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, Solu...

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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 border="0" cellpadding="0" cellspacing="0" style="width:671px;" width="669">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Motic SMZ168 BL</td>
			<td style="width:77px;">Motic</td>
			<td style="width:101px;">SMZ168BL</td>
			<td style="width:235px;">microscope for mouse surgery</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:257px;">KL1500LCD</td>
			<td style="width:77px;">Pulch and Lorenz microscopy</td>
			<td style="width:101px;">150500</td>
			<td>light for mouse surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rompun (Xylazin) 2%</td>
			<td style="width:77px;">Bayer</td>
			<td>PZN:01320422</td>
			<td>anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Microfederschere</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">FD100R</td>
			<td>fine scissors, for cut into the aorta</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Durotip Feine Scheren</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">BC210R</td>
			<td>for abdominal cut</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Anatomische Pinzette</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">BD215R</td>
			<td>for surgery until the abdomen is opened</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Pr&#228;parierklemme</td>
			<td style="width:77px;">Aesculap</td>
			<td style="width:101px;">BJ008R</td>
			<td>for surgery&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Seraflex</td>
			<td style="width:77px;">Serag Wiessner</td>
			<td style="width:101px;">IC108000</td>
			<td>silk thread</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Ketamine 10%</td>
			<td style="width:77px;">Medistar</td>
			<td style="width:101px;"></td>
			<td>anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rompun (Xylazin) 2%</td>
			<td style="width:77px;">Bayer</td>
			<td style="width:101px;"></td>
			<td>anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Fine Bore Polythene Tubing ID 0.28mm OD 0.61mm</td>
			<td style="width:77px;">Portex</td>
			<td style="width:101px;">800/100/100</td>
			<td style="width:235px;">Catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Fine Bore Polythene Tubing ID 0.58mm OD 0.96mm</td>
			<td style="width:77px;">Portex</td>
			<td style="width:101px;">800/100/200</td>
			<td style="width:235px;">Catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Harvard apparatus 11 Plus</td>
			<td style="width:77px;">Harvard Apparatus</td>
			<td style="width:101px;">70-2209</td>
			<td>syringe pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">EZ-link Sulfo-NHC-LC-Biotin</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">21335</td>
			<td>biotin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Dynabeads Untouched Mouse T-cells</td>
			<td style="width:77px;">Invitrogen</td>
			<td style="width:101px;">11413D</td>
			<td>to embolize glomeruli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Collagenase A</td>
			<td style="width:77px;">Roche</td>
			<td style="width:101px;">10103578001</td>
			<td>to digest kidney tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">DynaMag-2</td>
			<td style="width:77px;">Invitrogen</td>
			<td style="width:101px;">123.21D</td>
			<td>Magnet catcher</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">100&#181;m cell stainer</td>
			<td style="width:77px;">Greiner-bio</td>
			<td style="width:101px;">542000</td>
			<td>for glomerular isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Axiovert 40 CFL</td>
			<td style="width:77px;">Zeiss</td>
			<td style="width:101px;">non available</td>
			<td>to confirm glomerular purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">TissueRuptor</td>
			<td style="width:77px;">Quiagen</td>
			<td style="width:101px;">9002755</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">CHAPS</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">C3023</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Tris-HCL</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">T5941</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">NaCl</td>
			<td style="width:77px;">VWR chemicals</td>
			<td style="width:101px;">27810295</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">NaF</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">201154</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">EDTA</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">E5134</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">ATP</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">34369-07-8</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">23225</td>
			<td>Follow the manufacturer&#39;s instructions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">nephrin antibody</td>
			<td style="width:77px;">Progen</td>
			<td style="width:101px;">GP-N2</td>
			<td>for westernblot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Polyclonal goat anti-podocalyxin antibody</td>
			<td style="width:77px;">R&#38;D Systems</td>
			<td style="width:101px;">AF15556-SP</td>
			<td>for westernblot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Streptavidin Agarose Resin</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">20347</td>
			<td>for immunoprecipitation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">Protein A sepharose CL-4B</td>
			<td style="width:77px;">GE Healthcare</td>
			<td style="width:101px;">17096303</td>
			<td>for immunoprecipitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">polyclonal rabbit anti-p57 antibody</td>
			<td style="width:77px;">SCBT</td>
			<td style="width:101px;">sc-8298</td>
			<td>for Immunohistochemistry</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">mouse monoclonal anti-beta actin antibody, clone AC-74</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">A2228</td>
			<td>Western blot loading control</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">rabbit anti-p44/42</td>
			<td style="width:77px;">cell signalling</td>
			<td style="width:101px;">4695</td>
			<td>for westernblot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Pierce High sensitivity streptavidin-HRP</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">21130</td>
			<td>for westernblot</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polyclonal mouse ICAM-2 antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF774</td>
			<td>for westernblot</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polyclonal mouse anti-VE-cadherin</td>
			<td>R&#38;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 ...

