Lab #13: Kidney function and ion regulation
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Lab #12: Kidney function and ion regulation
Introduction
The excretory system consists of the kidneys, ureters, bladder
and urethra. The excretory organs play a major role in maintaining
homeostatic control of solute and water concentrations. The
functional unit of the kidney is the nephron that includes the
glomerulus (Bowman's capsule + capillary ball), proximal convoluted
tubule, loop of Henle, distal convoluted tubule and collecting
duct. The glomerulus is the site of blood filtration, where all but
the blood cells and the largest dissolved proteins are physically
filtered in to the cavity of Bowman's capsule. Therefore, the fluid
entering the tubule system is almost identical to blood plasma. The
rate at which plasma is filtered in the glomerulus is the
glomerular filtration rate (in ml/min). From there, while passing
through the tubule system, the filtrate is subjected to
reabsorption (selective re-uptake of molecules from the filtrate;
e.g. Na+, HCO3-, Ca++, glucose, amino acids) and tubular secretion
(addition of molecules to the filtrate; e.g. K+, H+). These
processes are so effective that, for example, no glucose is
excreted under normal circumstances. Similarly, up to 99% of the
volume of filtered water is reabsorbed. Unlike the former, the
degree of reabsorption of water, H+, Na+ and HCO3- are carefully
regulated and highly variable. Various mechanisms are in place to
ensure a homeostatically controlled concentration of these
molecules. The final product passing to the collecting duct can
very different from the initial filtrate.
Regulation of salt and water levels by the kidney involves
several negative feedback mechanisms:
a) Atrial natriuretic hormone (ANH; or atriopeptin, AP) is
released by the atria in response to increased stretch (presumably
by increased blood water volume) and causes increased glomerular
filtration rate and Na+ and water loss.
b) Antidiuretic hormone (ADH) is secreted by the posterior
pituitary in response to a decrease in plasma volume or increase in
plasma osmolarity and causes increased water reabsorption and
decreases urinary water loss. (Caffeine acts to inhibit the
secretion of this hormone.)
c) Aldosterone is secreted by the adrenal cortex in response to
a long sequence of steps initiated by a decrease in plasma Na+
concentration or plasma volume and causes increased reabsorption of
Na+.
The kidneys and respiratory system maintain acid-base balance.
Under normal conditions, almost all the bicarbonate ion in the
filtrate is reabsorbed. However, when a great deal of base is
ingested, plasma pH increases. The respiratory system responds
quickly by decreasing respiration, which increases plasma CO2 that
is interconverted to H+. The kidney, through a slower path, slows
the rate of bicarbonate reabsorption, resulting in more bicarbonate
lost and increased urine pH.
In this laboratory, you will investigate the response of the
kidney to changes in the volume, salt concentration and acidity of
the blood and to a substance that increases urine output (a
diuretic). There will be up to five treatments, in which each group
will drink different solutions- tap water ad lib, distilled water,
isoosmotic NaCl, hyperosmotic NaCl, basic sodium barcarbonate and
the diuretic drug caffeine. You will monitor changes in the
properties of the urine produced in response to these experimental
conditions- volume, pH, specific gravity, Na+ concentration,
glucose concentration and protein concentration.
Materials and Methods:
Part I- the preliminaries
The class will be divided in up to six treatment groups. For
each the experiment will be run as follows. First, urine is
collected and analyzed to produce baseline (control) data. Then, a
solution will be consumed and subsequent urine output collected and
analyzed (experimental data).
All data will be presented relative (higher, lower, the same) as
the initial control data.
The solutions are:
A. Tap water ad lib (that is, as often or as much as you like)-
this is one control
B. 750- 1000ml Tap water- this is another control
C. 750- 1000ml Isoosmotic (0.9%) NaCl
D. 75- 100ml Hyperosmotic (5.0%) NaCl)
E. 500ml Basic Sodium Barcarbonate (0.3% NaHCO3)
F. 250- 500ml Coffee- a potent diuretic
1. The class will divide up into up to six groups (GROUPS A-
F).
2. Pick up your urinalysis kit. It includes:
The test solutionCoffee and tap water treatment students will
provide their own solution
Plastic beakerFor urine collection
Rinse and dry after each use
Discard at end of the experiment
Graduated cylinderTo measure urine output
Hydrometer (with instructions)Measures specific gravity (related
to the amount of dissolved solutes present in the urine)
ThermometerMeasures urine temperature just after specific
gravity determination (for temperature correction of hydrometer
reading)
Dropper, bulb, 3 dropper bottles with solutions, test tubeFor
Cl- concentration determination
Bottle of "Combistix"Dipped in urine to determine pH, protein
and glucose concentrations
3. Set aside a four hour period to conduct the experiment. You
will need to be near a bathroom, so home is likely the place to
be.
4. Before starting, be certain you are fully familiar with the
procedure to be followed. It is very important to follow this
protocol closely.
Part II- urine collection schedule
1. Unless you are in the water ad lib group, do not drink
anything during the course of the experiment except for the
experimental solution. Avoid drinking a large amount of liquid
before beginning the experiment.
2. Note the Table provided below. You will fill this out using
the following sequence. I have provided a suggested start time of
1:00, just to make the timetable clearer. You can, of course, start
at any time you like.
