C H A P T E R
Assessment of Renal Function
Lesley A. Inker, Li Fan, and Andrew S. Levey
GLOMERULAR FILTRATION RATE
Glomerular filtration rate (GFR) is a product of the average filtration
rate of each nephron, the filtering unit of the kidneys, multiplied by
the number of nephrons in both kidneys. The normal level for GFR is
approximately 130ml/min/1.73m2 for men and 120ml/min/1.73m2
for women, with considerable variation among individuals according
to age, gender, body size, physical activity, diet, pharmacotherapy,
and physiologic states such as pregnancy. To standardize the function
of the kidney for differences in kidney size, which is proportional to
body size, GFR is adjusted for body surface area (BSA), computed
from height and weight, and is expressed per 1.73m2 BSA, the mean
BSA of young men and women. Even after adjustment for BSA, GFR
is approximately 8% higher in young men than in women and declines
with age; the mean rate of decline is approximately 0.75ml/min/yr
after age 40 years, but the variation is wide, and the sources of varia-
tion are poorly understood. During pregnancy, GFR increases by
about 50% in the first trimester and returns to normal immediately
after delivery. GFR has a diurnal variation and is 10% lower at mid-
night compared with the afternoon. Within an individual, GFR is
relatively constant over time but varies considerably among people,
even after adjustment for the known variables.
Reductions in GFR may result from a decline in the nephron
number or in the single-nephron (SN) GFR from physiologic or
hemodynamic alterations. An increase in SNGFR caused by increased
glomerular capillary pressure or glomerular hypertrophy can com-
pensate for a decrease in nephron number; therefore the level of GFR
may not reflect the loss of nephrons. As a result, there may be sub-
stantial kidney damage before GFR decreases.
MEASUREMENT OF THE GLOMERULAR
FILTRATION RATE
The GFR cannot be measured directly. Instead, it is measured as the
urinary clearance of an ideal filtration marker.
Concept of Clearance
Clearance of a substance is defined as the volume of plasma cleared
of a marker by excretion per unit of time. The clearance of substance
x (Cx) can be calculated as Cx = Ax/Px, where Ax is the amount of x
eliminated from the plasma, Px is the average plasma concentration,
and Cx is expressed in units of volume per time. Clearance does not
represent an actual volume; rather, it is a virtual volume of plasma
that is completely cleared of the substance per unit of time. The value
for clearance is related to the efficiency of elimination: the greater
the rate of elimination, the higher the clearance. Clearance of sub-
stance x is the sum of the urinary and extrarenal clearance; for
substances that are eliminated by renal and extrarenal routes, plasma
clearance exceeds urinary clearance.
30
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Urinary Clearance
The amount of substance x excreted in the urine can be calculated
as the product of the urinary flow rate (V) and the urinary concentra-
tion (Ux). Therefore urinary clearance is defined as follows:
C x = (U x V)/ Px
Urinary excretion of a substance depends on filtration, tubular
secretion, and tubular reabsorption. Substances that are filtered but
not secreted or reabsorbed by the tubules are ideal filtration markers
because their urinary clearance can be used as a measure of GFR.
For substances that are filtered and secreted, urinary clearance
exceeds GFR; and for substances that are filtered and reabsorbed,
urinary clearance is less than GFR.
Measurement of urinary clearance requires a timed urine collec-
tion for measurement of urine volume, as well as urine and plasma
concentrations of the filtration marker. Special care must be taken to
avoid incomplete urine collections, which will limit the accuracy of
the clearance calculation.
Plasma Clearance
Interest in measurement of plasma clearance continues because it
avoids the need for a timed urine collection. GFR is calculated from
plasma clearance (Cx) after a bolus intravenous injection of an exog-
enous filtration marker, with the clearance (Cx) computed from the
amount of the marker administered (Ax) divided by the average
plasma concentration (Px), which can be computed from the area
under the curve of plasma concentration versus time.
C x = A x / Px
The decline in plasma levels is secondary to the immediate disap-
pearance of the marker from the plasma into its volume of distribu-
tion (fast component) and to renal excretion (slow component).
Plasma clearance is best estimated by use of a two-compartment
model that requires blood sampling early (usually two or three time
points until 60 minutes) and late (one to three time points from 120
minutes onward). As with urinary clearance, plasma clearance of a
substance depends on filtration, tubular secretion, and tubular reab-
sorption, but in addition, extrarenal elimination.
Exogenous Filtration Markers
Inulin, an uncharged polymer of fructose with molecular weight of
approximately 5000 daltons (d), was the first substance described as
an ideal filtration marker and remains the reference (gold standard)
against which other markers are evaluated. The classic protocol for
inulin clearance requires a continuous intravenous (IV) infusion to
achieve a steady state and bladder catheterization with multiple
timed urine collections. Because this technique is cumbersome, and
inulin measurement requires a difficult chemical assay, this method
has not been used widely in clinical practice and remains a research
 CHAPTER 3 Assessment of Renal Function 31

