1. No category

Renal function in the laboratory rat: a student
exercise.
R L Walker and M E Olson
Advan in Physiol Edu 268:S49, 1995.
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Advances in Physiology Education is dedicated to the improvement of teaching and learning physiology, both in
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September and December by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991.
Copyright © 1995 by the American Physiological Society. ISSN: 1043-4046, ESSN: 1522-1229. Visit our website at
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FUNCTION
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THE
A STUDENT
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LABORATORY
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RAT:
EXERCISE
Richard L. Walker and Merle E. Olson
Department
of Biological
Sciences, Faculty
of Medicine,
Calgary,
Alberta
T2N lN4, Canada
University
of Calgary,
ecause of the increased
concern over use of human body fluids in
physiology
teaching laboratories,
we developed
an exercise in renal
function that utilizes laboratory rats. The purpose is to demonstrate
the
role of the kidneys in the homeostatic control of extracellular
fluid volume, plasma
ionic concentrations,
and osmolarity. Three treatment groups are utilized: a volumeexpanded (access to 1 g/l00 ml sucrose) group, a volume-expanded
and salt-loaded
(access to 0.9 g/l00 ml NaCl) group, and a volume-depleted
(water-deprived)
group.
A normovolemic
control group (access to tap water) is also included. Rats are housed
individually
in metabolic cages that allow accurate measurement
of fluid intake and
urine output. Blood samples are removed via cardiac puncture. The animals recover
from this procedure and can be reutilized within 2-3 wk. When class data are pooled,
clear trends are seen that demonstrate
the volume-, osmo-, and ionoregulatory
abilities of the kidneys.
B
Key words:
268
(ADV
PHYXOL.
EDUC.
kidney; laboratory
13): S49-S55,
teaching
One of the traditional laboratory exercises regarding extracellular fluid volume homeostasis and kidney function utilized in physiology courses is human
urine production in response to drinking a large
volume of fluid, either water or isotonic saline. In
our experience, this exercise clearly demonstrates
the volume-, osmo-, and ionoregulatory functions of
the kidney and hypothalamus; however, there are
concerns and issues related to human experimentation in physiology teaching laboratories. It is often
difficult to establish baseline normovolemic data
because of the variety of daily activities of students
before attending lab. Quite often students do not
follow instructions about exercise or eating and
drinking before lab, and it can be difficult to obtain
blood samples to demonstrate the regulation of
plasma ions and osmoticity. In addition, there is
increasing concern about the use of human body
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13 : NUMBER
1995.
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1 -ADVANCES
fluids in teaching laboratories and the ethical and
liability issuesregarding the intake of saline.
To circumvent these issues, we have retained the
basic protocols utilized in the renal function exercise but have utilized rats as subjects rather than
students. There are several advantages to using rats
rather than humans as subjects, including better
control of diet and fluid intake, 24-h urine collection
with use of metabolic cages, and the ability to obtain
blood samples without risk to the student, the
instructor, or the lab technician.
PROTOCOL
Our subjects are female Sprague-Dawley rats weighing 250-300 g. Although the experiment will work
with smaller animals, the larger ones produce a
greater urine volume, and it is easier to obtain an
o 1995
THE AMERICAN
IN PHYSIOLOGY
S49
PHYSIOLOGICAL
EDUCATION
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SOCIETY
1995
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AM. J. PHYiSIOL.
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MATERIALS
Metabolic
Bleeding
D
E
A
S
AND
METHODS
Cages
Technique
To measure the parameters
mentioned
in the protocol, it is necessary to obtain 0.75-1.0
ml of plasma,
which in our experience
necessitates
withdrawal
of
2 ml of blood. We have chosen to obtain this volume
of blood via cardiac puncture
by use of the following
procedure,
which conforms
with guidelines
of the
Canadian
Council on Animal Care and the National
Institutes
of Health.
Rats are anesthetized
with
halothane,
and the blood sample is removed by use
of a heparinized
21-gauge
needle and 3-ml syringe.
Once anesthetized,
the rat is held ventral
side up
with one hand while the other is used to insert the
needle beneath the sternum.
The needle is inserted
at a 45” angle - 1 cm into the thorax. Done properly, it is easy to obtain 2 ml within
a few seconds.
When the technique
is performed
by trained personnel, rats recover with no visual signs of distress. After
the cardiac
puncture
all animals
are given free
access to tap water and food. Our experience
has
been that the animals can be reutilized
within
2-3
wk of this procedure.
