Box Extension 29.2

Methods of Study of Kidney Function: Micropuncture and Clearance

Some of the methods used to study kidney function, although technically difficult, are intuitively easy to understand. A technique of this sort that has revolutionized renal physiology is micropuncture. Fine micropipettes are inserted into individual nephrons at identified points, permitting samples of tubular fluid to be withdrawn for analysis of composition. Such samples from amphibian nephrons reveal, for example, that the glucose concentration falls virtually to zero by the end of the proximal convoluted tubule. This is how we know that the proximal tubule is the site of glucose reabsorption.

A method that is not so intuitively simple to understand—but important in both physiological research and medical practice—is the study of renal clearance. Clearance studies are used to measure the glomerular filtration rate and can be used to quantify the reabsorption or secretion of solutes in the renal tubules. Box Extension 29.2 explains the principles and uses of renal clearance studies.

The most fundamental clearance concept is plasma clearance, defined to be the volume of blood plasma that would need to be completely depleted—or “cleared”—of a solute to yield the quantity of the solute excreted in the urine over a specified period of time. The plasma clearance can be different for different solutes and thus is solute-specific.

Before we examine how plasma clearance can be used to study kidney function, let’s establish how it is calculated. Suppose a toad excretes 100 mL of urine per day, and the urine contains 2 μmol Na⁺ per mL. The animal’s rate of excretion of Na⁺ would then be 2 × 100 = 200 μmol/day. Suppose we measure the animal’s plasma concentration of Na⁺ and find that it is 100 μmol/mL. We would then know that to obtain the quantity of Na⁺ excreted in a day, 2 mL of plasma would have to be completely cleared of Na⁺. The plasma clearance of Na⁺ would therefore be 2 mL/day. In algebraic form, if V is the volume of urine excreted per unit of time, U is the urinary concentration of the substance in question, and P is the plasma concentration, then the plasma clearance of the substance per unit of time, C, is computed as

C = UV/P

The way that plasma clearance is calculated should not be taken to imply that the kidneys obtain the quantity of a solute they excrete by completely clearing part of the plasma while leaving the rest untouched. Actually, of course, the quantity excreted is obtained by incompletely clearing the plasma at large. Although the plasma clearance is in this sense an artificial concept, it is also a powerful concept in the analysis of renal function.

One way in which the plasma clearance is used is to measure the glomerular filtration rate. To use the plasma clearance for this purpose, an artificial solute is introduced into the blood plasma (e.g., by injection). A solute useful for this purpose must meet three criteria: First, it must enter the nephrons by glomerular filtration and not be introduced into the urine by any other mechanism. Second, during ultrafiltration it must be freely filtered, so that its concentration in the filtrate is the same as its concentration in the blood plasma. Third, it must not be reabsorbed in the nephrons or elsewhere, so that whatever amount enters the nephrons by filtration is fully excreted. The most commonly used substance meeting these criteria is inulin, a fructose polysaccharide derived from Jerusalem artichokes. It is known to meet the required criteria in mammals, amphibians, and some (but not all) other vertebrates. In these animals, the glomerular filtration rate equals the plasma clearance of inulin. We can use algebra to demonstrate this equality. Suppose inulin has been introduced into the blood plasma of an animal at concentration P. If F is the animal’s glomerular filtration rate (volume of filtrate produced per unit of time), then the amount of inulin filtered per unit of time is FP, because the concentration of inulin in the filtrate equals the inulin concentration in the plasma. The amount of inulin excreted in urine per unit of time is UV (the concentration of inulin in the urine times the volume of urine excreted per unit of time). For a substance such as inulin that, following filtration, is neither added to the urine nor reabsorbed, the amount filtered and the amount excreted must be the same. Thus FP = UV. By rearrangement,

F = UV/P

Thus, as stated previously, the glomerular filtration rate equals the plasma clearance.

Most native plasma solutes that enter the nephrons by filtration (e.g., Na⁺ and glucose) undergo postfiltration exchange with the blood; a solute, for example, might be secreted into the urine across the walls of the nephrons following primary-urine formation by filtration, or it might be reabsorbed from the urine between filtration and excretion. Whether a solute is added or removed in net fashion following filtration can be determined by comparing its clearance with the GFR. For example, if the clearance of a solute exceeds the GFR, one knows that the volume of plasma being cleared of that solute per unit of time exceeds the volume of plasma filtered per unit of time. A difference of this sort indicates that the solute is added to the urine following filtration. To illustrate, pioneering studies of bullfrogs showed that their urea clearance can be several times higher than their GFR; this finding provided evidence that in bullfrogs, urea is transferred from the plasma into the urine by secretion across the walls of the nephrons in addition to being introduced by filtration at the Bowman’s capsules. Conversely, if the clearance of a freely filtered solute is less than the GFR, the difference indicates that the solute is reabsorbed after filtration. Consider, for example, results for toads (Rhinella marina [formerly called Bufo marinus]) living in distilled water: Their GFR (inulin clearance) was about 6 mL/h, but their Na⁺ clearance was far lower, about 0.04 mL/h. Because we know that Na⁺ is freely filtered, these data tell us that, every hour, the amount of Na⁺ contained in 6 mL of plasma entered the nephrons by filtration, yet only the amount contained in 0.04 mL of plasma was excreted. Most of the filtered Na⁺ must have been reabsorbed.

A useful quantitative measure is the relative clearance of a substance, defined to be the ratio of that substance’s plasma clearance to the GFR (e.g., inulin clearance). For a freely filtered substance, this ratio is the fraction of the amount filtered that is excreted. To illustrate, the relative Na⁺ clearance for the toads just discussed was the Na⁺ clearance (0.04 mL/h) divided by the GFR (6 mL/h), or 0.007 (0.7%). This value tells us that the kidneys of the toads were excreting only 0.7% of the amount of Na⁺ they filtered. When the toads were transferred to saline water, their relative Na⁺ clearance rose to 53%, indicating that under those conditions, their kidneys reduced their reabsorption of Na⁺ and excreted 53% of the amount they filtered.

Although substances such as inulin were introduced to the study of kidney physiology because their plasma clearance permits measurement of the GFR, such substances also have important applications in micropuncture studies. To illustrate, suppose that a frog is injected with inulin prior to a micropuncture study of its proximal tubular fluids. What will be found is that the inulin concentration of the tubular fluid increases as the fluid flows through the proximal tubule. This increase provides direct evidence that water is reabsorbed in the proximal tubule; because inulin enters nephrons only by glomerular filtration, the increase of its concentration from the beginning to the end of the proximal tubule must result from water removal, not inulin addition. In bullfrogs, micropuncture studies show that as tubular fluid flows through the proximal tubule, its concentration of urea increases even more than its concentration of inulin. This result is evidence that the proximal tubule is a site of urea secretion into the urine.