I. General Features of Renal Function -Objectives II. Blood Flow and Filtration -Objectives III. Tubular Anatomy and Function -Objectives IV.

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1 ORGANIZATION This syllabus is divided into ten sections or topics. Each section is divided into objectives. On the contents page, clicking on the section title takes you directly to that section. Clicking on the word objectives beneath each section s title provides a list of the objectives in that section and clicking on one of the objectives will provide the material on that objective (See Contents). Almost all the objectives contain one or more figures. Clicking on the figure will cause a full screen version of the figure to appear. That version may contain text that explains or emphasizes aspects of the subject matter (Fig. 2-1). Alternatively, clicking on a complex figure may lead to two or more figures that present the material in steps (Fig. 6-2). A listing of the figures is also provided and the figures can be directly accessed from that list (Figures). This may facilitate a review of a section or topics. Each objective ends with a set of study questions or problems. Each question is followed by a box which the student may use to type in an answer. Below the box is a mouse roll-over icon. Placing the mouse pointer there will cause the correct answer to appear. (For example, see Contents; click on Objectives under Section IV, select Objective 2 and go to the questions at the end of the text). This syllabus will be modified and expanded to include additional topics from time to time. Comments, questions and suggestions by users are solicited. Please contact the author by lsulliva@kumc.edu A special acknowledgment is due to Philip Wilhauk of the NetLearning Division at the University of Kansas Medical Center. He designed the web pages and devised the methods for presenting the questions and answers and the means for navigating through the material.

2 I. General Features of Renal Function -Objectives II. Blood Flow and Filtration -Objectives III. Tubular Anatomy and Function -Objectives IV. Measurement of Renal Function -Objectives V. Excretion of Organic Molecules -Objectives VI. Mechanisms of Salt and Water Reabsorption in the Proximal Tubule and to Understand Their Relationships -Objectives VII. Regulation of Water Balance -Objectives VIII. Control of Extracellular Fluid Volume -Objectives IX. The Role of the Kidney in Acid-base Regulation -Objectives X. Potassium Homeostasis -Objectives

3 In the process of evolution, the first simple forms of life developed in a fluid medium or environment of a constant composition, the sea. As time progressed, organisms evolved that were able to live first in fresh water and then on dry land. These organisms were able to face a hostile and ever changing external environment because they had developed mechanisms that enabled them to bathe their cells in a constant internal environment. To state it differently, these organisms could live relatively free and independent of changes in their external environment because of the constancy of the composition of their internal environment, their extracellular fluids. Organisms acquired this physiologic freedom principally because of the development of the kidney, the organ primarily responsible for the maintenance of the internal environment. Fig A cross-section of the renal architecture. The kidneys accomplish this vital task in the following way. From the large volume of plasma that the circulation brings to the kidney daily, the glomeruli filter a fluid almost identical in composition to plasma except for protein. This fluid then flows through the approximately 2,000,000 nephrons in the kidneys. The cells lining these nephrons reabsorb specific substances from this fluid in varying quantities and return them to the blood. These cells also extract additional substances from the blood and secrete them into the urine. As the kidneys perform their task, the process of glomerular filtration and all the myriad tubular mechanisms respond to a variety of factors to return to the circulation a fluid with the composition and volume required to maintain the constancy of the internal environment. In a day's time, the kidneys process the equivalent of the extracellular fluid volume of the body some 15 times by filtering approximately 40 gallons of fluid, reabsorbing from it the necessary amount of various substances and water, and adding other substances to urine. Less than half a gallon of fluid with a vastly different composition is finally excreted.

4 OBJECTIVE 1: TO GAIN A GENERAL UNDERSTANDING OF THE ANATOMY OF THE KIDNEY. A. The arterial network of the kidney can be characterized as a low resistance-high flow circulatory bed (Fig. 1-1). The renal artery branches into several interlobar arteries (not shown) and thence into arcuate arteries that run along the boundary between the cortex and medulla. The arcuate arteries send branches out through the cortex toward the surface of the kidney. These are the interlobular arteries from which are derived the short afferent arterioles that supply the glomeruli. The renal circulatory system possesses the unique feature of two capillary beds arranged in series and separated only by the efferent arteriole. The first in the series, the glomerular capillary bed forms a ball that is enclosed within Bowman's capsule. These capillaries and the capsule together form the glomerulus. The glomerular capillaries empty into the efferent arteriole which conveys the blood to the second capillary bed, the peritubular capillaries. In the superficial cortex and mid-cortex, the peritubular capillaries form networks that surround all the cortical tubular structures. In the juxtamedullary region, the efferent arterioles branch after leaving the glomeruli. Some of these branches immediately divide into peritubular capillary networks in the outer medulla where they surround loops of Henle and collecting tubules. Others form bundles of vessels, the vasa recta that penetrate deep into the medulla before branching. Fig Major sections of the nephrons and their position within the cortex and medulla. B. Urine formation begins in the glomerular capillaries. An ultrafiltrate of plasma crosses the capillary wall and enters the tubule. The glomerular filtrate flows first into the proximal tubule, which superficially is composed of two major sections: an initial convoluted portion, the pars convoluta, and then a straight segment, the pars recta, which descend toward the medulla (Fig. 1-2). The tubular fluid then enters the hairpin loop of Henle, which is divided into a thin segment and a thick segment. The loop of Henle conducts the urine flow toward or through the medulla and then directs it toward the surface of the cortex. At the end of the thick, ascending limb of the loop, the tubular fluid comes into contact through the tubular wall with the same glomerulus from which it originated and with the afferent and efferent arterioles attached to the glomerulus. The

5 confluence of these structures forms the juxtaglomerular apparatus (j.g. apparatus). This is the major site of control of the rates of renal blood flow, glomerular filtration and renin secretion. From the loop of Henle, the tubular fluid flows into the convoluted distal tubule and mixes with tubular fluid from other nephrons as several distal tubules join to form the collecting tubules. The collecting tubules carry the tubular fluid straight down from the cortex through the medulla into the papilla. The urine then exits into the renal pelvis. C. Differences exist between the cortical nephrons that originate near the surface of the kidney and the juxtamedullary nephrons that begin near the border between the cortex and medulla of the kidney (Fig.1-2). The major anatomic difference is the length of the loops of Henle. The thin segment of the loop in cortical nephrons is short and the thick segment often begins before the bend in the loop. These loops do not extend into the medulla. In addition, the proximal convoluted tubules of cortical nephrons are shorter and the glomeruli are smaller than those of juxtamedullary nephrons. Fig. 1-1 illustrates such nephrons as they might appear in situ in a section of the kidney. The glomeruli and the proximal and distal convoluted tubules of all nephrons are confined to the cortex, whereas only the loops of Henle of the juxtamedullary nephrons and the collecting tubules enter the medulla. QUESTIONS: 1. What is the unique feature of the circulatory anatomy in the kidney? 2. Where is the juxtaglomerular apparatus? 3. Compare the anatomy of the superficial cortical nephrons and the juxtamedullary nephrons

