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Articles |
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Laboratoire de Biochimie A,
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Service de Nephrologie et Transplantation Rénale, and
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Laboratoire de Biochimie B, Hôpital St-Louis, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France.
a Author for correspondence. Fax 33-1-42-49-92-47; e-mail Biochimie-b{at}chu-stlouis.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Renal function was evaluated in 30 adults receiving renal transplants (46 ± 9 years, mean ± SD) and in 56 healthy controls (38 ± 10 years) using cystatin C. Plasma cystatin C was determined daily starting the day of surgery and for 3 weeks after surgery by an immunonephelometric assay.
Results: Plasma concentration significantly decreased during the first week (-44% vs -29% for creatinine). Plasma cystatin C correlated with plasma creatinine (r = 0.741; P <0.0001) and the reciprocal of the creatinine clearance estimated by the Cockcroft-Gault formula (r = 0.882; P <0.001). In all three cases of acute renal impairment, the increase in plasma cystatin C values was more prominent than that of creatinine.
Conclusions: Plasma cystatin C is an alternative and accurate marker of allograft function in adult transplant patients. Increased sensitivity compared with creatinine for the detection of acute reduction in glomerular filtration rate allows in some cases a more rapid diagnosis of acute rejection or treatment nephrotoxicity.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since the introduction of sensitive automated immunoassays, cystatin C has been proposed as an alternative marker of GFR (5). Human cystatin C is a 122-amino acid basic low-molecular weight protein (Mr 13 300) that belongs to the superfamily of cysteine proteinase inhibitors (6). The product of a housekeeping gene (7), its plasma concentration in adults is relatively constant (~1 mg/L) and independent of gender and age, at least before the age of 50 years (8). Because of its low molecular weight and positive charge, cystatin C is freely filtered by the glomerular membrane before it is entirely catabolized in the proximal renal tubule (9)(10). In adults suffering from renal diseases, cystatin C more sensitively reflects a reduction of GFR evaluated by 99mTc-diethylenetriamine pentaacetic acid (DTPA) or 51Cr-labeled EDTA filtration clearance than creatinine (5). The diagnostic value of plasma cystatin C as a marker of GFR in renal transplantation patients, however, has not been yet fully investigated (11).
In this prospective study, cystatin C was evaluated as a marker of allograft function during the early postoperative transplantation period. Plasma cystatin C was determined by a recently developed particle-enhanced immunonephelometric assay (12)(13). Plasma cystatin C kinetics were compared with those of plasma creatinine used in our institution for estimation of GFR in transplant recipients. The results are discussed with the following clinical indicators of renal function: dialysis requirements, acute rejection, and treatment nephrotoxicity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Primary diagnosis was chronic interstitial nephropathy (n
= 4), diabetic glomerulopathy (n = 3), polycystic kidney disease
(n = 3), nephrosclerosis (n = 3), focal segmental
glomerulosclerosis (n = 2), IgA nephropathy (n = 3),
amyloidosis (n = 2), vascular nephropathy (n = 1),
Goodpasture syndrome (n = 1), systemic lupus erythematosus
(n = 1), uric acid nephropathy (n = 1), membranous
glomerulonephritis (n = 1), mesangiocapillary glomerulonephritis
(n = 1), and unknown (n = 4).
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Immunosuppressive regimen included steroids (methylprednisolone at the initial dose of 500 mg, followed by 1 mg · kg-1 · day-1, progressively tapered) and cyclosporine (initial dose of 8 mg · kg-1 · day-1, and then adjusted according to blood concentrations) or FK506 in cases of cyclosporine intolerance (at the dose of 0.1 mg · kg-1 · day-1). Delayed graft function (DGF) was defined as a requirement for dialysis during the first 2 weeks after transplantation. All patients except one (peritoneal dialysis) were on conventional dialysis. Episodes of acute rejection diagnosed by renal biopsy were treated with 5 days of intravenous methylprednisolone.
This study was in accordance with the ethics standards of the Helsinki Declaration of 1975, as revised in 1983. Results obtained in transplantation patients were compared with those of 56 age- and sex-matched healthy subjects (28 males and 28 females; mean age, 38 ± 10 years).
analysis
Sampling.
Allograft function was evaluated on a daily basis
starting the day of surgery (day 0) and for 3 weeks thereafter or until
hospital discharge, whichever occurred first. Blood (7 mL) was
drawn by venipuncture in a Vacutainer® Tube with
heparin as an anticoagulant (Becton Dickinson) before centrifugation
(1500g at 20 °C for 15 min) and analyzed for creatinine.
Before cystatin C analysis, samples were frozen at -20 °C, which is
considered the best condition to store samples before cystatin C
measurement (12)(13).
