the glomerular filtration rate ( gfr ) is the most widely used indicator of overall renal function .
the gfr can be measured by clearance of an ideal , usually exogenous , filtration marker such as inulin , iothalamate , edta , diethylene triamine pentaacetic acid , or iohexol .
the clearance of endogenous markers such as creatinine or blood urea nitrogen can overestimate or underestimate the gfr .
all these methods have the drawback to be complex and require 24-hour urine collection , which is not always practicable in day - to - day clinical practice .
a more pragmatic approach is to estimate gfr from equations based on serum creatinine [ 1 , 2 ] .
cystatin c is a nonglycosylated protein with low molecular mass ( 13.3  kda ) generated by all nucleated cells of the body at a constant rate , is freely filtered by the glomerulus , and is not secreted by renal tubules but completely reabsorbed with subsequent degradation by proximal tubular cells [ 4 , 5 ] . for those properties
compared with serum creatinine , cystatin c levels are less dependent on ethnicity , sex , age , and muscle mass or protein intake . moreover , as serum creatinine , cystatin c is an independent predictor of cardiovascular and overall mortality . compared to gfr measurement based on the renal clearance of exogenous markers , estimated
glomerular filtration rate ( egfr ) is more precise if derived from both cystatin c and creatinine levels in serum .
on the other hand , measurement of cystatin c is costly and egfr based on cystatin c needs further validation across a broad spectrum of populations with or without chronic kidney disease .
the purpose of our current study was to compare estimates of egfr based on cystatin c and creatinine in randomly recruited uruguayans , using egfr derived from cystatin c as reference .

 genotipo fenotipo y ambiente de la hipertension en uruguay ( gefa - ht - uy ) is a prospective cohort study started in april 2012 conducted by the unidad de hipertensin arterial , hospital de clnicas dr .
the ethics committee of the university hospital approved the study protocol and all participants gave informed written consent .
the aim of the study is to explore the relation of blood pressure with genetic and environmental factors in a representative uruguayan population sample .
nuclear families were randomly recruited from the inhabitants of a geographically defined area , the juana de amrica housing project , located about 10  km from downtown montevideo . a nuclear family had to include at least one parent and two siblings .
family members living at the same address or within a distance of no more than 10  km were eligible .
149 people had participated , of whom we excluded 30 from the present analysis , because either cystatin c or creatinine had not been measured ( n = 22 ) or because they had not yet completed the physical examination ( n = 8) .
trained observers administered a standardised questionnaire inquiring into each participant 's medical history , smoking and drinking habits , and intake of medications .
after participants had rested for 5 minutes in the sitting position , the observers obtained five consecutive blood pressure readings ( phase v diastolic pressure ) to the nearest 2  mm  hg , using mercury sphygmomanometers .
standard cuffs had a 12  24  cm inflatable portion , but if upper arm girth exceeded 31  cm , larger cuffs with 15  35  cm bladders were used .
hypertension was an office blood pressure of at least 140  mm  hg systolic or 90  mm  hg diastolic or use of antihypertensive drugs .
the observers measured body height to the nearest 0.5  cm with a pliable measurer and the participant standing against the wall .
venous blood samples were obtained after at least 12 hours fasting and were kept at 4c and within 2 hours period were analysed for serum levels of cystatin c , creatinine , cholesterol , and glucose .
diabetes mellitus was the use of anti - diabetic drugs or a fasting glucose  126  mg / dl ( 7 
after the participants had rested 15  min in the supine position , we recorded during an 8  s period the radial waveforms at the right side by applanation tonometry .
we used a high - fidelity spc-301 micromanometer ( millar instruments , houston , tx ) interfaced with a computer running sphygmocor software , version 8.2 ( atcor medical , west ryde , new south wales , australia ) .
we discarded recordings when the systolic or diastolic variability of consecutive waveforms exceeded 5% or the amplitude of the pulse wave signal was less than 80  mv .
we calibrated the radial pulse wave on the brachial blood pressure . from the radial signal ,
the sphygmocor software calculates the aortic pulse wave by means of a validated generalised transfer function [ 10 , 11 ] .
the augmentation index was the ratio of the second to the first peak of the pressure wave expressed as a percentage .
aortic pulse wave velocity was measured by sequential ecg - gated recordings of the arterial pressure waveform at the carotid and femoral arteries .
