In order for blood to perform its essential functions of bringing nutrients and oxygen to the cells of the body, and carrying waste materials away from those cells, the chemical composition of the blood must be carefully controlled. Blood contains particles of many different sizes and types, including cells, proteins, dissolved ions, and organic waste products. Some of these particles, such as proteins like hemoglobin, are essential for the body. Others, such as urea (a waste product from protein metabolism), must be removed from the blood or they will accumulate and interfere with normal metabolic processes. Still other particles, including many of the simple ions dissolved in the blood, are required by the body in certain concentrations that must be tightly regulated, especially when the intake of these chemicals varies. The body has many different means of controlling the chemical composition of the blood. For instance, you learned in the "Iron Use and Storage in the Body: Ferritin and Molecular Representations" tutorial that the ferritin protein can help to control the amount of free iron in the blood. As you will discover in the tutorial entitled, "Blood, Sweat, and Buffers: pH Regulation During Exercise", buffers dissolved in the blood can help regulate the blood's pH. But the largest responsibility for maintaining the chemistry of the blood falls to the kidneys, a pair of organs located just behind the lining of the abdominal cavity. It is the job of the kidneys to remove the harmful particles from the blood and to regulate the blood's ionic concentrations, while keeping the essential particles in the blood (Figure 1).
This is a schematic diagram illustrating the kidneys' ability to separate particles in the blood in order to maintain optimal body chemistry. Blood enters the kidney through the renal artery. In the kidney, the blood undergoes filtration and dialysis to separate the particles that will be removed from the body (through the ureter to the bladder) from those that will be returned to the circulating blood (through the renal vein).
The kidneys meet these challenges through a remarkably elegant system. Essentially, kidneys act like dialysis units for blood, making use of the different sizes of the particles and specially-maintained concentration gradients. Blood passes through the membrane-lined tubules of the kidney, which are analogous to the dialysis bags used in this Experiment. Particles that can pass through the membrane pass out of the tubules by diffusion, thus separating the particles that remain in the blood from those that will be removed from the blood and excreted. The dialysis mechanism used by the kidneys allows them to function effectively over a very wide range of conditions. For example, sodium intake can vary from one tenth to ten times the average consumption, with only minimal fluctuations in blood-plasma sodium concentrations. Even when the kidneys are severely damaged, the kidneys can still effectively maintain the body's chemistry as long as at least ten percent of their functional units are working. Nonetheless, damage to the kidneys can cause the functional capacity to drop below this level, and fatal illness will develop unless an artificial system is employed to perform the work of the kidneys.
The kidneys have three basic mechanisms for separating the various components of the blood: filtration, reabsorption, and secretion. These three processes occur in the nephron (Figure 2), which is the most basic functional unit of the kidney. Each kidney contains approximately one million of these functional units. The nephron contains a cluster of blood vessels known as the glomerulus, surrounded by the hollow Bowman's capsule. The glomerulus and Bowman's capsule together are known as the renal corpuscle. Bowman's capsule leads into a membrane-enclosed, U-shaped tubule that empties into a collecting duct. The collecting ducts from the various nephrons merge together, and ultimately empty into the bladder.
Glomerulus filters proteins and cells from the blood.
All other blood components pass into Bowman's capsule, then into the tubule.
Reabsorption and Secretion:
Semipermeable membranes surrounding the tubule allow selective passage of particles back into the blood (reabsorption), or from the blood into the tubule (secretion).
Collects all material that has not returned to the blood through the tubular membranes. This material exits the kidney as urine.
This is a schematic diagram of the nephron.
This table summarizes the essential functions of the major segments of the nephron.
Blood first enters the kidney through the renal artery (see Figure 1), which branches into a network of tiny blood vessels called arterioles. These arterioles then carry the blood into the tiny blood vessels of the glomerulus. It is here, in the renal corpuscle, where filtration occurs. The glomerulus filters proteins and cells, which are too large to pass through the membrane channels of this specialized component, from the blood. These large particles remain in the blood vessels of the glomerulus, which join with other blood vessels so that the proteins remain circulating in the blood throughout the body. The small particles (e.g., ions, sugars, and ammonia) pass through the membranes of the glomerulus into Bowman's capsule. These smaller components then enter the membrane-enclosed tubule in essentially the same concentrations as they have in the blood. Hence, the fluid entering the tubule is identical to the blood, except that it contains no proteins or cells.
