Kidney International, Vol. 42 (1992), pp. 663—667

Plasma tumor necrosis factor soluble receptors in chronic renal failure MANFRED BROCKHAUS, YAACOV BAR-KHAYIM, SWIETLANA GuRwlcz, ASHER FRENSDORFF,

and NuRIT HARAN Central Research Units F., Hoffmann-La Roche Ltd. Basel, Switzerland; Division of Nephrology, Kaplan Hospital, Rehovot, and Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel

Plasma tumor necrosis factor soluble receptors in chronic renal failure. Two soluble tumor necrosis factor receptors (sTNFRs) were detected in

the plasma of patients with different degrees of chronic renal failure (CRF) and of long-term hemodialysis (HD) patients. In uremic undialyzed patients, plasma levels of both sTNFRs increased progressively with declining renal function. A linear correlation was found between sTNFR plasma levels and plasma creatinine concentration. sTNFR levels in end-stage uremic patients shortly before commencement of first HD treatment were approximately tenfold higher than in normal subjects. Long-term HD patients showed a further increase in plasma sTNFRs. The origin of sTNFRs, as well as their physiological role remains to be elucidated.

of HD patients. Levels of TNFa before the start of HD sessions were either normal [34] or elevated [35], while HD treatment

appeared either to induce an increase [34] or to have no influence [35] on plasma TNFa levels. We have recently shown, however, that high levels of TNFBP present in the plasma and

urine of HD patients interfere with the determination of TNFa by immunoassays [36]. Although particularly high levels of sTNFRs were reported in plasma and urine of hemodialysis patients [17], the actual levels have not been reported. In the present study we employed a specific and sensitive immunoassay for measurement of the

concentrations of two sTNFRs in the plasma and urine of uremic patients. We also attempted to determine the extent to Tumor necrosis factor-a (TNFa) is produced primarily by which these levels are affected by uremia and hemodialysis. monocytes/macrophages, and participates in a wide range of biological activities. It is an important mediator of various host

Methods

responses to acute or chronic injury, infection and neoplasia [1—3]. When TNFa is produced in limited amounts it may have a beneficial role in controlling the outcome of infections, as well

Subjects

Included in the study were 25 patients (18 men and 7 women) as in modulating the metabolic activities of diverse tissues with chronic renal failure (CRF). Their ages ranged from 29 to [4—9]. However, when excessively produced, as in septic 75 years (mean 48.6 years). Of these 15 were long-term hemoshock, it can lead to host mortality [10—15]. Recently, TNF binding proteins (TNFBP) were purified from

dialysis (HD) patients, receiving between 4 to 13 years (mean urine [16—20] and from HL-60 cell lysates [21], and were 6.6 years) of HD treatment for four hours, three times a week. identified as soluble receptors (sTNFRs) of TNF [2 1—24]. sTN-

FRs bind TNF with high affinity and high specificity, thereby blocking the different biological activities induced by TNF in vitro [24, 25]. Recently, it has been shown that sTNFRs also confer some TNF-antagonizing effects in vivo: they have been reported to protect mice from LPS-induced mortality [26] and to prevent, in BALB/c mice, TNF-induced hemorrhagic necrosis of a transplanted Meth-A sarcoma [27]. Hemodialysis (HD) treatment is associated with activation of

All of them except one were anuric patients. HD was performed using cuprophane membranes. The other 10 patients had various degrees of renal failure, with plasma creatinine concentrations ranging from 0.19 to 0.8 mM/liter. Four of these patients

were in the end-stage of renal failure (ESRD) and started dialysis treatment within a month of the study. The agematched healthy controls (mean 46.0, ranged from 30 to 70 years) included two men and three women.

Samples

mononuclear cells [28—311. Such activation can stimulate mono-

Heparinized blood (10 U/mi) was withdrawn from each of mRNA coding for TNFa, without translation, occurs in subject. The blood samples were taken from the antecubital nuclear cells to produce cytokines [32]. Increased transcription mononuclear cells during HD [33]. Contradictory observations have been reported with regard to levels of TNFa in the plasma

vein of control subjects and undialyzed CRF patients, while in

HD patients they were taken from the arteriovenous fistula immediately before starting and at the end of dialysis session.

Received for publication September 4, 1991 and in revised form February 28, 1992 Accepted for publication April 13, 1992

© 1992 by the International Society of Nephrology

TNFa measurement TNFa was determined by using the T-Cell-Sciences ELISA kit. The assay is a "sandwich" immunoassay employing two

663

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Brockhaus ci a!: sTNFRs in uremia

different monoclonal anti-TNFa antibodies. The assay detects biologically active TNFa only, with a sensitivity of 10 pg/mi.