we measured the distances from the suprasternal notch to the carotid sampling site ( distance a ) and from the suprasternal notch to the femoral sampling site ( distance b ) .
pulse wave travel distance was calculated as distance b minus distance a. pulse transit time was the average of 10 consecutive beats .
pulse wave velocity was the distance in meters divided by the transit time in seconds .
serum cystatin c was measured by a particle - enhanced turbidimetric immunoassay ( petia ) , ( cobas , roche diagnostics , germany ) .
the latex enhanced particles coated with anticystatin c antibodies in the reagent agglutinate with the human cystatin c in the sample .
the degree of the turbidity caused by the aggregate was determined turbidimetrically at 546  nm .
this assay has a detection limit of 0.4  mg / l and a coefficient of variation of 1.3% .
serum creatinine was measured by modified kinetic jaff methods with the modifications described elsewhere [ 13 , 14 ] .
the detection limit is 0.17  mg / dl and the coefficient of variation was 1.6% .
we use a creatinine method that has calibration traceable to an idms reference measurement procedure according to present recommendations [ 15 , 16 ] .
first , we computed egfr from serum cystatin c egfrcys , as described by inker and colleagues .
next , we calculated egfr from serum creatinine according to the idms - traceable mdrd study equation ( mdrd ) [ 17 , 18 ] formula ( egfrmdrd ) or the chronic kidney disease epidemiology collaboration ( ckd - epi )   equation ( egfrepi ) .
finally , as proposed by inker and colleagues ,   we also derived egfr from both serum cystatin c and serum creatinine ( egfrmix ) .
all aforementioned estimates [ 13 ] account for sex and age and with the exception of egfrcys also consider ethnicity ( black versus nonblack ) .
this particular characteristic was irrelevant for our current study as our participants only included whites mainly of european descent .
table s1 in the online data supplement provides detailed information on each formula ( see table s1 in supplementary material available online at http://dx.doi.org/10.1155/2014/837106 ) . in our current analyses , we compared findings based on the various methods to estimate gfr against egfrcys as the referent method . low glomerular filtration rate ( l - gfr ) was an egfr < 60  ml / min/1.73  m based on a single determination of each biomarker . for database management and statistical analysis , we used sas software , version 9.3 ( sas institute , cary , nc ) . first , in exploratory analyses , we assessed the characteristics of participants by fourths of the distribution of egfrcys . for comparison of means and proportions , we applied student 's t - test ( or anova ) and the  statistic , respectively .
we assessed agreement between paired measurements on a continuous scale by bland and altman 's method . to allow comparison with literature data , we also computed correlation coefficients .
the national kidney foundation kdoqi guideline proposes a threshold of 60  ml / min/1.73  m to diagnose chronic kidney disease .
in categorical analyses , we , therefore , also assessed the agreement between equations to dichotomize subjects in l - gfr or not - l - gfr using cohen 's kappa statistic .
a kappa value of 0.20 or less indicates slight agreement , 0.20 to 0.40 fair agreement , 0.41 to 0.60 moderate agreement , 0.61 to 0.80 substantial agreement , and 0.81 to 1.00 almost perfect agreement .
we studied the association between the four definitions of egfr dichotomized at 60  ml / min/1.73  m in hypertensive and diabetic subjects by mcnemar 's test for paired comparisons of proportions . because of the low frequencies in some cells , we applied exact statistics in two - by - two tables .
finally , we assessed the added capacity of egfrcys to differentiate between normotension versus hypertension or between people with or without diabetes mellitus , using the integrated discrimination improvement ( idi ) and the net reclassification improvement ( nri ) [ 22 , 23 ] .
the 119 participants included 68 women ( 57.1% ) and 53 ( 44.5% ) hypertensive patients , of whom 35 ( 66.0% ) were on antihypertensive drug treatment . among 68 women and 51 men , 11 ( 16.2% ) and 5 ( 9.8% ) were smokers ; 21 women ( 30.1% ) and 32 men ( 62.7% ) reported intake of alcohol . in smokers ,
median tobacco use was 10 cigarettes per day ( interquartile range , 615 ) . in drinkers , the median alcohol consumption was 8  g per day ( interquartile range , 454 ) . in the whole study population , age ( sd ) averaged was 56.5 ( 17.3 ) years and systolic and diastolic blood pressure 126.5 ( 19.6 )  mm  hg and 79.6 ( 11.7 )  mm  hg , respectively .
