JoVE Journal > Biology
Summary
Abstract
Introduction
Protocol
Résultats
Discussion
Divulgations
Acknowledgements
Materials
References
Traduction automatique
Please note that all translations are automatically generated. Click here for the English version.
Biologie

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

Published: September 01, 2015
doi:
10.3791/53035
, , , , ,
1Department of Physiology,Medical College of Wisconsin, 2Department of Integrative Biology & Pharmacology,University of Texas Health Science Center at Houston

Summary

表现在肾小管上皮细胞离子通道在多囊肾病的病理一个显著的作用。这里，我们描述用于执行囊性上皮从啮齿动物肾脏新鲜分离的膜片钳分析和细胞内钙水平的测量的实验方案。

Abstract

期间多囊肾囊肿萌生和扩展是一个复杂的过程，其特征在肾小管细胞增殖，管腔流体积累和细胞外基质的形成异常。离子通道和细胞内钙信号的活动是决定的肾小管上皮细胞功能的关键生理参数。我们开发适合于实时观察离子通道的活性与在上皮单层从肾囊肿新鲜分离的细胞内Ca 2+水平膜片钳技术和登记的方法。 PCK大鼠的常染色体隐性遗传性多囊肾病（ARPKD）遗传模型，这里被用作离子通道和钙流的体外分析。这里描述的是详细的一步一步的步骤旨在隔离囊性单层和非扩张管从PCK或正常只SD（SD）大鼠，并监视单通道活性和细胞内Ca 2+动力学。此方法不需要酶加工，并允许在分析新鲜分离的上皮单层的天然环境。此外，这种技术是将细胞内钙的变化非常敏感，并生成高分辨率的图像进行精确的测量。最后，分离的囊性上皮可进一步用于与抗体或染料，制备原代培养物的纯化和关于各种生化分析染色。

Introduction

离子通道在许多生理功能，包括细胞生长和分化中发挥显著作用。常染色体显性和隐性多囊肾疾病（ADPKD和ARPKD，分别）是其特征在于，所述的肾小管上皮细胞起源的肾流体填充囊肿的发展遗传性疾病。多囊肾是由PKD1或PKD2编码polycystins 1和参与细胞增殖和分化的调节如图2所示，膜蛋白的突变引起的。 PKD2本身或作为与PKD1一个复杂也发挥作为钙 -permeable阳离子通道1。所述PKHD1基因编码fibrocystin（涉及小管和/或维持上皮极性的纤毛相关受体样蛋白）的突变是ARPKD 2的遗传动力。囊肿生长是一个复杂的现象，伴随着不安扩散3,4，5血管生成，分化和极性损失性肾小管细胞6-8。

有缺陷的再吸收和分泌增强囊性上皮有助于在管腔和囊肿膨胀9,10-流体积聚。受损的流量依赖性钙离子浓度我的信号也已PKD 11-15时挂cystogenesis。

这里，我们描述适用于单通道活性和细胞内Ca 2+水平的囊性上皮单层从PCK大鼠分离的膜片钳测量的方法。这种方法成功地被我们用于表征上皮细胞钠离子通道（ENaC的）10的活性和钙离子浓度依赖我引起的钙 -permeable TRPV4和嘌呤信号级联13处理 。

在这些研究中，我们使用的PCK大鼠，模型ARPKD引起的在PKHD1基因的自发突变。该PCK应变originallÿ从的Sprague-Dawley（SD）大鼠16从而SD大鼠被用作适当的控制用于 ​​与PCK应变比较而得。其结果是，既大鼠肾段和非扩张集合管从同一PCK大鼠分离可以作为两个不同的比较组对囊性上皮实验。

Protocol

下面描述的实验程序，在威斯康星州和德克萨斯大学健康科学中心休斯敦大学医学院获得批准的机构动物护理和使用委员会，并分别按照卫生指南实验动物的护理和使用国家研究院​​。 图1显示的组织分离和处理过程的主要步骤。简要地说，从PCK或SD大鼠的肾脏用于收集无论是从健康非拨小管或囊肿导管上皮单层的手动隔离。在这里，我们从4-16周龄PCK大鼠10,13研究肾脏。 …