6 OBJECTIVE 2: TO GAIN A GENERAL UNDERSTANDING OF HOW URINE IS FORMED. A. The process of urine formation begins with filtration into the glomerulus. In the glomerular capillaries, the hydrostatic pressure of the blood forces 18 to 20% of the plasma to filter through a complex membranous structure and enter the nephron (1 in Fig. 1-3). This ultrafiltrate contains all the substances that exist in the plasma, in the same concentration as in plasma except the plasma proteins, which are retained in the blood, and substances bound to the proteins. Table 1-1 lists some of the quantities filtered by all the glomeruli in the kidneys of an average individual weighing 70 kg. After plasma is filtered and the ultrafiltrate enters the nephron, a variety of forces operate to alter the concentration of substances in that fluid and the amount of each that is excreted (4 in Fig. 1-3). Fig A cartoon of the nephron and its associated blood supply. The basic steps in urine formation are illustrated. 1. Filtration. 2. Reabsorption. 3. Secretion. 4. Excretion. B. Most of the naturally occurring constituents of the filtrate are reabsorbed to varying extent across the tubular wall and returned to the blood stream (2 in Fig. 1-3). This reabsorption across the epithelial cell layer forming the tubular wall may occur passively down an electrochemical gradient or it may be accomplished actively by specific transport processes residing in the tubular cells. The amount of each of these substances that is excreted is always less than the amount filtered. Their concentration in the final urine may be greater or smaller than their concentration in the filtrate, depending on the relative degrees to which they and water are reabsorbed. Table 1-1 lists the amount filtered/day, the amount excreted, and the percent of the filtered amount that is reabsorbed for some of the common constituents of the filtered plasma. C. Some substances gain entrance to the tubule not only by filtration but by secretion, that is, by crossing the tubular wall from the blood perfusing the tubules (3 in Fig. 1-3). The concentration of these substances in the urine is usually greater than in the glomerular filtrate and

7 the amount excreted exceeds the amount that is filtered. Among such compounds are hippurate, penicillin, thiamine and saccharin. A few substances, such as potassium and uric acid, are both absorbed and secreted by the tubular epithelium, and their concentration in the urine and the amount excreted vary greatly. The tubular cells also can secrete into the tubular fluid the products of enzymatic reactions that take place within them. Two such substances are hydrogen ions and ammonia. The terms secretion and excretion are misused at times particularly in the older literature. The term secretion should be applied only to the processes that add substances to the tubular fluid by transport across the tubular wall. Excretion refers to what is delivered to the ureter. Table 1-1. Quantities involved in urine formation in the human. Average values for an individual weighing 70 kg. FLUID: Renal Blood Flow, RBF= 1200 ml/min (20-25% of cardiac output). Renal Plasma Flow, RBF= 660 ml/min. Glomerular Filtration Rate, GFR= 125 ml/min. Fraction of plasma flow filtered, GFR/RPF,= SOLUTES: Excreted per Plasma Conc. Filtered per day day mm mmoles g mmoles g Percent Absorbed Sodium , Chloride , Bicarbonate 25 4, Potassium Glucose ~0 ~0 100 Urea Urate WATER: 180L L 99+ D. The concentration of any substance in the final urine depends not only on the rate of transport of that substance by the nephron, but also on the rate of water reabsorption. In order to determine how the rate of transport of a particular substance is altered in various circumstances, one should consider the rate of excretion of that substance rather than its concentration. Moreover, because the intake of various substances, and particularly the intake of water by a normal individual, varies over a wide range, the concentrations of substances in the urine vary over a much wider range than do their concentrations in plasma.

8 QUESTIONS: 4. What are the four general processes involved in urine formation? 5. What fraction of the filtered sodium and chloride are excreted? What fraction of the filtered urea is excreted? What fraction of the filtered glucose is excreted? 6. When the urine flow equals 1 L/day, what would be the average concentrations of Na, urea and urate in the urine? What would be the average concentrations of these substances when the urine flow equals 5 L/day? "Recognizing that we have the kind of internal environment we have because we have the kind of kidneys we have, we must acknowledge that our kidneys constitute the major foundation of our physiological freedom. Only because they work the way they do has it become possible for us to have bones, muscles, glands and brains. Superficially it might be said that the function of the kidneys is to make urine; but in a more considered view one can say that the kidneys make the stuff of philosophy itself." Homer W. Smith: From Fish to Philosopher, The Story of Out Internal Environment, Little Brown & Co., Boston, 1953.

9 OBJECTIVE 1. TO DETERMINE THE SITES OF CONTROL OF BLOOD PRESSURE AND BLOOD FLOW WITHIN THE KIDNEY. A. The afferent and efferent arterioles are the major sites of control of renal blood flow.figure 2-1 illustrates the changes that occur in the hydrostatic pressure of blood as it flows from the renal artery through the arterioles and capillary beds into the renal vein. The largest falls in pressure occur in the afferent and efferent arterioles. These are the sites of greatest resistance to flow, and therefore, the major sites of control of blood flow. Fig. 2-1.Pressure gradients and control points in the renal circulation. H.P. = hydrostatic pressure. b = colloid osmotic pressure. B. The afferent and efferent arterioles maintain a high pressure in the glomerular capillary bed and control the rate of blood flow through the bed.the unique positioning of the glomerular capillary bed between the two major resistance sites, the afferent and efferent arterioles, permits the maintenance of a relatively high hydrostatic pressure in that bed and also provides a mechanism for close control of the pressure and flow. C. The afferent and efferent arterioles maintain a low pressure in the peritubular capillary bed. Hydrostatic pressure is low in this capillary bed because of the high resistance of the afferent and efferent arterioles upstream.

