ASSESSMENT OF RENAL FUNCTION IN RENAL TRANSPLANT PATIENTS USING CYSTATIN C. A COMPARISON TO OTHER RENAL FUNCTION MARKERS AND ESTIMATES

Abstract

To date, little evidence is available to define the role of cystatin C in patients with renal transplants. Thus, to assess, whether cystatin C (CysC) provides better information on renal function than other markers, CysC, creatinine clearance (CrCl), serum creatinine (SCr), β₂-microglobulin (β₂-M), and ¹²⁵I-Iothalamate clearance were determined in 30 patients. Correlation and ROC curves were obtained and characteristics like sensitivityand specificity were calculated. Further, to evaluate the usefulness of these markers for monitoring, intraindividual coefficients of variation for CysC and SCr measurements were compared in 85 renal transplant patients. CysC correlated best with GFR, whereas SCr, CrCl and β₂-M all had lower correlation coefficients. CysC was superior to SCr, even when renal function equations of were used. The diagnostic accuracy of CysC was significantly better than SCr, but did not differ significantly from CrCl and β₂-M. Together, our data show that in patients with renal transplants, CysC has a similar diagnostic value as CrCl. However, it is superior to determinations of SCr. The intraindividual variation of CysC is significantly greater than that of SCr. This might be due to better ability of CysC to reffect temporary changes especially in mildly impaired GFR, most critical for early detection of rejection and other function impairment. In conclusion, CysC allows for easy and accurate assessment of renal function (GFR) in steady state renal transplant patients and is clearly superior to the commonly used serum creatinine.

• Beta-2-microglobulin
• Creatinine
• Cystatin C
• Glomerular filtration rate
• Kidney transplantation
• Renal function equations

INTRODUCTION

In renal transplant patients, assessment of renal function is crucial, because planning pharmacotherapy, administration of fluid or contrast dye or the decision in performing a kidney biopsy partly depend on the degree of renal function impairment. Renal function commonly is assessed in measuring or estimating the glomerular filtration rate (GFR). For determination of GFR, veryaccurate clearance measures of exogenously administered substances, like Inulin, radiolabeled substances (¹²⁵I-Iothalamate, ⁵¹Cr-EDTA etc.) and Iohexol have been described. These are all slow, complicated, laborious, and costly and some of the methods require radiation exposure or maycause allergic reactions. In order to have less complicated and faster methods, results of endogenous markers are used. Creatinine clearance and serum creatinine therefore have become very common markers. Measuring serum creatinine is a cheap, fast and easy way to receive information on GFR, but the marker is plagued with several significant disadvantages including a lack of sensitivity to measure renal function impairments of less than 50% or the inability to detect rapid changes of GFR [[1]]. Furthermore, analytical interference with the widely used Jaffé method and its modifications result in a limited specificity [1-2]. Certain substances (ascorbate, bilirubin, uric acid, acetoacetate, pyruvate, cephalosporins, methyldopa etc.) can lead to an overestimation up to 20% of the real creatinine value [[1]]. In addition, muscle mass changes, nutrition, physical activity and inflammatory processes interfere with creatinine based estimation of GFR [[1]]. Together, serum creatinine can only be used as a crude indicator of significantly impaired renal function. In order to improve assessment of renal function with one serum creatinine measurement, renal function estimate equations have been validated in renal transplant patients [[3]]. These equations correct the serum creatinine value for age, gender, height, and, weight, respectively, and allow for estimation of creatinine clearance or GFR. However, included with these equations are all the previously mentioned disadvantages of the serum creatinine measurement. In addition, erroneous estimates are especially obtained in patients with rapidly changing renal function, edematous and obese patients and patients with profound muscle wasting [[1]].

