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1 Fall 12 OSMOTIC REGULATION OF THE RENAL SYSTEM: Effects of fasting and ingestion of water, coke, or Gatorade on urine flow rate and specific gravity Dorette Franks The purpose of the physiology experiment was to study the osmotic regulation of the kidneys in order to understand fluid balance and hormone regulation during fasting and fluid consumption. Urine flow rate and corrected specific gravity was observed and measured over the course of specific timed intervals by sixty- two students during fasting and following ingestion of water, coke, and Gatorade consumption. Further testing of blood ketone, glucose, protein, and ph with a Labstix strip was also performed. Physiology 611, San Francisco State University 2012 Dorette Franks

2 Introduction The human body contains two kidneys, a bean- like structure located in the upper dorsal compartment of the abdominal cavity. The kidney has numerous functions, but its main function is to regulate osmotic extra cellular fluid balance by way of sodium and water concentration, which correlates to plasma volume and blood pressure. The kidney also serves as regulation for other ions, such as potassium or chloride, excretion of metabolic end products and foreign wastes (urea and drugs), and acid- base maintenance. The kidneys are highly vascularized, receiving its blood supply from the renal artery. The functional unit of the kidney is the Nephron. Its role is to regulate plasma volume, osmolarity, and ph by way of glomerular filtration, tubular reabsorption, and tubular secretion. Unfiltered, oxygen rich blood passes through the kidney into the nephron via the glomerulus. The role of the glomerulus is to filter protein- free plasma to the renal tubules for reabsorption via the Bowman s capsule. Within the renal tubules, the proximal convoluted tubule (PCT) reabsorbs a majority of the filtrate into the peritubular capillaries. Reabsorption of larger molecules, such as glucose, amino acids, and creatine, occur by active transport, while reabsorption of water occurs through osmosis. Sodium ions are reabsorbed by active transport in the distal convoluted tubule (DCT). Reabsorbed solutes and water enter back into the blood stream via the peritubular capillaries and exit the nephron through the efferent arteriole. Substances the body does not reabsorb at the peritubular capillaries or distal convoluted tubules are actively secreted. Excess plasma fluid, such as water, sodium, and hydrogen ions that disturb the equilibrium and osmotic balance of the body will be excreted as urine. 2

3 Filtration, reabsorption, and secretion operate under hormonal regulation are one of the foundations of homeostasis that either reduces or increases blood pressure, volume, and plasma osmolarity relative to the external environment (of the extracellular fluid). The primary hormones and their pathways are: anti- diuretic hormone (ADH), aldosterone (regulated by the renin- angiotensin- aldosterone system [RAAS]), and atrial natriuretic peptide (ANP). If blood volume and blood pressure is low, the fist thing the kidneys want to do is correct plasma osmolarity. Baroreceptors on the aortic arch detect a change in blood pressure, and osmoreceptors in the hypothalamus detect changes in plasma osmolarity. When plasma osmolarity is low, the hypothalamus sends a signal to the post pituitary to release the hormone, ADH. ADH targets receptors on the DCT and collecting duct to increase water reabsorption by increasing aquaporins. As plasma is reabsorbed, urine flow rate decreases increasing blood volume and pressure and decreasing plasma osmolarity. When blood volume, pressure and osmolarity are returned to normal, the system shuts off (aquaporins are inhibited) and urine flow rate returns to normal. Aldosterone is released by the adrenal cortex of the kidneys. It is signaled by low blood volume, pressure and low extra cellular fluid sodium concentrations in the distal convoluted tubule. The RAAS pathway regulates release of aldosterone. The juxtaglomerular cells that trigger the release of renin sense low sodium concentrations in the DCT. Renin travels in the blood stream and targets the liver to convert angiotensinogen into angiotensin I and then into angiotensin II. Angiotensin II targets the adrenal cortex, releasing aldosterone. Aldosterone causes vasoconstriction on the afferent arterial and results as increase water reabsorption, increase blood pressure (thus increasing sodium concentration) and decreasing urine flow rate. The path is inhibited by the increase in pressure, plasma and sodium concentration, 3

4 regulated by ANP. An increase in blood pressure sends a response to the arterial cells to secrete ANP. ANP targets both the hypothalamus and post pituitary gland to decrease ADH secretion, increase water reabsorption, and increase water excretion. It also targets the juxtaglomerular cells to inhibit renin secretion and reduce sodium reabsorption. Further, the release of ANP decreases aldosterone to increase sodium excretion and promotes vasodilation of the afferent arteriole to increase glomerular water and sodium filtration. The primary purpose of this experiment is to observe the human body kidney function relative to osmotic regulation and fluid balance maintenance as it correlates to hormonal regulation. Additionally, analyzing and comparing the urine flow rate and specific gravity of San Francisco State University student subjects during a fasting state followed by ingestion of water, Coke or Gatorade leads to understanding the primary purpose. Finally, this experiment offered student subjects to test urine samples using a Labstix strip in order to determine urine ph and the presence of blood, glucose, ketone, and protein in the urine sample. My hypothesis is that the students who drank Gatorade upon a 5- hour fasting state will have the highest urine flow rate 90 minutes prior to ingestion (T=90). Methods Prior to the experiment, students randomly volunteered to participate in one of the following group in order to determine urine flow rate and specific gravity: non- drinking, water, coke, or Gatorade. Non- drinkers would remain in a fasting state and consume no other fluid during the course of the experiment, water drinkers would drink only water (which contains no solutes), coke drinkers consumed coke containing caffeine and sugar 4

