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

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 kidneys^2,3,4,5,6. 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 podocytes^8,9,10. Endocytic trafficking has previously been shown for nephrin and podocalyxin^{3,11,12,13,14}.

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 groups^18,19.

Previously, we and others have used ex vivo labeling of glomerular cell surface proteins by biotinylation^2,3,4,20,21. 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 literature^2,20,22. 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.

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.

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

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 ...

Materials

Name	Company	Catalog Number	Comments
Motic SMZ168 BL	Motic	SMZ168BL	microscope for mouse surgery
KL1500LCD	Pulch and Lorenz microscopy	150500	light for mouse surgery
Rompun (Xylazin) 2%	Bayer	PZN:01320422	anesthesia
Microfederschere	Braun, Aesculap	FD100R	fine scissors, for cut into the aorta
Durotip Feine Scheren	Braun, Aesculap	BC210R	for abdominal cut
Anatomische Pinzette	Braun, Aesculap	BD215R	for surgery until the abdomen is opened
Präparierklemme	Aesculap	BJ008R	for surgery
Seraflex	Serag Wiessner	IC108000	silk thread
Ketamine 10%	Medistar		anesthesia
Rompun (Xylazin) 2%	Bayer		anesthesia
Fine Bore Polythene Tubing ID 0.28mm OD 0.61mm	Portex	800/100/100	Catheter
Fine Bore Polythene Tubing ID 0.58mm OD 0.96mm	Portex	800/100/200	Catheter
Harvard apparatus 11 Plus	Harvard Apparatus	70-2209	syringe pump
EZ-link Sulfo-NHC-LC-Biotin	Thermo Scientific	21335	biotin
Dynabeads Untouched Mouse T-cells	Invitrogen	11413D	to embolize glomeruli
Collagenase A	Roche	10103578001	to digest kidney tissue
DynaMag-2	Invitrogen	123.21D	Magnet catcher
100µm cell stainer	Greiner-bio	542000	for glomerular isolation
Axiovert 40 CFL	Zeiss	non available	to confirm glomerular purity
TissueRuptor	Quiagen	9002755	Tissue homogenizer
CHAPS	Sigma-Aldrich	C3023	for lysis buffer
Tris-HCL	Sigma-Aldrich	T5941	for lysis buffer
NaCl	VWR chemicals	27810295	for lysis buffer
NaF	Sigma-Aldrich	201154	for lysis buffer
EDTA	Sigma-Aldrich	E5134	for lysis buffer
ATP	Sigma-Aldrich	34369-07-8	for lysis buffer
Pierce BCA Protein Assay Kit	Thermo Scientific	23225	Follow the manufacturer's instructions
nephrin antibody	Progen	GP-N2	for westernblot
Polyclonal goat anti-podocalyxin antibody	R&D Systems	AF15556-SP	for westernblot
Streptavidin Agarose Resin	Thermo Scientific	20347	for immunoprecipitation
Protein A sepharose CL-4B	GE Healthcare	17096303	for immunoprecipitation
polyclonal rabbit anti-p57 antibody	SCBT	sc-8298	for Immunohistochemistry
mouse monoclonal anti-beta actin antibody, clone AC-74	Sigma-Aldrich	A2228	Western blot loading control
rabbit anti-p44/42	cell signalling	4695	for westernblot
Pierce High sensitivity streptavidin-HRP	Thermo Scientific	21130	for westernblot
polyclonal mouse ICAM-2 antibody	R&D Systems	AF774	for westernblot
polyclonal mouse anti-VE-cadherin	R&D Systems	AF1002	for westernblot