In this study, we pooled data from 3418 patients with CKD in three research studies and one clinical population to develop and compare GFR estimating equations using serum creatinine, cystatin C or both. Strengths of the study include the large study population; calibration of the creatinine assays in each study to standardized values; measurement of cystatin C in a single laboratory; multiple period urinary clearances of validated filtration markers for measurement of GFR; and the use of a separate external validation dataset. The key findings are that in populations with CKD, cystatin C alone provides GFR estimates that are more accurate than serum creatinine alone and nearly as accurate as serum creatinine, age, sex and race, thus providing an alternative estimate of GFR which is not linked to serum creatinine and muscle mass. Nevertheless, the addition of age, sex and race to cystatin C reduced bias in some subgroups defined by these variables, and an equation that uses both serum creatinine and cystatin C with age, sex and race was better than equations that use only one of these markers.
Cystatin C is an endogenous, 13 kilodalton protein filtered by the glomeruli and reabsorbed and catabolized by the tubular epithelial cells with only small amounts excreted in the urine, and reported to be generated at a relatively constant rate irrespective of muscle mass. Thus, it was anticipated that cystatin C would provide a better estimate of GFR than estimating equations based on serum creatinine. We found a weaker association of age, sex and race with cystatin C than with serum creatinine, which is consistent with this a priori
hypothesis as well as with most published cystatin C based estimating equations that do not include terms for age, sex or race.4, 5, 21–23
Both markers provide independent information for the estimation of GFR. The combination of both markers in an equation with age, sex and race provided the most accurate estimate in our dataset, and was equivalent to the average of the estimates from the equation that used cystatin C alone and the MDRD Study equation. More work is needed to determine optimal use of the combination or sequential use of filtration markers, to provide even more accurate GFR estimates.
Several findings in our study suggest that cystatin C levels are affected by factors other than GFR. First, variation in cystatin C alone among subgroups defined by age, sex and race was observed. For the same level of eGFR, serum cystatin levels were 9% lower for women than men, 6% higher for blacks than for whites, and 9% lower for 40 year olds compared to 20 year olds. These differences may reflect differences in the genreration of cystatin among these groups. Accordingly, there was a small improvement in performance of the equation with the addition of these variables. Second, cystatin C based equations slightly overestimated measured GFR at eGFR greater than 90 ml/min/1.73 m2
), whereas the creatinine based equations remained unbiased. Finally, precision was lower for the cystatin C based estimating equations that did not include creatinine. Previous studies have also showed preliminary evidence for non-GFR determinants of cystatin C, including non-renal elimination as well differences in generation among individuals as related to such factors as inflammation, steroid use, thyroid disease, and some studies have shown an effect of body composition.4, 22, 24–28
Variation in these factors among individuals likely account for the lower precision of the cystatin based estimates. The absence of urinary excretion has made it difficult to rigorously evaluate cystatin C as a filtration marker and to examine its non-GFR determinants. The specific nature and magnitude of these factors is not known and requires further study.
The performance of equations using serum creatinine and cystatin C is known to vary among populations. For example, the MDRD Study equation performs well in patients with CKD but is less accurate in potential kidney donors, young people with type 1 diabetes and patients with substantially reduced muscle mass.2, 13, 29
Rule et al showed that a cystatin C based estimating equation performed differently among patients with native kidney disease, kidney transplant recipients and potential kidney donors; therefore, we cannot draw conclusions about the relative performance of serum cystatin C or creatinine in other populations.4
This has important implications for use of these equations in screening populations for CKD, as the equations have not been tested in populations without known CKD.