5 (glucose), and those who drank Gatorade ingested electrolytes and some glucose as contained within the beverage. Since the coke- study group was low, I decided to make this a personal experiment joined the coke drinkers. The amount of fluid intake was determined by individual body weight using calculations provided later in this study. To ensure accurate results, students were instructed to refrain from exercise, excessive, excessive alcohol, salt, caffeine, and sugar intake the night prior to the experiment. Students fasted five hours prior to the experiment and were permitted to drink one cup (8 oz.) of water during the first two hours of fasting in order to avoid prolong dehydration. One hour prior to class, students voided their bladders completely. Upon arriving at class at 2:10 PM, students obtained two urinary cups, went to the nearest bathroom and again void their bladders completely. This signaled the start of the experiment known as T = 0 (where T is time). Upon returning to class, individual urine specimens were briefly set aside and consumption of selected group fluids commenced. Individual fluid consumption is based upon body weight as demonstrated by the following equation: ml. of fluid intake = [Body weight (lb.) x 7 ml/lb.] x 0.80 Individual calculation: 784 ml. of fluid intake = [141 lb. x 7 ml.lb.] x 0.80 = 790 ml. of coke. After consumption of fluids, students in each group (non drinking, water, coke, Gatorade) measured their individual urine flow rate and calculated specific gravity follows every half hour until 120 minutes (as T = 0, T = 30, T = 60, T = 90, T = 12) and entered the results on a spreadsheet specific to the group and time interval. Individual urine flow rate is calculated as follows: Urine flow rate = milliliters urinated/minutes since last urination Individual calculation: 29 ml. urinated /66 min. = 0.44 ml/min (flow rate) 5

6 During each thirty- minute interval, students measured each new urine sample by determining temperature and calculating urine specific gravity. Specific gravity of a liquid is a unitless measurement used to determine the density of a substance as compared to the specific gravity of water. The specific gravity of water is Water containing solute will have a density greater than 1.000, where normal ranges for urine as between to To measure urine specific gravity, a urinometer (a hydrometer specific to measuring urine density) containing a scale of small markings was used. The urinometer sinks in the individual test urine proportional to the specific gravity of urine and a measurement can be read. The measurement obtained is used to calculate the urine specific gravity. However, since the urinometer is calibrated to 15 C, another equation was used to adjust for this calibration to obtain the corrected specific gravity of urine. For every 3 degrees above 15 C of the urine temperature, add A sample of this calculation is below: At T=0, urine temperature = 27 C; measured specific gravity = C - 15 C = 12 C 12 C 3 C = 4 C 4 C x = (specific urine gravity) = Corrected urine gravity = To determine the ph as well as the presence of blood, glucose, ketone, or protein the blood, a Labstix strip containing five separate reagent areas for each was immersed in fresh urine and then immediately removed. Immediate removal of the Labstix strip inhibits cross contamination of adjacent reagent areas. Excess urine was removed by placing the strip against a piece of paper towel. Results of the reagents were read by comparing the change in color of the reagent on the strip to the sample diagram on the Labstix bottle. Change in color varied between each reagent but indicated either negative or positive for the presence of the reagent in the urine. 6

7 Results Class Average Urine Flow Rate vs. Time Ave. URF (ml/min) T = 0 T = 30 T = 60 T = 90 T = 120 Time of Void (min.) Non- drinking Water Coke Gatorade Figure 1: Average urine flow rate versus time void; x- axis equal s time of void in 30- minute intervals; y- axis represents average urine flow rate (ml./min.). The class average urine flow rate versus time (shown in figure 1, above) indicates that the students who consumed water had overall highest overall, peaking at T = 90 over those who consumed nothing, coke or Gatorade. Class Average Corrected Specific Gravity vs. Time Ave. Corrected Speci7ic Gravity T=0 T=30 T=60 T=90 T=120 Time of Void (min.) Non- drinker Water Coke Gatorade Figure 2: Class average corrected specific gravity versus time; x- axis equals time of void in 30- minute intervals; y- axis represents average corrected specific gravity. 7

