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)
2. Preparing the Inverted Microscope Stage for Imaging/ Microscope Settings
3. Exposing the Kidney in a Munich Wistar Rat for Intravital 2-Photon Imaging
4. Placing the Munich Wistar Rat on the Stage for Imaging
5. Acquiring Images to Quantify Renal Permeability of Albumin
6. Calculating GSC for Fluorescent Albumin from 3D Volumes
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 stated earlier, the placement of the incision used in exteriorizing the kidney is the most crucial step in producing stable images free of motion artifact.
Figure 1. A detailed schematic of positioning an orientation of the rat on the microscope stage prior to imaging. Gently lay the rat kidney side down (as shown in A) over the Repti-Therm heating pads with the kidney laying in the coverslip dish. Roll the kidney outward (*) so that the ventral side touches the coverslip bottom and contact is made with the autoclave tape (AT). To minimize breathing induced motion, roll the rat forward (**) so that the thorax is out of the area immediately above the coverslip dish. Fill the dish with sterile 0.9% saline and cover with water jacket. Use a thermometer to monitor rectal temperature and turn the Repi-Therm pads on and off as needed. Click here to view larger figure.
Figure 2. Exteriorization of kidney in Munich Wistar Rat prior to placement on microscope stage. An anesthetized rat is placed with its left size up; area between the rib cage and upper thigh shaved (shown in gray, A). Once sequential incisions are made to cut through the skin, outer then inner muscle layer as shown by the line traversing the kidney in A. The kidney with associated peri-renal fat should be visible (B). Using a pair of forceps, the fat is pinched in a hand-over-hand fashion until the lower most pole is reached (B-E). Gently squeeze the area under the kidney while pulling on the fat to exteriorize. Click here to view larger figure.
Figure 3. Single plane images taken from 3D volumes showing a glomerulus of a Munich Wistar Rat. Panels A & B show background images and one taken ~12 min post infusion of Texas Red Rat Serum Albumin (TR-RSA) in black and white. Note the lack discernible intensity of capillary loops (CL) or Bowman’s space (BowSp) in the background image (A). Panel C shows a region taken from panel A in pseudocolor to better visualize the low intensity background levels of tissue. Here, a small region is drawn in a capillary loop (longer region) and within Bowman’s space to estimate pre-existing fluorescent intensity levels which must be subtracted from values obtained after infusion of fluorescent albumin. Panels D and E are taken from panel B and shown in pseudocolor. Three small regions of interest drawn in Bowman’s space are used to calculate the average intensity of fluorescent albumin that has moved across the filtration barrier (D). Average intensity values for the individual regions were reported in the highlighted area within the “Show Region Statistics” dialog box (panel D’). To calculate the circulating plasma intensity values within capillary loops (CL, in panel E) a large region is drawn over the brightest capillary loop and the bright values along the highlighted using a threshold function (shown a orange pixels). Only the intensity values of the pixels highlighted in orange will be reported regardless of the shape or size of the surrounding region. Panel E’ shows the raw average intensity values of the albumin within the capillary loops; note the checked “Use Threshold For Intensity Measurements” box that is checked. Panel F shows the progression of values form left to right starting with the background values for the Capillary Loops and Bowman’s space, which are subtracted from the raw values obtained within the images. Once the corrected values are derived, the Bowman’s space intensity value is divided by the Capillary Loop intensity value to yield the Glomerular Sieving Coefficient which is a ratio of permeability; impermeant molecules have a value of zero (0), while those that are freely filtered have a value of one (1). Bar = 20 μm. Click here to view larger figure.
Figure 4. Uptake of filtered fluorescent albumin occurs predominantly in the early segment of proximal tubules, the S1. Panel A shows a cross section of a glomerulus and S1 segment taken ~20 min post infusion of the initial Texas Red RSA bolus. The opening the Bowman’s space and avid uptake of the albumin (red) is show in the S1 segment. Panel B shows a shallow 20 μm 3D projection of the same data set. Panel C shows a 3D projection using a lower power 20x objective approximately 60 min post infusion. Note the same glomerulus seen in panels A & B shown in C (*) and the higher levels of albumin retrieval seen in that segment versus other proximal tubule segments. Bar = 20 μm. Click here to view larger figure.
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:
Validation of this technique occurs by imaging a freely filtered compound with a GSC of one (1). Here, concerns that consistently underestimating albumin plasma levels, due to optical limitations being responsible for overestimating albumin permeability, are nullified3. Additionally, having determined a GSC value for a 70kDa dextran that is virtually identical to that obtained by measuring urinary clearance/plasma levels and micropuncture prove intravital 2-photon microscopy is capable of correctly determining the permeability of fluorescent macromolecules2. Finally, the ability to rapidly visualize the glomerulus and tubular segments along the filtrate path, with a high degree of resolution, 2-photon microscopy stands uniquely poised to quantify the importance of the glomerular filtration barrier and PT in determining protienuria and albuminuria.
The protocols described herein are based on the use of a 2-Photon system with an inverted stage which has benefits in the simplicity of the surgical procedures involved and rat placement on the stage. For information on utilizing an 2-Photon system with an upright stage refer to the chapter by Dunn et al.7 in Current Protocols in Cytometry (2007).
The authors have nothing to disclose.
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.
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 |