Serum Cystatin C-Immunoglobulin High-Molecular-Weight Complexes in Kidney and Liver Transplant Patients

Kidney International, Vol. 60 (2001), pp. 1561–1564

Serum cystatin C-immunoglobulin high-molecular-weight complexes in kidney and liver transplant patients

JESU´ S HERMIDA,RAFAEL ROMERO, and J. CARLOS TUTOR

Laboratorio Central y Servicio de Nefrologı´a, Hospital Clı´nico Universitario de Santiago de Compostela, Santiago de Compostela, Spain

Serum cystatin C-immunoglobulin high-molecular-weight com- results obtained for renal transplant patients are still plexes in kidney and liver transplant patients. very limited [8] and even contradictory. Background. It has been suggested recently that the glomerular filtration rate (GFR) in renal transplant patients is under- Some authors found that serum cystatin C was a good estimated by serum cystatin C due to an impaired filtration of marker for GFR in renal transplant patients, and that it complexed cystatin C with immunoglobulins. Consequently, was clearly superior to the commonly used serum creati- serum cystatin C may not be a reliable marker of GFR in these nine [9–12]. However, others believe that cystatin C in patients. Our study was designed to determine whether this these cases would lead to a significant underestimation supposition is correct. Methods. In 87 serum samples from patients with various of the GFR [13–15] and, consequently, that it is not a kidney diseases, 182 samples from renal transplant patients, reliable marker in patients after renal transplantation and 72 samples from liver transplant patients, the concentra- [15]. Boke¨nkamp et al, after rejecting other possible tions of cystatin C and creatinine were determined, as well as causes (analytical interference of immunosuppressors, the residual concentration of cystatin C after precipitation of metabolic changes caused by immunosuppression, and macromolecules with polyethylene glycol (PEG; 6000 molecular weight). tubulointerstitial damage leading to a back-leak of intact Results. The residual concentration of serum cystatin C after cystatin C into circulation), suggested that it was due precipitation with PEG in all cases was much higher (70 to to an impaired glomerular filtration of the complexed 100%) than that expected in the case of the existence of cystatin cystatin C with immunoglobulins [14]. The immunoglob- C-immunoglobulin complexes. In the kidney and liver trans- ulins may form high molecular weight complexes with plant patients, there was no significant correlation between the residual concentration of cystatin C and the postoperative time normal serum constituents [16, 17], and the demonstra- (r ϭϪ0.098). tion of the existence of these circulatory complexes is of Conclusions. The results suggest that in renal or liver trans- obvious clinical interest. plant patients there is no formation of high molecular weight Macromolecules are known to precipitate selectively serum cystatin C-immunoglobulin complexes, regardless of the post-transplant period. in concentrated solutions of polyethylene glycol (PEG). Our study examined the possible existence of cystatin C-immunoglobulin serum complexes in kidney and liver transplant patients via precipitation with PEG. This tech- Human cystatin C is a nonglycosylated basic low molecular weight protein (Ϸ13.3 kD) that is produced by nique has been widely used in the case of other serum all nucleated cells with a stable production rate, and protein molecules [18–22]. therefore, its blood concentration should be determined mainly by the glomerular filtration rate (GFR) [1–3]. METHODS Serum cystatin C appears to meet the requirements of One hundred eighty-two serum samples were analyzed an ideal GFR marker [3] and has been suggested to be from 52 patients who had undergone kidney transplanta- more suitable than serum creatinine [4–7]. However, the tion (26 male and 26 female; mean age Ϯ SEM of 47.8 Ϯ 1.97 years; range 19 to 72 years) and 71 serum samples Key words: glomerular filtration rate, polyethylene glycol 6000, marker from 28 liver transplant patients (17 male and 11 female; for GFR, creatinine, end-stage renal disease. mean age 53.6 Ϯ 2.04 years; range 35 to 67 years). We also analyzed 87 samples from 66 patients (42 male and Received for publication February 19, 2001 and in revised form April 24, 2001 24 female) with different types of kidney disease, with Accepted for publication May 10, 2001 normal to severely impaired kidney function (mean age  2001 by the International Society of Nephrology 58.7 Ϯ 2.26 years; range 19 to 87 years). In cases in which

