Identification of Mg-transporting renal tubules and cells by ion microscopy imaging of stable isotopes

Subhash Chandra1, George H. Morrison1, and Klaus W. Beyenbach2

1 Ion Microscopy Laboratory, Department of Chemistry, and 2 Section of Physiology, Cornell University, Ithaca, New York 14853

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Sites of renal Mg transport were identified in seawater killifish (Fundulus heteroclitus) using a Cameca model IMS-3f ion microscope. Killifish were given an intraperitoneal injection of the stable isotope 26Mg (99.5% enrichment) to stimulate and trace renal Mg excretion. We identified two sites of 26Mg transport in frozen freeze-dried cryosections of kidney: the proximal tubule, known to secrete Mg, and the collecting duct, heretofore not known to handle Mg. In epithelial cells of the proximal tubule, the punctate distribution of injected 26Mg suggests transcytotic excretion of Mg in bound form. In collecting ducts, a subpopulation of Mg/Ca-rich cells was identified with high accumulations of injected 26Mg. Here, the punctate distribution of 26Mg decreased from the apical to the basal region of the cells, revealing a transcytotic gradient of apparently bound Mg. Since proximal tubules of fish are implicated with Mg secretion, Mg/Ca-rich cells in the collecting duct may reabsorb Mg, thereby providing the usual two-step of renal regulation, now also for Mg.

renal handling of magnesium; secondary ion mass spectrometry; proximal tubule; collecting duct; transcytotic transport

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

EVEN THOUGH MAGNESIUM is gaining attention in a variety of pathophysiologies (1), its normal physiology is not well understood. Analytical difficulties can largely be blamed. The utility of the only radiotracer of magnesium, 28Mg, is limited due to its short half-life (21.3 h) and poor availability. Electron probe analysis provides measures of total Mg (free and bound) in dry or frozen-hydrated tissue with excellent spatial resolution in intracellular compartments as small as the endoplasmic reticulum (18, 20, 29). Fluorescent indicators and Mg2+-selective electrodes are used in the measurement of free Mg2+ in live preparations with the advantage of realtime studies of changing concentrations of intracellular free Mg2+. These may come about from shifts between bound and free Mg2+ inside the cell and/or transport of Mg across the cell membrane (6, 25). To distinguish between these two mechanisms, the measurement of both total and free Mg2+ in cells is necessary, a requirement that rarely is met in studies of the physiology of intracellular Mg.

In the present study, we introduce a new method for investigating Mg transport: imaging of the tracer stable isotope 26Mg by ion microscopy and using enriched 26Mg as a tracer of 24Mg. Ion microscopy is based on secondary ion mass spectrometry (SIMS), and the technique is capable of detecting any element from hydrogen to uranium via isotopic detection based on their mass-to-charge ratio with detection limits in the range of parts per million to parts per billion (7, 11, 22). Moreover, with a spatial resolution of 0.5 µm, the Cameca model IMS-3f ion microscope used in this study is capable of producing visual images of isotopic gradients in relation to tissue morphology. Since ion microscopy analysis is made by continuously eroding (sputtering) the sample in the z-axis direction, it is possible to image, sequentially, the intracellular distribution of many different isotopes from the same cell.

