Mechanisms of urine formation

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Fig. 16.9. Distal tubule ion transport. In the distal convoluted tubule and collecting duct of the nephron, the cells secrete K⁺ into the filtrate. Na⁺∕K⁺ ATPase pumps in the basolateral membrane actively transport K⁺ into the cell where it then passes down its concentration gradient and exits from the apical end of the cell into the lumen. This allows the recovery of Na⁺essentially in exchange for elimination of K⁺.

then the activity of these pumps can control the uptake of glucose, K⁺, or Cl.

Figure 16.10 illustrates the histological appearance of renal corpuscle as well as several cross-sectioned profiles of parts of a nephron. At first it may seem confusing. The larger rounded structures are the renal corpuscles, and the various tubules are sections through proximal or distal convoluted tubules, perhaps an ascending or descending loop of Henle or collecting ducts. Other tubelike structures include the network of peritubular capillaries. Remember the name of the beginning and ending section of the nephron: convo­luted tubule. Because of its highly coiled, curving structure, a given histological section from the kidney might well exhibit many profiles of the same tubular structure as it is cut at various places. However, there are distinct structural features that allow the identifi­cation of proximal versus convoluted tubules, or dif­ferences between loops of Henle and collecting ducts, for that matter. One simple feature that allows a quick orientation is the presence of renal corpuscles. If these structures are present, then the tissue section was taken from either within the cortex or the tissue included the boundary between the cortex and medulla. The

Fig. 16.10. Nephron histology. Panel A gives a low-power survey image of the tissue from the cortex of the kidney. The larger rounded structures (arrows) are the renal corpuscles. The other abundant circular profiles are cross-sectioned areas of various segments of nephrons. Most of the profiles would be either proximal or distal convoluted tubules. Panel B shows some of the detail of a renal corpuscle (outlined by the brackets). Ultrafiltrate flows from this structure into the proximal convoluted tubule (PCT). In this case, by chance, a portion of the tubule that drains this renal corpuscle has been sectioned. The large arrow indicates the direction of flow.

appearance of long parallel arrays of simple tubes is a major indication that the tissue section was prepared from a sample collected deeper into the renal medulla, perhaps near the apex of a renal pyramid. These tubules likely represent the walls of the ascending or descending loops of Henle of the juxtamedullary nephrons, peritubular capillaries, or numerous collect­ing ducts.

As filtrate enters the PCT, forces begin the recovery of important nutrients, ions, and water. Because of these activities, the structure of the cells of PCT and distal convoluted tubule (DCT) are distinct. As shown in Figure 16.11, epithelial cells of both PCT and DCT are cuboidal, but the cells of the PCT typically have abundant microvilli and mitochondria. In paraffin

Fig. 16.11. Proximal and distal tubules. This image is taken from the renal cortex and shows profiles of proximal (PCT) and distal convoluted tubules (DCT). In both cases the cells are cuboidal, but cells of the PCT are larger and have evidence of stained material along the apical cell surface, evidence of abundant microvilli (arrows).

Table 16.3. Summary of functions of nephron regions.

Fig. 16.12. Flow of selected materials. This diagram illustrates the dramatic rate of removal of amino acids and glucose from the ultrafiltrate formed in the glomerulus. By the time fluid enters the loop of Henle, essentially all of the amino acids and glucose have been recovered in normal circumstances. When animals are given inulin or Itippuric acid, the rate of appearance of these substances can be used to monitor kidney health. For example, for inulin that enters the filtrate remains, it is neither reabsorbed nor is any transported from the peritubular blood (no secretion). This gives a measure of the glomerular filtration rate or GFR. Hippuric acid, on the other hand, is not recovered, but in addition, all of it in the blood that enters the glomerulus is transported into the filtrate (100% secretion). Thus, evaluation of Itippuric acid concentrations provides a measure of plasma flow rate to the kidney.

Table 16.4. Rate of fluid flow in regions of the nephron.

