Name: an efficient and fast method for labeling and analyzing mouse glomeruli
Uploaded: 2024-02-09T13:30:00.000+00:00
Duration: 9 min 50 s
Description: Watch this Scientific Journal Video about Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues at JoVE.com

This study was supported by grants from the National Natural Science Foundation of China (82204951) and Sichuan Science and Technology Program (2020JDRC0102).

Adult C57BL/6 mice (6 weeks of age, 25-30 g) were used in this study. All procedures were performed in compliance with local regulations of animal welfare and experimental ethics. The study was approved by the West China Hospital of Sichuan University Biomedical Research Ethics Committee.
1. Glomeruli labeling and tissue preparation
<ol>
	<li>Glomeruli labeling
	<ol>
		<li>Dissolve FITC-dextran (10 mg) in 1x phosphate-buffered saline (PBS) at a ratio of 1:1 (1 mg: 1 mL) to prepare the working probe solution. 
		NOTE: The working solution can be stored at 4 &#176;C for 1 month.</li>
		<li>Place the C57 mouse in a mouse tail vein fixator. Wet the gauze with hot water, wrap the gauze around the middle part of the mouse tail, and warm the tail for 1 min.</li>
		<li>Wipe the mouse tail with 75% ethanol until the left and right tail veins are visible.</li>
		<li>Draw 100 &#181;L of the working probe solution with a 1 mL insulin syringe and slowly inject the solution into the mouse tail vein.</li>
		<li>After injection, let the probe solution circulate for 30 min. Allow the mice to move freely in their cages.</li>
		<li>Once sufficient circulation is achieved, deeply anesthetize the miceby intraperitoneal injection of sodium pentobarbital (1%, 60-80 &#181;g/kg).</li>
	</ol>
	</li>
	<li>Kidney sampling and fixation
	<ol>
		<li>Fix the anesthetized mice to the anatomical plate in a supine position. Secure their paws with medical tape.</li>
		<li>After skin preparation, euthanize the mice with a lethal dose of sodium pentobarbital (1%, 10 mg/kg).</li>
		<li>Make a midline abdominal incision to expose the abdominal cavity.</li>
		<li>Reveal the kidneys and remove them with a scalpel and scissors in the fume hood. Remove the renal envelope gently.</li>
		<li>Collect the kidneys and fix them in 4% paraformaldehyde (PFA) at 4 &#176;C overnight.</li>
		<li>Wash the samples with PBS three times 1 h each with shaking at room temperature (RT).</li>
		<li>Prepare the kidney for microscopy.
		<ol>
			<li>Slice the kidney for confocal microscopy: Cut the kidney into 1 mm thick slices using the brain matrix (1 mm steel, 40-75 g) to standardize the slice thickness. Transfer the slices into 4% PFA and continue the fixation at 4 &#176;C overnight.</li>
			<li>Whole kidney for light-sheet microscopy: Fix the kidneys in 4% PFA at 4 &#176;C for 48 h.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Clearing
<ol>
	<li>Reagent preparation
	<ol>
		<li>Prepare CUBIC-L reagent by mixing 10% (wt/wt) N-butyldiethanolamine, 10% (wt/wt) Triton X-100 and 80% ddH2O.</li>
		<li>Prepare CUBIC-R reagent by mixing 45% (wt/wt) antipyrine, 30% (wt/wt) nicotinamide, 0.5% (vol/vol) N-butyldiethanolamine and 24.5% ddH2O. Ensure the solution has a pH of 9.6-9.8 and a refractive index (RI) of 1.522. 
		NOTE: CUBIC-L is used for delipidation, and CUBIC-R is used for refractive index matching.</li>
	</ol>
	</li>
	<li>Clearing procedure
	<ol>
		<li>Collect the kidney samples in a 50 mL centrifuge tube and then clear them in CUBIC-L with shaking at 60 rpm at 37 &#176;C. Replace CUBIC-L every 4 h (for kidney slices) or every 24 h (for a whole kidney) until achieving satisfactory optical transparency. 
		&#8203;NOTE: This process takes 1 day for kidney slices and 5 days for a whole kidney.</li>
		<li>Wash the samples three times in PBS with gentle shaking at RT for 1 h each.</li>
		<li>Apply CUBIC-R to the samples, shaking samples at 37 &#176;C, 60 rpm for 6 h.</li>
		<li>Observe the cleared samples directly or store them in black tubes filled with CUBIC-R at RT for 1 week.</li>
	</ol>
	</li>
</ol>
3. Image acquisition
<ol>
	<li>Confocal imaging 
	NOTE: Image the sliced kidney samples with confocal microscopy (Lenses: A Plan Apo &#955; 4&#215;/0.2 N1 objective or a Plan Apo VC 10&#215;/0.45 DIC N1 objective). Capture large images (e.g., 6052 &#181;m x 6052 &#181;m for the kidney slice) with z-stacks.
	<ol>
		<li>Turn on the confocal microscope in the following sequence: laser power, confocal control switch, computer, microscope, and confocal hardware. After running the self-test, turn on the confocal software.</li>
		<li>Select the appropriate lens. Remove the sample from the CUBIC-R reagent, place it in the confocal dish, and cover it to prevent the CUBIC-R reagent from drying out.</li>
		<li>Move the sample to the middle of the field of view and adjust the focal plane until it is in focus.</li>