70 -'———-- —

60

ii

sTNFRs measurement The assay is a slight modification of a procedure already published [37, 381. The assay is based on a sandwich between monoclonal antibodies raised against TNFR-A(p75) or TNFR30 B(p55) and 1251-TNF. Polyvinyl plates (96 well, Dynatech Alexandria, Virginia, USA) were first coated with affinitypurified rabbit-anti-mouse IgG (10 tg/m1 in PBS, 50 id/well), to 10increase attachment of the monoclonal antibody to the plastic. The sensitized plates were incubated overnight at 4°C with a 0 solution of mouse monoclonal antibody to either TNFR-A(p75) Normal ESRD ESRD-i-HD or TNFR-B(p55), (10 .tg/ml) in blocking buffer [Tris-Hcl (50 Fig. 1. Plasma immunoreactive levels of sTNFR-A(p75, ) and mM) pH 7.4, NaCI (140 mM), EDTA (5 mM), de-lipidated milk sTNFR -B (p55, 0) in normal controls (N = 5), undialyzed patients with renal disease (ESRD) (creatinine range 0.6 to 0.8 mM/liter) powder (1%)]. The monoclonal antibody utr-4 for TNFR-A(p75) end-stage (N = 4) and ESRD patients after long-term hemodialysis (HD) (N and htr-20 against TNFR-B(p55) are specific, noncrossreactive 13). Results are means SD.

and directed to non-inhibitory epitopes of the respective

TNFRs [39, 40]. The wells were washed once with PBS. Urine and plasma samples were diluted 1:20 in blocking buffer before being added to the plates in aliquots of 45 id/well. The plates were incubated for 14 hours at 4°C and washed once with PBS. '251TNF (450 Ci/mmol, 106 cpmlml) in blocking buffer, was then added to 50 p1/well aliquots. Plates were incubated for two

hours at 4°C, and washed four times with PBS. The bound radioactivity was measured in each well by gamma scintillation counting. Nonspecific binding of '251-TNF was determined in the absence of serum and was less than 5% of the total counts. The data was corrected for nonspecific binding. The specific binding of '251-TNF to sTNFRs could be prevented either by the addition of 20 ,tsg/ml of unlabeled TNFa, or by replacing the anti-TNFR monoclonal antibodies by nonspecific antibodies,

thus confirming the specificity of the assay. Linearity of the assay was verified using TNFRs from cell lysate of HL6O cells [21], as well as recombinant TNFR-A(p75) and TNFR-B(p55) [25, 41]. The concentration of sTNFR-B(p55) was calculated

from the amount of bound radioactivity using a 1:1 binding

tested immediately before a dialysis session was started. The mean concentrations of sTNFRs in the plasma of both groups of ESRD patients were considerably higher than that of the normal subjects [sTNFR-A(p75), 2.36 ng/ml 0.8; sTNFR-B(p55), 2.68 ng/ml 0.94]. Among the patients with ESRD, mean

plasma sTNFR levels in the long-term HD group [sTNFRA(p75), 29.6 ng/ml

9.5; 5TNFR-B(p55), 55.27 ng/ml

11.95]

were significantly higher (P < 0.001) than in the undialyzed group [sTNFR-A(p75), 19.57 ng/ml 7.5; sTNFR-B(p55), 27.0 ng/ml 7.7]. Moreover, as shown in Figure 1, in each of these

groups there was a marked difference in the level between the

two types of receptors: in HD patients the mean level of sTNFR-B(p55) in the plasma was approximately twice as high as that of sTNFR-A(p75) (P < 0.001), while in the undialyzed ESRD group the difference, though less pronounced, was still significant (P < 0.01). In contrast, no significant difference was

observed between the levels of the two types of sTNFR in normal subjects.

stoichiometry between '251-TNF and sTNFR-B(p55) [25]. The Relationship between renal failure and sTNFR levels concentrations calculated this way were consistent with those To establish the relationship between renal failure and circuobtained by using recombinant sTNFR-B(p55) as the standard. lating levels of sTNFRs, plasma sTNFR levels were determined We assumed the same stoichiometry for the calculation of the in 10 undialyzed patients with different degrees of CRF. Plasma concentration of sTNFR-A(p75) as well. Results are expressed creatinine levels in these patients ranged from 0.19 to 0.8

as ng/ml, assuming a molecular weight of 30 kDa for both mM/liter. The levels of sTNFRs in the plasma were found to

sTNFRs. increase with decreasing renal function. A strong linear correPost-HD levels were corrected for ultrafiltration using weight lation between plasma levels of sTNFR-A(p75) or sTNFRchanges, according to Bergstrom and Wehie [42]. B(p55) and plasma creätinine or urea concentrations was observed (Fig. 2). Also, in these patients plasma sTNFR-B levels Statistical analysis were consistently higher than those of sTNFR-A (P < 0.01). No All data were expressed as means SD. Data were analyzed such correlations between plasma sTNFR levels and plasma using the Student's t-test (paired or unpaired). A probability of creatinine or urea were obtained in HD patients, as shown in Figure 3. less than 0.05 was considered as significant.

