BIO 240 Cuyamaca College Renal System Physiology Biology Lab

BIO 240 Cuyamaca College Renal System Physiology Biology Lab BIO 240 Cuyamaca College Renal System Physiology Biology Lab Renal System Physiology T he kidneys are excretory and regulatory organs. By excreting water and solutes, the kidneys are responsible for ridding the body of waste products and excess water. The kidneys regulate 1) plasma osmolarity, or the concen- tration of a solution expressed as osmoles of solute per liter of solvent; 2) plasma volume; 3) acid-base balance; 4) electrolyte balance; 5) excretion of metabolic wastes and foreign materials; and 6) the production and secretion of hormones that regulate osmolarity and electrolyte balance. All these activities are extremely im- portant to maintaining homeostasis in the body. 3 The kidneys are located between the posterior abdominal wall and the ab- dominal peritoneum. Although many textbooks depict the kidneys directly across from each other, the right kidney is actually slightly lower than the left. Each human kidney contains approximately 1.2 million nephrons, the functional units of the kidney. Each nephron is composed of a renal corpuscle and a renal tubule. The renal corpuscle consists of a tuft of capillaries, called the glomeru- lus, which is enclosed by a fluid-filled capsule called Bowman’s capsule. An af- ferent arteriole supplies blood to the glomerulus. As blood flows through the glomerular capillaries, protein-free plasma filters into the Bowman’s capsule, a process called glomerular filtration. An efferent arteriole then drains the glomerulus of the remaining blood. The filtrate flows from Bowman’s capsule to the start of the renal tubule, called the proximal convoluted tubule, then on to the proximal straight tubule, followed by the loop of Henle, a U-shaped hairpin loop. The filtrate then flows into the distal convoluted tubule before reaching the con- necting tubule and the collecting duct, where urine collects. The distal tubule and collecting duct are composed of two cell types: principal cells and intercalated cells. Principal cells reabsorb Na + and water and secrete K + . Intercalated cells secrete either H + or HCO – and are, therefore, very important in the regulation of the acid/base balance. ORDER NOW FOR CUSTOMIZED AND ORIGINAL NURSING PAPERS Glomerular Filtration Let’s take a closer look at what happens during glomerular filtration. Blood en- ters the glomerulus from the afferent arteriole. Starling forces (hydrostatic and osmotic pressure gradients) drive protein-free plasma from the blood across the walls of the glomerular capillaries and into the Bowman’s capsule. The glomeru- lar filtration rate is an index of kidney function. In humans, the filtration rate 119 FIGURE 9.1 Opening screen of the Simulating Glomerular Filtration experiment. ORDER NOW FOR CUSTOMIZED AND ORIGINAL NURSING PAPERS ranges from 80 to 140 ml/min, so that in 24 hours as much as 180 liters of plasma is filtered by glomeruli. The filtrate formed is devoid of cellular debris and is essentially protein free. The concentration of salts and organic molecules are sim- ilar to that of blood. Normal urine output is 1–1.5 liters/24 hours. The difference is reabsorbed in the body. Normally, only about 20% of the blood that enters the nephron is filtered, due to the osmotic pressure of the blood (oncotic pressure) and the hydrostatic pressure from the fluids in Bowman’s capsule. The glomerular filtration rate can be altered by changing afferent arteriole resistance, efferent arteriole pressure, or the size of the filtration surface, or by a process called renal autoregula- tion. BIO 240 Cuyamaca College Renal System Physiology Biology Lab Once the filtrate is formed, the nephron must reabsorb materials that the body needs and excrete unneeded materials from the body. While as much as 180 liters are filtered each day, less than 1% of the filtered water, sodium chloride, and other solutes are excreted in the urine. More than 67% of this reabsorption takes place in the proximal convoluted tubule. The distal convoluted tubule and collecting duct reabsorb ap- proximately 7% of the filtered NaCl, secrete a variable amount of K + and H + , and reabsorb a variable amount of wa- ter. It is in this distal part of the nephron that hormones act to reabsorb water and electrolytes. Aldosterone regulates NaCl reabsorption (and thus NaCl excretion as well). ADH (anti- diuretic hormone) causes the permeability of the distal tubule and collecting duct to increase, promoting the uptake of wa- ter from the filtrate. ADH is considered the body’s most im- portant hormone for regulating water balance. In the first three activities you will be concentrating on how arterial diameter and pressure affect glomerular filtra- tion rate and urine