Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70

Summary

Here, we present a protocol to test glomerular permeability in mice using a highly sensitive, nonradioactive tracer. This method allows repetitive urine analyses with small urine volumes.

Abstract

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the glomerulus and reabsorbed in the tubular system up until the absorption limit is reached. Early increases in pathological albumin filtration may, thus, be missed by analyzing albuminuria. Therefore, the use of tracers to test glomerular permselectivity appears advantageous. Fluorescently labeled tracer fluorescein isothiocyanate (FITC)-polysucrose (i.e., FITC-Ficoll), can be used to study glomerular permselectivity. FITC-polysucrose molecules are freely filtered by the glomerulus but not reabsorbed in the tubular system. In mice and rats, FITC-polysucrose has been investigated in models of glomerular permeability by using technically complex procedures (i.e., radioactive measurements, high-performance liquid chromatography [HPLC], gel filtration). We have modified and facilitated a FITC-polysucrose tracer-based protocol to test early and small increases in glomerular permeability to FITC-polysucrose 70 (size of albumin) in mice. This method allows repetitive urine analyses with small urine volumes (5 µL). This protocol contains information on how the tracer FITC-polysucrose 70 is applied intravenously and urine is collected via a simple urinary catheter. Urine is analyzed via a fluorescence plate reader and normalized to a urine concentration marker (creatinine), thereby avoiding technically complex procedures.

Introduction

Functional or structural defects within the glomerular filtration barrier increase glomerular permeability to albumin, resulting in the detection of albumin in the urine (albuminuria). Albuminuria predicts cardiovascular outcome and is an important marker for glomerular injury¹. Even low levels of albuminuria, lying within the normal range, are associated with an increased cardiovascular risk¹.

Under physiological conditions, albumin is filtered through the glomerulus and is almost completely reabsorbed in the tubular system^2,3. In mic....

Protocol

The investigations were conducted according to the guidelines outlined in the Guide for Care and Use of Laboratory Animals (US National Institutes of Health Publication No. 85-23, revised 1996). All animal experiments were performed in accordance with the relevant institutional approvals (state government Landesamt für Natur, Umwelt und Verbraucherschutz [LANUV] reference number 84-02.04.2012.A397).

1. Preparation of instruments, solutions, and equipment

1. Reconstitute FITC-polysucrose 70 with 0.9% sterile sodium chloride (NaCl) to a final concentration of 10 mg/mL (i.e., 100 mg in 10 mL of NaCl).
2. Dialyze FITC-polys....

Results

As depicted in Figure 2, the method to test glomerular permeability in mice is built up in three phases. The first phase is called the preparation phase, in which a urinary catheter and a central venous catheter are placed. The second phase is called the equilibration phase, starting with an intravenous bolus injection of FITC-polysucrose 70 and followed by the continuous infusion of FITC-polysucrose 70 for 60 min. The last phase is called the experimental ph.......

Discussion

The presented method enables the investigator to test glomerular permeability in mice in a very sensitive manner using a tracer. With this method, short-term increases in glomerular permeability can be diagnosed using only small amounts of urine. The most critical steps for successfully mastering this technique are 1) developing manual expertise in mouse surgery, especially in the cannulation of a central vein, 2) placing the urinary catheter without harming the mucosa, and 3) manual expertise in handling 384-well plates.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Motic SMZ168 BL	Motic	SMZ168BL	microscope for mouse surgery
KL1500LCD	Pulch and Lorenz microscopy	150500	light for mouse surgery
Microfederschere	Braun, Aesculap	FD100R	fine scissors
Durotip Feine Scheren	Braun, Aesculap	BC210R	for neck cut
Anatomische Pinzette	Braun, Aesculap	BD215R	for surgery
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
BD Insyte Autoguard	BD	381823	urinary catheter
Multimode Detector DTX 880	Beckman Coulter		plate reader
384 well microtiterplate	Nunc	262260	384 well platte
Creatinine Assay Kit	Sigma-Aldrich	MAK080	to measure creatinine concentration
96 well plate	Nunc	260836	for creatinine assay
FITC-labeled polysuccrose 70	TBD Consultancy	FP70	FITC-ficoll
Angiotensin II	Sigma-Aldrich	A9525	used to test glomerular permeability
BP-98A	Softron		for blood pressure measurement
OTS 40.3040	Medite	01-4005-00	heating plate for mouse surgery
Instillagel 6mL	Farco-Pharma GmbH		for urinary catheter
Exacta	Aesculap	GT415	shaver