The initial ultrafiltrate in Bowman’s space has almost the same solute composition as the plasma. It only lacks albumin and other high-molecular-weight solutes in the bloodstream that could not pass through the glomerular filtration barrier. Note that the amount of these large solutes in the ultrafiltrate are very small in number compared with the total number of solutes that freely pass the barrier (see Table 4.2). Therefore, in Bowman’s space, the osmolality of the initial filtrate is the same as that of the plasma (≈300 mOsm) and is termed isosmotic. As discussed in Chapter 3, the solute composition (concentration and type) in the filtrate changes continuously throughout its passage through the tubules of the nephron. In contrast, the osmolality remains unchanged (isosmotic at ≈300 mOsm) until the filtrate reaches the thin descending limb of the loop of Henle (Fig. 4.2). Here, the countercurrent multiplier mechanism causes the filtrate in the loops of Henle to become progressively hyperosmotic in the descending limb and then hypo-osmotic in the ascending limb. When the filtrate enters the distal tubules, it is slightly hypo-osmotic (≈100 mOsm).

The final osmolality of urine is determined in the distal tubules and collecting ducts. The surrounding medullary interstitium has a hypertonic concentration gradient that facilitates the reabsorption of water when ADH is present. In the distal tubules with ADH present, the filtrate again becomes isosmotic (≈300 mOsm), matching the hypertonicity of the cortical interstitium (see Fig. 4.2). As the filtrate passes through the collecting tubules, the osmolality of the filtrate can continue to increase (as water is absorbed) until it matches that of the surrounding hypertonic medullary interstitium, provided ADH is present. Note that the maximum urine osmolality possible—1200 to 1400 mOsm/kg—is determined by the osmolality of the medullary interstitium. The collecting tubules do not exchange solutes but can passively reabsorb water until an osmotic equilibrium is attained.

The osmolality of a random urine specimen can be as low as 50 mOsm/kg or as high as 1400 mOsm/kg. Under normal circumstances, urine osmolality ranges from one to three times (275–900 mOsm/kg) that of serum (275–300 mOsm/kg). The urine-to-serum osmolality (U/S) ratio is a good indicator of the ability of the kidneys to concentrate the urine. In normal individuals with an average fluid intake, the U/S ratio is between 1.0 and 3.0.

It is important to note that in the bloodstream, the plasma osmolality remains relatively constant. In contrast, urine osmolality can vary greatly as the kidneys excrete unwanted solutes in the volume of water that the body does not need. With a typical American diet, 100 to 1200 mOsm of solutes require elimination each day. Other diets higher in salt and protein require a larger volume of water to excrete the increased solute load. Some disorders (e.g., diabetes mellitus) can produce as much as 5000 mOsm/day of solutes for elimination. Consequently, a large volume of water is required to excrete them in the urine. Because the kidneys have no direct means of replacing excessive water loss, adequate fluid intake is mandatory. These individuals experience intense thirst, known as polydipsia. It is only with an increased water intake that these individuals are able to excrete the excessive load of solutes.

Osmolality is determined by measuring a colligative property of the sample. Colligative properties of a solution depend only on the number of solute particles present. Particle size and ionic charge have no effect; only the number of particles present as ions or as undissociated molecules in the solution affects colligative properties. The four colligative properties are (1) depression of freezing point, (2) depression of vapor pressure, (3) elevation in osmotic pressure, and (4) elevation of boiling point. These properties are interrelated, and the value of one can be used to calculate each of the others. In the clinical laboratory, freezing point depression osmometry predominates for several reasons. This method can be used to detect the presence of volatile solutes (e.g., ethanol, methanol, ethylene glycol) and results are accurate even with lipemic serum samples.2 Table 4.3 summarizes osmolality methods and their limitations.

Freezing Point Osmometry

A contemporary freezing point osmometer consists of four principal components: (1) a mechanism to supercool the sample slowly to about −7°C, (2) a thermistor to monitor the temperature of the sample, (3) a means to initiate freezing (or “seeding”) of the sample, and (4) a direct readout display.

