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Technical Briefs |
1
Hennepin County Medical Center, Clinical Laboratories, Minneapolis MN 55415
2
Department of Physiology, Queens University, Kingston, Ontario, K7L 3N6 Canada
a author for correspondence: fax 612-904-4229; e-mail
fred.apple{at}co.hennepin.mn.us
Recent publications of consensus documents for the redefinition of myocardial infarction are heavily predicated on the role of increases in cardiac troponin (I or T) in serum in the setting of ischemic symptoms (1)(2)(3)). Cardiac troponin T (cTnT) has also been reported to be a predictor of mortality in patients with end stage renal disease (4)(5). An unexplained increase in the frequency of increased serum cTnT compared with serum cardiac troponin I has been described in these patients (6)(7). Explanations advanced for this difference include the possibility of nonspecific reactions in the cTnT assay or de novo expression of cTnT in skeletal muscle of renal diseased patients that is subsequently released into the serum (8). However, recent studies on skeletal muscle from renal disease patients have shown that although the two antibodies used in the cTnT diagnostic assay (M7 and M11.7) individually bind to muscle proteins, this would not cause false-positive results in the current-generation cTnT assay marketed by Roche (8). On the other hand, cardiac troponin I is not expressed in fetal or in healthy or diseased adult human skeletal tissue (9). An additional possibility is that cTnT or other immunoreactive proteins are being expressed in diseased renal tissue. In the current study, we report evidence, using the two monoclonal antibodies from the third-generation Roche cTnT immunoassay, that diseased renal tissue is not the tissue source of circulating cTnT in acute and chronic renal diseased patients.
This study used renal biopsy tissues obtained for histological analysis to assist in differential diagnosis of 10 patients with renal disease and without known cardiac disease (4 males and 6 females; age range, 21–43 years). The diagnoses of the patients included segmental glomerular sclerosis, membranous glomerulopathy, membranous lupus nephritis, necrosis, membranous nephropathy, interstitial fibrosis, and sclerosis of glomeruli. The serum creatinine in these subjects was 7–67 mg/L. All specimens were stored frozen (-80 °C) before processing. Biopsy samples were homogenized on ice in a buffer containing, per liter, 6 mol of urea, 20 mmol of Tris (pH 6.8), 50 mmol of NaF, 0.2 mmol of Na3VO4, 1 µmol of leupeptin, 1 µmol of pepstatin, 0.36 µmol of aprotinin, 2 mmol of sodium EDTA, and 0.25 mmol of phenylmethylsulfonyl fluoride. Under denaturing and reducing conditions, 5–10 µg of total protein determined by Lowry assay (10) was separated on 12% sodium dodecyl sulfate-polyacrylamide gels as described previously (11). Transfer of proteins onto nitrocellulose membranes and Western blot analysis using monoclonal anti-cTnT antibodies (M7 or M11.7; kindly provided by K. Hallermayer, Roche Diagnostics, Penzberg, Germany) in a concentration of 2 mg/L were performed (11). Comparison studies between the Roche serum cTnT assay and the Western blot technique were not carried out to determine the sensitivity of the gel system. However, we did compare more or less equal amounts of protein loading. Serum cTnT concentrations were determined using the third-generation Roche cTnT assay on the Elecsys 2010, with an upper 99th percentile reference limit of 0.01 µg/L.