Effect of a dialysis session In nearly all of the patients the plasma levels of both sTNFR Plasma sTNFR levels were higher at the end of a dialysis session than before it was Figure 1 compares the concentrations of the two types of started. Analysis of the data revealed that the increment (13.9% sTNFRs detected in the plasma of 13 long-term HD patients, 7.2) induced by HD treatment was significant (P < 0.01) and four ESRD patients shortly before the initiation of HD treat- correlated with weight loss occurring during a dialysis session ment (mean plasma creatinine 0.69 0.09 mM/liter, range 0.6 to [421. No increase beyond that accountable by weight loss was 0.8 mM/liter), and of five normal subjects. All HD patients were observed. Results

665

Brockhaus et at: sTNFRs in uremia

40

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zI.(0

0

0

0

100

200

300

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100

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200

300

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40

C

r=0.8

oo:

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20

I 0

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0

0.8

0.4 0.6 Creatinine, mM/liter

0.2

A

70

. .

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.

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100

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.

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.

.

35

Ic

80

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25

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60

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4

0.8

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35

0.4 0.6 Creatinine, mM/liter

0.2

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Fig. 2. Correlation between plasma concentrations of sTNFRs and urea (A, B) or creatinine (C, D) in 5 normal subjects and 10 undialyzed CRF patients.

0.9 0.7 Creatinine, mM/liter

1.1

0.5

1.1 0.9 0.7 Creatinine, mM/liter

sTNFRs in urine

Since urine (from normal, febrile, or HD patients) is the

Fig. 3. Relationship between plasma concentrations of sTNFRs and urea (A, B) or creatinine (C, D) in 13 HD patients. Blood was withdrawn immediately before dialysis treatment.

TNFa plasma concentrations Plasma levels of TNFa were determined using a T-Cell-

source from which TNF binding proteins were first isolated, we Sciences ELISA kit. In all plasma samples the concentration of measured the concentrations of sTNFRs in the urine of some of TNFa was less than 10 pg/mI, similar to the values obtained in

the uremic patients. The highest sTNFR concentrations were found in end-stage renal patients. Urine from these patients when concentrated 20-fold contained as much as 0.15 to 0.24 mg/liter sTNFR-A(p75) and 0.24 to 0.8 mg/liter sTNFR-B(p55). These remarkably high levels explain the TNFa binding activity observed in the urine of HD patients and validates their urine as

normal subjects in agreement with our previous observation [36].

Discussion

This study shows that two types of soluble TNF receptors are

significantly increased in the plasma of uremic patients. The a good source for these proteins. As in the plasma, the urine results are consistent with the observation of Peetre et a! that also contained higher levels of sTNFR-B(p55) than of sTNFR- the plasma of HD patients contains high levels of TNFa binding protein, which later was identified as a TNFa soluble receptor A(p75).

666

Brockhaus et a!: sTNFRs in uremia

[17, 241. Our present findings are complementary to our previ-

able by changes in body weight were observed. After onset of

display a TNFa binding activity which interferes with immunoassays for TNFa [36]. sTNFR levels were determined in this study using an imniunoassay that employs two different and non-cross-reacting monoclonal antibodies, each specific for a different receptor for TNFa [39, 40]. Our results show that plasma levels of sTNFRs are already high in the early stages of renal failure, and increase further with the development of uremia. Patients treated by HD over a long period show an additional increase in sTNFRs. Two distinct mechanisms, which are not necessarily mutually exclusive, could explain these observations: (1.) increased production of sTNFRs, and (2.) reduced clearance of sTNFRs due to renal insufficiency. With regard to the first, human polymorphonuclear cells (PMN)s, as well as mononuclear cells, were shown upon stimulation in vitro to shed TNF receptors, which

urea. This is most probably due to their differential clearance through the dialysis membrane. In conclusion, we have demonstrated here that uremia and dialysis treatment are associated with a considerable increase in plasma and urinary levels of two soluble TNF receptors. As to the origin and physiological role of these proteins, their elucidation must await further studies.

ous observation that the plasma and urine of HD patients RD there is no correlation between sTNFRs and creatinine and

Acknowledgments We thank Wolfgang Aufe for his technical assistance, and Michelle Landau for support.

