Name: isolation of glomeruli and in vivo labeling of glomerular cell surface proteins
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Description: Watch this Scientific Journal Video about Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins at JoVE.com

The authors thank Blanka Duvnjak for her exceptional technical assistance. This work was supported by Deutsche Forschungsgemeinschaft (www.dfg.de) WO1811/2-1 to M.W. and QU280/3-1 to I.Q. The funder had no role in the study design, data collection, data analysis, decision to publish, and preparation of the manuscript.

Mice were obtained as an in-house breed from the local animal care facility or from Janvier Labs in France. The investigations were conducted according to the guidelines outlined in the Guide for Care and Use of Laboratory Animals (U.S. National Institutes of Health Publication No. 85-23, revised 1996). All animal experiments were performed in accordance with the relevant institutional approvals (state government LANUV reference number AZ:84-02.04. 2016.A435).
1. Preparation of Instruments, Solutions, and Equipment
<ol>
	<li>Prepare 1 L of phosphate buffered saline supplemented with 1 mM magnesium chloride (MgCl2) and 0.1 mM calcium chloride (CaCl2) (PBSCM) and filter it through a sterile filter.</li>
	<li>Prepare 5 mL of sterile PBSCM per mouse for perfusion and place it on ice.</li>
	<li>For each mouse, prepare 5 mL of sterile PBSCM supplemented with 0.5 mg/mL biotin.</li>
	<li>For each mouse, prepare 5 mL of sterile PBSCM and add 0.8 x 108 magnetic beads (e.g., 200 &#181;L of a 4 x 108 beads/mL solution, without pretreatment) for embolization of the glomeruli. Prepare this solution under the cell culture bench to keep the magnetic beads in the original tube sterile. Place this solution on ice.</li>
	<li>Prepare a quenching solution by adding 100 mM glycine to PBSCM (5 mL per mouse) and keep it on ice.</li>
	<li>Make a collagenase solution (0.378 U/mL collagenase A in sterile PBSCM). Per mouse, pipette 1 mL of the collagenase solution into a 2 mL tube and place it on ice.</li>
	<li>For washing, prepare sterile PBSCM (see step 1.1) in a 50 mL tube and place it on ice.</li>
	<li>For perfusion, use a syringe pump with a flow of 2.0 mL/min.</li>
	<li>Prepare a 10 mL syringe with a 21G needle. Place the tip of the needle in a 20-30 cm long catheter (inner diameter, ID = 0.58 mm). Connect the catheter (ID 0.58 mm) with a 10 cm short catheter (ID 0.28 mm) and cut the tip of the smaller catheter oblique for an easy insertion into the mouse aorta.</li>
	<li>For ligature procedures during the surgery, cut three 5-7 cm silk threads (4-0 to 6-0) per mouse.</li>
	<li>For the surgery, prepare 3 surgical clamps, 2 surgical scissors, 2 tweezers, 2 fine tweezers, 1 fine scissor, and swabs. Prepare the anesthesia (e.g., intraperitoneal anesthesia ketamine 100 mg/kg bodyweight and xylazine 5 mg/kg bodyweight).</li>
	<li>For isolation of the glomeruli of two kidneys, use a 100 &#181;m cell strainer, two 50 ml tubes, and a magnet catcher.</li>
	<li>Prepare CHAPS lysis buffer: 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS); 20 mM Tris(hydroxymethyl)-aminomethan (Tris) pH 7.5; 50 mM sodiumchorlide (NaCl); 50 mM sodiumfluoride (NaF); 15 mM Na4P2O7; 0.1 mM Ethylenediaminetetraacetic acid (EDTA) pH 8.0; 2 mM sodiumorthovanadate; and 2 mM adenosinetriphosphat (ATP).</li>
	<li>Cool centrifuges to 4 &#176;C.</li>
</ol>
2. Surgery Under the Microscope
<ol>
	<li>Anesthetize the mouse (e.g., with ketamine/xylazine intraperitoneally, see step 1.11). Perform the toe-pinch test to confirm proper anesthesia.</li>
	<li>Disinfect the ventral side of the mouse with 70% isopropanol.</li>
	<li>Perform a median cut through the skin from the pelvis to the sternum and remove the skin from the abdominal fascia using tweezers. Cut the skin on both sides in the middle of the abdomen with surgical scissors.</li>