Exogenous Filtration Markers for Estimation Relationship of GFR and Non-GFR

of Glomerular Filtration Rate Determinants to Serum Levels
Method of
Marker Administration Comments G (cells) UV=
Inulin Continuous IV Gold standard GFR P TR + TS
infusion
G (diet) UV GE=
Iothalamate Bolus IV Can be administered as P
(kidney) GFR P TR + TS
injection or radioactive compound with
subcutaneous iodine 125 (125I) as the tracer
injection or in nonradioactive form, with
GFR =
assay using HPLC methods. In E (gut, liver) (G + TR TS E) / P
radioactive form, potential
Figure 3-1 Relationship of glomerular filtration rate and non-GFR
problem of thyroid uptake
determinants to serum levels. G, Generation; E, extrarenal elimination;
of 125I. Iothalamate is secreted,
P, plasma; TR, tubular reabsorption; TS, tubular secretion. (Modified from
leading to overestimation
reference 1.)
of GFR
99m
Tc-DTPA Bolus IV injection Dissociation of 99mTc leads to
plasma protein binding and
underestimation of GFR
Estimated Glomerular Filtration Rate
51
Cr-EDTA Bolus IV injection 10% lower clearance than inulin from Plasma Levels
Iohexol Bolus IV injection Low incidence of adverse effects; Figure 3-1 shows the relationship of plasma concentration of sub-
comparable to inulin; expensive stance x to its generation (Gx) by cells and dietary intake, urinary
and difficult to perform assay excretion (Ux V), and extrarenal elimination (Ex) by gut and liver.
Table 3-1 Exogenous filtration markers for estimation of glomerular The plasma level is related to the reciprocal of the level of GFR, but
filtration rate. 51Cr-EDTA, Chromium 51labeled ethylenediaminetetraace- it is also influenced by generation, tubular secretion and reabsorp-
tic acid; GFR, glomerular filtration rate; HPLC, high-performance liquid tion, and extrarenal elimination, collectively termed non-GFR deter-
chromatography; IV, intravenous; 99mTc-DTPA, technetium 99mlabeled minants of the plasma level.1
diethylenetriaminepentaacetic acid.
In the steady state, a constant plasma level of substance x is main-
tained because generation is equal to urinary excretion and extrare-
tool. Alternative exogenous substances include iothalamate, iohexol, nal elimination. Estimating equations incorporate demographic and
ethylenediaminetetraacetic acid, and diethylenetriaminepentaacetic clinical variables as surrogates for the non-GFR determinants and
acid, often chelated to radioisotopes for ease of detection (Table 3-1). provide a more accurate estimate of GFR than the reciprocal of the
Alternative protocols to assess clearance have also been validated, plasma level alone. Estimating equations are derived from regression
including subcutaneous injection and spontaneous bladder empty- of measured GFR on measured values of the filtration marker and
ing. There are advantages to alternative exogenous filtration markers observed values of the demographic and clinical variables. Estimated
and methods, but also limitations. Understanding the strengths and GFR (eGFR) may differ from measured GFR in a patient if a discrep-
limitations of each alternative marker and each clearance method ancy exists between the true and average values for the relationship
will facilitate interpretation of measured GFR.1 of the surrogate to the non-GFR determinants of the filtration
marker. Other sources of errors include measurement error in the
filtration marker (e.g., failure to calibrate assay for filtration marker
Endogenous Filtration Markers to assay used in development of equation), measurement error in
Endogenous filtration markers are substances generated in the body GFR in development of the equation, and regression to the mean.
at a relatively constant rate and eliminated largely by glomerular In principle, all these errors are likely to be greater at higher values
filtration. Therefore, the serum level correlates highly with measured for GFR.2
GFR after accounting for factors other than GFR that influence the
non-GFR determinants. Currently identified endogenous filtration
markers include low-molecular-weight metabolites and serum pro- CREATININE
teins. Filtered metabolites may undergo reabsorption or secretion,
which contribute to their urinary excretion. Comparison to urinary Metabolism and Excretion
clearance of exogenous filtration markers enables inferences about Creatinine is a 113-d end product of muscle catabolism. Advantages
the renal handling of endogenous filtration markers. By contrast, of creatinine include its ease of measurement and the low cost and
filtered serum proteins are reabsorbed and degraded within the widespread availability of assays. Disadvantages include the large
tubule with minimal appearance in the urine. For filtration markers number of non-GFR determinants, leading to a wide range of GFR
excreted in the urine, urinary clearance can be computed from a for a given serum creatinine level (see Table 3-2). For example, a
timed urine collection and a single measurement of serum concen- serum creatinine level of 1.5mg/dl (132 mol/l) may correspond to
tration. If the serum level is not constant during the urine collection, a GFR from approximately 20 to 90ml/min/1.73m2.
as in acute kidney disease or when residual kidney function is Creatinine is derived by the metabolism of phosphocreatine in
assessed in dialysis patients, it is necessary to obtain additional blood muscle as well as from dietary meat intake or creatine supplements.
samples during the urine collection to estimate the average serum Creatinine generation is proportional to muscle mass, which can be
concentration. estimated from age, gender, race, and body size. Table 3-3 lists factors
Creatinine is the most frequently used endogenous filtration that can affect creatinine generation.3
marker in clinical practice. Urea was widely used in the past, and Creatinine is released into the circulation at a constant rate during
cystatin C presently shows great promise (Table 3-2). normal physiologic conditions. It is not protein bound and is freely