Under normal circumstances, nearly all of the glucose and amino acids are recovered from the filtrate before it reaches the loop of Henle. The significance of mea­suring of inulin and hippuric acid to understanding kidney function is also indicated. Table 16.3 summa­rizes major functions associated with each of the seg­ments of the nephron.

In addition to the recovery of dissolved substances, the rate of fluid flow also is dramatically reduced as each of the progressive segments of the nephron is traversed. As indicated earlier, the capacity to regulate the volume of urine produced is critical. As shown in Table 16.4, despite an average rate of filtrate formation of ~125mL∕min in a 70-kg primate, urine production is typically only about 1 mL per minute. Just how this regulation takes place will be discussed in subsequent sections.

sections of fixed kidney tissue the Iumenal spaces of the PCT sometimes appear as if the apical ends of the cells are painted. This color and thickness is an indica­tion of stain accumulation on the proteins that coat the microvilli.

The remarkable capacity of the nephron to recover important nutrients is illustrated in Figure 16.12.

Countercurrent mechanisms and medullary osmotic gradient

The mammalian kidney has a remarkable ability to regulate the concentration of urine to maintain the water content of the body so that the osmolarity of body fluids remains within very narrow boundaries.

Box 16.1 Alcohol and urination

While not an issue for our animals, it is often observed that drinking beverages containing alcohol seems to induce more frequent urination. Certainly, consumption of additional liquids has an impact on kidney function, but it also turns out that ethanol inhibits the secretion of antidiuretic hormone or ADH. Can you use this knowledge to figure a mech­anism to explain why this might occur?

For most animals there is a very real danger of desic­cation so the kidney is designed to reabsorb nearly all of the water that is filtered. However, if necessary, the kidney is also capable of responding to produce very hypotonic urine to eliminate water. For example, con­sider that a 10-kg dog likely produces more than 50 L of glomerular filtrate per day but only 0.2-0.25 L of urine. If the dog is water deprived it can produce a very low volume of urine whose osmolarity is 10 times as great as blood plasma or, with water loading, urine with an osmolarity of only 100mOsm∕L. How does the kidney manage these remarkable shifts (Box 16.1)?

The answer to this question is reflected in the complex but elegant structure of the nephron, variable function of cells within different nephron segments, and a unique arrangement of blood vessels within the tissue surrounding the nephrons. Three key elements are involved in determining if concentrated or dilute urine will be produced. First, there is the presence of an osmotic gradient that is generated and maintained in the tissue surrounding the juxtaglomerular neph­rons whose loops of Henle dip from the cortex of the kidney, deep into the medulla. Second, the fluid of the tubule becomes progressively dilute as it passes through the loop of Henle into the distal convoluted tubule. Third, the permeability of the collecting duct cells to water can be directly regulated by the action of antidiuretic hormone (ADH). As the dilute filtrate fluid passes from the distal convoluted into the col­lecting ducts, it again traverses from the region of the cortex down through the renal medullary tissue to the apex of the renal pyramids for release. As illustrated in Figure 16.13, the osmolarity of the tissue fluid becomes progressively greater, ~1200mOsm∕L, near the lower regions of the medulla, so that given the opportunity to respond to the osmotic pressure, water will pass across the cells of the collecting ducts into the interstitial fluid and be recovered in surrounding capillaries. However, this does not occur automati­cally. In the absence of sufficient secretion of ADH, the water is retained within the tubular fluid and a more copious and dilute urine is produced. Water recovery or excretion within this region of the nephron is some­times referred to as facultative reabsorption. That is,

Fig. 16.13. Kidney osmotic gradient. The difference between the osmolarity of interstitial fluid in the cortex compared with the medulla is dramatic. The development and maintenance of this gradient is important as it allows the production of either dilute or concentrated urine.

water recovery does not occur unless ADH is secreted to facilitate this process. This is contrasted with reab­sorption of water that happens within the PCT. In these areas of the nephron the cells are always perme­able to water. This means, for example, that when sodium is reabsorbed from the tubular fluid the osmotic difference that is created allows water to follow. Thus, water reabsorption in these regions is referred to as obligatory transport. Since the tubule is permeable to water the removal of sodium or other solutes create an osmotic drive that water is "obligated" to follow. Figure 16.14 illustrates factors that create and help maintain the interstitial osmotic gradient.

