Expt. 29 Urine Formation

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I. Introduction Urine formation begins with the movement of plasma ultrafiltrate into the kidneys. This plasma ultrafiltrate is an essentially protein-free fluid which passively passes from the glomerular capillaries into the Bowman's space. This process is driven by Starling forces. Glomerular filtration is followed by reabsorption of water and solutes from the different parts of the renal tubules, then by the secretion of selected solutes into the renal tubules. URINE FORMATION The ability of the kidneys to selectively clear waste products from the blood and simultaneously maintain the bodys essential water and electrolyte balances is controlled in the nephron by the following functions: renal blood flow, glomerular filtration, tubular reabsorption, and tubular secretion.

RENAL BLOOD FLOW The renal artery supplies blood to the kidneys. Blood enters the capillaries of the nephron through the afferent arteriole. It then flows through the glomerulus and into the efferent arteriole. Before returning to the renal vein, blood from efferent arteriole enters the peritubular capillaries and the vasa recta and flows slowly through the cortex and medulla of the kidney close to the tubules. The proximal convoluted tubule provides the immediate reabsorption of essential substances from the fluid, and final adjustment of the urinary composition in the distal convoluted tubule. The vasa recta are located adjacent to the ascending and descending loop of Henle in the juxtamedullary nephrons. In this area, the major exchanges of water and salts take place between the blood and the medullary interstitium, which maintains the osmotic gradient in the medulla that is necessary for renal concentration.

GLOMERULAR FILTRATION The glomerulus consists of a coil approximately eight capillary lobes. It serves as a nonselective filter of plasma substances with molecular weights of less than 70,000. Actual filtration process involves several factors including cellular structure of the capillary walls and Bowmans capsule, hydrostatic and oncotic pressures, and the feedback mechanism of the rennin-angiotensin-aldosterone system. TUBULAR REABSORPTION

The cellular mechanisms involved in tubular reabsorption are active and passive transport. Active Transport SUBSTANCE LOCATION Glucose, amino acids and Proximal convoluted tubule salts Chloride Ascending loop of Henle Sodium Distal convoluted tubule Water Urea Sodium Proximal convoluted tubule, descending loop of Henle, and collecting tubules Proximal convoluted tubule and ascending loop of Henle Ascending loop of Henle

Passive Transport

TUBULAR SECRETION Tubular secretion serves two major functions: elimination of waste products not filtered by the glomerulus and regulation of the acid-base balance in the body through the secretion of hydrogen ions.

Foreign substances which cannot be filtered by the glomerulus because they are bound to plasma proteins enter the peritubular capillaries, where they dissociate from their carrier proteins because of strong affinity for tubular cells. The major site for removal of the nonfiltered substances is the proximal convoluted tubule. CONCENTRATION AND DILUTION OF URINE The regulation of plasma osmolarity is accomplished by varying the amount of water excreted by the kidneys. It is due to the response to water deprivation or to water intake. When the osmolality is too low, nervous and hormonal feedback mechanisms cause the kidneys to excrete a great excess of water in urine causing a dilute urine, but removes water from the body to increase the body fluid osmolality back to normal. When the osmolality of body fluids is too great, the kidneys excrete an excess of solutes to reduce the body fluid osmolality again back to normal, but at the same time excreting a concentrated urine. OSMOLAL CONCENTRATION CHANGES IN THE DIFFERENT SEGMENTS OF THE TUBULES Proximal Tubule Osmolality of the fluid remains almost exactly equal to that of the glomerular filtrate, 300 mOsm/L throughout the entire extent of the proximal tubule. Loop of Henle The osmolality rises rapidly because of the countercurrent mechanism. During high concentration of ADH, the loop of Henle osmolality rises much higher than when a dilute urine is being formed because of large quantity of urea that is passively reabsorbed into the medullary interstitium from the collecting ducts. Thick Ascending Limb The osmolality falls to a very low level usually about 100 mOsm/L. Late Distal Tubule, Cortical Collecting Duct, and Collecting Duct The osmolality depends entirely on ADH. In the absence of ADH, very little water is reabsorbed, osmolality remains less than 100 mOsm/L, very dilute urine is formed. In the presence of excess ADH, these segments become highly permeable to water, most of the water is reabsorbed, thus producing a very concentrated urine. TESTS DONE ON URINE SPECIMENS VOLUME Urine volume depends on the amount of water that the kidneys excrete. The amount excreted is usually determined by the bodys state of hydration. Factors that influence urine volume include fluid intake, fluid loss from nonrenal sources, variations in the secretion of antidiuretic hormone and the