Representative Results

潜在的ENaC参与cystogenesis已经证明了几项研究观察到了破坏表皮生长因子（EGF）在PKD进展22-25和异常钠重吸收在ARPKD鼠模型和组织培养26-28信令。例如，Veizis 等人表明，阿米洛利敏感的 Na +吸收在光盘细胞ARPKD 29的非直系同源BPK小鼠模型下降。我们最近证明，受损的钠和水的重吸收的孢囊是在加重cystogenesis 10的一个重要因素。具体来说，我们采用电生理…

Discussion

我们在这里描述的传统膜片钳技术和表面荧光钙成像的应用程序从ARPKD的鼠的遗传模型导出囊性上皮单层。该协议包括三个步骤，其中最应引起重视的囊肿（协议节的步骤1.5）的隔离和电生理研究。这些关键步骤需要大量的训练和耐心，读者不应该立刻感到沮丧。

首先，最要注意的囊肿单层隔离的过程。制备的这一部分需要手工技巧和显著影响进一步的工作，作为检体和其连…

Divulgations

The authors have nothing to disclose.

Acknowledgements

作者想感谢格伦·斯洛克姆（威斯康星医学院）和科琳A.拉文（尼康仪器公司）与显微镜实验优秀的技术援助。这项研究是支持由美国国立卫生研究院授予R01 HL108880（以AS），R01 DK095029（以OPO）和K99 HL116603（以TSP），全国肾脏基金会IG1724（以TSP），美国心脏协会13GRNT16220002（以OPO）和本·J.里普斯研究奖学金由美国肾脏病学会（至DVI）。

Materials

Fura-2 AM Life Technologies F-14185
Flou-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
DOWNLOAD MATERIALS LIST