The vasa recta, the network of capillaries that follow the course of the loop of Henle, are also essential in maintenance of the osmotic gradient. This is because this arrangement acts as a countercurrent exchange mechanism to minimize alterations in the concentra­tions of solutes within the interstitial fluid. Essentially, this allows the recycling of salt. Without this arrange­ment, if ordinary vessels paralleled the loop of Henle the medullary gradient would quickly be dissipated. Specifically, as large amounts of Na⁺ were absorbed, water would follow and the gradient would essen­tially be flushed away. In contrast, the vasa recta func­tion to minimize disruption. First, these capillaries get only about 10% of the blood flow. This acts to make blood flow relatively slow in comparison with other capillaries. In addition, throughout the course of the vessels, the cells are freely permeable to both water and sodium. Consequently, the blood makes passive exchanges with the tubular fluid to maintain equilib­rium with the surrounding interstitial fluid. For example, as the blood flows toward the apex of the

Fig. 16.14. Role of loop of Henle. The thin limbs of the loops of Henle and the DCT generate and maintain the osmotic gradient between the cortex and medulla. The osmolarity of the tubule fluid leaving the PCT is about 300mOsm∕L. It becomes progressively more concentrated so that osmolarity increases as water leaves in response the tonicity of the surrounding fluids. This is because the descending limb is permeable to water but not sodium. After the hairpin turn, the fluid becomes progressively more dilute because the cells are impermeable to water but not sodium. The differences in permeability of the descending and ascending limbs and the countercurrent flow act to create and maintain the interstitial tissue osmotic gradient. In the upper regions of the ascending loop (indicated by the thick lines) the cells are impermeable to water, but active transport of sodium (chlorine follows via electrochemical attraction) indicated by the black circles and arrows occurs. This creates dilute tubular fluid (~100mOsm∕L) as the fluid enters the collecting ducts. As indicated, the permeability of the collecting ducts to water is controlled by secretion of ADH. Control of permeability allows either diluted (low ADH secretion) or concentrated (high ADH secretion) urine to be created as needed to maintain homeostasis.

renal pyramid, it loses water and gains salt because of the increasing osmolarity. In other words, the blood becomes hypertonic. However, after the vessels bend in the region of the hairpin loop, the process is reversed. As the blood leaves the medulla and enters the cortex the osmolarity is essentially the same as when it entered the descending side of the system. These countercur­rent exchanges protect the osmotic gradient that is created by the selective actions of the descending and ascending limbs of the loop of Henle. Figure 16.15 illustrates the idea of a countercurrent system gener­ally and of the vasa recta specifically (Box 16.2).

While the structure-function relationships among parts of the nephron explain much of the action of the kidney with respect to the capacity of the kidney to control production of either dilute or concentrated

Fig. 16.15. Countercurrent exchange in the vasa recta. The vessels of the vasa recta follow the curved course of the loop of Henle. Since the cells are freely permeable to both sodium and water, osmolarity of the blood (values within the vessel) can reach equilibrium with the surrounding interstitial fluid during transit. However, the blood has essentially the same osmolarity when it enters and leaves the system. These transient shifts allow the osmotic gradient in the surrounding interstitial fluid to be maintained.

Box 16.2 Kangaroo rats

What are some adaptations that animals in very, very harsh climates have completed to avoid dehy­dration or other osmotic disasters? Desert-dwelling kangaroo rats rarely drink. They get about 90% of daily water needs from tissue metabolism and 10% from food (seeds and vegetation). There are both behavioral and physiological adaptations. They stay in cool burrows in the day. They have no sweat glands, but most importantly, they have very long loops of Henle. So they are able to concentrate urine to an extreme degree. For example, they can concentrate urea to 3500 mmol ∕L, compared with 400mmol/L in humans. Overall urine produced is 20 times more concentrated than body fluids. Feces are also extremely dry.

urine, what are the factors that determine which type of urine should be produced? To understand this regu­lation we need to appreciate the fact that the kidney, specifically specialized cells within the glomerulus, afferent arteriole, and distal convoluted tubule, are important in the long-term chronic control of blood pressure. A second key element is the fact that main­tenance of blood and interstitial fluid osmolarity, while sensed by cells of the hypothalamus, requires changes in kidney function to effect homeostatic control of body fluid osmolarity.