		<li>Apply 3D reconstruction (step 3.1.4.1) and large image reconstruction (steps 3.1.4.2-3.1.4.3).
		<ol>
			<li>3D reconstruction (confocal): Switch the zoom plane in one direction until no fluorescence is visible; this position is defined as the top. Then, switch the zoom plane in the opposite direction until no fluorescence is visible; this position is defined as the bottom. Click the Run button in the lower-right corner of the dialog box to start the scanning.</li>
			<li>Large image reconstruction (confocal): Move the field of view to the middle of the sample, and set the current position as the center. Select a 2 x 2 field of view.</li>
			<li>In the ND multifunction interface, select the Z stack to reconstruct large images and set different options in the panel. Then press the Run butto n to start the scanning.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Light sheet fluorescence microscopy (LSFM) imaging 
	NOTE: Image the whole kidneys with a light sheet fluorescence microscope (Lens: EC Plan-Neofluar 5x/0.16 (Medium n = 1.529) objective).
	<ol>
		<li>Sample attaching (LSFM):
		<ol>
			<li>Glue the transparent kidney to the sample fixing adapter and wait until the kidney is firmly attached.</li>
			<li>Soak the kidney in the sample bin and push the sample bin into the microscope system. Close the hatch.</li>
		</ol>
		</li>
		<li>Observe and scan (LSFM):
		<ol>
			<li>In the Locate interface, move the sample to a suitable observation position. Switch to the Acquisition interface, set the optical path, select 488 nm laser, LBF 405/488/561/640, optical splitting filter block SBS LP 490, select Camera 2, and change the pseudo-color to green.</li>
			<li>Fill in the Left/Right Z Offset value recorded earlier in the Channel submenu. Select the smallest zoom (0.36) and fine-tune it for maximum clarity.</li>
		</ol>
		</li>
		<li>Find the X\Y boundary and Z range of the entire organ. Adjust the laser intensity and exposure time (preferably less than 10 ms), and click Run to start.</li>
		<li>If the tissue is too big, merge two or more images with image stitching. Open the captured file using the microscopy software (here, ZEISS ZEN 3.7). Select Processing &#62; Method &#62; Stitching &#62; Method Parameter &#62; Apply to complete image stitching.</li>
	</ol>
	</li>
</ol>
4. Data processing and quantification
NOTE: Process the image stacks with Imaris (image analysis) software, using the Surface function to label the glomeruli and perform analysis.
<ol>
	<li>Count of glomeruli
	<ol>
		<li>Import large images (confocal microscopy) or stitched images (light-sheet microscopy) into the image analysis software.</li>
		<li>Open the files in the image analysis software. Click the Surface button to create a new Surface. Follow the guide at the bottom left part of the screen.
		<ol>
			<li>Click the Next button (right arrow button) without ticking anything.</li>
			<li>Tick the box before the Smooth option and enter a proper number (e.g., 14.5) to control the detail of the Surface. Select Background Subtraction in Thresholding and fill the box with the approximate average diameter of the glomeruli. Click the Next button.</li>
			<li>Adjust if needed, and click the Next button. Select the proper range and click the Next button to create a Surface.</li>
		</ol>
		</li>
		<li>Click Filter, then Add button to add Sphericity to filter out non-spherical objects. Click the Duplicate button to copy this new surface.</li>
		<li>Click the Statistics button, then click the Overall button; the software will present the number of glomeruli as the Total Number of Surface.</li>
	</ol>
	</li>
	<li>Volume of glomeruli
	<ol>
		<li>Create a surface of the glomeruli as above.</li>
		<li>When the Surface is completed, click the Selection button to select the target objects and present the volume of every object. Click the Save button, and export statistics to a spreadsheet for further analysis.</li>
	</ol>
	</li>
	<li>Volume of the slice or whole kidney
	<ol>
		<li>Import large images (confocal microscopy) or stitched images (light-sheet microscopy) into the image analysis software.</li>
		<li>Open files in the image analysis software. Click the Surface button to create a new Surface. Follow the guide at the bottom left part of the screen.
		<ol>
			<li>Click the Next button (right arrow button) without ticking.</li>
			<li>Tick the box before the Smooth option and enter a proper number (e.g., 50) to control the detail of the Surface. Select Absolute Intensity in Thresholding. Click the Next button.</li>
			<li>Adjust the Surface until it covers the whole tissue, and click the Next button. Click the Next button to create the Surface.</li>
		</ol>
		</li>
		<li>Click the Selection button to select the Surface of the whole tissue and present the volume of the tissue. Click the Save button to export statistics to a spreadsheet.</li>
	</ol>
	</li>
</ol>