	<li>Change surgical tools. Apply a median cut from xiphoid to the bladder through the abdominal muscle layer and divide them into four quadrants using tweezers and surgical scissors. Attach the upper two aside with clamps toward the neck of the mouse with two surgical clamps. 
	NOTE: The abdomen is now opened.</li>
	<li>Put visceral organs aside with a sterile swab and cut the hepatic-phrenic ligament with fine scissors.</li>
	<li>Prepare a ligation (with a silk thread 4-0 to 6-0) around the aorta proximal of the renal arteries on the height of the adrenal gland with two fine tweezers.&#160;Tighten the proximal aortic ligation to abolish blood flow with tweezers.</li>
	<li>Free the distal aorta from fascia, fat, and other tissues and prepare a ligation around the hepatic/mesenteric artery cranial of the renal arteries using fine surgical tweezers.</li>
	<li>Prepare a ligation (with a silk thread 4-0 to 6-0) around the aorta distal of the renal arteries and clamp the vena cava and aorta at the height of the bifurcation.</li>
	<li>Cut a small hole into the aorta distal to the renal arteries so that the hole is half the diameter of the aorta. Put the catheter into the aorta and fix it with the prepared ligature. 
	CAUTION: Avoid bubbles in the perfusion system to prevent air embolism.</li>
	<li>Start perfusion with the ice-cold PBSCM at a flow rate of 2 mL/min.</li>
	<li>Cut a hole into the renal vein at the level of the renal arteries with fine surgical scissors and tighten the ligation around the hepatic and mesenteric arteries. 
	NOTE: Kidneys should turn pale after starting the perfusion.</li>
</ol>
3. In Vivo Biotinylation
<ol>
	<li>Change the syringe bubble-free to ice-cold PBSCM supplemented with 0.5 mg/mL biotin for surface labelling, and perfuse kidneys with 5 mL at a flow rate of 2 mL/min. 
	NOTE: Mix the PBSCM solutions (e.g., by turning the 50 mL tubes) gently to avoid forming bubbles within the solution. After aspiration of the solution within the syringe, evacuate any bubbles or air from the syringe. Use an extra cannula (21G) on the syringe to fill the space in the cannula that is connected to the catheter system. Finally, stick the syringe into the cannula with the catheter without bubbles.</li>
	<li>Change the syringe bubble-free to ice-cold PBSCM supplemented with 100 mM glycine to quench the glomeruli and perfuse with 5 mL at a flow of 2 mL/min.</li>
	<li>Change the syringe bubble-free to ice-cold PBSCM supplemented with 200 &#956;L magnetic beads/mL and perfuse kidneys at 2 mL/min. 
	NOTE: On the kidney surface, embolization of glomeruli with brown magnetic beads will be visible.</li>
</ol>
4. Isolation of Glomeruli from Two Kidneys
<ol>
	<li>Remove kidneys at the hilum and remove the capsule. Place the kidneys on ice in 15 mL of PBSCM in a 10 cm cell culture dish.&#160;Euthanize the mouse with cervical dislocation under anesthesia after harvesting the kidneys. The surgery and perfusion procedures last approximately 20-22 min.</li>
	<li>Cut kidneys into the smallest pieces possible with a new double-edge blade. Move the tissue into a 2 mL tube with 1 mL of collagenase A (see step 1.6) and digest at 37 &#176;C for 30 min. Before and after digestion, mix gently with a 1000 &#956;L cut pipette.</li>
	<li>Rinse the digested tissue through a 100 &#956;m cell strainer using ice-cold PBSCM. Gently use the cell-scraper to mince the remaining tissue trough the cell strainer and rinse it with ice-cold PBSCM afterwards to a total volume of 50 mL.</li>
	<li>Centrifuge the suspension at 4 &#176;C 500 x g for 5 min. Remove the supernatant and resuspend the pellet with slightly less than 1.5 mL of ice-cold PBSCM while vortexing the pellet. Afterwards, put the glomerular suspension into a new 2 mL tube.</li>
</ol>
5. Washing
<ol>