Endogenous creatinine clearance provides more accurate information on renal function. However, because of the 24 hour collection period, testing creatinine clearance is laborious and slow. In addition, according to Perrone et al., creatinine clearance is not veryuseful in determining the exact level of renal function [[1]]. Sources of inaccuracy consist of preanalytical (inaccurate urine collection), analytical (interference with endogenous and exogenous substances in the Jaffé method) and physiological (tubular secretion of creatinine in patients with decreased GFR, making it a non ideal filtration marker) problems [[1]]. Together, creatinine clearance is only of use in determining whether renal function is nearlynormal, moderatelyor severely reduced [[1]]. As neither serum creatinine nor creatinine derived renal function estimates or creatinine clearance fulfill the profile of an ideal marker of GFR (endogenous marker, measured in plasma, produced at constant rate, freely filtered in glomerulus, no extrarenal elimination, no secretion in tubulus, no renal reabsorption into plasma), new accurate, fast and cheap markers are wanted [[4]]. Several reports have shown, that serum cystatin C is an accurate marker of GFR, superior to serum creatinine [5-10]. Cystatin C is a low molecular weight basic protein (13.36 kD), synthesized by nucleated cells as a housekeeping gene product of the cystatin gene superfamily of cystein proteinase inhibitors [[11]]. The endogenous production rate is constant, and is not affected by inflammatory processes, malignancy, changes of body mass, nutrition, fever or gender [10-11]. It is mainly freely filtered in the glomeruli and subsequently reabsorbed and catabolized in the proximal tubule [[11]]. The availability of fast and automated methods for determination has made cystatin C a favorable marker in determining GFR, especially in situations where accurate, and fast estimations of GFR are needed (i.e., transplantation, oncology, pediatrics and intensive care) [[11]]. However, cystatin C has mainly been established in healthy humans and in patients with renal problems caused by a variety of different diseases [6-8], [12-13]. So far, only few data are available on the performance of cystatin C as a marker of GFR in patients with renal transplants [[14]]. Thus, in order to assess whether cystatin C and other markers would be reliable indicators of GFR in this patient group, a comparison with a gold standard measurement was established in a group of 30 stable renal transplant patients. Further, to demonstrate the characteristics as markers for monitoring, intraindividual variability of cystatin C and creatinine was compared in 85 renal transplant patients.

PATIENTS AND METHODS

Thirty renal transplant patients in a steady state condition (15 male/15 female), aged 49 ± 15.5 years (mean ± SD), were included in the study. Steady state was defined as lack of acute rejection periods during the past 6 months and stable cyclosporin A medication during the past 2 weeks. Time since transplantation was 6 ± 4.5 years. Immunosuppression was carried out with cyclosporin A and, in some patients was supplemented with prednisone. After informed consent had been obtained, determinations of ¹²⁵I-Iothalamate, serum creatinine, creatinine clearance, cystatin C and β₂-microglobulin were performed on the occasion of a routine follow-up appointment. To assure correct sample collection procedure, the patients were hospitalized in the Department of Nephrology. In order to calculate the intraindividual variability of cystatin C and serum creatinine, at least 6 measurements, obtained during a 13 months follow up were used. Creatinine was measured on a Dimension (Dupont, Wilmington, DE) using a modified Jaffé method to avoid interference by bilirubin [[15]]. Creatinine was determined in serum and 24 hr urine to calculate the creatinine clearance related to 1.73 m² body surface. In addition, estimation of the creatinine clearance was done according to the equations of Jelliffe and Cockroft-Gault, respectively, and estimate of GFR was obtained according to the equation of Walser [[3]]. Cystatin C was determined by particle-enhanced turbidimetric immunoassay (PETIA) (Dako, Glostrup, Denmark) using a Cobas Mira (Roche Diagnostics Basel, Switzerland) [[5]]. β₂-microglobulin was measured on a Cobas Core using an enzyme immunoassay (EIA) (Roche Diagnostics, Basel, Switzerland) [[16]]. ¹²⁵I-Iothalamate clearance, as a gold standard measurement, was determined according to the method described by Israelit et al. [[17]]. The ¹²⁵I-Iothalamate clearance was compared with estimated and measured creatinine clearance as well as with the reciprocal values of cystatin C, creatinine and β₂-microglobulin. Correlations were calculated according to Pearson. In order to quantitate the diagnostic value of the individual parameters, receiver operating characteristics (ROC) curves were calculated and sensitivity, specificity, likelihood ratios and predictive values were obtained and analyzed. The cut-off for the gold standard measurements was set at 60 mL/min. Comparison of the ROC curves was performed according to Hanley and McNeil [[18]]. Intraindividual coefficients of variation were compared using the Mann Whitneyrank sum test. P-values < 0.05 were considered significant. All data were calculated using MS Excel (Microsoft Corp., Seattle, WA) and Analyze-it (Analyze-it Software Ltd, Leeds, UK).