An added advantage of isotopic detection is the use of stable isotopes as tracers of transport (9, 10). The stable isotopes of magnesium are 24Mg, 25Mg, and 26Mg with natural abundances of 78.70%, 10.13%, and 11.17%, respectively. Thus stable 26Mg in high enrichment can be used as a tracer of 24Mg in ways similar to the use of radioactive 28Mg. This was done in the present study, where seawater killifish were given an intraperitoneal load of stable 26Mg isotope in quantities sufficient to stimulate renal Mg excretion. We subsequently traced the renal handling of 26Mg in frozen, freeze-dried cryosections of the kidney by SIMS ion microscopy. We identified two tubular sites based on tracer 26Mg localization: 1) the proximal tubule, which is known to net secrete Mg into the tubule lumen, and, surprisingly, 2) the collecting duct, which in fish had not been known to handle divalent cations. Seawater killifish were chosen for this study because the kidneys of seawater fish possess the most powerful Mg transport systems known. They are capable of generating urine Mg concentrations in excess of 100 mM from plasma concentrations little over 1 mM (3, 5, 19, 26).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Experimental animals and Mg loading. Killifish, Fundulus heteroclitus, weighing between 11 and 27 g, were purchased from the Marine Biological Laboratory (Woods Hole, MA), maintained in artificial sea water at 15°C (Instant Ocean, Eastlake, OH), and fed TetraMin staple fish food (Tetra Werke, Melle, Germany) every other day. Killifish were anesthetized in sea water containing 3-aminobenzoic acid methyl ester (MS-222, 1:10,000; Sigma, St. Louis, MO) and subsequently injected intraperitoneally with 100 µl of water containing 3 µmol of 26MgCl2 · 6H2O (99.5% 26Mg enrichment; Oak Ridge National Laboratory, TN). This 26Mg load is sufficient to raise plasma Mg concentrations from 1.2 mM to fatal concentrations over 8 mM. To prevent such a rapid rise in plasma Mg while presenting a substantial Mg challenge, the Mg load was injected into the peritoneum. The kidneys of killifish and other teleost fish are known to respond to even micromolar increases in peritubular (13, 14, 26) or plasma Mg concentrations (19). Killifish receiving sham injections of 100 µl water served as controls. The fish were then returned to sea water where they recovered from anesthesia 1-2 min later. Thirty or sixty minutes postinjection, the killifish were overanesthetized with MS-222 (10:10,000), decapitated, and pithed, and their kidneys were removed for cryopreservation (Institutional Animal Care and Use Committee, Cornell University, Protocol 86-37-96: Renal Electrolyte and Fluid Transport in Fish).

Preparation of tissue for SIMS analysis. Upon removal from the body, the kidney was immediately quick-frozen in supercooled isopentane. Frozen cryosections were cut to a thickness of 3-4 µm at -25 to -30°C using a cryostat (model HM 505E; Micron, Walldorf, Germany). The frozen cryosections were pressed onto chilled indium substrates to adhere them flat to an electrically conducting surface for ion microscopy analysis and then freeze-dried at -30°C overnight (28). The frozen, freeze-dried sections were subsequently coated with gold to enhance electrical conductivity for ion microscopy measurements. Cryosections adjacent to those used for ion microscopy were stained with hematoxylin and eosin for the morphological identification of renal tubules. According to Edwards and Schnitter (17), the nephron of killifish consists of a well-vascularized glomerulus, a short neck segment, a proximal tubule, and a collecting duct. The proximal tubule constitutes ~90% of the renal tubule (17). An intermediate segment and distal convolution are lacking.

Ion microscopy and image processing. A Cameca model IMS-3f ion microscope, operating with a 5.5 keV mass-filtered 250 nA O+2 primary ion beam with a focused diameter of ~75 µm, was used to monitor positive secondary ions. A contrast aperture of 60 µm in diameter was used in the imaging mode for the entire study. Isotopic images of masses 12, 23, 24, 26, 39, and 40 Da were recorded from freeze-dried kidney cryosections to reveal the intracellular distributions of 12C, 23Na, 24Mg, 26Mg, 39K, and 40Ca, respectively. Isotope images were digitized directly from the microchannel plate/fluorescent screen assembly of the ion microscope using a charge-coupled device (CCD) camera (Photometrics, Tucson, AZ) and digitized to 14 bits/pixel with a Photometrics camera controller. The image integration times for the 39K, 23Na, 24Mg, and 26Mg images were 0.4, 0.4, 60, and 120 s, respectively. Images of 12C and 40Ca were integrated for 120 s. High-mass resolution analyses were performed to confirm the purity of positive secondary ion signals from isotopes in the freeze-dried kidney cryosections. The image processing was performed using DIP Station (Hayden Image Processing Group) on an Apple Macintosh Quadra 840AV.

