Tracking Glomerular Fate Over Long Time Distances

Serial multiphoton microscopy helps to understand the dynamic processes in kidney disease and regeneration in vivo

The glomerular filtration barrier (GFB) is a complex spatial structure within the kidney glomerulis where ultrafiltration takes place. Podocytes are critical elements of the GFB and take part in the filtration process. They have been shown to be involved in the development of kidney diseases. However, due to technical limitations, the mechanism of glomerular pathology is not well understood. János Peti-Peterdi’s group at the University of Southern California developed a new method called serial multiphoton microscopy (MPM) that allows to track the fate of a single glomerulus over several days. Recently it was published as the cover story in nature medicine. In this interview he comments on how serial MPM affected his research.



Your paper made it to the title page of nature medicine. What is shown in the picture, and why was it chosen for the cover? What makes it special/interesting?

The cover page shows a visually appealing, representative multi-color image of the surface region of the living mouse kidney. The image was acquired by using in vivo multiphoton fluorescence imaging of newly generated transgenic mice that we named Podocin-Confetti mice. The mouse underwent unilateral ureter obstruction which is a classic, common model of kidney fibrosis. Podocytes, one of the most important cell types in the kidney which form and maintain the glomerular filter, are labeled in one of four colors, based on cell-specific expression of genetically encoded membrane-targeted CFP (blue), nuclear GFP (green), cytosolic YFP (yellow) or cytosolic RFP (red). Labeled dextran (plasma dye) illuminates the renal vasculature. This new technology helps to identify and track the fate of the same single podocytes in the intact living kidney over the course of disease. This improved method for identifying and tracking single podocytes in the intact kidney could improve our understanding of the mechanisms of glomerular injury and regeneration. We believe that the novelty and significance of this approach helped this image and our report make it to the cover.

Timing may helped also. This was the December issue of the journal and therefore the holiday season. The multi-color labeled glomeruli at the end of the vascular tree look similar to Christmas balls on a Christmas tree.

Please describe the method of serial MPM that you have developed with regard to study intact kidney tissue. How does it work?

To better understand the dynamics of podocyte migration we developed serial MPM imaging of the same glomerulus over time in the intact mouse kidney in vivo, once in every 24 hours. Serial MPM requires multiple surviving, minimally invasive surgeries of the same animal. In anesthetized mice the left kidney was gently exteriorized from a dorsal flank incision which is well tolerated by animals compared to the traditional ventral approach, and mice were placed on a heated microscope stage for MPM imaging using an inverted microscope. An area of the kidney suitable for imaging was identified and the position of the kidney was noted for identical placement on the following days.

After acquiring z-stacks of the glomerulus a small distant area in the field of view was marked by shortly focusing the laser beam on this area with high power. This maneuver generated an easy to find highly fluorescent spot (reference point) which remained there for 3–5 days. The position of the mark relative to the glomerulus of interest was documented. After imaging, the kidney was placed back into the retroperitoneum and the flank cut was closed with sutures. This procedure was repeated in the same animal/glomerulus 24, 48, 72, etc. hours later by removing the sutures and exteriorizing the kidney again. With this technique we were able to subsequently find approximately 70 % of the glomeruli that were marked in the first imaging session. Z-stacks of marked glomeruli were acquired with identical imaging settings as the day before.

The images were acquired using a Leica TCS SP5 multiphoton confocal fluorescence imaging system with a 63x Leica glycerine-immersion objective (NA 1.3) powered by a Chameleon Ultra-II MP laser at 860 nm (Coherent) and a Leica DMI6000 B inverted microscope’s external nondescanned detectors. Short pass filters (680 nm for blue and red, 700 nm for green and yellow), dichroic mirrors (cut off at 515 nm for green and yellow, 560 nm for blue and red) and bandpass filters were specific for detecting CFP/GFP/YFP/RFP emission (473 nm / 514 nm / 545 nm / 585 nm) (Chroma).

Fig. 1: Identification and tracking of single podocytes in the intact kidney of a Podocin-Confetti mouse using in vivo multiphoton fluorescence imaging. Podocytes outside the glomerular capillary loops are labeled in one of four colors, either by membrane-targeted CFP (blue), nuclear GFP (green), cytosolic YFP (yellow) or cytosolic RFP (red). Labeled dextran (plasma dye, greyscale) illuminates the renal vasculature.

What were the difficulties before? What is new about serial MPM?