The tubule functions as a dialysis unit, in which the fluid inside the tubule is the internal solution and the blood (in capillaries surrounding the tubule) acts as the external solution. Particles may pass through the membrane and return to the blood stream in the process known as reabsorption, which is analogous to the movement of particles from the internal to the external solution in the dialysis experiment you performed in lab. The reabsorption of many blood components is regulated physiologically, as discussed below. Alternatively, particles may pass through the membrane from the blood into this tubule in the process known as secretion, which is analogous to the movement of particles from the external solution into the dialysis bag in the experiment you performed in lab. The most important particles that are secreted from the blood back into the tubules are H+ and K+ ions, as well as organic ions from foreign chemicals or the natural by-products of the body's metabolism.
The blood components that remain in the nephron when the fluid reaches the collecting duct are excreted from the body.The collecting duct from one nephron meets up with many others to feed into the ureter. The ureters (one from each kidney) enter the bladder, which leads to the urethra, where the liquid waste is excreted from the body. Hence, the material that is filtered and secreted from the blood into the tubule, less the amount that is reabsorbed into the blood, is ultimately excreted from the body.
The localization of each of these processes within specific components of the nephron is summarized in Table 1, above.
From the overview of kidney function above, it is clear that blood components (e.g., water, ions, sugars) must be able to pass between the nephron tubules and the blood-filled capillaries surrounding them. But recall from the Introduction to this experiment (in the lab manual) that phospholipid-bilayer membranes are not permeable to polar molecules, because the interior lipid region of the membrane is nonpolar. Thus, the polar components of blood could not cross the membranes surrounding the tubules (Figure 3), unless these membranes contained special channels to allow the passage of polar species.
This is a schematic diagram of a segment of a nephron tubule with no protein channels (unlike a real tubule segment, which contains channels) in the phospholipid-bilayer membrane surrounding the tubule, shown as a lengthwise slice through the tubule segment. Polar molecules (green) cannot travel out of the tubule to the blood in the capillaries, because they are insoluble in the hydrophobic (nonpolar) lipid interior of the membrane. To permit passage of polar and charged species between the capillaries and the nephron, the membrane must have protein channels embedded in it, as discussed below. Phospholipid-bilayer membranes are discussed further in the Introduction to the experiment in the lab manual.
Note: For simplicity, the tubule is depicted here as being enclosed by a single membrane. In fact, the tubule and capillaries are lined with cells that are surrounded by membranes. Thus, a particle must travel across several membranes in order to move between the interior of the tubule and the blood-containing capillaries. This figure is not drawn to scale.
The channels required to allow the passage of polar blood components are formed by proteins embedded in the phospholipid-bilayer membrane (Figure 4). Proteins that form channels in the membrane typically have membrane-spanning cylindrical shapes: there is a hydrophobic surface that can interact with the tail region of the phospholipid-bilayer membrane and a hollow internal core that forms the pore. These proteins form a "tunnel" from the aqueous phase on one side of the membrane to the aqueous phase on the other side of the membrane. The size of the tunnel determines the size of the particles that will be able to pass through the channel.
This is a CPK representation of a potassium channel embedded in a schematic phospholipid-bilayer membrane. (The pale yellow circles represent the polar head groups and the gray lines represent the nonpolar tails.) This channel is composed of four polypeptide chains (shown in different colors) that span the width of the membrane, with a hollow space through which potassium ions may pass (like a tunnel). Some of the amino acids have been removed to reveal the space occupied by the potassium ion as it crosses the membrane from the aqueous phase on one side to the aqueous phase on the other side.
Note: The coordinates for this protein were determined by x-ray crystallography, and the protein component of this image was rendered using SwissPDB Viewer and POV-Ray (see References).
If the internal core of the protein channel is lined with hydrophilic amino-acid residues, then the channel allows passage of polar or charged particles between the two aqueous sides of the membrane. Figure 5 shows a representative ion channel, with hydrophilic residues lining the internal core and hydrophobic residues lining the regions of the protein that contact the lipid tails in the interior of the membrane.
This is a view through the opening of the same potassium channel shown in Figure 4. Notice that the inner core is lined with hydrophilic amino-acid residues (blue) that interact favorably with the charge on the ion (yellow). The outer areas of the channel contain hydrophobic amino-acid residues (plum), which interact favorably with the hydrophobic lipids in the membrane.
Note: The coordinates for this protein were determined by x-ray crystallography, and the image was rendered using SwissPDB Viewer and POV-Ray (see References).
These channels may be left open continuously, or they may be opened and closed by elaborate cellular gating mechanisms, as we will see below for three representative cases in the kidneys. In either case, passage of particles through the membrane is dictated by the size, shape, and polarity of the channel.
- Briefly, explain how the structure of the channel might allow the channel to discriminate between these two anions. HINT: You may find it helpful to consult the three-dimensional CPK representations in the on-line table of polyatomic ions.