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32 SECTION II Investigation of Renal Disease

Comparison of Creatinine, Urea, and Cystatin C as Filtration Markers

Variable Creatinine Urea Cystatin C
Molecular Properties
Weight (d) 113 60 13,000
Structure Amino acid derivative Organic molecular product of Nonglycosylated basic protein
protein metabolism
Physiologic Determinants of Serum Level
Generation Varies, according to muscle mass Varies, according to dietary Thought to be mostly constant
and dietary protein; lower in protein intake and catabolism by all nucleated cells; increases
elderly persons, women, and in hyperthyroid state and with
whites steroid use; lower in elderly
persons and women
Handling by kidney Filtered, secreted, and excreted in Filtered, reabsorbed, and excreted Filtered, reabsorbed, and
urine in urine catabolized
Extrarenal elimination Yes; increases at reduced GFR Yes; increases at reduced GFR Preliminary evidence of increases
at reduced GFR
Use In Estimating Equations for GFR
Demographic and clinical Age, gender, and race; related to Not applicable Age, gender
variables as surrogates for muscle mass
physiologic determinants
Accuracy Accurate for GFR <60ml/min/1.73m2 Not applicable Unknown
Assay
Method Colorimetric or enzymatic Direct measurement, enzymatic PENIA, PETIA, or ELISA
colorimetric and electrochemical
Assay precision Very good except at low range Precise throughout range Precise throughout range
Clinical laboratory practice Multiple assays; widely used Multiple assays; enzymatic and Not on most autoanalyzers; not
nonstandard calibration colorimetric more common standardized
Standardized recommendation SRM 967 SRM 912a ERM-DA471/IFCC
materials (SRMs)
Reference assay IDMS IDMS PENIA, PETIA, or ELISA

Table 3-2 Comparison of creatinine, urea, and cystatin C as filtration markers. ELISA, Enzyme-linked immunosorbent assay; GFR, glomerular
filtration rate; IDMS, isotope-dilutionmass spectroscopy; PENIA, particle-enhanced nephelometric immunoassay; PETIA, particle-enhanced turbidimetric
immunoassay. (Modified with permission from reference 2.)

filtered across the glomerulus and secreted by the tubules. Several
medications, such as cimetidine and trimethoprim, competitively
inhibit creatinine secretion and reduce creatinine clearance. These
medications thus lead to a rise in the serum creatinine concentration
without an effect on GFR (Table 3-3).
In addition, creatinine is contained in intestinal secretions and
can be degraded by bacteria. If GFR is reduced, the amount of cre-
atinine eliminated through this extrarenal route is increased. Anti-
biotics can raise serum creatinine concentration by destroying
intestinal flora, thereby interfering with extrarenal elimination, as
well as by reducing the GFR. The rise in serum creatinine concentra-
tion after inhibition of tubular secretion and extrarenal elimination
is greater in patients with a reduced GFR. Clinically, it can be difficult
to distinguish a rise in serum creatinine concentration caused by
inhibition of creatinine secretion or extrarenal elimination from a
decline in GFR. However, processes other than a decrease in GFR
should be suspected if serum urea concentration remains unchanged
despite a significant change in serum creatinine concentration in a
patient with an initially reduced GFR.
Creatinine clearance is usually computed from the creatinine
excretion in a 24-hour urine collection and single measurement of
serum creatinine in the steady state. Creatinine excretion rates vary
with age, gender, and race and are approximately 20 to 25mg/kg/day
and 15 to 20mg/kg/day in a complete collection in healthy young
men and women, respectively. Equations are available to estimate the
creatinine excretion from age, gender, weight, and other variables.4
Deviations from these expected values can give some indication of
errors in timing or completeness of urine collection. Creatinine
clearance systematically overestimates GFR because of tubular cre-
atinine secretion. In the past the amount of creatinine excreted by
tubular secretion at normal levels of GFR was thought to be relatively
small (10% to 15%), but with newer, more accurate assays for low
values of serum creatinine, this difference may be substantially
greater. At low values of GFR, the amount of creatinine excreted by
tubular secretion may exceed the amount filtered.2
Creatinine Assay
Historically, the most common assay for measurement of serum
creatinine was the alkaline picrate (Jaffe) assay that generates a color
reaction. Chromogens other than creatinine are known to interfere
with the assay, giving rise to errors of up to approximately 20%
in normal individuals. Modern enzymatic assays do not detect non-
creatinine chromogens and yield lower serum levels than with the
alkaline picrate assays. Until recently, calibration of assays to adjust
for this interference was not standardized across laboratories, thereby
limiting the estimation of GFR from serum creatinine concentra-
tions, especially at higher GFR.
To address the heterogeneity in creatinine assays, fresh-
frozen serum pools with known creatinine levels traceable to an

