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Micropuncture of the Kidney: A Primer on Techniques

John N. Lorenz

10.1002/cphy.c110035

Source: Volume 2, Issue 1, January 2012

Published online: January 2012

Full Article on Wiley Online Library



Abstract

Techniques to evaluate renal function at the single nephron level have been instrumental and indispensible in furthering our understanding of the mammalian kidney. Techniques that were first introduce in the 1920s, and later refined in the 1950s and 1960s, permit sophisticated interrogation of glomerular filtration and hemodynamics, and tubular epithelial transport activity. Much of what we know about the physiology and pathophysiology of the kidney has been produced or, to some degree, confirmed by renal micropuncture. While micropuncture is perhaps not as widely employed as before, it remains an essential tool for comprehensive evaluation of kidney function, particularly in this age of genetically pliable experimental models. This review aims to provide a introduction to common methodologies and approaches used to conduct micropuncture experiments. Topics covered include instrumentation and equipment, pipet fabrication techniques, animal preparation, and experimental procedures for evaluating single nephron hemodynamics and tubular function. © 2012 American Physiological Society. Compr Physiol 2:621‐637, 2012.

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Figure 1. Figure 1.

Custom grinding station for beveling micropuncture pipets. Dissecting microscope is mounted horizontally so as to observe the grinding of the pipet tip at an oblique angle. The grinding wheel is a modified computer hard drive with the cover removed, and the disk is covered with a fine‐grit abrasive film [(A) 0.3 μm grit, Thomas Scientific]. The disk spins at a high rate (4,200 rpm) and is very flat. The pipet is mounted in a holder (B), and is lowered slowly onto the surface of the spinning disk using a fine micrometer (C). The pipet is illuminated from behind (D), and the wheel is lubricated with a constant water drip (E).
Figure 2. Figure 2.

Examples of different pipets made for renal micropuncture. The wax block pipet has a large opening to allow injection of the viscous wax, and to vent the upstream segment if necessary. Sample collection pipets are usually finely tapered and range in diameter from 5 to 15 μm; this pipet is suitable for a distal tubular collection. Pressure pipets have very fine tip diameters of 2 to 3 μm; the shank is generally finally tapered for a short distance and then steeply tapered to minimize electrical resistance. The filament on the lumen wall of the pressure pipet can be seen.
Figure 3. Figure 3.

Completed micropuncture setup for mouse, which is positioned on the heated table in a right lateral decubitus lie to expose the left flank, with head to the right and tail to the left. Left kidney is immobilized in a Lucite cup anchored from the rear, and is illuminated with a fiber optic light source from the front. The Leitz manipulator on the left is holding the marking pipet, and the Leitz manipulator on the right is holding the pressure pipet. The wax‐block/paraffin press can be seen behind the pressure pipet and is attached to a Narishige hydraulic manipulator with controller at the far lower right.
Figure 4. Figure 4.

Surface of a Munich‐Wistar rat kidney prepared for micropuncture measurement. The pipet, at right, has been inserted into a surface glomerulus (G), and dyed artificial tubular fluid has been injected and allowed to flow downstream; in this image, the dye fills part of the late proximal labyrinth, and the last surface loop of the proximal tubule is identified (LP). Adapted, with permission, from Vallon V. Micropuncturing the nephron. Pflügers Archiv European Journal of Physiology 458: 189‐201, 2009.
Figure 5. Figure 5.

Pipet configuration for fluid collections from the proximal and distal tubules, and from the peritubular capillary (star vessel). Pipets for fluid collections are back‐filled with stained oil, and a mobile oil block is inserted into the tubule before aspiration of tubular fluid begins in a timed collection. Fluid segments can be identified using a marking pipet to inject dyed fluid and allowing it to flow downstream. GFR can be measured by analyzing inulin concentration in fluid and plasma. The capillary pipette is filled with stained oil to prevent evaporation and to aid visualization, but a block is not inserted since the blood is not collected quantitatively. (Artwork in this and following figures, with permission, by W. H. Beierwaltes).
Figure 6. Figure 6.

Arrangement for efficient transfer and handling of micropuncture samples. On the right, the sampling pipet is mounted in a holder on a micromanipulator that allows adjustment in three axes. On the left, oil‐filled microcaps (1 μl) are positioned on a modified microscope stage to also allow for movement in three axes. Under a dissecting microscope, the pipet tip is advanced into the column of oil (here stained with Oil Red‐O dye), and pressure applied to expel the micropuncture sample into the oil column (see inset). This sample is approximately 30 nl.
Figure 7. Figure 7.

Pipet configuration for proximal tubule microperfusion. The upstream segment is blocked with wax, and an oil block is injected downstream for quantitative collection, while perfusing the intervening segment with artificial tubular fluid.
Figure 8. Figure 8.

Pipet configuration for analysis of tubuloglomerular feedback by orthograde perfusion of the loop of Henle. An early proximal segment is blocked with wax, and pressure is measured upstream while perfusing the loop of Henle at varying rates. As alternative to pressure measurement, early proximal flow rate or SNGFR can be measured.
Figure 9. Figure 9.

Pipet configuration for analysis of tubuloglomerular feedback by retrograde perfusion of the loop of Henle. The nephron is blocked at proximal and distal sites, and pressure is measured upstream as in Figure 8. The loop of Henle is perfused from the distal segment with fluid of varying ionic composition, directing flow proximally.
Figure 10. Figure 10.

Pipet configuration for analysis of tubuloglomerular feedback using the closed‐loop approach. Native tubular flow is not blocked and is evaluated videometrically by injecting fluorescent dye pulses upstream and interrogating transit time in a downstream segment. Changes in flow rate to the loop of Henle are imposed by injecting or aspirating fluid to or from a late proximal site.