We begin by considering the role of sodium chlo­ride. More than 90% of the osmotic activity of extracel­lular fluid is directly related to concentrations of NaCl, especially Na. In most situations, reabsorption or movement of salt across an epithelial or cell mem­brane also results in the movement of water because of osmosis. Thus, the amount of salt in the body is a very important determinant of extracellular fluid volume and consequently blood volume and blood pressure. In short, all things being equal, reabsorption of more sodium from the glomerular filtrate of the kidney leads to greater reabsorption of water. Thus, there is a direct relationship between kidney function and control of blood pressure via control of extracel­lular fluid volume. Certainly, something as important as control of blood pressure (volume) and osmolarity cannot be left to chance. The first component involved is the renin-angiotensin system.

Figure 16.6 shows cells that make up the juxtaglo­merular apparatus (JGA). The JGA is a combination of

Fig. 16.16. Renin-angiotensin system. The secretory cells of the juxtaglomerular apparatus (JGA) indicated in the boxed area release renin in response to low blood pressure in the afferent arteriole and/or low sodium in distal CT. In a cascade of reactions, renin first promotes the cleavage of the serum protein angiotensinogen (synthesized in the liver). This action produces angiotensin I, which is modified by angiotensinogen-converting enzyme (ACE) by removal of two additional amino acids to form the eight amino acid peptide, angiotensin II, as the blood passes through the lungs. Angiotensin Il has two major actions. First, it stimulates a general vasoconstriction, which increases blood pressure rather quickly, and second, it promotes secretion of aldosterone from the adrenal cortex. As described previously, aldosterone promotes increased reabsorption of sodium, which leads to increased water recovery, increased extracellular fluid volume, and, ultimately, increased blood volume and therefore increased blood pressure. Finally, increased angiotensin Il also increases synthesis of antidiuretic hormone in the hypothalamus. ADH increases water permeability of the collecting ducts to also increase blood volume and pressure. the cells of the wall of the afferent arteriole and a segment of the distal convoluted tubule. Secretory cells within the JGA respond to decreases in blood pressure and/or very low concentrations of Na within the fluid of the distal CT by secreting the enzyme renin. Renin acts to restore blood pressure in two ways. Most rapidly, as outlined in Figure 16.16, increased blood renin causes an increase in the blood concentra­tion of angiotensin II. This agent has two actions to help restore blood pressure. First, it causes a general­ized vasoconstriction of capillary sphincters through­out the body. This acts to reduce blood flow through many capillary beds, increases vessel resistance, and thereby increases venous return to the heart. The increased volume produces increased cardiac output and therefore an increase in blood pressure. In addi­tion, angiotensin II also promotes the secretion of the steroid hormone aldosterone from the adrenal cortex. Aldosterone acts to promote increased reabsorption of Na from the distal CT. As indicated previously, enhanced recovery of Na within this region of the nephron leads to simultaneous reabsorption of water. The reabsorbed water enters the capillaries and con­sequently the vascular system, which also increases blood pressure. Furthermore, the angiotensin II also promotes the secretion of ADH. This is called the renin-angiotensin system.

The mechanism aldosterone uses to increase sodium reabsorption in the distal CT is not fully understood. Like other steroid hormones, aldosterone diffuses across the cells of its target cells and binds to receptors in the cytoplasm. These activated receptor-hormone complexes migrate to the nucleus to ultimately pro­duce transcription of specific genes. These newly minted proteins are responsible for the effects of aldosterone. However, three detailed mechanisms have been proposed to explain the increased sodium reabsorption.

