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High Nerve Growth Factor Blood Concentration In Renal Transplantation: A New Prognostic Marker?
High Nerve Growth Factor Blood Concentration In Renal Transplantation: A New Prognostic Marker? P. Gigliotti, D. Lofaro, F. Leone, A. Perri, D. Vizza, T. Papalia, and R. Bonofiglio ABSTRACT Background. Nerve growth factor (NGF) belongs to the family of neurotropic proteins NGF is markedly expressed in proteinuric renal diseases and in end-stage renal disease; it might be involved in kidney physiopathology. To date, little is known about NGF concentrations in kidney transplant recipients (KTRs). Because NGF exerts its action on cell survival and differentiation, tissue repair, and inflammatory responses, it may also be implicated in the pathogenesis of chronic allograft nephropathy. The aim of this study was to determine circulating NGF concentrations in KTRs and to ascertain their use as a prognostic marker for kidney transplant outcomes. Methods. Using enzyme-linked immunosorbent assay, we performed quantification of NGF in the serum of 40 prevalent KTRs at baseline and at 6 months. Results. NGF concentrations in KTRs averaged 1.16 0.67 ng/mL. They negativelinearly correlated with recipient age. Logistic multivariate regression analysis showed NGF to be independently associated with increased proteinuria over the 6-month follow-up. Conclusions. Our data demonstrated that serum concentrations of NGF in KTRs were elevated and that they could be considered to be a prognostic marker in kidney transplantation.
N
ERVE GROWTH FACTOR (NGF) belongs to the family of neurotropic proteins that includes brain-derived neurotropic factor, neurotrophin-3, and neurotrophin-4/5. NGF contributes to maintaining the critical balance between cell survival and death both during development and in adulthood.1e3 NGF which is produced and released by a variety of mammalian cells, acts via a highaffinity cell surface receptor, tyrosine kinase receptor A (TRKA), and a low-affinity receptor, p75 neurotrophic receptor (p75NTR), a member of the tumor necrosis factor receptor superfamily.4e9 on the one hand NGF may exert its cell protective actions through the TRKA receptor; on the other hands it triggers, apoptosis in some cells through the p75NTR receptor. NGF is not only a promoter of the growth and survival of nerve cells but also of other cell types,2,4 including renal cells.10,11 To date, little is known about NGF concentrations in kidney transplant recipients (KTRs). Because NGF exerts actions on cell survival and differentiation, tissue repair, and inflammatory responses, it might be implicated in the pathogenesis of chronic allograft nephropathy. The aim of the present study was to determine the concentration of circulating NGF among a population of
KTRs and to verify whether it could be considered to be a prognostic marker for kidney transplant outcomes. MATERIAL AND METHODS Patients The subjects eligible for this study were recruited from a cohort of 200 KTRs regularly followed in the transplant ambulatory unit over a 6-month period. We applied the following criteria to select the study sample: (a) age 18e64 years, seeking to minimize age-related variations in NGF levels; (b) serum creatinine <2.0 mg/dL; (c) 24-hour proteinuria <3 g; (d) no clinical or laboratory evidence of diabetes, according to the American Diabetes Association criteria12;
From the Research Center “Rene e Trapianto,” Department of Nephrology, Dialysis, and Transplantation, “Annunziata” Hospital, Cosenza, Italy. Address reprint requests to Dr Renzo Bonofiglio, Department of Nephrology, Dialysis, and Transplantation, “Annunziata” Hospital, via F. Migliori, 87100 Cosenza, Italy. E-mail: rbonofi@ tin.it
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.07.020
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2654
Transplantation Proceedings, 45, 2654e2656 (2013)
NGF LEVEL IN RENAL TRANSPLANTATION
2655
(e) arterial blood pressure levels <140/90 mm Hg on 3 separate occasions and in the presence of antihypertensive treatment; (f) normal serum lipid profile defined by plasma levels of cholesterol and triglycerides <200 mg/dL and <150 mg/dL, respectively, and in the presence of lipid-lowering treatment; (g) absence of obesity, with a body mass index <30 kg/m2; (h) absence of drug or alcohol dependency; (i) no smoking habits in the previous 2 years; (f) immunosuppressive therapy with steroids mycophenolate mofetil, and calcineurin inhibitor, and steroid and antieinterleukin-2 antibody prophylaxisi; (g) >1 year after transplantation; and (h) hemoglobin >10.5 g/dL, and in the presence of erythropoietin treatment. We enrolled 40 KTRs who met these criteria for the study. Each of the participants gave informed consent. The study was approved by the Ethical Committee of our hospital.