Figure 1.

Custom grinding station for beveling micropuncture pipets. Dissecting microscope is mounted horizontally so as to observe the grinding of the pipet tip at an oblique angle. The grinding wheel is a modified computer hard drive with the cover removed, and the disk is covered with a fine‐grit abrasive film [(A) 0.3 μm grit, Thomas Scientific]. The disk spins at a high rate (4,200 rpm) and is very flat. The pipet is mounted in a holder (B), and is lowered slowly onto the surface of the spinning disk using a fine micrometer (C). The pipet is illuminated from behind (D), and the wheel is lubricated with a constant water drip (E).



Figure 2.

Examples of different pipets made for renal micropuncture. The wax block pipet has a large opening to allow injection of the viscous wax, and to vent the upstream segment if necessary. Sample collection pipets are usually finely tapered and range in diameter from 5 to 15 μm; this pipet is suitable for a distal tubular collection. Pressure pipets have very fine tip diameters of 2 to 3 μm; the shank is generally finally tapered for a short distance and then steeply tapered to minimize electrical resistance. The filament on the lumen wall of the pressure pipet can be seen.



Figure 3.

Completed micropuncture setup for mouse, which is positioned on the heated table in a right lateral decubitus lie to expose the left flank, with head to the right and tail to the left. Left kidney is immobilized in a Lucite cup anchored from the rear, and is illuminated with a fiber optic light source from the front. The Leitz manipulator on the left is holding the marking pipet, and the Leitz manipulator on the right is holding the pressure pipet. The wax‐block/paraffin press can be seen behind the pressure pipet and is attached to a Narishige hydraulic manipulator with controller at the far lower right.



Figure 4.

Surface of a Munich‐Wistar rat kidney prepared for micropuncture measurement. The pipet, at right, has been inserted into a surface glomerulus (G), and dyed artificial tubular fluid has been injected and allowed to flow downstream; in this image, the dye fills part of the late proximal labyrinth, and the last surface loop of the proximal tubule is identified (LP). Adapted, with permission, from Vallon V. Micropuncturing the nephron. Pflügers Archiv European Journal of Physiology 458: 189‐201, 2009.



Figure 5.

Pipet configuration for fluid collections from the proximal and distal tubules, and from the peritubular capillary (star vessel). Pipets for fluid collections are back‐filled with stained oil, and a mobile oil block is inserted into the tubule before aspiration of tubular fluid begins in a timed collection. Fluid segments can be identified using a marking pipet to inject dyed fluid and allowing it to flow downstream. GFR can be measured by analyzing inulin concentration in fluid and plasma. The capillary pipette is filled with stained oil to prevent evaporation and to aid visualization, but a block is not inserted since the blood is not collected quantitatively. (Artwork in this and following figures, with permission, by W. H. Beierwaltes).



Figure 6.

Arrangement for efficient transfer and handling of micropuncture samples. On the right, the sampling pipet is mounted in a holder on a micromanipulator that allows adjustment in three axes. On the left, oil‐filled microcaps (1 μl) are positioned on a modified microscope stage to also allow for movement in three axes. Under a dissecting microscope, the pipet tip is advanced into the column of oil (here stained with Oil Red‐O dye), and pressure applied to expel the micropuncture sample into the oil column (see inset). This sample is approximately 30 nl.



Figure 7.

Pipet configuration for proximal tubule microperfusion. The upstream segment is blocked with wax, and an oil block is injected downstream for quantitative collection, while perfusing the intervening segment with artificial tubular fluid.



Figure 8.

Pipet configuration for analysis of tubuloglomerular feedback by orthograde perfusion of the loop of Henle. An early proximal segment is blocked with wax, and pressure is measured upstream while perfusing the loop of Henle at varying rates. As alternative to pressure measurement, early proximal flow rate or SNGFR can be measured.



Figure 9.

Pipet configuration for analysis of tubuloglomerular feedback by retrograde perfusion of the loop of Henle. The nephron is blocked at proximal and distal sites, and pressure is measured upstream as in Figure 8. The loop of Henle is perfused from the distal segment with fluid of varying ionic composition, directing flow proximally.



Figure 10.

Pipet configuration for analysis of tubuloglomerular feedback using the closed‐loop approach. Native tubular flow is not blocked and is evaluated videometrically by injecting fluorescent dye pulses upstream and interrogating transit time in a downstream segment. Changes in flow rate to the loop of Henle are imposed by injecting or aspirating fluid to or from a late proximal site.

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Drafting of this article drew heavily on previous chapters on micropuncture in the Handbook of Physiology: Renal Physiology.  These previous chapters offer numerous insights regarding the methodology and evolution of this valuable experimental approach.

Gottschalk CW and Lassiter WE. Micropuncture methodology. In: Handbook of Physiology: Renal Physiology, edited by Orloff J and Berliner RW. Washington: American Physiological Society, 1973, p. 129-143.

Velazquez H and Wright FS. Renal micropuncture techniques. In: Handbook of Physiology: Renal Physiology, edited by Windhager EE. New York: Oxford University Press for the American Physiological Society, 1973, p. 249-269.

A more recent review discussing modern micropuncture techniques is also excellent.

Vallon V. Micropuncturing the nephron. Pflügers Archiv European Journal of Physiology 458: 189-201, 2009.

Supplementary Information

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Article Title: Micropuncture of the Kidney: A Primer on Techniques
 

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John N. Lorenz. Micropuncture of the Kidney: A Primer on Techniques. Compr Physiol 2012, 2: 621-637. doi: 10.1002/cphy.c110035