volume. Follow the instructions for start- ing PhysioEx in the Getting Started section at the front of this manual. From the drop-down menu, choose Exercise 9: Re- nal System Physiology and click GO. Then click Simulat- ing Glomerular Filtration. You will see the screen shown in Figure 9.1. Click Help at the top of the screen and then select Bal- loons On/Off. Now move your mouse around the simulated nephron in the yellow section of the screen. Labels will appear for the various parts of the nephron as you roll over them. Note in particular the glomerulus and the glomerulus capsule. Also note the “afferent tube” and “efferent tube” to the left of the glomerulus—these represent the afferent and efferent arterioles delivering and draining blood from the glomerulus. You may adjust the radius of either of these tubes by clicking the ( + ) and ( – ) buttons next to the respective tubes. You may also adjust the blood pressure of the source beaker by clicking the ( + ) and ( – ) buttons next to the “Pres- sure (mmHg)” display. Once you have identified all the equipment on screen, 8.Reduce the afferent arteriole radius to 0.30 mm, and click Start. Under these conditions, does the fluid flow through the nephron? click Help again and select Balloons On/Off (you cannot proceed with the experiment unless the labels are turned off). At the bottom left of the screen are two beakers. The left beaker, which we call the “source beaker,” represents the blood supply being delivered to the nephron. When the Start button is clicked, blood will flow from the source beaker to What is the glomerular filtration rate? How does it compare to your baseline data, and why? the afferent arteriole and then to the group of small tubes rep- resenting the glomerulus. As blood flows through the glomerulus, you will see ultrafiltration occur. (Ultrafiltration means filtration from the plasma of everything except pro- teins and cells.) Blood will then be drained from the glomeru- lus to the “drain beaker” next to the source beaker. At the end of the nephron tube, you will see the formation of urine in a small beaker at the lower right of the screen. To watch a test run of this process in action, click the Start button. At the end of the run, click Refill underneath the drain beaker before you begin the activities that follow. 9.Using the simulation, design and carry out an experi- ment for testing the effects of increasing or decreasing the efferent radius. How did increasing the efferent radius affect glomerular filtration rate? A C T I V I T Y 1 Effect of Arteriole Diameter on Glomerular Filtration How did decreasing the efferent radius affect glomerular filtration rate? In this activity you will investigate how the diameters of the afferent and efferent arterioles leading to and from the glomerulus can affect the glomerular filtration rate. 1.The afferent radius display should be set at 0.50 mm, and the efferent radius at 0.45 mm. If not, use the ( + ) or ( – ) but- tons next to the afferent and efferent radius displays to adjust accordingly. Physiologically, what could be the cause of a change in affer- ent or efferent arteriole radius? 2.Be sure the left beaker is full. If not, click the Refill ? button. 3.The pressure gauge above the left beaker should read 90 mm Hg. If not, click the ( + ) or ( – ) buttons next to the pressure display to adjust accordingly. 4.Click the Start button. As the blood flows through the nephron, watch the displays for glomerular pressure and glomerular filtration rate at the top right of the screen, as well as the display for urine volume at the bottom right of the screen. 5.After the drain beaker has stopped filling with blood, click Record Data. This will be your baseline data for this activity. 6.Click the Refill button. 7.Increase the afferent radius by 0.05 mm and repeat steps 3–6, making sure to click Record Data at the end of each run. Keep all the other variables at their original settings. Continue repeating the activity until you have reached the maximum afferent radius of 0.60 mm. Compare this data with your baseline data. How did increasing the afferent arteriole radius affect glomerular filtration rate? A C T I V I T Y 2 Effect of Pressure on Glomerular Filtration Next you will investigate the effect of blood pressure on glomerular filtration rate. 1.Under the Data Sets display, highlight Pressure. This will allow you to save data in a new data set window. You can always retrieve your data from the previous activity by high- lighting the Afferent data set. 2.Make sure that the source beaker is filled with blood, and that the drain beaker is empty. If not, click Refill. 3.Adjust the pressure gauge (on top of the source beaker) to 70 mm Hg. Set the afferent radius at 0.50 mm and the efferent radius at 0.45 mm. 4.Click the Start button. Watch the Glomerular Pressure and Glomerular Filtration Rate displays at the top right of the screen. 