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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 20  |  Issue : 6  |  Page : 1000-1004
Evaluation of serum tumor necrosis factor α and its correlation with histology in chronic kidney disease, stable renal transplant and rejection cases

1 Department of Pathology, Division of Immunopathology, UGC Advanced Immunodiagnostic Training and Research Center, Varanasi, India
2 Department of Nephrology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India

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Date of Web Publication27-Oct-2009


Tumor necrosis factor alpha (TNF α) is a cytokine secreted by macrophages, helper T cells, Natural Killer cells, B lymphocytes and non lymphoid cells e.g. endothelial cells, fibroblast and tumor cell lines. Aim of the study was to find the utility of TNF α in diagnosing renal transplant rejection among the renal transplant cases (n=29), and comparison with the levels in patients on maintenance hemodialysis (n=21) and healthy controls (n=20). TNF α in healthy controls varied from 2 to 15 pg/mL. In chronic renal failure and renal transplant rejection cases TNF α was above 45 pg/mL. In stable renal transplant patients it was higher than normal (16 to 30 pg/mL). In both acute and chronic transplant rejection TNF α increase correlated well with histology. Thus our study suggests that TNF a level more than 45 pg/mL can be taken as an immunological marker of renal transplant rejection.

How to cite this article:
Sonkar GK, Usha, Singh R G. Evaluation of serum tumor necrosis factor α and its correlation with histology in chronic kidney disease, stable renal transplant and rejection cases. Saudi J Kidney Dis Transpl 2009;20:1000-4

How to cite this URL:
Sonkar GK, Usha, Singh R G. Evaluation of serum tumor necrosis factor α and its correlation with histology in chronic kidney disease, stable renal transplant and rejection cases. Saudi J Kidney Dis Transpl [serial online] 2009 [cited 2023 Feb 4];20:1000-4. Available from: https://www.sjkdt.org/text.asp?2009/20/6/1000/57253

   Introduction Top

Tumor necrosis factor α was identified in 1975 as an endotoxin-induced glycoprotein. It was originally described as a circulating factor that can cause necrosis of tumors, but has since been identified as a key regulator of the infla­mmatory response. [1] Tumor necrosis factor α (Cachectin) is a 17 kilodalton, nonglycosylated protein. The biologically active form of tumor necrosis factor α (TNF α) is a trimer. It is ele­vated in plasma during kidney transplant rejection [2] and is involved in many inflamma­tory phenomenon. [3],[4] The secretion of TNF α in allograft rejection largely results from acti­vation of immune cells triggered by alloantigen presentation to immunocompetent cells of host origin. [3],[5] Macrophages, CD4+ T cells, Natural Killer (NK) cells, neutrophils, T and B lym­phocytes fibroblasts, endothelial cells and va­riety of tumor cell lines have been shown to produce TNF α[2],[1] Elevation of circulating TNFα have been reported in allograft rejection [6],[7] and in patients undergoing dialysis as com­pared with healthy controls. [8]

Scarcity of data exists comparing the levels of TNF α in renal failure, renal transplant pa­tients and healthy controls from India. Hence the main aim of this study was to evaluate serum TNF α as an immunological marker for assessing levels in chronic kidney disease (CKD) Stage V on maintenance hemodialysis and renal transplant rejection (Tx Rej) cases due to cost and being non invasive. Attempt has been made to correlate the serum TNF α with histological findings of rejection.

   Methods Top

A total of 80 subjects were enrolled in this study over one and a half year and included the following:

  1. 21 patients with CKD undergoing mainte­nance hemodialysis
  2. 29 post transplant serum samples which included 18 stable grafts (serum creatinine less than 2.0) and 11 biopsy proven graft rejection and
  3. 20 normal healthy controls (NHC).

Histopathological grading of Tx Rej was done according to Banff 2003 criteria update. [9] In post transplant cases serum creatinine (SCr) was assayed every month for one year follow up and their mean value was taken while se­rum TNF α was assayed at an average of 4.3 months post transplant during follow up. Serum TNF α was assayed using sandwich ELISA (kit of Beckman Coulter, France, supplied by Awadh Scientific Ltd, Lucknow, India). The study was approved by local ethical commi­ttee and informed consent was obtained from all patients enrolled in the study.

Manufacturer's instruction was as follows: 100 μL of conjugate per well and 100 μl of calibrators (1000, 250, 62.5, 15.6 and 0 pg/mL) or 100 μL serum samples were added in each well of ELISA plate and incubated for 2 hours at room temperature on a shaker. After wa­shing the wells with wash solution for three times, 200 μL of pNPP substrate solution was added to each well and incubated for 45 mi­nutes in dark while shaking. 50 μl of stop so­lution was added and the optical density was read at 405 nm. Values of unknown samples were calculated using the standard graph plotted using the 5 calibrator values. SCr was assayed by autoanalyser, using kits supplied by Tulip diagnostics (P) Ltd, Goa, India.

