URINE FORMATION, CREATININE CLEARANCE AND SODIUM CLEARANCE
BACKGROUND
Changes in the rate of urine production can occur by several mechanisms: for example, an increase in the
rate of urine formation could occur as a result of a decrease in the water permeability of the collecting duct,
an increase in the filtered fraction, or an increase in the blood flow to the kidney. The last two changes
would affect the glomerular filtration rate (GFR). The glomerular filtration rate is measured by measuring
the clearance of a substance that is freely filtered and neither actively secreted nor reabsorbed from the
tubular urine. Several substances fulfill these criteria: inulin (a fructose polysaccharide) is perhaps the best
one, but sucrose, creatinine, ferrocyanide and other substances fulfill them more or less well, depending on
the species of animal involved. We will use creatinine to measure GFR.
Creatinine Clearance = rate of urinary creatinine loss
plasma creatinine concentration
The clearance of creatinine can be compared to the clearance of Na + , a substance that is characteristically
highly reabsorbed by the kidney.
The rate of urine flow in the rabbit under anesthesia is generally quite low, even when the extracellular
volume is supplemented with the IV drip. The IV infusion will contain creatinine, in addition to the regular
Locke’s solution ingredients, to supplement the animal's endogenous production of creatinine. To establish
the control conditions, the animal will be maintained on this solution from the beginning of the experiment.
Then an additional slow injection of this creatinine solution will be made into the IV line to help saturate
the animal. The first half-hour sample interval will serve as a control. During the second half-hour we will
measure the effects of the diuretics furosemide and acetazolamide on urine production and GFR.
Furosemide (Lasix) is a loop diuretic - it inhibits salt reabsorption in the thick ascending limb of the Loop
of Henle. Acetazolamide (Diamox) inhibits carbonic anhydrase and, thus, inhibits reabsorption of HCO 3across the proximal tubule.
Each group will have designated one of its members as a chemist to perform the creatinine and Na+ assays.
PROCEDURE
1. Anesthetize the rabbit with urethane and shave the neck and abdomen.
2. Insert the tracheal cannula.
3. Fill the IV drip vessel with Locke's-creatinine solution and clear the line of bubbles. Insert the IV drip
line in the jugular. Start the drip at a rate of 10 drops/minute.
4. Cannulate the carotid with a piece of PE tubing attached to a syringe containing heparinized Locke's
solution. The carotid cannula is for collection of blood to determine the plasma creatinine and Na+
concentrations. The cannula needs to be kept clot-free until the blood samples are taken. If the blood
pressure tends to push the syringe plunger out, clip off the cannula with a hemostat between flushings of
heparinized Locke’s.
5. Make a lower abdominal incision. Move the viscera out of the way and find the urinary bladder. Keep
the viscera wet with sponges of warm Locke’s-creatinine. Use a desk lamp to warm the abdomen.
6. Remove excess urine from the bladder with a larger gauge needle and a large syringe. Cannulate the
bladder with the large diameter PE tubing provided. Make certain that you tie the cannula into the bladder
posterior to the two ureters, which empty into the bladder on the posterodorsal surface. Position the
cannula so that it drains downward into a 100 ml graduated cylinder for urine collection. It may help to
prop the front of the rabbit board up so that urine drains more freely from the bladder. Carefully staple the
abdominal incision back together as best you can to keep the viscera from getting cold or drying out. Urine
flow may or may not begin now.
7. Inject additional Locke’s-creatinine solution through the IV line. The volume of solution injected
should equal 3% of the animal's body weight. Inject this slowly over a period of 10-15 minutes.
8. Immediately after injecting the additional solution, adjust the IV drip to deliver 20 drops/minutes. This
should saturate the animal with fluid. Urine flow should begin fairly soon after the injection is completed.
If it does not, consult the TA. Wait about 10 minutes for the animal to equilibrate.
9. Empty the graduate of any urine that may have collected, discard this urine, and start the control halfhour collection interval.
1. At ten minutes after the start of the first interval, take a blood sample. This is done as follows:
1) Have two vessels ready; the one is a small beaker for blood to be discarded, the other is a heparinized
centrifuge tube for the sample. 2) Remove the syringe from the carotid cannula. 3) Collect the first
milliliter or so of blood in the discard vessel. This is a mixture of Locke's solution and blood that is coming
from the dead space of the carotid and cannula and is not representative of the rest of the blood in the body.
4) Quickly switch the tube to the centrifuge tube and collect 2 ml of blood. The chemist will assay this for
creatinine and Na+. Remember to save any unused plasma until the assays have been concluded.
11. At the end of the half-hour, record the volume of urine that has accumulated. The chemist of each
group will then begin the creatinine and Na+ assay on the urine sample. Save any leftover urine in case of
failure or accident.
12. Your TA will assign your group to administer either furosemide or acetazolamide to your animal. Inject
the dose provided into the IV line over a 5-10 minute period.
13. Empty the urine graduated cylinder, discard the urine, and start a new half-hour collection interval.
14. Ten minutes after the start of the second interval, take a blood sample using the same procedure as in
step 10. Assay for creatinine.
15. At the end of the diuretic interval, measure the volume of urine that has collected and assay for
creatinine and Na+.
16. If you got this far successfully, congratulations! Check with the instructor before sacrificing the
animal.
ANALYSIS
Blood Assay
Follow the same procedure as for the practice session.
Urine Assay
1. Urine does not need to be deproteinated. Pipet 0.5 ml of urine into a 100 ml volumetric and make volume
up to the line with distilled water.
2. Treat the diluted sample as if it were protein-free plasma sample, following the directions for the blood
assay from that point. The total dilution factor for the urine will be 200 X 1.5 = 300X
Clearance Calculations
Ccreatinine = (2 X urine vol.)(300)(urine absorbance)
(plasma absorbance)(15)
This simplifies to (40)(urine vol.)(urine absorbance)
(plasma absorbance)
CNa = (urine Na+ concentration)(2 X urine volume)
(plasma Na+ concentration
The units of both clearances are ml/hour.
PREPARING YOUR REPORT
You now have the following pieces of data: the urine flow rates for the two intervals, the urine creatinine
concentrations for the two intervals, the urine Na+ concentrations for the two intervals and the
corresponding plasma creatinine and Na+ concentrations. Your report should explain the changes, if any,
in urine volume flow, GFR and Na+ clearance that took place as a result of the diuretic.