Blood Samples Blood collected in the morning and was separated for serum by centrifugation at 3,500 rpm for 15 minutes.
NGF Protein Determination NGF concentrations were determined with the use of a Promega enzyme-linked immunosorbent assay kit (NGF Emax Immunoassay System, no. G7631; Promega, Madison, Wisconsin) according to the manufacturer’s instructions at enrollment and after 6 months. NGF concentrations were expressed as ng/mL; all assays were performed in triplicate.
RESULTS
The basal clinical and laboratory features of these patients are presented in Table 1. The mean concentration of NGF in the KTR serum was 1.16 0.67 ng/mL. No significant differences in NGF levels were observed between men and women. There were no significant linear correlations between NGF and glomerular filtration rate (GFR), creatinine, and proteinuria, and only a linear negative correlation between NGF and patient age (R2 ¼ .218; P < .05). To investigate the possible role of NGF on kidney transplant outcomes after a 6-month follow up, we first stratified patients according to the GFR decline: Dþ GFR (stable GFR over 6 months) versus D GFR (loss of GFR over 6 months). This was also done according to the increase in proteinuria: Dþ proteinuria (increasing proteinuria over 6 months) and D proteinuria (stable proteinuria over 6 months). We observed NGF concentrations to be higher in the D GFR group (1.26 0.78) versus the Dþ GFR group (0.85 0.24; P ¼ .02) and in the Dþ proteinuria group (1.39 0.79) versus D proteinuria group (0.87 0.31; P ¼ .02). We then performed a logistic regression analysis including univariate and multivariate (adjusted for age, sex,
and GFR) models, which showed NGF concentrations to be independently associated with proteinuria worsening. (odds ratio, 1.03; 95%, confidence interval [CI], 1.01e1.05). The data were confirmed by a receiver operating characteristic (ROC) analysis, showing an area under the ROC curve of 0.751 (95% CI, 0.575e0.927; Fig 1) with 1.15 ng/mL as the best prognostic threshold (negative predictive value ¼ 61.1; positive predictive value ¼ 83.3). DISCUSSION
The aim of this study was to investigate the behavior of NGF among a population of KTRs. Previously we also investigated NGF serum concentrations in 2 inflammatory models; glomerulonephritis end-stage renal disease during hemodialysis and treatment.13e15 We demonstrated elevated NGF concentrations among those subjects compared with healthy subjects. Those findings and the observations that NGF and NGF receptors are expressed in developing, healthy, and diseased human adult kidney cells11 led to the hypothesis that NGF is involved in renal physiopathology. In the present work, we demonstrated that KTRs also displayed increased NGF levels, which were predictive of kidney injury. In fact, NGF levels were independently associated with the development of proteinuria in KTR. Proteinuria is one of the best predictors of chronic kidney disease progression and kidney transplant failure. We think that the NGF increase is not implicated in kidney damage that successively leads to proteinuria development, but rather that it could represent a protective signal against injury. It may be part of the responsive mechanism to the kidney cell damage leading to an altered glomerular filtration barrier causing the onset of proteinuria. In fact, based on
Table 1. Clinical Parameters of the Study Population mean SD
Male:female ratio Age (y) Serum creatinine (mg/dL) eGFR (mL/min/1.73 m2) Proteinuria (g/24 h) CPR (mg/L)
28:12 43 8 1.3 0.4 98.2 12.1 0.5 0.35 1.4 0.4
Fig 1.