   Results Top

In NHC all the 20 cases (100%) had TNF α level below 15 pg/mL. Mean value of serum TNF α in the NHC was 7.45 ± 4.61 pg/mL and range varied from 2 to 15 pg/mL [Table 1]. The TNF α level in CKD patients were markedly raised to a minimum of 3 times the NHC (P< 0.05) [Table 1]. In stable renal transplantation (Tx Stb) cases majority (66.7%) had mild rise of TNF α between 16-30 pg/mL and only one patient had TNF α above 30 pg/mL. In contrast all patients with renal Tx Rej cases had very high TNF α level, all 11 patients had TNF α above 45 pg/mL [Table 1]. Rise of TNF α in both stable and rejection cases was statistically significant as compared to NHC [Table 1].

Correlation of TNF α with histological fin­dings showed that it was increased above 45 pg/mL in all acute cellular rejection, chronic rejection, interstitial nephritis with acute tubular necrosis, membranous glomerular nephritis with thrombotic microangiopathy [Table 2].

There was no positive correlation of serum creatinine with TNF α. SCr in NHC was less than 1.5 mg/dL compared to above 4 mg/dL in CKD patients [Table 3]. In Tx Stb cases only in 3 patients had mild rise of SCr was noted (1.6, 1.8 and 1.9 mg/dL) in contrast to trans­plant rejection where all cases had SCr above 2.4 mg/dL (2.4-5.7 mg/dL) [Table 3].

   Discussion Top

In our study we found low levels of TNF α in NHC and significantly higher levels in CKD and renal transplant patients similar to earlier studies. [2],[6],[7],[8]

The diagnosis of transplant rejection by non-invasive means has been a goal for many years because kidney biopsies are invasive, costly, and can be associated with morbidity and even death from bleeding. Rejection of renal allograft begins with the immunological mechanism starting from the recognition of graft antigens and the recruitment of cytotoxic T lymphocytes. Then together with the release of inflammatory cytokines such as TNF α and IFN γ, the proliferation and differentiation of T and B lymphocytes occurs leading to mac­rophage activation and further increase in TNF α leading to graft rejection.

All patients of renal Tx Rej in our study had very high levels of TNF α of more than 45 pg/mL where as in renal Tx Stb cases majority had the level between 15-30 pg/mL. Ficek et al. [10] have reported marked increase of TNF α in acute renal failure patients (70 pg/mL) and an even significantly higher levels in hemo­dialyzed patients (216 pg/mL). Contrary to it, study done by Powell et al reported that plasma TNF α are not chronically elevated in chronic renal failure, peritoneal dialysis or hemodia­lyzed patients. [11] Abdallah et al [7] noted eleva­tion of TNF α in the majority patients of heart transplantation especially moderate and severe rejection. In our study of renal transplant re­jection, TNF α was markedly raised with a mean value of 71.45 ± 22.27 pg/mL.

The increased level of TNF α supports that this cytokine is a mediator of immune process during rejection. Study conducted by Bukan et al [12] on 30 end stage renal failure (ESRF) pa­tients have reported that there was an increase in serum TNF α before hemodialysis and after hemodialysis and contributed to possible arthe­rosclerotic process. The TNF α raised from 73.49 ± 21.01 to 109.02 ± 3.09 pg/mL which was found to be statistically significant. It has been suggested that impaired monocyte func­tion leads to changes in immune response to infectious agents and overproduce proinfla­mmatory cytokines such as IL-1β, TNF α and IL-6. Cottone et al. from Italy [13] measured se­rum TNF α in 35 renal transplant recipients, 35 CKD and 26 healthy controls and found similar results. In CKD group as well as trans­planted group, the TNF α was higher than in controls. However contrary to our study, the TNF α levels in CKD and transplant groups were statistically insignificant.

Al-Lamki et al [14] studied TNF receptors 1 and 2 (TNF R1, R2) in normal kidney and acute transplant rejection by immunohistochemistry and immunogold electron microscopy. They demonstrated TNF R1 within endothelium of glomeruli of normal kidney that was lost in acute renal transplant rejection and detected in abundance on infiltrating leukocytes in the interstitium. Contrary to it TNF R2 was de­monstrated predominantly on epithelial cells of distal tubules of kidney. TNF was also ab­sent in normal kidney but present on infiltra­ting leukocytes of rejecting grafts. This study thus implicates TNF in acute allograft kidney rejection. Macrophage appearance, as a result of cell mediated immunity, in the interstitium of rejecting kidneys secretes TNF α. This is evident in our cases also where all transplant rejected kidneys whether acute or chronic, showed mononuclear cell (MNC) infiltration.

