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1 Department of Anatomy, University College London, UK2 Centre for Nephrology and Department of Physiology, Royal Free & University College Medical School, London, UK3 Department of Nephrology, Second University of Naples, Italy
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Abstract |
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(Received 7 August 2003;
accepted after revision 15 December 2003)
Corresponding author D.G Shirley: Centre for Nephrology, Institute of Urology and Nephrology, Middlesex Hospital, Mortimer Street, London, W1W 7EY. E-mail: david.shirley{at}ucl.ac.uk
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Introduction |
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Despite the problems, several attempts have been made to adapt confocal microscopy for use in vivo (Petroll et al. 1994; Andrews, 1994); and in this context we have previously reported a confocal system for the study of renal tubules in anaesthetized rats (Boyde et al. 1998). The present paper describes an improved video-rate scanning confocal microscopy (VRSCM) system that provides enhanced structural and functional imaging at high temporal resolution, thus allowing visualization of morphological changes in response to acute physiological events. This innovation was made possible through modification of a commercial video-rate laser scanning confocal microscope to operate with a 633 nm HeNe laser. Using red light minimizes irradiation damage, reduces light scattering, and improves the depth to which we can image live tissues. Other significant modifications include an improved digital acquisition system. In addition, the use of a specially designed operating table and kidney dish allowed a much more stable animal preparation than was previously possible.
The aim of the present study was to assess the efficacy of this system in the investigation of dynamic changes in nephron structure. This was done by administering three classes of diuretics with well-defined tubular actions, and monitoring in real time the consequent changes in proximal and/or distal tubular diameter.
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Methods |
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200 g) were anaesthetized with sodium thiopentone (Byk Gulden, Konstanz, Germany; 100 mg kg–1, I.P.) and placed on a heated blanket so as to maintain rectal temperature at 37°C. The right jugular vein was cannulated and 0.9% NaCl solution infused at 2 ml h–1; a tracheotomy was performed; and the bladder was catheterized. The rat was then placed on a specially designed platform that had the double function of being a thermostatically controlled heated dissection table and a microscopy stage. The left kidney was exposed by a lateral incision, freed of perirenal fat, placed on to a specially designed Perspex dish that suppressed transmission of breathing movements to the kidney while not interfering with the renal blood supply, and kept moist with saline. Surface tubules were observed using a video-rate scanning confocal microscope (Odyssey, Noran Inc., Middleton, WI, USA) retro-fitted with a 633 nm HeNe red laser and seated on an antivibration table. It was used in reflection mode. A further procedure for dampening down kidney movements was the use of an exact thickness of ‘coverslip’ glass between the objective and the kidney capsule: a sandwich of 410 µm of glass, made of two coverslips with this net thickness, was placed between a Zeiss 40/1.0 oil immersion objective lens and the kidney capsule, held in place with immersion oil. In most cases, this gave a very stable field of view and allowed high resolution optical sectioning of the living tissue at 10–40 µm below the renal capsule.
Resolving power was virtually unaffected by kidney movements, because of the high temporal resolution of the scanning system. Measurements were made from single fields of video recordings, covering a time interval of 0.02 s. The amplitude of any respiration-derived motion was generally less than 2 µm, and tubules have internal diameters of 15–25 µm. Moreover, we were able to select fields in which there was hardly any motion. Blood pulsation did not give rise to detectable motion, because of the stabilization given by the lens/coverslips arrangement contacting the kidney surface.
Fields for study were selected on the basis that they contained both proximal and distal tubular segments, together with peritubular capillaries. Red blood cells could be easily identified because they are highly reflective; white cells were recognized by their slower motion and their tendency to adhere to the capillary wall and occasionally to move against the flow. Proximal tubules could be recognized by their intensely scattering brush borders.
Images were recorded on videotape, with accompanying audio-commentary from the operator. Measurements were made from representative fields showing both proximal and distal tubular segments. One group of rats (n= 3) was given an acute intravenous injection of the osmotic diuretic mannitol (500 mg (kg body wt)–1); a second group (n= 4) was given an acute intravenous injection of the loop diuretic frusemide (2 mg (kg body wt)–1); and a third group (n= 3) was given an acute intravenous injection of the thiazide diuretic hydrochlorothiazide (25 mg (kg body wt)–1). Selected image frames were grabbed from video recordings during baseline conditions and at 5, 10, 15 and 20 min after the administration of diuretic.
The software used for digital acquisition and improvement, and for metric and statistical off-line analysis of grabbed sequences of frames, was Lucida Analyse (Kinetic Imaging Ltd, Cairn Research, Faversham, Kent). The internal diameter of proximal and distal tubular segments was measured on each picture at three different points along the segment and the mean calculated. These measurements were facilitated by a sharp reflective gradient at the luminal border. The percentage change of averaged diameter against baseline was then calculated for each class of diuretics tested. Images were digitized to 512 pixels wide. With an image field width of 150 µm, this gave a pixel width of 0.3 µm, which is in close agreement with the calculated lateral resolution. A change in luminal diameter of 1 µm could be reliably detected.
