Lab 18 Excretory System and Urinalysis

Activity 1: Anatomy of the Excretory System

1.      Obtain torso model, stickers and felt pen.
2.      Write the organs and structures that you should be able to identify on the stickers
        Lungs, Liver, Kidney, Ureter, Urinary Bladder, Urethra
3.      Take turns within your group to label the structures. As you label indicate the function of the organ or structure for the excretory system

4.      Have your instructor check your labeling before moving to Activity 2.

Activity 2: Anatomy and Dissection of the Kidney

1.      Obtain kidney model, stickers and felt pen
2.      Write the structures that you should be able to identify on the stickers
3.      Take turns within your group to label the structures.
4.      Have your instructor check your labeling before moving to the dissection.

a.       Once the model has been labeled, move to the side to allow for dissection of the kidney.

Dissection of Kidney:

1.      Obtain the Sheep’s kidney, Dissection Tray and Tools from your instructor
2.      Review how to hold dissection tools and select members of your group that will dissect and members that will be responsible for identifying structures
3.      Find the Ureter, Renal Artery and Vein
4.      Using the scalpel. Hold the kidney so that the hilum is facing you
5.      Start at hilum and make coronal incision towards the apex of the kidney

a.       Make sure the cut is with some pressure as the capsule may be difficult to cut through.

6.      As you reach the apex, turn the kidney so that hilum is facing the dissection mat

a.       Continue incision along the lateral margin of the kidney
b.      Your partner will need to apply pressure to “spread” the incision allowing you dissect through the cortex and into the medulla of the kidney

7.      Continue incision past the base of the kidney and flip the kidney so that the hilum is facing you once again
8.      Once back at the hilum, lay kidney flat and reflect the kidney

a.       Note that you may need to repeat the incision made around the lateral margin and through the entirety of the medulla to allow for the kidney to be reflected in two

9.      After reflecting the kidney

a.       Find a renal pyramid and at the base of the pyramid, make a transverse incision on one-side of the reflected kidney

a)      This incision will free a region of one lobe of the kidney from the rest of the organ

10.    Inspect the kidney and take turns within your group to identify the various anatomical regions, structures and blood vessels of importance that you are responsible for knowing.
11.    Place the kidney under the dissection microscope and examine the kidney looking for blood vessel and renal interactions.

a.       Can you see the glomerulus and Bowman’s capsule? Why or Why not? Discuss in your group and once you have an answer call your instructor over to see how correct your thinking might be.

Activity 3: Urinalysis

Purpose
The purpose of the lab is to examine 6 different urine samples from individuals that have all changed something within their diet, level of hydration or exercise over the last 24 hours. Where you are going to attempt to determine if by urinalysis you are able to determine what changes were made by each person being analyzed.

Background
Analysis of urine is used to determine the chemical composition of excretions (typically collected over 24-hour period OR at the second voiding of bladder) found in urine. Observation can reveal a lot about the chemistry of the urine and the chemistry of the fluids of the body for the person based on their hydration and metabolic status. A person that is normally hydrated and following a normal diet (not restricting any macronutrients or over consuming drugs or alcohol) should produce about 500-1500 mL urine per day that is a color of pale to straw yellow with no turbidity (able to see through the liquid). Whereas those that are dehydrated can produced ½ to ¼ that volume of urine with a color may become darker yellow or amber in color and those that are overconsuming alcohol or drugs may have a turbidity, or cloudiness, that hinders the ability to see through the urine sample. Regardless of the total volume of urine being produced 95-96% will be water (H₂O). Even though yellow is generally seen as normal, many things that are eaten or drunk can cause the color to change to every hue of color in the rainbow. In addition to color and volume, things that we consume can impact the concentration of volatile chemicals being excreted in urine.  These volatile chemicals being excreted within the urine can be observed in the odor of the urine, the most common volatile chemical that can be observed would be ammonia, a chemical excreted as a buffer to help stabilize metabolic acids that are being excreted.  Along with the general observations of urine, urinalysis can provide insight into the metabolic state of the person through chemical analysis of the urine sample.  This chemical analysis will examine pH, Urine Specific Gravity, concentrations of wastes (i.e. urea, creatinine, acids and nitrites), concentration of metabolites (i.e. ketones, glucose, and proteins) or electrolytes (i.e. Na⁺, K⁺, Cl^-, Mg²⁺, Ca²⁺, PO₄^3-). When discussing these chemical values there are two units given, either millimolarity (mmol/L, mM) or milliequivalent (mEq)/L.  The mEq is the indication of the ionic charge provided by the ion in the solution relative to the total amount of ion present (mg of salt).