- Which, if any, of the following ions would you expect to be able to pass through this channel: bicarbonate (HCO3-), hydroxide (OH-), or permanganate (MnO4-)? Briefly, explain your reasoning. HINT: You may find it helpful to consult the three-dimensional CPK representations in the on-line table of polyatomic ions.
The direction of the passage of particles through the channel is also dependent on concentration gradients. A concentration gradient exists whenever a concentrated solution is in contact with a less concentrated solution. Because the solutions are in contact, particles may flow between the two solutions (or between two regions of the same solution) by the process known as diffusion. Diffusion is a term used to describe the mixing of two different substances that are placed in contact. The substances may be gases, liquids, or solids. Diffusion is the migrating by random motion of these different particles.Although particles move in every direction, there is a net flow from the more concentrated solution to the less concentrated solution ("down the concentration gradient"). As the number of particles in the more concentrated solution diminishes and the number of particles in the less concentrated increases, the difference in concentration between the two solutions decreases. Hence, the concentration gradient is said to get smaller (Movie 1). All else being equal, the concentrations of the solutions change more rapidly when the difference in their concentrations is greater. This diffusion process continues until the concentrations of the two solutions are equal. This state is known as dynamic equilibrium. When the two solutions are in dynamic equilibrium, particles continue to move between the two solutions, but there is no net flow in any one direction, i.e., the concentrations do not change.
The graph at the top of this figure plots the time course of the changes in concentration that occur after a solution (A) with a 1.0 M concentration of some particle is placed in contact (via a semipermeable membrane) with another solution with a 0.0 M concentration of the particle. The blue line represents the concentration of the particle in solution A, and the magenta line represents the concentration of the particle in solution B. Over time, the concentrations become equal and no longer change; at this point, the solutions are said to be in dynamic equilibrium.
The schematic at the bottom shows the two solutions approximately 2 seconds after the solutions are placed in contact with one another.
To view a QuickTime movie showing the movement of the particles by diffusion between these two solutions, please click on the pink button below. Click the blue button below to download QuickTime 4.0 to view the movie.
In biological systems such as the kidney, the two solutions are often separated by a membrane. Protein channels in the membrane allow particles to cross the membrane, flowing "down the concentration gradient" until equilibrium is reached. Sometimes these channels may be closed, so that particles will not travel across the membrane, even if there is a strong concentration gradient. (In effect, the two solutions are no longer in contact when the channels are closed.) In other cases, the proteins in the membranes act like "pumps," using energy to move particles "against the concentration gradient" (i.e., so the more concentrated solution becomes even more concentrated); examples are the light-driven proton pump that occurs in the photosynthetic thylakoid membrane discussed in the introduction to the Experiment, the proton pumps used in the synthesis of ATP, the body's energy currency (which you will encounter in the tutorial entitled "Energy for the Body: Oxidative Phosphorylation"), and the sodium pumps discussed below.
- At what point is the rate of change in the concentrations of the two solutions greatest?
- Briefly, explain why the rate of concentration change is greatest at this point.
- Does diffusion occur across the membrane? Briefly, explain your answer.
- Does the concentration of Na+ or Cl- change in neither, either, or both of the two solutions? Briefly, explain your answer.
How do the kidneys actually filter the blood to remove the necessary particles in the proper amounts? Each component of the nephron contains specialized semipermeable membranes that filter molecules and maintain tightly-regulated concentration gradients. The selectivity of the membrane acts in conjunction with the concentration gradients to perform the dialysis functions of the kidney.
Lipid-soluble substances can easily pass through the phospholipid membrane, and so these substances tend to be readily reabsorbed into the blood, even without protein channels. This can be a problem, because many drugs and toxins, such as the pesticide DDT, are lipid-soluble, and hence are reabsorbed into the blood. Thus, it is very difficult to remove these toxins.
Most of the components of the blood, however, are polar or charged and hence require protein channels to cross the membrane (i.e., through the hydrophobic membrane interior). The channels in the nephrons are specialized to allow only the passage of particular types of particles, based on size, shape, and charge interactions with the amino acids lining the channel interior. The number and regulation of these specialized channels allow the kidneys to control the amount of each polar (or charged) species in the blood that is excreted. Most waste products undergo only partial reabsorption, so that large amounts of the substance remain in the tubule and are thus removed from the body in the urine. In contrast, useful plasma components, such as water, nutrients, and inorganic ions, are reabsorbed completely or nearly completely.
To demonstrate how the specialized membranes of the kidneys work to maintain the blood's chemistry properly, we shall consider three different blood-plasma components (Na+, H2O, and urea), and how the flow of each component between the nephron tubule and the surrounding blood-containing capillaries is controlled.
