Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy
October 11th, 2018
We present use of 2-photon microscopy to place a micropipette within Bowman's urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.
This technique can help answer key questions on single-nephron physiology, including the glomerular filtration rates of systemic proteins and metabolites and their contributions to tubular physiology. The main advantage of this micropuncture technique is that it allows access to Bowman's space and the cortical nephron in all mice. Generally, individuals new to this method would struggle because it requires correctly executed preparation steps.
After confirming a lack of response to toe pinch, apply ointment to the eyes and use tape to immobilize the extremities of a 20 to 25 gram adult mouse. Use a depilatory cream to remove all of the hair on the left side of the animal, and use the spleen to locate the left kidney through the skin on the dorsal and caudal side of the spleen. Make a 0.5 centimeter incision in the skin, followed by a smaller incision in the peritoneum just large enough for the kidney to be pushed through.
Extrude the kidney with gentle pressure and place a polysiloxane kidney stabilizer form around the tissue. Line the kidney up with the spacer such that the lateralmost surface of the kidney extends beyond the stabilizer by about one millimeter, and fix the kidney to the form with cyanoacrylate adhesive. Glue a head plate to the stabilizer form and mount the head plate to mounting bars on the base plate.
Next, fill the well in the polysiloxane support with one percent agarose solution and hold a 10 millimeter cover slip on top of the form until the agarose is firm. Seal the cover slip to the head plate with glue and create a ring around the cover slip with dental cement. Then inject 100 to 150 microliters of FITC-Dextran retro-orbitally and quickly move the mouse and fixation plate to the two-photon microscope stage.
After manually focusing on the kidney surface, switch to the non-scanning two-photon mode and explore the imaging window to locate a target glomerulus that is greater than 30 micrometers below the cover slip and less than 400 micrometers from the lateral kidney capsule. Record the lateral and vertical distance to the target glomerulus. Then record the x, y, and z stage coordinates for the glomerulus.
And lastly, raise the objective focal point about one millimeter into the water column without changing the x and y stage coordinates. Drive the pipette tip into the water column and turn on the DAPI excitation. Move the pipette in the x and y dimensions to the point of maximal fluorescence of the tip.
This will be the center of the objective. Change the x citation setting to red fluorescent protein and visualize the pipette with the ocular to allow precise centering in the ocular view. Switch back to two-photon to find the pipette under the live two-photon view and place the tip precisely in the center of the image, this is the registration position.
Then save an image of the pipette and register the stage and the micropipette controller coordinates. Remove the pipette from the water column in the x-axis without moving the y and z-axis and move the pipette z to the target glomerulus z-coordinate and the edge of the kidney. Note the stage x and calculate the kidney edge pipette x using the offset from the registration stage x.
Increase the stage x to move the stage toward the pipette until the edge of the kidney is far to the left of the screen, while remaining visible. Quickly advance the pipette to about 100 micrometers away from the kidney edge pipette X has just calculated. Increase the red gain and begin advancing the pipette tip slowly to the kidney edge under live two-photon imaging while monitoring the red pixel histogram.
Drive the pipette in the x-axis slowly to the glomerulus target pipette x, keeping an eye on the stage x. Upon reaching the glomerulus, document the position with a z-stack. With the pipette in position, set the micropump to inject 100 nanoliters of perfluorodecalin over two minutes.
To ensure patency of the pipette, and to reduce confounding from pipette plugging during entry. After four to six minutes of filtration, set the micropump to aspirate up to 300 nanoliters at a rate of up to 50 nanoliters per minute. This beautiful, near surface glomerulus demonstrates favorable imaging due to the surface position of the glomerulus at 20 micrometers below the renal capsule.
The glomerulus is too close to the surface for microinjection however, as the pipette would hit the cover slip. These glomeruli are optimally positioned with the lateral edges 250 micrometers to the right and appearing less sharp because of the refraction caused by their 70 micrometer depth from the capsule. Both factors that make the glomeruli accessible.
In this typical renal entry image, a mean intensity projection from a z-stack with orthogonal views reveals the pipette tip within Bowman's space, note the red pipette tip spectral artifact from the extremely bright fluorescence of the quantum dots arranged on the conical section of the tip. Here, a volume rendering from a z-stack acquired after positioning a pipette in Bowman's space, shows the pipette tip within the space abutting the capillary tuft. If a pipette with a too large opening is used, the renal capsule can break causing subcapsular bleeding and entry of the blood into the pipette lumen.
