This technique can help answer key questions on single-nephron physiology, including the glomerular filtration rates of systemic proteins and metabolites and their contributions to tubular physiology. The main advantage of this micropuncture technique is that it allows access to Bowman's space and the cortical nephron in all mice. Generally, individuals new to this method would struggle because it requires correctly executed preparation steps.
After confirming a lack of response to toe pinch, apply ointment to the eyes and use tape to immobilize the extremities of a 20 to 25 gram adult mouse. Use a depilatory cream to remove all of the hair on the left side of the animal, and use the spleen to locate the left kidney through the skin on the dorsal and caudal side of the spleen. Make a 0.5 centimeter incision in the skin, followed by a smaller incision in the peritoneum just large enough for the kidney to be pushed through.
Extrude the kidney with gentle pressure and place a polysiloxane kidney stabilizer form around the tissue. Line the kidney up with the spacer such that the lateralmost surface of the kidney extends beyond the stabilizer by about one millimeter, and fix the kidney to the form with cyanoacrylate adhesive. Glue a head plate to the stabilizer form and mount the head plate to mounting bars on the base plate.
Next, fill the well in the polysiloxane support with one percent agarose solution and hold a 10 millimeter cover slip on top of the form until the agarose is firm. Seal the cover slip to the head plate with glue and create a ring around the cover slip with dental cement. Then inject 100 to 150 microliters of FITC-Dextran retro-orbitally and quickly move the mouse and fixation plate to the two-photon microscope stage.
After manually focusing on the kidney surface, switch to the non-scanning two-photon mode and explore the imaging window to locate a target glomerulus that is greater than 30 micrometers below the cover slip and less than 400 micrometers from the lateral kidney capsule. Record the lateral and vertical distance to the target glomerulus. Then record the x, y, and z stage coordinates for the glomerulus.
And lastly, raise the objective focal point about one millimeter into the water column without changing the x and y stage coordinates. Drive the pipette tip into the water column and turn on the DAPI excitation. Move the pipette in the x and y dimensions to the point of maximal fluorescence of the tip.
This will be the center of the objective. Change the x citation setting to red fluorescent protein and visualize the pipette with the ocular to allow precise centering in the ocular view. Switch back to two-photon to find the pipette under the live two-photon view and place the tip precisely in the center of the image, this is the registration position.
Then save an image of the pipette and register the stage and the micropipette controller coordinates. Remove the pipette from the water column in the x-axis without moving the y and z-axis and move the pipette z to the target glomerulus z-coordinate and the edge of the kidney. Note the stage x and calculate the kidney edge pipette x using the offset from the registration stage x.
Increase the stage x to move the stage toward the pipette until the edge of the kidney is far to the left of the screen, while remaining visible. Quickly advance the pipette to about 100 micrometers away from the kidney edge pipette X has just calculated. Increase the red gain and begin advancing the pipette tip slowly to the kidney edge under live two-photon imaging while monitoring the red pixel histogram.
Drive the pipette in the x-axis slowly to the glomerulus target pipette x, keeping an eye on the stage x. Upon reaching the glomerulus, document the position with a z-stack. With the pipette in position, set the micropump to inject 100 nanoliters of perfluorodecalin over two minutes.
To ensure patency of the pipette, and to reduce confounding from pipette plugging during entry. After four to six minutes of filtration, set the micropump to aspirate up to 300 nanoliters at a rate of up to 50 nanoliters per minute. This beautiful, near surface glomerulus demonstrates favorable imaging due to the surface position of the glomerulus at 20 micrometers below the renal capsule.
The glomerulus is too close to the surface for microinjection however, as the pipette would hit the cover slip. These glomeruli are optimally positioned with the lateral edges 250 micrometers to the right and appearing less sharp because of the refraction caused by their 70 micrometer depth from the capsule. Both factors that make the glomeruli accessible.
In this typical renal entry image, a mean intensity projection from a z-stack with orthogonal views reveals the pipette tip within Bowman's space, note the red pipette tip spectral artifact from the extremely bright fluorescence of the quantum dots arranged on the conical section of the tip. Here, a volume rendering from a z-stack acquired after positioning a pipette in Bowman's space, shows the pipette tip within the space abutting the capillary tuft. If a pipette with a too large opening is used, the renal capsule can break causing subcapsular bleeding and entry of the blood into the pipette lumen.
Using this method, 17 proteins, primarily of low molecular weight, have been identified from a minimum of two unique peptides per protein. Coupled with this method, other methods such as maspectroscopy, ion sensitive electrode measurements, and fluorescent antibody injection can be used to answer questions about the role of luminal solutes in tubular and glomerular physiology.