Fluorescent Labeling of Glomeruli in Cleared Mouse Kidneys for Microscopy

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All authors declare no conflict of interest.

Tissue-clearing technologies can be classified into 3 or 4 groups29,30,31. Organic solvent-based tissue clearing (e.g., DISCO and PEGASOS), aqueous-based tissue clearing (e.g., CUBIC), and hydrogel embedding tissue clearing (e.g., CLARITY) have all been applied in kidney clearing 25,26,28,32. CUBIC, as we have demonstrated, works best when the glomeruli (vessels) are labeled with FITC-Dextran. In addition, CUBIC has a relatively more convenient operation process and requires no more than ordinary microscopic lenses27, as the processed samples do not need to be soaked in organic reagents33. However, different clearing methods and vessel label methods have their own advantages and disadvantages; researchers may need to experiment with different approaches to meet their specific needs.
Previous studies noticed that the glomeruli could be labeled in cleared kidneys when blood vessels in the kidneys were labeled13,25,26,28. A thorough explanation for this phenomenon still needs to be further investigated, but it can be deduced that a good labeling method for the vascular system also effectively labels the glomeruli. Based on the experimental results, we would recommend the intravascular injection of FITC-Dextran with a medium molecular weight (such as 150 kDa). However, since labeling relies on blood circulation, this method may fail to label the glomeruli when they lack circulation.
LSFM permits large-scale tissue observation, making it more suitable for whole kidney glomeruli detection. However, light-sheet microscopes may not be available in every laboratory. So, we are providing an alternative set of protocols using ordinary confocal microscopes. Although the kidney needs to be sliced into multiple sections, obtaining data such as the counts and volume of glomeruli in the whole kidney is feasible. Besides, confocal microscopy provides a smaller volume of data, which is more data processing friendly.
Imaris provides direct data output. However, care should be taken to match the analog signal to the original closely. Additionally, the software may take a long time to process large files, which can be frustrating. As long as the glomeruli can be clearly labeled and visualized, there is more than one unique solution for data analysis. Researchers from different laboratories could work with their own familiar software and programming languages.

The number and volume of glomeruli are very important for the diagnosis and treatment of various kidney diseases1,2,3,4,5. The golden standard of glomeruli number estimation is the physical dissector/fractionator combination. However, this method requires special reagents and equipment, making it slow and expensive6,7,8,9. Biopsy provides a wealth of information, but obviously, this method is only suitable for rough estimations10,11. Medical imaging technologies, including magnetic resonance imaging (MRI), computed tomography (CT), and X-ray, are also widely used in glomerular detection12,13,14,15, but such technologies require bulky instruments. New methods, such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometer16 or the thick and thin section method17, have also been used in glomerular detection, though they remain tedious and laborious.
With the help of transparency technologies, it is possible to observe deeper depths and obtain richer and more complete information from thick tissues or even whole organs18,19,20,21,22,23. Therefore, transparency technologies have been widely used in kidney research24. The observation and detection of glomeruli in the cleared kidneys are also involved. However, these published articles either only briefly referred to glomerular detection25 or used difficult-to-achieve labeling methods such as transgenic animals26, self-produced dyes13, or high-concentration antibody incubation27 to label the glomeruli. In addition, although studies had analyzed glomeruli in cleared kidneys, the analyses were always limited13 or relied on analysis algorithms established by the authors themselves26.
We have previously demonstrated a more convenient way to label the glomeruli in mice kidneys28. By using Imaris, we found that glomeruli count, frequency, and volume could be quickly obtained. Thus, here we present a more accessible, comprehensive, and simplified set of methods to label and analyze the glomeruli of mice kidneys.

