor. Mean SD.
FENA
123 þ 184 97 þ 164 96 þ 162
URINARY EXOSOMES
3721
Fig 1. The NGAL abundance is elevated in urine exosomes from a KT patient. Western blot determination of NGAL in cellular and exosomal fraction of urine from a KT patient. Abbreviations: H, HEK293 cells; W, whole urine from a healthy individual.
using enhanced chemiluminescence techniques (PerkinElmer, Life Sciences, Boston, Mass, United States). The blots were scanned and densitometric analysis was performed using the public domain NIH Image program v1.61 (US National Institutes of Health, http://rsb. info.nih.gov/nih-image).
Data Analysis In each Western blot 100 mg of cell lysate of HEK293 cells were incorporated as an internal control. The cellular or exosomal urine fraction (100 mg) was loaded in the gel. Densitometry was made to each band and the values were expressed as arbitrary units (AU) and normalized with HEK293 signal.
Statistical Analysis Exploratory and descriptive analyses were conducted about the characteristics of subjects studied. Median test was used to statistically compare differences on NGAL levels between different patient categories.
RESULTS
The studied patients showed a positive evolution of renal function, estimated from serum creatinine (SCr). The average SCr level (15 patients) during postoperative days (POD) was reduced from first (6.2 3.6) to third (3.0 2.9) POD (Table 2). As expected, the SCr evolution was better in patients with LD (POD1 5.2 2.6 to POD3 2.2 1.8; Table 2) compared with DD (POD1 8.2 5.4 to POD3 5.6 4.3; Table 2). We did not find significant differences in the FENa evolution during the POD in each group of patients (LD and DD) (Table 2). However, the FENa levels of FENa in DD was significantly higher than in LD patients. Western blot analysis of NGAL on cellular and exosomal fraction from urine showed the existence of 2 bands, 23 (NGAL-23) and 46 (NGAL-46) kd, suggesting the expression of monomer and dimmer forms of NGAL (Fig 1). We studied both isoforms of NGAL and the results were expressed separately and as total NGAL. We found that NGAL protein was expressed in the exosomal fraction of urine in all KT patients. We also found that NGAL abundance was higher in the exosomal than cellular fraction (Fig 1 shows a representative blot). We also compared the levels of NGAL in LD and DD patients in the cellular and exosomal fractions of urine. In the cellular fraction, the results showed that the NGAL levels were similar in LD and
Fig 2. Levels of NGAL abundance in cellular (A) and exosomal (B) fraction of the urine. Total NGAL ¼ NGAL23 þ NGAL46.
DD patients, except for DD patients on POD1, when significant high levels of NGAL were observed (P ¼ .013; Fig 2A). However, the analysis of exosomal fraction showed higher levels of NGAL in DD than LD patients in the 3 PODs analyzed (P < .05; Fig 2B). The sum of the NGAL abundance during the 3 days after allograft transplantation was significantly higher in DD than LD patients (P < .001; data not shown). Three patients (3/15) developed DGF after transplantation (2 DD and 1 LD patient). Table 3 shows that age and gender distribution of DGF and non-DGF were similar
3722
ALVAREZ, SUAZO, BOLTANSKY ET AL
Table 3. Demographic Information (A) and Evolution of SCr and FENa (B) in DGF and non-DGF Patients in the POD A
DGF Non-DGF
Age
Gender
Ischemia (min)
46 12.50 34 11.97
M 100% M 75%
776 616 165 213
B SCr mg/dL POD
1 2 3
FENa
DGF
Non-DGF
DGF
Non-DGF
11.6 2.0 12.1 4.7 8.6 1.3
4.7 2.3 2.5 1.5 1.7 0.8
119 187 98 163 96 162
8.5 7.5 2.1 1.3 2.3 1.4
Note: Mean SD.
and that CIT was higher in DGF patients. The SCr level was significantly higher in DGF than non-DGF during the 3 PODs (P < .001; Table 3). Table 3 also shows that the FeNa values were significantly higher in the DGF group at POD2 (P < .038). Fig 3A shows that NGAL expression in cellular fraction was similar in the DGF and non-DGF groups. However, as Figure 3B shows, in the exosomal fraction the 2 NGAL forms (NGAL-23 and NGAL-46) were significantly higher in the DGF than the non-DGF group during POD1, 2, and 3.