Although
other methods
of
obtaining
blood are available,
we have found that
Each pair of students
is assigned
to calculate
the
24-h fluid intake and urine production
for two rats
(same or separate treatment
groups)
by measuring
the drinking
fluid remaining
in the calibrated
water
bottles and the urine in the collection
flask. Each
student
pair then analyzes the urine and plasma
samples
of those two rats for sodium,
chloride,
osmolarity,
and creatinine
(see Analytic
Procedures).
Because
of our time constraints
(3-h lab
sessions),
it is difficult
for the average student
to
analyze
more than two urine
or blood
samples;
therefore
class data are pooled,
and a summary table
is made available
at the end of the week. From the
urine flow rates and urine and plasma ion, osmotic,
and creatinine
concentrations,
students
are expected to calculate
the sodium and chloride
clearances, creatinine
clearance
[as a measure of glomerular filtration
rate (GFR)],
osmotic
and free-water
clearance,
and fractional
ion excretion
(see Calculations) .
1 - ADVANCES
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At the beginning
of lab, the instructor
or technician
draws a blood sample (see MATERIALS
AND METHODS)
from each rat. The blood samples are transferred
to
labeled microcentrifuge
tubes and spun for 2-3 min
at 13,000 rpm (- 13,OOOg),
and the plasma is then
transferred
to another set of labeled tubes.
: NUMBER
D
Animals are housed individually
in 17 x 17 x 25-cm
stainless steel cages with wire mesh flooring,
which
allows urine to pass through
but not feces or food
particles
(Fence Cage Products,
Boston, MA; Allentown Caging Equipment,
Allentown,
NJ; Nalge, Rochester,
NY). As shown
in Fig. 1, each cage is
equipped
with a food hamper
and water
bottle
holder. Graduated
glass water bottles are used, each
holding
up to 100 ml of fluid. Erlenmeyer
flasks or
large (50-ml)
test tubes serve as urine collection
vessels. These are attached
by fiber tape to the
funnel-shaped
cage bottom.
To retard evaporation,
a thin layer of paraffin oil can be added to the flask
or tube.
On the morning
before
the laboratory,
rats are
individually
housed
in metabolic
cages (see details
in MATERIALS
AND METHODS).
The cage bottom funnels
urine
into a collection
flask. Drinking
fluids are
placed in calibrated
water bottles within
easy access
of the cage interior,
and each rat also has access to
rat chow.
13
N
We assume volume expansion
or depletion
to have
taken place as a result of these treatments.
No effort
is made to quantify the changes in extracellular
fluid
volume;
however,
this may be possible
by the
measurement
of the inulin
space, which
would
require
intravenous
injection
of inulin and an assay
or the use of radiolabeled
inulin.
adequate
blood
sample without
harming
the animal. Rats are assigned
to one of three treatment
groups
and a control
group.
1) The volumeexpanded
(water-loading)
group is given access to 1
g/100
ml sucrose
as the drinking
fluid for 24 h,
which results in hypoionic
expansion
of body fluid
compartments.
2) The volume-expanded
and saltloaded group is given access to isotonic
(0.9 g/100
ml) NaCl as the drinking
fluid for 24 h, resulting
in
isotonic
expansion
of body fluid compartments.
3)
The volume-depleted
group
is given no access to
water or other drinking
fluid for 24 h. 4) The control
group is given free access to tap water.
VOLUME
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A
graduated
water
bottle
food
hamper
wire mesh floor
stainless
steel
cage with wire
mesh front
17 cm
4
+
collection
funnel
graduated
conical
test tube for
urine
collection
FIG. 1.
Metabolic
cage: frontal
view, A; top view, B. Attachments
include
a graduated
water bottle (100 ml) and urine collection
tube. Fecal material
is trapped
in the wire screen that slides beneath
the wire mesh floor of the cage. Urine passes
through
the screen and is funneled
into the urine collection
tube.
cardiac puncture is much less stressful for the
animal when this volume of blood must be drawn.
Analytic
titration (model CMTlO
chloride titrator, Radiometer, Copenhagen, Denmark). Osmolarity is measured by freezing point depression (model 3MO
microsmometer, Advanced Instruments, Needham
Heights, MA).
Procedures
Students measure the rat urine and plasma osmolality and the sodium and chloride concentrations
during the lab period. Sodium is measured via flame
emission (digital flame photometer model 265500, Cole-Parmer, Chicago, IL). Plasma samples are
diluted 1:200 before measurement. The sodium
concentration in urine samples will vary depending
on the animal treatment group. Urine from control
and water-loaded animals is diluted 1:200, but urine
from dehydrated or salt-loaded rats often requires
1: 500 dilution before measurement. Undiluted
samplesof urine and plasma are used in the measurement of chloride concentration via coulombmetric
VOLUME
13
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Previous investigators have shown that creatinine
clearance is a valid measure of GFR in rats (4).