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 CHAPTER 3 Assessment of Renal Function 33

Factors Affecting Serum Creatinine Concentration

Factors Effect on Creatinine Mechanism/Comment
Age Decrease Reduced creatinine generation caused by age-related decline in muscle mass
Female gender Decrease Reduced creatinine generation caused by reduced muscle mass
Race
African American Increase Higher creatinine generation caused by higher average muscle mass in African
Americans; not known how muscle mass in other races compares with that
of African Americans or Caucasians
Diet
Vegetarian Decrease Decrease in creatinine generation
Ingestion of cooked meats and Increase Transient increase in creatinine generation, although this may be blunted by
creatinine supplements transient increase in GFR
Body Habitus
Muscular Increase Increased muscle generation caused by increased muscle mass and/or increased
protein intake
Malnutrition, muscle wasting, Decrease Reduced creatinine generation caused by reduced muscle mass and/or reduced
amputation protein intake
Obesity No change Excess mass is fat, not muscle mass, and does not contribute to increased
creatinine generation.
Medications
Trimethoprim, cimetidine, fibric acid Increase Reduced tubular secretion of creatinine
derivatives other than gemfibrozil
Keto acids, some cephalosporins Increase Interference with alkaline picrate assay for creatinine

Table 3-3 Factors affecting serum creatinine concentration. (Modified from reference 3.)

isotope-dilutionmass spectrometry (IDMS) reference are available
for instrument manufacturers to standardize creatinine measure-
ments.5 Use of standardized assays is recommended.6 Standardiza-
tion will reduce, but not completely eliminate, the error in estimating
GFR at higher levels (Table 3-3).
Estimated Glomerular Filtration Rate
from Serum Creatinine
Again, GFR can be estimated from serum creatinine by equations
that use age, gender, race, and body size as surrogates for creatinine
generation.1 Despite substantial advances in the accuracy of estimat-
ing equations based on creatinine during the past several years, GFR
estimates remain imprecise, and no equation is likely to overcome
the limitations of creatinine as a filtration marker. None of the equa-
tions is expected to work as well in patients with extreme levels for
creatinine generation, such as amputees, large or small individuals,
patients with muscle-wasting conditions, or people with high or low
levels of dietary meat intake (Table 3-3). Because of differences
among racial and ethnic groups according to muscle mass and diet,
equations developed in one racial or ethnic group are unlikely to be
accurate in multiethnic populations. As discussed later, further
improvements will probably require additional filtration markers.
Cockcroft-Gault Formula
The Cockcroft-Gault formula estimates creatinine clearance from
age, gender, and body weight, in addition to serum creatinine (Box
3-1).7 An adjustment factor for women is based on a theoretical
assumption of 15% lower creatinine generation because of lower
muscle mass. Comparison to normal values for creatinine clearance
requires computation of BSA and adjustment to 1.73m2. Because of
the inclusion of a term for weight in the numerator, this formula
systematically overestimates creatinine clearance in edematous or
obese patients.
The Cockcroft-Gault formula has three main limitations. First,
it is not precise, in particular in the GFR range above 60ml/min.
Second, it estimates creatinine clearance rather than GFR and thus
is expected to overestimate GFR, while normal values for creatinine
secretion are not well known. Third, the formula was derived by
older assay methods for serum creatinine, which cannot be cali-
brated to newer assay methods, which would be expected to lead to
a systematic bias in estimating creatinine clearance.
Importantly, before standardization of creatinine assays, the
Cockcroft-Gault formula was widely used to assess pharmacokinetic
properties of drugs in patients with impaired kidney function. The
accuracy of drug dosing recommendations based on the Cockcroft-
Gault formula using creatinine values from modern assays remains
controversial. One study suggested that drug dosage adjustment
guided by the Cockcroft-Gault formula is slightly less accurate than
adjustments based on more accurate estimating equations.8
Modification of Diet in Renal Disease Study
The Modification of Diet in Renal Disease (MDRD) study equation
uses age, gender, and race (African American vs. Caucasian or other)
and standardized serum creatinine to estimate GFR9 (Box 3-1). It
was derived from a study population with chronic kidney disease
(CKD) and underestimates the measured GFR in populations with
higher levels of GFR (Fig. 3-2). It has not been validated in children
or pregnant women. The MDRD study equation had greater preci-
sion and greater overall accuracy than the Cockcroft-Gault formula.
Modifications of the MDRD have now been reported in racial and
ethnic populations other than African American and Caucasian,
including those in China, Japan, Thailand, and South Africa.10,11 In
general, these modifications improve the accuracy of the MDRD
equation in the study population but do not generalize well to other
populations.
Organizations in several countries now recommend GFR esti-
mates (eGFR) as the primary method of clinical assessment of