Estimates of concentrations of 24Mg, 26Mg, and 39K were obtained using relative sensitivity factors (RSF) of these isotopes referenced to the tissue matrix element 12C (2). The RSF for a particular element is a calibration factor used to convert the ratio of the analyte signals to the 12C signals from a region of interest into a dry weight concentration. The generation of concentration information from ion microscopy images required the following three steps, described previously by Ausserer et al. (2): 1) spatial registration of the analyte image and the 12C+ image to permit ratioing of signals from exactly corresponding areas, 2) calculation of the mean CCD pixel values per second for identical sample features of interest in the analyte image (CCDX) and the 12C+ reference image (CCDC), and 3) conversion of the signal ratio CCDX/CCDC to analyte dry weight isotopic concentration CX via the equation
C<SUB>X</SUB> = [CCD<SUB>X</SUB>/CCD<SUB>C</SUB>] ⋅ [1/MCP<SUB>X/C</SUB>] ⋅ [1/RSF<SUB>X/C</SUB>]
where MCPX/C, a constant, is the sensitivity of the microchannel plate for the analyte normalized to the sensitivity of the microchannel plate for carbon, and RSFX/C is the relative sensitivity factor of the analyte referenced to carbon. The MCPX/C terms and the RSF values of elements discussed here have been determined from homogenates of several cell culture cell lines, including normal rat kidney cells (2).

Isotope images of the cells and tubules in the renal tissue were quantified by using the above approach. As an example of the calculation of the dry weight concentration of 24Mg in cells of the proximal tubule of the control fish, the observed CCDX and CCDC for 24Mg were 58.90 and 7.66, respectively. The MCPX/C and RSFX/C for 24Mg have been determined to be 0.84 and 3.93 × 10-3 (2). Therefore
[<SUP>24</SUP>Mg] = (58.90/7.66)(1/0.84)(1/3.93 × 10<SUP>3</SUP>)
[<SUP>24</SUP>Mg] = 2.33 × 10<SUP>−3</SUP> or 2.33 parts per thousand dry weight
The wet weight molar concentration can be estimated from the dry weight value by assuming 85% cell water content. Thus the conversion of the 2.33 parts per thousand dry weight concentration of 24Mg into wet weight molar concentration (24Mgwwc, in M) is
<SUP>24</SUP>Mg<SUB>wwc</SUB> = (2.33)(0.15/24)
where 0.15 represents the dilution factor of 85% cell water content, and 24 is the atomic mass in daltons for 24Mg. Therefore
<SUP>24</SUP>Mg<SUB>wwc</SUB> = 0.01456 M or 14.56 mM
Quantitative concentrations of isotopes reported here should be considered as estimates rather than the absolute values because of two main reasons: 1) the RSFs we are applying here were generated from cell culture homogenates rather than from the kidney tissue itself, and 2) the assumed water content of 85% in cellular compartments is uncertain.

Imaging of transported 26Mg. The natural abundances of the 24Mg and 26Mg isotopes are 78.70% and 11.17%, respectively. Therefore, 24Mg is 7.046 times more abundant than 26Mg. Since the spatial distribution of both naturally abundant isotopes in the kidney is expected to be the same (see RESULTS), the contribution of naturally abundant 26Mg was corrected from the ion microscopy mass 26 image by dividing the mass 24 image by 7.046 and then digitally registering (pixel-by-pixel overlaying) and subtracting this image from the mass 26 image. This correction provided the distribution of mostly exogenous tracer 26Mg that had been injected into the fish and processed by the epithelial cells of the kidney.

Statistical analysis of the data. The data were analyzed with the Student's t-test for the significance (P < 0.05) of the difference between two sample means. They are expressed as means ± SD.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Ion microscopy imaging of control kidneys. Figure 1 shows six isotope images in the same renal cryosection prepared from a killifish sham injected with water (control). The isotope images reveal the cross-sectional shape and contour of two proximal tubules, a portion of collecting duct, and interrenal tissue. The level of brightness within an image is proportional to the total concentration (free plus bound) of the isotope. Therefore, visual comparisons of concentrations are valid within an image only.


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Fig. 1.   Ion microscopy images of 24Mg (a), 26Mg (b), 39K (c), 23Na (d), 40Ca (e), and 12C (f) from a renal cryosection of a control seawater killifish. PT, proximal tubule; CD, collecting duct; IT, interrenal tissue (endocrine, hematopoietic). Arrows, punctate distribution of 24Mg in epithelial cells of the proximal tubule.

Figure 1a reveals the distribution of 24Mg to be diffuse as well as punctate (arrows) in epithelial cells of the proximal tubule. In interrenal tissue containing lymphoid, hematopoietic, chromaffin, and endocrine cells, 24Mg is rather homogeneously distributed with intensities lower than those in epithelial cells of the proximal tubule.