This method can help answer key questions in the nephrology field about the role of cell surface protein trafficking in proteinuric and not proteinuric kidney disease. The main advantages of this technique are that it facilitates the study of cell surface protein trafficking in-vivo and that more than one protein can be analyzed per experiment. Though this method can provide insight into glomerular cell surface trafficking, can also be applied to other organ systems that are perfused and are accessible by isolation techniques.Begin by confirming a lack of response to toe pinch and disinfecting the ventral side of the anesthetized mouse with 70%isopropyl. Make a median cut through the skin from the pelvis to the sternum and use tweezers to remove the skin from the abdominal fascia. Cut the skin on both sides of the abdominal midline and make a medium through the abdominal muscle layer from the bladder to the xiphoid.Use the scissors and forceps to divide the muscles into four quadrants And use two surgical clamps to secure the upper two quadrants towards the neck of the animals. Place the animal under a dissecting microscope and use a sterile swap to move aside the visceral organs. Using fine scissors, cut the hepatic phrenic ligament.Using fine surgical tweezers, place one suture around the hepatic mesenteric artery branial of the renal arteries. And around the aorta, proximal to the renal arteries, at the adrenal gland. Free the distal aorta from the fascia, fat and other tissue.Then, clamp the vena cava and the aorta at the height of the bifurcation and place another suture around the aorta distal of the renal arteries. Use tweezers to tighten the proximal aortic suture, attenuating the blood flow and cut a small hole half of the diameter of the aorta into the aorta, distal to the renal arteries. Insert a 10 centimeter catheter attached to a 10 milliliter syringe containing five milliliters of ice cold PBS plus calcium and magnesium into the aorta.And fix the catheter with the prepared ligatures. Then begin perfusing the kidneys at a two milliliter per minute flow rate. Using fine scissors to cut a hole into the renal vein at the renal arteries and tightening the suture around the hepatic and mesenteric arteries.After the entire volume of PBS has been delivered replace the perfusate with five milliliters of ice cold PBS plus calcium and magnesium supplemented with 0.5 milligrams per milliliter biotin for surface labeling. Continue to perfuse the kidneys at a two milliliter per minute flow rate. When the entire volume has been delivered, attach a syringe containing five milliliters of ice cold PBS plus calcium and magnesium supplemented with 100 millimolar glycine to the catheter.Quench the glomeruli with all five milliliters of the glycine solution at a two milliliter per minute flow rate. Then perfuse the kidneys with five milliliters of ice cold PBS plus calcium and magnesium supplemented with 200 microliters of magnetic beads per milliliter at a two milliliter per minute flow rate. The embolization of the glomeruli with the brown magnetic beads will be visible on the kidney surface.When all of the beads have been delivered remove the capsule of the kidneys and harvest the kidneys at the hilum. Place the kidneys in a 10 centimeter cell culture dish containing 15 milliliters of PBS plus calcium and magnesium. Use a new double edged blade to cut the kidneys into the smallest pieces possible.Transfer the tissue into a two milliliter tube containing one milliliter of collagenase A for 30 minutes at 37 degrees Celsius, mixing the tissue solution gently before and after the digestion with a modified 1000 microliter pipette tip. At the end of the incubation, filter the digested tissue through a 100 micron cell strainer. Use a cell scraper and ice cold PBS plus calcium and magnesium to mash the remaining tissue through the cell strainer.Bring the final volume of the kidney single cell suspension up to 50 milliliters with fresh ice cold PBS plus calcium and magnesium and collect the kidney cells by centrifugation. Remove the supernatant then re-suspend the pellet with slightly less than 1.5 milliliters of ice cold PBS plus calcium and magnesium while vortexing and transfer the glomerular suspension into a new two milliliter tube. To wash the glomeruli, place the tube into a magnet to aggregate the glomeruli.After one minute, use a 1000 microliter pipette to remove the supernatant. And remove the tube from the magnet. Add one milliliter of PBS plus calcium and magnesium to the tissues.Pipette the glomeruli up and down one time and vortex the cells. Return the tube to the magnet and wash the glomeruli again as just demonstrated. Until a sample purity has reached at least 90%by light microscope analysis at a 40 to 100 X magnification.Then collect the magnetic bead purified glomeruli by centrifugation. To achieve a greater than 95%purity, the glomeruli needed to be washed thoroughly as demonstrated. Biotin perfusion facilitates labeling of the capillary loops in biotin but not controlled perfused mouse kidneys.Immune precipitation of the biotin fraction of glomerular extracts reveals that the glomerular transmembrane proteins nephrin and podocalyxin are immunoprecipitated with the biotin but not with the control fraction. Staining of the immunoprecipitated fraction with streptavidin further confirms the presence of biotin laded nephrin in the biotin perfused but not control perfused animals. Endothelial protein vascular endothelial cadherin and intracellular adhesion molecule two are also immunoprecipitated within the biotin fraction.In the early phase of nephrotoxic nephritis a reduced expression of cell surface nephrin is observed in nephrotoxic nephritis animals compared to controls. Indeed densitometic analysis shows a significant reduction of biotin laded nephrin in nephrotoxic nephritis animals compared to controllers. Quantitative analysis of the total nephrin to actin ratio reveals no significant differences between the control and nephrotoxic nephritis mice.In addition, equal amounts of podocytes are observed in nephrotoxic nephritis and control animals. In late nephrotoxic nephritis, nephrotoxic nephritic animals exhibit a recovery of nephrin expression with no significant differences in cell surface nephrin measured between control and nephrotoxic nephritic mice and an overall total nephrin reduction in nephrotoxic nephritic animals. Further, podocyte numbers are decreased compared to control animals.While attempting this procedure, it's important to remember to keep the syringes bubble free during their connection in order to avoid air embolism of the glomeruli and to work under ice cold conditions once kidney perfusion has started. Following this procedure other methods like the isolation of glomeruli RNA and protein can be performed to answer additional questions about protein expression or interactions in healthy and diseased kidneys.

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JoVE Journal

Biochemistry

8.9K visualizações.  Heinrich-Heine-University. Este método pode ajudar a responder a perguntas-chave no campo da nefrologia sobre o papel do tráfico de proteínas superficiais celulares na doença renal proteinúrica e não proteinúrica. As principais vantagens dessa técnica são que facilita o estudo do tráfico de proteínas superficiais celulares in vivo e que mais de uma proteína pode ser analisada por experimento. Embora este método possa fornecer informações sobre o tráfico de superfície celular glomerular, também pode se...