URINALYSIS LAB

ZOOLOGY 142L

INTRODUCTION

The urinary system is composed of the kidneys, the bladder, and ducts (ureters and urethra). Although the urinary system is primarily associated with the production of urine with its excretion of nitrogenous waste (urea, uric acid), it serves many other functions. These include regulating blood volume and pressure, pH (hydrogen ion concentration), osmolarity of body fluids (overall concentration of all solutes), and secretion of regulating hormones (erythropoietin, calcitriol), and enzymes (rennin). Examining urine supplies a broad range of information about the normalcy of function for both this system and others systems of the body. Urinalysis is one of the most important and widely used clinical assessment methods employed in the medical field.

The kidney and its blood supply are the main functional components of the renal (urinary) system. The renal artery delivers the blood, and the materials put in the blood by all the tissues of the body (water, plus nutrients, waste products, hormones, etc.), to the kidney for processing (refer to figure below). The material processing is provided by the million microscopic nephrons (functional units) that are located partly both in the cortex and medullary (pyramid) regions of the kidney. The materials flowing into the nephron that are still needed by the body are returned to the blood, and the waste products are allowed to flow from the nephron into the renal pelvis, and then into the ureters, bladder, urethra, and out (in a controlled manner).

The nephron has three basic functional areas along a continuous tube, the glomerulus, tubules (very small tubes), and collecting duct. Associated with the nephron are vessels that first deliver blood to the nephron, participate in the processing actions, and then retrieve the materials that are still needed in the body. The nephron processes the material from the blood with three different functions, filtration, reabsorption, and secretion. The glomerulus filters materials smaller than an albumin protein (the smallest protein in the blood) from the blood into the tubules. The tubules (proximal convoluted, loop of the nephron, and distal convoluted tubules, and collecting duct) reabsorb all the water and molecules in the filtrate that are still needed in the body. The tubules cells (that are the walls of the tubules) can also secrete some molecules from the adjacent blood vessels (peritubular capillaries) into the tubules, and so add them to the filtrate. The water and molecules that remain in the tubules and collecting ducts pass out into the renal calycies and pelvis on the way out of the body.

The processes of the nephron at first seem inefficient, filtering out a lot of water and materials, and then reabsorbing about almost all of it. However, reabsorbing the proper (still needed) materials (molecules, ions, atoms) requires recognition by receptor proteins that are on the tubule cell membranes. It is obvious that cells will make receptors for the molecules they use all the time, but much less likely that tubule cells will make receptors for things they do not need, or have never encountered before.

The first step in processing materials by the nephron starts with filtration by the glomeruli (~ one million per kidney). About 16% of the blood that passes into the afferent arteriole of the glomerulus is actually filtered into the glomerular capsule (the filtration fraction). The remaining 84% of the blood continues out through the efferent arteriole, into the peritubular and vasa recta capillaries, and back into the general circulation. The 16% of blood volume that is filtered by the glomerulus turns out to be about 180-200 liters per day, or 125 ml/min, and is called the Glomerular Filtration Rate (GFR), which is the volume that enters the proximal convoluted tubule (PCT). Since the urine production rate is only about 2 liters per day that means 99% (~198 l/d) of the filtrate is taken back into the blood before it reaches the end of the collecting ducts. How much water and material that is taken back into the blood depends on the action of the cells that make up the tubule walls (tubule cells).

Of the ~125 ml/min (= GFR,~200 l/day) of the filtrate that enters the PCT about 90% (~114 ml/min, ~180 l/day) is reabsorbed by the PCT, and Distal Convoluted Tubule (DCT) cells, and so only about 10% (~12.5 ml/min,~20 l/day) reaches the beginning of the collecting duct. This is accomplished by “obligatory water reabsorption”, which occurs when solutes (mainly Na+) are reabsorbed by tubule cells, and water follows by osmosis. The fraction of the ~12.5 ml/min (20 l/day) of water that becomes urine (usually ~ 2 l/day) depends on the action of Antidiuretic Hormone (ADH) on the ‘principle’ cells that make up the walls of the collecting duct (CD).