First, the sodium pump hypothesis suggests that the activity of the Na⁺/ K⁺ pumps in the basolateral membrane is simply stimulated. A second, metabolic hypothesis suggests that aldosterone increases pro­duction of ATP, perhaps due to enhanced fatty acid oxidation, and that making more ATP available simply powers the Na⁺/ K⁺ membrane pumps more effec­tively. A third hypothesis is that aldosterone increases the synthesis of sodium channel proteins that are deposited in the apical membranes of the cells. Fur­thermore, animals are stimulated by thirst to increase water intake as illustrated by Figure 16.17.

There are also elements to "adjust" the activity of the renin-angiotensin system to prevent extremes. For example, atrial natriuretic peptide (ANP) is released by cells in the atrium of the heart in response to increased venous blood pressure. ANP acts to increase urine production and sodium excretion by inhibiting

Fig. 16.17. Thirst reactions. In addition to the kidney control of urine production, drinking must also be regulated. Multiple elements (dry mouth, reduced blood osmolarity, and increased blood angiotensin II) act via osmoreceptors in the hypothalamus to elicit the sensation of thirst that leads to drinking. Ingestion of water moistens mucus membranes and absorption increases osmolarity of fluid bathing osmolarity-sensitive cells in the hypothalamus, reducing the sensation of thirst and along with kidney action restoring homeostasis.

the release of ADH and renin and thus secretion of aldosterone by the adrenal gland.

Renal clearance

Data illustrated in Figure 16.12 show absorption and secretion patterns for some selected substances as these materials pass through regions of the nephrons. Measuring changes in the concentrations of two of these substances, inulin and hippuric acid (specifi­cally aminohippuric acid or para-aminohippuric acid [PAH]), are very valuable tools to evaluate kidney function. To determine effectively if plasma is cleared or "cleaned" of unwanted waste products, two vari­ables related to kidney action must be measured. First, we need a measure of the rate of blood or plasma flow to the kidneys, and second, we need to determine how much of the blood plasma is filtered. You might recall from our description of blood flow to the renal glom­erulus that as blood enters via the afferent arteriole, some fluid is lost to create the ultrafiltrate that enters

the lumen of the nephrons, and the remainder exits via the efferent arteriole.

Inulin, a sugar that is isolated from the tubers of dahlias, is filtered from the blood, but it is neither reabsorbed from the filtrate nor is any additional inulin secreted into the lumen of the nephrons. For this reason, measuring the concentrations of inulin in samples from the renal vein and artery following injection into systemic circulation gives a measure of the volume of blood plasma that is filtered by the kidney. This is called the GFR. This determination of the GFR is based on the idea of clearance, which is the rate at which the plasma is cleared or cleaned of a given substance. The clearance rate (RC) is determined by the rate of elimination divided by the plasma con­centration of the substance in question, as follows:

where RC_x is the volume of plasma cleared of sub­stance ? per unit time, U_x is the urine concentration of substance X, V is the volume of urine collected divided by the time period of the collection, and P_x is the plasma concentration of X. GFR is considered the best single parameter for assessing overall renal function because its value is directly related to the functional mass of kidney tissue. In veterinary practice, measure­ment of blood levels of urea nitrogen (BUN) and cre­atine concentration are often used as screening tools to detect renal dysfunction. Unfortunately, values typ­ically rise markedly only when 75% or more of the nephrons no longer function.

The total clearance of a particular substance is the sum of the rates of filtration and secretion with the rates of reabsorption subtracted. To determine the fil­tration rate accurately, the effects of secretion and reabsorption have to be taken into account. In the case of inulin, since it is freely filtered but is neither secreted nor reabsorbed and is not produced in animals, it is ideal for calculating GFR. Indeed, the equation for calculation of inulin clearance is in effect an equation for calculation of GFR as shown in the following equa­tion, where GFR is in milliliters per minute, C_tauι_ta is the rate of clearance of inulin from the plasma in mil­liliters per minute, U_tauι_ta is the inulin concentration in a urine sample collected over a period of time T in minutes; V is the volume of urine collected over time T; and P_tau]_ta is the average plasma inulin concentration during time T.