DISCUSSION
Considering that NGAL determination (in the whole urine using an enzyme-linked immunosorbent assay [ELISA]) has been associated with acute renal injury after 2 hours of cardiopulmonary bypass33 and early, predictive biomarker of DGF following KT,12e16 we decided to study if different urine fractions (cellular or exosomal fraction) have different NGAL expression in a group of patients subjected to KT. The results showed that NGAL is detectable by Western blot in the cellular and exosomal fraction (Fig 1). The cellular fraction expressed lower levels of NGAL compared with the exosomal fraction (Fig 1). In the cellular fraction, NGAL abundance was similar to the studied patients (in general less than 50 AU; Fig 2A and 3A). However, the NGAL abundance was different in the exosomal fraction in the studied patient, suggesting that NGAL in urine exosomes might be a better source to study the sensitivity and specificity of this marker in kidney damage. The higher levels of NGAL in the exosomal fraction in DD patients suggest that this protein could be a marker of allograft damage, considering the potential kidney deterioration in this kind of donor (higher CIT). Our analysis showed that the highest differences in exosomal NGAL were observed in the abundance of 46 kd conformation, suggesting that this protein structure could be more relevant in the evaluation of allograft damage, although the differences observed with NGAL-23 correlate with the NGAL-46 abundance.
Fig 3. Comparison of NGAL abundance among DGF and nonDGF in the cellular (A) and exosomal (B) fraction of the urine.
The incidence of DGF in the analyzed patients was 20% (3/15). The comparison between DGF and non-DGF patients displayed that NGAL abundance in the cellular fraction was similar in DGF and non-DGF patients. However, in the exosomal fraction, the NGAL level was higher in DGF than non-DGF patients (Fig 3B) during the 3 PODs analyzed. The exosomal NGAL correlates strongly with the SCr level in the DGF and non-DGF groups (Table 3 and Fig 3). Table 3 shows that the differences in SCr between the DGF and non-DGF groups were more clearly observed from POD2, however, exosomal NGAL started from POD1. So, these results suggest that the analysis of exosomal urinary fraction could be a better substrate to investigate as a biomarker, providing more
URINARY EXOSOMES
information about the physiopathology of the kidney. We speculate that NGAL in the exosomal fraction could be more specific to evaluate renal damage because exosomes should be a representation of the physiological state of the renal cell. These results confirmed the published data suggesting that urine NGAL determination is a biomarker of DGF in renal transplantation.12e16 Our results showed that NGAL is concentrated in the exosomal fraction of urine, suggesting that this fraction becomes a more sensitive source to analyze NGAL than cell fraction urine. Time is limited to introduce proper treatment after the initiating insult, therefore, the determination of NGAL in the exosomal fraction of the urine could be a more reliable early marker related to DGF, especially in the settings of early dialysis treatment or antirejection therapy. In conclusion, our findings might impact positively the clinical management of patients undergoing KT, especially because NGAL is a biomarker of damage more than functionality. REFERENCES 1. Laupacis A, Keown P, Pus N, et al. A study of the quality of life and cost-utility of renal transplantation. Kidney Int. 1996;50: 235e242. 2. Russell JD, Beecroft ML, Ludwin D, Churchill DN. The quality of life in renal transplantation: a prospective study. Transplantation. 1992;54:656e660. 3. Wolfe RA, Ashby VB, Milford EL, et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N Engl J Med. 1999;341:1725e1730. 4. Merion RM, Ashby VB, Wolfe RA, et al. Deceased-donor characteristics and the survival benefit of kidney transplantation. JAMA. 2005;294(21):2726e2733. 5. Danovitch GM. How should the immunosuppressive regimen be managed in patients with established chronic allograft failure? Kidney Int. 2002;61:S68eS72. 6. Taylor AL, Watson CJ, Bradley JA. Immunosuppressive agents in solid organ transplantation: mechanisms of action and therapeutic efficacy. Crit Rev Oncol Hematol. 2005;56(1):23e46. 7. 2008 OPTN/SRTR Annual Report: Transplant Data 1998e2007. 