The image of stable isotope 26Mg (Fig. 1b) is essentially a low-intensity duplicate of the image of 24Mg (Fig. 1a). The high-intensity punctate distribution of 26Mg (arrows) is found in the same spatial location as 24Mg (Fig. 1, a and b).

Images of 39K, 23Na, 40Ca, and 12C shown in Fig. 1, c-f, respectively, reveal distinct distributional differences from 24Mg or 26Mg (Fig. 1, a and b). The interrenal tissue shows speckles of high 39K distributed randomly (Fig. 1c). The cellular 23Na distribution is rather homogeneous throughout the tissue (Fig. 1d). The 40Ca image reveals a ring near the basal pole with concentrations higher than elsewhere in the cell. In addition, high-intensity saturated regions of 40Ca are observed in interrenal tissue and in some cells of the collecting duct (Fig. 1e). The 12C image represents the distribution of the major tissue matrix element carbon. As expected, carbon is distributed relatively homogeneously throughout the tissue matrix.

In control fish, estimates of total 24Mg concentration in epithelial cells of the proximal tubule that include diffuse and punctate distributions ranged from 12 to 20 mM with an average of 17.0 mM (n = 12). Concentrations of 26Mg ranged from 1.52 to 2.90 mM with an average of 2.55 ± 0.39 mM (n = 12). Accordingly, the isotope ratio 24Mg/26Mg measured in epithelial cells of proximal tubules is 6.67, which falls within 10% of 7.046, the ratio of the natural abundances of 24Mg and 26Mg. Thus, as expected, epithelial cells of the proximal tubule do not discriminate between these isotopes of Mg.

The mean concentrations of 39K and 23Na were 159.3 mM and 37.3 mM (n = 12) in epithelial cells of the proximal tubule (Table 1). Concentrations of electrolytes observed here are in general agreement with the electron probe observations of total K, Na, and Mg concentrations in proximal tubule cells of dogfish (18).

                              
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Table 1.   Estimates of intracellular isotopes in renal proximal tubule epithelial cells in control and 26Mg-loaded killifish

It should be noted that the presence of voids (empty spaces) in the lumen of tubules, peritubular space, and interstitial space reflects the lack of organic tissue matrix in these regions, as these regions are primarily occupied by fluids (tubular, peritubular, and interstitial). However, in freeze-dried cryosections, the thin powdery layer of these fluids quickly sputters away under the primary ion beam bombardment, leaving void spaces in areas where extracellular fluid is expected. This situation is similar to the previous ion microscopy observations of the faster sputtering and removal of the extracellular nutrient growth medium compared with cells in cultures (12). Isotope concentrations in extracellular compartments such as the tubule lumen, interstitial fluid, and plasma were not of interest. Instead, the cellular handling of Mg was of interest in the present study, and, therefore, what is important for intracellular measurements is that small intracellular regions occupying the organic matrix do not suffer from differential sputtering artifacts. Indeed, the relative homogeneity of the 12C signal in the epithelial cells of the proximal tubule (Fig. 1f) reflects the lack of significant differential sputtering artifacts, which is in agreement with the previous evaluation of differential sputtering of intracellular compartments of normal rat kidney cells grown in vitro (8).

Ion microscopy imaging of kidneys from fish loaded with stable 26Mg. Figure 2 shows six isotope images from the same region of renal cryosection prepared from a seawater killifish 30 min after the intraperitoneal injection of stable 26Mg tracer. Three partial cross sections of the renal proximal tubule occupy the field of view, allowing comparisons between different segments of the proximal tubule. Proximal tubules of marine fish, including killifish, are known to net secrete Mg into the tubule lumen (3-5, 13, 14, 18, 19, 26). Since killifish proximal tubules do not discriminate between the natural isotopes of Mg (see above), loading them with 26Mg is expected to stimulate transepithelial secretion of both 24Mg and 26Mg.


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Fig. 2.   Ion microscopy images of endogenous 24Mg (a), 26Mg-raw image (b, which is a sum of both endogenous and transported 26Mg-tracer), 26Mg-corrected image to reveal only the injected exogenous stable 26Mg-tracer (c), 40Ca (d), 39K (e), and 23Na (f) from a renal cryosection of a seawater killifish 30 min after intraperitoneal loading with stable 26Mg (99.5% enrichment) to stimulate renal Mg excretion. Three partial cross sections of proximal tubule occupy the field of view.