RESULTS

In order to evaluate the correlation of the investigated markers with GFR comparison with ¹²⁵I-Iothalamate clearance was accomplished. One patient dropped out due to technical problems. Figure 1a–1d demonstrate the measured parameters: reciprocals of cystatin C (Figure 1a), serum creatinine (Figure 1b) and β₂-microglobulin (Figure 1c) as well as the creatinine clearance (Figure 1d) correlate with GFR. The correlation coefficient (r) for l/cystatin C was 0.83. Correlation coefficients for l/serum creatinine (r = 0.67), β₂-microglobulin (r = 0.58) and creatinine clearance (r = 0.57) were lower than the value found for l/cystatin C. Surprisingly, the serum creatinine derived estimations of creatinine clearance and GFR did not enhance validity of serum creatinine values. Estimation of creatinine clearance according to Jelliffe (r = 0.25) (Figure 1e) and Cockroft-Gault (r = 0.10) (graph not shown), respectively, and calculation of the GFR according to Walser (r = 0.22) (Figure 1f) all revealed a weak correlation with ¹²⁵I-Iothalamate clearance.

Figure 1. Correlations of the investigated parameters with a gold standard measurement (¹²⁵I-Iothalamate clearance) of GFR. a) l/cystatin C, b) 1/creatinine, c) 1/β₂-microglobulin, d) creatinine clearance, e) Walser equation for estimation of GFR, f) Jelliffe equation for estimation of creatinine clearance. Correlation coefficients and linear regression line equations are shown within the graphs.

As correlations are not really useful in determining, whether a marker provides accurate results, ROC analysis was utilized. To compare the accuracy of the investigated parameters, the analysis was performed on the ability of the different markers to detect a decreased GFR below 60 mL/min. As seen in Figure 2, l/cystatin C had a significantly higher diagnostic accuracy than l/ serum creatinine (p = 0.024). Surprisingly, no significant difference of the areas under the ROC curves comparing l/cystatin C with creatinine clearance and 1/β₂-microglobulin (not shown on graph) could be observed.

Figure 2. ROC curves for diagnostic accuracy of 1/cystatin C, 1/creatinine and creatinine clearance in discriminating between mildlyand moderatelyreduced GFR (≥ 60 mL/min and < 60 mL/min). Area under the 1/cystatin C curve (AUC) exceeds AUC of 1/creatinine significantly(p = 0.024). AUC of 1/cystatin C and creatinine clearance show no significant difference (p > 0.5).

At a cutoff of 1.69 mg/L, cystatin C had a 100% specificity and 65% sensitivity, while a 100% sensitivity and 22% specificity was seen at a cut-off of 1.27 mg/L. Serum creatinine, at a cut-off of 158 μmol/L reached 100% specificity and 35% sensitivity, whereas 90% sensitivity and 11% specificity was observed at a cut-off of 99 μmol/L. At a cut-off of 4.35 mg/L, β₂-microglobulin showed a 100% specificity and 60% sensitivity, whereas a 90% sensitivityand 11% specificity could be seen at a cut-off of 2.38 mg/L. Finally, at a cut-off of 33 mL/min, creatinine clearance exhibited a 100% specificity and 45% sensitivity, whereas a 100% sensitivityand a 11% specificity was achieved at a cut off of 171 mL/min.

At last, to describe the characteristics as monitoring markers, intraindividual coefficients of variation of 85 patients revealed significantly lower intraindividual coefficients of variation for serum creatinine than for cystatin C (p < 0.001). This indicates, that serum creatinine is a more constant monitoring parameter than cystatin C.

DISCUSSION

Management of patients with renal transplants requires periodic assessment of renal function. An ideal marker of GFR should provide results, which are accurate, sensitive in detecting mild renal function impairments, and further show a low variability over time, when compared to a gold standard. Although clearance determinations of exogenously administered substances are accurate, all these methods are time consuming and cumbersome for both patients and staff. Using endogenous markers is much simpler. To date, creatinine clearance has been felt to be the best method available. However, the method brings along problems with patient compliance as well as analytical problems. Further problems arise because of biological variation of creatinine synthesis and elimination [[1]]. The use of serum creatinine as a rough estimate of GFR is plagued by interference through muscle mass changes, nutrition, physical activity and inflammatory processes [[1]]. The serum concentration of β₂-microglobulin, another endogenous marker, is influenced byits production rate and the GFR [[19]]. Its production rate, however, is different in patients with lymphoproliferative syndromes, infections and autoimmune-diseases [[16]]. Furthermore, immunosuppressive drugs alter the β₂-microglobulin production rate, making it unreliable for estimating GFR in renal transplant patients.

