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Method Article

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

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DOI:

10.3791/58542

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January 18th, 2019

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Nephrology, Medical Faculty, Heinrich-Heine-University, 2Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf

In This Article

Summary

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.

Abstract

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).

Introduction

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.

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Protocol

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

  1. 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.
  2. Prepare 5 mL of sterile PBSCM per mouse for perfusion and place it on ice.
  3. For each mouse, prepare 5 mL of sterile PBSCM supplemented with 0.5 mg/mL biotin.
  4. For each mouse, prepare 5 mL of sterile PBSCM and add 0.8 x 108 magnetic beads (e.g., 200 µL 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.
  5. Prepare a quenching solution by adding 100 mM glycine to PBSCM (5 mL per mouse) and keep it on ice.
  6. 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.
  7. For washing, prepare sterile PBSCM (see step 1.1) in a 50 mL tube and place it on ice.
  8. For perfusion, use a syringe pump with a flow of 2.0 mL/min.
  9. 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.
  10. For ligature procedures during the surgery, cut three 5-7 cm silk threads (4-0 to 6-0) per mouse.
  11. 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).
  12. For isolation of the glomeruli of two kidneys, use a 100 µm cell strainer, two 50 ml tubes, and a magnet catcher.
  13. 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).
  14. Cool centrifuges to 4 °C.

2. Surgery Under the Microscope

  1. Anesthetize the mouse (e.g., with ketamine/xylazine intraperitoneally, see step 1.11). Perform the toe-pinch test to confirm proper anesthesia.
  2. Disinfect the ventral side of the mouse with 70% isopropanol.
  3. 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.
  4. 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.
  5. Put visceral organs aside with a sterile swab and cut the hepatic-phrenic ligament with fine scissors.
  6. 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. Tighten the proximal aortic ligation to abolish blood flow with tweezers.
  7. 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.
  8. 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.
  9. 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.
  10. Start perfusion with the ice-cold PBSCM at a flow rate of 2 mL/min.
  11. 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.

3. In Vivo Biotinylation

  1. 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.
  2. 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.
  3. Change the syringe bubble-free to ice-cold PBSCM supplemented with 200 μL 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.

4. Isolation of Glomeruli from Two Kidneys

  1. 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. Euthanize the mouse with cervical dislocation under anesthesia after harvesting the kidneys. The surgery and perfusion procedures last approximately 20-22 min.
  2. 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 °C for 30 min. Before and after digestion, mix gently with a 1000 μL cut pipette.
  3. Rinse the digested tissue through a 100 μm 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.
  4. Centrifuge the suspension at 4 °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.

5. Washing

  1. Use the magnet catcher to pull the glomeruli to one side. Wait for 1 min before the glomeruli have moved toward the magnet.
  2. Remove the supernatant with a 1000 μL pipette while the 2 mL tube remains in the magnet catcher. During the first and second washing steps (see step 5.3), 250 μL 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.
  3. Remove the 2 mL tube from magnet catcher and add 1 mL of PBSCM, pipette up and down (very important), then vortex.
  4. Put the tube back into the magnet catcher and start over with step 5.2.
  5. 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.
  6. 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 μL 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 μL aliquot x 100.
  7. Successful isolation of glomeruli leads to a range of 10,000-40,000 glomeruli.

