LAB 10: RENAL FUNCTION

Transcription

1 Page 10.1 LAB 10: RENAL FUNCTION The renal system is comprised of the kidneys, ureters, urinary bladder and urethra. This system has many functions, the most obvious are the creation, storage and excretion of urine. To understand the functions of this system, one must explore how and why urine a waste product is made. The kidneys make the urine and are located retroperitoneal which means their posterior side is next to the posterior abdominal wall (against the back ) and there anterior side is covered with peritoneum. The by-product of urine is made as the kidney s filter blood plasma. In filtering blood plasma, the kidneys control blood volume (which ultimately controls the body s levels of extracellular fluid), regulate erythrocyte production and acid/base balance, and help to rid the body of toxins and waste products. Renal function can be evaluated using simple urine tests. These tests can yield a lot of data about the health of the body and they are non-invasive which makes them important tools in health care. Objectives At the conclusion of this laboratory the student will understand and be able to describe 1. The functions of the kidney based on certain urinalysis determinants. 2. The normal and abnormal constituents of urine. 3. The physical characteristics of urine. This lab consists of 3 activities which students will do with a partner. There will be no human blood or urine used in this lab. ACTIVITY 1. Urinalysis of the student s own urine (outside of lab) ACTIVITY 2. Urinalysis of artificial urine ACTIVITY 3. Prediction and analysis of data from a fluid consumption simulation BACKGROUND AND REFERENCES Some of the functions of the kidneys include (1) filtration of the blood plasma, (2) reabsorption back into the blood of water, ions and reusable molecules (e.g., glucose) and (3) secretion and elimination of waste products that were not removed during the initial filtration process. The final products of kidney function are eliminated in the urine during the process of micturition. The processes by which the kidneys perform these functions are extremely complex. Within each 24 hour period 180 liters of filtrate is processed from the blood passing through the kidneys. This equates to the total plasma volume being filtered approximately 60 times per day.

2 Page 10.2 Filtration: The kidneys filter the blood plasma. The blood components that are not filtered are RBCs, WBCs, platelets and most plasma proteins. Filtration occurs at the renal corpuscle. Fluid and small substances move from the glomerulus to the Bowman s capsule to form the ultrafiltrate. Reabsorption: Many plasma components that are filtered in the kidneys are still useful for body functions, and, thus, are reabsorbed back into the vascular system. Some of the components of plasma that are reabsorbed are ions (Na+, K+, Ca++), water, glucose, amino acids, etc. Secretion: All blood plasma is not filtered during a single passage through the kidneys, yet, certain waste products in the blood may still be eliminated from the blood by selective secretion into the kidney tubules. Some products that are secreted are urea, creatinine and hydrogen ions. Erythropoiesis: This function is not demonstrated in this lab. The kidneys produce a hormone, erythropoietin, which stimulates the production of red blood cells in the bone marrow. For example, when an individual goes from sea level to a higher altitude erythropoietin production increases, due to the decreased po2, to stimulate increased RBC production for greater oxygen carrying capacity of the blood. The efficiency of kidney function may be determined by an analysis of the products of kidney function. Analysis of the production and composition of urine is known as urinalysis. There are a number of compounds that may be administered to a subject to determine the efficiency of filtration, reabsorption and secretion. For example, inulin is a plant product that is filtered by the kidney but is neither reabsorbed nor secreted. By measuring the amount of inulin that appears in the urine after injecting inulin into a person s bloodstream, one can determine the amount of plasma that is filtered per unit time. This amount is known as the glomerular filtration rate (GFR). A normal GFR is 125 ml/min. Some of the analysis methods are beyond the capabilities of this lab, however, considerable information is available from more basic techniques. Checkpoint: Why is the process in the kidneys called reabsorption and not just absorption (such as in the digestive system)?