The function of the ADH system is to control (regulate) the blood volume, osmolarity, and consequently blood pressure (which produces blood flow throughout the body). The blood pressure depends on the volume of blood (mainly water) pushing against the vessel walls. The volume of blood in the vessels depends on the concentration of all the solutes (osmolarity) that stay only in the blood vessels (mainly albumin proteins). Remember, water moves (diffuses) to the area that has the highest concentration of solutes by osmosis (diffusion of water). The total number (moles) of all dissolved atoms and molecules (solutes) is called osmolarity (moles solute/l = OsM, millimoles solute/l = mOsM). Note, that concentration (moles solute/l) is the proportion, the fraction, the ratio, of the amount of solute (moles) to the amount of water (liters). When the water moves into an area by osmosis it increases the volume and pressure (= osmotic pressure), that stretches the walls of the vessel, and so increases the blood pressure. Blood vessels can also actively constrict on the blood volume with smooth muscle to increase pressure.

The ADH system is primarily an interaction between the hypothalamus and collecting duct cells of the kidney, and also includes drinking behavior, as well as actions on blood vessels and sweat glands. All of these interactions can affect the blood volume and pressure. Diuresis is the production of urine volume, so Anti-Diuretic Hormone works against (anti-) the production of urine volume (by reabsorbing the filtrate, and preventing it from exiting the collecting duct as urine). The “controlled condition” of the ADH system is the blood osmolarity and pressure. 300 mOsM (300 millimoles of solute/liter = osmotic pressure value) is the optimum osmolarity of solutes in the blood for the best (optimum) operation of the body as a whole. The blood pressure (~90 mmHg average) is also a directly controlled condition, and is reciprocally related to osmolarity, in that if osmolarity is high (due to low water volume, in the moles solute/liters water, ratio) the blood pressure is low. However, the body is more sensitive to changes in osmolarity, and so regulates it more tightly (smaller variations). As a result, the actual blood pressure also has less variation (on the average) around the optimum value.

The following are the components and effects of the Anti-Diuretic-Hormone (ADH) negative feedback control (regulatory) system (see figure below)in response to dehydration:

1)controlled condition=blood osmolarity (mOsM) [and blood pressure]

2)receptors = hypothalamic osmoreceptor [baroreceptor] cells

3)input=changes inside the osmoreceptor [baroreceptor] cells

4)control center=hypothalamic osmoreceptor [baroreceptor] cells

5)output=ADH (from posterior pituitary gland)

6)effectors=kidney collecting duct principal cells

7)effect=a)principal cells become more permeable with high ADH, and more water in CD is reabsorbed back into blood, and less water is lost in urine (= anti-diuresis)

b)relatively more solute is excreted than water, [solute] > 300 mOsM

8) result=the amount of solute decreases more than the amount of water [moles solute/l water] and so the osmolarity [moles solute/l water] eventually returns to 300 mOsM = controlled condition returns to optimum level

6)effectors=hypothalamic Thirst Center cells

7)effect=a)high ADH increases the sense of thirst

b)more water is consumed (behavior)

8) result=the amount of solute remains about the same (> 300 millimoles), but the amount of water increases [~moles solute/l water] and so the osmolarity [moles solute/l water] eventually returns to 300 mOsM, controlled condition returns to optimum level

6)effectors=sweat glands

7)effect=a)high ADH inhibits perspiration

b)less water is lost through sweat

8) result=more water is retained in the body, which limits increases in blood solute concentration

6)effectors=vascular smooth muscle cells

7)effect=a)high ADH stimulates contraction of vascular smooth muscles

b)vessels constrict on the existing blood volume

8) result=blood pressure rises back toward the optimum level (~90 mmHg), controlled condition returns to optimum level

There are three overall conditions that the ADH system responds to;

1)over-hydration ([solute]<300mOsM = low; BP>90mmHg = high)

2)eu-hydration([solute]=300mOsM = normal; BP=90mmHg = normal)

