Renal Micropuncture Techniques
This essay will critically review some of the major uses of renal micropuncture techniques, focusing in particular on the stationary method, free-flow micropuncture and renal microperfusion, the latter including both in vivo and in vitro methods. The essay will conclude with a short review of other uses of micropuncture. Wearn and Richards (1924) were the first people to use micropuncture successfully on the kidney. They successfully inserted a micropipette into the glomerulus of a single amphibian nephron and their results provided conclusive evidence that ultrafiltration of the blood occurred at this site. Walker, Bott, Oliver, and MacDowell (1941) later extended the technique to the proximal and distal convoluted tubules. They demonstrated isosmotic reabsorption in the proximal convoluted tubule and an above plasma concentration of chloride ions. This latter result implied that reabsorption in the kidney was not a simple passive affair. Gottschalk and Mylle (1958) micropuncture the loop of Henle providing conclusive evidence for Kuhn’s theoretical countercurrent multiplier system, and Sakai, Jamison, and Berliner (1965) micropuncture the collecting duct. A problem with micropuncture techniques is that they require exceptionally fine and skilled micromanipulation to avoid puncturing the other side of the tubule. This is why the original experiments, up until Gottschalk (1956), were done on amphibian nephrons. The problem with mammalian nephrons was that they were too small, had an absence of glomeruli on the surface of the kidney, and tended to move due to respiratory movements. These problems were overcome by a combination of two improvements. Alexander and Nastuk (1953) developed the micropipette puller, which mechanically “pulled” capillary tubes overheat, instead of by hand. This produced micropipettes with much smaller diameters (8×10
Gottschalk (1956) developed a method whereby the kidney is isolated in a double cup, thus decreasing any movements due to the animal’s respiration. All the above experiments used the free-flow micropuncture technique. A micropipette is inserted through the tubule wall and fluid enters the pipette spontaneously due to intratubular hydrostatic pressure. One problem with this technique is that if too much fluid is withdrawn from the tubule, either by negative pressure or simply by capillarity, a tubular flow will change affecting tubular pressure, decreasing transit time and increasing the glomerular filtration rate. Analytical data from such samples is then of little use. To avoid this firstly, fluid should be allowed to enter only very slowly (therefore a little positive counter pressure should be applied to the pipette). Secondly, tubular diameter should be closely monitored to avoid collapse. Thirdly the sample size should not be greater than 10-20% of the total volume flow past the puncture site. Finally, intratubular pressure should be measured before and during sampling and kept constant. Later experiments blocked the nephron with a lump of mercury allowing all the fluid passing the puncture site to be collected. This allowed the glomerular filtration rates of single nephrons to be calculated. Mercury was originally used to block the nephron, although a high pressure was required to inject it, and problems such as filling the whole nephron with mercury, or breaking pipette seals often arose. Kennedy’s method of staining mineral oil provided a superior substitute. The next technique to be developed, by Windhager and Schatzmann (1953), and later improved upon by Gertz (1963), was the stationary perfusion, also known as the “split-drop” technique. A droplet of oil was injected into the tubule, followed by the test solution which split the oil drop. Time lapse photography was used to measure the changes in the droplet length and thus demonstrate reabsorption or secretion along discrete tubule lengths.
-6m approx.) allowing more accurate micromanipulation.
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