Example TimeTable Line Number(s)Note(s)
1:001.Note the time when you begin the experiment in the "Time
when collected" column. Empty your bladder, but do NOT collect the
urine at this time; you are simply noting the time when you start
the experiment
Wait 30 minutes
1:302.Note the time, empty your bladder and perform the analyses
on your first control sample
Wait 30 minutes
2:003.Note the time, empty your bladder and perform the analyses
of your second control sample
4.Now you begin the experiment. Drink the solution, note the
time (in the "time when collected" column) and note the volume of
solution consumed
DO NOT collect any additional urine; you merely drink the
solution, note the volume of solution consumed and the time
Wait 30 minutes
2:305.Note the time, empty your bladder and perform the analyses
on your first experimental sample
Wait 30 minutes
3:00- 5:00 at 0:30 intervals6.- 10. Note the time, empty your
bladder and perform the analyses on experimental samples two
through six
Wait 30 minutes between sequential samples
Part III- treatment groups
1. Treatment groups are as follows:
a) GROUP A: does not drink a test solution. Drink water only
when thirsty, just as you normally would.
b) GROUP B: drinks 750- 1000ml distilled water after the control
samples. You drink no more after that.c) GROUP C: drinks 750-
1000ml 0.9% NaCl after the control samples.
d) GROUP D: drinks 75- 100ml 5.0% NaCl after the control
samples. This will be difficult to get down; don't gulp it. Eating
something salty and doughy such as pretzels or crackers might help.
If you begin to feel nauseated, stop drinking (note how much you
did drink) and continue with the experiment.
e) GROUP E: drinks 500ml 0.3% sodium barcarbonate after the
control samples.
f) GROUP F: drinks 250- 500ml coffee after the control samples.
Drink no coffee before the experiment. Try to drink the solution as
quickly as you would any of the other test solutions.
Part IV- analyses
1. Temperature: Determine as soon as practical. Immerse the
thermometer to the 25o mark, if possible. This is the depth at
which it is calibrated for accuracy at the factory.2. Volume:
Determined using the graduated cylinder. Rate of urine formation
(ml/min) is determined by dividing volume produced (in ml) by the
number of minutes since the last collection.
3. Specific gravity: Determined using the hydrometer. (This is
the solution's density relative to distilled water.)
i. Fill the urinometer (or graduated cylinder) with enough urine
for the float to be able to bob freely without touching the bottom.
To be sure the float does not cling to the side, spin the
hydrometer gently.
ii. When it stops spinning, note the line on the float
calibration that is exactly at the surface of the fluid. The float
floats higher in solutions with more dissolved solutes. Note the
scale indicates specific gravities of 1.000 and higher. Only the
last two digits may be seen and these represent the last two of the
three significant digits; e.g. 1.0XX. This is the "measured
specific gravity". It will typically range from 1.010 to 1.025
where pure water is 1.000.
iii. Measure the temperature.
iv. Determine the "temperature corrected" specific gravity of
the urine:
For every 3oC above 15oC, add 0.001 to the specific gravity
reading.
v. If the output of urine is insufficient to float the
hydrometer, save the urine from one sample and pool it with the
next sample. However, do all the other measurements on each sample
collected.
3. Cl-: It is difficult to measure Na+ concentration directly.
We will measure Cl- instead. As Na+ is reabsorbed in the kidney,
Cl- follows by electrostatic attraction. Thus, the concentration of
Na+ excreted will be roughly equivalent to the concentration of Cl-
excreted. We will measure the latter (Cl-) to estimate the
concentration of the former (Na+).
i. Measure 10 drops of urine into the test tube. (One drop is
approximately 0.05ml.)
ii. Add one drop of 20% potassium chromate.
iii. Add 2.9% silver nitrate one drop at a time, while shaking
continuously. Count the minimum number of drops that are just
sufficient to change the color of the solution from yellow to
brown.
iv. Calculate Cl- concentration: number of drops of silver
nitrate X 61 = ____ mg Cl- / 100ml urine
4. Combistix:
i. Dip a stick in the urine and tap off the excess.
ii. Determine pH and protein concentration immediately by
comparing the color the two patches against the colored scales on
the side of the bottle:
protein-
NORMAL- negative to trace
pH-
NORMAL- 5- 9 (depending on treatment)
iii. In the period of time 10- 30 sec after dipping the stick,
determine the glucose concentration in the same manner:
glucose-
NORMAL- negative to trace
iv. Readings out of normal range should be redone immediately
and monitored closely throughout the remainder of the
experiment
Part V- after you are done
1. Discard the test tube, dropper and plastic beaker.
2. Please carefully wash the equipment to be returned (graduated
cylinder, hydrometer and tube, and thermometer).
3. Deliver you data to Dr. Baube by deadline given.
4. Dr. Baube will provide a complete set of data to each student
authoring a report and to anyone else
interested.Results/Discussion-
a) Calculate mean values of all variables at each time period
for each treatment group.
b) Plot time vs. the mean value. Create a different graph for
each of the significant variables (volume, specific gravity, Cl-
concentration (as an estimate of relative Na+ concentration) and
pH). Interpret.
c) Include a consideration of possible hormone(s) and
respiratory system involved in the responses documented. (Students
in the sodium bicarbonate treatment group (GROUP E) might be polled
on their experiences with respect to the latter variable.)
Kidney function experiment data sheet-
Name: ___________________________________
Experimental solution used:_________________________
Volume consumed (ml): _____
volume of urine collectedTime when collected
(example time)time since last voidingRate of urine formation
(ml/min)pHmeasured specific gravitytemperature at measurementtemp.
corrected specific gravity[Cl-] (mg Cl-/100ml)glucoseprotein
1. control(1:00)NOTE TIMEYOU BEGIN EXPERI-MENTING:VOID
ANDDISCARDWITHOUTTEST-ING
2.(1:30)30 minutes
3.(2:00)30 minutes
4. experimental(2:00)DRINK
THESOLU-TIONXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
5.(2:30)30 minutes
6.(3:00)30 minutes
7.(3:30)30 minutes
8.(4:00)30 minutes
9.(4:30)30 minutes
10.(5:00)30 minutes