Certain segments of the nephron tubule contain proteins that act as pumps for sodium ions. These pumps use energy from the body to pump sodium ions out of the tubule into the blood (Figure 7). In this manner, Na+ is actively reabsorbed into the blood. As more and more ions are pumped out of the tubule, a concentration gradient builds up, with greater Na+ concentration outside the tubule than inside the tubule. However, because this reabsorption is achieved by active pumping, rather than passive diffusion, sodium ions continue to leave the tubule. The amount of sodium ions that are reabsorbed can be controlled by the hormone aldosterone. When large quantities of aldosterone are present, sodium reabsorption into the blood is enhanced, and so very little sodium is excreted from the body. When aldosterone levels are low, the pumps are less active, so more sodium remains in the tubules and is excreted. Hence, the body can maintain the optimal blood concentrations of sodium ions by secreting aldosterone in response to low sodium levels or decreasing aldosterone secretion in response to high sodium levels.
This schematic diagram shows the reabsorption of Na+ ions (pink) and water (blue). Na+ crosses the tubular membrane into the blood outside the tubule by a pump, against the concentration gradient. H2O crosses the tubular membrane into the blood outside the tubule by passive diffusion through a channel, down the concentration gradient.
The active pumping of sodium ions out of the tubule and into the blood causes the water concentration inside the tubule to increase (since the number of water molecules is the same, but the total number of particles (Na+ and H2O) is smaller), while the water concentration in the blood decreases. Hence, a water-concentration gradient is established. Portions of the tubular membrane are impermeable to water, but other portions contain hydrophilic channels through which water can flow. Water exits the tubule and enters the blood through these hydrophilic (polar) channels by passive diffusion down the concentration gradient (Figure 7).
In the collecting duct (see Figure 2), the permeability of the membrane is subject to being altered in response to the hormone vasopresin, also known as antidiuretic hormone (ADH). When the body needs to retain water, as in dehydration situations, the concentration of ADH increases, and the high ADH level causes the water-permeability of these membranes to be great. Therefore, large amounts of water are reabsorbed into the blood, and only a little water will be excreted in the urine. However, when the body has plenty of water, the level of ADH drops, causing this portion of the membrane to become relatively impermeable to water. In this case, a larger amount of water remains in the nephron (in the collecting duct) to be excreted.
Urea is a waste product formed in the liver during the metabolic breakdown of proteins. The body does not use urea, and so the kidney's aim is to remove this metabolite through the blood. As the glomerular filtrate enters the tubule, it is rich in urea, because the urea freely passes through the membranes of the glomerulus. Although it might seem as though all of this urea would thus be excreted in the urine, in fact only about half of it is. The tubular membranes are freely permeable to urea. Water reabsorption raises the concentration of urea inside the tubules, since the urea in the tubules is now diluted with less water. Hence, urea will flow down the concentration gradient, out of the tubules and into the surrounding blood-containing capillaries. As more urea exits the tubules, the difference in urea concentration between the tubules and the capillaries decreases, until the two solutions are at equilibrium. Then, although urea may continue to pass between the fluids, there is no longer any net flow of urea out of the tubules. The remaining urea in the tubules will be excreted from the body.
- Based on the changes in the concentrations of sodium and water in the blood that would result from drinking so much water, predict what will happen to the levels of aldosterone and ADH in the body. Briefly, explain your answer.
- What effect will these hormonal changes have on sodium and water reabsorption (i.e., how will the amount of these substances excreted through the bladder be affected by drinking a large amount of water)?
When the kidneys do not function properly, dialysis must be performed artificially. Without this artificial kidney dialysis, toxic wastes build up in the blood and tissues, and cannot be filtered out by the ailing kidneys. This condition is known as uremia, which means literally "urine in the blood." Eventually this waste build-up leads to death.
The artificial kidney uses cellulose membranes in place of the phospholipid-bilayer membranes used by real kidneys to separate the components of blood. This cellulose membrane is the same type of membrane that you used in this experiment. Cellulose is a polymer of glucose molecules that form long, straight chains (Figure 8). Parallel chains form linkages with one another by hydrogen bonding to make strong fibers. These fibers in turn interact to form the strong, sheet-like structure of the membrane.
This is a two-dimensional ChemDraw representation of a cellulose chain (polymer strand). One of the glucose units is shown in blue.
The arrangement of the cellulose fibers may contain gaps, creating tunnels through the membrane (Figure 9). These form the pores through which particles may pass from one side of the membrane to the other. The size of the gaps determines the size of the particles that will be able to pass through the membrane (i.e., the molecular weight cut-off, as described in the Introduction to the experiment in the lab manual).