The image of 24Mg reveals heterogeneous intensities of 24Mg in different cross sections of the proximal tubule (Fig. 2a), suggesting different Mg transport capacities along the length of the tubule. The distribution of 24Mg is again diffuse and punctate, confirming the distribution observed in Fig. 1a.

The image of 26Mg in Fig. 2b reflects endogenous 26Mg already present in the kidney plus exogenous 26Mg that had been injected into the fish 30 min earlier to stimulate renal Mg excretion. Since endogenous 26Mg in Fig. 2b is equal to 1/7.046 of 24Mg in Fig. 2a, endogenous 26Mg can be subtracted, pixel by pixel, from the "26Mg-raw" image (Fig. 2b) to reveal exogenous 26Mg that had been injected into the peritoneum of the fish 30 min earlier (Fig. 2c). Accordingly, the subtraction reveals that exogenous 26Mg accumulates in proximal epithelial cells consistent with tubular secretion (Fig. 2c). Moreover, this subtraction reveals exogenous 26Mg that has entered the cell to be associated with punctate focal points of 0.5 to 3 µm in diameter, in contrast to the diffuse distribution of endogenous Mg (Figs. 1a and 2a).

Thirty minutes after peritoneal injection of 26Mg, the concentration of 24Mg in epithelial cells of the proximal tubule had significantly (P < 0.001) increased from 17.0 mM in control fish to 38.3 mM in injected fish (Table 1), indicating that the injected load of 26Mg stimulated secretory transport of both 24Mg and 26Mg. Correcting for endogenous 26Mg yields 1.2 mM, the concentration of exogenous 26Mg that entered proximal epithelial cells, consistent with transepithelial secretion (Table 1).

Figure 2d shows the distribution of 40Ca in proximal tubules. Intracellular 40Ca reveals a different distribution than 24Mg in general (Fig. 2a) and exogenous 26Mg in particular (Fig. 2c). Again, higher 40Ca intensities are observed at the basal pole of epithelial cells (Fig. 2d).

The images of 39K and 23Na in renal tubules of Mg-loaded fish are shown in Fig. 2, e and f, respectively. On average, the 39K and 23Na concentrations have remained unchanged, even though concentrations of Mg have increased significantly after 30 min postinjection of the tracer (Table 1).

Isotope measurements of proximal tubules reported above for 30 min postinjection of 26Mg tracer were qualitatively similar to those for 60 min postinjection. However, there were quantitative differences that reflect the kinetics expected from the renal excretion of the injected 26Mg load. At 60 min postinjection of 26Mg tracer, epithelial cells of the proximal tubule contained 14.6 mM 24Mg and 0.74 mM of exogenous 26Mg tracer (Table 1).

An interesting pattern of the handling of Mg by the cells of the proximal tubules emerges from Table 1. The endogenous 24Mg content of the epithelial cells of the proximal tubules approximately doubles after 30 min postinjection of the tracer, and it returns to its control value at 60 min postinjection of the tracer. Quantitatively, between 30 and 60 min postinjection of the tracer, there is a decrease of 62% of the endogenous 24Mg from the epithelial cells of the proximal tubules. In contrast, a modest 38% decrease in mean is observed for the exogenous 26Mg tracer between these time points (P < 0.05). This implies that a significant exchange of the tracer 26Mg is occurring with the cellular endogenous 24Mg as magnesium is secreted across the epithelium.

Mg in cells of the collecting duct. Figure 3 shows isotope images from a killifish kidney 30 min after the intraperitoneal injection of the stable isotope 26Mg. Both proximal tubules and collecting ducts are captured in the field of view. In isotope images of the kidney, proximal tubules had an outer diameter of 43.1 ± 6.6 µm (mean ± SD) and an inner diameter of 17.7 ± 3.6 µm in 27 proximal tubules. In contrast, outer diameter was 93.3 ± 29.7 µm and inner diameter was 42.5 ± 11.7 µm in 10 collecting ducts. Next to the obvious geometric differences in proximal tubules and collecting ducts that aid in their identification, large collecting ducts often revealed a peritubular sheath of fibrous connective tissue as does, for example, the collecting duct in the 39K image (Fig. 3a). Furthermore, there are obvious differences in the intensity of 39K signals from proximal tubules and collecting ducts (Fig. 3a), indicating significantly higher concentrations in epithelial cells of collecting ducts than in proximal tubules (P < 0.05, Tables 1 and 2). This pattern, however, is not followed for 23Na concentrations, as there is no significant difference in Na between the cells of proximal tubules and collecting ducts (Tables 1 and 2).


