Inulin

The renal clearance of inulin remains the gold standard for measuring GFR. Inulin is an uncharged 5.2 kDa polymer of fructose that possesses many characteristics of an ideal filtration marker. It is not protein-bound and is freely filtered at the glomerulus without being reabsorbed, secreted, or metabolized by the kidney. Further, it is eliminated exclusively by the kidney.¹⁴ The filtered load of inulin (GFR × P_in) is therefore equal to its urinary excretion (U_in × V) where P_in and U_in are the plasma and urine concentrations of inulin (mg/dL), respectively, and V is the urine flow rate (mL/min). The renal clearance of inulin (C_in) can be calculated:

The classic protocol for inulin clearance is cumbersome, involving an intravenous infusion of inulin over several hours and serial timed blood and urine specimens. Properly done, inulin clearances provide the most accurate measure of GFR; however, the complexity of the protocol introduces a potential for significant error. Incomplete urine collections, for example, diminish the accuracy of the test and can be particularly problematic in children who are not yet toilet-trained or who have urologic disease (e.g., vesicoureteral reflux, bladder dyssynergia) affecting their ability to spontaneously void to completion. Placement of a bladder catheter or use of a bladder scanner may therefore improve accuracy, although the former may pose a small risk of infection and the latter may be of limited benefit in children with vesicoureteral reflux. Limited availability of inulin, technical issues pertaining to its assay, and the laborious nature of the protocol make it impractical for routine clinical use.

Iothalamate

Iothalamate (MW 614 Da) is also freely filtered by the glomerulus and has been studied extensively as an alternative exogenous marker for GFR measurements. Unlike inulin, however, iothalamate has some protein binding (<8%) and proximal tubular secretion (10%) raising concern for overestimation of GFR. Reported correlations between iothalamate and inulin renal clearances in the literature have been variable, with some studies demonstrating good correlation, whereas most suggest that iothalamate overestimates inulin clearance.^15–17

Creatinine Clearance

Clearance studies that make use of endogenous markers such as serum creatinine obviate the need for a constant infusion and make the study more amenable to clinical practice. However, the problems associated with timed urine collections persist and accordingly limit the usefulness of this technique, particularly in children. Further, creatinine is a flawed filtration marker. Although it is predominantly eliminated by glomerular filtration, a small but variable amount (~10%) is eliminated by tubular secretion. As renal function deteriorates, the proportion of secreted to filtered creatinine progressively increases, leading to an overestimation of GFR and making creatinine clearance a more unreliable measure of GFR.¹¹ Administration of oral cimetidine beginning 2 days prior to the study can at least partially circumvent this issue by inhibiting the tubular secretion of creatinine.¹⁸ Although timed urine collections are still necessary, they are typically obtained over a 2-hour period facilitating performance in a monitored clinic setting.

Guidelines from the National Kidney Foundation no longer recommend routine performance of creatinine clearance studies to estimate GFR, as prediction formulas are now felt to be more accurate.¹⁹ Nonetheless, creatinine clearance-based studies can still be helpful when assessing renal function in individuals with atypical body composition (e.g., anorexia, malnutrition) or dietary intake (e.g., vegetarian diet).²⁰

Radioisotopes

Plasma clearance studies are most commonly performed with radioisotopes that are more readily available and easier to assay than inulin. Their major disadvantage is, of course, radiation exposure, which limits their use, particularly in children.^{27 51}Cr-EDTA, ^99mTc-DTPA, and ¹²⁵I-Iothalamate have been extensively studied and compared to inulin renal clearance.^15,28 All are low-molecular-weight compounds that are freely filtered by the glomerulus but which differ slightly in protein binding and renal handling. ⁵¹Cr-EDTA (292 Da), appears to have little protein binding, and its plasma clearance correlates well with inulin clearances. It is commonly used in Europe but is unavailable in the United States.^{25 99m}Tc-DTPA (393 Da) is most frequently used in the United States but demonstrates variable accuracy in GFR measurements based on product source, differences in protein binding, and potential dissociation of the chelate (t_1/2 = 6 hours) during the study.^{29 125}I-Iothalamate (614 Da) is a high-osmolar anionic contrast agent with some protein binding that also undergoes active secretion by the proximal tubule, contributing to overestimation of plasma clearance compared to inulin or EDTA.¹⁷ Nuclear GFR studies can also be performed using a gamma camera, which measures the renal uptake of the tracer 2 to 3 minutes after injection and which can provide information regarding differential kidney function; however, the estimated GFR obtained this way is not as accurate as with the plasma-sampling technique.²⁵ To reduce radiation exposure, plasma clearance studies can also be performed using nonradiolabeled iothalamate which can be measured by high-performance liquid chromatography (HPLC).^{30, 31}