Table 1. Standard norm urine concentration of electrolyte, metabolic wastes, metabolites and other factors typically seen in urinalysis

Urine Dipstick Chemical Analysis

pH: The glomerular filtrate of blood plasma is usually acidified by renal tubules and collecting ducts from a pH of 7.4 to about 6 in the final urine. However, depending on the acid-base status, urinary pH may range from as low as 4.5 to as high as 8.0. The change to the acid side of 7.4 is accomplished in the distal convoluted tubule and the collecting duct.  In general, people with high protein (meat) diets, low carbohydrate diets, or who are being metabolically stressed should have a urine pH in the acid range and a person who is a vegetarian may have a urine pH in the alkaline range.

Urine Specific Gravity (USG): Urine Specific Gravity (USG) is a measure of density that is proportional to urine osmolality which measures solute concentration relative to di-water. This measure indicates the ability of the kidney to concentrate or dilute the urine relative to plasma, thereby providing a solution that is closer to di-water (1.00) or more solutes that di-water (>1.00) per volume, typically 10 µL on the laboratory urine-refractometer, that uses the refraction of light as a measure of solids in the urine (thus providing density)  In this lab we will use a urometer/hydrometer to measure USG, where we will use a float to determine the relative density of the sample of urine to di-water. If USG is not > 1.022 after a 12-hour period without food or water, renal concentrating ability is impaired and the patient either has generalized renal impairment or nephrogenic diabetes insipidus. Any urine having a USG over 1.035 may be contaminated or possibly contains very high levels of glucose or other dissolved substances.

Table 2. Relationship of Urine Specific Gravity to Hydration Status. Euhydrated is normally hydrated. Hyperhydrated is an indication of too much water consumption. Dehydrated is an indication of acutely under consuming water, while Hypohydrated is a chronic state of under consuming water.

Protein: Normally, only small plasma proteins filtered at the glomerulus are reabsorbed by the renal tubule. However, a small amount of filtered plasma proteins and protein secreted by the nephron. Normal total protein excretion does not usually exceed 150 mg/24 hours or 10 mg/100 ml in any single specimen. More than 150 mg/day is defined as proteinuria. Proteinuria > 3.5 gm/24 hours is severe and known as nephrotic syndrome. Albumin is a protein that can cause turbidity of the urine and can be tested to provide positive results (which represent a slightly hazy appearance in urine) are equivalent to 10 mg/100 ml or about 150 mg/24 hours (the upper limit of normal). 1+ corresponds to about 200-500 mg/24 hours, a 2+ to 0.5-1.5 gm/24 hours, a 3+ to 2-5 gm/24 hours, and a 4+ represents 7 gm/24 hours or greater.

Glucose: Less than 0.1% of glucose normally filtered by the glomerulus appears in urine (< 130 mg/24 hr.). Glycosuria (excess sugar in urine) can be thought of as a symptom of diabetes mellitus, yet consumption of a large sugar meal prior to providing the urine sample or if the person is sick or excessively stressed can also result in an indication of glycosuria.

Ketones: Ketone bodies (acetone, acetoacetic acid, β-hydroxybutyric acid) resulting from someone following a ketotic diet (low carbohydrate) or some other form of calorie deprivation (starvation), are easily detected using urine dipsticks.  Ketonuria may result from starvation or diets low in carbohydrates since the body is forced to use fat stores.  Ketonuria and glycosuria together, is one of the diagnostic indicators for a condition associated with diabetes mellitus known as diabetic ketoacidosis.