Biochemical analysis.
Plasma creatinine was
enzymatically assayed on a Vitros 750 apparatus (Ortho-Clinical
Diagnostics). The sample volume was 10 µL, and the assay was
performed at 37 °C; total analysis time was 7 min. In our hands, the
interassay imprecision (CV; n = 31) was <3% (low control, 90
µmol/L; high control, 737 µmol/L).
Plasma cystatin C was measured using a latex particle-enhanced immunonephelometric assay on a BN100 nephelometer (Behring). The assay procedure has been described in detail recently by others (12)(13). Briefly, the assay is performed at room temperature with a six-point calibration covering the range of 0.23–7.25 mg/L. The calibrator used is a purified cystatin C from human urine (1.45 mg/L). The sample volume is 80 µL. The time for analysis is 6 min, each subsequent sample reading being available after 8 s. In our hands, the interassay CV (n = 20) was <4% for both the low (1.4 mg/L) and high (2.8 mg/L) controls.
Calculations.
The percentage of discordant changes in
plasma cystatin C and creatinine concentrations was calculated on a
day-to-day basis after transplantation. A change in opposite direction
(increase/decrease) of >10% between the two markers was considered as
discordant. On hospital discharge or at the end of the 3-week period,
creatinine clearance was estimated from plasma creatinine using the
formula of Cockcroft and Gault (1). This formula developed
for adults previously was validated against
99mTc-DTPA-measured GFR in renal transplantation
patients at steady-state renal function (14). Calculations
of estimated clearance by this formula were not done for patients
requiring hemodialysis during the last week of the study (n = 8).
A cutoff of 80 mL/min was selected for normal estimated creatinine
clearance (15).
statistical analysis
Data are presented as mean ± SD or as median and range when
appropriate after checking for gaussian distribution. Differences
between two groups were evaluated by the Wilcoxon signed-rank test.
Multiple comparisons were performed by the Friedman repeated-measure
ANOVA on ranks followed by the Dunn test. Correlation between
techniques was evaluated by linear regression and ANOVA. Results with
P <0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Cystatin C plasma concentrations were not influenced by age
(r = 0.230; not significant) or by gender (males, 7%
higher vs females; not significant). The calculated reference interval
(based on mean ± 1.96 SD) for creatinine was 0.46–0.94 mg/L. The
calculated sex-specific reference intervals for plasma creatinine were
60–100 µmol/L for females and 64–109 µmol/L for males.
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transplant patients
In patients without DGF (n = 16), a significant decline in
plasma concentration was more rapidly obtained for cystatin C (day 1:
-62% vs day 0; P <0.05) than for creatinine (day 1:
-25%; not significant; Fig. 2
). Starting on day 4 posttransplantation (and until the end of
the study period), the decrease in the plasma concentration of
patients without DGF was more pronounced for creatinine than for
cystatin C (Fig. 2
). In patients with DGF (n = 14), the reduction
in the plasma concentration was not significant until day 14 (-34% vs
day 0) for creatinine and day 18 (-22%) for cystatin C (Fig. 2
). At
the end of the 3-week study period, a 52% reduction from the initial
plasma creatinine concentration was observed in patients with DGF vs
33% for cystatin C. The frequency of discordant daily changes in
cystatin C and creatinine plasma values was significantly higher in
patients with DGF (35%) than in those without DGF (19%; P
<0.01). Overall, plasma cystatin C significantly correlated with
creatinine (r = 0.741; P <0.0001; n =
530; day 0 to day 20). The correlation was better in patients without
DGF (r = 0.818; P <0.0001; n = 255)
than in those with DGF (r = 0.429; P
<0.0001; n = 275; Fig. 3
).
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There were four biopsy-documented acute rejection episodes (incidence,
13%) during the 3-week study period (at days 10, 14, 17, and 18
posttransplantation). In two cases, acute rejection was suspected by
prolonged DGF, as demonstrated by persistent increases in both plasma
creatinine and cystatin C concentrations (case 1; Fig. 4
A). An acute rise in plasma creatinine was attributed to acute
rejection in two patients, as illustrated in Fig. 4B
(case 2). There
was one case of acute treatment (FK506) nephrotoxicity at day 9
postsurgery, with a 44% increase of plasma creatinine over 2 days
(Fig. 4C
). Plasma cystatin C gradually rose (125%) during 7 days
before diagnosis. Treatment nephrotoxicity regressed spontaneously.
