Laboratory 10 The primary function of the kidney is to maintain a constant level of blood solute concentration (or blood osmolarity). Additionally, the kidneys are responsible for excretion of metabolic wastes (urea, ketones, and metabolic acids) and reabsorption of essential molecules (i.e. glucose, calcium, amino acids). The way the kidney maintains blood osmolarity is complex and requires both an understanding of kidney microanatomy and of basic principles of osmosis. The following three pictures outline the salient features of kidney anatomy. Collectively this is the NEPHRON The physiological process that generates URINE starts in the CORTEX. Urine then passes to the MEDULLA (the collecting ducts of the pyramids) then to the renal pelvis then to the URETER then it is stored in the URINARY BLADDER (not pictured) until you MICTURATE, then urine gets outside through your URETHRA! The physiology of Urine production Focus on the third picture (the Nephron) for the following discussion. The picture of the NEPHRON contains three basic parts 1) Glomerulus 2) The Renal Tubule (with many named parts) 3) The Peritubular capillary system (unlabelled but it is the black tube -circulatory system- wrapped around the renal tubule!) 1
The GLOMERULUS is a tightly woven capillary bed stuffed within the RENAL CAPSULE. Thus, the glomerulus is the interface between the renal arterial supply and the renal tubule where urine is formed. An extreme amount of pressure in the glomerulus results in the squeezing of blood FROM THE CAPILLARY TO THE CAPSULE! The liquid/fluid entering the capsule consists of water and dissolved solutes; plasma proteins and blood cells remain in the capillaries because they are too big to fit through the capillary walls. The liquid/fluid entering the capsule is called FILTRATE and is isotonic to normal blood (300 mosm/l)! The filtrate moves down the length of the renal tubules where the NEPHRON allows reabsorption of some molecules and secretion of others. Make sure you understand the difference between reabsorption and secretion! Reabsorption occurs when a molecule moves FROM the renal tubule TO the peritubular capillaries! Secretion occurs when molecules move FROM the interstitial fluid (located between the peritubular system and the renal tubule) INTO the renal tubule! When the filtrate reaches the end of the renal tubule and collecting tubule, it changes names to URINE and then flows into renal pelvis and on from there! Because both reabsorption and secretion occur along the length of the renal tubule the osmolarity of the FILTRATE is very, very different from the osmolarity of the URINE. Which molecules are reabsorbed, how are they reabsorbed, and why does this result in concentrated urine? Many different ions and molecules are reabsorbed during the process of urine formation; however, those with a major role include Sodium (Na + ), Chloride (Cl - ), and Water (H 2 O). The following diagram shows how ions/molecules move during urine formation. Focus on how NaCl (actually moving as Na + and Cl -!) and water move! 2
As the filtrate (300 mosm/l) moves into the proximal convoluted tubule, Na + ions are actively transported into the interstitial fluid. Three things happen as a consequence of this active Na + transport: 1) Cl - follows Na + due to electrical attraction; 2) With Na + and Cl - movement, the interstitial fluid becomes more concentrated and 3) Water molecules leave the renal tubule and enter the interstitial fluid via Osmosis. Thus, active transport of Na + and the following of Cl - dfiffussion create a hypertonic interstitial fluid that helps the body reabsorb water (passively via OSMOSIS). When the filtrate reaches the descending limb of the loop of Henle, 70% of the filtrate volume was reabsorbed in the proximal convoluted tubule and the filtrate is isosmotic to the interstitial fluid at this point! Since water continues to move out as the filtrate travels through the descending limb of the loop of Henle the filtrate becomes more concentrated as it approaches the bent portion (the loop ) of the Loop of Henle. Next, the concentrated filtrate (up to 1200 mosm/l) moves into the ascending limb of the loop of Henle. Since the filtrate is SO concentrated here, Na + diffuses from the ascending limb of the loop of Henle into the interstitial fluid (Cl - follows due to electrical attraction) and both ions are then reabsorbed by the peritubular capillary! Movement of the filtrate through the loop of Henle ensures that ions are reabsorbed and water is conserved! Desert animals and human from arid environments have a REALLY REALLY long loop of Henle to increase water and ion reabsorption! The ascending limb of the loop of Henle is impermeable to water, so water does not follow the ions into the interstitial fluid. Therefore, by the end of the loop of Henle the filtrate is dilute (100 mosm). So why