5.When the run has finished, click the Record Data but- ton. This is your baseline data. What happened to the glomerular filtration rate and urine vol- ume after you reduced the pressure? 6.Increase the pressure by 5 mm Hg and repeat the experi- ment. Continue increasing the pressure by 5 mm and repeat- ing the experiment until you have reached the maximum pressure of 100 mm Hg. Be sure to click Record Data and Refill after each experimental run. As pressure increased, what happened to the pressure in the glomerulus? How could you adjust the afferent or efferent radius to com- pensate for the effect of the reduced pressure on glomerular filtration rate and urine volume? Use the simulation to deter- mine your answer. What happened to the glomerular filtration rate? Compare the urine volume in your baseline data with the urine volume as you increased the pressure. How did the urine volume change? 8.Next, click the square valve button (currently reading “valve open”) above the collecting duct. The valve should now read “valve closed.” 9.Click Start. At the end of the run, click Record Data. What changes are seen in nephron function when the valve is closed? How could increased urine volume be viewed as being bene- ficial to the body? Why were these changes seen? ? BIO 240 Cuyamaca College Renal System Physiology Biology Lab In the first activity you looked at arteriole diameter and its Is the kidney functional when the glomerular filtration rate is zero? Explain your answer. role in glomerular filtration. Next you examined the effect of pressure on glomerular filtration. In the human body, both of these effects are occurring simultaneously. In this activity you will investigate the combined effects of arteriole diame- ter and pressure changes on glomerular filtration. 1.Under the Data Sets window, highlight Combined. This What is the major “ingredient” that needs to be removed from the blood? will allow you to save data in a new data set window. You can always retrieve your data from previous activities by high- lighting the Afferent data set or the Pressure data set. 2.Set the pressure at 90 mm Hg, the afferent arteriole at 0.50 mm, and the efferent arteriole at 0.45 mm. Studies on aging have demonstrated that some nephrons may fail as we get older. Will this be a problem regarding urine formation? 3.Click the Start button and allow the run to complete. Then click Record Data. This is your baseline data. 4.Click Refill. 5.Lower the pressure to 80 mm Hg. Leave afferent arteri- ole at 0.50 and efferent arteriole at 0.45 mm. 6.Click the Start button and allow the run to complete. If blood pressure went down—for example, as the result of blood loss—what changes would the kidney need to make to maintain its normal filtration rate? Then click Record Data. 7.Click Refill. 10. Click Tools ® Print Data to print your data. ? FIGURE 9.2 Urine Formation. (a) Opening screen of the Simulating Urine Formation experiment. Part (b) follows. Simulating Urine Formation Reabsorption is the movement of filtered solutes and water from the lumen of the renal tubules back into the plasma. Without reabsorption, we would excrete the solutes and wa- ter that our bodies need. In the next activity you will examine the process of passive reabsorption that occurs while filtrate travels through a nephron and urine is formed. Click Experiment at the top of the screen and select Simulating Urine Formation. You will see the screen shown in Figure 9.2. The light yellow space surrounding the dark yellow nephron represents interstitial space between the nephron and peritubular capillaries that branch out from the efferent arteriole. The movement of solutes and water from the renal tubules to the interstitial space is dependent on the concentration gradient —that is, the difference between the concentration of solutes in the tubules and the concentration of solutes in the interstitial space. Notice the display for Conc. Grad. (mosm) near the bottom of the screen. By click- ing the (+) and (-) buttons next to this display and then clicking Dispense, you can adjust the solute concentration of the interstitial space. When you click Start, filtrate will begin flowing through the nephron, and solute and water will move from the tubules to the interstitial space to the peritubular capillaries, completing the process of reabsorption. Note that the capillaries are not shown on screen. Note also the two dropper bottles at the right side of the screen, which contain the hormones aldosterone and ADH (antidiuretic hormone). For the first activity you will be deal- ing with ADH only, which increases the water permeability of the distal convoluted tube and the collecting