The glomerular filtration barrier (GFB) has a very complex and dynamic 3D structure formed by several different cell types including special interstitial cells called mesangial cells, endothelial cells, and podocytes. Podocytes are critical elements of the GFB, a complex vascular unit in the glomerular capillaries that performs plasma ultrafiltration. Podocytes are highly differentiated cells which form long primary and secondary extensions that wrap around the outside of the capillary loops. Between their interdigitating foot processes the slit diaphragm is formed which is a key component of the GFB. Recent genetic and cell biological studies highlighted the critical importance of podocytes in the development of glomerular diseases. Still, a critical barrier in understanding the mechanistic details of glomerular pathology has been the technical limitation to study the GFB in its native environment. Due to the lack of in vivo data, there are still significant gaps in our understanding of podocyte dynamics and motility and their link to albuminuria and glomerulosclerosis. However, this insight would be needed for the development of new treatment strategies.

The application of high resolution imaging tools, particularly this serial MPM approach may provide the long missing in vivo technology for podocyte research. MPM is a revolutionary, minimally invasive optical sectioning technique which allows the imaging of the mouse kidney including glomeruli in vivo. Serial MPM represents a new technical advance which allowed us, for the first time, to visualize the exact same podocyte/glomerular region (tissue volume) over several days in the intact living kidney during the course of glomerular disease. No other current technology is capable of achieving this. Advantages include (i) overcoming glomerular heterogeneity issues, (ii) establishing the dynamics and pattern of cell migration, tissue remodeling and (iii) combination with functional measurements.

Which new insights did you obtain by serial MPM that you were not able to get otherwise?

Serial MPM imaging was instrumental in depicting, for the first time, the dynamics and the dramatic changes in glomerular morphology, cellular composition, and podocyte projections which often occurred within 24 hours of the previous imaging session. We found evidence for the migration of podocytes away from their usual anatomical location, from the glomerular tuft. Our data support the highly dynamic (new paradigm) rather than static nature (current dogma) of the glomerular environment and cellular composition. Moreover, a new anatomical discovery was the first visualization of nanotubules connecting the visceral (podocytes) and parietal epithelial layers (PECs) which may participate in cell-to-cell communication and cell migration.

How has serial MPM changed your research?

Serial MPM of novel mouse models with fluorescent cell lineage tags is a novel, unique, state-of-the-art imaging approach that we will continue to use in our research to directly and quantitatively visualize the recruitment and fate of different renal cell types in vivo and to address their functions and roles in renal vascular and glomerular remodeling in health and disease. So far, we have developed several new transgenic mouse models in which seven different renal cell types can be labeled specifically by the expression of genetically encoded, multi-color fluorescent proteins.

This new toolbox in combination with the technological breakthrough of serial, intravital MPM imaging is a tremendous technical advance for our research program and has allowed us, and will continue to allow us, to study the dynamics of normal and diseased kidney functions in the living kidney in unprecedented detail. The application of this new technology allowed us to generate several highly innovative concepts and approaches including the identification and characterization of new, non-traditional mechanisms and roles for many different renal cell types.

New advances in stem/progenitor cell research has brought a new perspective and focus on tissue regeneration and cell fate changes in several areas of the biomedical sciences including renal and cardiovascular (patho)physiology. Novel technologies have become available for fluorescent tagging of cell lineage and individual cell fate tracking in various organs. We will apply these amazing research tools in combination with serial MPM to advance our understanding of the functions and fate of specific renal cell types (such as podocytes, cells of the macula densa, renin producing cells, tubular epithelial cells, capillary and lymphatic endothelium, mesenchymal progenitor cells, etc) which are important regulators of renal and glomerular functions (such as glomerular filtration, renal blood flow, tubular secretion and reabsorption), critical components of the renin-angiotensin system, and also important players in renal pathologies.

What are the future possibilities of the approach?

In addition to tracking the temporal dynamics of cell migration and fate changes, tissue remodeling during kidney disease, we can use serial MPM imaging to learn about the changes in intracellular and cell-to-cell signaling during the course of disease. For example, serial MPM imaging of mice with cell-specific expression of the genetically encoded calcium indicator GCaMP3 in podocytes was recently established in our laboratory. This new approach allowed us to quantitatively visualize calcium elevations in podocytes during the development of glomerulosclerosis and kidney fibrosis, and their causative role in pathological changes in glomerular filtration, vascular permeability, and propagation of podocyte injury and pathology.

The paper describing this technical advance, podocyte calcium imaging in vivo is currently in press in the Journal of Clinical Investigation. In combination with serial MPM imaging of kidney structure and function, the novel Cre/lox-based transgenic approaches will help us in future work to specifically label, manipulate (ablate) cells in vivo in the intact kidney, knock out specific genes specifically in certain cell types (conditional knockout mice) and analyze their role in kidney function in health and disease and in renal vascular and glomerular remodeling. The use of future, ever-developing imaging techniques and approaches (e.g. long wavelength infrared lasers, extremely sensitive detectors, super-resolution nanoscopy) are expected to further push the limits of intravital, functional imaging of many renal cell types including the podocyte.

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