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Fig. 3.   Ion microscopy images of a renal cryosection from a seawater killifish 30 min after intraperitoneal loading with stable 26Mg (99.5% enrichment) to stimulate renal Mg excretion: 39K (a), 23Na (b), native 24Mg (c), corrected image of 26Mg (d, revealing mostly foreign 26Mg that was injected into the fish to stimulate renal transport of Mg), 40Ca (e), and 12C (f).

                              
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Table 2.   Estimates of intracellular isotopes in collecting duct epithelial cells of 26Mg-loaded (30 min ip) seawater killifish

The image of 24Mg (Fig. 3c) reveals remarkable heterogeneities in the distribution of Mg in cells of the collecting duct. In particular, there is a subpopulation of epithelial cells, usually appearing as clusters, with high concentrations of 24Mg (Fig. 2c; Table 2). The same clusters of cells are also seen in Fig. 2d from where native, endogenous 26Mg has been subtracted to reveal 26Mg that had been injected into the peritoneum of the fish to stimulate renal handling of Mg. The concentration of tracer 26Mg in cell clusters is approximately fivefold greater (P < 0.01) than in other epithelial cells of the collecting duct (Table 2). Furthermore, cell clusters with high Mg concentrations also revealed elevated 40Ca signals (Fig. 2e), with Ca concentrations ~1/20th the concentrations of Mg. In contrast, the images of 39K and 23Na do not reveal preferential accumulation of these ions in cells with high concentrations of Mg and Ca (Fig. 3, a and b; Table 2). In view of the high concentrations of Mg and Ca in this subpopulation of collecting duct cells, these cells are henceforth called Mg/Ca-rich cells. They make up 27 ± 6% of the epithelial cell population of collecting ducts, a relative abundance not unlike that of intercalated cells in mammalian collecting duct (21).

The 12C image (Fig. 3f) does not reveal enhancements of carbon signals in Mg/Ca-rich cells, supporting the conclusion that the isotopic distributions observed in Mg/Ca-rich cells are real and do not represent some unknown tissue matrix-dependent SIMS artifact.

Transcellular profile of Mg in collecting ducts. To further investigate the handling of divalent cations in Mg/Ca-rich cells of collecting ducts, we examined serial cryosections with the light microscope (Fig. 4) and the ion microscope (Fig. 5). The hematoxylin and eosin stain of the cryosection aided the identification of renal proximal tubules and collecting ducts by light microscopy (Fig. 4). A peritubular sheath of fibrous connective tissue is characteristic of large collecting ducts (Fig. 4). The boxed region in Fig. 4 represents the approximate region that was analyzed with the ion microscope in the next serial cryosection (Fig. 5). The Mg/Ca-rich cells are easily recognized by 1) intense signals of endogenous 24Mg and 40Ca emitting from these cells (Fig. 5, c and e) and 2) intense signals of exogenous 26Mg that had been injected into the peritoneum of the fish 30 min previously (Fig. 5d). Once again, Mg/Ca-rich cells do not show elevated levels of 39K (Fig. 5a) or 23Na (Fig. 5b), ruling out the artifactual precipitation of ions in cells enriched with Ca and Mg. Furthermore, the image of 12C is rather homogenous for tubular elements and does not reveal enhancements of carbon signals in the Mg/Ca-rich cells, which is indicative of no major SIMS artifacts (Fig. 5f).


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Fig. 4.   Optical micrograph of a renal cryosection stained with hematoxylin and eosin from a seawater killifish 30 min after intraperitoneal loading with stable 26Mg (99.5% enrichment) to stimulate renal Mg excretion. Boxed region illustrates the region examined in detail in an adjacent frozen freeze-dried cryosection with the ion microscope (Fig. 5). BV, blood vessel; FCT, fibrous connective tissue; RS, renal sinus.


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Fig. 5.   Ion microscopy images of part of a large collecting duct (boxed region of Fig. 4) from a seawater killifish 30 min after intraperitoneal loading with stable 26Mg (99.5% enrichment) to stimulate renal Mg excretion: 39K (a), 23Na (b), native 24Mg (c), corrected image of 26Mg (d, revealing mostly foreign 26Mg that was injected into the fish to stimulate renal transport of Mg), 40Ca (e) and 12C (f).