	<li>Use the magnet catcher to pull the glomeruli to one side. Wait for 1 min before the glomeruli have moved toward the magnet.</li>
	<li>Remove the supernatant with a 1000 &#956;L pipette while the 2 mL tube remains in the magnet catcher. During the first and second washing steps (see step 5.3), 250 &#956;L of supernatant remains in the tube to avoid the loss of glomeruli. Perform this procedure quickly to avoid letting tubular structures sink to the bottom of the tube. 
	NOTE: The washing procedure will last longer otherwise.</li>
	<li>Remove the 2 mL tube from magnet catcher and add 1 mL of PBSCM, pipette up and down (very important), then vortex.</li>
	<li>Put the tube back into the magnet catcher and start over with step 5.2.</li>
	<li>Repeat the washing steps until the purity of glomeruli has reached 90%. For this, examine a representative aliquot of the supernatant under a microscope (40-100X). 
	NOTE: Glomeruli will appear as round structures containing brown magnetic beads. Elongated structures are tubular fragments, and free magnetic beads appear as brown round dots. Cell debris might appear as bulky structures.</li>
	<li>If purity is reached, estimate the count of glomeruli by dissolving the glomeruli in 1 mL of PBSCM. After mixing, take an aliquot of 10 &#956;L and count the glomeruli under the microscope. Calculate the number of glomeruli with the following equation: final number of glomeruli = number of glomeruli in 10 &#956;L aliquot x 100.</li>
	<li>Successful isolation of glomeruli leads to a range of 10,000-40,000 glomeruli.</li>
</ol>
6. Protein Extraction and Immunoprecipitation (IP)
<ol>
	<li>Collect glomeruli by centrifugation at 4 &#176;C at 6800 x g for 5 min. Remove the supernatant while using a magnet catcher and resuspend the pellet in ice-cold lysis buffer (e.g., 30,000 glomeruli in 300 &#956;L; CHAPS, see step 1.11). Homogenize the samples with a tissue homogenizer at the highest speed for 30 s and lyse them on ice for 30 min.</li>
	<li>Remove insoluble material by centrifugation at 15,000 x g for 30 min for 4 &#176;C. Pipette the supernatant which includes the cell lysate into a new 1.5 mL tube. Discard the pellet.</li>
	<li>Measure the protein concentration of the supernatant by using a bicinchoninic (BCA)-based method following the manufacturer&#39;s instructions. A successful lysis of 30,000 glomeruli yields to a glomerular protein amount of 700-1,000 &#181;g/mL. Adjust to equal protein amounts with lysis buffer.</li>
	<li>Take an aliquot of 10% of the total volume for glomerular lysate and add 2x Laemmli + dithiothreithol (DTT) before incubation for 5 min at 95 &#176;C.</li>
	<li>For IP, incubate the rest of the lysate with streptavidin agarose beads overnight at 4 &#176;C on an overhead shaker.</li>
	<li>Centrifuge agarose beads at 1000 x g for 3 min at 4 &#176;C, remove the supernatant, and add 800 &#956;L of lysis buffer to wash the beads for unspecific protein binding.</li>
	<li>Repeat the wash 3 times, and remove the supernatant completely.</li>
	<li>Add 30 &#956;L of 2x Laemmli + DTT and incubate at 95 &#176;C for 5 min.</li>
	<li>Load the lysate and IP probes on a 10% SDS gel and run the SDS gel at 70 V for 30 min, then at 20 mA per gel for 1.5 h. Afterwards, blot the gel for 2 h at 200 mA on a nitrocellulose membrane.</li>
	<li>Block the nitrocellulose membrane overnight at 4 &#176;C with 5% bovine serum albumin (BSA) in washing buffer.</li>
	<li>Incubate the membrane with the antibody of interest overnight at 4 &#176;C. Wash with washing buffer 3 times for 5 min on a shaker and incubate the membrane with the HRP-tagged secondary antibody for 1 h at room temperature. Repeat the washing step.</li>
	<li>Visualize the lysate and immunoprecipitation probes on the membrane by using a super-resolution chemiluminescent agent with a CCD camera.</li>
</ol>

