Evaluation
of Renal Function:
Removal of Phenol Red from the
Circulation
Introduction
Renal function is dependent upon blood flow,
glomerular blood flow, glomerular filtration rate, etc. It is usually
evaluated by measuring the renal clearance of substances from the plasma.
This requires collection of arterial and venous blood and urine samples.
This is not possible in our experimental animals (rats) due to their small blood
volume and their relatively low rate of urine formation.
You will employ a different approach to examine
renal function. You will monitor the removal of phenol red, a substance
that is filtered and secreted with an Extraction Ratio of
approximately 0.67, from the plasma by measuring the amount remaining in the
plasma over a 30 minute post-injection period. You will then repeat the
procedure after treating the animal with exogenous agents that may or may not be
expected to alter the rate of removal. These agents of interest will alter
systemic blood pressure, cardiac output, renal blood flow and pressure, blood
volume, plasma ion concentrations, and/or plasma osmotic pressure.
Experimental Procedures
Animal preparation - Following the protocols and methods
described in previous exercises, anesthetize an animal with urethane and
cannulate the trachea, carotid artery, and jugular vein.
Connect the jugular cannula to a 1 cc syringe as usual
that will later be used to inject the phenol red. You also might choose to use a
somewhat shorter piece of tubing for carotid cannulation since you will have to
withdraw blood samples through this piece of tubing and longer pieces make for
greater difficulty in withdrawing the blood (greater L = greater R!!!!)
Taking blood samples - Remove a 0.5 ml blood sample from
the carotid cannula into a syringe containing 0.3 ml of heparinized saline
attached to the blood pressure transducer (Remember to turn the stopcock off to
the transducer prior to attempting to withdraw the blood sample). You might
choose to remove the blood directly into the 1 cc syringe containing 3 cc of
heparinized saline. Mix the sample gently and centrifuge at speed 4 in the
microcentrifuge for 3 min. in order to pellet out the red blood cells.
Take 0.4 ml of the supernatant (top layer of liquid which=plasma and heparinized
saline) using an automatic pipet with small tip and dilute it with 2.5 ml of
distilled water. Save this sample as your spectrophotometric "blank" (see
below). As soon as you have withdrawn this blood sample and all future samples,
flush the cannula with 0.5 ml heparinized saline. (Why should you do this ?)
Now, inject 1.0 ml of a 7.5 mg/ ml phenol red solution
into the jugular vein. Quickly flush the cannula with 0.5 ml of
heparinized saline. Exactly 3.0 min after flushing the cannula, withdraw
0.5 ml (This volume is critical, it must be the same throughout the
experiment!!!) of blood into a syringe containing 0.3 ml of heparinized saline.
Rinse the cannula with 0.5 ml heparinized saline. Again, gently mix the
withdrawn blood with the heparinized saline, centrifuge, and dilute 0.4 ml of
the supernatant with 2.5 ml of dH2O. Label this sample as TIME 0 and set
it aside.
Repeat this procedure at 5, 10, 20 and 30 min after
this first withdrawal. Also be careful to note any changes in BP, HR, or
other variable during the course of the experiment.
Experimental agents - You may now be supplied with a
solution containing an agent that may be expected to alter blood pressure or
flow (What agents might you choose?). You will be instructed in the use of an
infusion pump. The infusion pump provides a steady, continuous dose of the drug
at extremely slow flow rates. (Why is a continuous dose at a very low flow rate
necessary?) Set up the infusion pump with the agent of choice and continuously
monitor the rat's BP using the blood pressure transducer set-up. Inject 1.0 ml
of phenol red. Withdraw blood samples at the correct times as described
above. Be certain to monitor BP, HR, and respiration during the course of
the experiment! Remember that you must turn the stopcock on the blood pressure
transducer "off" to the transducer when you withdraw blood samples.
But wait, we may be removing a portion of the rat's blood
volume. Might this have an effect on kidney function and renal clearance in
addition to the effects of the experimental agent?? Can you think of a way to
deal with this problem? In other words, we need to consider some appropriate
"controls". You can assign different lab groups to different parts of the
experiment if you wish.
Spectrophotometric determination or [phenol red] - After
collecting all of your blood samples, you must quantify the amount of phenol red
found in the plasma at each time point. Your laboratory instructor will
provide an absorption spectrum for phenol red as well as the phenol red stock
solution. Each lab (this means one group can perform this for the entire lab
with the gratitude of the entire lab of course) must construct a phenol red
standard curve using phenol red concentrations from 0.001 mg/ ml to 0.075mg/ ml.
This will enable you to correlate phenol red samples of KNOWN CONCENTRATION with
their absorbencies read from the spectrophotometer. You will then be able to
determine the concentration of phenol red in your experimental samples by
comparing each of their absorbencies read from the spec, with the standard
curve.
Use your pre-phenol red injection sample as the "blank"
and zero the spectrophotometer. Now record the absorbency of the other
samples. Using the extinction coefficient (the slope) of the standard
curve, calculate the [phenol red] in the blood samples. Some easy
calculations will yield the [phenol red] in the blood at various times.
Data analysis - You will now construct a plot of [phenol
red] in the blood vs. time (0 to 30 min) without and with the drug. Compare the
rates of removal and explain and discuss any differences induced by the
exogenous agent. Remember to consider the results of the "control" experiments.
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