<table><tbody><tr><td>4% PFA</td><td>Biosharp</td><td>7007171800</td><td>Fixation reaagen</td></tr><tr><td>502 Glue&amp;nbsp;</td><td>Deli</td><td>7146</td><td>For fixing the kidney to the sample fixing adapter&amp;nbsp;</td></tr><tr><td>Antipyrine</td><td>Aladdin</td><td>A110660</td><td>Clearing reagent</td></tr><tr><td>Brain Matrix</td><td>RWD Life Science</td><td>1mm 40-75</td><td>Tissue slicing</td></tr><tr><td>Confocal microscopy</td><td>Nikon</td><td>A1plus</td><td>Image acquisition</td></tr><tr><td>FITC-Dextran</td><td>Sigma-Aldrich</td><td>FD150S</td><td>Labeling reagent</td></tr><tr><td>Light sheet fluorescence microscopy&amp;nbsp;</td><td>Zeiss</td><td>Light sheet 7&amp;nbsp;</td><td>Image acquisition</td></tr><tr><td>Mice</td><td>Ensiweier</td><td></td><td>Adult C57BL/6 mice (6 weeks of age, 25&amp;ndash;30 g)&amp;nbsp;</td></tr><tr><td>N-Butyldiethanolamine</td><td>Aladdin</td><td>B299095</td><td>Clearing reagent</td></tr><tr><td>Nicotinamide</td><td>Aladdin</td><td>N105042</td><td>Clearing reagent</td></tr><tr><td>Pentobarbital Natriumsalz</td><td>Sigma-Aldrich</td><td>P3761</td><td></td></tr><tr><td>Tail vein fixator</td><td>JINUOTAI</td><td>JNT-FS35</td><td>Fix the mouse for vail injection</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Clearing reagent</td></tr></tbody></table>

an efficient and fast method for labeling and analyzing mouse glomeruli

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological imaging provides intuitive information; thus, it is of great significance to label and observe the glomeruli. However, the glomeruli observation methods currently in use require complicated operations, and the results may lose label details or three-dimensional (3D) information. The clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) tissue clearing technology has been widely used in renal research, allowing for more accurate detection and deeper detection depth. We found that mouse glomeruli can be rapidly and effectively labeled by tail vein injection of medium molecular weight FITC-Dextran followed by the CUBIC clearing method. The cleared mouse kidney could be scanned by a light-sheet microscope (or a confocal microscope when sliced) to obtain three-dimensional image stacks of all the glomeruli in the entire kidney. Processed with appropriate software, the glomeruli signals could be easily digitized and further analyzed to measure the number, volume, and frequency of the glomeruli.