The specimen, urine or serum, in a sample chamber is placed into the osmometer. The instrument begins the cooling sequence depicted in Fig. 4.3. The initial supercooling process (segment AB, Fig. 4.3) proceeds slowly to prevent premature freezing of the sample. As the sample temperature approaches −7°C, freezing of the sample is induced (seeded) by the instrument (point B). As ice crystals form, the heat of fusion released to the sample (segment BC) is detected by a thermistor. The sample temperature increases until an equilibrium between the solid (ice) and liquid phases is reached, which by definition is the freezing point (segment CD). This temperature plateau is maintained for approximately 1 minute or longer before it again decreases (segment DE). Using the freezing point obtained, the instrument calculates and then displays the osmolality of the sample using the proportionality formula given in Equation 4.1.

1000 mOsm  particles−1.86°C=χ mOsm  particlesmeasured  freezing  point  of sample Equation 4.1

Equation 4.1

Measurement of freezing point depression is based on the fact that pure water freezes at 0°C, and that adding 1 mole (1000 mOsm) of solute particles to 1 kg pure water causes the freezing point to decrease by 1.86°C. This relationship is constant and enables the use of a simple proportionality formula. For example, assume that the thermistor probe measures the freezing point of a urine specimen as −1.20°C. By inserting this value into the equation and solving for x, the osmolality of the urine sample is found to be 645.2 mOsm/kg. To achieve the precision of ±2 mOsm/kg, as is seen with freezing point osmometers, accurate temperature measurements are crucial. The thermistor obtains these temperature measurements accurately and rapidly.

For osmolality results to be read directly from the instrument readout, the microprocessor of the osmometer must be calibrated using sodium chloride standard solutions of known osmolality. These sodium chloride solutions are available commercially in concentrations ranging from 50 to 2000 mOsm or are prepared by the laboratory. After calibration, the osmometer measures the freezing point, converts it to the corresponding osmolality value, and displays it on the direct readout.

The sample size necessary for osmolality determinations varies from 20 μL to 2.0 mL, depending on the osmometer used. A problem occasionally encountered with freezing point osmometry is premature freezing, which can be caused by particulate matter in the sample that prevents proper supercooling. This is usually overcome by simply repeating the determination.

Because density depends on two variables—the number of solutes present and their relative mass—the relationship of SG to osmolality is close but not linear (see Fig. 4.4). This relationship is relatively constant in health. However, with some conditions this relationship is nonexistent, as high-molecular-weight solutes such as glucose, urea, or proteins are being excreted in the urine. Table 4.4 shows SG values of water with NaCl, urea, and glucose added. Each solute addition represents essentially the same number of particles added to the solution—approximately 5.2 × 10²³ particles. In each solution, despite the presence of the same number of particles, the values for “true” density and SG by refractive index differ, demonstrating the effect of solute mass. In other words, the presence of large-molecular-weight solutes (e.g., glucose, protein, radiographic media) will dramatically increase urine density more so than an equivalent number of small-molecular-weight solutes such as sodium or chloride ions. Note that this effect is not observed with the reagent strip SG method, which detects only ionized solutes.

In summary, osmolality and SG are physical properties used to assess urine concentration. Osmolality measurements detect only the number of solutes present, regardless of solute type or mass. In contrast, SG determined by refractometry reflects the collective number and mass of solutes in the urine, whereas the reagent strip SG method detects only ionic solutes. See Chapter 5 for a detailed discussion of the SG methods most commonly used when performing a routine urinalysis.

Although SG determinations are easier and require less time to perform, osmolality determinations are preferred when evaluating renal concentrating ability. Osmolality measurements are considered a more accurate assessment because, as previously discussed, each solute particle contributes equally to the osmolality value. In contrast, SG measurements are a density comparison that is affected more by some solutes than others. Recall from Table 4.2 that the three most prevalent urine solutes are urea, chloride, and sodium. Chloride and sodium are reabsorbed selectively throughout the nephrons by active and passive tubular transport mechanisms. Therefore, monitoring the concentration of chloride and sodium in urine reveals the kidney’s ability to process and concentrate the ultrafiltrate. In contrast, urea is not an accurate indicator of the kidney’s ability to concentrate urine because it is only passively processed in the nephrons (i.e., urea cycle), and the magnitude of this exchange varies owing to several factors (e.g., tubular flow rate).