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34 SECTION II Investigation of Renal Disease

Equations for Estimating Glomerular Filtration Rate

Cockroft-Gault Formula6
Male (140 Age) Weight or (140 Age) Weight
Ccr (ml / min) = Ccr (ml / min) =
72 Scr (mg / dl) 0.814 Scr (mol / l)
Female (140 Age) Weight 0.85 or (140 Age) Weight 0.85
Ccr (ml / min) = CCr (ml / min) =
72 Scr (mg / dl) 0.814 Scr (mol / l)
MDRD Study Equation for Use with Standardized Serum Creatinine (Four-Variable Equation)8
GFR (ml / min / 1.73 m2 ) = 175 Standardized Scr (mg / dl)1.154 Age00.203 0.742 (if female) 1.210 (if black )
GFR (ml / min / 1.73 m2 ) = 30, 849 Standardized Scr (mol / l)1.154 Age0.203 0.742 (if female) 1.210 (if black )
CKD-EPI Equation for Use with Standardized Serum Creatinine11
GFR (ml / min / 1.73 m2 ) = 141min (Scr / , 1) max(Scr / , 1)1.209 0.993Age 1.018 (if female) 1.157 (if black )

where is 0.7 for females and 0.9 for males, is 0.329 for females and 0.411 for males, min indicates the minimum of Scr/ or 1,
and max indicates the maximum of Scr/ or 1.

Female 0.7 GFR = 144 (Scr / 0.7)0.329 (0.993)Age 1.157 (if black)

> 0.7 GFR = 144 (Scr / 0.7)1.209

Male 0.9 GFR = 141 (Scr / 0.9)0.411

> 0.9 GFR = 141 (Scr / 0.9)1.209

CKD-EPI Equation for Use with Standardized Serum Cystatin C19
GFR (ml / min / 1.73 m2 ) = 133 min(Scys / 0.8, 1)0.499 max(Scys / 0.8, 1)1.328 0.996Age 0.932 (if female)

where min indicates the minimum of Scys/0.8 or 1, and max indicates the maximum of Scys/0.8 or 1.

0.8 GFR = 133 (Scys / 0.8)0.499 0.996Age 0.932 (if female)

> 0.8 GFR = 133 (Scys / 0.8)1.328

CKD-EPI Equation for Use with Standardized Serum Creatinine and Cystatin C19
GFR (ml / min / 1.73 m2 ) = 135 min(Scr / , 1) max(Scr / , 1)0.601 min(Scys / 0.8, 1)0.375 max(Scys / 0.8, 1)0.711 0.995Age
0.969 (if female) 1.08 (if black )

where is 0.7 for females and 0.9 for males; is 0.248 for females and 0.207 for males; min indicates the minimum of Scr/ or 1,
or of Scys/0.8 or 1; and max indicates the maximum of Scr/ or 1, or of Scys/0.8 or 1.

Female 0.7 0.8 GFR = 130 (Scr / 0.7)0.248 (Scys / 0.8)0.375 0.995Age 1.08 (if black)

> 0.8 GFR = 130 (Scr / 0.7) 0.248

(Scys / 0.8) 0.711

>0.7 0.8 GFR = 130 (Scr / 0.7)0.601 (Scys / 0.8)0.375

> 0.8 GFR = 130 (Scr / 0.7)0.601 (Scys / 0.8)0.711

Male 0.9 0.8 GFR = 135 (Scr / 0.9)0.207 (Scys / 0.8)0.375

> 0.8 GFR = 135 (Scr / 0.9)0.207 (Scys / 0.8)0.711

>0.9 0.8 GFR = 135 (Scr / 0.9)0.601 (Scys / 0.8)0.375

> 0.8 GFR = 135 (Scr / 0.9)0.601 (Scys / 0.8)0.711

Box 3-1 Equations for estimating glomerular filtration rate. Age in years; weight in kg; Scr, serum creatinine; Scys, serum cystatin C. The Modification
of Diet in Renal Disease (MDRD) study and Chronic Kidney Disease Epidemiology (CKD-EPI) equation calculator can also be found online at http://www.
kidney.org/professionals/kdoqi/gfr_calculator.cfm.