Since there is only one single layer of tall columnar epithelial cells with basal nuclei in collecting ducts (Fig. 4), the cryosection reveals transcellular Mg and Ca gradients with concentrations highest at the apical pole and lowest at the basal pole of epithelial cells (Fig. 5, c-e). Examination of the distribution of injected, exogenous 26Mg (Fig. 5d) shows that the transcellular gradient of transported 26Mg consists of punctate focal points, suggesting a gradient of bound Mg.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Advantages and limitations of ion microscopy. Multi-isotopic detection with high sensitivity and the capability of subcellular analysis via imaging of several isotopes from the same cell make ion microscopy a powerful technique for ion transport studies in both in vitro (9) and animal models (10). Because of the expensive instrumentation of ion microscopy and its limited availability for biological research, the technique remains grossly underexplored in biology. The present study is the first physiological application of 26Mg stable isotope for understanding the renal structures implicated with Mg handling. The technique is not suitable for live cell analysis due to high-vacuum requirements. A careful sample preparation is required to fix the specimen and immobilize diffusible species in their native state for ion microscopy analysis. The detection system of the ion microscope cannot discriminate between the free and the bound forms of an isotope, and, therefore, isotope images represent the distribution of total (both free and bound forms) concentration of the isotope.

The application of ion microscopy has provided new insights into sites and mechanisms of renal Mg transport from images of stable isotopes. We observed evidence consistent with transcellular transport of Mg across renal epithelial cells, and we discovered a new site of Mg transport in fish, the collecting duct.

Secretion of Mg by renal proximal tubules. Proximal tubules of seawater fish in general (19) and killifish in particular (13) are known to net secrete Mg. Since Mg secretion across fish proximal tubules contributes more than 90% of the Mg excreted in the final urine (19), the Mg images of the proximal tubule taken in this study largely reflect transepithelial secretion of Mg expected after challenging the fish with a Mg load. We made the following observations. First, in the primary segment of Mg transport in the fish kidney, i.e., the proximal tubule, Mg transport takes a transcellular route (Fig. 2c). A paracellular route, as in the mammalian thick ascending limb (16), is unlikely in view of the large transepithelial electrochemical Mg potential generated by secretory Mg transport (5). Second, Mg transport capacity changes with length along the proximal tubule of killifish, consistent with known functional heterogeneities along the length of the vertebrate nephron (Fig. 2). However, at any level of proximal tubule, the epithelial cells of the tubule appear to share the same transport capacity (Fig. 2c). Third, the intracellular distribution of the exogenous 26Mg load, which is expected to be removed from the renal circulation by tubular secretion, is punctate rather than diffuse (Fig. 2c). Furthermore, 30 min after giving the peritoneal load of stable 26Mg tracer, the average concentration of the 26Mg tracer in the regions of punctate distribution is 1.2 mM (Table 1), which is in excess of typical concentrations of free Mg2+ (0.5 to 1 mM) measured in eukaryotic cytoplasm (15, 25, 27). Since the concentration of transported magnesium (26Mg and 24Mg) must be much greater than 1.2 mM, it is unlikely that magnesium is moved through the cell as ionic Mg2+ free in the cytoplasm. Instead, Mg is probably transported through the cell bound to some as yet unknown ligand. Binding to a ligand would be expected to give rise to the punctate intracellular distribution of Mg (Figs. 1, 2) and allow the transport of large quantities of Mg through the cell without raising cytoplasmic free Mg2+ to concentrations that might interfere with normal cell functions. Using electron probe X-ray microanalysis, Hentschel and Zierold (18) made observations in shark proximal tubules also consistent with transepithelial transport of Mg in bound form. Apical vacuoles in epithelial cells of the proximal tubule were found to have Mg concentrations as high as 229 mmol/kgH2O that clearly cannot be free Mg (18). Hentschel and Zierold (18) suggest that these high vacuolar Mg concentrations reflect transepithelial transport of Mg in bound form. Although we did not observe Mg-rich vacuoles in the apical region of killifish proximal tubules using ion microscopy, we nevertheless find evidence for the punctate distribution of exogenous 26Mg suggestive of transport in bound form.