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At the end of the study (day 20 or hospital discharge), plasma
creatinine was 180 µmol/L (79–602 µmol/L) vs 2.59 mg/L (1.04–6.54
mg/L) for cystatin C. Both markers were significantly correlated
(r = 0.890; P <0.001). Estimated creatinine
clearance by the Cockcroft and Gault formula (1) in patients
with stable renal function (no hemodialysis during the last week,
n = 22) was 45 mL/min (18–76 mL/min), and no patient was within
the reference interval (>80 mL/min). Plasma creatinine was
within the reference interval for five patients (79–96 µmol/L)
compared with none for cystatin C. The relationship between estimated
creatinine clearance and the plasma concentrations of creatinine and
cystatin C in patients with stable renal function (n = 22) is
represented in Fig. 5
. There was a significant correlation between estimated
creatinine clearance and the reciprocal of plasma cystatin C
(r = 0.882; P <0.001).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the last decade, substantial technical improvements, especially concerning sensitivity and practicability, have been made in the measurement of plasma cystatin C. In the present study, we measured the plasma cystatin C concentration with a fully automated latex particle-enhanced immunonephelometry assay (12)(13). This technique has been shown to correlate well with automated latex particle-enhanced immunoturbidimetry assays and displayed excellent analytical performance (12)(13). Turbidimetric assays (18)(19) and nephelometric assays are not subject to the same analytical interferences (such as bilirubin or hemolysis) as plasma creatinine. The analytical precision was excellent (CV <4%), and the total analysis time was comparable to that of creatinine (~5 min); thus this assay is well suited for routine use in clinical laboratories. The cost of this assay is ~15-fold higher than that of creatinine determination by the Vitros technology, which in terms of cost-effectiveness remains acceptable if significant clinical benefits can be obtained with this new marker.
The mean cystatin C plasma concentration in our group of healthy adults was similar to that reported in a group of healthy subjects (n = 12; mean age, 40 years), using an immunoturbidimetric assay (0.65 ± 0.05 mg/L) (20). Slightly higher values (mean cystatin C, 0.80 mg/L) were reported by others (13) in a group of 52 adults with normal renal function (ages, 21–79 years). These data are in agreement with an increase in plasma cystatin C values with age, especially after the age of 50 years (8). Like others (8)(13)(20), we did not find the sex difference reported previously (21)(22). Plasma cystatin C correlated poorly with plasma creatinine, as reported previously in subjects with GFR >80 mL/min (13) or GFR >70 mL · min-1 · 1.73 m-2 (11). These data suggest that different physiological factors (such as sex, dietary factors, or body composition) influence cystatin C and creatinine plasma concentrations in healthy adults.
In renal transplant patients, plasma cystatin C concentrations paralleled those of creatinine regardless of graft function (absence or presence of DGF). Consequently, plasma cystatin C and creatinine significantly correlated over the postoperative study period (r = 0.741; P <0.0001), as observed previously in adult renal transplant patients (11) and subjects suffering from chronic renal disease (13). Some differences, however, were apparent in their respective plasma kinetics. During the first 4 days posttransplantation, the cystatin C plasma concentration decreased more rapidly than that of creatinine. Transtubular leakage of the low-molecular weight (Mr 100) creatinine (23) has been reported in acute renal failure, thus leading to high plasma creatinine values. Starting on day 4 posttransplantation, the decrease in plasma concentration became more prominent for creatinine than for cystatin C, which might be attributable to an underestimation of GFR by plasma cystatin C, an overestimation of GFR by plasma creatinine, or both. A stronger correlation between cystatin C than between creatinine and the measured GFR has been reported in adults suffering from renal diseases (5)(19)(24). The sensitivity to detect a reduction in GFR is also better for cystatin C than for creatinine when cutoffs at 70 mL · min-1 · 1.73 m-2 (11), 72 mL · min-1 · 1.73 m-2 (16), or 80 mL · min-1 · 1.73 m-2 (13)(21) are considered. Better accuracy of plasma cystatin C for estimation of GFR would need to be confirmed by a gold standard technique for measuring GFR, such as 99mTc-DTPA, 51Cr-labeled EDTA, or inulin clearance.
For patient follow-up, the ability to detect rapid changes in GFR is
clinically more important than accuracy itself. With diminished GFR, a
significant increase in plasma concentration of cystatin C and
creatinine will depend on the rate of its accumulation in plasma, which
depends on its production rate and distribution volume, but also on its
biological intraindividual variation. Repeated measures obtained in
healthy subjects (20) suggested that intraindividual
variation might be more important for cystatin C (13.3%) than for
creatinine (4.9%). If true, cystatin C would be less sensitive for the
detection of acute rejection episodes for a given individual than
creatinine (20). In all four episodes of acute rejection and
one treatment acute nephrotoxicity in our study, the plasma cystatin C
concentration broadly paralleled that of creatinine. Interestingly, the
rise in the plasma cystatin C concentration was more prominent than
that of creatinine. Diagnosis of acute rejection could have been
anticipated by ~3 days with cystatin C in patient 2 (Fig. 4B
), and
treatment acute nephrotoxicity could have also been detected earlier
(patient 3; Fig. 4C
).