6. Protein Extraction and Immunoprecipitation (IP)

  1. Collect glomeruli by centrifugation at 4 °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 μL; 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.
  2. Remove insoluble material by centrifugation at 15,000 x g for 30 min for 4 °C. Pipette the supernatant which includes the cell lysate into a new 1.5 mL tube. Discard the pellet.
  3. Measure the protein concentration of the supernatant by using a bicinchoninic (BCA)-based method following the manufacturer's instructions. A successful lysis of 30,000 glomeruli yields to a glomerular protein amount of 700-1,000 µg/mL. Adjust to equal protein amounts with lysis buffer.
  4. 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 °C.
  5. For IP, incubate the rest of the lysate with streptavidin agarose beads overnight at 4 °C on an overhead shaker.
  6. Centrifuge agarose beads at 1000 x g for 3 min at 4 °C, remove the supernatant, and add 800 μL of lysis buffer to wash the beads for unspecific protein binding.
  7. Repeat the wash 3 times, and remove the supernatant completely.
  8. Add 30 μL of 2x Laemmli + DTT and incubate at 95 °C for 5 min.
  9. 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.
  10. Block the nitrocellulose membrane overnight at 4 °C with 5% bovine serum albumin (BSA) in washing buffer.
  11. Incubate the membrane with the antibody of interest overnight at 4 °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.
  12. Visualize the lysate and immunoprecipitation probes on the membrane by using a super-resolution chemiluminescent agent with a CCD camera.

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Results

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

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Discussion

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 ...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Motic SMZ168 BLMoticSMZ168BLmicroscope for mouse surgery
KL1500LCDPulch and Lorenz microscopy150500light for mouse surgery
Rompun (Xylazin) 2%BayerPZN:01320422anesthesia
MicrofederschereBraun, AesculapFD100Rfine scissors, for cut into the aorta
Durotip Feine ScherenBraun, AesculapBC210Rfor abdominal cut
Anatomische PinzetteBraun, AesculapBD215Rfor surgery until the abdomen is opened
PräparierklemmeAesculapBJ008Rfor surgery 
SeraflexSerag WiessnerIC108000silk thread
Ketamine 10%Medistaranesthesia
Rompun (Xylazin) 2%Bayeranesthesia
Fine Bore Polythene Tubing ID 0.28mm OD 0.61mmPortex800/100/100Catheter
Fine Bore Polythene Tubing ID 0.58mm OD 0.96mmPortex800/100/200Catheter
Harvard apparatus 11 PlusHarvard Apparatus70-2209syringe pump
EZ-link Sulfo-NHC-LC-BiotinThermo Scientific21335biotin
Dynabeads Untouched Mouse T-cellsInvitrogen11413Dto embolize glomeruli
Collagenase ARoche10103578001to digest kidney tissue
DynaMag-2Invitrogen123.21DMagnet catcher
100µm cell stainerGreiner-bio542000for glomerular isolation
Axiovert 40 CFLZeissnon availableto confirm glomerular purity
TissueRuptorQuiagen9002755Tissue homogenizer
CHAPSSigma-AldrichC3023for lysis buffer
Tris-HCLSigma-AldrichT5941for lysis buffer
NaClVWR chemicals27810295for lysis buffer
NaFSigma-Aldrich201154for lysis buffer
EDTASigma-AldrichE5134for lysis buffer
ATPSigma-Aldrich34369-07-8for lysis buffer
Pierce BCA Protein Assay KitThermo Scientific23225Follow the manufacturer's instructions
nephrin antibodyProgenGP-N2for westernblot
Polyclonal goat anti-podocalyxin antibodyR&D SystemsAF15556-SPfor westernblot
Streptavidin Agarose ResinThermo Scientific20347for immunoprecipitation
Protein A sepharose CL-4BGE Healthcare17096303for immunoprecipitation
polyclonal rabbit anti-p57 antibodySCBTsc-8298for Immunohistochemistry
mouse monoclonal anti-beta actin antibody, clone AC-74Sigma-AldrichA2228Western blot loading control
rabbit anti-p44/42cell signalling4695for westernblot
Pierce High sensitivity streptavidin-HRPThermo Scientific21130for westernblot
polyclonal mouse ICAM-2 antibodyR&D SystemsAF774for westernblot
polyclonal mouse anti-VE-cadherinR&D SystemsAF1002for westernblot

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Tags

Glomerular IsolationIn Vivo BiotinylationCell Surface Protein LabelingKidney PerfusionMagnetic Bead PurificationNephrin Podocalyxin AnalysisImmunoprecipitation MethodProtein Trafficking StudyPrimary Podocyte Culture
Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins
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