2656
recent studies, we suppose that up-regulation of NGF represents a protective signal against potential damage to kidney cells. In a recent report, Aloe et al16 showed a marked increase in serum NGF levels and NGF expression in the kidneys of rats displaying streptozotocin-induced type 1 diabetes. Subcutaneous administration of NGF caused a marked uptake of this cytokine by kidney cells without proapoptotic factor expression. This hypothesis is also supported by the evidence that injured diabetic skin, cardiac ischemia, corneal inflammation, peripheral neuropathy, and retinal and pancreatic cell damage are all characterized by NGF up-regulation and would benefit from local NGF treatment.17e21 However, the possibility that NGF acts with other growth factors known to be involved in kidney cell physiopathology remains to be investigated. In conclusion, NGF may be considered to be a marker for transplant outcomes. REFERENCES 1. Connor B, Dragunow M. The role of neuronal growth factors in neurodegenerative disorders of the human brain. Brain Res Rev. 1998;27:1e39. 2. Levi-Montalcini R. The nerve growth factor 35 years later. Science. 1987;237:1154e1162. 3. Sofroniew MV, Howe CL, Mobley WC. Nerve growth factor signaling, neuroprotection, and neural repair. Annu Rev Neurosci. 2001;24:1217e1281. 4. Aloe L, Tirassa P, Bracci-Laudiero L. Nerve growth factor in neurological and non-neurological diseases: basic findings and emerging pharmacological prospectives. Curr Pharm Des. 2001;7: 13e23. 5. Aloe L, Skaper SD, Leon A, Levi-Montalcini R. Nerve growth factor and autoimmune diseases. Autoimmunity. 1994;19: 141e150. 6. Flugel A, Matsumuro K, Neumann H, et al. Anti-inflammatory activity of nerve growth factor in experimental autoimmune encephalomyelitis inhibition of monocyte transendothelial migration. Eur J Immunol. 2001;31:11e22. 7. Kohyama T, Liu X, Wen FQ, Kobayashi T, et al. Nerve growth factor stimulates fibronectininduced fibroblast migration. J Lab Clin Med. 2002;140:329e335.
GIGLIOTTI, LOFARO, LEONE ET AL 8. Tuveri M, Generini S, Matucci-Cerinic M, Aloe L. NGF, a useful tool in the treatment of chronic vasculitic ulcers in rheumatoid arthritis. Lancet. 2000;356:1739e1740. 9. Villoslada P, Hauser SL, Bartke I, et al. Human nerve growth factor protects common marmosets against autoimmune encephalomyelitis by switching the balance of T helper cell type 1 and 2 cytokines within the central nervous system. J Exp Med. 2000;191:1799e1806. 10. Sariola H, Saarma M, Sainio K, et al. Dependence of kidney morphogenesis on the expression of nerve growth factor receptor. Science. 1991;254:571e573. 11. Alpers CE, Hudkins KL, Ferguson M, et al. Nerve growth factor receptor expression in fetal, mature, and diseased human kidneys. Lab Invest. 1993;69:703e713. 12. Gibbons RJ, Chatterjee K, Daley J. ACC/AHA/ACP-ASIM guidelines for the management of patients with chronic stable angina: executive summary and recommendations. A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. Circulation. 1999;99:2829e2848. 13. Bonofiglio R, Caroleo MC, Antonucci MT, et al. Transforming growth factor beta and nerve growth factor renal disease progression. Biomed Rev. 2005;16:23e34. 14. Bonofiglio R, Antonucci MT, Papalia T, et al. Nerve growth factor (NGF) and NGF-receptor expression in diseased human kidneys. J Nephrol. 2007;20(2):186e195. 15. Antonucci MT, Bonofiglio R, Papalia T, et al. Nerve growth factor and its monocyte receptors are affected in kidney disease. Nephron Clin Pract. 2009;111(1):c21ec28. 16. Aloe L, Rossi S, Manni L. Altered expression of nerve growth factor and its receptors in the kidneys of diabetic rats. J Nephrol. 2011;24(6):798e805. 17. Reimer MK, Mokshagundam SP, Wyler K, et al. Local growth factors are beneficial for the autonomic reinnervation of transplanted islets in rats. Pancreas. 2003;26:392e397. 18. Vidaltamayo R, Mery CM, Angeles-Angeles A, et al. Expression of nerve growth factor in human pancreatic beta cells. Growth Factors. 2003;21:103e107. 19. Salis MB, Graiani G, Desortes E, et al. Nerve growth factor supplementation reverses the impairment, induced by type 1 diabetes, of hind limb postischaemic recovery in mice. Diabetologia. 2004;47:1055e1063. 20. Lambiase A, Rama P, Bonini S, et al. Topical treatment with nerve growth factor for corneal neurotrophic ulcers. N Engl J Med. 1998;338:1174e1180. 21. Reinshagen M, Rohm H, Steinkamp M, et al. Protective role of neurotrophins in experimental inflammation of the rat gut. Gastroenterology. 2000;119:368e376.