Because creatinine is a natural product of creatine
catabolism, GFR can be estimated if plasma creatinine concentration and the rate of creatinine excretion are known (see Calculations
below). Students
measure plasma and urine creatinine via the alkaline
picrate method (kit 555-A, Sigma Chemical, St.
Louis, MO). This measurement requires 0.3 ml of
undiluted plasma and a urine sample diluted 1:20
(1: 50 for dehydrated rat urine).
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4-
fecal
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Calculations
[Uionl
. I-W
Lpionl
where [Uion] is urine ion concentration, [Pion] is
plasma ion concentration, and UV is urine flow rate.
Pcrl
and
Free-water clearance = UV - Co,,
Fractional ion excretion.
rate of ion excretion
rate of ion filtration
LUionl
= GRF * [Pion]
Fractional ion reabsorption.
Fra
--
GRF
’ LPionl
GFR*
=l
-
IJVa
LUionl
LPionl
- fractional ion excretion
Ion clearance and fractional ion excretion or reabsorption are ways of expressing the renal processing
of ions. Osmotic and free water clearances are
indicative of the concentrating power of the kidneys. If free-water clearance is negative, then the
VOLUME
13
: NUMBER
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S
Volume expansion by ingestion of 1 g/100 ml
sucrose has a significant effect on fluid intake and
urine output (ml/24 h) compared with the control
group. It appears that rats in the sucrose group
drink more than the necessary amount to replace
fluid loss, and the urine flow rate reflects this
increased intake. Because the excess fluid taken in is
hypoionic to intra- and extracellular fluids, the
volume expansion serves to dilute the body fluid ion
concentrations (1, 3). The physiological response is
a reduction in antidiuretic hormone (ADH) release
from the hypothalamus, thus decreasing water reabsorption. Inhibition of ADH release may result from
a slight reduction in plasma osmolarity (detected by
hypothalamic osmoreceptors) or increased blood
volume (detected by stretch receptors in the systemic circulation). No changes in sodium or chloride clearances are noted because there are no
changes in the total body mass of these ions,
although, as one might expect, there are significant
reductions in urinary ion concentrations and osmolarity. Free-water clearance becomes positive, thus
reflecting the attempts to excrete the excess water
while retaining valuable salt ions.
where UosMis urine osmolarity, PoSM
is plasma osmolarity, and CosMis osmotic clearance.
IJV’
E
I - ADVANCES
In contrast to the results of rats drinking sucrose
solution, volume expansion and salt loading via
ingestion of isotonic (0.9 g/100 ml) NaCl result in
IN PHYSIOLOGY
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U0sM.u-v
p
osM
Fractional ion excretion =
D
We have conducted this exercise successfully for the
past 7 years in a 3rd-year undergraduate physiology
course with consistent results. Although the means
may vary from year to year within treatment groups,
the trends are unequivocal. The data in Figs. 2-4 and
Table 1 were collected by last year’s class. A statistical comparison of means was performed by one-way
analysis of variance.
Osmotic and free-water clearances.
Osmotic clearance =
I
Experimental Results: Renal Regulation of
Extracellular Fluid Volume and Composition
IYcl--w = GFR
where [U,,] is urine creatinine concentration,
[Pcrl is plasma creatinine concentration.
D
RESULTS AND DISCUSSION
Creatinine clearance.
Creatinine clearance =
N
urine is being concentrated to a value greater than
the osmotic concentration of the plasma. Positive
free-water clearances indicate dilution of the urine
(urine osmotic concentration less than that of the
plasma), usually as a result of reduced water reabsorption by the distal tubules and collecting ducts.
Ion (Na+ or Cl-) clearances.
Ion (Na+ or Cl- ) clearances =
A
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N
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V
A
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N
S
A
N
D
400
A
*
I
D
E
A
*
8
300
.A
z
s
502
0G E
O 200
UF:
E
5
.M
%
m
100
control
dehydr
salt
S
q
urine
n
plasma
Na+
NC-L+
0
sucrose
con lrol
treatment
dchydr
salt
sucrose
treatment
2000
*
B
z?.gs
z g 1000
OE
go
q
urine
q
plasma
osmol.
osmol.
500
control
salt
dehydr
0
sucrose
control
treatment
FIG. 2.