based on a self - report of the main maternal and paternal background , 37 participants ( 31.1% ) reported a mixture of caucasian , african , or native - american , while 82 participants ( 68.9% ) reported coincident caucasian background . among all participants , serum cystatin c and serum creatinine averaged from 0.99 ( 0.22 ) mg / l to 0.81 ( 0.21 ) mg / dl with no difference between women and men for cystatin c ( 0.99 versus 1.00  mg / l ; p = 0.77 ) , whereas women had lower serum creatinine than men had ( 0.72 versus 0.93  mg / dl ; p < 0.0001 ) . in all participants ,
mean values were 80.0 ( 23.8 )  ml / min/1.73  m for egfrcys , 89.7 ( 22.5 )  ml / min/1.73  m for egfrmdrd , 91.5 ( 19.0 )  ml / min/1.73  m for egfrepi , and 85.6 ( 20.2 )  ml / min/1.73  m for egfrmix , with no sex differences ( p  0.23 ) . 

table 1 provides the characteristics of participants by fourths of the distribution of egfrcys , which was used as reference . the prevalence of hypertension ( p = 0.027 ) , age ( p < 0.0001 ) , and systolic blood pressure ( p = 0.0097 ) , but not diastolic blood pressure ( p = 0.79 ) or mean arterial pressure ( p = 0.18 ) increased ( p = 0.027 ) with lower egfrcys category . the central systolic augmentation index and aortic pulse wave velocity also rose ( p <
0.0001 ) across decreasing fourths of the egfrcys distribution . trends in egfrmdrd , egfrepi , and egfrmix ran in parallel with the distribution of egfrcys . 

figure 1 shows the bland and altman plots comparing egfrmdrd , egfrepi , and egfrmix , with egfrcys as the referent method .  
table 2 shows the mean deviations of egfrmdrd , egfrepi , and egfrmix from egfrcys . in all participants , egfrmdrd , egfrepi , and egfrmix
were , respectively , 9.7 , 11.5 , and 5.6  ml / min/1.73  m higher than egfrcys .
the corresponding 2 sd intervals expressed in ml / min/1.73  m ( figure 1 ) ranged from 38.5 to + 57.9 for egfrmdrd , 25.5 to + 48.5 for egfrepi , and 10.2 to + 21.4 for egfrmix , and the corresponding correlation coefficients were 0.04 ( p = 0.69 ) , 0.26 ( p = 0.0046 ) , and 0.40 ( p < 0.0001 ) , respectively .
analyses stratified according to sex , age , normotension versus hypertension , and absence versus presence of diabetes mellitus were consistent with those in all participants ( table 2 ) .  
figure 2 shows that across fourths of the distribution of egfrcys , egfrmdrd , egfrepi , and egfrmix were consistently higher ( p < 0.002 ) than egfrcys except in the highest category of egfrcys ( p = 0.25 ) .
the prevalence of l - gfr was the highest for egfrcys , intermediate for egfrmix , and the lowest for egfrmdrd and egfrepi ( table 3 ) .
using egfrcys as reference , cohen 's kappa statistic was 0.230 ( 95% confidence interval [ ci ] , 0.036 to 0.427 ; p = 0.0005 ) for egfrmdrd , 0.151 ( ci , 0.021 to 0.322 ; p = 0.032 ) for egfrepi , and 0.587 ( ci , 0.399 to 0.775 ; p <
the prevalence of l - gfr was higher ( p < 0.0001 ) if patients were categorized based on egfrcys ( 17 patients , 32.1% ) compared with egfrmdrd ( 4 patients , 7.5% ) , egfrepi ( 3 patients , 5.7% ) , or egfrmix ( 8 patients , 15.1% ) . among 20 diabetic patients , we observed a similar trend .
the prevalence of l - gfr was 8 patients ( 40.0% ) based on egfrcys , 2 patients ( 10.0% ) based on egfrmdrd , 2 patients ( 10.0% ) based on egfrepi , and 3 patients ( 15% ) based on egfrmix .
however , the differences with egfrcys did not reach formal statistical significance ( p  0.075 ) . finally , we explored whether an egfrcys below 60  ml / min/1.73  m improved the differentiation between normotension versus hypertension or between people without or with diabetes mellitus based on the other estimates of gfr .