1561 1562 Hermida et al: Serum cystatin C in transplantation these patients underwent periodic hemodialysis, samples were taken immediately beforehand. Similarly, 14 serum samples were studied from patients with rheumatoid arthritis (4 male and 10 female; mean age of 55.0 Ϯ 2.8 years; range 23 to 78 years). Serum cystatin C was determined using a particle- enhanced immunoturbidimetric assay [23] in a Cobas Fara II Analyser (Roche Diagnostics, Basel, Switzer- land) using reagents from Dako Diagnostics (Copenha- gen, Denmark); the reference interval considered in our laboratory was 0.6 to 1.4 mg/L. Investigation of cystatin C-immunoglobulin complexes was carried out by deter- mining the residual concentration of cystatin C after precipitation with PEG (molecular weight 6000), according to the procedure described by Levitt and Ellis [18]. Se- rum samples were mixed with equal volumes of 240 g/L PEG solution (final concentration 120 g/L), incubated at 37ЊC for 10 minutes and centrifuged; the cystatin C concentrations in the supernants were determined, and the relative residual concentrations, with respect to the Fig. 1. Residual concentrations of recombinant human cystatin C (ᮀ), same samples mixed with equal volumes of water, were serum cystatin C (᭿), IgA (᭡), IgG (᭺), and IgM (᭹) after precipitation with different concentrations of polyethylene glycol (PEG). calculated. The possible analytical interference from PEG on the cystatin C turbidimetric determination was studied using a recombinant human cystatin C solution (4.5 mg/L) supplied by Dako Diagnostics. Serum creati- patients. No statistically significant difference was found nine was determined using a kinetic alkaline picrate between the different groups of patients studied. We method, which significantly reduces the interference of also obtained an analogous interindividual variation for non-creatinine chromogens, in an Advia 1650 Analyser the residual concentrations of cystatin C in the groups (Bayer Diagnostics, Mu¨ nchen, Germany). of patients with kidney disease (CV ϭ 7.3%), liver trans- Statistical analysis of the data was carried out using plantation (CV ϭ 8.2%), and kidney transplantation the Microsoft-Excel package (version 5.0). The Kolmo- (CV ϭ 8.2%). In the 14 patients with rheumatoid arthri- gorov-Smirnov test was applied to check for normality. tis, with a concentration of serum cystatin C of 1.63 Ϯ Parametric tests were used when the data had a Gaussian 0.11 mg/L (range 1.0 to 2.1 mg/L), there was a mean distribution (Student t test and Pearson correlation coef- residual concentration of this protein, after precipitation ficient); otherwise, nonparametric tests were used (Wil- with PEG, of 82.5 Ϯ 2.9% (range 61 to 100%). coxon test and Spearman correlation coefficient). The In the kidney and liver transplant patients, no signifi- results are expressed as mean Ϯ SEM and median. Sig- cant correlation was found between the residual concen- nificance was defined as a P value of less than 0.05. tration of cystatin C and post-transplant time (Fig. 2). Likewise, a significant correlation between these vari- ables was not achieved when the kidney (r ϭϪ0.059) RESULTS or liver (r ϭϪ0.109) transplant patients were considered Figure 1 shows the results corresponding to the resid- separately. ual concentrations of recombinant human cystatin C, serum cystatin C, and immunoglobulins A, G, and M, after precipitation with different final concentrations of DISCUSSION PEG (0 to 190 g/L) by means of the general procedure Precipitation with PEG has been shown to be a valid indicated in the Methods section. The results indicated screening test to detect serum immunoglobulin com- for serum cystatin C and immunoglobulins are the aver- plexes with several enzyme proteins [18–21] and prolac- age results of those obtained in four serum samples from tin [22]. However, it cannot be used for all analytical healthy controls and kidney disease patients. methods because of a possible interference from PEG Table 1 shows the results obtained for the serum con- [24]. The addition of PEG led to a very discreet decrease centrations of cystatin C and creatinine in kidney disease, in the concentration of recombinant human cystatin C and liver and kidney transplant patients. Table 2 shows (Fig. 1), which suggests that PEG, at least at a final the residual concentrations of cystatin C after precipita- concentration of 120 g/L in the samples, does not produce tion with PEG (final concentration 120 g/L) in these a significant analytical interference in the turbidimetric Hermida et al: Serum cystatin C in transplantation 1563