Western blotting for all 10 renal tissues using both M7 and M11.7
antibodies to detect cTnT expression is shown in Fig. 1
. The control (human heart) demonstrated one major 42-kDa band
(intact cTnT) with both antibodies and several degradation products of
minor intensity and lower molecular masses. When we used the
M11.7 antibody, no 42-kDa cTnT protein was detected in any of the 10
renal tissues. However, a 36-kDa protein band was detected in 6 of 10
samples. The M7 antibody detected small quantities of a 42-kDa protein
in two renal tissue specimens (patients 4 and 9), but it did not detect
bands at lower molecular masses. When we used four other anti-TnT
antibodies [polyclonal anti-TnT P1 and P3 (BiosPacific), monoclonal
anti-TnT JKT-12 (Sigma), and monoclonal anti-TnT 4D-11 (Biodesign)]
that bind to epitopes different from those bound by M7 and M11.7, none
of these bands was confirmed as cTnT, nor did we find evidence that
these bands would be skeletal TnT (data not shown). Time-matched serum
specimens, available for 2 of the 10 renal disease patients (patients 3
and 7), gave cTnT concentrations of 0.06 and 0.10 µg/L, respectively.
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Our findings demonstrate that M7 and M11.7 individually detected at
least two proteins in diseased renal tissues (Fig. 1
). However, because
only 2 of the 10 tissues demonstrated a 42-kDa protein with only the M7
antibody, it would appear that the 42-kDa protein expressed in renal
tissue is different from the intact, control heart cTnT protein
detected by both M7 and M11.7 and the other four antibodies used. These
findings were very similar to those demonstrated in skeletal muscle
specimens obtained from patients with renal disease (8).
Therefore, if the 42-kDa protein is released from injured renal tissue
into the circulation, it would not lead to a false-positive result in
blood. The same holds true for the 36-kDa protein detected by M11.7 but
not M7. It should be noted that the molecular mass of human cTnT is
34.5 kDa. Depending on the electrophoretic conditions and which
molecular markers are used, the molecular mass of intact cTnT may seem
to vary, e.g., 42 kDa in the present study vs 39 kDa in a previously
published study (8). Contamination of renal biopsy samples
with plasma positive for cTnT (not tested for in patients 4 and 9) is
highly unlikely because only M7 detected the 42-kDa protein, and tissue
homogenates were highly diluted before Western blot analysis.
Our findings further support the tissue source of circulating cTnT found in plasma specimens (0.06 and 0.10 µg/L) from the two patients with renal disease in the present study as not the kidney. However, it should be noted that a limitation of the current study was that it represented a small study size. The mechanisms involved in the release of cTnT in patients with renal disease have not been determined. However, increasing evidence now clearly supports the fact that increased serum/plasma cTnT concentrations in patients with acute or chronic renal disease are associated with increased coronary disease risk factors (11) and increased mortality (4)(5)(12)(13).
References
The following articles in journals at HighWire Press have cited this article:
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  M. S. Willis, J. A. Snyder, R. H. Poppenga, and D. G. Grenache Bovine cardiac troponin T is not accurately quantified with a common human clinical immunoassay J Vet Diagn Invest, January 1, 2007; 19(1): 106 - 108. [Abstract] [Full Text] [PDF]
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 N. A. Khan, B. R. Hemmelgarn, M. Tonelli, C. R. Thompson, and A. Levin Prognostic Value of Troponin T and I Among Asymptomatic Patients With End-Stage Renal Disease: A Meta-Analysis Circulation, November 15, 2005; 112(20): 3088 - 3096. [Abstract] [Full Text] [PDF]
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 D. Ritter, P. A. Lee, J. F. Taylor, L. Hsu, J. D. Cohen, H. D. Chung, and K. S. Virgo Troponin I in Patients without Chest Pain Clin. Chem., January 1, 2004; 50(1): 112 - 119. [Abstract] [Full Text] [PDF]
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 B. J. Freda, W. H. W. Tang, F. Van Lente, W. F. Peacock, and G. S. Francis Cardiac troponins in renal insufficiency: Review and clinical implications J. Am. Coll. Cardiol., December 18, 2002; 40(12): 2065 - 2071. [Abstract] [Full Text] [PDF]
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A. S. Jaffe Testing the wrong hypothesis: the failure to recognize the limitations of troponin assays J. Am. Coll. Cardiol., October 1, 2001; 38(4): 999 - 1001. [Full Text] [PDF]
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