N
ERVE GROWTH FACTOR (NGF) belongs to the family of neurotropic proteins that includes brain-derived neurotropic factor, neurotrophin-3, and neurotrophin-4/5. NGF contributes to maintaining the critical balance between cell survival and death both during development and in adulthood.1e3 NGF which is produced and released by a variety of mammalian cells, acts via a highaffinity cell surface receptor, tyrosine kinase receptor A (TRKA), and a low-affinity receptor, p75 neurotrophic receptor (p75NTR), a member of the tumor necrosis factor receptor superfamily.4e9 on the one hand NGF may exert its cell protective actions through the TRKA receptor; on the other hands it triggers, apoptosis in some cells through the p75NTR receptor. NGF is not only a promoter of the growth and survival of nerve cells but also of other cell types,2,4 including renal cells.10,11 To date, little is known about NGF concentrations in kidney transplant recipients (KTRs). Because NGF exerts actions on cell survival and differentiation, tissue repair, and inflammatory responses, it might be implicated in the pathogenesis of chronic allograft nephropathy. The aim of the present study was to determine the concentration of circulating NGF among a population of
KTRs and to verify whether it could be considered to be a prognostic marker for kidney transplant outcomes. MATERIAL AND METHODS Patients The subjects eligible for this study were recruited from a cohort of 200 KTRs regularly followed in the transplant ambulatory unit over a 6-month period. We applied the following criteria to select the study sample: (a) age 18e64 years, seeking to minimize age-related variations in NGF levels; (b) serum creatinine <2.0 mg/dL; (c) 24-hour proteinuria <3 g; (d) no clinical or laboratory evidence of diabetes, according to the American Diabetes Association criteria12;
From the Research Center “Rene e Trapianto,” Department of Nephrology, Dialysis, and Transplantation, “Annunziata” Hospital, Cosenza, Italy. Address reprint requests to Dr Renzo Bonofiglio, Department of Nephrology, Dialysis, and Transplantation, “Annunziata” Hospital, via F. Migliori, 87100 Cosenza, Italy. E-mail: rbonofi@ tin.it
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.07.020
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2654
Transplantation Proceedings, 45, 2654e2656 (2013)
NGF LEVEL IN RENAL TRANSPLANTATION
2655
(e) arterial blood pressure levels <140/90 mm Hg on 3 separate occasions and in the presence of antihypertensive treatment; (f) normal serum lipid profile defined by plasma levels of cholesterol and triglycerides <200 mg/dL and <150 mg/dL, respectively, and in the presence of lipid-lowering treatment; (g) absence of obesity, with a body mass index <30 kg/m2; (h) absence of drug or alcohol dependency; (i) no smoking habits in the previous 2 years; (f) immunosuppressive therapy with steroids mycophenolate mofetil, and calcineurin inhibitor, and steroid and antieinterleukin-2 antibody prophylaxisi; (g) >1 year after transplantation; and (h) hemoglobin >10.5 g/dL, and in the presence of erythropoietin treatment. We enrolled 40 KTRs who met these criteria for the study. Each of the participants gave informed consent. The study was approved by the Ethical Committee of our hospital.
Blood Samples Blood collected in the morning and was separated for serum by centrifugation at 3,500 rpm for 15 minutes.