10 QUESTIONS: 1.What are the two major sites of resistance to blood flow in the kidney?

11 OBJECTIVE 2: TO UNDERSTAND THE MAGNITUDE AND ROLE OF THE STARLING FORCES IN EACH CAPILLARY BED. A. The afferent and efferent arterioles maintain the hydrostatic pressure in the glomerular capillary bed higher than the colloid osmotic pressure. Therefore plasma is filtered into the nephron (figs 2-1 and 2-2). The arterioles control the rate of filtration by controlling blood pressure and flow in the glomerular capillary bed. Normally, 18% to 20% of the plasma flowing through the capillary bed is filtered into the tubules. The glomerular capillary membrane is relatively impermeable to protein, so the loss of the protein-free filtrate raises the protein concentration and thus the colloid osmotic or oncotic pressure, b, of the 80 to 82% of the plasma remaining in the circulation, as illustrated in Figure 2-2. Fig Balance between hydrostatic and colloid osmotic pressures in the two capillary beds. B. The hydrostatic pressure is low in the peritubular capillaries because of the resistance of the two arterioles upstream. Oncotic pressure in the peritubular capillaries is high because of glomerular filtration. Therefore fluid is absorbed into the capillaries (fig. 2-2). The balance of the Starling forces causes fluid to move from the interstitial space surrounding the tubules into the blood stream. This is fluid that was reabsorbed by the tubular epithelial cells. Absorption of that protein-free fluid into the capillaries reduces the plasma colloid osmotic pressure back down to that of arterial plasma (Fig. 2.1) QUESTIONS: 2. Which of the two major forces causing fluid movement across capillary walls is dominant in the glomerular capillary bed: In the peritubular capillary bed? 3. Why does the colloid osmotic pressure change in each of the two capillary beds?

12 OBJECTIVE 3: TO UNDERSTAND THE ANATOMY AND FUNCTION OF THE GLOMERULUS. A. The glomerulus consists of a group or tuft of specialized capillaries situated between an afferent and an efferent arteriole. The capillary loops are held together by mesangial cells. The capillary tuft projects into Bowman's capsule, which is a hollow sphere lined by a single layer of epithelial cells that are continuous with the layer of cells forming the proximal tubule. A complex interdigitating network of epithelial cells lines the outer surface of each glomerular capillary. These epithelial cells, called podocytes, send out foot processes which interdigitate with foot processes from neighboring cells. The endothelium lining the inner surface of the capillaries is penetrated by openings or fenestrations of about 600 to 1000 angstroms in diameter. These fenestrations take up a large portion of the surface area. Fig The filtration pathway. B. Fluid leaving the capillary and entering Bowman's capsule must pass through the three layers that comprise the capillary wall: the capillary endothelium, a basement membrane, and the epithelial cell layer (Fig. 2-3). Fenestrations in the capillary endothelium are easily penetrated by compounds of large molecular weight but not by the cellular elements of the blood. The basement membrane is composed of a dense layer, the lamina densa, sandwiched between

13 two less dense layers, the lamina rara interna and externa. The foot processes of the epithelial cell layer are separated by channels or slits that may be approximately 240 angstroms in width and 3000 to 5000 angstroms in height. Neighboring foot processes are connected at their base by a slit membrane, which is also the outer limiting membrane of the basement membrane. The foot processes also contain contractile elements. The basement membrane consists largely of glycoproteins and collagen. Glycoproteins also coat both the endothelial and the epithelial cell membranes, cover the endothelial fenestrations, and fill the slits or channels between the foot processes. The glycoproteins contain sialic acid, which confers a strong negative charge to the cell coating and the basement membrane. The filtration pathway is through the fenestrations in the endothelium, across the basement membrane and the slit membrane, and through the slits between the foot processes into Bowman's capsule. C. The fluid crossing the glomerular membrane and entering the capsular space is an ultrafiltrate of plasma, containing all the substances that exist in plasma except the proteins. The concentrations of these substances in the filtrate are the same as in plasma except for a few substances that are bound to the plasma proteins to a varying extent (many drugs fall into this category). These exceptions are minor, and it is emphasized that the composition of the ultrafiltrate is almost identical to the composition of plasma except for the proteins. D. The filtration pathway bars most proteins from passing through. The filtration pathway provides little hindrance to the passage of solutes with a molecular weight up to 5000 (molecular radius, 14 angstroms). Above that point, the ability of macro-molecules to penetrate the barrier is a function of their shape, size and ionic charge. Neutral macromolecules, up to a radius of 20 angstroms, are not hindered in crossing the glomerular membrane, but the penetrating ability of larger molecules decreases rapidly with size. Negatively charged macromolecules are evidently repelled by the fixed negative charges present in the basement membrane and the glycoprotein cell coating. Table 2-1 compares the fractional clearance of the plasma protein albumin with dextran molecules of the same molecular radius. Dextran sulfate is negatively charged and its fractional clearance is much less than that of neutral dextran. Albumin is a polyanion at plasma ph, and its low fractional clearance is due not only to its size but also to its negative charges. Table 2-1. Fractional clearance of albumin and selected dextran molecules. Macromolecule Molecular Radius Fractional Clearance (M) (A) (U/P) m /(U/P) in Normal Values Albumin 36 <0.001 Neutral Dextran Dextran Sulfate Experimental glomerular nephritis Neutral Dextran Dextran Sulfate

14 E. Disease processes affect the glomerulus in several different ways. In glomerulonephritis, immune complexes are deposited in the glomerular capillary walls. The deposits interact with complement in the blood and then attract leukocytes, which in turn, release proteolytic enzymes that produce minute holes in the capillaries. Erythrocytes and plasma proteins can then leak into Bowman's space and, eventually into the final urine. Some diseases evidently change the net charge on the glycoproteins coating the glomerular capillaries, allowing proteins like albumin to cross the glomerular barrier more easily and leak into the urine. In these states, one sees excessive protein but not erythrocytes in the urine. Table 2-1 illustrates how one form of glomerulonephritis, induced experimentally in rats, reduces to some extent the ability of neutral dextran with the same molecular radius as serum albumin to penetrate the glomerular capillary. This suggests that the disease may cause the loss of capillary surface area through which protein can enter Bowman's space. In contrast, the fractional clearance of negatively charged dextran sulfate of the same radius is greatly increased. Evidently, the disease process has caused the loss of fixed negative charges from the membrane, enabling negatively charged macromolecules like albumin to penetrate the membrane more easily and appear in the urine to a much greater extent. QUESTIONS: 4. What is the path taken by filtered substances from the glomerular capillary lumen to Bowman's space? What are the constraints that limit the penetration of albumin through that path? 5. Compare and contrast the concentration of Na, glucose, albumin and Ca in the following three fluids: Afferent arteriolar plasma, efferent arteriolar plasma, the glomerular filtrate.