8. Taylor DO, Edwards LB, Boucek MM, et al. Registry of the International Society for Heart and Lung Transplantation: twentyfourth official adult heart transplant report e 2007. J Heart Lung Transplant. 2007;26(8):769e781. 9. Devarajan P. Neutrophil gelatinase-associated lipocalin (NGAL). A new marker of kidney disease. Scand J Clin Lab Invest. 2008;241(suppl):89e94. 10. Devarajan P. Neutrophil gelatinase-associated lipocalin an emerging troponin for kidney injury. Nephrol Dial Transplant. 2008;23:3737e3743. 11. Mori K, Lee HT, Rapoport D, Drexler IR, Foster K, Yang J. Endocytic delivery of lipocalinsiderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest. 2005;115: 610e621. 12. Parikh CR, Jani A, Mishra J, Ma Q, Kelly C, Barasch J, Edelstein CL, Devarajan P. Urine NGAL and IL-18 are Predictive Biomarkers for Delayed Graft Function Following Kidney Transplantation. Am J Transplant. 2006;6:1639e1645. 13. Kusaka M, Kuroyanagi Y, Mori T, et al. Serum neutrophil gelatinase-associated lipocalin as a predictor of organ recovery from
3723 delayed graft function after kidney transplantation from donors after cardiac death. Cell Transplant. 2008;17:129e134. 14. Lebkowska U, Malyszko J, Lebkowska A, et al. Neutrophil gelatinase-associated lipocalin and cystatin C could predict renal outcome in patients undergoing kidney allograft transplantation: a prospective study. Transplant Proc. 2009;41(1):154e157. 15. Malyszko J, Malyszko JS, Mysliwiec M. Serum neutrophil gelatinase-associated lipocalin correlates with kidney function in renal allograft recipients. Clin Transplant. 2009;23(5):681e686. 16. Hall IE, Yarlagadda SG, Coca SG. IL-18 and urinary NGAL predict dialysis and graft recovery after kidney transplantation. J Am Soc Nephrol. 2010;21(1):189e197. 17. Smith ER, Zurakowski D, Saad A, Scott RM, Moses MA. Urinary biomarkers predict brain tumor presence and response to therapy. Clin Cancer Res. 2008;14:2378e2386. 18. Manfredi MA, Zurakowski D, Rufo PA, Walker TR, Fox VL, Moses MA. Increased incidence of urinary matrix metalloproteinases as predictors of disease in pediatric patients with inflammatory bowel disease. Inflamm Bowel Dis. 2008;14:1091e1096. 19. Fernández CA, Yan L, Louis G, Yang J, Kutok JL, Moses MA. The matrix metalloproteinase-9/neutrophil gelatinaseassociated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients. Clin Cancer Res. 2005;11:5390e5395. 20. Yilmaz A, Sevketoglu E, Gedikbasi A, et al. Early prediction of urinary tract infection with urinary neutrophil gelatinase associated lipocalin. Pediatr Nephrol. 2009;24(12):2387e2392. 21. Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol. 2005;17:879e887. 22. Taylor DD, Akyol S, Gercel-Taylor C. Pregnancy-associated exosomas and their modulation of T cell signaling. J Immunol. 2006;176:1534e1542. 23. Sabapatha A, Gercel-Taylor C, Taylor DD. Specific isolation of placenta derived exosomes from the circulation of pregnant women and their immunoregulatory consequences. Am J Reprod Immunol. 2006;56:345e355. 24. Admyre C, Johansson SM, Qazi KR, et al. Exosomes with immune modulatory features are present in human breast milk. J Immunol. 2007;179:1969e1978. 25. Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood. 1999;94:3791e3799. 26. Skriner K, Adolph K, Jungblut PR, Burmester GR. Association of citrullinated proteins with synovial exosomes. Arthritis Rheum. 2006;54:3809e3814. 27. Admyre C, Grunewald J, Thyberg J, et al. Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid. Eur Respir J. 2003;22: 578e583. 28. Andre F, Schartz NE, Movassagh M, et al. Malignant effusions and immunogenic tumour-derived exosomes. Lancet. 2002;360:295e305. 29. Mears R, Craven RA, Hanrahan S, et al. Proteomic analysis of melanoma-derived exosomes by twodimensional polyacrylamide gel electrophoresis and mass spectrometry. Proteomics. 2004;4: 4019e4031. 30. Pisitkun T, Shen RF, Knepper MA. Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A. 2004;101:13368e13373. 31. Simpson RJ, Jensen SS, Lim JW. Proteomic profiling of exosomes: current perspectives. Proteomics. 2008;8:4083e4099. 32. Zhou H, Cheruvanky A, Hu X, et al. Urinary exosomal transcription factors, a new class of biomarkers for renal disease. Kidney Int. 2008;74(5):613e621. 33. Mishra J, Dent C, Tarabishi R, et al. Neutrophil gelatinaseassociated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. The Lancet. 2005;365:1231e1238.