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The authors have nothing to disclose.

The presented method enables successful isolation of glomeruli to investigate glomerular RNA or protein. In addition, primary glomerular cell cultures can be performed from the isolated glomeruli. If biotin is applied before glomerular isolation, labeling of glomerular cell surface proteins can be performed. With this method, in vivo glomerular cell surface protein trafficking can be studied, and semi-quantitation of protein abundance is possible. The most critical steps for successfully testing glomerular cell ...

Proteinuria is a hallmark of glomerular injury and usually accompanies disruption of the glomerular filter1. The glomerular filter is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes. The delicate molecular structure of the glomerular filter is highly dynamic and subject to cell surface protein trafficking in both healthy and diseased kidneys2,3,4,5,6. Endocytosis of cell surface proteins has been shown to be essential for the survival of podocytes7. Nephrin and podocalyxin are transmembrane proteins expressed on podocytes. Nephrin is the backbone of the glomerular slit diaphragm, while podocalyxin is a sialoglycoprotein coating the secondary foot processes of podocytes8,9,10. Endocytic trafficking has previously been shown for nephrin and podocalyxin3,11,12,13,14.
To the best of our knowledge, endocytosis of cell surface proteins has not yet been described in glomerular endothelial cells in the literature. However, endothelial cells in general express all necessary proteins for the different types of endocytosis (i.e., clathrin-dependent, raft-dependent endocytosis)15,16. Therefore, endothelial cell surface trafficking may be studied with this method using, for example, vascular endothelial (VE)-cadherin and intracellular adhesion molecule (ICAM-2) as a cell surface marker protein for glomerular endothelial cells17.
Unfortunately, there is no accurate in vitro model for the delicate three-layered glomerular filter in which cell surface protein trafficking can be studied. The goal of this method is thus to study glomerular protein trafficking in vivo. In addition, this protocol contains information on how to isolate glomeruli, enabling further analysis of glomerular RNA, proteins, or cells. Similar glomerular isolation techniques have been described by different groups18,19.
Previously, we and others have used ex vivo labeling of glomerular cell surface proteins by biotinylation2,3,4,20,21. However, in this ex vivo method, isolated glomeruli were exposed to mechanical stress, which may influence endocytic trafficking. Alternatively, immunofluorescence labeling of glomerular cell surface proteins has extensively been used in the literature2,20,22. With this method, however, only a small number of proteins can be analyzed within one slide, and quantitation of immunofluorescence images is often difficult.
This novel in vivo method offers a reliable tool to study glomerular cell surface protein abundance and trafficking accurately in healthy and diseased kidneys, and it can be used as an addition to immunofluorescence tests.

<table><tbody><tr><td>Motic SMZ168 BL</td><td>Motic</td><td>SMZ168BL</td><td>microscope for mouse surgery</td></tr><tr><td>KL1500LCD</td><td>Pulch and Lorenz microscopy</td><td>150500</td><td>light for mouse surgery</td></tr><tr><td>Rompun (Xylazin) 2%</td><td>Bayer</td><td>PZN:01320422</td><td>anesthesia</td></tr><tr><td>Microfederschere</td><td>Braun, Aesculap</td><td>FD100R</td><td>fine scissors, for cut into the aorta</td></tr><tr><td>Durotip Feine Scheren</td><td>Braun, Aesculap</td><td>BC210R</td><td>for abdominal cut</td></tr><tr><td>Anatomische Pinzette</td><td>Braun, Aesculap</td><td>BD215R</td><td>for surgery until the abdomen is opened</td></tr><tr><td>Pr&amp;auml;parierklemme</td><td>Aesculap</td><td>BJ008R</td><td>for surgery&amp;nbsp;</td></tr><tr><td>Seraflex</td><td>Serag Wiessner</td><td>IC108000</td><td>silk thread</td></tr><tr><td>Ketamine 10%</td><td>Medistar</td><td></td><td>anesthesia</td></tr><tr><td>Rompun (Xylazin) 2%</td><td>Bayer</td><td></td><td>anesthesia</td></tr><tr><td>Fine Bore Polythene Tubing ID 0.28mm OD 0.61mm</td><td>Portex</td><td>800/100/100</td><td>Catheter</td></tr><tr><td>Fine Bore Polythene Tubing ID 0.58mm OD 0.96mm</td><td>Portex</td><td>800/100/200</td><td>Catheter</td></tr><tr><td>Harvard apparatus 11 Plus</td><td>Harvard Apparatus</td><td>70-2209</td><td>syringe pump</td></tr><tr><td>EZ-link Sulfo-NHC-LC-Biotin</td><td>Thermo Scientific</td><td>21335</td><td>biotin</td></tr><tr><td>Dynabeads Untouched Mouse T-cells</td><td>Invitrogen</td><td>11413D</td><td>to embolize glomeruli</td></tr><tr><td>Collagenase A</td><td>Roche</td><td>10103578001</td><td>to digest kidney tissue</td></tr><tr><td>DynaMag-2</td><td>Invitrogen</td><td>123.21D</td><td>Magnet catcher</td></tr><tr><td>100&amp;micro;m cell stainer</td><td>Greiner-bio</td><td>542000</td><td>for glomerular isolation</td></tr><tr><td>Axiovert 40 CFL</td><td>Zeiss</td><td>non available</td><td>to confirm glomerular purity</td></tr><tr><td>TissueRuptor</td><td>Quiagen</td><td>9002755</td><td>Tissue homogenizer</td></tr><tr><td>CHAPS</td><td>Sigma-Aldrich</td><td>C3023</td><td>for lysis buffer</td></tr><tr><td>Tris-HCL</td><td>Sigma-Aldrich</td><td>T5941</td><td>for lysis buffer</td></tr><tr><td>NaCl</td><td>VWR chemicals</td><td>27810295</td><td>for lysis buffer</td></tr><tr><td>NaF</td><td>Sigma-Aldrich</td><td>201154</td><td>for lysis buffer</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E5134</td><td>for lysis buffer</td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>34369-07-8</td><td>for lysis buffer</td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td>Follow the manufacturer&#039;s instructions</td></tr><tr><td>nephrin antibody</td><td>Progen</td><td>GP-N2</td><td>for westernblot</td></tr><tr><td>Polyclonal goat anti-podocalyxin antibody</td><td>R&amp;amp;D Systems</td><td>AF15556-SP</td><td>for westernblot</td></tr><tr><td>Streptavidin Agarose Resin</td><td>Thermo Scientific</td><td>20347</td><td>for immunoprecipitation</td></tr><tr><td>Protein A sepharose CL-4B</td><td>GE Healthcare</td><td>17096303</td><td>for immunoprecipitation</td></tr><tr><td>polyclonal rabbit anti-p57 antibody</td><td>SCBT</td><td>sc-8298</td><td>for Immunohistochemistry</td></tr><tr><td>mouse monoclonal anti-beta actin antibody, clone AC-74</td><td>Sigma-Aldrich</td><td>A2228</td><td>Western blot loading control</td></tr><tr><td>rabbit anti-p44/42</td><td>cell signalling</td><td>4695</td><td>for westernblot</td></tr><tr><td>Pierce High sensitivity streptavidin-HRP</td><td>Thermo Scientific</td><td>21130</td><td>for westernblot</td></tr><tr><td>polyclonal mouse ICAM-2 antibody</td><td>R&amp;amp;D Systems</td><td>AF774</td><td>for westernblot</td></tr><tr><td>polyclonal mouse anti-VE-cadherin</td><td>R&amp;amp;D Systems</td><td>AF1002</td><td>for westernblot</td></tr></tbody></table>

