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Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Published: April 17th, 2013



1Medicine/Nephrology, Indiana University School of Medicine

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

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.

1. Conjugation of Rat Serum Albumin to Sulfo-Rhodamine 101 Sulfonyl Chloride (Texas Red)

  1. Dissolve 100 mg of Rat Serum Albumin (RSA) in 6.667 ml of 100 mM Sodium Bicarbonate pH 9.0; final concentration 15 mg/ml in a 50 ml conical tube.
  2. Place solution in ice/water beaker and cool to between 0 to 4 °C.
  3. Add 200 μl of high quality anhydrous Dimethyl Formamide (DMF) to a 10 mg vial of Texas Red Sulfonyl Chloride (TRSC); vortex on medium for 15 sec.
  4. Vortex RSA solution on medium .......

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Figure 3 shows an example of an image taken from a surface glomerulus of a Munich Wistar Frömter rat and the steps taken to determine the permeability of fluorescent albumin. The GSC value for albumin of 0.0111 derived for this individual glomerulus fall within the range seen in this strain of Munich Wistar rats when in the fed condition3. The stability seen in these images is due to the careful planning and execution of instructions depicted in Figures 1 and 2. As .......

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The steps highlighted here represent what we feel to be ones that will produce consistent and accurate permeability values because they circumvent the following pitfalls:

  1. Scattering: The use of a red emitting fluorophores allow for more efficient collection of light since longer wavelength photons are less prone to scatter. Using either green or blue emitting fluorophores will introduce greater variation in GSC's because of the increased variability in intensity values from the capillary loops and.......

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The authors would like to thank Drs Silvia B Campos-Bilderback and George J Rhodes for completing surgical procedures involving placement of venous access lines. They would also like to thank Sara E Wean for maintaining the Munich-Wistar colonies consisting of both Simonsen and Frömter strains. This work was supported by funding provided to the Indiana Center for Biological Microscopy, and the National Institutes of Health grants P30-DK079312, and 5RO1-DK091623 awarded to Bruce A Molitoris.


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Name Company Catalog Number Comments
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

  1. 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).
  2. 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).
  3. Sandoval, R. M., et al. Multiple factors influence glomerular albumin permeability in rats. Journal of the American Society of Nephrology. 23 (3), 447 (2012).
  4. Tojo, A., Endou, H. Intrarenal handling of proteins in rats using fractional micropuncture technique. American Journal of Physiology. 263, 601 (1992).
  5. 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).
  6. Sandoval, R. M., Molitoris, B. A. Quantifying endocytosis in vivo using intravital two-photon microscopy. Methods in Molecular Biology. 440, 389 (2008).
  7. Dunn, K. W., et al. Live-animal imaging of renal function by multi-photon microscopy. Curr. Protoc. Cytom. Chapter 12, Unit 12.9 (2007).

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