K
IDNEY transplantation (KT) is the treatment of choice for end-stage renal disease (ESRD) because it prolongs patient survival, improves quality of life, and is less costly than dialysis.1e4 The use of newer immunosuppressive agents has decreased considerably the acute rejection.5,6 Unfortunately, a substantial proportion of grafts still fail within a decade.7 The allograft failure due to chronic allograft dysfuntion still remains as one of the leading causes of organ loss and death.8 It is necessary to explore new tools
From the Transplantation Unit, Davila Clinic (S.A., A.B., M.U., D.C., G.I., A.V., M.H.), and Laboratory of Molecular Physiology, University of Los Andes (C.S., S.V., J.E.C, A.R., C.E.I.), Santiago, Chile. Supported by FAI-MED-00309/University of Los Andes-Chile and FONDECYT-1100885. Address reprint requests to Carlos E. Irarrazabal, PhD, Molecular Physiology Laboratory, Universidad de los Andes, S. Carlos Apoquindo 2200-Las Condes, Santiago, Chile E-mail: cirarrazabal@ uandes.cl
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.08.079
Transplantation Proceedings, 45, 3719e3723 (2013)
3719
3720
ALVAREZ, SUAZO, BOLTANSKY ET AL Table 1. Demographic Information
Patient
Age
Gender
Ischemia (min)
Donor
DGF
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
42 41 44 60 24 57 40 18 40 42 38 34 16 36 22
M M M M F M F M M M F M M M M
67 35 32 1182 80 27 60 ND 92 720 60 120 300 1080 420
L L L D L L L L L D L L L D D
Y N N Y N N N N N N N N N Y N
Abbreviations: M, male; F, Female; L, living; D, deceased; DGF, delayed graft function; N, No; Y, Yes.
for early detection of renal injury in the post-transplantation period to decide the right therapeutic strategy. Neutrophil gelatinase-associated lipocalin (NGAL) is emerging as a novel biomarker of acute kidney injury (AKI) from several etiologies, such as cardiac surgery, contrast nephropathy, KT, and sepsis.9 This protein is produced in several human tissues and particularly in the distal nephron. It has siderophore-chelating property and acts as an irontransporting shuttle.9,10 NGAL increases in both serum and urine 48 hours before the increase of creatinine, and shows a strong correlation with changes in creatinine concentrations.11 NGAL has been found to work as an early, predictive biomarker of delayed graft function (DGF) following KT.12e16 However, urinary NGAL along with other urine metalloproteinases are also promising biomarkers for the presence of brain tumor,17 inflammatory bowel diseases,18 breast cancer,19 and urinary tract infection.20 Additional studies are necessary to validate the sensitivity and specificity of urine NGAL in kidney injury in patients with other comorbidities. We hypothesize that vesicles present in the urine could be a better source to explore as kidney injury biomarkers. The exosomes are nanovesicles (40e100 nm of diameter), released from different types of cells and have been found in fluids such as blood,21 amniotic fluid,22,23 breast milk,24 platelets,25 synovial fluid,26 bronchoalveolar lavage fluid,27 malignant ascites,28,29 and urine.30 The exosomas are produced by the endocytic and exocytic activity in physiological and physiopathologic conditions.31 The study of the exosome composition has demonstrated that it contain
proteins from different cell compartments, such us plasmatic membrane (NKCC2 and CD64),30 cytoplasmatic glyceraldehyde-3-phosphate dehydrogenase (GAPDH),30 and nuclear (AFT3 and WT-1)32 compartments. The protein composition of the exosomal fluid constitutes a potential new method to evaluate biomarkers of kidney disease. The aim of the present study was to evaluate the NGAL abundance in the urine exosome and to study its relationship with renal injury after KT. MATERIALS AND METHODS Patients We analyzed 15 patients during 2009 from the Transplant Unit of the Davila Clinic: 11 living donors (LD) and 4 deceased donors (DD). The mean age was 37 years old (range, 16e60 years) and 75% were male. The average cold ischemia time (CIT) was 14 hours to DD (n ¼ 4) and 1.3 hours to LD (n ¼ 11; Table 1). The immunosuppression was standard based on Thymoglobulin and Simulect for induction, and tacrolimus, mycophenolate mofetil, and steroids for long-term therapy. All patients signed an informed consent form. DGF was considered whenever hemodialysis was required in the post-KT period.