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Performance of GFR Estimating Equation
90
Measured-Estimated GFR (ml/min/1.73 m2)
60
30
0 The CKD-EPI creatinine equation now allows reporting of eGFR
-30
-60
-90
30 60 90
Estimated GFR (ml/min/1.73 m2)
Measured-Estimated GFR (ml/min/1.73 m2)
90
60
30
0
-30
-60
-90
30 60 90
Estimated GFR (ml/min/1.73 m2)
Figure 3-2 Comparison of performance of Chronic Kidney Disease
Epidemiology Collaboration (CKD-EPI) and Modification of Diet in
Renal Disease (MDRD) study equations. Difference between measured
GFR (mGFR) and estimated GFR (eGFR) versus eGFR for CKD-EPI equation
(top panel) and MDRD equation (bottom panel), showing smoothed regres-
sion line and 95% confidence interval (CI, computed from lowest smoothing
function in R) and using quantile regression, excluding lowest and highest
2.5% of eGFR values. For the two equations, median bias (percentage of
estimates within 30% of measured GFR, P30) is 2.5 (84) and 5.5 (81), respec-
tively. To convert GFR from ml/min/1.73m2 to ml/s/m2, multiply by 0.0167.
(Modified from reference 12.)
kidney function.6 Because of limitations in accuracy at higher levels,
recommendations include reporting eGFR as a numerical value only
if the GFR estimate is less than 60ml/min/1.73m2 and reporting
eGFR as greater than 60ml/min/1.73m2 for higher values.6
Chronic Kidney Disease Epidemiology Collaboration
The 2009 Chronic Kidney Disease Epidemiology Collaboration
(CKD-EPI) creatinine equation (Box 3-1) has been developed from
a large database of participants in research studies and patients from
clinical populations with diverse characteristics, including those
with and without kidney disease, diabetes, and a history of organ
transplantation, to overcome the limitations of the MDRD Study
equation.12 The CKD-EPI equation is based on the same four vari-
ables as the MDRD equation but uses a two-slope spline to model
the relationship between GFR and serum creatinine, which partially
corrects the underestimation of GFR at higher levels seen with the
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CHAPTER 3 Assessment of Renal Function 35
MDRD study equation. The CKD-EPI creatinine equation also
incorporates slightly different relationships for age, gender, and race.
As a result, the CKD-EPI equation is as accurate as the MDRD at
eGFR of less than 60ml/min/1.73m2 and is more accurate at higher
levels (Fig. 3-2). The CKD-EPI is also more accurate across a wide
range of characteristics, including age, gender, race, body mass
Underestimate
index, and presence or absence of diabetes or history of organ trans-
plantation. As with the MDRD, modifications of the CKD-EPI equa-
tions in Japan improve accuracy in these study populations.11
across the entire range of values, without substantial bias. It is cur-
Overestimate
rently reported by the two major nationwide laboratories in the
United States, as well as by laboratories in France. The 2012 Kidney
Disease: Improving Global Outcomes (KDIGO) guidelines recom-
mend that clinical laboratories report eGFR in all adults using
CKD-EPI creatinine equations, or using other equations if shown to
be superior to CKD-EPI equation in that population.13
120 150
UREA
The serum urea level has limited value as an index of GFR, in view
Underestimate
of widely variable non-GFR determinants, primarily urea generation
and tubular reabsorption (see Table 3-2).
Urea is a 60-d end product of protein catabolism by the liver.
Factors associated with the increased generation of urea include
protein loading from hyperalimentation and absorption of blood
after a gastrointestinal hemorrhage. Catabolic states caused by infec-
Overestimate
tion, corticosteroid administration, or chemotherapy also increase
urea generation. Decreased urea generation is seen in patients with
severe malnutrition and liver disease.
Urea is freely filtered by the glomerulus and then passively
reabsorbed in both proximal and distal nephrons. As a result of
tubular reabsorption, urinary clearance of urea underestimates GFR.
120 150
Reduced kidney perfusion in the patient with volume depletion and
states of antidiuresis are associated with increased urea reabsorption.
This leads to a greater decrease in urea clearance than the concomi-
tant decrease in GFR. At GFR of less than about 20ml/min/1.73m2,
the overestimation of GFR by creatinine clearance resulting from
creatinine secretion approximates the underestimation of GFR by
urea clearance from urea reabsorption; thus the average of creatinine
and urea clearance approximates the measured GFR.
CYSTATIN C
Metabolism and Excretion
Cystatin C is a 122amino acid protein with molecular weight of
13kd (see Table 3-2). Its multiple biologic functions include extra-
cellular inhibition of cysteine proteases, modulation of the immune
system, antibacterial and antiviral activities, and modification of the
bodys response to brain injury. The serum concentration of cystatin
C remains constant from about 1 to 50 years of age. In analyses of
the Third National Health and Nutrition Examination Survey
(NHANES III), the median and upper 99th percentile levels of serum
cystatin C for people age 20 to 39 without history of hypertension
and diabetes were 0.85mg/l and 1.12mg/l, respectively, with levels
lower in women than in men, higher in non-Hispanic whites than
in blacks and Mexicans, and increasing steeply with age.14
Cystatin C has been thought to be produced at a constant rate by
a housekeeping gene expressed in all nucleated cells. It is freely
filtered at the glomerulus because of its small size and basic pH.
Approximately 99% of the filtered cystatin C is reabsorbed by the
36 SECTION II Investigation of Renal Disease

