Taze İzole glomerulusların Podositler Tek kanallı Analizi ve Kalsiyum Görüntüleme
Summary
Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.
Abstract
Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.
Introduction
Böbrekler, çeşitli maddeler için homeostatik dengeyi korumak ve toplam kan basıncını belirleyen bir şekilde kan hacmini düzenler. Hiper veya hipotansiyon arasında değişen renal filtrasyon, geri emiliminin veya salgı kurşun bozukluklar veya eşlik patolojik durumlar, sonunda böbrek nakli gerektiren son dönem böbrek hastalığı için. kapiller endotel, bazal membran ve epitel hücreleri tek-hücre tabakası – – yarık diyafram bütünlüğü ve işlevi 1 korunmasında önemli bir rol oynamaktadır Podositler renal filtreleme birimi (glomerulus) üç tabakadan oluşur. Permselektif glomerüler filtre Disfonksiyon gibi proteinüri olarak makromoleküllerin, idrar kaybına neden olur. Çeşitli ajanlar glomerüller filtrasyon bariyerinin bütünlüğünü belirleyen Podositler ve ayak süreçleri, yapısını etkileyebilir.
Podositler glom bakım katılaneruli filtrasyon işlevi. Bu Podosit tarafından uygunsuz kalsiyum taşıma hücre hasarı yol açar ve nefropatilerin 2,3 çeşitli biçimlerde ilerlemesinde önemli bir rol oynadığı tespit edilmiştir. Bu nedenle, podosit fonksiyonunun çalışmalar için etkili olacaktır hücre içi kalsiyum konsantrasyonu değişiklikleri, doğrudan ölçümü için izin veren bir modelinin geliştirilmesi. İzole glomerüller önce albümin yansıma katsayısı ölçümü 4 ve bütün hücre elektrofizyolojik patch-kelepçe ölçümlerde 5,6 integral hücresel akımların değerlendirmesini değiştirir dahil çok sayıda çalışmalarda kullanılmıştır. Mevcut yazıda, farmakolojik ajanların uygulamalarına tepki olarak hücre içi kalsiyum konsantrasyonu değişiklikleri ölçmek hücrelerin içinde kalsiyum bazal düzeylerini tahmin ve bireysel kalsiyum kanalları aktivitesini değerlendirmek için araştırmacı izin veren protokol açıklar. Ratometric kalsiyum konsantrasyonu ölçümleri ve patch-kelepçe electrophysiology sırasıyla podosit ve kanal aktivitesi olan hücre içi kalsiyum konsantrasyonu değişiklikleri belirlemek için kullanıldı.
Protocol
Representative Results
Discussion
Burada tarif edilen bir yaklaşım, kemirgen glomerül Podositler kalsiyum işleme analizi sağlar. Bu teknik, patch-kelepçe tek kanallı elektrofizyoloji ve floresan rasyometrik konfokal görüntüleme uygulama sağlar. Bununla birlikte, her iki yaklaşım da kendi ayrı ayrı kullanılabilir. Önerilen protokol 1) böbrek temizleme dahil olmak üzere birçok nispeten basit adımları vardır; 2) diferansiyel eleme ile glomerül izolasyonu; 3) patch-kelepçe elektrofizyolojik deneyler veya hücre içi kalsiyum değiş…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Yazarlar mikroskobu deneyleri ile mükemmel teknik yardım için Glen Slocum (Wisconsin Tıp Koleji) ve Colleen A. Lavin (Nikon Instruments, Inc.) teşekkür etmek istiyorum. Gregory Blass yazının kritik redaksiyon için kabul edilmektedir. Bu araştırma (AS) 1-15-BS-172, ve (DVI) Nefroloji American Society Ben J. Lipps Araştırma Bursu hibe Sağlık hibe HL108880 ve American Diabetes Association Ulusal Sağlık Enstitüleri tarafından desteklenmiştir.
Materials
| Fluo4 AM | Life Technologies | F14217 | 500µl in DMSO |
| FuraRed AM | Life Technologies | F-3020 | |
| Poly-L-lysine | Sigma-Aldrich | P4707 | |
| Pluronic acid | Sigma-Aldrich | F-68 | solution |
| Ionomycin | Sigma-Aldrich | I3909-1ML | |
| Tube rotator | Miltenyi Biotec GmbH | 130-090-753 | Germany |
| Nikon confocal microscope (inverted) | Nikon | Nikon A1R | Laser exitation 488nm. Emission filters 500-550nm and 570-620nm |
| Objective | Nikon | Plan Apo 60x/NA 1.4 Oil | |
| Cover Glass | Thermo Scientific | 6661B52 | |
| High vacuum grease | Dow Corning | Silicone Compound | |
| Software | Nikon | Nikon NIS-Elements | |
| 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 | |
| Nicardipine | Sigma-Aldrich | N7510 | |
| Iberiotoxin | Sigma | I5904-5UG | |
| Niflumic acid | Sigma-Aldrich | N0630 | |
| DIDS | Sigma-Aldrich | D3514-25MG | |
| TEA chloride | Tocris | T2265 | |
| RPMI 1640 | Life Technologies | 11835030 | without antibiotics |
| BSA | Sigma-Aldrich | A8327 | 30% albumin solution |
| Temperature controlled surgical table | MCW core | for rodents | |
| Steel sieves: | #100 (150 μm), 140 (106 μm) | ||
| Gilson, Inc SIEVE 3 SS FH NO200 | Fisher Sci | 50-871-316 | |
| Gilson, Inc SIEVE 3 SS FH NO270 | Fisher Sci | 50-871-318 | |
| Gilson, Inc SIEVE 3 SS FH NO400 | Fisher Sci | 50-871-320 | |
| mesh 200 | Sigma-Aldrich | s4145 | screen for CD-1 |
| Binocular microscope | Nikon | Eclipse TS100 | |
| Binocular microscope | 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 |
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