Table 1. Serum cystatin C and creatinine concentrations in the groups of patients studied Cystatin C mg/L Creatinine lmol/L N MeanϮSEM Median Range MeanϮSEM Median Range Kidney disease 87 4.58Ϯ0.21 4.97 0.84–8.48 510.95Ϯ34.47 512.72 70.72–1175.7 Renal transplantation 182 3.07Ϯ0.13 2.38 1.16–10.27 289.95Ϯ18.56 185.64 61.88–1264.12 Liver transplantation 71 2.62Ϯ0.11 2.49 1.13–5.22 129.95Ϯ8.84 106.08 53.04–380.12

Table 2. Cystatin C residual concentration after precipitation with PEG 6000 (ﬁnal concentration 120 g/L) MeanϮSEM N%Median Range Kidney disease 87 86.8Ϯ0.68 87.6 70–100 Renal transplantation 182 84.8Ϯ0.52 84.2 70–100 Liver transplantation 71 88.2Ϯ0.87 88.0 71–100

method used. With this final concentration of PEG, as used in the precipitation procedure of Levitt and Ellis [18], we found a mean residual concentration for serum cystatin C of 86% (Fig. 1), which may be due to a nonspe- cific partial co-precipitation with other serum proteins with a greater molecular mass. The results obtained for amylase by other authors support this suggestion [18]. The results obtained for IgG and IgM, as shown in Figure 1, may be superimposed on those of Luraschi et al [21] and demonstrate the near-total precipitation of Fig. 2. Relationship between residual concentration of serum cystatin these immunoglobulins for a final concentration of 120 C and post-surgery time in kidney (᭺) and liver (᭹) transplant patients g/L PEG. Unfortunately, those authors did not evaluate (r ϭϪ0.098). the precipitation of IgA through the action of PEG [21], whose residual concentration in the same test conditions was, according to our results, approximately 30% (Fig. serum cystatin C concentrations with GFR within the 1). In any case, this fact does not appear to affect the reference limits, one of which was from a renal transplant discriminatory capacity of the test decisively [18–22], nor, recipient and the other two with systemic sclerosis or therefore, the interpretation of our results. rheumatoid arthritis [6]. An increased serum level of No significant difference was found for the residual cystatin C was found in patients with autoimmune dis- concentrations of cystatin C after precipitation with PEG eases [25], and a significant increase of cystatin C was in the three groups of patients (Table 2), which present found in a group of patients with rheumatoid arthritis, an analogous interindividual variation for this variable whereas creatinine did not differ significantly from the (CV Ϸ8%). Bearing in mind the results obtained for healthy control group [26]. Because the grafted kidney other serum components [18–22], the residual concentra- induces an antibody response it has been suggested re- tion of cystatin C after precipitation with PEG was, in cently, without definitive proof, that there is an impaired all cases, much higher than that expected in the case of filtration of cystatin C-immunoglobulin complexes in re- there being complexes with immunoglobulins. There- nal transplant recipients [14]. However, we were not able fore, it does not appear that in renal or liver transplants, to confirm the formation of these high molecular mass circulatory cystatin C forms high-molecular-weight com- complexes in our patients, either those with renal or plexes with immunoglobulins, regardless of the post- hepatic transplants or in the patients studied with rheu- transplant time (Fig. 2). As a result, it was not possible matoid arthritis. to confirm the hypothesis of Bo¨ kemkamp et al that in In accordance with our experience, elevated cystatin renal transplant patients cystatin C has an impaired glo- C concentrations with normal creatinine values in adult merular filtration due to the formation of these macro- kidney transplant recipients after discharge from the hos- complexes [14]. pital have been reported by other authors [27]. 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Le Bricon T, Thervet E, Froissart M, et al: Plasma cystatin C is also by additional factors that differ for cystatin C and superior to 24-h creatinine clearance and plasma creatinine for creatinine [28]. However, serum cystatin C appears to estimation of glomerular filtration rate 3 months after kidney transplantation. Clin Chem 46:1206–1207, 2000 be a better marker of GFR than serum creatinine in 13. Bo¨ kenkamp A, O¨ zden N, Dieterich C, et al: Cystatin C and creati- adults with normal to impaired kidney function [4–7, 28]. nine after successful kidney transplantation in children. Clin Ne- Therefore, cystatin C determination may be useful in phrol 52:371–376, 1999 14. Bo¨ kenkamp A, Domanetzki M, Zinck R, et al: Cystatin C serum kidney and liver transplant patients, although neither concentrations underestimate glomerular filtration rate in renal cystatin C nor creatinine reliably permits an estimation transplant recipients. Clin Chem 45:1866–1868, 1999 of the exact GFR. 15. 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