proximal tubular cells, where it is almost completely catabolized,
with the remainder eliminated in the urine largely intact.15 Some
evidence suggests the existence of tubular secretion as well as extra-
renal elimination, the latter estimated at 15% to 21% of renal
clearance.16
Because cystatin C is not excreted in the urine, it is difficult to
study its generation and renal handling. Thus, understanding
non-GFR determinants of cystatin C relies on epidemiologic associa-
tions. Some suggest that inflammation, adiposity, thyroid diseases,
certain malignant neoplasms, and use of glucocorticoids may
increase cystatin C levels. Two studies found that key factors leading
to higher cystatin C levels after adjustment for creatinine clearance
or measured GFR were older age, male gender, fat mass, white race,
diabetes, higher C-reactive protein level, increased white blood cell
count, and lower serum albumin level.17,18 Therefore factors other
than GFR must be considered in interpreting cystatin C levels.
Cystatin C Assay
Available assays to analyze cystatin C all can result in different values.
The International Federation of Clinical Chemists (IFCC) made a
reference material for standardization of cystatin C, but international
standardization of the assay is still in process.19 The assays are con-
siderably more expensive than those for creatinine determination.
Estimated Glomerular Filtration Rate
from Serum Cystatin C
Numerous studies have found that serum cystatin C levels are a
better estimate of GFR than serum creatinine concentration because
cystatin C is less affected than creatinine by age, gender, or race.
However, cystatin C or equations based on cystatin C are not more
accurate than creatinine-based estimating equations, due to varia-
tion in non-GFR determinants of serum cystatin C.20 Several studies,
though, have demonstrated that equations combining both these
filtration markers with age, gender, and race appear to be more
precise than equations using either marker alone, probably because
of smaller effects of the non-GFR determinants of both markers
when used in combination. The 2012 CKD-EPI cystatin C and
creatininecystatin C equations (see Box 3-1) are expressed for use
with standardized serum creatinine and cystatin C and are recom-
mended by the 2012 KDIGO guidelines (Fig. 3-3).20 The equation

Performance of Three CKD-EPI Equations for Estimating Glomerular

Filtration Rate
25
eGFRcr
eGFRcys
20 eGFRcr,cys
Median Difference

15

10

0
<15 1529 3059 6089 90120 >120
5 Estimated GFR (ml/min/1.73 m2)

60
eGFRcr
eGFRcys
50 eGFRcr,cys

40
1 - P30

30

20

10

0
<15 1529 3059 6089 90120 >120
Estimated GFR (ml/min/1.73 m2)
Figure 3-3 Performance of three equations for estimating GFR. GFR was estimated using the Chronic Kidney Disease Epidemiology estimating equa-
tions. Top, Median difference between measured GFR and estimated glomerular filtration rate (eGFR). The bias is similar with the equation using creatinine
alone (eGFRcr), that using cystatin C alone (eGFRcys), and the combined creatininecystatin C equation (eGFRcr,cys). Bottom, Accuracy of the three equations
according to percentage of estimates greater than 30% of measured GFR (1 P30). I bars indicate 95% CI. (Modified from reference 20.)

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 CHAPTER 3 Assessment of Renal Function 37