Using this method, 17 proteins, primarily of low molecular weight, have been identified from a minimum of two unique peptides per protein. Coupled with this method, other methods such as maspectroscopy, ion sensitive electrode measurements, and fluorescent antibody injection can be used to answer questions about the role of luminal solutes in tubular and glomerular physiology.
Overview
This study investigates single-nephron physiology using a unique 2-photon microscopy technique to access Bowman's space in mice. The findings demonstrate the potential of this approach to measure glomerular filtration rates of proteins and metabolites, offering insights into tubular physiology.
Key Study Components
Research Area
- Single-nephron physiology
- Renal physiology
- Glomerular filtration
Background
- Understanding kidney function at a single-nephron level is critical for physiological research.
- Traditional microscopy has limitations in micropuncture techniques.
- 2-photon microscopy provides improved imaging capabilities.
Methods Used
- Micropuncture technique with 2-photon microscopy
- Mouse model system
- Fluorescence imaging with FITC-Dextran and DAPI
Main Results
- The technique allows for measurement of glomerular filtration rates in real time.
- Mitigated common issues like pipette plugging during entry.
- Identified 17 proteins from the overflow fluid sampled.
Conclusions
- This method enhances the ability to study renal physiology in live animals.
- It opens avenues for further research into the role of solutes in renal function.
Frequently Asked Questions
Executive Industry Relevance
This technique enables direct access to Bowman's space in mice, overcoming limitations of conventional micropuncture and 2-photon imaging by allowing single-nephron-level interventions and measurements. It supports target validation and mechanistic de-risking in renal physiology research by providing quantitative aspirate analysis from glomerular filtrate. The method enhances predictive confidence in preclinical models by enabling real-time measurement of pressure, charge, flow, and molecular composition within the nephron.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of glomerular filtration rates of systemic proteins and metabolites to validate therapeutic targets.
- Operational Value: Provides access to Bowman's space and cortical nephron in all mice, supporting reproducible target engagement studies.
Screening & Assay Development
- Scientific Value: Facilitates preparation of validated biological systems for downstream assays by enabling recovery of filtrate from Bowman's space and nephron segments.
- Operational Value: Supports assay standardization through quantitative liquid chromatography/mass spectrometry readouts of aspirated proteins and metabolites.
Translational & Preclinical Research
- Scientific Value: Advances disease-relevant system studies by allowing measurement of luminal solutes' role in tubular and glomerular physiology.
- Operational Value: Ensures continuity from discovery through preclinical validation by enabling repeated access to single-nephron physiology in live animals.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from target validation to preclinical research by providing direct nephron access for mechanistic studies and biomarker-aligned readouts.
- Discovery Biology: Supports hypothesis testing and pathway clarification by enabling micropuncture of Bowman's space to assess filtration and reabsorption processes.
- Screening: Enhances assay readiness and reproducibility through stereotactic guidance and quantum dot-coated pipette visualization for precise glomerulus targeting.
- Analytics: Delivers quantitative measurements of pressure, charge, flow, and molecular composition (e.g., 17 low molecular weight proteins via LC/MS) to compare experimental conditions.
- Translational Research: Connects to preclinical continuity by allowing longitudinal studies of solute handling in disease models.
- Enterprise Reuse: Establishes a reusable platform for renal physiologic investigation across multiple projects due to its adaptability to various readouts (ion-sensitive electrodes, fluorescent antibodies, mass spectrometry).
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by reducing mechanistic ambiguity in single-nephron physiology through direct aspirate analysis.
- Operational Value: Improves standardization and scalability via stereotactic kidney stabilization and imaging-guided pipette navigation.
- Strategic Value: Enhances go/no-go decisions by providing early, nephron-level data on protein and metabolite filtration, reducing late-stage biological risk.
- Portfolio Impact: Enables risk-adjusted prioritization of renal targets based on quantitative flux and composition data from Bowman's space.
Implementation Considerations
- Requires expertise in renal surgery, 2-photon microscopy, and micropipette handling.
- Depends on two-photon microscope, quantum dot-coated pipettes, stereotactic apparatus, and agarose stabilization systems.
- Necessitates cross-team standardization of surgical preparation, imaging parameters, and aspirate collection protocols.
- Involves adaptation considerations for different mouse strains and disease models affecting glomerular depth and accessibility.
- Limited by imaging depth (~100 μm below capsule) and potential vascular trauma from pipette insertion, which may cause subcapsular bleeding if not carefully managed.
