Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

In the data presented in this study show the dramatic effects of unilateral ureteral obstruction on kidney function, inflammation, and fibrosis. It helps us to understand the pathophysiologic mechanisms of unilateral ureteral obstruction. The main advantage of this technique is the ability to study cell-cell interactions, subcellular events, and associate structure and function in a dynamic fashion within a functioning kidney.

Demonstrating the ureteral procedure will be done by Dr.Silvia Campos-Bilderback who's a surgeon in our lab. To begin, anesthetize the rat, then make an incision along the midline and locate the left kidney to separate it from the surrounding peritoneal organs. After locating the renal pedicle, comprising the renal artery, renal vein, and ureter, separate the ureter without damaging the delicate structure.

Use forceps to loop and tie a 3-0 suture around the ureter, then tie a knot a few millimeters on either side of the first knot to assure complete obstruction. Once the ureter is tied, carefully close the successive muscle layers, then close the abdomen completely. Close the outer skin with surgical staples.

For surgical preparation, place the anesthetized rat with an indwelling venous access line on the side with the shaved left flank facing up flat and straight on the table. Ensure that the front paws of the rat touch each other as should the rear paws. Once the rat is positioned, palpate the left flank just below the ribs to feel for the kidney and determine the natural position in the abdomen, then draw a line using a permanent marker along the shaved area, bisecting the kidney center in a nose-to-tail orientation.

Use a pair of toothed forceps to grasp and lift the skin upward and pinch the permanent marker line with a pair of hemostats to crush the underlying vasculature and prevent bleeding. Repeat the procedure for the thin outer muscle layer to minimize bleeding. To make the final incision of the thin inner abdominal muscle layer, palpate the kidney to estimate the size and position and lift the inner muscle layer with a pair of forceps, then crush a line that bisects the skin above the kidney with the hemostats that is approximately 1/3 the estimated size of the kidney.

While maintaining the grip on the muscle layer with the forceps, make the final incision. Next, use forceps in each hand to grip and hold the kidney fat at the lower pole of the kidney with a hand-over-hand technique working downward. Having a firm grip on the fat with one hand, pull the fat and very gently squeeze the kidney through the incision.

If the kidney does not pass through, easily widen the incision. To identify the white blood cells or WBCs lodged in the capillary loops, administer Hoechst 33342 nuclear stain at 8 micrograms per kilogram of rat weight via an indwelling venous access line. On the microscope, place the exposed kidney against the edge of the dish with a slight rotation so the abdominal side of the kidney is contacting the cover slip and the dorsal side is facing away from the edge.

To further minimize motion, pack two sterile 2-by-2 gauze pads moistened with saline against the dorsal side of the kidney reinforcing the contact of the abdominal side of the kidney to the edge. When the sample is positioned, look through the microscope eyepiece under epifluorescence illumination using a dual-pass rhodamine FITC cube. If a movement is detected in the sample position, make minor adjustments by adjusting the gauze ensuring it does not push under the kidney.

Roll the rat over slightly so the thorax is further away from the dish. Scan the surface of the kidney using epifluorescence illumination and mark the glomeruli positions using the software associated with the motorized stage controller. For each color channel under 2-photon illumination, take a shallow 3D volume of the upper portion of each marked glomerulus to serve as the background image.

In the display option of the imaging software, use a pseudo color palette to visualize the faint intensities of the background fluorescence of the glomerular capillary loops. Using a superficial blood vessel as the focal point, slowly infuse the fluorescent Texas Red RAP serum albumin or TRRSA while observing the rise and fall of fluorescence due to the systemic distribution until an intensity in the peritubular vasculature and capillary loops is just below saturation. After 10 minutes, acquire 3D volumes for all marked and imaged glomeruli at 1 micrometer intervals.

Find an appropriate vessel, either a capillary loop or a peritubular vessel, then rotate the image using the Rotate function as the Line Scan function in the image acquisition software requires the vessel to be perpendicular. Once the vessel is rotated and lying flat, select the XT function on the Acquisition menu and set it up to scan 4, 000 lines. Place the line across the vessel to be examined with the focal plane at the maximum diameter of the segment.

Next left-click on the color composite image and select the Take Snapshot tab to generate a reference image of the area where the line scan was taken. Immediately click the Start button to capture the line scan of the vessel. Later, import the line scans into the image processing software to determine the RBC flow rate.

Under the Measure dropdown menu, open the Show Region Statistics dialogue box and select the Single Line Drawing tool to draw a line that matches the slope of the RBC shadows. After noting the width and height of the line, use an equation to calculate the speed, then obtain an average of five calculations to report the speed for each line scan. Center a glomerulus in the imaging field and take a 3D data set starting at the glomerular surface and ending at 30 to 35 micrometers.

Use a 1-micrometer step size in the Z direction. Identify the WBCs by comparing the blue Hoechst channel with the Texas Red albumin channel by looking for exclusion of red dye in the capillary loop and a corresponding nuclear stain. If the WBCs appear static over three optical sections, define the cells as adhered, then report the values as occurrence per 10 optical sections from the top of the glomerulus taken at 1-micrometer intervals.

The number of glomerular per field in Munich Wistar Fromter or MWF rats was increased in the 5-week UUO group than in untreated rats. Sprague-Dawley rats, or SD rats, went from having no surface glomeruli to 2.02 per field following five weeks of the unilateral ureteral obstruction. Three-dimensional reconstructed images were observed to view the renal surface.

The 5-week unilateral uretal obstruction in the SD rats did not result in areas resembling normal tubular epithelia as seen in the MWF, untreated, and partially in 5-week UUO MWF rats. In the study of the renal vascular dynamics, renal blood flow was significantly reduced in the 5-week UUO MWF, and SD groups compared to untreated rats. The RBC speed within the glomerular capillary loops was significantly decreased in the 5-week UUO MWF, and SD rats compared to untreated MWF rats.

Additionally, untreated MWF rats had fewer WBCs per 10 optical sections from the top while the number increased in 5-week UUO MWF and 5-week UUO SD rats. With unilateral uretal obstruction, an increase in albumin permeability was seen. An albumin accumulation within the Bowman space was intense after unilateral uretal obstruction.

The S1 segment endocytosis large quantities of albumin as observed with physiologic conditions in untreated MWF rats. The same uptake could not be seen in the MWF or SD rats following 5 weeks of unilateral uretal obstruction. Many different methods such as repeat imaging to study a process over time, fixation of a specific area for imaging, and subsequent utilization of the fixed tissue to carry out higher resolution microscopy can be done in conjunction with this technique.

The technique is considered disruptive technology. It is enabled investigators to answer many new questions. In doing so, new insights have been provided and paradigm shifting data have been obtained.