This study provides a simple and efficient method for labeling and analyzing the glomeruli in mice kidneys.
Glomeruli (blood vessels) can be well labeled by intravascularly injected FITC-Dextran. After the clearing process, the kidney became transparent (Figure 1A), and the glomeruli could be clearly observed by using light-sheet microscopy (Figure 1B) or confocal microscopy (Figure 1C). Confocal microscopy has a limited scanning depth, so kidneys should be cut into approximately 1 mm-thick slices. If a light-sheet microscope is used, the whole kidney can be scanned directly.
With the clear signals, it was easy to count the number of glomeruli. In fact, the labeling was so effective that the glomeruli could be counted with the naked eye. The volume of glomeruli could also be directly measured. Of course, software such as Imaris greatly accelerated this process. Using the Surface function, all the glomeruli in a kidney slice (Figure 2), in an entire kidney (Figure 3), or in a strip (Figure 4)&#160;could be selected, and the number and volume of the glomeruli could be obtained directly (Figure 2C, Figure 3C, Figure 4C). The volume of the selected region could also be measured (Figure 2B, Figure 3B, Figure 4B), so the glomeruli volume ratio and frequency in a certain region or in the whole kidney could be calculated (Figure 2D, Figure 3D, Figure 4D). An area of the kidney could be selected to perform calculations in specific regions. We presented a strip similar to a biopsy. The number, volume, and frequency of the glomeruli in the strip could also be obtained easily (Figure 4).
As shown in the figures, the results presented in this study are (N = number, F = frequency, V = volume, V(a) = average volume, V(t) = total volume):
Nglomeruli in slice = 1128, Nglomeruli in whole kidney = 14006, Fglomerular in slice = 65 per mm3, Fglomerular in whole kidney = 105 per mm3, Vtotal glomeruli in slice/Vslice = 2.24%, Vtotal glomeruli in whole kidney/Vkidney = 5.54%, V(a)glomeruli in slice = 373654 &#181;m3, V(a)glomeruli in whole kidney = 521627 &#181;m3, V(t)glomeruli in slice = 421481724 &#181;m3, V(t)glomeruli in whole kidney = 7305386256 &#181;m3 (Figure 2D, Figure 3D).
Nglomeruli in strip = 63, Fglomeruli in strip = 72 per mm3, Vtotal glomeruli in strip/Vstrip = 2.23%, V(a)glomeruli in strip = 307698 &#181;m3, V(t)glomeruli in strip = 19384977 &#181;m3 (Figure 4D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65973/65973fig01.jpg" /> 
Figure 1: Kidney tissue transparency and FITC-dextran-labeled glomeruli. (A) Kidney before and after transparency treatment. (B) The image of a whole kidney scanned with LSFM. (C) The image of a slice of kidney scanned with the confocal microscope (left: 4x, right: 10x). scale bar = 300 &#181;m (4x, confocal); scale bar = 100 &#181;m (10x, confocal).; scale bar = 700 &#181;m (5x, zoom 0.36, LSFM). Glomeruli were labeled with FITC-Dextran. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65973/65973fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65973/65973fig02.jpg" /> 
Figure 2: The function Surface applied to the confocal scanned kidney slice. (A) Surface of the slice and all glomeruli are created. Pink = glomeruli, blue = out Surface of the kidney slice, green = original label of vessels (glomeruli and some big vessels). Scale bar = 300 &#181;m. (B) When the Surface of the slice (left) or the glomeruli (right) are selected (selected objects would turn yellow), data could be obtained directly (dotted box highlights where it shows the data). (C) The number, the volume of every single glomerulus, and the volume of total glomeruli could be directly obtained as well as the volume of the slice. (D) Exported data could be further analyzed so the calculation, such as the average volume of the glomeruli and volume ratio of glomeruli and selected area, could be worked out. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65973/65973fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65973/65973fig03.jpg" /> 
Figure 3: The function Surface applied to the LSFM scanned whole kidney. (A) Surface of the whole kidney and all glomeruli are created. Pink = glomeruli, blue = out Surface of the whole kidney, green = original label of vessels (glomeruli and some big vessels). Scale bar =1000 &#181;m. (B) When the Surface of the kidney (left) or the glomeruli (right) are selected (selected objects would turn yellow), data could be obtained directly (dotted box highlights where it shows the data). (C) The number, the volume of every single glomerulus, and the number of the glomeruli could be directly obtained, as well as the volume of the kidney. (D) Exported data could be further analyzed so the calculation, such as the average volume of glomeruli and volume ratio of glomeruli and the whole kidney, could be worked out. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65973/65973fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65973/65973fig04.jpg" /> 
Figure 4: The function Surface applied to the selected kidney strip. (A) Surface of the strip and all glomeruli are created. Pink = glomeruli, blue = out Surface of the kidney strip, green = original label of vessels (glomeruli and some big vessels). Scale bar =150 &#181;m. (B) When the Surface of the strip (left) or the glomeruli (right) are selected (selected objects would turn yellow), data could be obtained directly (dotted box highlights where it displays the data). (C) The number, the volume of every single glomerulus, and the volume of total glomeruli could be directly obtained, as well as the volume of the strip. (D) Exported data could be further analyzed so the calculation, such as the average volume of the glomeruli and volume ratio of glomeruli and selected area, could be worked out. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65973/65973fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues at JoVE.com

Author Spotlight: Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues 

This study presents an easy-to-use, complete, and simple set of methods to label and analyze glomeruli from CUBIC-cleared mouse kidneys. Data such as glomerulus number and volume can be obtained easily and reliably using fluorescein isothiocyanate (FITC)-Dextran, light sheet fluorescence microscopy (LSFM), or common confocal microscopy and software such as Imaris.