3)dehydration ([solute]>300mOsM = high; BP<90mmHg = low)

Response to Over-hydration

In over-hydration the amount of solute is too low, and/or the amount of water is too high ([solute]<300mOsM), and the BP is too high (>90mmHg) because the increased volume of water stretches the vessels (like a balloon). The kidneys need to get rid of more water and reabsorb more solute from the CD so that the ratio of solute to water ([solute]) will return to normal (300mOsM). The kidneys can do this by producing a higher volume (and rate, ml/min) of urine, with a solute concentration of that is less than in the blood ([solute]urine < [solute]blood < 300mOsM). This is referred to as the Production of Dilute Urine (<300mOsM). This way, each ml of urine has more water in it than a ml of blood, so more water than solute is being dumped. Therefore, in the blood the amount of solute is decreasing slowly, whereas the amount of water is decreasing faster, and eventually the ratio goes back to 300mOsM. This process is necessary since the body cannot get rid of water without any solute, or solute without water. As the volume of water decreases the blood pressure (BP) also decreases.

Production of Dilute Urine (<300mOsM); Process

All the processes working to get the body to go from a state of over-hydration back to normal hydration (get rid of more water than solute) can be summarized by the LOW FIVE:

1)[solute]blood < 300mOsM = LOW (over-hydration)

2)ADH output from posterior pituitary = LOW (less than normal)

3)permeability of the collecting ducts (CD) to water = LOW

4)reabsorption of water from the CD = LOW (more water stays in the CD to become the urine)

5)[solute]urine < 300mOsM = LOW < [solute]blood

Production of dilute urine is possible because more solute is reabsorbed from the filtrate by the Ascending Loop tubule cells than water, so the osmolarity entering the DCT is ~100 mOsM (see figure below). This is very dilute compared to the osmolarity of the blood (300mOsM normally). If none of the water is reabsorbed as the filtrate passes through the CD (permeability to water is low), then when the filtrate passes out the end of the CD as urine it will have the same solute concentration (~100 mOsM). If no water, but some solute, is reabsorbed as the filtrate passes through the CD then the urine can be even more dilute (~65 mOsM).

The lower the ADH, the less of the 12.5 ml/minute (~20 l/day) of water exiting the DCT is reabsorbed as it passes through the CD to become urine, and therefore the higher the urine production rate (ml/min), with a maximum of 12.5 ml/min.

Production of Concentrated Urine ([solute]>300mOsM)

In dehydration the amount of solute is too high, and/or the amount of water is too low ([solute]>300mOsM), and the BP is too low (<90mmHg, due to low water volume). The kidneys need to get rid of more solute, and reabsorb more water from the CD so that the ratio of solute to water ([solute]) will return to normal (300mOsM).

All the processes working to get the body to go from a state of dehydration back to normal hydration (get rid of more solute than water) can be summarized by the HIGH FIVE:

1)[solute]blood > 300mOsM = HIGH (dehydration)

2)ADH output from posterior pituitary = HIGH (more than normal)

3)permeability of the collecting ducts (CD) to water = HIGH

4)reabsorption of water from the CD = HIGH (less water stays in the CD to become the urine)

5)[solute]urine > 300mOsM = HIGH > [solute]blood

Production of concentrated urine is possible even though the osmolarity entering the DCT is ~100-200 mOsM (see figure below). If a lot of the water is reabsorbed as the filtrate passes through the CD (permeability to water is high), then when the filtrate passes out the end of the CD as urine it will have a higher solute concentration (>100-200 mOsM). A concentration gradient of solutes is maintained in the renal pyramid just outside of the CD, by what is called the “counter-current mechanism”. The solute concentration gradient is about 300 mOsM on the renal cortex side of the pyramid, and increases to as much as 1200 mOsM at the lower, apex, where the end of the CD is located (papillary duct, see figure below).