Serum cystatin C has been shown to have a stronger association with mortality and cardiovascular disease than serum creatinine, particularly in studies of older adults.30–33
In the Cardiovascular Health Study (CHS), participants with eGFR greater than 60 ml/min/1.73 m2
) (based on the MDRD Study equation) and cystatin C greater than 1.0 mg/L (75 nmol/L) had a worse outcome than participants with cystatin C less than 1.0 mg/L (75 nmol/L). A cystatin C of 1.0 mg/L (75 nmol/L) corresponds to an eGFR of 77 ml/min/1.73 m2
), according to the cystatin equation developed here31
The similarity of the performance of equations based on serum creatinine and cystatin C at this level of eGFR in the current study suggests that the stronger association of cystatin C to adverse outcomes in CHS may be due to factors other than GFR that effect the level of serum cystatin C or creatinine.34
An alternative explanation is that the study populations in this report differ from CHS. CHS participants were recruited from Medicare eligibility lists and therefore reflect the general population of older adults, including the frail elderly, whereas the studies included here mostly included patients with CKD who were not frail; therefore, the factors that may confound the relationship between serum creatinine or cystatin C to GFR may not be reflected in the current estimating equations. Studies of GFR measurements in older adults with and without CKD and across the range of health and functional status are required to determine whether cystatin C is a better filtration marker in this population.
Previous studies have shown a wide variability in eGFR for the same level of cystatin C.35
For example, cystatin C levels equivalent to eGFR of 60 ml/min/1.73 m2
) using the equations published by Rule and Grubb would be 1.09 mg/L (81.6 nmol/L) and 1.40 (105 nmol/L) respectively, compared to 1.23 mg/L (92.1 nmol/L) derived from our equation that uses cystatin C alone. These differences may be related to the variation among populations discussed above, or to differences among assays or GFR measurement methods. The high level of variation in the cystatin C assay is beginning to be recognized, and standardization and calibration of clinical laboratories will be important to obtain accurate GFR estimation using cystatin C, as has been shown for creatinine.36
There are several limitations to this analysis. First, as discussed above, the study population was composed mainly of patients with CKD; however, the intent of the current work is to compare estimating equations based on cystatin C to the best available equation based on serum creatinine. Indeed, to maximize this comparison, we did not test all possible transformations of cystatin C or other variables besides age, sex and race (e.g. diabetes), or their interactions. Second, only one external validation dataset was used; therefore, our findings may not be applicable to other populations. In particular, the small bias observed in the external validation dataset could be related to differences between the exogenous filtration markers used for clearance measurements, iothalamate for the development dataset, and EDTA for the validation dataset. Previous studies have shown both filtration markers to provide similar values to inulin clearance; however, a direct comparison cannot be performed as the two radioactive tracers can interfere with one another in the counting, EDTA is not approved for use in the United States, and iothalamate is not available in Europe.10–12
As well, differences between markers are likely to be manifested by bias‥ Since a bias is expected when applying equations in new dataset, the presence of a small bias in the external validation dataset suggests that the difference between filtration markers for clearance measurements is not likely to an important factor in the result. Use of only one external validation dataset also means that we were unable to test if the coefficient for black race differs between blacks in the United States and Europe. Third, the equation coefficients were derived from a pooled analysis of individual studies, rather than from a representative population, with some studies representing a substantial portion of certain demographic groups (e.g. Blacks in AASK). Therefore, it is possible that findings observed within a demographic group may reflect study differences and not characteristics of that group. Indeed, study participants were likely selected on the basis of previous serum creatinine values and given the colinearity between creatinine and cystatin C, this may lead to bias in the coefficients in all equations. However, all studies were of CKD populations and previous studies have suggested that differences among subgroups based on demographic characteristics are minimal for populations with native kidney disease.2, 13
Pooling across studies is probably preferable to using a single study in the absence of data from large representative samples. Fourth, equations were not compared with respect to classification of patients with measured GFR less than versus greater or equal to 60 ml/min/1.73 m2
. Our study population has a mean GFR that is well below 60 ml/min per 1.73 m2, thus, these analyses would not be very sensitive to differences in accuracy of the equations. In addition, these comparisons would not take into account error in measured GFR. Finally, these equations were not tested for assessment of change in GFR over time.
GFR estimating equations using cystatin C have the promise to provide more accurate estimates of GFR than equations using serum creatinine. Implementation of these equations in routine clinical practice requires standardization of the cystatin C assay, further investigation of the factors other than GFR that influence the level of cystatin C, and availability of wide-spread and cost-effective assays for additional markers. At the current time, the equations developed here may provide more accurate estimates in people in whom estimates based on serum creatinine are likely to be inaccurate due to conditions affecting muscle mass or diet or to estimate change in GFR over time in people with changing muscle mass or diet.