Again, since infused inulin (typically a standard dose to achieve an initial concentration of ~lmg∕mL of plasma or 3000 mg / m² body surface area) is elimi­nated only in the urine, its clearance rate value is equal

Fig. 16.18. Relationship between GRF and serum creatine. The vertical line indicates the lower value for GFR that is still considered normal. The horizontal line indicates a normal upper limit for serum creatine. Animals in the upper left quadrant of the graph have higher than normal serum creatine concentrations and correspondingly reduced kidney function as evidenced from the reduced GFR (adapted from Haller et al., 2003).

to the GFR. Measured values for inulin in humans are U = 125mg∕mL, V = ImL/min, and P = lmg∕mL. Thus, the calculated value for RC for inulin = (125 ? 1)/ 1 = 125mL∕min. This means that in 1 minute, the kidneys have removed or cleared the amount of inulin that would have been in 125 mL of blood plasma. Typical values for healthy cats, for example, average about 2.7mL∕min∕kg or ~12mL∕min for a typical adult cat. Figure 16.18 illustrates the relationship between BUN and GFR in a population of cats. In this case, GFR was estimated from the clearance of injected inulin. It was also found that the serum inulin value after a single time period (180 minutes) corresponded well with more extensive blood sampling. In practical terms this would minimize stress associated with mul­tiple sampling. Since some of the animals in this trial were suspected of being in early stages of renal disease, it is also apparent that several of the cats with elevated serum creatine levels have correspondingly reduced GRF values. This supports the predictions of impaired kidney function.

Although the standard method to calculate GFR is by measuring the clearance rate of inulin from the blood, GFR can be estimated by measuring other endogenous substances. In clinical veterinary situa­tions, this can be done by measuring the clearance of creatine. Creatine is a by-product of muscle metabo­lism that is handled by the kidney (at least in dogs) much like inulin; that is, it is freely filtered, not reab­sorbed, and not secreted into the tubular lumen. This, however, may not be true in all species. For example, in some animals, about 10% of the creatine in urine occurs because of secretion. In practical terms, the usual procedure is to collect the urine that is produced over 24 hours (either catheterization or collection in a metabolic cage). The volume produced is recorded, and the creatine concentration is measured. The con­centration of creatine in blood collected at the start and end of the collection period is typically measured and averaged to provide a measure of plasma concen­tration. These values are used in the clearance equa­tion to give an estimate of GFR. When many different animals are evaluated, it is better to express the GFR on a body weight or body surface basis (milliliters per minute per kilogram or milliliters per minute per square meter) to account for the large variation between species. Note the example given in Figure 16.18.

Para-aminohippuric acid is useful because it is fil­tered as it passes into the glomerulus. Moreover, the remaining portion that passes out of the efferent arte­riole is secreted into the tubular fluid. Thus, all of the material that is in the blood plasma that enters the kidney appears in the filtrate and ends up in urine. This property allows a calculation of plasma flow to the kidney. This can be very useful to evaluate kidney as well as cardiovascular health.

If less of a substance appears in the urine than was initially produced at the time of filtration, then some reabsorption of the substance must have occurred in the tubule. In a human, the GFR averages 125 mL per min (mostly water), but urine production is only about 1 mL per min. Clearly, most of the water is recovered. For important nutrients (Fig. 16.12), reabsorption is essentially complete in most circumstances. For example, consider glucose; unless blood concentrations are abnormally elevated, all of the filtered glucose is recovered so that the clearance is zero. There is, however, a maximum rate at which glucose can be recovered from the filtrate. In humans, for example, the transport maximum (T_max) is about 320mg∕min. As long as plasma glucose remains below about 1.8mg∕mL (180mg∕dL), all of the glucose appearing in the filtrate is recovered. At about 300mg∕dL of plasma, the transport mechanism becomes completely saturated so that glucose is lost in the urine. Exact transport values likely differ between species, but the idea remains the same. Specifically, elevated blood glucose occurs with diabetes so that appearance of glucose in the urine is an initial indication of diabetes in both humans and other animals.

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