15 OBJECTIVE 4. TO UNDERSTAND THE FORCES INVOLVED IN FILTRATION. A. The glomerular capillary hydrostatic pressure, P gc, is the major force inducing filtration across the glomerular capillary bed. This is opposed by the tubular pressure, P t (Fig. 2-4). B. Filtration is also opposed by the colloid osmotic pressure in the glomerular capillary plasma, b (Fig. 2-4). Since the filtering surface is permeable to all substances in plasma except protein and substances bound to protein only the protein exerts an effective osmotic pressure. The tubular fluid usually contains only trace amounts of protein so it exerts no effective osmotic force on the glomerular membrane. Fig Pressures involved in filtration. C. The net filtration pressure, P f = P gc - P t - b. Fig. 2-4 lists approximate values for the relevant pressures involved in filtration in the human. D. The surface area (A) and the hydraulic permeability (Lp) of the filtering membrane must also be considered in order to account for the volume filtered. The two values are difficult to determine separately but their product, the filtration coefficient, k f, can be calculated from measurements of GFR and P f. GFR = k f P f. The hydraulic permeability of the glomerular capillary is 10 to 100 times greater than that of capillaries elsewhere in the body. Fig Hydrostatic and colloid osmotic pressure profiles in the glomerular capillary bed. E. The net filtration pressure is higher at the beginning of the glomerular capillary bed than it is at the end because of a rise in

16 b (Fig 2-5). The hydrostatic pressure gradient, P ( P = P gc - P t ), across the glomerular membrane drops only slightly because of the high resistance downstream in the efferent arteriole. b increases along the length of the bed because the filtration of a protein-free fluid from the filtrate increases the protein concentration in the plasma remaining in the capillary. This causes the net filtration pressure, P f, to fall along the length of the bed. F. Changes in the hydrostatic pressure gradient, in renal blood flow and k f can result in changes in mean P f and thus in glomerular filtration rate. 1. A fall in P will reduce mean P f (Fig. 2-6B). This could result from a fall in P gc caused by a fall in arterial pressure or by changes in resistance in afferent and efferent arteriolar resistance. A fall in P could also result from an increase in P t. Mean P f for the entire capillary bed is reduced and that causes a fall in GFR. 2. A fall in RBF will reduce mean P f by causing an increase in mean b (Fig. 2-6C). This could occur by simultaneous constriction of the two arterioles which would preserve P. In the initial sections of the capillaries, P f would be the same. Thus, in those sections, the same volume of a protein-free fluid is removed from a smaller volume of plasma. That results in a greater rise in protein concentration, and thus in b, in the plasma remaining in the capillary. In the late sections of the capillaries, b approaches P and P f approximates zero. Mean b for the entire capillary bed is higher, thus mean P f is less and GFR is reduced. A rise in RBF will have the opposite effect. Fig Effect of factors altering pressures in the glomerular capillaries on mean filtration rate. 3. A fall in the filtration coefficient, k f, will tend to reduce GFR (Fig. 2-4) but cause an increase in mean P f (Fig. 2-6D). A fall in k f could result from a fall in capillary surface area or a reduction in the hydraulic permeability of the membrane. This will reduce filtration. However the reduction in filtration of a protein-free fluid in the initial sections of the bed results in a smaller rise in b. The fall in mean b tends to maintain P f in the later sections of the capillary

17 bed and the higher pressure there partially compensates for the fall in k f and tends to prevent a large drop in GFR. (Remember: GFR = k f x P f ). QUESTIONS: 6. How will GFR vary in response to the following? Rise in P t, fall in P gc, rise in afferent arteriolar b, rise in blood flow? 7. Define the following terms, L p, L p A, k f. What might cause a fall in k f? What would be the effect on GFR?

18 OBJECTIVE 5: TO UNDERSTAND HOW CHANGES IN CONSTRICTION OF THE AFFERENT AND EFFERENT ARTERIOLES ALTER RBF AND GFR. A. Changes in afferent arteriolar resistance will produce changes in P gc that will cause large changes in GFR. 1. Increased resistance to flow in the afferent arteriole (R aff, Fig. 2-7A) will lower pressure downstream in the glomerular capillaries (P gc ) and reduce GFR. The rise in R aff will reduce RBF. 2. In contrast, a fall in R aff (Fig. 2-7B) will cause a rise in pressure downstream and increase GFR. The fall in R aff will increase RBF. 3. The changes in RBF will cause changes in mean b that will magnify the effect of the changes in P gc on GFR. For example a rise in R aff drops P gc in the glomerular capillaries and, by reducing RBF, maintains b high. Both factors contribute to the drop in GFR. Fig Effect of changes in afferent and arteriolar resistance on GFR and RBF. B. Changes in efferent arteriolar resistance (R eff ) may have opposite effects on RBF and P gc. This may result in only small changes in GFR.

19 1. A rise in R eff (Fig. 2-7C) will increase pressure upstream in the glomerular capillaries but it will also reduce RBF. The rise in P gc will tend to increase GFR but the increase in mean b will tend to counter that and the increase in GFR will be small. 2. A fall in R eff (Fig. 2-7D) will drop P gc upstream but it will increase RBF. The fall in P gc will tend to reduce GFR but this is counteracted by the fall in mean b. Thus the fall in GFR will be small. C. Frequently R aff and R eff may change in the same direction. For instance an increase in both will reduce RBF with little alteration in P gc. The fall in RBF will reduce GFR but to a lesser extent than if P gc also fell. Thus GFR is affected to a much lesser extent than is RBF. QUESTIONS: 8. How will the forces involved in filtration and GFR change with the following: fall in afferent arteriolar resistance, rise in efferent arteriolar resistance? 9. Why does a rise in afferent arteriolar resistance cause a fall in GFR whereas a rise in efferent arteriolar resistance has the opposite effect? 10. Why do changes in afferent arteriolar resistance result in bigger changes in GFR than changes in efferent arteriolar resistance.