In conclusion, our study suggets that TNF α has a role in diagnosing transplant rejection. TNF α more than 45 pg/mL might suggest rejection in renal transplant patients. However further studies in a larger number of sample size is required to validate the result.

   Acknowledgement Top

We are thankful to UGC Advanced Immuno­diagnostic Training and Research Centre, De­partment of Pathology, Institute of Medical Sciences, Banaras Hindu University for its financial support. We would also like to thank Miss Sangeeta Singh, Junior Research Fellow for helping us in preparation of the manuscript.

   References Top

1.Bradley JR. TNF-mediated inflammatory disease. J Pathol 2008;214:149-60.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]  
2.Dorge SE, Roux-Lombard P, Dayer JM, Koch KM, Frei U, Lonnemann G. Plasma levels of tumor necrosis factor (TNF) and soluble TNF receptors in kidney transplant receptors.Transplantation 1994;58(9):1000-8.  Back to cited text no. 2      
3.Morel D, Normand E, Lemoine C, et al. Tumor necrosis factor alpha in human kidney transplant rejection - in situ hybridization. Transplantation 1993;55(4):773-7.  Back to cited text no. 3      
4.Wiggins MC, Bracher M, Mall A, Hickman R, Robsen SC, Kahn D. Tumour necrosis factor levels during acute rejection and acute tubular necrosis in renal transplant recipients. Trans­plant Immunology 2000;8:211-5.  Back to cited text no. 4      
5.Tracey KJ, Cerami A. Tumour necrosis factor, other cytokines, and disease. Annu Rev Cell Biol 1993;9:317-43.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]  
6.Maury CP, Teppo AM. Raised Serum Levels of Cache tin /Tumor Necrosis Factor Alpha in Renal Allograft Rejection. J Exp Med 1987; 166(4):1132-7.  Back to cited text no. 6      
7.Abdallah AN, Billest MA, Attia Y, Doutre­mepuich C, Cassaigne A, Iron A. Evaluation of plasma levels of tumor necrosis alpha and interleukin-6 as rejection markers in a cohort of 142 heart grafted patients followed by endomyocardial biopsy. Eur Heart J 1997;18: 1024-9.  Back to cited text no. 7      
8.Borazan A, Ustu H, Yucel Ustundag Y, et al. The effects of peritoneal dialysis and hemo­dialysis on serum tumor necrosis factor-alpha, interleukin-6, interleukin-10 and C-reactive­protein levels. Mediators Inflamm 2004;13(3): 201-4.  Back to cited text no. 8      
9.Racusen LC, Colvinb RB, Solezc K, et al. Antibody-Mediated Rejection Criteria - an Addition to the Banff '97 Classification of Renal Allograft Rejection. Am J Transplant 2003;3:708-14.  Back to cited text no. 9      
10.Ficek R, Kokot F, Chudek J, Adamczak M, Ficek J, Andrzej W. Plasma concentration of tumor necrosis factor alpha may predict the outcome of patients with acute renal failure. Kidney Blood Press Res 2006;29:203-9.  Back to cited text no. 10      
11.Powell AC, Bland LA, Oettinger CW, et al. Lack of plasma interleukin-1(3 or tumor necro­sis factor a elevation during unfavorable hae­modialysis conditions. J Am Soc Nephrol 1991;2:1007-13.  Back to cited text no. 11      
12.Bukan N, Sancak B, Pasaoglu H, Elbeg S, Unal A, Erten Y. Serum homocysteine, lipo­protein (a), tumor necrosis factor-alpha, total cholesterol and triglyceride levels in haemo­dialysis patients. Turkiye Klinikleri J Med Sci 2004;24:435-9.  Back to cited text no. 12      
13.Cottone S, Palermo A, Vaccaro F, et al. Infla­mmation and endothelial activation are linked to renal function in long term kidney trans­plantation. Transplant Int 2007;20(1):82-7.  Back to cited text no. 13      
14.Al-Lamki RS, Wang J, Skepper JN, Thiru S, Pober JS, Bradley JR. Expression of Tumor Necrosis Factor Receptors in Normal Kidney and Rejecting Renal Transplants. Lab Invest 2001;81:1503-15.  Back to cited text no. 14  [PUBMED]    

Correspondence Address:
UGC Advanced Immunodiagnostic Training and Research Center, Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005
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Source of Support: None, Conflict of Interest: None

PMID: 19861860

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  [Table 1], [Table 2], [Table 3]

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