Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Summary

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Abstract

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca²⁺ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca²⁺ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Introduction

Ion channels play a significant role in many physiological functions, including cell growth and differentiation. Autosomal dominant and recessive polycystic kidney diseases (ADPKD and ARPKD, respectively) are genetic disorders characterized by the development of renal fluid-filled cysts of the tubular epithelial cell origin. ADPKD is caused by mutations of PKD1 or PKD2 genes encoding polycystins 1 and 2, membrane proteins involved in the regulation of cell proliferation and differentiation. PKD2 by itself or as a complex with PKD1 also function as a Ca²⁺-permeable cation channel¹. Mutations of the PKHD1 gene encoding fibrocystin (a cilia-associat....

Protocol

The experimental procedures described below were approved by the Institutional Animal Care and Use Committee at the Medical College of Wisconsin and University of Texas Health Science Center at Houston and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Figure 1 demonstrates main steps of the tissue isolation and processing procedure. Briefly, kidneys from PCK or SD rats are used for manual isolation of epithelial monolayers of collecting ducts either from healthy non-dialed tubules or cysts. Here we studied kidneys from 4-16 weeks old PCK rats^10,13.

1. Is....

Results

Potential ENaC involvement in cystogenesis has been demonstrated by several studies that observed disrupted epidermal growth factor (EGF) signaling in PKD progression^22-25 and abnormal sodium reabsorption in ARPKD murine models and tissue cultures^26-28. For example, Veizis et al. showed that amiloride-sensitive Na⁺ absorption is decreased in CD cells from the non-orthologous BPK mouse model of ARPKD²⁹. We recently demonstrated that impaired sodium and water reabsorptio.......

Discussion

We described here applications of conventional patch-clamp technique and epifluorescence calcium imaging to cystic epithelial monolayers derived from a murine genetic model of ARPKD. The protocol consist of three steps, of which the most attention should be paid to the isolation of the cysts (step 1.5 of the protocols section) and to the electrophysiological studies. These key procedures require extensive training and patience, and the reader should not be frustrated at once.

First of all, the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fura-2 AM	Life Technologies	F-14185
Fluo-8	AAT Bioquest	21091
Poly-L-lysine	Sigma-Aldrich	P4707
Pluronic acid	Sigma-Aldrich	F-68	solution
Shaker	Boekel Scientific	260350
Light source	Sutter Instrument Co	Lambda XL	with integrated shutter/filter wheel driver
Neutral density filters	Nikon		ND4, ND8
Objective	Nikon	SFluo	40/1.3 DIC WD 0.22   oil
Camera	Andor Technologies	Zyla sCMOS
Nikon  microscope (inverted)	Nikon	Nikon Eclipse TE2000-S
Cover Glass	Thermo Scientific	6661B52
Diamond pencil	Fisher Scientific	22268912
Image acquisition software	Nikon	Nikon NIS-Elements
Image analysis software	ImageJ	http://imagej.nih.gov/	ND Utility plugin allows to import images in the native Nikon Instruments .nd2 format
Recording/perfusion chamber	Warner Instruments	RC-26
Patch Clamp amplifier	Molecular Devices	MultiClamp 700B
Data Acquisition System	Molecular Devices	Digidata 1440A	Axon Digidata® System
Low Pass Filter	Warner Instruments	LPF-8	8 pole Bessel
Borosilicate glass capillaries	World Precision Instruments	1B150F-4
Micropipette Puller	Sutter Instrument Co	P-97	Flaming/Brown type micropipette puller
Microforge	Narishige	MF-830	Japan
Motorized Micromanipulator	Sutter Instrument Co	MP-225
Inverted microscope	Nikon	Eclipse Ti
Microvibration isolation table	TMC		equipped with Faraday cage
Multichannel valve perfusion system	AutoMake Scientific	Valve Bank II
Recording/perfusion chamber	Warner Instruments	RC-26
Software	Molecular Devices	pClamp 10 . 2
Temperature controlled surgical table	MCW core		for rodents
Binocular stereomicroscope	Nikon	SMZ745
Syringe pump-based perfusion system	Harvard Apparatus
polyethylene tubing	Sigma-Aldrich	PE50
Isofluorane anesthesia	http://www.vetequip.com/	911103
Other basic reagents	Sigma-Aldrich