20 OBJECTIVE 6: TO UNDERSTAND THE ANATOMY OF THE JUXTAGLOMERULAR APPARATUS AND ITS ROLE IN THE CONTROL OF GFR AND RBF. Fig The juxtaglomerular apparatus. A. Each nephron forms a loop so that the beginning of the distal tubule comes into close contact with the glomerulus of the same nephron and its associated afferent and efferent arterioles (Fig. 1-2). This conjunction of the arterioles, glomerulus, and distal tubule of one vascular-tubular unit is called the juxtaglomerular apparatus (Fig. 2-8). Specialized cells appear in the wall of the tubule and the arterioles at the site of their contact. Large columnar cells, called the macula densa cells, in the wall of the distal tubule are in contact with the granular cells in the wall of the afferent arteriole. The granular cells produce and release the enzyme renin. Another group of cells, the lacis cells appears between the two arterioles. The fluid absorbed by macula densa cells bathe the lacis cells. It is postulated that changes in the composition of that fluid result in changes in secretion of paracrine factors that alter the degree of contraction of the smooth muscle in the walls of the arterioles, and the rate of renin secretion by the granular cells. B. The J.G. Apparatus is a Major Control Center. 1. The j.g. apparatus receives a variety of signals (Fig.2-9). The j.g. apparatus responds to signals carried by the nerves innervating it, to humoral factors arriving via the blood stream, to changes in pressure in the afferent arteriole, and to changes in solute delivery to the macula densa via the tubular fluid. 2. Changes in signal input to the j.g. apparatus cause changes in RBF, GFR and renin secretion. These inputs affect renal blood flow (RBF), glomerular filtration rate (GFR) and renin

21 secretion. In turn, these three factors exert a profound influence on blood pressure, blood volume, and extracellular fluid volume throughout the body (Fig. 2-9). Fig The juxtaglomerular apparatus is a major control point. 3. The secretion of renin and the formation of angiotensin has multiple effects. The enzyme, renin, splits a decapeptide from the alpha-2-globulin fraction of plasma protein. This decapeptide is angiotensin I, which loses two amino acids in the presence of a converting enzyme (angiotensin converting enzyme, ACE) to become the octapeptide angiotensin II. This is the active form. Most of the angiotensin I formed in the blood is converted to angiotensin II by the lungs, which contain large amounts of the converting enzyme. However, small amounts of the converting enzyme are also present in renal tissue and in plasma. The drug saralasin, which is a closely related octapeptide, blocks receptors of angiotensin II. Captopril is a drug that blocks the angiotensin converting enzyme (ACE) and prevents the conversion of angiotensin I to angiotensin II. The renin system plays a role in the maintenance of arterial pressure both by the vasoconstrictor effect of angiotensin II and by its effect on blood volume. Angiotensin II directly stimulates tubular salt and water reabsorption and indirectly stimulates it by causing the secretion of the mineralocorticoid hormone, aldosterone, by the adrenal cortex. C. The role of the j.g. apparatus in modulating the signal inputs and regulating RBF and GFR will be discussed below. Its role in regulating renin secretion and the changes that occur in NaCl and water excretion and blood volume will be described in Section IX. QUESTIONS: 11. What are the components of the juxtaglomerular apparatus? 12. Why is this a major site of control of extracellular fluid volume and blood pressure? 13. What types of signals are received by the juxtaglomerular apparatus and by what routes? What changes occur as a result of these signals?

22 OBJECTIVE 7: TO DETERMINE THE SIGNALS THAT ACT ON THE JUXTAGLOMERULAR APPARATUS TO ALTER ARTERIOLAR CONSTRICTION. A. In the healthy individual at rest, the resistance to flow by the arterioles is quite low compared to other organs and the blood flow per gram of tissue is high. That low resistance may be due to a continual formation of nitric oxide by the endothelial cells lining the arterioles and its vasodilating effect on the arteriolar smooth muscle. B. RBF and GFR can be altered by extrinsic neural and humoral factors arising outside the kidney, by intrinsic humoral factors generated locally and by internal autoregulating mechanisms. The autoregulating mechanisms will be discussed under objective 8. C. Sympathetic vasoconstrictor fibers innnervate both arterioles within the juxtaglomerular apparatus. A moderate increase in sympathetic firing usually reduces RBF, whereas the change in GFR is relatively small, reflecting an effect on both arterioles. A large increase in sympathetic input will reduce RBF greatly and may have a major impact on GFR. However other buffering mechanisms, described below may modify the effect of the sympathetic input. The vasoconstrictor fibers also reach the initial segments of the vasa recta which contain smooth muscle cells. There are no sympathetic vasodilator fibers entering the kidney. D. A variety of humoral factors can be carried to the arterioles. Both and receptors for catecholamines are present. The receptors greatly outnumber receptors however, and all dose levels of either epinephrine or norepinephrine cause only vasoconstriction. Acetylcholine causes vasodilation. Pyrogens, produced as a result of infection by bacteria, viruses, molds, or yeast also cause vasodilation. E. The kidney also produces a number of vasoactive substances that can influence arteriolar constriction. 1. The release of renin from the granular cells of the j.g. apparatus results in the formation of angiotensin II, a vasoconstrictor. 2.Prostaglandin E 2 (PGE 2 ) and I 2 (PGI 2 ) are vasodilators that are produced in the cortical and medullary areas of the kidney. The substrate for prostaglandin synthesis is arachidonic acid. The synthesis can be blocked by indomethacin and meclofenamate. They primarily affect the afferent arteriole; their effect on the efferent arteriole is much less. PGE 2 synthesis is stimulated by angiotensin II, by vasoconstriction and by extracellular fluid (ECF) volume depletion. Their effect buffers afferent arteriolar resistance and serves to prevent large drops in P gc and thus in GFR that would otherwise occur with vasoconstriction.

23 3. Bradykinin, adenosine, leukotrienes, dopamine and endothelin are also produced within the kidney and exert vasoactive effects. Their physiological importance is uncertain. QUESTIONS: 14. What is the effect of activation of sympathetic neural fibers to the kidney on arteriolar constriction? 15. What is the effect of an increase in circulating levels of epinephrine and norepinephrine? Of angiotensin II? 16. What is the effect of increased production of PGE 2 and PGI 2? What is the nature of the interaction between angiotensin II and the prostaglandins? Between the vasoconstriction induced by sympathetic nerves or the catecholamines and the prostaglandins?

24 OBJECTIVE 8: TO UNDERSTAND HOW THE KIDNEY AUTOREGULATES RBF AND GFR. A. The kidney is able to maintain both RBF and GFR relatively constant in the face of changes in arterial pressure from about 80 to 200 mm Hg in the absence of changes in sympathetic neural input and in circulating levels of vasoactive substances (Fig. 2-10). Below that range changes in arterial pressure may have large effects on RBF and GFR. The fact that GFR is maintained constant indicates that P gc, as well as glomerular plasma flow, is kept constant. It is apparent then, that resistance upstream from the glomerular capillaries in the afferent arteriole must change as renal artery pressure changes. Efferent arteriolar resistance may change also, but it is obvious that the major change occurs in the afferent arteriole. Fig Autoregulation. The effect of changes in arterial pressure on RBF and GFR. B. This autoregulation is the product of two different mechanisms, the myogenic response and the tubuloglomerular feedback mechanism (TGF). 1. The smooth muscle within the walls of the arterioles respond directly to the stretching effect of a rise in pressure within the arteriole by increasing the contraction of their smooth muscle cells, thereby reducing the diameter of the arteriole and increasing its resistance to flow. This is called the intrinsic myogenic response.

