The overall goal of this procedure is to show other investigators how to determine kidney function in awake mice. This is accomplished by first dialyzing, fluorescein isothiocyanate, or FSE inulin for 24 hours and determining its concentration for injection. The second step is to weigh the mouse, prepare accurate volumes of fitzy inulin for retrobulbar injection, and anesthetize the mouse.
Next, the Retrobulbar Fitzy inulin injection is performed and eight blood samples are taken over a 75 minute period. The final step is to measure Fitz Nulin using a micro volume fluoro spectrometer and to calculate kidney function by employing a two-phase exponential decay model. Ultimately combining the sensitivity of the Fiti Nulin single bolus injection method with the micro volume capability of the fluoro spectrometer allows for detecting differences in kidney function caused by genetic modifications in mice, dietary changes or drug applications.
The main advantages of this technique over existing methods like continuous infusion and anesthetized conditions, implantation of osmotic mini pumps are using radioactive inulin are that no surgery is needed. It is not a terminal experiment. No urine has to be collected, and it is possible to measure up to 24 M per day.
I will be demonstrating this procedure together with Maria Azimo, a research associate from my laboratory To prepare the Fitzy Nulin injection solution. Weigh enough ZI inulin for two milliliters of a 5%solution, dissolve the labeled inulin in 0.85%sodium chloride solution by heating to 90 degrees Celsius until completely dissolved. Place a piece of 20 centimeter dialysis membrane in double distilled water for 30 minutes to remove residual sodium acid from the membrane, flush the membrane a few times afterwards, fill the dialysis membrane with dissolved fitzy inulin seal properly with closures, and then weigh the entire membrane.
Allow the dissolved fitzy inulin to dialyze in one liter of 0.85%sodium chloride solution and stir slowly protected from light for 24 hours at room temperature following dialysis, weigh the entire dialysis membrane again and calculate the new concentration. Water will osmotically move into the membrane and unbound FSI will move out of the membrane. Thus, the concentration of FSI inulin will decrease substantially.
Calculate the new fit e inulin concentration using this formula where C is the new concentration. N is the initial fit E inulin amount, and V is the new volume, which is the difference in weight of dialysis tubing before and after dialysis, plus the volume of initial fitzy inulin solution. Next, sterilize the dialyzed solution by filtration through a 0.22 micron syringe filter.
Remember to always keep fitzy inulin protected from light to avoid photobleaching prior to the experiment. Take the body weight of the mice after anesthetizing the mice briefly with four to 5%ISO fluorine. Check anesthesia depth by toin reflex aspirate two microliters per gram of body weight of dialyzed fitzy nulin using a 100 microliter Hamilton syringe with a half inch long 26 gauge needle to remove air bubbles, then switch to a half inch long, 30 gauge needle for injection.
Proceed to inject the dialyzed fitzy inulin into the retroorbital plexus. Allow mice to recover spontaneously in their home cage. Cup one millimeter of mouse tail with scissors once and collect blood at multiple time points after injection in sodium heparin mini capillaries.
Seal mini capillaries after blood collection with cha seal and keep the samples protected from light. Put the sealed mini capillaries inside hematocrit capillaries and centrifuge for five minutes following centrifugation. Break mini capillaries by using a diamond cutter and transfer entire plasma by pipetting into a 0.2 milliliter tube.
Make a one to 10 dilution by using two microliters of plasma and 18 microliters of 0.5 moles per liter heaps in a new 0.2 milliliter tube. Next, measure two microliters of the diluted sample with the NanoDrop 3, 300 in duplicates for high throughput of six mice. Please follow the flow chart protocol provided in the text protocol to measure whole kidney GFR by the single bolus injection method eight blood samples must be collected at 3 5 7 10 15 35 56, and 75 minutes after FSE nulin injection.
In this flow chart, the numbers indicate collection time points while the numbers in parentheses indicate sample number time points left of the vertical red line indicate injection time points. Each mouse is marked with a different color. To get sequential blood collections follow the arrows.
Gray boxes indicate when the next mouse must be prepared for isof fluorine anesthesia prior to injection using this protocol. The minimum time between two blood collections from different mice is one minute dilute fitzy nulin injection solution with diluted mouse plasma to get the standard dilutions listed in the text protocol. The concentration of the standards will depend on the dialysis and will always be different for each preparation of FSI nulin.
Therefore, it is necessary to enter the concentration for the standard curve each time. Analyze data to calculate the glomerular filtration rate or GFR with appropriate software by using a two-phase exponential decay function as described in detail in the text protocol to measure GFR in conscious mice. Plasma kinetics of ZI inulin following a single dose intravenous injection is used for calculation of GFRA two compartment model is employed in the two compartment model.
The initial rapid decay phase represents redistribution of fitzy inulin from the intravascular compartment to the extracellular fluid. The later phase with slower decay in fitzy inulin concentration predominantly reflects systemic clearance from the plasma. Type one diabetes mellitus was induced by intraperitoneal application of streptozotocin.
GFR was determined before and five weeks after induction of diabetes type one diabetic animals clearly show glomerular hyperfiltration. After watching this video, you should have a good understanding of how to measure GFR in a big mice by using a single bolus injection technique and employing a two-phase exponential DK model.