8 The class average for corrected specific gravity versus time (shown in figure 2, above) indicates that the students who remained fasting during the study and remained in a fasting state had highest urine specific gravity over students who consumed water, coke, or Gatorade. Reagent/Indicator Labstix Result Blood Negative Ketone Negative Glucose Negative Protein Trace ph 6.5 Table 1: Individual Labstix strip test for the presence of reagent in a fresh sample of urine. Discussion Given that Gatorade contains both electrolytes (sodium) and sugar, which make for a highly concentrated solution as compared to water or coke, I hypothesized that those whom consumed Gatorade upon a five- hour fasting state would have the highest urine flow rate at T = 90. However, according to the class experiment, at T = 90, those who drank water contained the highest average urine flow rate. Despite the outcome of my hypothesis being incorrect, my hypothesis is correct and I will demonstrate the validity of my hypothesis and also explain the outcome of the class average results as indicated by Figure 1. At T = 0, the class average of urine flow rate measured between ml./min. with a difference of 0.57 ml./min. Over the duration from T = 0 to T = 90, the difference in class average urine flow rate continued to increase as follows: T = 30, 1.29 ml./min.; T = 60, 5.79 ml./ min.; T = 90, 7.66 ml./min. As of the increase in urine flow rate from T = 0 to T = 8

9 90 indicates that at T = 0, students entered the experiment under the very similar fasting states, which serves as the baseline for this experiment. At T = 0, without fluid ingestion for five hours, the hormone ADH is high and water being reabsorbed in the PCT (proximal convoluted tubule) via aquaporins. Fasting induced a decrease in plasma volume, a decrease in blood pressure and an increase in plasma osmolarity. Prior to ADH levels increasing, baroceptros and osmoreceptors, which regulate plasma osmolarity and blood pressure, received a stimulus indicating low blood pressure and high osmolarity. Osmoreceptors in the hypothalamus send signals to the post pituitary to secrete ADH, which targets the DCT and collecting duct to increase aquaporins for water reabsorption. Due to the attraction in opposite charges, sodium ions from the DCT follow the water and blood pressure decreases. Gatorade, a liquid high in sodium and glucose, is ingested directly after the first excretion of urine measurement at T = 0. At the time Gatorade was ingested, plasma volume and blood pressure were low due to the hormone regulation during fasting. The decrease in blood plasma, plasma volume, and sodium levels stimulated the release of aldosterone from the adrenal cortex to increase blood pressure and plasma, and increase sodium concentration levels. The decrease in blood pressure signals a release of renin from the juxtaglomerular cells into the blood stream to turn on the RAAS pathway. Renin travels through the blood to the liver where it converts angiotensinogen to angiotensin I then to angiotensin II, which additionally increases water reabsorption and increases blood pressure. Over T = 0 to T = 60, the body continues to absorb fluid At T = 75, high glucose and sodium concentration levels are detected in the blood for those who drank Gatorade. Gatorade increases osmolarity, blood pressure and volume 9

10 because it is highly concentrated, more so than coke and water, due to its sodium and glucose content. The increase in osmolarity sends signals to the post pituitary to continue secreting ADH, thus increasing aquaporins and water reabsorption. The increase in blood pressure and volume signals the secretion of ANP, which is stimulated when the heart is stretched as a result of high blood volume. ANPs job is suppress the release of ADH, renin, and aldosterone, and to inhibit sodium reabsorption. ANP will promote sodium secretion and causes vasodilation on afferent arterioles. At T = 90 for Gatorade, urine flow rate is at its highest, the result of the ANP pathway turning off ADH, renin, and aldosterone. Although, the group that drank coke also had a high urine flow rate, because urine does not contain sodium, blood pressure would not have been raised as high as it was for sodium. Further, the concentration of coke is lower than that of sodium, which stands to reason that Gatorade would have a higher urine flow rate over coke. The results of the student experiment indicated that at T = 90, water, then coke, followed close by Gatorade contained the highest urine flow rate. Non- drinker urine flow rate remained very low due to the continued fasting state and reabsorption of water. The effects that could have caused the outcome of water having a higher urine flow rate over Gatorade could be many. First, when calculating the amount of fluid to ingest, students could have calculated their weight too high that would result in an increase in water consumption relative to body weight. Secondly, maybe some students exercised vigorously which would cause an increase in water loss upon entering a fasting state. Further, calculation errors can occur due to human mistakes if rushed to input information, or by putting information on the wrong line. As the results of this study shoes, there were mistakes made, but the overall objective of the study was met to study osmotic regulation 10

11 of the renal system and the effect of fasting and ingestion of water, coke, and Gatorade on urine flow rate and specific gravity. 11

12 References Howley, E. T., & Powers, S. K., (4 th Ed). (2009). Exercise Physiology: Theory and Application to Fitness and Performance. New York, NY: McGraw Hill Vingerhoets, V.,& de Tribolet, N.,(1988). Hyponatremia Hypo- osmolarity in Neurosurgical Patients. "Appropriate Secretion of ADH" and "Cerebral Salt Wasting Syndrome" Retrieved on October 16, 2012 from

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