Nitrite: A positive nitrite test indicates that bacteria may be present in significant numbers in urine. Gram negative rods such as E. coli are more likely to give a positive test.  Presence of bacteria in urine (bacteriuria) may be confirmed by observing the urine microscopically.

Leukocyte: A positive leukocyte esterase test results from the presence of white blood cells either as whole cells or as lysed cells. Pyuria can be detected even if the urine sample contains damaged or lysed WBC's. A negative leukocyte esterase test means that an infection is unlikely and that, without additional evidence of urinary tract infection, microscopic exam and/or urine culture need not be done to rule out significant bacteriuria.

Erythrocytes (Blood/Hemoglobin): The urine dipstick can detect whole red blood cells as well as lysed RBC’s.  A positive result for blood may be verified by observing the urine specimen under the microscope.

References:

Armstrong LE. (2007) Assessing hydration status: the elusive gold standard. J Am Coll Nutr. 26: 575S-584S

Bouatra S, Aziat F, Mandal R, Guo AC, Wilson MR, et al. (2013) The human urine metabolome. PLOS ONE 8(9): e73076. https://doi.org/10.1371/journal.pone.0073076

Materials:
Urine Collection Cup
Urometer/Hydrometer
15 mL Conical Tube
Urine Test strip and Reference

Methods:
1.      Obtain urine collection cup
2.      Using the restroom, collect urine sample and return to lab with collection cup sealed
3.      Carefully pour sample of urine into the hydrometer container and obtain the Urine Specific Gravity (USG) for sample by reading the line of the hydrometer at the bevel of the liquid within container. Record results.
4.      Examine the physical characteristics of the urine (volume, color, and odor) and record results.

a.       For testing of odor, follow the wafting technique that is demonstrated by your instructor

5.      Using a 1-mL transfer pipette, carefully transfer 5-mL of sample of urine using into the appropriate test tube
6.      Dispose of urine in collection cup in the urinal of the restroom and wash hydrometer and hydrometer container with soap and water and then rinse with deionized water
7.      Using a white piece of paper assess sample for turbidity. Record results

a.       To test turbidity, draw a large + on the piece of paper and then hold it behind your test tube of urine

i.     A positive test for turbidity will result in urine that you are unable to see the + that you drew on the piece of paper due to a cloudiness of the urine.
ii.     A negative test for turbidity will result in you being able to see the + that you drew on the piece of paper

8.      Remove a test strips from the canister of urinalysis reagent strips, placing on paper towel on your lab bench
9.      Place test strip into test tube of urine, making sure that each of the reagent area of the strip is submerged
10.    Allow strip to react with urine for 20-seconds and then remove from the urine sample
11.    Carefully remove the strip and allow any excess urine to drip back into test-tube. Place strip on a piece of paper towel (do not dry the test strip)
12.    Note the color changes that occur on each reagent area of the strip and compare them to the indications on the canister (be certain to read reactions within 60-seconds of removal from the urine). Record results. Note: leukocytes may take up to 120-seconds for reaction to be readable.
13.    Dispose of urine from test-tube in urinal in the restroom.
14.    Obtain the survey results for group members to see dietary and hydration history for the last 24 hours.

Results:

Table 1. Urinalysis results of examination of physical and chemical characteristics of urine samples

Dietary Changes for samples:

#1:                                                                            

#2:                                                                            

#3:                                                                            

#4:                                                                            

#5:                                                                            

#6:                                                                            

Discussion:

Summarize the findings for the urinalysis of the members of your group. In this summary indicate the continuum of hydration status from the most hypohydrated (dehydrated) to the closet to being euhydrated (normal hydration), provide evidence for your statement of hydration. And discuss how the urinalysis values changed based on the hydration of the person or the diet that each person followed for the day prior to urine collection.