Another reason that osmolality determinations are better than SG determinations for assessing the concentrating ability of the kidneys is that small quantities of high-molecular-weight solutes (e.g., glucose, protein) will affect SG measurements but do not affect osmolality measurements. Glucose and protein are solutes that are actively and essentially completely reabsorbed in the proximal tubules. It is important to note that their presence in urine indicates a disease process, not a change in kidney function (i.e., concentrating ability). In contrast to SG measurements, the osmolality value of urine is not affected by the presence of glucose and protein. In such urine, the density (SG) is significantly increased because of the high mass of glucose and protein molecules, but the actual increase in particle numbers is small compared with the total solutes present (Fig. 4.4). Another advantage of osmolality measurements is that results increase or decrease in direct proportion to the solute number, regardless of the solute type.

With some chronic renal diseases, the renal tubules are no longer able to selectively reabsorb and secrete solutes and water from the ultrafiltrate as it passes through the nephron, and the solute concentration remains unchanged. Consequently, regardless of the patient’s hydration status, the urine osmolality and SG are the same as that of the initial ultrafiltrate in Bowman’s space—namely, the urine has a fixed SG of 1.010 and an osmolality of approximately 300 mOsm/kg (i.e., the same as protein-free plasma). Isosthenuria is the term used to describe an unchanging SG (or osmolality) regardless of hydration. It implies significant renal tubular dysfunction, is a feature of end-stage renal disease, and is also associated with polyuria and nocturia—excessive urination at night.

In summary, SG and osmolality are nonspecific tests used to determine the concentration of urine. They can only suggest or support a suspected decrease in renal function. The underlying problem—whether it is renal disease, diabetes insipidus, overhydration, or the effect of diuretic therapy—cannot be discerned using these tests.

Polyuria due to water diuresis can result from excessive water intake or from disorders involving ADH (vasopressin) see Table 4.1. When ADH production or secretion is defective, this indicates neurogenic diabetes insipidus. When the problem is lack of renal response to ADH, it is called nephrogenic diabetes insipidus. To differentiate the cause of water diuresis, a fluid deprivation test can be performed.

A fluid deprivation test evaluates the ability of renal tubular cells to selectively absorb and secrete solutes. In other words, it assesses the renal concentrating ability of the kidneys. During this test, water consumption by the patient is restricted and the concentration of the urine is evaluated at timed intervals. In a typical protocol, the patient eats a normal evening meal, and then from 6 PM until 8 AM the next day he or she drinks no water or other fluids. At 8 AM, a urine specimen is collected and the osmolality determined. If the urine osmolality is greater than 800 mOsm/kg, the renal concentrating ability of the kidneys is considered normal and the test is ended.

If the osmolality is less than 800 mOsm/kg, fluid deprivation continues. At 10 AM, both serum and urine specimens are collected for osmolality determinations. If the urine osmolality is greater than 800 mOsm/kg or the U/S ratio is greater than 3.0, normal renal concentrating ability is demonstrated and the test is ended. If neither condition is met, ADH (vasopressin) is administered subcutaneously, and at 2 PM and 6 PM serum and urine specimens are collected for osmolality testing. Note that regardless of a patient’s response, the test is terminated at 6 PM (i.e., after 24 hours). A positive response to ADH administration is a urine osmolality greater than 800 mOsm/kg or a U/S ratio of 3.0 or greater. These results indicate that the patient’s kidneys can respond to ADH but that inadequate ADH is produced by the patient (i.e., a neurogenic problem). In contrast, a negative response to ADH indicates a nephrogenic problem—the renal receptors for ADH are dysfunctional (see Fig. 4.1).

Other tests that assess renal concentrating ability use urine SG measurements. In the Fishberg concentration test, the patient undergoes the same fluid deprivation regimen as previously described. At each timed interval, a urine specimen is collected and the SG determined. If the urine SG becomes 1.025 or greater, renal concentrating ability is normal and the test ends.

Differing from the tests already discussed, Mosenthal’s test allows patients to maintain their normal diet and fluid intake and requires a special 24-hour urine collection. The 24-hour collection is unique in that it is collected as two separate 12-hour urine collections—a 12-hour day portion and a 12-hour night portion. The volume and SG of each 12-hour urine collection are determined. A normal Mosenthal’s test is indicated by a daytime urine volume that exceeds the nighttime volume and by a nighttime urine SG that is 1.020 or greater.