Download Lab 10 - Creighton Biology

Biology 450 - Animal Physiology
Fall 2007
Lab 10 – Renal Physiology
In this lab, we will again make use of our most convenient experimental animals to
examine renal responses to the intake of various solutions. Changes in urine
composition will be analyzed in humans after the consumption of a salt solution, an
alkaline solution, or different volumes of pure water.
Background
The kidneys play a vital role in homeostasis, or the maintenance of constant
conditions within the body. They regulate the chemical content, the pH, and the
osmotic pressure of the blood.
The kidneys form urine by the combined processes of glomerular filtration, tubular
reabsorption, and tubular secretion. Water and many chemical substances whose
molecular sizes are not too great filter out of the glomeruli and move through
nephrons and into collecting ducts. As the filtrate passes through the nephrons
some substances, such as sodium, calcium, glucose, and amino acids, are
reabsorbed, whereas other substances, such as potassium and hydrogen ions, are
secreted by the cells of the nephron tubule.
The reabsorption of glucose is usually so complete that sugar is not found in the
urine. However, the capacity of the kidneys to conserve glucose is not unlimited.
If the blood sugar level exceeds a value known as the renal threshold, the kidneys
cannot reabsorb all the glucose in the glomerular filtrate and some sugar will
appear in the urine. Many substances are normally reabsorbed in the kidney
tubules, but most are not reabsorbed as completely as sugar.
Water is reabsorbed both in the nephrons and collecting ducts of the kidneys to
such an extent that the volume of urine excreted may represent less than 1% of
the volume of glomerular filtrate. Water reabsorption is enhanced by a hormone,
antidiuretic hormone (ADH), which is released from the posterior pituitary. A
change in fluid intake, or a fluid loss from the body, can change the rate of ADH
release and consequently change the rate of water excretion.
Although the kidneys are not solely responsible for regulation of blood pH, they aid
in maintenance of the blood at a pH of 7.4. A change in kidney function, with a
resultant change in pH of the urine excreted, can allow blood pH to remain
unchanged despite altered metabolic activity or the intake of acidic or alkaline
substances.
For the composition of the blood to be held relatively constant despite the varied
intake and utilization of substances by the body, it is apparent that urine
composition must vary. This exercise will demonstrate the alteration in urine
composition and the rapidity with which kidney function can change following
consumption of a hypotonic solution of large volume, an isosmotic solution, or a
bicarbonate solution.
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Pre-Lab Procedures – Important!
Starting at dinner the night before lab, please attempt to consume “normal”
amounts of liquid. On the day of the lab, try to have a glass/can/bottle of
something between one and two hours before lab. Try also not to intake unusual
amounts of caffeine or (ahem) alcohol in the few hours before lab. We want
everyone to be normally hydrated with normal kidney function at the beginning of
the experiment.
Lab Procedures
Four subjects with normal kidney function will be required for the experiment.
Subjects 1-3 (see below) represent the experimental treatments while Subject 4
acts as the control. Urine samples will be taken before and after these treatments,
and the samples analyzed to determine renal function.
Important – Analyze only your own urine! Wear gloves.
Treatments
The four treatments are:

Subject 1 – Drinks 700* ml of water

Subject 2 – Drinks 350* ml of 300 mOsm NaCl solution (isosmotic to ECF)

Subject 3 – Drinks 350* ml of 3% (350 mM) NaHCO3

Subject 4 – Drinks 350* ml of water
* These volumes are adjusted for body size (see Intake Volume Table below)
Although subject 4 acts as a control for the group, each individual also provides his
or her own control by sampling urine production before and after the above
treatments.
Intake Volume Table
Weight
(lb)
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
Normal
volume
(ml)
225
250
275
300
325
325
350
375
400
425
450
475
500
525
550
2
High
volume
(ml)
450
500
550
600
625
650
700
750
800
850
900
950
1000
1050
1100
Urine collection and fluid intake schedule
Urine samples should be collected every thirty minutes, as described below.
Analyze each sample as soon as is convenient – avoid letting too many samples
accumulate!
1. At the beginning of the lab period, each subject should empty his or her
urinary bladder and note the time. Do not collect this sample.
2. After thirty minutes, again empty the bladder, this time collecting the full
sample. This sample will serve as a pre-treatment control.
3. Immediately after step 2, subjects 1-3 should drink the fluid appropriate to
their treatments as quickly as possible. If the sample cannot be finished
before the first post-treatment sample is taken (below), record the volume
that was drunk.
4. Thirty minutes after step 2, each subject should again empty his or her
bladder, collecting the full sample. Repeat this step twice more, every thirty
minutes.
If your samples will not be on the thirty-minute mark, be sure to record the exact
time between samples.
Measurements
For each urine sample, you will collect data allowing you to determine the rate of
urine formation, the concentration of urinary solids, and the pH of the urine:
1. Volume measurement: Use a graduated cylinder to measure the volume of
each urine sample collected.
2. Urine pH determination: Check the pH of each urine sample using the
common pH meter. Be sure to rinse the probe with distilled water after use!
3. Specific gravity measurement: Use a
refractometer to measure the specific gravity of
the urine samples. This device allows
measurement of the concentration of a solution
by the change in angle of light passing through
the sample – solutions with a higher specific
gravity refract light to a greater degree.

Open the “door” of the refractometer, place a
drop of urine on the surface, and close the
door.

Hold the refractometer to your eye and
determine the reading at which there is a
change in color in the background (see figure).

Rinse the refractometer with distilled water and dry it after each use.
4. Chloride concentration: Chloride ion concentration will be measured directly
to serve as an indication of salt excretion. Total sodium chloride
concentration will be calculated using a formula provided during lab. The
assay is performed spectrophotometrically in a 96-well plate. Each lab group
will share one tray to be used for all time points. For each sample:
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
Mix 20 μl of urine and 980 μl of distilled water in a clean tube

Place 4 μl of the diluted urine in a well in a 96-well plate. (Make certain
to note the well number!) Add 160 μl of chloride assay reagent.

Run the plate through the spectrophotometer once for each time point,
after all group members have added their sample.
Calculations
Calculate the following variables:

Urine production rate in ml/min.

Urinary solids concentration (the concentration of solutes in the urine) in
g/liter.
Concentration = (Specific gravity - 1)  1000  2.66g

Urinary solids excretion rate (the mass of solutes appearing the urine per
unit time) in g/min.
Rate = Urine production rate (ml/min)
 Urinary solids concentration (g/L)  (1.0 L / 1000 ml)

Sodium chloride content (the concentration of NaCl in the urine) in g/L
[NaCl] = [(Abs  Slope) – Int]  50  1.65
where
Abs is absorbance of the sample
Slope and Int are the are the slope and intercept relating Abs to [Cl-].
Use Slope = 1.61 and Int = 0.16 unless otherwise instructed.
50 is the dilution factor (20 μl in 1000 μl)
1.65 is the multiplier to get [NaCl] from [Cl-]

Sodium chloride excretion rate (the mass of NaCl appearing the urine per
unit time) in g/min.
Rate = Urine production rate (ml/min)  NaCl concentration (g/L)
 (1.0 L / 1000 ml)
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