NGF Protein Determination NGF concentrations were determined with the use of a Promega enzyme-linked immunosorbent assay kit (NGF Emax Immunoassay System, no. G7631; Promega, Madison, Wisconsin) according to the manufacturer’s instructions at enrollment and after 6 months. NGF concentrations were expressed as ng/mL; all assays were performed in triplicate.
RESULTS
The basal clinical and laboratory features of these patients are presented in Table 1. The mean concentration of NGF in the KTR serum was 1.16 0.67 ng/mL. No significant differences in NGF levels were observed between men and women. There were no significant linear correlations between NGF and glomerular filtration rate (GFR), creatinine, and proteinuria, and only a linear negative correlation between NGF and patient age (R2 ¼ .218; P < .05). To investigate the possible role of NGF on kidney transplant outcomes after a 6-month follow up, we first stratified patients according to the GFR decline: Dþ GFR (stable GFR over 6 months) versus D GFR (loss of GFR over 6 months). This was also done according to the increase in proteinuria: Dþ proteinuria (increasing proteinuria over 6 months) and D proteinuria (stable proteinuria over 6 months). We observed NGF concentrations to be higher in the D GFR group (1.26 0.78) versus the Dþ GFR group (0.85 0.24; P ¼ .02) and in the Dþ proteinuria group (1.39 0.79) versus D proteinuria group (0.87 0.31; P ¼ .02). We then performed a logistic regression analysis including univariate and multivariate (adjusted for age, sex,
and GFR) models, which showed NGF concentrations to be independently associated with proteinuria worsening. (odds ratio, 1.03; 95%, confidence interval [CI], 1.01e1.05). The data were confirmed by a receiver operating characteristic (ROC) analysis, showing an area under the ROC curve of 0.751 (95% CI, 0.575e0.927; Fig 1) with 1.15 ng/mL as the best prognostic threshold (negative predictive value ¼ 61.1; positive predictive value ¼ 83.3). DISCUSSION
The aim of this study was to investigate the behavior of NGF among a population of KTRs. Previously we also investigated NGF serum concentrations in 2 inflammatory models; glomerulonephritis end-stage renal disease during hemodialysis and treatment.13e15 We demonstrated elevated NGF concentrations among those subjects compared with healthy subjects. Those findings and the observations that NGF and NGF receptors are expressed in developing, healthy, and diseased human adult kidney cells11 led to the hypothesis that NGF is involved in renal physiopathology. In the present work, we demonstrated that KTRs also displayed increased NGF levels, which were predictive of kidney injury. In fact, NGF levels were independently associated with the development of proteinuria in KTR. Proteinuria is one of the best predictors of chronic kidney disease progression and kidney transplant failure. We think that the NGF increase is not implicated in kidney damage that successively leads to proteinuria development, but rather that it could represent a protective signal against injury. It may be part of the responsive mechanism to the kidney cell damage leading to an altered glomerular filtration barrier causing the onset of proteinuria. In fact, based on
Table 1. Clinical Parameters of the Study Population mean SD
Male:female ratio Age (y) Serum creatinine (mg/dL) eGFR (mL/min/1.73 m2) Proteinuria (g/24 h) CPR (mg/L)
28:12 43 8 1.3 0.4 98.2 12.1 0.5 0.35 1.4 0.4
Fig 1.