Urine Samples We collected urine samples from patients undergoing renal transplantation at 24, 48, and 72 hours post-transplantation. The samples were stored at -80 C. To 10 mL of urine a protease inhibitor mix was added (Complex Mini, Roche) and processed to the purification of urinary fractions. The cell fraction was obtained after 17,000g centrifugation for 15 minutes at 4 C. The overfluid was further centrifuged at 200,000g for 1 hour at 4 C to obtain the fraction rich in urinary exosomes. Both fractions were treated with lysis buffer (100 mmol/L Tris-HCl, pH 6.8, 500 mmol/L NaCl, 10% Tween 20), plus protease inhibitor (Complete Mini, Roche Applied Science, Indianapolis, Ind, United States). The protein concentration was determined in each sample using the commercial kit BCA Protein Assay Reagent (Pierce) and the lysates were stored at -80 C.
Western Blot The soluble proteins were separated on 7.5 or 10% Tris-Glycine gels and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, Calif, United States). Western blot analysis was performed according to standard conditions. In brief, nonspecific binding was blocked with 5% nonfat milk for 45 minutes at room temperature. Then membranes were incubated with mouse antiLipocain-2/NGAL (R&D Systems, Inc) overnight at 4 C. After washing with 0.1% Tween-20 in PBS, blots were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at room temperature. Proteins were detected
Table 2. Evolution of SCr and FENA in the POD Total (n ¼ 15)
LD (n ¼ 11)
DD (n ¼ 4)
POD
SCr mg/dL
FENA
SCr mg/dL
FENa
SCr mg/dL
1 2 3
6.2 3.6 4.3 4.4 3.0 2.9
30.6 þ 84.54 21.2 þ 73.45 22.3 þ 75.27
5.2 2.6 3.2 2.7 2.2 1.8
7.6 þ 5.0 2.3 þ 1.3 2.6 þ 2.0
8.2 5.4 7.6 7.2 5.6 4.3
Note: POD, Postoperative days; SCr, Serum Creatinine; FENa, Fractional Excretion of Sodium; LD, Living donor; DD, deceased donor. Mean SD.
FENA
123 þ 184 97 þ 164 96 þ 162
URINARY EXOSOMES
3721
Fig 1. The NGAL abundance is elevated in urine exosomes from a KT patient. Western blot determination of NGAL in cellular and exosomal fraction of urine from a KT patient. Abbreviations: H, HEK293 cells; W, whole urine from a healthy individual.
using enhanced chemiluminescence techniques (PerkinElmer, Life Sciences, Boston, Mass, United States). The blots were scanned and densitometric analysis was performed using the public domain NIH Image program v1.61 (US National Institutes of Health, http://rsb. info.nih.gov/nih-image).
Data Analysis In each Western blot 100 mg of cell lysate of HEK293 cells were incorporated as an internal control. The cellular or exosomal urine fraction (100 mg) was loaded in the gel. Densitometry was made to each band and the values were expressed as arbitrary units (AU) and normalized with HEK293 signal.
Statistical Analysis Exploratory and descriptive analyses were conducted about the characteristics of subjects studied. Median test was used to statistically compare differences on NGAL levels between different patient categories.