At the end of each experiment, the rat was killed with an overdose of anaesthetic.
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Results |
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The acute intravenous administration of mannitol (500 mg (kg body wt)–1) caused rapid increases in the diameter of both proximal and distal tubules (Fig. 1). Proximal tubular diameter had already increased maximally (by an average of 15%) at 5 min; thereafter, it fell towards control values. Distal tubular diameter increased more gradually, reaching a 55% increase at 20 min (Fig. 2).
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Figure 3 shows an example of an experiment in which a single dose of frusemide (2 mg (kg body wt)–1) was given intravenously. Figure 4 shows the percentage changes in proximal and distal tubular diameters. Within 5 min of frusemide administration, an increase in distal tubular diameter was clearly visible; by 15 min it was maximal (average increase 95%). However, at no time was an increase seen in proximal tubular diameter.
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As with frusemide, the effect of intravenous hydrochlorothiazide (25 mg (kg body wt)–1) on tubular diameter was restricted to the distal tubules. Figure 5 shows the typical pattern of dilation of a distal tubular segment which can easily be recognized in the centre of a field that includes a number of unaffected proximal segments. By 20 min after administration of the drug, distal tubular diameter had increased by an average of 52% (Fig. 6).
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Discussion |
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The Odyssey confocal system described in the present paper was able to provide clear images of dynamic changes in nephron diameter in response to the three classes of diuretics used. The principal site of action of mannitol is the proximal tubule (Lang, 1987), where it remains in the lumen as an unreabsorbed osmolyte and thereby reduces net fluid reabsorption. Thus, the rapid dilation of the proximal convoluted tubule observed here was to be expected. In addition to its proximal action, mannitol reduces reabsorption in the loop of Henle (Lang, 1987). It is believed that this action follows from an increase in medullary blood flow and consequent ‘washout’ of the medullary osmotic gradient (Okusa & Ellison, 2000). Such an effect would reduce water reabsorption from the thin descending limb and limit sodium reabsorption from the ascending limb (the latter being gradient-limited). The result is an increased delivery of both sodium and water into the distal tubule (Buerkert et al. 1981), which accords with our observation of a delayed distal tubular dilation after administration of this diuretic. Furthermore, there is evidence that mannitol can impair sodium and water reabsorption in the late distal tubule/collecting duct (Leyssac et al. 2000), which would also contribute to dilation in the distal tubule.
Loop diuretics, such as frusemide and bumetanide, have their principal site of action in the thick ascending limb of Henle, where they inhibit the apical Na+-K+-2Cl– cotransporter by binding to one of the Cl– sites (Okusa & Ellison, 2000). The resulting abolition of the so-called ‘single effect’ (NaCl reabsorption without water reabsorption) leads to rapid dissipation of the medullary osmotic gradient, with the consequence that water reabsorption from the thin descending limb is abolished, and large volumes of virtually isotonic fluid are therefore delivered to the distal tubule. At high doses, loop diuretics also inhibit proximal tubular reabsorption (Greenwood et al. 1990; Walter & Shirley, 1991). However, micropuncture studies have shown that the dose we employed here (2 mg (kg body wt)–1) is too low to affect the proximal tubule (Shirley et al. 1992). Thus, in agreement with our findings, no change in proximal tubular diameter would have been expected, whereas the marked increase in fluid delivery into the distal tubule should bring about significant dilation at this site. Indeed, distal tubular diameter was found to increase almost two-fold.
Unlike some thiazide diuretics, hydrochlorothiazide (as used in the present study) has very little carbonic anhydrase inhibitory activity and consequently is virtually without effect on the proximal tubule (Velázquez, 1987), which accords with the unchanged proximal tubular diameter recorded here. Hydrochlorothiazide's site of action is primarily the distal convoluted tubule (early distal tubule), where it interferes with the apical Na+-Cl– cotransporter (by binding to the Cl– site) and thereby reduces NaCl reabsorption (Okusa & Ellison, 2000). The resulting failure to lower intraluminal solute concentration will reduce osmotic water reabsorption in the late distal tubule, which will account for our observation of distal tubular dilation, albeit less marked than that seen with frusemide.
In summary, we conclude that the VRSCM system used in the present study is capable of detecting and quantifying predicted dynamic changes in renal tubular diameter. The method holds promise for future investigations of real-time changes in tubular morphology in vivo.
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Supplementary material |
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References |
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Okusa MD & Ellison DH (2000). Physiology and pathophysiology of diuretic action. In The Kidney. Physiology and Pathophysiology, ed. Seldin DW and Giebisch G, pp. 2877–2922. Lippincott Williams & Wilkins, Philadelphia.
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