This is a CPK representation of a cellulose membrane. Each cellulose fiber is colored to show the interactions of the fibers to form a sheetlike structure. Note the gaps between fibers that form pores in the membrane.
Note: The coordinates for this model were determined using molecular-mechanics calculations, and the image was rendered using the Insight II molecular-modeling system from Molecular Simulations, Inc. (see References).
Two types of artificial kidney dialysis are used clinically. Hemodialysis uses a cellulose-membrane tube that is immersed in a large volume of fluid. The blood is pumped through this tubing, and then back into the patient's vein. The membrane has a molecular-weight cut-off that will allow most solutes in the blood to pass out of the tubing but retain the proteins and cells. The external solution in which the tubing is immersed is a salt solution with ionic concentrations near or slightly lower than the desired concentrations in the blood. Recall that if the external concentration of a particular species is lower than the internal concentration, then that species will pass through the cellulose membrane by diffusion into the external solution. In this manner, excess substances in the blood are removed from the body. To maintain the blood's concentration of a species, the external solution is made to have the same concentration of that species as the blood. In such a case, the two solutions are in dynamic equilibrium, and so the blood's concentration does not change.
Peritoneal dialysis does not use an artificial membrane, but rather uses the lining of the patient's abdominal cavity, known as the peritoneum, as a dialysis membrane. Fluid is injected into the abdominal cavity, and solutions diffuse from the blood into this fluid. After several hours, the fluid is removed with a needle and replaced with new fluid. The patient is free to perform normal activities between fluid changings.
Thus, artificial kidney dialysis uses the same chemical principles that are used naturally in the kidneys to maintain the chemical composition of the blood. Diffusion across semipermeable membranes, polarity, and concentration gradients are central to the dialysis process for both natural and artificial kidneys.
Blood contains particles of many different sizes, shapes, and polarity. Some of these particles (e.g., proteins) are essential for the body; some (e.g., urea) must be removed from the blood and the body; others (e.g., many ions) must be maintained at certain concentrations. The body has many different methods for controlling the composition of the blood (including hemoglobin, ferritin, and buffers, which are discussed in other tutorials), but the kidneys have the primary responsibility for controlling the blood's chemical composition.
By filtration, reabsorption, and secretion mechanisms, the kidneys separated and regulate the components of the blood. Some of these components (e.g., proteins) are filtered from the fluid entering the tubule at the glomerulus, and remain in the blood. Other particles (e.g., water, ions, and sugar) are reabsorbed by the blood or secreted from the blood to maintain the proper concentrations; these processes occur while the fluid is flowing through the tubule. Any blood components that remain in the nephron when the fluid reaches the collecting duct (e.g., waste products such as urea) are excreted from the body.
The reabsorption and secretion of the blood components depend on the ability of these blood components to cross the nonpolar interior of the membrane surrounding the nephron tubule. The polar blood components can only pass through the membrane via special protein channels. These protein channels in the membrane are polar on the inside, to allow the passage of polar or charged particles through the membrane, and nonpolar on the outside, to interact with the nonpolar membrane interior. The size of the channel can determine which polar or charged particles will be able to cross the membrane through the channel.
The concentrations of the blood components are maintained by diffusion through the membrane (via the protein channels if the component is polar) and concentration gradients. Depending on the component, the concentration is maintained by passive diffusion (going "down" (with) the gradient), or by pumping (going "against" the gradient). Hence, the ability of the kidneys to remove harmful particles from the blood, and to regulate the concentration of other particles in the blood, depends on the chemical concepts of diffusion, polarity, and concentration gradients.
Doyle, D.A. et al. "Potassium Channel (KCSA) From Streptomyces Lividans," (1998) Science, 280, 69. Potassium channel PDB coordinates, Brookhaven Protein Data Bank.
Guex, N. and Peitsch, M.C. Electrophoresis, 1997, 18, 2714-2723. (SwissPDB Viewer) URL: http://www.expasy.ch/spdbv/mainpage.htm.
Insight II graphical program; Molecular Simulations, Inc. URL: http://www.msi.com.
Persistence of Vision Ray Tracer (POV-Ray). URL: http://www.povray.org.
Vander, A. et al. Human Physiology, 7th ed. WCB McGraw-Hill, Boston, 1998, p. 503-533, 547-548.
Stryer, L. Biochemistry, 4th ed. W.H. Freeman and Co., New York, 1995, p. 261-278, 299-301.
The authors thank Dewey Holten, Michelle Gilbertson, Jody Proctor and Carolyn Herman for many helpful suggestions in the writing of this tutorial.
The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71199-502008 to Washington University.