RESULTS
The studied patients showed a positive evolution of renal function, estimated from serum creatinine (SCr). The average SCr level (15 patients) during postoperative days (POD) was reduced from first (6.2 3.6) to third (3.0 2.9) POD (Table 2). As expected, the SCr evolution was better in patients with LD (POD1 5.2 2.6 to POD3 2.2 1.8; Table 2) compared with DD (POD1 8.2 5.4 to POD3 5.6 4.3; Table 2). We did not find significant differences in the FENa evolution during the POD in each group of patients (LD and DD) (Table 2). However, the FENa levels of FENa in DD was significantly higher than in LD patients. Western blot analysis of NGAL on cellular and exosomal fraction from urine showed the existence of 2 bands, 23 (NGAL-23) and 46 (NGAL-46) kd, suggesting the expression of monomer and dimmer forms of NGAL (Fig 1). We studied both isoforms of NGAL and the results were expressed separately and as total NGAL. We found that NGAL protein was expressed in the exosomal fraction of urine in all KT patients. We also found that NGAL abundance was higher in the exosomal than cellular fraction (Fig 1 shows a representative blot). We also compared the levels of NGAL in LD and DD patients in the cellular and exosomal fractions of urine. In the cellular fraction, the results showed that the NGAL levels were similar in LD and
Fig 2. Levels of NGAL abundance in cellular (A) and exosomal (B) fraction of the urine. Total NGAL ¼ NGAL23 þ NGAL46.
DD patients, except for DD patients on POD1, when significant high levels of NGAL were observed (P ¼ .013; Fig 2A). However, the analysis of exosomal fraction showed higher levels of NGAL in DD than LD patients in the 3 PODs analyzed (P < .05; Fig 2B). The sum of the NGAL abundance during the 3 days after allograft transplantation was significantly higher in DD than LD patients (P < .001; data not shown). Three patients (3/15) developed DGF after transplantation (2 DD and 1 LD patient). Table 3 shows that age and gender distribution of DGF and non-DGF were similar
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ALVAREZ, SUAZO, BOLTANSKY ET AL
Table 3. Demographic Information (A) and Evolution of SCr and FENa (B) in DGF and non-DGF Patients in the POD A
DGF Non-DGF
Age
Gender
Ischemia (min)
46 12.50 34 11.97
M 100% M 75%
776 616 165 213
B SCr mg/dL POD
1 2 3
FENa
DGF
Non-DGF
DGF
Non-DGF
11.6 2.0 12.1 4.7 8.6 1.3
4.7 2.3 2.5 1.5 1.7 0.8
119 187 98 163 96 162
8.5 7.5 2.1 1.3 2.3 1.4
Note: Mean SD.
and that CIT was higher in DGF patients. The SCr level was significantly higher in DGF than non-DGF during the 3 PODs (P < .001; Table 3). Table 3 also shows that the FeNa values were significantly higher in the DGF group at POD2 (P < .038). Fig 3A shows that NGAL expression in cellular fraction was similar in the DGF and non-DGF groups. However, as Figure 3B shows, in the exosomal fraction the 2 NGAL forms (NGAL-23 and NGAL-46) were significantly higher in the DGF than the non-DGF group during POD1, 2, and 3.
DISCUSSION
Considering that NGAL determination (in the whole urine using an enzyme-linked immunosorbent assay [ELISA]) has been associated with acute renal injury after 2 hours of cardiopulmonary bypass33 and early, predictive biomarker of DGF following KT,12e16 we decided to study if different urine fractions (cellular or exosomal fraction) have different NGAL expression in a group of patients subjected to KT. The results showed that NGAL is detectable by Western blot in the cellular and exosomal fraction (Fig 1). The cellular fraction expressed lower levels of NGAL compared with the exosomal fraction (Fig 1). In the cellular fraction, NGAL abundance was similar to the studied patients (in general less than 50 AU; Fig 2A and 3A). However, the NGAL abundance was different in the exosomal fraction in the studied patient, suggesting that NGAL in urine exosomes might be a better source to study the sensitivity and specificity of this marker in kidney damage. The higher levels of NGAL in the exosomal fraction in DD patients suggest that this protein could be a marker of allograft damage, considering the potential kidney deterioration in this kind of donor (higher CIT). Our analysis showed that the highest differences in exosomal NGAL were observed in the abundance of 46 kd conformation, suggesting that this protein structure could be more relevant in the evaluation of allograft damage, although the differences observed with NGAL-23 correlate with the NGAL-46 abundance.
