The overall goal of this procedure is to successfully visualize the filtration unit of the nephron and quantify filtration of fluorescent macromolecules across the filtration barrier into the urinary space in vivo. This is accomplished by first preparing the microscope stage for the live animal. Next, the kidney is exposed and the rat is placed on the stage.
Then individual glomeruli are identified and mapped and background 3D images are acquired. Finally, fluorescent albumin is infused. 3D volumes of individual glomeruli are acquired, and permeability is quantified.
Ultimately, results can be obtained that show the filtration coefficient of macromolecules across the glomeruli in the kidney through the use of intra vial two photon microscopy. The main advantage of two photon microscopy is that it allows for the simultaneous quantification of both glomerular filtration and proximal tubular reabsorption and transcytosis in the animal. At the same time, the implications of the technique extend beyond mechanistic into diagnostic and therapeutic as the importance of understanding both the role of the glomerular filtration and proximal tubular.
Reabsorption of albumin is critical in determining targeting of therapy. After anesthetizing the rat, insert an indwelling venous access line through either the jugular or femoral vein and shave the left flank from just below the rib cage to just above the left thigh. This is a non survival surgery.
Place the rat flat on its right side so that the left shaved side is facing up. Make sure it is flat on the bench with its posture elongated and not crouched with front paws touching each other and rear paws touching each other very gently. Palpate to feel the kidney to determine where it naturally lays within the retroperitoneum, and use a sharpie to draw a straight line parallel to the body with a pair of tooth forceps.
Pick up the skin and use a pair of hemostats to pinch the skin along the drawn line to crush the tissue and prevent bleeding using a pair of surgical scissors. Cut along the incision. Use the same procedure for cutting the outer muscle layer, which is thin as needed.
Use hemostats to prevent bleeding for the incision into the inner muscle layer, which will expose the peritoneum. Re palpate the kidney to estimate size. Pinch an incision line smaller than the kidney assuring the incision is just over the kidney.
It is best to make this incision too small and make it larger if needed. Locate the kidney and use forceps to grip the surrounding fat working towards the bottom pole of the kidney in a hand over hand fashion. Once the lower pole of the kidney is reached, gently pull the kidney through the incision while very gently squeezing below the kidney to exteriorize it.
If the incision is too small, crush the muscle layer and cut to widen it. Repeat the procedure to exteriorize the kidney after exteriorizing the rat's kidney and placing the rat on the microscope stage for imaging. If your microscope is equipped with a motorized stage capable of marking locations, using a dual pass fluorescein rod, domine filter, and a low powered objective, find and center individual glomeruli, which will appear as empty circular structures surrounded by proximal tubules.
Having an inherent yellow orange autofluorescence mark each location, switch the turret to a higher power water immersion objective, and take 3D data sets of each glomeruli. Making sure the capillary loops and Bowman space are clearly visible. Using a pseudo color palette will help to visualize these structures.
Next, focusing on a superficial blood vessel slowly infuse fluorescent albumin, making sure time is given to allow for systemic distribution for molecules with a low glomerular cing coefficient or GSC, it is essential to maximize the intensity values in the plasma, but not to reach levels that will saturate the photo detectors in the microscope. This increases detectability of filtered molecules After waiting approximately 10 minutes to allow any potential small molecular weight fragments to clear, acquire 3D volumes at one micron intervals to be used in calculating albumin's using metamorph image processing software. Load the 3D data sets along with the background images for each glomeruli in the volume containing the fluorescent albumin.
Locate a superficial capillary loop with enough empty space between its defined margins and the edge of the Bowman's capsule. In the background volume. Locate the same focal plane, which should contain all the visual cues of the albumin containing image.
Select a region within the capillary loop of interest and note the average intensity reading. Next, select a region within the Bowman space and note the average intensity reading. These will be used as background values for quantitation.
Select the similar region within the Bowman space in the albumin containing image. Do this for at least two other regions to acquire a value for the average intensity within Bowman's space. Select the capillary loop with the brightest plasma intensity and draw a region around it.
Next, using the threshold function, highlight the bright values within the circulating plasma, avoiding the dark streaks that are circulating red blood cells. Note the average intensity values of the selected plasma space. It is important to preferentially select the bright areas of the plasma because factors within the blood will only serve to cause an underestimation of plasma fluorescence levels.
Enter the values into an Excel spreadsheet to calculate the GSC where GSC equals the difference of the raw bowman space. Intensity minus background Bowman space intensity divided by the difference of the raw capillary loop intensity minus background capillary loop intensity. Uptake of filtered fluorescent albumin occurs predominantly in the early segment of proximal tubules.
The S one, this panel shows a cross section of a glomerulus and an S one segment taken from a Munich Wistar prompter rat, roughly 20 minutes post infusion of an initial Texas red RSA bolus. The opening of the Bowman space and avid uptake of the albumin in red is shown in the S one segment. This panel shows a shallow 20 micron 3D projection of the same dataset, and shown here is a lower power projection taken approximately 60 minutes post infusion.
These images demonstrate that intra vital microscopy can allow visualization of the fate of infused material after filtration corroborating to some degree, the filtration coefficient observed shown here are images taken from the surface glomerulus. This panel is a background image and this image was taken about 12 minutes post infusion of Texas Red rat serum albumin. These two panels are drawn in pseudo color.
Three small regions of interest drawn in Bowman's space are used to calculate the average intensity of fluorescent albumin that has moved across the filtration barrier. Average intensity values for the individual regions were reported in the highlighted area within the show region Statistics dialogue box. The GSC value for albumin of 0.0111 derived for this individual glomerulus falls within the range scene in this strain of Munich star rats when in the fed condition.
Following this procedure, other methods such as quantitative and qualitative analysis can be performed on other renal structures to answer additional questions such as the eventual trafficking of the macromolecule after filtration into the urinary space After its development. This technique allowed researchers in renal physiology to explore dynamic physiologic and pathophysiologic processes, not just once but longitudinally. Over time, it was.