however , the classification based on egfrcys did not improve idi ( p  0.53 ) or nri ( p  0.24 ) for hypertension or idi ( p  0.37 ) or nri ( p  0.24 ) for diabetes mellitus .
in our current analysis , we compared the performance of the equations based on cystatin c and creatinine to estimate gfr in a uruguayan population sample .
the uruguayan population has been considered as mainly european descent , with a negligible native american or african contributions .
however , based on serological and molecular markers , recent studies demonstrate that native american and african had an important influence in the conformation of the present one [ 2426 ] .
the key finding was that egfrcys provides lower estimates in comparison with creatinine based equations ( egfrmdrd and egfrepi ) .
mean egfr was 80.0 , 89.7 , 91.5 , and 85.6  ml / min/1.73  m for egfrcys , egfrmdrd , egfrepi , and egfrmix , respectively .
thus , the prevalence of l - gfr , was higher when derived from equations involving cystatin c ( egfrcys and egfrmix ) than when derived from creatinine based equations .
the prevalence of l - gfr using egfrcys , egfrmdrd , egfrepi , and egfrmix was 21.8% , 5.9% , 3.4% , and 11.8% , respectively .
in categorical analysis , the agreement between cystatin - based ( egfrcys ) equations and creatinine - based equations ( egfrmdrd and egfrepi ) to detect egfr under 60  ml / min/1.73  m was low . over the past
, many reports highlighted the ability of cystatin c to detect renal disease early in different settings [ 2731 ] .
several researchers reported cystatin c outperforms serum creatinine in the early diagnosis of acute kidney injury [ 32 , 33 ] or its prognosis . in line with our results ,
other investigators reported lower estimates of egfr using cystatin c as biomarker [ 6 , 35 , 36 ] .
in contrast , in a belgian population sample [ n = 4,189 ] , delanaye and colleagues report that the prevalence of egfr below 60  ml / min/1.73  m using egfrcys , egfrmdrd , egfrepi , and egfrmix was 4.7% , 13.0% , 9.8% , and 5.0% , respectively . in a recent report , among 394 old ( > 74  y )
subjects and patients from england , kilbride and colleagues describe lower egfrcys ( 55.2  ml / min/1.73  m ) in comparison with creatinine based equations ( egfrmdrd , 57.6  ml / min/1.73  m ; egfrepi , 57.0  ml / min/1.73  m ) .
grams and colleagues , analyzing representative subsamples of the adult participants in the national health and nutrition examination surveys ( nhanes ; 19881994
[ n = 15,133 ] and 19992002 [ n = 8,238 ] ) , observed slightly higher egfr using cystatin c than creatinine equations ( 102.9 versus 99.4  ml / min/1.73  m ) .
however , in line with our results , the proportion of subjects with egfr below 60  ml / min/1.73  m was larger if derived from cystatin c than creatinine ( 5.5% versus 4.7% ) .
more recently , tsai and colleagues   reported similar results in diabetic patients enrolled in nhanes .
the mean egfrcys was 100.7  ml / min/1.73  m and the egfrepi was 95.1  ml / min/1.73  m while the proportions of patients with egfr below 60  ml / min/1.73  m were 22.0% and 16.5% , respectively .
pattaro and colleagues also found similar results in a cross - sectional analysis of a population from three alpine villages .
finally , in a recent meta - analysis shlipak and colleagues noticed similar mean egfrcys and egfrepi estimates while the proportion of subjects under the threshold to define ckd was 13.7 and 9.7  ml / min/1.73  m using cystatin and creatinine based equations , respectively . in line with our current results
, delanaye described low agreement between creatinine and cystatin c based formulas ( kappa statistic 0.32 [ egfrcys versus egfrmdrd ] and 0.39 [ egfrcys versus egfrepi ] ) . in this belgian study , the percentage of discordant subjects estimated by egfrmdrd , egfrepi , and egfrmix was 11.2% , 8.29% , and 2.71% , taking egfrcys as reference . how to explain the discrepancies between cystatin c and creatinine based equations ?
we analysed creatinine in fresh serum samples and cystatin c in samples that were kept frozen at 80c . over a period of 10 years
, a decay in cystatin c levels occurs using a particle - enhanced nephelometric assay ( penia ) .
such decay is not observed if one uses the more robust particle - enhanced turbidimetric assay ( petia ) .
furthermore , our samples were analysed within one year after blood collection and were processed with calibration each time .
we used a validated method [ 40 , 41 ] and reagents ( tina - quant cystatin c gen .