Fluid
intake
(A) and urine
output
(B) of volumeexpanded,
volume-depleted,
and normovolemic
(control) rats measured
over 24 h. Volume
expansion
was
accomplished
by feeding
rats a solution
of 1 g/100 ml
sucrose
or 0.9 g/100
ml saline (salt). Volume
depletion (dehydr)
resulted
when rats were denied
access
to water for 24 h. Values are means & SE (n = S/group).
* Significantly
diRerent
from control
(P c 0.05).
13
: NUMBER
1 - ADVANCES
sucrose
treatment
FIG. 3.
Urine and plasma Na+ concentration
(A) and osmolarity (B) from volume-expanded
(sucrose
or salt), volume-depleted
(dehydr),
and normovolemic
(control)
rats. Values are means
f SE (n = S/group).
*Significantly different
from control
(P < 0.05).
motic concentration
of the intra- and extracellular
fluids (1, 3). Consequently,
the increase in Na+ and
Cl- clearances
may reflect a reduction
in aldosterone and/or
increased
atria1 natriuretic
peptide
(ANP) release, along with a drop in plasma ADH.
The increase
in fractional
sodium
excretion
and
osmotic
clearance
corresponds
to the increase
in
ion clearances.
The free-water
clearance
value is
very low because of the large osmotic clearance.
significant
increases in NaS and Cl- clearances
and
urinary salt and osmotic concentrations
well above
those in the control
group.
Urine output
is also
elevated
compared
with the controls
but is only
about one-half the volume
excreted by the sucrosetreated animals, reflecting
the fact that fluid intake is
less than that seen with the sucrose-treated
rats.
Volume
expansion
due to the intake of isotonic
saline serves to increase
the total body mass of
sodium
and chloride
without
alteration
in the os-
VOLUME
salt
dehydr
Volume
depletion
(dehydration)
duces urinary output and increases
IN PHYSIOLOGY
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significantly
reurine osmolarity
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*
1500
I
N
N
0
V
A
T
I
0
N
S
A
N
D
Measurements
dehydr
Treatment
Group
n
Na+ clcarancc
q
Cl- clearance
I
D
A
TABLE 1
of osmotic
and free-water
fractional
sodium
excretion
Osmotic
Clearance,
kl+rninP1.
100 g-l
Control
Sucrose
Salt
Dehydr
E
S
clearance
Free-Water
Clearance,
pl.rnin-l
* 100 g-l
- 3.8
+ 1.0
6.9
8.2
17.5
7.0
- 12.6
- 5.6
and
Fractional
Na+ Excretion
0.0063
0.0057
0.0546
0.0028
Values are estimates calculated
from mean plasma and urine ion
concentrations,
osmolarities,
urine flows, and glomerular
filtration rate measurements
in control,
volume-expanded
(sucrose),
volume-expanded
and salt-loaded
(salt), and volume-depleted
(dehydr)
rats.
salt
treatment
Student Assignments
control
dchydr
salt
We encourage
our students
to use a computer
spreadsheet
for data analysis and graphics;
consequently
the class data are available
on computer
disk. Trends in the data become very apparent,
and,
if the students
are familiar with standard
statistical
analyses, means can be compared
on a statistical
basis.
sucrose
treatment
FIG. 4.
Ion (A) and creatinine
(B) clearances
in +rnirP
100
g-l for volume-expanded
(sucrose
or salt), volumedepleted
(dehydr),
and normovolemic
(control)
rats.
Creatinine
clearance
is used as a measure
of glomerular filtration
rate (GFR).
Values are means + SE (n =
S/group).
*Significantly
different
from
control
(P <
l
Each student is expected
to write a scientific
report
that includes
an introduction,
a complete
results
section with sample calculations,
and a discussion
of
the physiological
control mechanisms
demonstrated
in the results. The major points we look for in the
discussion
are control
of ADH, aldosterone,
and
ANP in response
to volume
depletion
and volume
expansion,
and how the data reflect the regulation
of these hormones.
0.05).
about twofold more than that of the control animals.
This reflects an increase in hypothalamic
release of
ADH. The mechanisms
involved
may include
the
response
of the hypothalamic
osmoreceptors
to a
slight increase
in blood osmolarity,
and of stretch
receptors
in the systemic circulation
to decreased
blood volume. Although
GFR and sodium clearance
appear to be reduced
compared
with control values,
they are not statistically
significant
changes.