Together, neither creatinine, nor creatinine clearance or β₂-microglobulin meet the criteria of a practical and ideal marker of GFR. Our results clearly demonstrate that of all available markers, cystatin C shows the best correlation with GFR. Like Keevil et al. who described, that cystatin C is superior to serum creatinine, we demonstrate with these data the usefulness of cystatin C as a test for detecting renal function impairment [[20]]. Cystatin C is superior to serum creatinine even when the renal function estimate equations of Cockroft-Gault, Walser and Jelliffe are used. In comparison to the other investigated parameters, these equations all showed weak correlations and therefore seem to be overrated when estimating GFR in renal transplant patients (Table 2). When comparing cystatin C with creatinine clearance, no significant differences in accuracy were found. However, due to the fact that our patients were in steady state, it can be assumed that the studywas biased against cystatin C, because serum concentrations of cystatin C, in contrast to creatinine clearance values, are not affected by inflammatory conditions. Therefore, it can be expected that during common inflammatory conditions like acute rejection or infections, cystatin C would continue to provide accurate assessment of GFR, while creatinine clearance would vary substantially. Additional problems with creatinine clearance arise because of the often-encountered low compliance in 24 hr urine collection, especially when performed under outpatient conditions. Taken together, cystatin C is superior to creatinine because of accuracy reasons, which can not be made up by using renal function estimate equations. The two other measured parameters exhibited a similar diagnostic accuracy than cystatin C, but they both show significant sources of interference, such as disorders and medication influencing the immune system (β₂-microglobulin) or compliance problems as well as a prolonged time period until results are available (creatinine clearance in 24 hr urine). To be of use for monitoring renal function, anyparameter is expected to have low intraindividual variability. Keevil et al. reported that cystatin C has a larger intraindividual variation than creatinine, as evaluated in 12 healthy volunteers [[20]]. As shown with our data, this finding also holds true for renal transplant patients. As cystatin C is a more sensitive marker of changes in GFR than creatinine, the broader variability of cystatin C could be attributable to this fact, reffecting small temporary changes more accurately [[6]]. It seems, that creatinine is not sensitive enough to assess transient changes in mild renal function impairment, thus incorrectly indicating a low variability. However, intraindividual variability of creatinine and cystatin C at different degrees of renal function impairment merits further investigation.

Table 2. Correlation coefficients According to Pearson for the Investigated Parameters, When Compared to ¹²⁵I-Iothalamate Clearance

Parameter	r (Pearson)
1/Cystatin C	0.83
1/Creatinine	0.67
1/β2-microglobulin	0.58
Creatinine clearance	0.56
Jelliffe equation	0.25
Walser equation	0.22
Cockroft-Gault equation	0.10

In conclusion, in steady state renal transplant patients, cystatin C was found to be more accurate than serum creatinine in detecting renal function impairment. Whereas accuracy of cystatin C was not significantly different from creatinine clearance and β₂-microglobulin, practicability was clearly superior to creatinine clearance. Less sources of interference make cystatin C a more suitable GFR marker in renal transplant patients than β₂-microglobulin. However, further studies are needed to evaluate the diagnostic value of cystatin C in renal transplant patients that are not in steady state.

Figure 3. Intraindividual coefficients of variations (CV's) for cystatin C and creatinine in 85 renal transplant patients, obtained during 1 year follow up. CV's for creatinine are significantly lower than for cystatin C (p < 0.001).

Table 1. Diagnostic Test Characteristics of Cystatin C, Serum Creatinine, Creatinine Clearance and β-2-microglobulin in Renal Transplant Patients at Given Cut-Offs

Parameter	Cut-off	+LR	−LR	Spec	Sens	+PV	−PV
Cystatin C	1.64 mg/L	6.3	0.34	89%	70%	93%	57%
Creatinine	125 μmol/L	1.4	0.45	44%	80%	76%	50%
Creatinine Clearance	66 mL/min	7.7	0.17	89%	85%	94%	73%
β2-microglobulin	3.57 mg/L	1.7	0.37	67%	75%	83%	55%

LR = likelihood ratio. Spec = specificity. Sens = sensitivity. PV = predictive value.