duct of the nephron (Figure 9.2b and c). Near the bottom left of the screen you will notice a Probe. When the probe turns red, you can click and drag it over various parts of the nephron and over the beaker to measure the solute concentration pres- ent. Finally, notice the equipment at the very top of the screen for adding glucose carriers. We will explain this equipment in Activity 5, when you will be studying the reabsorption of glu- cose. Descending limb of loop of Henle 100 Collecting duct 300 Descending limb of loop of Henle Collecting duct 100 150 H2O 300 Cortex H2O Outer medulla 300 600 NaCI NaCI 100 400 DCT 100 100 300 600 Cortex H2O Outer medulla 300 600 NaCI NaCI 100 400 DCT H2O H2O H2O 300 300 400 600 H2O 300 600 NaCI Urea 900 NaCI Urea 900 H2O Inner medulla 900 700 1200 Urea 100 1200 H2O Inner medulla 900 700 1200 H2O Urea H2O 900 1200 1200 (b) Absence of ADH Key: Active transport Passive transport (c) Presence of ADH FIGURE 9.2 (continued) Urine Formation (b) Mechanisms for forming dilute urine (c) and concentrated urine. Effect of Solute Gradient on Urine Concentration 1.Make sure Gradient is highlighted within the Data Sets window. 2.Click and drag the dropper top from the bottle of ADH and release it on top of the gray cap directly above the right side of the nephron. The cap will open, and ADH will be dropped onto the collecting duct. The cap will then close. 3.The Conc. Grad. (mosm) window should read 300. If not, adjust the (+) or (-) buttons accordingly. 4.Click Dispense to apply the 300 mosm concentration to it turns red, click and drag it over to the urine collecting beaker to measure the urine solute concentration. The value will appear in the Concentration window next to the probe’s original location. 6.At the end of the run, click Record Data. 7.Increase the concentration gradient by 300 mosm (i.e., set it at 600 mosm) and repeat the experiment. Remember to add ADH to the collecting duct before clicking Start. Con- tinue increasing the gradient by 300 mosm and repeating the experiment until you reach 1200 mosm, the normal value for interstitial solute concentration in the kidney. Be sure to click Record Data after each run. How did the urine solute concentration change as the concen- tration gradient of the interstitial fluid increased? the interstitial fluid. It is important to note that this is also the typical value of solute concentration in the blood. 5.Click Start and allow the blood to flow through the sys- tem. Watch the Probe at the bottom left of the screen. When What happened to the urine volume as the concentration gradient increased? Why? What happens to the glucose concentration as you add glucose carriers to the system? By increasing the concentration gradient, what are you doing to the urine that is formed? Predict what would happen to urine volume if you did not add ADH to the collecting duct. At what point does the glucose concentration in the urine be- come zero? A person with type I diabetes cannot make insulin, and a per- son with type II diabetes does not respond to insulin that is made. In either case, the diabetic person is unable to absorb glucose into the body. What would you expect to find in the urine of a diabetic person? Why? ? ? A C T I V I T Y 5 Reabsorption of Glucose Glucose is not very large, and as such it is easily filtered out of the plasma into Bowman’s capsule as part of the filtrate. To ensure that glucose is reabsorbed into the body so that it can form the starting material of metabolism, glucose carriers are present in the nephron. There is a finite number of carriers per cell, so if too much glucose is taken in, not all of it will be reab- sorbed into the body. Glucose is absorbed by secondary active transport, the “push” of which comes from the gradient created by the transport of sodium. The carriers that transport these molecules across the membrane are proteins embedded in the cell membrane. In this activity you will examine the effect of glucose carriers on glucose reabsorption. BIO 240 Cuyamaca College Renal System Physiology Biology Lab 1.Highlight Glucose within the Data Sets window. 2.Set the Conc. Grad. (mosm) to 1200 and click Dis- pense. Recall that 1200 is the normal value for solute concentration in the kidney. 3.Click Start. 4.Click Record Data at the end of the run. This run will serve as a “control” run, with no glucose carriers present. Note that no ADH has been added, either—your focus will be on the absence or presence of glucose carriers. 5.At the top of the screen, click the (+) button until the Glucose Carriers window reads 100. Then click Add Car- riers. 6.Click Start. 