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological ...

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Nanyang Technological University

Research

JoVE Journal

Biology

1.2K Views.  Sichuan University. Het labelen en tellen van glomeruli met behulp van conventionele methoden kan ingewikkeld, vervelend, tijdrovend en omvangrijk zijn. Onze methode biedt een onmiddellijke en intuïtief nauwkeurige etiketteringsmethode voor analyse van de glomeruli in de gehele nier. Onze techniek is een snelle, gemakkelijke, nauwkeurige en goedkope methode om glomeruli in de hele nier te analyseren.Er is geen speciale apparatuur voor nodig, dus het kan in elk laboratorium worden uitgevoerd. Dit is een b...

Video: Auteur in de schijnwerpers: onderzoek naar nierbiologie ondersteunen door glomeruli in geklaarde weefsels te labelen 

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Biochemistry

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Medicine

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular deoxyribonucleic acid (ecDNA) is released during different forms of cell death including apoptosis, necrosis, necroptosis, neutrophil extracellular traps (NETs) and pyroptosis. Measurement of this cell death is time consuming with several different biomarkers required to identify the different biochemical forms of cell death. Measurement of ecDNA is generally conducted in serum and urine as a surrogate for renal damage, not in the actual target organ where the pathological injury occurs. The current difficulty in investigating ecDNA in the kidney is the lack of methods for formalin fixed paraffin embedded tissue (FFPE) both experimentally and in archived human kidney biopsies. This protocol provides a summary of the steps required to stain for ecDNA in FFPE tissue (both human and murine), quench autofluorescence and measure the ecDNA in the resulting images using a machine learning tool from the publicly available open source ImageJ plugin trainable Weka segmentation. Trainable Weka segmentation is applied to ecDNA within the glomeruli where the program learns to classify ecDNA. This classifier is applied to subsequent acquired kidney images, reducing the need for manual annotations of each individual image. The adaptability of the trainable Weka segmentation is demonstrated further in kidney tissue from experimental murine anti-MPO glomerulonephritis (GN), to identify NETs and ecMPO, common pathological contributors to anti-MPO GN. This method provides objective analysis of ecDNA in kidney tissue that demonstrates clearly the efficacy in which the trainable Weka segmentation program can distinguish ecDNA between healthy normal kidney tissue and diseased kidney tissue. This protocol can easily be adapted to identify ecDNA, NETs and ecMPO in other organs.

Extracellular DNA (ecDNA) released during cell death is proinflammatory and contributes to inflammation. Measurement of ecDNA at the site of injury can determine the efficacy of therapeutic treatment in the target organ. This protocol describes the use of a machine learning tool to automate measurement of ecDNA in kidney tissue.

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular ...

Immunology and Infection

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

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

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Glomeruli Isolation: A Technique to Obtain Intact Glomeruli from Murine Kidney

Source: Eymael, J. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60324" target="_blank">Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation.</a> J. Vis. Exp. (2020)
This video describes the method to isolate intact glomeruli from mouse kidneys. The encapsulated and decapsulated glomeruli thus obtained can further be used for culturing parietal epithelial cell outgrowths from the murine kidney.

Source: Eymael, J. et al. Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Cancer Research

Urinary Tract Cancer

In vitro studies

Fluid Aspiration from Mouse Glomeruli: A Method to Aspirate Glomerular Fluid from Mouse Using Two-Photon Mediated Micropuncture

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 lateral-most surface of the kidney extends beyond the stabilizer by about 1 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 1% agarose solution and hold a 10-millimeter coverslip on top of the form until the agarose is firm. Seal the coverslip to the head plate with glue and create a ring around the coverslip 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 coverslip 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 1 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 excitation 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 axes, 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 as 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 2 minutes to ensure patency of the pipette and to reduce confounding from pipette plugging during entry. After 4 to 6 minutes of filtration, set the micropump to aspirate up to 300 nanoliters at a rate of up to 50 nanoliters per minute.

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

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