3 Page 10.3 The functional units of the kidneys are the nephrons. Figure 1: the nephron is the functional unit of the kidney (source: Wikimedia Commons) The following are simplified discussions of the structure and basic function(s) of the parts of the nephron See Figure 1 1) Proximal Convoluted Tubule: This initial portion of the nephron is comprised of Brush Border Cells Simple Cuboidal with LOTS of microvilli on the apical (luminal) membrane and a folded basolateral membrane. They have lots of mitochondria because they need tons of ATP to power the transporters on the membranes. This is the location of the majority of reabsorption

4 Page 10.4 What is a Transport Maximum (TM)? The limiting factor on the reabsorption of substances is how many and how fast the types of transporters can work. For example: Diabetes Mellitus. One of the key symptoms of this disease is the excretion of glucose in the urine. Normally glucose is reabsorbed in the proximal convoluted tubule. However, since this disease impedes cells ability to take in glucose the blood glucose levels are abnormally high. Because there is so much glucose from the blood that gets filtered that all the glucose transporters working as fast as they can go can t work fast enough to reabsorb it all so you excrete it because you ve exceeded your transport maximum! 2) Descending Loop of Henle: This location in the nephron is comprised of Simple Squamous Epithelium, with no microvilli. There are not many transporters of any kind (besides the basic leaks and pumps) but it allows the water to flow out of the tubule and down its concentration gradient (which is made by the ascending Loop of Henle) but the solutes can t move so this is where there is a great increase in the concentration of filtrate in the tubule What is the Countercurrent Multiplier? This is a physiological example of countercurrent exchange and is another name for the loop of Henle. The Descending Loop is only permeable to water the ascending Loop actively transports solutes out. The bottom of the Loop is around 1200 mosm whereas the top (cortex) is only around normal body osmolarity: mosm How does this differ from the Vasa Recta? The vasa recta is a special set up of capillaries around the Loop of Henle it allows the capillaries to feed the medulla of the kidney without ruining the osmotic gradient. Blood moving down the descending portion loses water, glucose and O2 to the tissues and gains NaCl and CO2. Blood moving up the ascending portion regains water by osmosis and loses NaCl thereby maintaining the high osmolarity of the medullary tissue without a change in the osmolarity of the venous blood leaving the kidney 3) Ascending Loop of Henle and Early Distal Convoluted Tubule: This part of the nephron is composed of Simple Cuboidal cells with very few, small, microvilli on the luminal surface but with highly folded basolateral membranes (with lots of mitochondria and transport proteins). The luminal membrane is covered in a special glycoprotein matrix that is totally impermeable to water. This is the only place in the human body that osmosis is restricted. 4) Juxtaglomerular Apparatus: This structure is the major intrinsic controller of blood flow to the nephron and is made up of two distinct cell types 1) The Juxtaglomerular Cells (JG Cells): Specialized smooth muscle cells surrounding the afferent arteriole. They act as baroreceptors.

5 Page ) The Macula Dense (MD): Specialized cells that are part of the DCT and lie against the glomerular arterioles. These act as osmolarity receptors to monitor the osmolarity of the filtrate. 5) Late Distal Convoluted Tubule and Cortical Collecting Duct: These two structure are both composed of simple cuboidal cells. 1) Principle Cells: This cell type s permeability to water and solutes is under hormonal control: a) Aldosterone (Released through the Renin-Angiotensin-Aldosterone System or from the release of ACTH from the anterior pituitary stimulate the reabsorption of Na+ ions and secretion of K+ ions) b) ADH (Released by the Hypothalamus or because of presence of Angiotensin 2 causes insertion of Aquaporins into the cells which increases water reabsorption. Diabetes Insipidus is the Hypothalamus not releasing enough of this {central DI} or the kidneys not having the receptors to respond to this {nephrogenic DI} which causes the sufferer to produce large amounts of urine.) c) Atrial Natriuretic Peptide (Released by atrial myocardial cells when blood volume or blood pressure are too high cause decrease in rate of Na+ reabsorption in the DCT, causing you to excrete Na+ in the urine, the dilation of the glomerular capillaries which causes an increase in GFR and urinary water loss and inactivation of renin, aldosterone and ADH secretion so overall you pee more and pee more Na+ which decreases blood volume and consequentially blood pressure] 2) Intercalated Cells: Major responsibility to transport H+ and HCO3- to maintain acid/base balance 6) Medullary Collecting Duct: This structure is composed of simple cuboidal cells with less microvilli and folds of the basolateral membrane (compared to the proximal and distal tubules) This part of the nephron is mostly Principle cells so their permeability to water and other solutes (urea etc.) is regulated by hormones Checkpoint: List the parts of the nephron from the point of filtration through the collecting ducts. What is/are the function(s) of each of these parts?

