Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The overall goal of the following experiment is to assess nephron segment composition and functionality in the adult zebra fish kidney. This is accomplished by first injecting fluorescently labeled dextrin into an nest that ties zebra fish. The second step is to remove and fix the kidney to prepare tissue for subsequent labeling methods.

Next, the kidney is bleached to remove pigmentation and then stained to fluorescently labeled the desired nephron segments. Results are obtained using immunofluorescence, microscopy and or bright field imaging that enable visualization of kidney anatomy based on the selected stain or stains. The following labeling methods can help answer key questions in the kidney field regarding organ anatomy and can be used to study nephron composition and evaluate renal functionality both before and after an injury event.

The Implications of these techniques extend toward the study of genetic anomalies in adult kidney formation and could also be applied to assess renal status during chronic disease modeling. To begin decant the solution from an anesthetized fish and carefully place the animal on a wet sponge mold ventral side. Up next, fill an insulin syringe with 20 microliters of thaw dextrin stock solution.

Place the needle so that insertion occurs at a shallow angle at the ventral midline of the abdomen. To avoid injecting the organs slightly, raise the needle just after injecting to lift the body wall and create a space in which to inject the solution. After the injection, gently return the fish to the tank to recover from anesthesia.

To begin decant away any extra solution from an anesthetized fish and place it on a tissue or paper towel. After removing the head and internal organs, use super fine dissection needles to pin open the body walls. Locate the kidney organ which is adherent to the dorsal wall of the animal.

Use fine forceps to detach the kidney from the dorsal wall. Gently place the kidney into a five milliliter glass vial containing one XPBS and wash three times for five minutes each. Remove the kidney with a transfer pipette and place it onto a clean glass Slide with one to two drops of one XPBS.

Use fine forceps to flatten the kidney on the slide, making sure no tissue has curled or otherwise overturned on itself. A tungsten wire tool can be used to make small incisions in the connective tissue to facilitate the flat positioning of the kidney. Next place small pieces of modeling clay on each corner of an 18 by 18 millimeter glass cover slip.

Slowly set the cover slip onto the kidney, keeping a slight angle. To minimize trapping air bubbles, add an additional drop of one XPBS to fill the space between the cover slip and slide if necessary. A separate group of zebrafish are euthanized and soaked in fixative overnight.

When ready, use fine forceps to carefully detach the kidney from the dorsal body wall and place the organ into a glass vial. Wash the dissected kidney three times with three to five milliliters of one XPBS with 0.05%tween for five minutes each. Next, remove the PBS solution and rinse the kidney with three milliliters of a 5%sucrose solution for 30 minutes.

After this time, replace the solution with three milliliters of 30%sucrose and store overnight at four degrees Celsius the following day. Remove the solution and wash the kidney two times before adding three to five milliliters of bleaching solution. To remove the melanocyte pigmentation present on the kidney organ, place the glass vial on a rotator and watch carefully as the pigmentation disappears.

Depigmentation typically takes approximately 20 minutes, but occasionally can take as long as 60 minutes. If the bleaching solution is left on too long, disintegration can occur and it is therefore advised to monitor the sample every 10 to 15 minutes to check integrity when the pigmentation has been removed from the kidney wash twice and incubate with 4%PFA solution for one hour at room temperature. Next, remove the 4%PFA solution and wash the kidney three times after the last wash at three to five milliliters of blocking solution and incubate the kidney at room temperature for two hours.

When blocking is complete, proceed directly to the selected staining protocol. Remove the blocking solution and wash the kidney three times before letting the kidney soak in one XPBS for at least 10 minutes. During this time, transfer the kidney to a 12 well or 24 well culture dish.

Replace the one XPBS with sufficient working alkaline phosphatase substrate solution to cover the sample and place the sample on a rotator for 30 minutes at room temperature. After this time, stop the reaction by washing the kidney in alkaline phosphatase wash buffer solution. Rinse the kidney with three changes of wash buffer solution over 10 to 15 minutes.

Next, remove the wash buffer solution and mount the kidney onto a clean glass. Slide in the phosphatase mounting medium included in the labeling kit. Visualize the alkaline phosphatase stained kidney using a stereo microscope or compound microscope with a hooked to Debbie filter set.

First, prepare a 200 microliter working DBA solution by diluting DBA in one XPBS. Replace the PBS solution with 200 microliters of DBA solution and place the sample on a rotator for one hour at room temperature. After this time, remove the DBA solution and wash the kidney three times with three to five milliliters of one XPBS for five minutes each.

Remove the one XPBS and mount the kidney on a clean glass slide. As demonstrated earlier, visualize the DBA stained distal segments of the kidney using a standard Trixie or Texas red filter set on a fluorescent stereo microscope or compound microscope. In this brightfield image of an unbleached kidney, the black pigmentation corresponds to the scattered population of melanocytes.

Here nephron PCT segments are visualized three days following dextrin injection. The inset contains an image of a single nephron with melanocytes. This image shows an adult kidney after removal of melanocyte pigmentation.

These representative images depict slight morphological variations between PCT segments, while many nephron PCTs are tightly coiled. Other nephrons contain PCT regions that have minimal coiling dextrin cascade blue dextrin, Lucifer yellow, and Dextrin fluoro Ruby all preferentially label the PCT segments in renal nephrons, both dextrin Cascade Blue and Lucifer Yellow show some non-specific labeling with much higher background observed in Lucifer yellow treated kidneys. In contrast, dextrin fluoro Ruby showed dramatically reduced background with intense PCT labeling.

An adult kidney stained by wish to detect a specific marker of the PCT transporter cell type shows an expression pattern characteristic of dextrin uptake with a distribution of various tightly coiled looped PCT domains, as well as more elongated PCT stretches that show a consistent wide diameter. After watching this video, you should have a good understanding of how to successfully label nephron segments within the adult zebrafish kidney, such as the proximal tubule with alkaline phosphatase and medic distal tubule with DBA. Don't forget that working with 4%paraldehyde can be extremely hazardous, and precautions such as wearing a coat, gloves and eyewear should always be taken while performing this procedure.