is urine so concentrated? The interstitial fluid is concentrated with lots of Na + and Cl -. As the dilute filtrate passes through the distal convoluted tubule and into the collecting tubule, water moves into the interstitial fluid by OSMOSIS. The interstitial fluid is hypertonic to the filtrate in the tubule.thus by Osmosis water will move towards the hypertonic solution! This makes the end product of the collecting tubule SUPERCONCENTRATED URINE! The body has internal mechanisms to alter water and ion balance in response to dehydration and/or exercise. We will not focus on these in detail. However, diuretics (such as Caffeine!) are drugs that mimic the effects of water balance within the body. Specifically, diuretics enter the proximal tubule and PREVENT Na + FROM BEING REABSORBED! Think about the cascading effects of this diuretic. Increasing Na + solute concentration in the proximal tubule will DECREASE the movement of water to the interstitial fluid (less water and Cl - will follow the active transport of Na +, thus more will be retained in urine). This will result in less concentrated urine and a more dehydrated internal environment of the body (less water reabsorbed). This is why you NEVER NEVER NEVER drink pop when you are dehydrated! When you drink different fluids, you body releases hormones which alter the normal function of the kidney! In this lab, you will examine how different fluids alter normal kidney function by examining 1) amount of urine output and 2) the characteristics of urine output. By seeing how urine changes when consuming different beverages you should be able to work back to the level of the nephron and figure out how normal kidney function was altered! Objectives: Based on these results you should: 1) know how caffeine and NaCl alters urine output, and 2) understand why changes in renal function GENERATE the observed changed in urine output For example, when you drink Gatorade, you should see a decrease in urine output.based on your understanding of renal function you should be able to reason HOW RENAL FUNCTION CHANGES TO RESULT IN DECREASED URINE OUTPUT and changes in chloride clearance!!! 3
Each group of 4 will take test beverages 1-4. These beverages contain the following 1) DIET Caffeinated Pop (I think you southerners call this stuff Soda?) 2) DIET Decaffeinated Pop 3) Water 4) Gatorade At your bench you should have four graduated cups. You will urinate into these! In addition, you should have a rack of test tubes. You will transfer urine into these, as necessary, to examine different properties. Procedure: 1) You will urinate at 0, 30, 60, and 90 minutes. 0 minutes will be urination BEFORE you drink your test beverage. Each group member will drink ONLY ONE test beverage. You MUST drink it ALL during the five minutes after you come back from the restroom with your sample (time 0). Then you will collect as much urine as possible at each of these times! 2) You should measure the following for each time a.- Color Describe the color you see when placing the graduated cup on a white piece of paper. b.- Urine Volume Use the graduated cylinder to measure the volume of urine output Make sure to respect the MENISCUS! c.- Specific Gravity with Urinometer and Float 1) Fill urinometer cylinder ~ 2/3 full, remove foam with filter paper 2) Figure out how to read the marking on the float! 3) Insert the float into the urine and make sure it is free floating 4) Read the marking on the float at the level of the meniscus d.- ph 1) Dip the ph paper in the urine 3 times and then shake away any excess urine 2) Let sit for ~ 1 minute and compare color on wheel to color of paper e.- Chloride concentration 1) Save ~ 10 drops in a test tube of urine from each test time! 2) Add 1 drop of Potassium Chromate to each tube 3) Gently Swirl 4) Add silver nitrate drop by drop to each tub while swirling 5) Count the number of drops required to get a BRICK RED PRECIPITATE. The number of drops is equal to the grams of NaCl per Liter of Urine! f.- Clearance Use the following equation to measure clearance of Chloride: Plasma Chloride Concentration is normally 3.5 g/l (Urine Volume)(Chloride Concentration) Clearance of Chloride = ---------------------------------------------------------- (Plasma Chloride Concentration)(Time) 4
Clearance tells you how much plasma can be cleared of the substance over time! Its an important clinical tool to test for normal renal function!!!!!!!! Make sure all you units Jive! Liters, grams, and minutes! I drank and this is what I got: 0 min 30 min 60 min 90 min Color Volume Specific Gravity ph Chloride Conc (g/l) Chloride Clearance Graph Volume and Chloride Clearance VS. time 5
Results of each Treatment, note how each variable changed over time in each treatment! Color Diet Caffeinated Diet Decaff. Water Gatorade Volume Specific Gravity ph Chloride Conc (g/l) Chloride Clearance 6