Iohexol

Iohexol is emerging as an excellent alternative to inulin and radioisotopes for clearance studies. Iohexol (Omnipaque) is a low-molecular-weight (821 Da), nonionic, low-osmolar intravenous contrast agent used routinely in the United States for radiologic studies at doses appreciably higher than that required for GFR studies.^32,33 It is almost entirely eliminated through glomerular filtration and is not reabsorbed, secreted, or metabolized by the kidney, thus fulfilling many of the desired traits of an ideal filtration marker. Further, it has minimal protein binding and negligible extrarenal elimination even in advanced chronic kidney failure.^32,34 Though it can easily be measured by x-ray fluorescence or high-performance liquid chromatography, the latter is more sensitive and, therefore, requires a significantly lower dose of iohexol for the study.^33,35 Iohexol has been safely used for clearance studies for many years in Scandinavia.^32,36,37 Its excellent safety profile has recently been confirmed in the United States in over 1000 GFR estimations performed as part of the Chronic Kidney Disease in Children (CKiD) study, a multicenter, prospective, observational, cohort study of the progression of chronic renal failure.^26,38 (G.J.S., unpublished observations). Iohexol plasma clearance results appear to be comparable to renal inulin clearance and plasma EDTA disappearance studies across a broad range of GFR.^33,39,40

Creatinine

Serum creatinine is the most commonly used laboratory study to assess renal function in clinical practice. It is simple, convenient, and practical—requiring a single blood sample—and therefore well suited for serial examination. However, the relationship between serum creatinine and GFR is quite complex, being influenced by several factors other than GFR. Therefore, at best, creatinine provides a crude estimate of the GFR. As illustrated in Figure 69-2, serum creatinine bears an inverse, nonlinear relationship with GFR and lacks sensitivity to acute and small changes in GFR. Notably, at low levels of serum creatinine corresponding to normal renal function, a substantial decrease in GFR may occur before being reflected by even a small increase in serum creatinine. In contrast, at higher levels of serum creatinine associated with renal failure, the same absolute rise in creatinine reflects a much smaller loss of remaining renal function. To a first approximation, every doubling of the serum creatinine represents a 50% decline in remaining GFR.

Figure 69–2 Idealized relation between the steady-state levels of serum creatinine and GFR in adults. When renal function is normal, a marked decrease in GFR can be associated with only a mild increase in serum creatinine.

Ideally, an endogenous marker such as creatinine can serve as a useful surrogate for GFR if it is produced at a constant rate and eliminated only via the kidney at a rate equivalent to its production rate such that a steady state exists. Figure 69-3 illustrates the relationship between the plasma level of an endogenous filtration marker, its generation (from cells and diet), and its elimination (renal and extrarenal). Ideally, elimination of the marker must occur exclusively by glomerular filtration. The serum level of the marker would then be expected to rise with renal impairment. Serum creatinine, however, does not strictly fulfill these criteria. Although creatinine production is relatively constant, it varies amongst and within individuals.⁴¹ It is derived from the nonenzymatic dehydration of muscle creatine and hence is highly dependent on muscle mass. Consequently, in children, creatinine generation fluctuates with growth in addition to diet and illness, as seen with adults.⁴¹ The reference range for serum creatinine levels representing normal GFR will thus vary with age, size, and gender after puberty. The relationship between GFR and serum creatinine is therefore particularly complex in children. Maturational changes in serum creatinine and GFR do not parallel one another. GFR is physiologically low at birth, whereas serum creatinine is elevated; however, because of fetal-maternal placental equilibration of creatinine, the elevated creatinine is not indicative of the infant’s renal function but rather the mother’s. Following birth, GFR steadily increases, reaching adult levels over the next 2 years. Serum creatinine, on the other hand, declines over the first few weeks, becoming reflective of the infant’s renal function. The creatinine level then remains stable until approximately 2 years of age as muscle mass accrues proportionally to the increase in GFR. Beyond 2 years of age, when GFR per BSA has fully matured, the ongoing accretion of muscle results in a progressive rise in serum creatinine until adolescence, when adult levels are achieved (0.7 mg/dL for adolescent females and 0.9 mg/dL for adolescent males). Superimposition of a severe or chronic illness associated with malnutrition and muscle wasting makes the interpretation of GFR from serum creatinine alone even more difficult in the pediatric patient. For example, at first glance, maintenance of a stable creatinine in a patient with a prolonged ICU course may be reassuring for preservation of renal function; however, if significant muscle atrophy has occurred, this actually suggests deterioration of renal function.