Fig 3. Comparison of NGAL abundance among DGF and nonDGF in the cellular (A) and exosomal (B) fraction of the urine.
The incidence of DGF in the analyzed patients was 20% (3/15). The comparison between DGF and non-DGF patients displayed that NGAL abundance in the cellular fraction was similar in DGF and non-DGF patients. However, in the exosomal fraction, the NGAL level was higher in DGF than non-DGF patients (Fig 3B) during the 3 PODs analyzed. The exosomal NGAL correlates strongly with the SCr level in the DGF and non-DGF groups (Table 3 and Fig 3). Table 3 shows that the differences in SCr between the DGF and non-DGF groups were more clearly observed from POD2, however, exosomal NGAL started from POD1. So, these results suggest that the analysis of exosomal urinary fraction could be a better substrate to investigate as a biomarker, providing more
URINARY EXOSOMES
information about the physiopathology of the kidney. We speculate that NGAL in the exosomal fraction could be more specific to evaluate renal damage because exosomes should be a representation of the physiological state of the renal cell. These results confirmed the published data suggesting that urine NGAL determination is a biomarker of DGF in renal transplantation.12e16 Our results showed that NGAL is concentrated in the exosomal fraction of urine, suggesting that this fraction becomes a more sensitive source to analyze NGAL than cell fraction urine. Time is limited to introduce proper treatment after the initiating insult, therefore, the determination of NGAL in the exosomal fraction of the urine could be a more reliable early marker related to DGF, especially in the settings of early dialysis treatment or antirejection therapy. In conclusion, our findings might impact positively the clinical management of patients undergoing KT, especially because NGAL is a biomarker of damage more than functionality. REFERENCES 1. Laupacis A, Keown P, Pus N, et al. A study of the quality of life and cost-utility of renal transplantation. Kidney Int. 1996;50: 235e242. 2. Russell JD, Beecroft ML, Ludwin D, Churchill DN. The quality of life in renal transplantation: a prospective study. Transplantation. 1992;54:656e660. 3. Wolfe RA, Ashby VB, Milford EL, et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N Engl J Med. 1999;341:1725e1730. 4. Merion RM, Ashby VB, Wolfe RA, et al. Deceased-donor characteristics and the survival benefit of kidney transplantation. JAMA. 2005;294(21):2726e2733. 5. Danovitch GM. How should the immunosuppressive regimen be managed in patients with established chronic allograft failure? Kidney Int. 2002;61:S68eS72. 6. Taylor AL, Watson CJ, Bradley JA. Immunosuppressive agents in solid organ transplantation: mechanisms of action and therapeutic efficacy. Crit Rev Oncol Hematol. 2005;56(1):23e46. 7. 2008 OPTN/SRTR Annual Report: Transplant Data 1998e2007. 8. Taylor DO, Edwards LB, Boucek MM, et al. Registry of the International Society for Heart and Lung Transplantation: twentyfourth official adult heart transplant report e 2007. J Heart Lung Transplant. 2007;26(8):769e781. 9. Devarajan P. Neutrophil gelatinase-associated lipocalin (NGAL). A new marker of kidney disease. Scand J Clin Lab Invest. 2008;241(suppl):89e94. 10. Devarajan P. Neutrophil gelatinase-associated lipocalin an emerging troponin for kidney injury. Nephrol Dial Transplant. 2008;23:3737e3743. 11. Mori K, Lee HT, Rapoport D, Drexler IR, Foster K, Yang J. Endocytic delivery of lipocalinsiderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest. 2005;115: 610e621. 12. Parikh CR, Jani A, Mishra J, Ma Q, Kelly C, Barasch J, Edelstein CL, Devarajan P. Urine NGAL and IL-18 are Predictive Biomarkers for Delayed Graft Function Following Kidney Transplantation. Am J Transplant. 2006;6:1639e1645. 13. Kusaka M, Kuroyanagi Y, Mori T, et al. Serum neutrophil gelatinase-associated lipocalin as a predictor of organ recovery from
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