25 2. The TGF mechanism involves the juxtaglomerular apparatus and the close anatomical relationship among the macula densa, the glomerulus and the arterioles of the same nephron (Fig 2-11). The macula densa senses changes in the composition of tubular fluid that occur when the tubular fluid flow rate changes and signals the afferent arteriole to alter constriction. Changes in the tubular fluid flow rate are brought about by changes in GFR and/or the rate of reabsorption in the proximal tubule. For example a rise in GFR increases the tubular fluid flow rate at the macula densa. That triggers an increase in afferent arteriolar resistance that reduces pressure in the glomerular capillaries and brings GFR back down. The rise in resistance also reduces RBF. Fig The tubuloglomerular feedback mechanism. 3. The macula densa apparently senses changes in the delivery of Cl - ([Cl] in tubular fluid x tubular fluid flow rate) that occur as flow rate changes. The macula densa cells absorb Cl - and the rate of reabsorption is dependent on the rate of delivery. It is thought that an increase in reabsorption causes the release of a paracrine, perhaps adenosine, that causes the afferent arteriolar smooth muscle cells to contract. C. Both the myogenic mechanism and the TGF mechanism contribute to autoregulation but the extent of the participation of each and how each responds to various situations that alter RBF and GFR is still largely unknown. QUESTIONS: 17. In the absence of changes in extrinsic factors, the kidney autoregulates RBF and GFR. To regulate both it seems obvious that the major change in resistance must occur in which arteriole? Why? 18. On what primary anatomical feature is the tubuloglomerular feedback mechanism based? What does this mechanism primarily regulate, GFR or RPF? In this feedback mechanism, what is the signal, the sensor, the effector? 19. Considering the TGF mechanism, what would be the effect on GFR of a fall in the rate of fluid reabsorption in the proximal tubule?

26 20. What other mechanism may be invoved in autoregulation? How would this mechanism respond to maintain GFR and RPF constant when arterial blood pressure is increased? OBJECTIVE 9. TO UNDERSTAND THE INTERRELATIONSHIPS BETWEEN EXTRINSIC NEURAL AND HUMORAL FACTORS AND THE AUTO-REGULATORY MECHANISMS. A. The autoregulatory mechanisms control RBF and GFR but extrinsic neural and humoral mechanisms can alter the 'set point' of these mechanisms. For instance an increase in sympathetic nerve activity can shift the curves in Fig down and to the right so that the plateau GFR and RBF will be lower than otherwise. B. The autoregulatory mechanisms will buffer the effect of extrinsic neural and humoral mechanisms on afferent arteriolar resistance so that it changes less than the efferent resistance. The result of this is that fractional changes in GFR tend to be less than the change in RBF. For instance, an increase in sympathetic firing by itself will cause constriction of both the afferent and efferent arteriole and both GFR and RBF will be reduced. The increase in pressure within the afferent arteriole and the fall in GFR will trigger the autoregulatory mechanisms so that the afferent resistance is reduced. This reduces the effect of the sympathetic firing on GFR and RBF. Efferent arteriolar resistance is not buffered and this maintains a reduced RBF but tends to maintain P gc high so that GFR is affected much less. Fig illustrates these relationships and includes the effect of PGE 2 as well. Fig The effect of the autoregulatory mechanisms and PGE 2 on the response of the renal arterioles to extrinsic factors. QUESTIONS: 21. What is the relationship between intrinsic and extrinsic regulation of RBF and GFR? In many situations in which RBF is reduced the fall in GFR is much less. This indicates that the primary change in constriction has occurred in which arteriole?

27 OBJECTIVE 1: TO DETERMINE THE GENERAL FEATURES OF A TRANSPORTING EPITHELIUM. A. A transporting epithelium usually consists of a single layer of polarized cells arranged in a hexagonal array. 1. Around each cell near its luminal or apical face is a circumferential belt, the zonula occludens (tight junction) which connects it with neighboring cells. The basal surface of each cell is embedded in a basement membrane. The lateral surfaces of neighboring cells are separated by a fluid filled space. This paracellular space is separated from the lumen by the zonula occludens (Fig 3-1). Fig The basic structure of epithelial cell layers. The width of the zonula occludens and the paracellular space have been greatly exaggerated for purposes of emphasis. 2. The microanatomy of the apical membrane may differ dramatically from that of the basolateral membrane, for instance, the brush border of the apical face and the extensive folding of the basolateral membrane of the proximal tubular cells. 3. The protein or molecular complexes within the apical and basolateral membranes differ in type and quantity, for instance, Na-K-ATPase is present in basolateral membranes of tubular cells but is not found in apical membranes. Receptors for antidiuretic hormone (ADH) are located in the basolateral but not the apical membranes of collecting tubular cells. B. The functional features of the epithelium are determined by the molecular complexes found in the apical and basolateral membranes and by the permeability of the zonula occludens. The asymmetrical features of the apical and basolateral membranes determine the direction of transport across the cell and, in part, the rate of transport. For instance, Na + channels

28 exist in the apical membrane of principal cells in the collecting tubule that allow Na + to enter the cell from the tubular fluid. Na-K-ATPase in the basolateral membrane pumps Na out into the extracellular fluid. Thus there is a net reabsorptive transport of Na + across the cell. The zonula occludens surrounding these cells has a low permeability to Na + so there is little passive diffusion back into the lumen and the cells can reduce the urine Na + concentration to very low levels. C. The paracellular space constitutes a micro environment which may differ in composition with the interstitial fluid. Its composition is affected by transport across the surrounding basolateral membranes and diffusion across the zonula occludens. In turn its composition affects the rate of that transport. D. Solutes may cross the epithelium via the cellular pathway or via the paracellular pathway. Transport through the cellular pathway usually involves specific carriers and channels. Transport through the paracellular pathway occurs by diffusion driven by electrical and chemical gradients between the tubular fluid and the interstitial fluid. The permeability or conductance of the paracellular pathway for passive movement is higher than the path through the cells. But active transport rates through the cell usually exceed the passive transport rates through the paracellular path. QUESTIONS: 1. What are the major anatomical features of an epithelial cell layer? 2. What is the essential feature of epithelial cells that allows net transport of solutes and water in one direction across the cell layer? 3. What paths may solutes take across the epithelial cell layer?