After renal transplantation, hemodialysis was required in almost 50% of our patients. We found a higher degree of discrepancy (35% of discordant results; P <0.01) between cystatin C and creatinine plasma kinetics in patients requiring hemodialysis than in those with a normal course (19%). In addition, cystatin C and creatinine weakly correlated in hemodialyzed patients (r = 0.429). The molecular weight of cystatin C is 13 300 with an Einstein-Stokes radius of 30–40 Å, which is much higher than creatinine (Mr 100 and 3 Å) (2)(25). In a large study of 112 patients on stable maintenance hemodialysis, a 30% reduction in serum cystatin C was observed after dialysis with mostly AN69 high-flux membranes (10). As expected, the elimination of cystatin C during dialysis increased with the ultrafiltration coefficient (UFC) of the membrane, an estimate of the permeability: 0% (vs 40% for creatinine) for UFC <15 mL · h-1 · m2 · mmHg and ~ 60% (as for creatinine) for UFC >15 mL · h-1 · m2 · mmHg (10). Thus, removal of cystatin C by hemodialysis seems highly dependent on the type of membrane selected. This is an important issue in renal transplantation because it could limit the use of plasma cystatin C as a marker of graft function in patients with DGF. On the other hand, if poorly filtered by dialysis membranes, plasma cystatin C could be used by the nephrologist to monitor appropriate duration of hemodialysis in patients with DGF. Prolonged unnecessary hemodialysis could be avoided as soon as a significant decrease in plasma cystatin C is obtained by dialysis.
In conclusion, plasma cystatin C is as an alternative and probably more accurate marker of GFR than creatinine in adult transplantation. In some cases, a more prominent rise in plasma cystatin C values allows a more rapid diagnosis of acute rejection or treatment nephrotoxicity. Cystatin C does not, however, appear clearly superior to plasma creatinine, at least during the early postoperative period (first 3 weeks). Limits to its routine use in transplantation include cost, which is much higher for an immunonephelometric assay than for a creatinine determination; absence of clearance measurements; and poorly documented behavior during hemodialysis. Further prospective studies are needed to evaluate this last issue and the potential of plasma cystatin C in the long term follow-up of graft function in renal transplantation.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  U. Poge, T. Gerhardt, A. Bokenkamp, B. Stoffel-Wagner, H.-U. Klehr, T. Sauerbruch, and R. P. Woitas Time course of low molecular weight proteins in the early kidney transplantation period--influence of corticosteroids Nephrol. Dial. Transplant., November 1, 2004; 19(11): 2858 - 2863. [Abstract] [Full Text] [PDF]
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 A. Bokenkamp, J. A.E. van Wijk, M. J. Lentze, and B. Stoffel-Wagner Effect of Corticosteroid Therapy on Serum Cystatin C and {beta}2-Microglobulin Concentrations Clin. Chem., July 1, 2002; 48(7): 1123 - 1126. [Full Text] [PDF]
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O. F. Laterza, C. P. Price, and M. G. Scott Cystatin C: An Improved Estimator of Glomerular Filtration Rate? Clin. Chem., May 1, 2002; 48(5): 699 - 707. [Abstract] [Full Text] [PDF]
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G. Filler, F. Priem, N. Lepage, P. Sinha, I. Vollmer, H. Clark, E. Keely, M. Matzinger, A. Akbari, H. Althaus, et al. {beta}-Trace Protein, Cystatin C, {beta}2-Microglobulin, and Creatinine Compared for Detecting Impaired Glomerular Filtration Rates in Children Clin. Chem., May 1, 2002; 48(5): 729 - 736. [Abstract] [Full Text] [PDF]
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L. Risch, R. Herklotz, A. Blumberg, and A. R. Huber Effects of Glucocorticoid Immunosuppression on Serum Cystatin C Concentrations in Renal Transplant Patients Clin. Chem., November 1, 2001; 47(11): 2055 - 2059. [Full Text] [PDF]
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T. Le Bricon, E. Thervet, M. Froissart, M. Benlakehal, B. Bousquet, C. Legendre, and D. Erlich Plasma Cystatin C Is Superior to 24-h Creatinine Clearance and Plasma Creatinine for Estimation of Glomerular Filtration Rate 3 Months after Kidney Transplantation Clin. Chem., August 1, 2000; 46(8): 1206 - 1207. [Full Text] [PDF]
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