using cystatin C without creatinine does not appear to require speci- Current estimating equations will be less accurate in people with
fication of race. Also, in patients with reduced muscle mass (e.g., factors affecting serum creatinine concentration other than GFR (see
neuromuscular or liver disease, low body mass index) or in patients Table 3-3). In these patients, more accurate GFR estimates require
with diabetes, cystatin C may result in more accurate GFR estimates additional testing, such as measurement with an endogenous filtra-
than creatinine. tion marker (e.g., cystatin C, 2M, TP), a timed urine creatinine
Some studies show that a lower eGFR based on serum cystatin C clearance measurement, or clearance measurement using an exoge-
is a better predictor of the risk of cardiovascular disease and total nous marker.
mortality than is a lower eGFR based on serum creatinine concentra-
tion.21 Whether this is caused by its superiority as a filtration marker
or the confounding by non-GFR determinants of cystatin C and Acute Kidney Injury
creatinine remains to be determined. In the future, GFR estimating In the nonsteady state, there is a lag before the rise in serum level
equations using the combination of serum cystatin C and creatinine because of the time required for retention of an endogenous filtration
may be useful as a confirmatory test for CKD. However, this is fea- marker (Fig. 3-4). Conversely, after recovery of GFR, there is a lag
sible only after standardization, widespread availability, and cost before the excretion of the retained marker. During this time, neither
reductions of cystatin C assays, as well as further investigation of the serum level nor the GFR estimated from the serum level accu-
non-GFR determinants of serum cystatin C. rately reflects the measured GFR. Nonetheless, a change in the eGFR
in the nonsteady state can be a useful indication of the magnitude
and direction of the change in measured GFR. If the eGFR is decreas-
OTHER FILTRATION MARKERS ing, the decline in eGFR is less than the decline in measured GFR.
Conversely, if the eGFR is increasing, the rise in eGFR is greater
2-Microglobulin (2M) and -trace protein (TP) are two other than the rise in measured GFR. The more rapid the change in eGFR,
low-molecular-weight serum proteins being evaluated as filtration the greater is the change in measured GFR. When eGFR reaches a
markers for estimating GFR and for their role in prognosis. However, new steady state, it more accurately reflects measured GFR. In
2M and TP are not recommended for use at this time. An 11.8-kd patients with acute kidney injury, serum cystatin C appears to
subunit of major histocompatibility complex (MHC) class I mole- increase more rapidly than serum creatinine.30 More data are required
cules, 2M is present on all nucleated cells and plays a central role in to establish whether changes in serum cystatin C are a more sensitive
cellular immunology. TP, also known as lipocalin prostaglandin indicator of rapidly changing kidney function than changes in serum
D2 synthase, is a 168amino acid glycoprotein of 23 to 29kd. As creatinine.
with cystatin C, 2M and TP are freely filtered by the glomerulus
and extensively reabsorbed and degraded by the proximal tubule,
with only small amounts excreted in the urine under normal
conditions.
In NHANES-III analyses, the median (upper 99th percentile) Effect of a Sudden Decrease in Glomerular
levels of serum 2M and TP for people age 20 to 39 without history
of hypertension or diabetes were 0.52mg/l (0.81mg/l) and 1.59mg/l
Filtration Rate on Endogenous Marker
(2.80mg/l), respectively, with levels lower in women than in men Acute GFR decline
and higher in non-Hispanic whites than in blacks and Mexicans;22
levels were higher in older people. Several studies found correlations
of serum 2M and TP levels with directly measured GFR that were 120 GFR 120
better or similar to those observed with creatinine and that were 90 90
similar to cystatin C.23-26 In addition, studies have shown that 2M 60 60
and TP are better predictors of adverse health outcomes than cre- Marker generation
atinine and are potentially as accurate as cystatin C in the general
population and in patients with CKD.23,27,28 However, some factors
may limit their use as a filtration marker; serum 2M concentration
Marker filtration Day Pmarker eGFR
may be increased in several malignancies and inflammatory and excretion 1.0 1.0 120
states, and corticosteroid administration may decrease serum TP 1.5 1.6 79
concentration.28,29 2.0 1.8 69
2.5 1.9 65
3.0 2.0 60
CLINICAL APPLICATION OF ESTIMATED Cumulative marker
balance
GLOMERULAR FILTRATION RATE
Chronic Kidney Disease 2.0 2.0
Estimation of GFR is necessary for the detection, evaluation, and 1.5 1.5
management of patients with CKD. Current guidelines recommend 1.0 Plasma marker 1.0
testing of patients at increased risk of CKD for albuminuria, as a concentration
marker of kidney damage, or a reduced eGFR to assess kidney func- 0 1 2 3 4
tion and staging of disease severity by eGFR level. Use of serum Day
creatinine alone as an index of GFR is unsatisfactory and can lead
Figure 3-4 Sudden decrease in glomerular filtration rate. Graphs
to delays in detection of CKD and misclassification of the severity of show effect of acute GFR decline (top) on generation, filtration/excretion,
CKD. Use of estimating equations allows direct reporting of eGFR and balance of endogenous marker (middle) and concentration of plasma
by clinical laboratories whenever serum creatinine is measured. marker (bottom). (Modified from reference 1.)

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38 SECTION II Investigation of Renal Disease
MARKERS OF TUBULAR DAMAGE
The urinary excretion of low-molecular-weight serum proteins may
increase when proximal tubular reabsorption is impaired, which
may serve as a marker of proximal tubular damage. Examples
include cystatin C and 2M, as previously described, as well as
interleukin-18 (18,000d), retinol-binding protein (21,000d) and 1-
macroglobulin (33,000d). In contrast, other markers of tubular
damage are produced in the kidney in response to injury, such as
N-acetyl--glucosaminidase (NAG) and urinary kidney injury mol-
ecule 1 (KIM-1). Increased excretion of neutrophil gelatinase
associated lipocalin (NGAL), a 25,000-d protein in kidney disease,
may reflect impaired reabsorption of filtered NGAL or increased
production by the kidney. These and other urinary markers of
tubular damage under investigation are discussed further in
Chapter 71.
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