29 OBJECTIVE 2: TO DETERMINE THE ANATOMICAL AND GENERAL FUNCTIONAL FEATURES OF THE VARIOUS TUBULAR EPITHELIA. A. The proximal tubule reabsorbs 60 to 70% of the filtrate but causes only minor changes in its composition. Fig Changes in the urine to plasma concentration ratio (U x /P x ) of various substances along the length of the nephron. Note that the ordinate is a log scale. 1. There is a huge multiplication of the apical membrane surface area by microvilli and of the basolateral area by folds and fingers. Thus the ratio of surface area to tubular fluid volume is very large. This greatly increases the efficiency of the membrane transport mechanisms in reabsorbing the filtrate. 2. The permeability of the cell membranes to water is very high because of the presence of large numbers of the water channel, aquaporin I, in both the apical and basolateral membrane.

30 Fig Changes in the fraction of the filtered amount of substances remaining in the tubular fluid along the length of the nephron. 3. The tight junction joining the cells has a very high conductance to small ions. Thus, the permeability of the paracellular pathway to these solutes is high. 4. The transport systems located here reabsorb electrolytes at a high rate but, because of the high water permeability and the high conductance of the paracellular pathway, these systems can establish only small gradients across the tubular epithelium (Figs 3-2, 3-3). These transport systems can be classified as high rate-low gradient mechanisms. 5. A variety of organic substances are transported by the proximal tubule. Glucose and amino acids are reabsorbed here. Uric acid and a large variety of drugs are secreted into the tubular lumen. The tubular epithelium is poorly permeable to most of these organic solutes. Thus, in contrast to the electrolyte transport mechanisms, the organic solute transport systems can establish large gradients. B. The loop of Henle consists of three distinct segments and the epithelia of these segments have differing characteristics. 1. No active transport occurs in the thin descending limb. The epithelium is permeable to water and to small ions and passive transport occurs between the tubular fluid and the hyperosmotic interstitium. The cells are flat except for bulges in the cell that accommodate the nuclei. Very few mitochondria are present.

31 2. The thin ascending limb has a low permeability to water. The presence of electrolyte transport is debatable. 3. The thick ascending limb actively reabsorbs NaCl across an epithelium that has a very low permeability to water. The surface area of the basolateral membrane is greatly multiplied by the presence of folds. Large numbers of mitochondria are present. The epithelium has a low permeability to water and the paracellular pathway has a moderate permeability to electrolytes. The transport of salt into the medullary interstitium raises its osmotic concentration. Salt transport mechanisms may be classified as moderate rate-moderate gradient systems. C. The distal tubule is a zone of transition. Structure and function gradually change along its length from that of the thick ascending limb to that of the collecting tubule. Salt is actively reabsorbed. Water permeability is variable: In the initial segment it is low, in the later segment it varies from low in the absence of antidiuretic hormone, ADH, to high in its presence. Permeability of the paracellular pathway to electrolytes is low. The electrolyte transport mechanisms may be classified as low-rate, high gradient mechanisms. D. Two major cell types are present in the collecting tubule, the principal cells which reabsorb salt and secrete potassium and the intercalated cells which secrete protons and bicarbonate. Aldosterone stimulates this transport. Permeability of the paracellular pathway to electrolytes is very low. Permeability to water is low in the absence of ADH, high in its presence. The electrolyte transport mechanisms may be classified as low-rate, high gradient mechanisms. E. Summary (Table 3-1). The anatomically complex cells of the very permeable proximal tubule reabsorb the greater mass of the filtrate and transport a large variety of organic and inorganic substances without causing any changes in the osmotic concentration of the tubular fluid. Only minor changes in the concentrations of the major ions occur. The electrolyte and water reabsorptive systems in the proximal tubule can be classified as high rate, low gradient systems. The anatomy of the medulla and the transport characteristics of the loop of Henle permit the establishment of a hypertonic environment which in turn osmotically concentrates the urine in the collecting ducts. The transport systems in the thick ascending limb can be classified as moderate rate, moderate gradient systems. The more impermeable distal tubule and collecting duct reabsorb smaller fractions of the filtered water and solute than does the proximal tubule but, under the influence of hormones and other factors, they can vary the ratio of salt to water in the reabsorbed fluid. Thus, the osmotic concentration and the concentration of individual solutes in the final urine can be made to vary over a wide range. The electrolyte and water transport systems in the distal tubule and collecting tubule, in contrast to the proximal tubule, are low rate, high gradient systems. Table 3-1. Summary of the functional properties of the various sections of the nephron.

32 PROXIMAL TUBULE HENLE'S LOOP: DESCENDING LIMB ASCENDING LIMB DISTAL TUBULE COLLECTING TUBULE PROPERTIES TRANSPORT TRANSPORTED CHARACTERISTICS SOLUTES High LpA High g Moderate LpA High g Very low LpA, moderate g Low/variable LpA Variable LpA, low g High rate - low gradient Little change in [Osm] Little change in [Electrolyte] No active transport; passive equilibration with medullary ISF Moderate rate-moderate gradient Fall in [Osm], [NaCl] Low rate - high gradient ADH & Aldosterone effect on latter half Low rate - high gradient. Aldosterone & ADH effect Bulk reab. of NaCl, NaHCO 3, K, H 2 O, all glucose, AA reabsorbed, secretion of organic acids and bases Diffusion of NaCl, H 2 O, urea NaCl, K reab., no H 2 O reab.. NaCl reab., K reab. & sec., some H 2 O reab. NaCl reab., K sec., H sec., HCO3 reab. & sec., variable H 2 O reab. Almost all the tubular transport mechanisms are regulated in such a way as to minimize any changes that may occur in the volume of the organism's extracellular fluid, its osmotic concentration, the concentration of major solutes and its acid-base status. QUESTIONS: 4. What are the particular anatomical characteristics that distinguish the proximal tubule from other epithelia? What are the principal properties of the proximal tubular epithelium that permit bulk reabsorption of the major inorganic ions and water with little change in their concentration? 5. What changes occur in the composition of the glomerular filtrate as the fluid flows through the proximal tubule? What changes occur in the volume of the filtrate? What are the general features of the composition of the reabsorbed fluid? What effect does the return of that fluid to the circulation have on the composition of plasma? On its volume?

33 6. Compare and contrast the major anatomical and functional features of the thin descending limb and the thick ascending limb of the loop of Henle. What are the principal anatomical and functional differences between the proximal tubule and the thick ascending limb? 7. What are the principal differences in function between the proximal tubule and the cortical collecting tubule? How are these functional differences mirrored in their structural differences? 8. What is the major functional difference in the paracellular pathways in the proximal tubule and collecting tubule? What is the major consequence of this in terms of ion concentration gradients across the two epithelial layers? On the electrical gradients? 9. If the composition of the filtrate and the extracellular fluid surrounding the proximal tubule are almost identical, how can the process of passive diffusion bring about significant net transport in one direction? What is the route of least resistance for passive diffusion of ions?