2656
recent studies, we suppose that up-regulation of NGF represents a protective signal against potential damage to kidney cells. In a recent report, Aloe et al16 showed a marked increase in serum NGF levels and NGF expression in the kidneys of rats displaying streptozotocin-induced type 1 diabetes. Subcutaneous administration of NGF caused a marked uptake of this cytokine by kidney cells without proapoptotic factor expression. This hypothesis is also supported by the evidence that injured diabetic skin, cardiac ischemia, corneal inflammation, peripheral neuropathy, and retinal and pancreatic cell damage are all characterized by NGF up-regulation and would benefit from local NGF treatment.17e21 However, the possibility that NGF acts with other growth factors known to be involved in kidney cell physiopathology remains to be investigated. In conclusion, NGF may be considered to be a marker for transplant outcomes. REFERENCES 1. Connor B, Dragunow M. The role of neuronal growth factors in neurodegenerative disorders of the human brain. Brain Res Rev. 1998;27:1e39. 2. Levi-Montalcini R. The nerve growth factor 35 years later. Science. 1987;237:1154e1162. 3. Sofroniew MV, Howe CL, Mobley WC. Nerve growth factor signaling, neuroprotection, and neural repair. Annu Rev Neurosci. 2001;24:1217e1281. 4. Aloe L, Tirassa P, Bracci-Laudiero L. Nerve growth factor in neurological and non-neurological diseases: basic findings and emerging pharmacological prospectives. Curr Pharm Des. 2001;7: 13e23. 5. Aloe L, Skaper SD, Leon A, Levi-Montalcini R. Nerve growth factor and autoimmune diseases. Autoimmunity. 1994;19: 141e150. 6. Flugel A, Matsumuro K, Neumann H, et al. Anti-inflammatory activity of nerve growth factor in experimental autoimmune encephalomyelitis inhibition of monocyte transendothelial migration. Eur J Immunol. 2001;31:11e22. 7. Kohyama T, Liu X, Wen FQ, Kobayashi T, et al. Nerve growth factor stimulates fibronectininduced fibroblast migration. J Lab Clin Med. 2002;140:329e335.
GIGLIOTTI, LOFARO, LEONE ET AL 8. Tuveri M, Generini S, Matucci-Cerinic M, Aloe L. NGF, a useful tool in the treatment of chronic vasculitic ulcers in rheumatoid arthritis. Lancet. 2000;356:1739e1740. 9. Villoslada P, Hauser SL, Bartke I, et al. Human nerve growth factor protects common marmosets against autoimmune encephalomyelitis by switching the balance of T helper cell type 1 and 2 cytokines within the central nervous system. J Exp Med. 2000;191:1799e1806. 10. Sariola H, Saarma M, Sainio K, et al. Dependence of kidney morphogenesis on the expression of nerve growth factor receptor. Science. 1991;254:571e573. 11. Alpers CE, Hudkins KL, Ferguson M, et al. Nerve growth factor receptor expression in fetal, mature, and diseased human kidneys. Lab Invest. 1993;69:703e713. 12. Gibbons RJ, Chatterjee K, Daley J. ACC/AHA/ACP-ASIM guidelines for the management of patients with chronic stable angina: executive summary and recommendations. A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. Circulation. 1999;99:2829e2848. 13. Bonofiglio R, Caroleo MC, Antonucci MT, et al. Transforming growth factor beta and nerve growth factor renal disease progression. Biomed Rev. 2005;16:23e34. 14. Bonofiglio R, Antonucci MT, Papalia T, et al. Nerve growth factor (NGF) and NGF-receptor expression in diseased human kidneys. J Nephrol. 2007;20(2):186e195. 15. Antonucci MT, Bonofiglio R, Papalia T, et al. Nerve growth factor and its monocyte receptors are affected in kidney disease. Nephron Clin Pract. 2009;111(1):c21ec28. 16. Aloe L, Rossi S, Manni L. Altered expression of nerve growth factor and its receptors in the kidneys of diabetic rats. J Nephrol. 2011;24(6):798e805. 17. Reimer MK, Mokshagundam SP, Wyler K, et al. Local growth factors are beneficial for the autonomic reinnervation of transplanted islets in rats. Pancreas. 2003;26:392e397. 18. Vidaltamayo R, Mery CM, Angeles-Angeles A, et al. Expression of nerve growth factor in human pancreatic beta cells. Growth Factors. 2003;21:103e107. 19. Salis MB, Graiani G, Desortes E, et al. Nerve growth factor supplementation reverses the impairment, induced by type 1 diabetes, of hind limb postischaemic recovery in mice. Diabetologia. 2004;47:1055e1063. 20. Lambiase A, Rama P, Bonini S, et al. Topical treatment with nerve growth factor for corneal neurotrophic ulcers. N Engl J Med. 1998;338:1174e1180. 21. Reinshagen M, Rohm H, Steinkamp M, et al. Protective role of neurotrophins in experimental inflammation of the rat gut. Gastroenterology. 2000;119:368e376.