Organic anions, including fluorescent dyes, which proximal tubules of killifish secrete in addition to Mg, also present a punctate distribution in proximal epithelial cells, which has been interpreted to reflect compartmentalization in vesicles (23, 24). This vesicular compartmentalization is considered to be important in transepithelial secretion, because intracellular movement of fluorescein-loaded vesicles and transepithelial secretion of organic anions are inhibited in the presence of nocodazole, a specific disrupter of microtubules (23, 24). Whether the punctate distribution of transported 26Mg also reflects cytoplasmic transport in vesicular compartments remains to be determined.

The evidence for transcellular secretion in killifish proximal tubules raises the question of why transcellular mechanisms of Mg transport have not received much attention in mammalian kidneys. One reason is that the major site of Mg transport in the mammalian kidney is the thick ascending limb of the loop of Henle (15, 16). Another reason is the usual focus on intracellular ionized Mg2+ in studies of mammalian systems. Yet the large number of reports in which cytosolic free Mg2+ has been measured, despite some discrepancies in the absolute value, all concur in finding small variations in concentration over long periods of time, even under stimulatory conditions (27). This should hardly be surprising, since more than 90% of intracellular Mg is bound in most eukaryotic cells (27). Thus free Mg2+ concentrations are not likely to change a great deal with rates of transcellular Mg transport in the kidney, especially not in the case of the apparent compartmentalization of transported Mg observed in killifish proximal tubules. Accordingly, the evaluation of bound Mg (or total Mg) is as important as the measurement of free Mg2+ in studies of renal Mg transport and its regulation.

A role for collecting ducts in renal homeostasis of divalent cations? Our discovery of a group of Mg/Ca-rich cells in collecting ducts labeled with high concentrations of tracer 26Mg after an intraperitoneal 26Mg load was surprising because this portion of the fish nephron was not thought to be engaged in transepithelial transport of divalent cations. Furthermore, collecting ducts with heavy investments of peritubular connective tissue (Figs. 4 and 5) are thought to serve primarily as conduits to the ureter and urinary bladder and to have little transport activity. Mg/Ca-rich cells exhibited transcellular 26Mg gradients with concentrations highest at the apical pole of the cell near the tubule lumen. Transcellular gradients, as well as high concentrations of divalent cations (Table 2), suggest transport through the cytoplasm in sequestered, compartmentalized form. Whether Mg or Ca is reabsorbed from or secreted into the tubule lumen of collecting ducts cannot be discerned from the present images (Figs. 3 and 5) and requires further exploration. As a first hypothesis, we suggest that Mg is reabsorbed from the tubule lumen for the following reason. In fish adapted to freshwater, the kidney must net reabsorb filtered Mg if the fish is to remain in Mg balance. Furthermore, conventional wisdom has it that proximal tubules change from net secretion of Mg in seawater to net reabsorption in freshwater (19). However, this is not so, at least not in isolated proximal tubules of killifish (13). Proximal tubules isolated from killifish adapted to freshwater surprisingly continue to secrete Mg (13). Thus, in freshwater killifish, Mg is delivered to the tubule lumen by glomerular filtration and tubular secretion. Where then is the site of reabsorption that is required for renal Mg balance? Indeed, tubular sites for Mg reabsorption in fish kidneys have heretofore been unknown. Using the ion microscope, in the present study, we believe we have identified a potential site for tubular Mg reabsorption, a group of specialized cells in the collecting duct. Reabsorption of Mg here would equip the kidney of fish with the usual two-step of bidirectional renal regulation, now also for Mg, i.e., proximal delivery (filtration and secretion) and distal reabsorption.

    ACKNOWLEDGEMENTS

We gratefully acknowledge C. A. Smith for cryosectioning, Dr. C. Wahl for morphological evaluations of cryosections, and D. R. Lorey II for digital image processing and for the critical review of the manuscript. P. Chandra is gratefully acknowledged for statistical analysis of the data.

    FOOTNOTES

This work was supported by the National Institutes of General Medical Sciences Grant GM-24314 (G. H. Morrison and S. Chandra) and National Science Foundation Grant SF-IBN-9220464 (K. W. Beyenbach).

Address for reprint requests: K. W. Beyenbach, Section Physiology, VRT 926, Cornell Univ., Ithaca, NY 14853.

Received 31 January 1997; accepted in final form 7 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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