6 Page 10.6 Normal Characteristics of Urine Urinalysis is a quick way to evaluate the function of the kidneys. In order to determine if there is disease present, one must understand the characteristics of normal kidneys (kidneys in homeostasis) Yellow, clear color The color is due to a pigment (urobilinogen) which is a breakdown product from the heme groups of red blood cells. The first breakdown product (bilirubin) is first excreted with the bile, modified, and then reabsorbed in the intestines to be excreted in the urine. The color of urine will vary with urine volume, diet, and some disease states. If urine is allowed to stand, it will become cloudy. Bacterial contamination (indicative of a urinary tract infection) of the urine will also make freshly collected urine cloudy. Aromatic odor this odor becomes more ammonia-like if the urine is allowed to stand. The odor of urine can be affected by diet (i.e. Aspargus) and by some disease states. ph The average ph is 6, but the ph can range from This ph is affected by diet and by some disease states. If the kidneys are compensating for respiratory acidosis, the ph will be low. If the kidneys are compensating for respiratory alkalosis, the ph will be high. If an individual is taking excess amounts of vitamin C (ascorbic acid), the ph will be low. Specific gravity The specific gravity of urine ranges from to The specific gravity can be affected by diet, fluid intake, and some disease states. The more concentrated the urine is, the higher the specific gravity is. Specific gravity is a measure of the density of a liquid compared to the density of water. Water is defined as having a specific gravity of 1. Waste Chemicals Urea, Creatinine, uric acid, hippuric acid, Indican, ketone bodies, and other substances, depending on diet, are normal constituents of urine. Various inorganic salts are also normally present in urine, the most abundant of these is Na+ and Cl-.

7 Page 10.7 Checkpoint: From memory, List and Explain at least 3 normal characteristics of urine. Abnormal Conditions that can be tested for by Urinalysis: Proteinuria = Proteins in the urine (usually the plasma protein albumin so this is sometimes called albuminuria). The presence of protein in the urine is indicative of injury to the glomerular capillary beds or increased blood pressure this means that there is damage to the kidney filters). These proteins can sometimes cause the urine to be foamy (although a lot of bilirubin in the urine can cause this also). This process can also be caused by consuming too much protein in one s diet (over time). Glucosuria = Glucose in the urine. The presence of glucose in the urine is indicative of diabetes mellitus (but NOT an indication of diabetes insipidus). This can also be present in a kidney that has trauma that damages its filters. Hematuria = Red blood cells in the urine. If you can see the blood in the urine this is called gross hematuria, if you can t it is called microhematuria. Hematuria can be caused by a variety of disease states including kidney trauma, kidney stones, kidney infection, renal cancers, etc. It is quite common for blood to be present in the urine post exercise in marathon runners (this is most often caused by rubbing of the bladder or urethra walls and not damage to the kidney itself). Women who are menstruating will also have blood present in their urine. Eating a lot of beet roots can cause a false positive for hematuria because of the presence of the red pigment betanin Pyruia = White blood cells in the urine. Pyruia is indicative of kidney, bladder and/or urethral infection. Ketosis = An abnormally high amount of ketone bodies in the urine. Ketosis occurs in uncontrolled diabetes mellitus, starvation (or repeated fasting), and/or a chronic low carbohydrate (ketogenic) diet. Ketosis causes a sweet or acetone smell in the urine. Urobilinogenuria = An abnormally high amount of urobilinogen in the urine. Urobiliongen is a normal breakdown product of hemoglobin. Excess amounts of

8 Page 10.8 urobilinogen can occur in the urine due to hemolytic anemia (which can lead to jaundice), liver cirrhosis, or other disease states. Casts = Hardened masses that can occur in the urine. Casts usually form in the distal convoluted tubules and collecting ducts of the nephrons. This process can be exacerbated or accelerated through the presence of excess protein in the urine (proteinuria). There are many many types of casts (waxy, fatty, hyaline, bacterial, epithelial etc.) and the presence of particular casts can be indicative of various disease states. Checkpoint: From memory, List and Explain at least 3 abnormal conditions that can be tested for by urinalysis. Renal Plasma Clearance In order to maintain homeostasis, the kidneys must maintain the ability to clear the plasma of unwanted substances. The clearance of a substance(x) is defined as the volume of plasma that would have to be completely cleared ( i.e. the concentration of the substance in the plasma reduced to zero) of that substance to account for the observed amount of that substance in the urine. The calculation of clearance is based on the equation that Plasma Volume x Concentration(x) in Plasma = Urine Volume x Concentration(x) in Urine So... The Clearance of a substance (x) is calculated as: Urine Volume x Concentration(x) in Urine Concentration(x) in Plasma The clearance of a substance depends on the (1) Glomerular Filtration Rate (GFR), (2) the rate at which the substance enters the urine via secretion, and (3) the rate at which the substance leaves the urine via excretion. The GFR of a healthy individual is ml/min and it can be estimated by the Clearance of Creatinine (CrCl). Creatinine