2 ) standardized to the international reference material erm - da 471/ifcc , as currently recommended for the use of ckd - epi equations [ 3 , 7 ] .
studies of bias ( mean difference from reference method ) usually overestimated gfr compared with the reference when using mdrd ( range 1.0 to + 3.5 ) or ckd - epi ( range 0.23 to + 4.4 ) and underestimated when using cystatin c based equations ( range 5.7 to 1.2 ) [ 38 , 4244 ] .
fewer studies showed a lower positive bias of cystatin c than ckd - epi based equations [ 7 , 45 ] .
several epidemiological studies showed that cystatin c is a better predictor of outcomes in coronary heart disease , acute coronary syndrome , and heart failure , independently of serum creatinine and gfr estimation [ 6 , 4649 ] .
furthermore , peralta et al . demonstrated in a large and ethnically diverse population that subjects with decreased egfrcys had elevated risk of death , cardiovascular disease , and heart failure and had an elevated risk of kidney failure [ 50 , 51 ] . in keeping with these studies ,
we found a higher prevalence of l - gfr among hypertensive subjects ( p < 0.001 ) if they were categorized based on egfrcys ( 32.1% ) than on egfrepi ( 5.7% ) with a similar but not significant trend among the few diabetics patients .
however , when computing idi and nri we did not observe significant differences between the various estimates of egfr for hypertension or diabetes ( p  0.24 ) .
in accordance with previous reports [ 48 , 5254 ] , the prevalence of hypertension ( p = 0.27 ) and age ( p < 0.0001 ) , systolic blood pressure ( p < 0.01 ) , cholesterol ( p = 0.004 ) , aortic pulse wave velocity ( p < 0.0001 ) , and the central augmentation index ( p < 0.0001 ) increased with lower categories of egfrcys ( table 1 ) . our results should be interpreted within the limitation of the study .
first , we did not have a reliable  gold standard  due to the variability in 24  h urine collections . performing inulin or
iothalamate clearance implies invasive and tedious procedures that are not suitable for our population study .
second , participants of the study may not be representative of the uruguayan population , because we randomly sampled a neighbourhood and our participants had a higher cardiovascular risk profile than the general population of uruguay .
finally , the small sample size of our population is a limiting factor to analyse specific subgroups of participants .
however , the number of participants is large enough to describe the difference between egfr estimating equations . in conclusion , to our knowledge , this is the first report based on a population from south america comparing to the performance of egfr equations based on cystatin c and creatinine .
we confirm discrepancies in egfr using equation based on different biomarkers , particularly in the range of gfr under 60  ml / min/1.73  m. generally , the equation based on cystatin c , compared with creatinine , results in lower egfr values and , therefore , higher estimates of the prevalence of egfr below 60  ml / min/1.73  m.
 
 background . estimation of glomerular filtration rate ( egfr ) from biomarkers has evolved and multiple equations are available to estimate renal function at bedside . methods . in a random sample of 119 uruguayans ( 54.5% women ; 56.2 years ( mean ) ) , we used bland and altman 's method and cohen 's kappa statistic to assess concordance on a continuous or categorical ( egfr < 60 versus 60  ml / min/1.73  m2 ) scale between egfrcys ( reference ) and egfr derived from serum creatinine according to the modification of diet in renal disease ( egfrmdrd ) or the chronic kidney disease epidemiology collaboration equations ( egfrepi ) or from both serum cystatin c and creatinine ( egfrmix ) . results . in all participants , 
 egfrmdrd , egfrepi , and egfrmix were , respectively , 9.7 , 11.5 , and 5.6  ml / min/1.73  m2 higher ( p < 0.0001 ) than egfrcys . the prevalence of egfr < 60  ml / min/1.73  m2 was the highest for egfrcys ( 21.8% ) , intermediate for egfrmix ( 11.8% ) , and the lowest for egfrmdrd ( 5.9% ) and egfrepi ( 3.4% ) . using egfrcys as reference , we found only fair agreement with the equations based on creatinine ( cohen 's kappa statistic 0.15 to 0.23 ) . 
 conclusion . using different equations we reached clinically significant differences in the estimation of renal function . 
 egfrcys provides lower estimates , resulting in higher prevalence of egfr < 60  ml / min/1.73  m2 . 
1. Introduction
2. Materials and Methods
3. Results 
4. Discussion 