When the ADH levels are high, the permeability of the CD wall is high, and so water diffuses by osmosis from the lower concentration area of solute in the CD (~100-200 mOsM) to the area of higher solute concentration (~300-1200 mOsM) just outside the CD. The higher the ADH levels the higher the permeability of the CD wall to water, the faster water can diffuse out of the CD, and so the solute inside can come to equilibrium with a higher solute concentration outside CD, as the filtrate passes along the CD. The water that is reabsorbed enters the vasa recta capillaries and is returned to the general circulation to decrease the solute concentration back toward normal (300mOsM).

The higher the ADH, the more of the 12.5 ml/minute (~20 l/day) of water exiting the DCT is reabsorbed as it passes through the CD to become urine, and therefore the lower the urine production rate (ml/min).

URINALYSIS EXPERIMENT QUESTION

A 53 year sedentary male, has a BMI of 32, a 37% body fat, a blood pressure of 142/92, a total cholesterol level of 230 mg/dl, a HDL cholesterol level of 27 mg/dl, a LDL cholesterol level of 175 mg/dl, and Triglyceride level of 130 mg/dl. The urinalysis measured a positive presence of glucose in the urine, positive for ketones, a specific gravity of 1.042, a pH of 4.6, and a urine production rate of 16.7 ml/min. Are the measured values normal? What do the measurements indicate?
URINALYSIS METHODS

Urinalysis is the physical, chemical, and microscopic examination of urine. The protocols are designed to detect and measure various structural elements, physical properties, and chemical compounds that are in the urine and thus urinalysis may be used as a screening procedure for disease or dysfunction. Urinalysis may be clinically recommended as part of a physical examination, if there are signs or history of diabetes or kidney disease, evidence of blood in the urine, if there are symptoms of a urinary tract infection, or dehydration. Dehydration can be caused by losing too much fluid, not drinking enough water or fluids, or both. Vomiting and diarrhea are also common causes of fluid loss, which can also be caused by excessive sweating, perhaps due to exercise. When severe, dehydration is a life-threatening emergency.

A typical urinalysis includes the following tests:

1)The physical color and appearance of urine is assessed including color density and turbidity.

2)A special urinalysis strip ("dipstick", a commonly used product named Multistix) tests for various substances in the urine. The strip contains small pads of chemicals that change color when they come in contact with specific substances, and the degree of color change is proportional to the amount of each specific substance.

3)The microscopic structures of certain materials found in the urine sample are examined. This is done to look at cells, urine crystals, mucus, and other substances, and also to identify any bacteria or other microorganisms that might be present. We will not be doing this evaluation in this lab.

4)Urine samples will also be evaluated for volume voided and urine production rate. Changes in urine volume produced per unit time (ml/min) depend primarily on the hydration state of a normal healthy test subject and any diuretics consumed (caffeine, certain drugs, etc.).

5)The urine specific gravity reveals how concentrated or dilute the urine is (moles of solute/liter).

6)Another method to measure how concentrated or dilute the urine is, is called conductivity. Conductivity measures the ability of a substance to carry electrical current. In urine, salt ions (Na+, Cl-) are the electrolytes, the primary conductors, since they are electrically charged (+,-). Thus conductivity may be used to measure the salt concentration of urine. Though not exactly the same, both specific gravity and conductivity can be used to assess the hydration state of a subject (see journal article describing using of conductivity measure as a way of tracking athlete hydration state). Specific gravity measures the presence of both ionic (like Na+& Cl-) and non-ionic compounds (like urea). However for any one subject, the proportion of ionic and non-ionic compounds should remain stable over the course of the experiment so that relative changes in specific gravity and conductivity are comparable.

CAUTION !!!!!!

Remember, urine is a body fluid. Students completing this lab should take every precaution (as determined by biosafety guidelines) when collecting, and handling urine samples to minimize any direct exposure to urine. Obtain and wear goggles and disposable gloves. Dispose of urine samples upon completion of testing in the restroom. All equipment and work surfaces coming in contact with urine should be disinfected upon completion of the lab exercise. If disposable supplies are used, they should be in sealed in a plastic bag and thrown in the trash or as specifically directed by your instructor.