9 Page 10.9 enters the urine via filtration and is then only slightly affected by secretion and reabsorption. Thus, while almost all of the creatinine that is found in the urine is a result of filtration, sometimes this measurement can be an overestimation due to active secretion or underestimation due to reabsorption of creatinine. A more accurate determination of GFR is made with the plant product inulin because the nephron neither secretes nor reabsorbs this substance. Urea enters the urine by both filtration and secretion. Some urea leaves the urine by reabsorption. Under normal circumstances, about 60% of the urea that is filtered is ultimately reabsorbed. The clearance of urea can be calculated by measuring the concentration of urea in both the urine and the plasma and by measuring the volume of urine produced in a specified period of time. Checkpoint: Explain why a healthcare provider would use an inulin test rather than a CrCl or urea clearance test to assess the filtration capacity of the kidneys? *Adapted from Laboratory Guide to Human Physiology, Stuart Ira Fox, WCBrown Normal urea clearance is 64-99ml/min normal urea plasma conc. is 5-25 mg/dl EXPERIMENTAL PROCEDURES Activities 1 & 2 Urinalysis of your own and artificial urine 1. Activity 1: Obtain a urinalysis test strip, and color analysis chart from your lab instructor. Note the time for reading each measurement (make sure you understand what each piece of the test strip is measuring). Take the strip and chart with you to the Toilette, begin urination, place the test stick under the stream of urine so that all of the analysis areas are touched by the urine. Blot excess urine from the stick with some toilette paper. Critically time and take note of the color of each test area compare them to the color chart you were given. When you are done, dispose of your urine stick in the trash can (as well as the chart if soiled). Do not forget to wash your hands when you finish! Analyze your urine measurements what does it indicate about your urinary health?

10 Page Activity 2: You will have access to six unknown urine samples. These unknowns are not real urine, they are simulated urine made in the lab. However, please make sure to wash your hands before and after these tests. You will be provided with Chemstrip urine test strips. For each urine sample, immerse the test strip into the urine sample for no longer than one second. Draw the test strip along the rim of the specimen container to remove excess urine. 3. Apply the test strip to a portion of filter paper (or paper towel) to remove excess urine. 4. For each urine sample compare and record the color values (and corresponding numerical values) indicated on the strip for -specific gravity -ph -glucose -ketones -blood -protein 5. Observe the demonstration of the hydrometers to determine the specific gravity. See Lab 10 report for questions Activity 3. Simulated experiment with urinalysis. A class was divided into the following groups that drank the designated solutions. Group 1: This is the control group would consume no fluids. Group 2: Members of this group consumed 500 ml of distilled water. Group 3: Members of this group consumed 500 ml of a 5% sucrose (table sugar, a dimer of fructose and glucose) solution. Group 4: Members of this group consumed 500 ml of 1 % sodium bicarbonate (also known as baking soda = NaHCO3) solution. Group 5: Members of this group consumed 500 ml of regular coffee (not decaffeinated). - Sample Collection: The members of each group voided (emptied their bladders) prior to beginning the experiment. Some urine from this sample was saved for microscopic analysis described below. At minute intervals following the consumption of the designated fluids members of each group collected the maximal urine volumes possible and performed the required analyses. - Urinalysis 1. With the volumetric containers provided, the volume of each urine sample was determined and recorded. The subjects then performed urinalysis on urine samples at 0 minutes, 30 minutes, 60 minutes, and 90 minutes after the fluids were consumed.

11 Page You are to predict the results of urinalysis for each group (indicate a up for increased amount, a down for a decreased amount and no for no change) -volume -specific gravity -ketones -ph -blood -glucose -protein - Note: we are not looking for right answers here, we are more interested in the logic that you used to come up with your answers (see Lab 10 report)