necessity to excrete increased amounts of dissolved solids, such as glucose or salts. The normal daily output is usually 1200 to 1500 ml, a range to 600 to 2000 ml is may be considered normal. SPECIFIC GRAVITY Defined as the density of a solution compared with the density of a similar volume of distilled water at a similar temperature. Because urine is actually water that contains dissolved chemicals, the specific gravity of urine is a measure of the density of the dissolved chemicals in the specimen. The kidneys produce urine with specific gravity that ranges from 1.005-1.035. VALUES: 1.106-1.022 normal adults with normal diets and normal fluid intake (24 hr. period) 1.023 random specimen 1.022 no fluids for 12 hrs. overnight 1.026 or higher 24 hrs. without fluid Less than 1.003 minimum specific gravity after a standard water load. pH Along with the lungs, the kidneys are the major regulators of the acid-base content in the body. They do this through the secretion of hydrogen in the form of ammonium ions, hydrogen phosphate and weak organic acids, and by the reabsorption of bicarbonate from the filtrate in the convoluted tubules. A healthy individual will usually produce a first morning specimen with a slightly acidic pH of 5.0 to 6.0. The pH of normal random samples can range from 4.5 to 8.0. Consequently, no normal values are assigned to urinary pH, and it must be considered in conjunction with other patient information. Protein Of the routine chemical tests performed on urine, the most indicative of renal disease is the protein determination. The presence of proteinuria is often associated with early renal disease. Normal urine contains very little protein, usually less than 10mg/dl or 100 mg per 24 hrs. excreted. This protein consist primarily of low molecular weight serum proteins that have been filtered by the glomerulus and proteins produced in the genitourinalry tract. Glucose Determination of glucose in urine is valuable in the detection and monitoring of diabetes mellitus. Under normal circumstances, almost all the glucose filtered b the glomerulus is reabsorbed in the proximal convoluted tubule. Should the blood level of glucose become elevated, the tubular transport of glucose ceases and glucose appears in urine. The renal threshold for glucose is approximately 160-180 mg/dl. II. Materials Wide-Mouth Collecting Bottles (1L capacity) Graduated Cylinders 1L 0.9% NaCl Solution

1L 5% Glucose 1L Distilled Water Urine dipstick determination for pH and specific gravity III. Methodology Preparation Four subjects from the group were assigned to do the procedure. They were advised to eat a light meal the evening prior to the experiment, after which they will be placed on NPO (nothing per orem) eight hours prior to the start of the experiment. The types of food and fluid taken in prior to the experiment were noted. The materials used were prepared. Wide mouth collecting bottles or beakers with one liter capacity were used for urine collection. Graduated cylinders were used for approximation of the urine volume. Urine reagent strips were used for determination of pH and specific gravity. Specimen Collection Specimens must be collected in clean, dry, leak- proof containers. It should have a wide mouth to facilitate collections from female patients and a wide, flat bottom to prevent overturning. They should be made of clear materials to allow for determination of color and clarity. All specimens must be properly labeled with the subjects name with the date and time of collection in it. Specimen Handling The fact that a urine specimen is so readily available and easily collected often leads to laxity in the treatment of the specimen after its collection. Changes in urine composition take place not only in vivo but also in vitro, thus necessitating correct handling procedures after the specimen is collected. A specimen must be tested within two hours. First Sample The four subjects were assigned to collect the first urine sample. It was collected at the start of the laboratory procedure. The first voided morning urine was not used in the experiment. The time of collection was noted. The first sample of urine collected was subjected for physical and chemical examination such as: volume determination, specific gravity, pH, sugar content and protein content. Second Sample After thirty minutes, the second sample was collected by the same subjects. The samples were also subjected to physical and chemical examination that was done on the first sample. As soon as the second sample was collected, the subjects drink one liter of the fluid assigned to them: distilled water, 5% glucose, 0.9% sodium chloride. The fourth subject will drink nothing. Third Sample The third sample was c

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