34 OBJECTIVE 3: TO UNDERSTAND THE GENERAL ELECTRICAL PROPERTIES OF THE TUBULAR EPITHELIA. A: The transport and permeability properties of the basolateral membrane, the apical membrane and the paracellular pathway interact in a complex manner to set up a transepithelial electrical gradient (V te ) that varies along the length of the nephron. B. The major conductance present in almost all tubular cell membranes is a potassium channel. That, together with the Na-K-ATPase, establishan electrical gradient such that the interior of the cell is electrically negative with regard to the exterior. C. The presence of channels for other ions and electrogenic transport mechanisms in the apical and basolateral membranes modify the transmembrane electrical gradients. Since the presence of these channels and mechanisms differ between the two membranes of each cell, the electrical gradients across the apical and basolateral membrane differ and this creates a transepithelial electrical gradient. For example, the principal cells in the collecting tubule possess K + channels in both the apical and basolateral membrane and Na-K-ATPase in the basolateral membrane. There is also a Na + channel in the apical membrane only (Fig. 3-4). The Na + channel permits Na + to enter the cell and this tends to depolarize the apical membrane to a voltage that is less negative (-30 mv in Fig. 3-4) than that of the basolateral membrane (-80 mv). This results in a transepithelial potential difference (-50 mv) with the lumen more negative than the interstitium. D. The conductance of the paracellular pathway is higher than that of the cellular pathway, thus the transepithelial electrical gradient tends to drive a current flow (ion flux) through the paracellular pathway. In the example in Fig. 3-4, an anion flux out of the lumen or a cation flux into the lumen would occur. This passive flux tends to minimize the transepithelial potential difference and also would tend to increase the apical potential difference and reduce the basolateral potential difference. E. The magnitude of V te depends upon the individual properties of the apical and basolateral membrane and the magnitude of the conductance of the paracellular pathway. The conductance of that pathway is very high in the proximal tubule and falls progressively from there to the collecting duct. Basically, the lower the conductance of the paracellular path, the higher the electrical gradient, in other words, current flow through the paracellular pathway tends to shunt V te. Thus, the proximal tubule with its high conductance has only a small transepithelial electrical gradient. The distal tubule and collecting tubule have a high electrical gradient partly because of the low conductance of the paracellular path.

35 Fig An example of the generation of a transepithelial electrical gradient by a tubular cell. F. The magnitude of the passive flux of ions driven by the electrical gradient is proportional to the product of the gradient (V te ) and the conductance (g te ). This product and thus the passive flux of ions through the paracellular path is much higher in the proximal tubule than in the distal or collecting tubule. G. An ionic chemical gradient across the tubular epithelium can also drive a passive flux through the paracellular pathway and this will affect V te. QUESTIONS: 10. Why may the apical membrane potential differ from the basolateral membrane potential? What is the origin of the transepithelial electrical gradient.? 11. What effect does V te have on ion fluxes through the paracellular pathway? What is the effect of those fluxes on V te and on V a and V b? 12. In the late proximal tubule the tubular fluid Cl concentration exceeds that of the interstitium and drives a passive flux of Cl through the paracellular pathway. What will be the effect of this flux on V te?

36 OBJECTIVE 4: TO UNDERSTAND THE TYPES OF TRANSPORT MECHANISMS PRESENT IN TUBULAR EPITHELIA. A. Net transport by passive diffusion can occur across a tubular cell membrane or across the tubular epithelium when a favorable chemical (concentration) or electrical gradient is present and the barrier has a finite permeability to the substance. Since the composition of the filtrate entering the tubule initially has practically the same composition as that of the plasma in the peritubular capillaries, active transport of some type is required to establish a gradient for passive diffusion. For example, the active reabsorption of salt in the proximal tubule drives water reabsorption which causes the concentration of urea in the tubular fluid to increase. That chemical gradient then drives passive urea reabsorption. B. Active transport mechanisms which involve the interaction of a membrane component with the transported substance can be categorized in a number of ways: 1. Capacity-limited systems exhibit the characteristics of specificity, competition and saturation. The glucose reabsorptive system is a classic example. The system will transport only a few other sugars of similar size and they will compete among themselves for transport by the system. The capacity of the transport system is limited so that it can be easily saturated. 2. Gradient-limited systems are limited not so much by the capacity of the pump but by the chemical gradient the pump establishes. Active transport of an ion out of the tubule tends to reduce the concentration of that ion in the tubular fluid, establishing a chemical gradient for passive back-diffusion. The rate of that passive back-diffusion is affected by the electrical gradient and by the conductance or permeability of the epithelium. The difference between the rate of pumping and the rate of passive back-diffusion is the rate of net transport (Fig. 3-5).

37 Fig An illustration of a gradient-limited transport system. The rate of passive back-diffusion is also affected by the rate of water reabsorption. A high rate tends to maintain the concentration of the ion in the tubular fluid high, retarding or preventing the establishment of a chemical gradient for an ion that is being reabsorbed. Thus, the net rate of transport will increase. A high tubular fluid flow rate past the site of transport also tends to prevent the establishment of a chemical gradient and, therefore, permits a high rate of net transport. 3. Active transport systems can also be categorized as primary or secondary systems. A transport mechanism which directly utilizes metabolic energy is called a primary active mechanism. The Na-K pump for instance utilizes the metabolic energy stored in ATP. These systems may operate either as capacity-limited systems or as gradient-limited systems. A secondary active system utilizes the chemical or electrical energy resulting from the work of a primary transport system. The Na-H exchange mechanism for instance moves Na into the cell as a result of the Na concentration (chemical) gradient (low cell Na) established by the Na-K pump. In doing so it also transports protons out of the cell against its gradient. Actually the direction and rate of transport is governed by the algebraic sum of the gradients for both substances. The Na-H exchange mechanism is electrically neutral so the electrical gradient across the membrane has no effect on the transport. The Na-glucose cotransporter transfers net charge so that the total gradient for the system includes the sum of the chemical gradients for the two substances plus the electrical gradient for Na. These systems may also operate either as capacity-limited systems or gradient-limited systems. 4. There are additional terms applied to transport systems. Symports move two or more substances in the same direction, e.g., the Na-glucose symport operating in the apical membrane of the proximal tubule. Antiports move two substances in opposite directions, e.g., Na-H antiport.