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).

To isolate glomeruli accurately, it is necessary to perfuse murine kidneys with PBSCM first. Perfusion with PBSCM turns kidneys pale (Figure 1A). Embolization of glomeruli with magnetic beads will be visible as brown dots on the kidney surface (Figure 1B). Isolation of glomeruli with the magnet catcher may show contamination with renal tubuli (Figure 1C</stro...

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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.

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 ...

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Research

JoVE Journal

Biochemistry

9.0K Views.  Heinrich-Heine-University. 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.

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

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Neuroscience

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 ...

Medicine

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

Biology

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 ...

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria&#39;s distribution, fusion, and fission events without staining the cells.

The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative ...

Fluorescent Labeling of Glomeruli in Cleared Mouse Kidneys for Microscopy

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.

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.

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

Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for isolating, purifying, and culturing glomerular MCs have been developed and optimized since the 1980s for use in biomedical research, particularly in the field of nephrology. Mice are the most frequently used experimental animal models in research on renal diseases. In this study, we developed an optimized protocol for murine MCs isolation and ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression. This optimized approach significantly reduces the study duration for researchers and enables long-term cell preservation. The necessary equipment for the procedures is easily accessible in a basic biomedical laboratory, and the procedural steps are straightforward. The acquisition of target cells requires only 2-3 weeks, a reduction of at least 1 week compared to existing methods.

This study developed an optimized protocol for the isolation of murine mesangial cells (MCs) and their ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression.

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for ...

Isolation of Cell-Surface and Intracellular Proteins from an Astrocyte Culture

Source: Tham, D. K. L., et al. <a href="https://app.jove.com/v/55974">Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation.</a> J. Vis. Exp. (2017).
This video demonstrates the isolation of cell-surface and intracellular proteins from an astrocyte culture. The astrocyte culture is placed on ice to inhibit endocytosis, and then the cell-surface proteins are labeled with a biotinylation reagent. The cells are lysed to release the biotinylated cell-surface proteins and non-biotinylated intracellular proteins. Finally, streptavidin-coated beads are used to separate the biotinylated cell-surface proteins from the intracellular proteins.

1. Determining Relative Cell-surface Protein Expression in Astrocytes by Biotinylation
NOTE: Here, we illustrate the application of this biotinylation ...

JoVE Encyclopedia of Experiments

Neuropathology

Infectious Diseases

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

Source: Eymael, J. et al. <a href="https://www.jove.com/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. ...

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

Summary

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Chapters in this video

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

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

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

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

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

Isolation of Cell-Surface and Intracellular Proteins from an Astrocyte Culture

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