References

  1. Torres, V. E., Harris, P. C., Pirson, Y. Autosomal dominant polycystic kidney disease. Lancet. 369 (9569), 1287-1301 (2007).
  2. Zhang, M. Z., et al. PKHD1 protein encoded by the gene for autosomal recessive polycystic kidney disease associates with basal bodies and primary cilia in renal epithelial cells. Proc. Nat. Acad. Sci U.S.A. 101 (8), 2311-2316 (2004).
  3. Chang, M. Y., et al. Haploinsufficiency of Pkd2 is associated with increased tubular cell proliferation and interstitial fibrosis in two murine Pkd2 models. Nephrol. Dial. Transpl. 21 (8), 2078-2084 (2006).
  4. Park, F., Sweeney, W. E., Jia, G., Roman, R. J., Avner, E. D. 20-HETE mediates proliferation of renal epithelial cells in polycystic kidney disease. J. Am. Soc. Nephrol. 19 (10), 1929-1939 (2008).
  5. Huang, J., Woolf, A., Long, D. Angiogenesis and autosomal dominant polycystic kidney disease. Ped. Nephrol. 28 (9), 1749-1755 (2013).
  6. Wilson, P. D. Apico-basal polarity in polycystic kidney disease epithelia. Bioch Biophys Acta. 1812 (10), 1239-1248 (2011).
  7. Wilson, P. D. Epithelial cell polarity and disease. Am. J. Physiol. Renal Physiol. 272 (4 Pt 2), F434-F442 (1997).
  8. Wilson, P. D., et al. Reversed polarity of Na(+) -K(+) -ATPase: mislocation to apical plasma membranes in polycystic kidney disease epithelia. Am. J. Physiol. Renal Physiol. 260 (3 pt 2), F420-F430 (1991).
  9. Murcia, N. S., Sweeney, W. E., Avner, E. D. New insights into the molecular pathophysiology of polycystic kidney disease. Kidn. Intern. 55 (4), 1187-1197 (1999).
  10. Pavlov, T. S., Levchenko, V., Ilatovskaya, D. V., Palygin, O., Staruschenko, A. Impaired epithelial Na+ channel activity contributes to cystogenesis and development of autosomal recessive polycystic kidney disease in PCK rats. Ped. Res. 77 (1), 64-69 (2014).
  11. Siroky, B. J., et al. Loss of primary cilia results in deregulated and unabated apical calcium entry in ARPKD collecting duct cells. Am. J. Physiol. Renal Physiol. 290 (6), F1320-F1328 (2006).
  12. Hovater, M. B., et al. Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals. Purin. Signal. 4 (2), 155-170 (2008).
  13. Zaika, O., et al. TRPV4 Dysfunction Promotes Renal Cystogenesis in Autosomal Recessive Polycystic Kidney Disease. J. Am. Soc. Nephrol. 24 (4), 604-616 (2013).
  14. Rohatgi, R., et al. Mechanoregulation of intracellular Ca2+ in human autosomal recessive polycystic kidney disease cyst-lining renal epithelial cells. Am. J. Physiol. Renal Physiol. 294 (4), F890-F899 (2008).
  15. Xu, C., et al. Attenuated, flow-induced ATP release contributes to absence of flow-sensitive, purinergic Cai2+ signaling in human ADPKD cyst epithelial cells. Am. J. Physiol. Renal Physiol. 296 (6), F1464-F1476 (2009).
  16. Katsuyama, M., Masuyama, T., Komura, I., Hibino, T., Takahashi, H. Characterization of a novel polycystic kidney rat model with accompanying polycystic liver. Exp. Animals. 49 (1), 51-55 (2000).
  17. Ilatovskaya, D., Staruschenko, A. Single-channel analysis of TRPC channels in the podocytes of freshly isolated glomeruli. Methods Mol. Biol. 998, 355-369 (2013).
  18. Pavlov, T. S., et al. Deficiency of renal cortical EGF increases ENaC activity and contributes to salt-sensitive hypertension. J. Am. Soc. Nephrol. 24, 1053-1062 (2013).
  19. Mironova, E., Bugay, V., Pochynyuk, O., Staruschenko, A., Stockand, J. Recording ion channels in isolated, split-opened tubules. Methods Mol. Biol. 998, 341-353 (2013).
  20. Pavlov, T. S., et al. Endothelin-1 inhibits the epithelial Na+ channel through betaPix/14-3-3/Nedd4-2. J. Am. Soc. Nephrol. 21 (5), 833-843 (2010).
  21. Sun, P., et al. High Potassium Intake Enhances the Inhibitory Effect of 11,12-EET on ENaC. J. Am. Soc. Nephrol. 21 (10), 1667-1677 (2010).
  22. Zheleznova, N. N., Wilson, P. D., Staruschenko, A. Epidermal growth factor-mediated proliferation and sodium transport in normal and PKD epithelial cells. Biochim. Biophys. Acta. 1812 (10), 1301-1313 (2011).
  23. Sweeney, W. E., von Vigier, R. O., Frost, P., Avner, E. D. Src inhibition ameliorates polycystic kidney disease. J. Am. Soc. Nephrol. 19 (7), 1331-1341 (2008).
  24. Sweeney, W. E., Avner, E. D. Functional activity of epidermal growth factor receptors in autosomal recessive polycystic kidney disease. Am. J. Physiol. Renal Physiol. 275 (3 Pt 2), F387-F394 (1998).
  25. Orellana, S. A., Sweeney, W. E., Neff, C. D., Avner, E. D. Epidermal growth factor receptor expression is abnormal in murine polycystic kidney. Kidn. Intern. 47 (2), 490-499 (1995).
  26. Rohatgi, R., et al. Cyst fluid composition in human autosomal recessive polycystic kidney disease. Ped. Nephrol. 20 (4), 552-553 (2005).