Figure 69–3 Determinants of the serum level of endogenous filtration markers. The plasma level (P) of an endogenous filtration marker is determined by its generation (G) from cells and diet, extrarenal elimination (E) by gut and liver, and urinary excretion (U + V) by the kidney. Urinary excretion is the sum of filtered load (GFR × P), tubular secretion (TS), and reabsorption (TR). In the steady state, urinary excretion equals generation and extrarenal elimination. By substitution and rearrangement, GFR can be expressed as the ratio of the non-GFR determinants (G, TS, TR, and E) to the plasma level.

(Modified from Stevens LA and Levey AS: Measured GFR as a confirmatory test for estimated GFR, J Am Soc Nephrol 20:2305-2313, 2009.)

The second requirement, that excretion of the marker occurs only by glomerular filtration, is also flawed in the case of creatinine. Although the majority of serum creatinine is eliminated by glomerular filtration, a small but unpredictable amount is eliminated by tubular secretion and gastrointestinal degradation. Proximal tubular secretion of creatinine typically accounts for approximately 10% of its elimination, although considerable inter- and intra-individual variability exists.⁴² At normal levels of GFR, the impact of tubular secretion on GFR is minimal. However, with deteriorating renal function, the proportion of secreted versus filtered creatinine increases, resulting in a lower serum creatinine than predicted for the true level of GFR, thus decreasing the sensitivity for serum creatinine to detect mild decreases in renal function.⁴² A similar phenomenon occurs in the setting of moderate-to-severe renal failure, when the bacterial degradation of creatinine within the gastrointestinal tract can become clinically significant, leading to a decrease in serum creatinine concentration.^43,44 Failure to recognize the influence of tubular secretion and gastrointestinal elimination on serum creatinine values can result in overestimation of renal function and may lead to higher, inappropriate dosing of medications. Conversely, in patients with advanced kidney failure, administration of medications (e.g., cimetidine, trimethoprim) that inhibit the tubular secretion of creatinine or administration of antibiotics that mitigate the gastrointestinal degradation of creatinine may result in an elevation of serum creatinine and subsequent underestimation of GFR without any true change in renal function. If not appreciated, this may be misconstrued as worsening renal function and lead to potential underdosing of medications.

Third, the requirement for a steady state cannot be overemphasized. When GFR abruptly declines with AKI, the production rate of creatinine exceeds its clearance rate leading to a gradual rise in serum creatinine that lags behind the true GFR. (Figure 69-4) During this period, serum creatinine does not accurately reflect the true GFR, which is unknown. Nonetheless, serum creatinine is still typically used to crudely estimate renal function as better alternatives do not currently exist. However, it must be recognized that use of the serum creatinine or creatinine-based estimating equations (see below) to estimate GFR prior to the establishment of a new steady state will result in overestimation of the renal function. Similarly, during the recovery phase of AKI, serum creatinine cannot be used to estimate GFR as it will again lag behind the true GFR until the kidney clears the accumulated creatinine and reaches a new steady state. Use of serum creatinine to estimate GFR during the recovery phase of AKI will therefore result in an underestimation of true GFR.

Figure 69–4 Following an acute decline in GFR, filtration and excretion of an endogenous marker (e.g., creatinine) decrease while production of the marker remains constant, leading to a gradual rise in serum marker concentration over several days. During this period, use of the serum level of the marker to assess GFR will result in an overestimate, as illustrated above (where eGFR is expressed in mL/min/1.73 m²). The increase in serum marker level results in a higher filtered load until a new steady state is achieved such that the production and filtration rates of the marker are again equivalent, assuming tubular secretion and extrarenal elimination are negligible. Once a new steady state is achieved, eGFR approaches GFR.

(Modified from Stevens LA, Levey AS: Measured GFR as a confirmatory test for estimated GFR, J Am Soc Nephrol 20:2305-2313, 2009; and Kassirer JP: Current concepts: clinical evaluation of kidney function: glomerular function, N Engl J Med 285[7]:385-389, 1971.)