The
control
sodium excretion
under these conditions
is
complicated
by the inhibitory
action of elevated
plasma osmolarity
on the adrenal release of aldoste-
VOLUME
13
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Conclusions
The ability of the mammalian
nephron,
in conjunction with the hypothalamus
and adrenal
cortex, to
maintain
plasma
ion concentrations,
osmolarity,
IN PHYSIOLOGY
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rone (2, 5, 6). Normally,
the reduction
in plasma
volume
due to water deprivation
would
stimulate
increased
release of renin and angiotensin
II, which
would
enhance
aldosterone
release,
but a slight
elevation
in plasma osmolarity
counteracts
the effects of angiotensin
on the adrenal
cortex,
and
aldosterone
levels may actually fall, thus increasing
sodium clearance.
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T
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N
S
and extracellular fluid volume is emphasized in this
laboratory exercise. The regulation of GFR and
plasma Na+, Cl-, and osmotic concentrations can be
demonstrated through the use of volume-loaded
and volume-depleted rats simply by adjusting the
salt and sucrose content of the drinking water. All of
the trends apparent in the classic human renal
function labs are reproducible in this exercise, with
the advantage that blood and urine samples can be
collected and analyzed accurately without health
concerns for students or ethical issues concerning
the ingestion of salt- or sucrose-ladened solutions.
Address for reprint
Sciences, University
6 September
1994;
Pbysiol.
in final
form
and Disease.
2. Childers,
J. W.,
enhanced
B., F. L. Coe,
and
natriuresis
and
F. C. Rector.
Suggested
Fluid
D
E
Electrolyte
in
Philadelphia,
PA: Saunders,
1987.
E. G. Schneider.
Aldosterone
and the
of hypertonic
infusions
in the dog. Am.J.
Knox, F. G., and J. P. Granger.
Control of sodium excretion: the
kidney produces under pressure. NIPS 2: 26-29,
1987.
B. Natriuretic hormone: a contribution to the development of a hypothesis. NIPS 6: 114-118,
1991.
Lichardus,
M. An important region for osmoregulation:
anterior wall of the third ventricle. NIPS 2: 13-16, 1987.
McKinley,
the
B. M., and S. J. Cannizzo.
Establishment of optimal
juxtaglomerular cell models. NIPS 9: 188-192,
1994.
Rayson,
VOLUME
13
: NUMBER
Water channels in renal and nonrenal
1995.
I - ADVANCES
11):
F30-F37,
Readings
G. F. Role of renal nerves in edema formation. NIPS 9:
183-lSS,1994.
I., and D. Brown.
Physiol.
Norwalk,
DiBona,
Sabolic,
S
M. G. Fluid
Teachers and their students may find the following articles from
News in Physiological
Sciences useful when exploring the kidney’s role in body fluid homeostasis:
tissues. NIPS 10: 12-17,
A
1. Pitts,
R. F. Physiology
of the Kidney
and Body Fluids (3rd
ed.). Chicago: Year Book Medical Publishers,
1974.
2. Rose, B. D. Clinical
Physiology
of Acid-Base and Electrolyte
Disorders
(2nd ed.). New York: McGraw-Hill,
1984.
3. Stricker,
E. M., and J. G. Verbalis.
Hormones
and behavior:
the biology
of thirst
and sodium
appetite.
Am. Sci. 76:
261-267,1988.
4. Vander,
A. J. Renal Physiology
(5th ed.). New York: McGrawHill, 1995.
5. Windhager,
E. E. (Editor).
Handbook
of Physiology.
Renal
Physiology.
Bethesda,
MD: Am. Physiol. Sot., 1992, sect. 8.
28 February
RenalPhysiology
242 (Renal
I
and Electrolytes:
Physiology
and PathoCT: Appleton
and Lange, 1991, p. l-38.
Harvey,
A. M., and R. L. Malvin.
Comparison
of creatinine
and inulin clearances
in male and female rats. Am. J. Physiol.
209: 849-852,1965.
Merrill,
D. C., M. M. Skelton,
and A. W. Cowley,
Jr. Humoral
control
of water
and electrolyte
excretion
during
water
restriction.
Kidney ht. 29: 1152-1161,
1986.
Schneider,
E. G. In water deprivation,
osmolality
becomes an
important
determinant
of aldosterone
secretion.
News Pbysiol.
Sci. 5: 197-201,
1990.
Cogan,
physiology.
References
1. Brenner,
Health
D
1982.
R. L. Walker,
Dept. of Biological
Calgary, Alberta T2N lN4, Canada.
accepted
N
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Received
1995.
requests:
of Calgary,
A