7.At the end of the run, click Record Data. 8.Continue to increase the number of glucose carriers by 100 at a time and repeat the experiment until you have reached the maximum number of carriers, 500. Be sure to dispense the concentration gradient before beginning each run, and to click Record Data after each run. BIO 240 Cuyamaca College Renal System Physiology Biology Lab A C T I V I T Y 6 Effect of Hormones on Reabsorption By now you should understand how filtration occurs and how it is controlled. You should understand passive solute move- ment and the role of glucose carriers on glucose reabsorption. Next you will examine the actions of hormones on the nephron, most of which occur in the collecting duct. Antidiuretic hormone (ADH) is influenced by the osmo- lality (the concentration of a solution expressed in osmoles of solute particles per kilogram of solvent) of body fluids as well as the volume and pressure of the cardiovascular system. A 1% change in body osmolality will cause this hormone to be se- creted. The primary action of this hormone is to increase the permeability of the distal tubule and the collecting duct to water so that more water is taken up into the body. ADH binds to re- ceptors in the principal cells to cause this reaction by opening aquaporins, or water channels in the apical membrane. With- out water uptake, the body would quickly dehydrate. The kid- ney tightly regulates the amount of water excreted under nor- mal conditions to maintain water balance in the body. Water intake must precisely match water loss from the body. If wa- ter intake is down, or if there has been a fluid loss from the body, the kidneys work to conserve water by making the urine very hyperosmotic (having a relatively high solute con- centration) to the blood. If there has been a large intake of fluid, the urine is more hypo-osmotic. In the normal individ- ual, urine osmolarity varies from 50 to 1200 milliosmoles/kg water. The osmolality of the body must be maintained within very narrow limits. BIO 240 Cuyamaca College Renal System Physiology Biology Lab Aldosterone is an adrenal cortical hormone under the control of the body’s renin-angiotensin system. A decrease in blood pressure is detected by cells in the afferent arteriole and triggers the release of renin. Renin acts as a proteolytic enzyme, causing angiotensinogen to be converted into an- giotensin I. Endothelial cells have an enzyme, called the con- verting enzyme, which converts angiotensin I into an- giotensin II. Angiotensin II works on the adrenal cortex to induce it to secrete aldosterone. Aldosterone acts on the col- lecting duct of the kidney to promote the uptake of sodium Which hormone has the greater effect on urine volume? Why? from filtrate into the body and the release of potassium from the body. Coupled with the addition of ADH, this electrolyte shift also causes more water to be reabsorbed into the blood, resulting in increased blood pressure. 1.Highlight Hormone within the Data Sets window. 2.Set the number of Glucose Carriers to zero and click How does the addition of aldosterone affect the concentration of potassium in the urine? Dispense to ensure that no carriers from the previous activity remain in action. 3.Set the Conc. Grad. (mosm) to 1200 and click Dis- pense. 4.Click Start. At the end of the run, click Record Data. This is your “control” run and baseline data. How does the addition of ADH affect the concentration of potassium in the urine? How does this compare to the effect of adding aldosterone, with respect to potassium concentra- tion in the urine? Baseline Urine Volume = 5.Click and drag the dropper top from the bottle of aldo- sterone and release it on top of the gray cap directly above the right side of the nephron. The cap will close, and aldosterone will be dropped onto the collecting duct. How does the addition of both hormones affect 1) urine con- centration, 2) urine volume, and 3) potassium concentration? 6.Click Start. At the end of the run, click Record Data. Urine volume with aldosterone present = 7.Click and drag the dropper top from the bottle of ADH and release it on top of the gray cap directly above the right side of the nephron. The cap will close, and ADH will be If ADH were not used, how would the urine concentration vary? Explain your answer. dropped onto the collecting duct. 8.Click Start. At the end of the run, click Record Data. 9.For your fourth run, dispense both ADH and aldosterone onto the collecting duct. You should see a yellow outline ap- pear around the collecting duct. 10.Click Start. At the end of the run, click Record Data. 11.Click Tools ® Print Data to print your data. ? Histology Review Supplement For a review of renal tissue, go to Exercise H: Histology At- las & Review on the PhysioEx website to print out the Re- nal Tissue Review worksheet. Get a 10 % discount on an order above $ 100 Use the following coupon code : NURSING10

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