title: Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium.
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.
authors.affiliation: Department of Physiology, Medical College of Wisconsin.
authors.forename: Tengis S
authors.initials: TS
authors.lastname: Pavlov
authors.affiliation: Department of Physiology, Medical College of Wisconsin.
authors.forename: Daria V
authors.initials: DV
authors.lastname: Ilatovskaya
authors.affiliation: Department of Physiology, Medical College of Wisconsin.
authors.forename: Oleg
authors.initials: O
authors.lastname: Palygin
authors.affiliation: Department of Physiology, Medical College of Wisconsin.
authors.forename: Vladislav
authors.initials: V
authors.lastname: Levchenko
authors.affiliation: Department of Integrative Biology & Pharmacology, University of Texas Health Science Center at Houston.
authors.forename: Oleh
authors.initials: O
authors.lastname: Pochynyuk
authors.affiliation: Department of Physiology, Medical College of Wisconsin; staruschenko@mcw.edu.
authors.forename: Alexander
authors.initials: A
authors.lastname: Staruschenko
mesh_terms: D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation / Q000502:physiology; D004195:Disease Models, Animal; D062686:Epithelial Sodium Channel Blockers / Q000494:pharmacology; D053503:Epithelial Sodium Channels / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism / Q000473:pathology; D008856:Microscopy, Fluorescence / Q000379:methods*; D018408:Patch-Clamp Techniques / Q000379:methods*; D017044:Polycystic Kidney, Autosomal Recessive / Q000378:metabolism / Q000473:pathology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction
chemical_list: D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; C013407:benzamil; D000584:Amiloride; D002118:Calcium