  27. Rohatgi, R., Greenberg, A., Burrow, C. R., Wilson, P. D., Satlin, L. M. Na transport in autosomal recessive polycystic kidney disease (ARPKD) cyst lining epithelial cells. J. Am. Soc. Nephrol. 14 (4), 827-836 (2003).
  28. Olteanu, D., et al. Heightened epithelial Na+ channel-mediated Na+ absorption in a murine polycystic kidney disease model epithelium lacking apical monocilia. Am. J. Physiol. Cell Physiol. 290 (4), C952-C963 (2006).
  29. Veizis, I. E., Cotton, C. U. Abnormal EGF-dependent regulation of sodium absorption in ARPKD collecting duct cells. Am. J. Physiol. Renal Physiol. 288 (3), F474-F482 (2005).
  30. Wilson, P. D. Polycystic kidney disease. NEJM. 350 (2), 151-164 (2004).
  31. Hillman, K. A., et al. P2X(7) receptors are expressed during mouse nephrogenesis and in collecting duct cysts of the cpk/cpk mouse. Exp. Nephrol. 10 (1), 34-42 (2002).
  32. Turner, C. M., Ramesh, B., Srai, S. K., Burnstock, G., Unwin, R. J. Altered ATP-sensitive P2 receptor subtype expression in the Han:SPRD cy/+ rat, a model of autosomal dominant polycystic kidney disease. Cells Tissues Organs. 178 (3), 168-179 (2004).
  33. Hillman, K. A., et al. The P2X7 ATP receptor modulates renal cyst development in vitro. Biochem. Biophys. Res. Commun. 322 (2), 434-439 (2004).
  34. Wilson, P. D., Hovater, J. S., Casey, C. C., Fortenberry, J. A., Schwiebert, E. M. ATP release mechanisms in primary cultures of epithelia derived from the cysts of polycystic kidneys. J. Am. Soc. Nephrol. 10 (2), 218-229 (1999).
  35. Schwiebert, E. M., et al. Autocrine extracellular purinergic signaling in epithelial cells derived from polycystic kidneys. Am. J. Physiol. Renal Physiol. 282 (4), F763-F775 (1152).
  36. Stockand, J. D., et al. Purinergic inhibition of ENaC produces aldosterone escape. J. Am. Soc. Nephrol. 21 (11), 1903-1911 (2010).
  37. Pochynyuk, O., et al. Paracrine Regulation of the Epithelial Na+ Channel in the Mammalian Collecting Duct by Purinergic P2Y2 Receptor Tone. J. Biol. Chem. 283 (52), 36599-36607 (2008).
  38. Zaika, O., Mamenko, M., Boukelmoune, N., Pochynyuk, O. IGF-1 and insulin exert opposite actions on ClC-K2 activity in the cortical collecting ducts. Am. J. Physiol. Renal Physiol. 308 (1), F39-F48 (2015).
  39. Lalo, U., Pankratov, Y., Kirchhoff, F., North, R. A., Verkhratsky, A. NMDA receptors mediate neuron-to-glia signaling in mouse cortical astrocytes. J. Neurosci. 26 (10), 2673-2683 (2006).
  40. Lalo, U., Andrew, J., Palygin, O., Pankratov, Y. Ca2+-dependent modulation of GABAA and NMDA receptors by extracellular ATP: implication for function of tripartite synapse. Biochem. Soc. Trans. 37 (Pt 6), 1407-1411 (2009).
  41. Li, D., et al. Inhibition of MAPK stimulates the Ca2+ -dependent big-conductance K channels in cortical collecting duct). Proc. Nat. Acad. Sci U.S.A. 103 (51), 19569-19574 (2006).
  42. Bugaj, V., Mironova, E., Kohan, D. E., Stockand, J. D. Collecting duct-specific endothelin B receptor knockout increases ENaC activity. Am. J. Physiol. Cell Physiol. 302 (1), C188-C194 (2012).
  43. Pavlov, T. S., et al. Regulation of ENaC in mice lacking renal insulin receptors in the collecting duct. FASEB J. 27 (7), 2723-2732 (2013).
  44. Gleason, C. E., et al. mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity. J. Clin. Invest. 125 (1), 117-128 (2015).
  45. Frindt, G., Palmer, L. G. Acute effects of aldosterone on the epithelial Na channel in rat kidney. Am. J. Physiol. Renal Physiol. , (2015).
  46. Ilatovskaya, D. V., et al. Angiotensin II has acute effects on TRPC6 channels in podocytes of freshly isolated glomeruli. Kidn. Int. 86 (3), 506-514 (2014).
  47. Ilatovskaya, D. V., Palygin, O., Levchenko, V., Staruschenko, A. Pharmacological characterization of the P2 receptors profile in the podocytes of the freshly isolated rat glomeruli. Am. J. Physiol. Cell Physiol. 305 (10), C1050-C1059 (2013).

Tags

Keywords: Patch ClampLive Fluorescence MicroscopyPolycystic Kidney DiseasePKDARPKDIon ChannelsCalcium SignalingEpithelial MonolayerEx Vivo AnalysisPCK RatSprague Dawley RatNative SettingEpithelial IsolationImmunostainingPrimary CultureBiochemical Assays
check_url/fr/53035?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citer Cet Article
Pavlov, T. S., Ilatovskaya, D. V., Palygin, O., Levchenko, V., Pochynyuk, O., Staruschenko, A. Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium. J. Vis. Exp. (103), e53035, doi:10.3791/53035 (2015).
Télécharger la Citation
Réimpressions et Autorisations

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image