Notes added in proof Since the submission of this manuscript, three publications reported

could be detected in the medium [24, 43]. Elastase from increased plasma levels of sTNFRs in several pathological situations, azurophilic granules of PMNs was shown in vitro to act on the including renal failure: 75 kDa TNFR, to release a 32 kDa soluble fragment [371. It has 1. ADERKA D, ENGELMANN H, HORNIK V. SIZ0RNIcK Y, LEVO Y, WALLACH D, KUSHTAI G: Increased serum levels of soluble recepbeen shown that uremia affects PMNs oxidative metabolism tors for tumor necrosis factor in cancer patients. Cancer Res and phagocytic activity in vitro [44—48]. Oxidative metabolism

of PMNs in the resting state displayed a correlation with creatinine clearance similar to that found by us for sTNFRs

51:5602—5607, 1991

2. ADOLF GR, APFLER I: A monoclonal antibody-based enzyme im-

munoassay for quantitation of human tumor necrosis factor binding

with creatinine levels [48]. It is possible that reactive oxygen protein I, a soluble fragment of the 60 kDa TNF receptor, in biological fluids. J Immuno! Meth 143:127—136, 1991 metabolites may, directly or indirectly, contribute to sTNFR production in CRF patients. An alternative mechanism that 3. LANTZ M, THYSELL H, NILssoN E, OLSSON I: On the binding of tumor necrosis factor (TNF) to heparin and the release in vivo of the could account for increased sTNFR levels is the effect of TNF TNF-binding protein I by heparin. J Clin Invest 88:2026—2031, 1991 itself. TNF has been shown to cause an increase in sTNFR levels in the serum of TNF-treated cancer patients [49]. Reprint requests to Dr. Nurit Haran, Division of Nephro!ogy, Kaplan With regard to the second mechanism, some proteins are Hospital, 76100 Rehovot, Israel. cleared from the plasma by the kidney, where they are filtered, absorbed by tubular epithelium, and eventually hydrolyzed References within renal cells to their constituent amino acids. As a consequence, when renal function is impaired the plasma half life and I. TORT! FM, DIECKMANN B, BEUTLER B, CERAMI A, RINGOLD GM: plasma levels of these proteins are markedly elevated [reviewed A macrophage factor inhibits adipocyte gene expression: An in in 50]. If sTNFRs are normally cleared from the plasma mainly vitro model of cachexia. Science (Wash. DC) 229:867—869, 1985 through the kidneys, CRF could be expected to lead to the 2. BEUTLER B, CEit,&M! A: Tumor necrosis, cachexia, shock, and inflammation: A common mediator. Ann Rev Biochem 57:505—5 18, accumulation of these proteins in the plasma. As the degree of 1988 renal failure increases, smaller amounts of sTNFR would be 3. BEUTLER B, CERAMI A: The biology of cachectin/TNF—A primary cleared by the kidney and more retained in plasma. Such a mediator of the host response. Ann Rev Immuno! 7:625—655, 1989 mechanism could explain the linear correlation observed here 4. TRACEY KJ, CERAMI A: Metabolic responses to cachectin/TNF: A brief review. Ann NYAcad Sd 587:325—331, 1990 between plasma sTNFR levels and plasma creatinine, as well as the additional increase observed in HD patients, when residual 5. TRACEY KJ, WE! H, MANOGUE KR, FONG Y, HES5E DG, NGUYEN I-IT, Kuo GC, BEUTLETR B, COTRAN RS, CERAMI A, LOWRY SF: renal function is further reduced. In this case the elevated levels Cachectin/tumor necrosis factor induces cachexia, anemia and of sTNFRs would merely be an additional expression of reinflammation. JExp Med 167:1211—1227, 1988 duced renal function. In patients with renal disease, as the 6. FONG Y, MOLDAWER LL, MARANO MA, WE! H, BARBER A, MANOGUE KR, TRACEY KJ, Kuo G, FISCHMAN DA, CERAMI A, selectivity of the glomerular semi-permeable barrier is altered, LOWRY SF: Cachectin/TNF or IL-la induces cachexia with redisconsiderable amounts of these proteins may be filtered and tribution of body proteins. Am J Physiol 256:R659—R665, 1989 secreted in the urine. This could explain the high concentrations 7. PLATA-SALAMAN CR, OOMURA Y, KAI Y: Tumor necrosis factor of sTNFRs found in urine of CRF patients, although shedding and interleukin 1-13: suppression of food intake by direct action in the central nervous system. Brain Res 448:106—1 14, 1988 of sTNFRs from damaged renal cells cannot be excluded as a 8. JOHNSON RA, WADDELOw TA, CARO J, OLIFF A, ROODMAN GD: source of urinary sTNFRs. At the present stage we do not have Chronic exposure to tumor necrosis factor in vivo preferentially sufficient data to differentiate the possibilities we mentioned. inhibits erythropoiesis in nude mice. Blood 74:130—138, 1989 The observation that plasma levels of sTNFRs are consider9. TRACEY KJ, MORGELLO S, KOPLIN B, FAHEY TJ Ill, Fox J, ALEDO A, MANOGUE KR, CERAMI A: Metabolic effects of cachectin/tumor ably higher in long-term HD patients (treated with cuprophane necrosis factor are modified by site of production. J Clin invest dialyzers) than in undialyzed ESRD patients, can equally be

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