म्यूनिख Wistar चूहों में 2 फोटॉन माइक्रोस्कोपी का उपयोग फ्लोरोसेंट अणुओं की ग्लोमेरुलर पारगम्यता बढ़ाता
Summary
2 फोटॉन माइक्रोस्कोपी उच्च संकल्प intavital उपयोग एक तकनीक सीधे कल्पना और सतह ग्लोमेरुली में gloemrular निस्पंदन यों को. इस विधि में सामान्य और रोगग्रस्त दोनों राज्यों में बड़े अणुओं की पारगम्यता विशेषताओं के प्रत्यक्ष निर्धारण के लिए अनुमति देता है.
Abstract
Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman’s space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.
Protocol
Representative Results
Discussion
यहाँ पर प्रकाश डाला कदम हम वे निम्नलिखित नुकसान को दरकिनार क्योंकि सुसंगत और सटीक पारगम्यता मूल्यों का उत्पादन होगा कि लोगों को हो क्या महसूस प्रतिनिधित्व करते हैं:
- छितराया: अब तरंगदैर्य फोटॉनो?…
Declarações
The authors have nothing to disclose.
Acknowledgements
लेखकों शिरापरक पहुँच लाइनों की नियुक्ति से जुड़े सर्जिकल प्रक्रियाओं को पूरा करने के लिए डीआरएस सिल्विया बी कैम्पोस-Bilderback और जॉर्ज जे रोड्स को धन्यवाद देना चाहूंगा. उन्होंने यह भी Simonsen और Frömter उपभेदों दोनों से मिलकर म्यूनिख Wistar कालोनियों को बनाए रखने के लिए सारा ई छुड़ाना धन्यवाद देना चाहूंगा.
Materials
| Olympus Floview 1000 confocal/Multi-photon microscope | Olympus America | Filters for detectors: Blue 430/100, Green 525/50, Red 605/90 | |
| Mode-Locked Ti:Sapphire Mai Tai Laser | Spectra-Physics | Tunable excitation wavelengths: ~750-1150 nm | |
| Gallium arsenide phosphide photodetectors | Hamamatsu Corp | Note: Front or Side mounted configurations available. | |
| Metamorph Image processing Software | Molecular Dynamics | Note: Version 6.1r1 | |
| Microsoft Excel | Microsoft Corportation | 2007 version | |
| Handling Forceps | Electron Microscopy Sciences | Cat# 78266-04 | |
| Mayo Dissecting Scissors | Electron Microscopy Sciences | Cat# 72962 | |
| CA Micro scissors Model 1C300 | Electron Microscopy Sciences | Cat# 78180-1C3 | |
| Kelly Hemostatic Forceps (straight) | Electron Microscopy Sciences | Cat# 72930 | |
| Water Jacket Blanket + Heating Pad | Gaymar | T/Pump PN 11184-000 Blanket-66N111CC | |
| Repti-Therm Under Tank Heater | ZooMed | RH-4 | |
| Texas Red Sulfonyl Chloride | Invitrogen/Molecular Probes | Cat# T-353 | |
| Rat Serum Albumin | Sigma Aldrich | Cat# A-6272 | |
| High Quality Anhydrous DMF | Sigma Aldrich | Cat# 270547 | |
| Strate-Line Autoclave Tape | Fisher Scientific | Cat# 11-889-1 | |
| Willco-dish Coverslip Bottom Dishes (50 mm/40 mm coverslip) | Electron Microscopy Sciences | Cat# 70665-07 |
Referências
- Russo, L. M., et al. The normal kidney filters nephritic levels of albumin retrieved by proximal tubule cells; retrieval is disrupted in nephritic states. Kidney International. 71, 504 (2007).
- Russo, L. M., et al. Impaired tubular uptake explains albuminuria in early diabetic nephropathy. Journal of the American Society of Nephrology. 20 (3), 489 (2009).
- Sandoval, R. M., et al. Multiple factors influence glomerular albumin permeability in rats. Journal of the American Society of Nephrology. 23 (3), 447 (2012).
- Tojo, A., Endou, H. Intrarenal handling of proteins in rats using fractional micropuncture technique. American Journal of Physiology. 263, 601 (1992).
- Asgeirsson, D., et al. Glomerular sieving of three neutral polysaccharides, polyethylene oxide and bikunin in rat: Effects of molecular size and conformation. Acta Physiologica. 191 (3), 237 (2007).
- Sandoval, R. M., Molitoris, B. A. Quantifying endocytosis in vivo using intravital two-photon microscopy. Methods in Molecular Biology. 440, 389 (2008).
- Dunn, K. W., et al. Live-animal imaging of renal function by multi-photon microscopy. Curr. Protoc. Cytom. Chapter 12, Unit 12.9 (2007).
