JOURNAL OF MORPHOLOGY 206:1-11(1990)
Cellular Basis of an Avian Countercurrent Multiplier System MARC POST, LEWIS GREENWALD, AND DAVID STETSON Department of Zoology, The Ohio State Uniuersity Columbus, Ohio 43210
ABSTRACT A kidney from the budgerigar (budgie,parakeet;Melopsittacus undulutus) is composed of cortical reptilian-type nephrons (without loops of Henle) and mammalian-type nephrons (with loops) grouped together in medullary cones. The loop of the mammalian-type nephrons has a descending segment composed of thin and highly interdigitated cells. These thin limb cells have few mitochondria (15% of cell volume), undetectable Na+,K+-ATPase activity, and virtually no basolateral surface amplification. Prior to the hairpin turn, the descending limb thickens, but the cells continue to lack basolateral amplification. Cells just prior to and within the hairpin turn resemble cells of the entire ascending limb. These cells are thick (there is no thin ascending segment in the avian loop), with extensive infoldings of the basolateral membrane surrounding numerous mitochondria (45?& of cell volume). The area of basolateral membrane is 25 times that of the apical membrane. The basolateral membrane (but not the apical membrane) is enriched in Na ,K+-ATPase activity. The structure of the avian mammalian-type nephron (as epitomized by the budgie nephron) and the fact that NaCl accounts for over 90 % of the osmotic activity of avian urine leads to the conclusion that the countercurrent multiplier of the avian kidney functions by active NaCl transport from the entire ascending limb. No explanation is offered for the transport specializationsfound in the thick descending segment of the loop,just prior to the hairpin turn. +
Among the vertebrates, only mammals and birds are able to form a urine that is more osmotically concentrated than the blood. The mammalian urinary-concentratingmechanism, the countercurrent multiplier system, has been the subject of numerous studies (reviewed in Jamison and Kriz, ’82; de Rouffignac and Jamison, ’87). Although the avian concentrating mechanism has been studied less, and is less well understood, there is little doubt that the bird kidney also uses some form of countercurrent multiplier system to form a concentrated urine. The evidence for this conclusion takes three forms. First, in addition to having nephrons lacking loops of Henle (reptilian-typenephrons),avian kidneys also possess nephrons with loops of Henle, the so-called mammalian-type nephrons, organized in groups c d e d medullary cones. These medullary cones are analogousto the mammalian medulla (Braun and Dantzler, ’72). Second, Poulson (’65) found that, as the number of mammalian-type nephions decreased in various birds, so did the urine concentration. At the extrapolated value of zero mammalian-type nephrons, the extrapolated urine osmotic concentration was the same as that of the blood, suggesting that the mammalian-type nephrons (those with loops of Henle) 0
1990 WILEY-LISS, INC.
are responsible for urine to plasma osmotic ratios ( U p ) ofgreater than one. Finally, the microcryoscopic analysis by Emery et al. (’72) showed that filtrate in the hairpin turns of mammaliantype nephrons had a greater osmolality than filtrate in tubules closer to the cortex, where the osmolality approached that of plasma. Osmotic concentration increased along most of the length of the collecting duct. These observationsare all consistent with a countercurrent concentrating mechanism. Although the above evidence suggeststhat the avian kidney uses a countercurrent multiplier system, as Braun and Reimer (’88) note, there are differences between the avian and mammalian systems. These differences may derive from the virtual lack of urea in avian urine (Skadhauge and Schmidt-Nielsen,’67).As is discussed below, urea is an important solute in the mammalian countercurrent system, and its absence in bird urine must have profound functional and perhaps structural consequences. Given that the avian and the mammal kidneys both use “mammalian-type nephrons” and countercurrent multiplication to form a concentrated urine, we sought to examine the extent to which the mechanisms are indeed similar, using
M. POST ET AL.
2
ultrastructural analyses and cytochemical localization of Na+,K+-ATPase,the sodium P U P enzyme. The subject of our study was the common parakeet (budgerigar),Melopsittacus undu&us, a domesticated species originallyfrom arid regions of Australia. The budgie has been shown to possess a number of physiologicaladaptations to its arid habitat, all of which contribute to the budgie's ability to survive in the laboratory without drinking water (Greenwald,et al., '67). Avian mammalian-type nephrons differ from true mammalian long-looped nephrons in that the avian descending thin limb thickens before, rather than after, the hairpin turn. Thus, there is a thick descending segment just prior to the turn, and the entire ascending limb of the loop of Henle is thick (reviewed in Braun and Dantzler, '72). There is no thin ascendmg limb. This arrangement resembles that of mammalian shortlooped nephrons. In the Results section, we discuss the structure of the avian mammalian-type nephrons starting from the thin descendinglimb, progressing to the transition where the thin limb thickens, then to the hairpin turn, and finally to the ascendinglimb. h4ATEFWK.S AND METHODS
Animals Budgies were obtained from local pet stores, were fed (ad libitum) commercialparakeet seed, and were provided continuously with drinking water. Electron microscopy To fix renal tissue, we decapitated birds quickly and removed their kidneys to a dish of fixative. The kidney was then teased apart under fixative until medullary cones were found. The cones, which contain the mammalian-type nephrons, were removed, and fixation was continued for a total of 1hr. For studies involving general electron microscopy or morphometry, the fixative contained 2.5% glutaraldehyde, 1%(v/v) acrolein, 0.1 M N42-hydroxyethyl) piperazineN-(2-ethanesulfonic acid) (HEPES), 5 9% (v/v) dimethyl sulfoxide (DMSO),0.2 M sucrose, and 0.5% (v/v) H202 The pH was adjusted to 7.4. After fixation and buffer rinse, the tissue was osmicated (1%OsO, for 1hr), dehydrated in a graded ethanol series, and embedded in Spurr's medium (Spurr, '69). Sections were stained with Reynold's lead citrate (Hayat, '81). Na+,K+-ATPasecytochemistry Tissue to be used for localization of Na+,K' ATPase was processed according to the method of Ueno et al. ('83). The medullary cones were
fixed for 20 min in 2 76 paraformaldehyde,0.25 % glutaraldehyde,0.25 M sucrme,5 % (v/v)DMSO, and 0.1 M sodium cacodylate, pH 7.4,4"C. Following fixation, the tissue was rinsed twice in cold buffer [0.1 M sodium cacodylate, 0.25 M sucrose, 10% (v/v) DMSO, pH 7.41. After the second rinse, the cones were embedded in agar and chopped into 75-pm-thick cross sectionswith a Sorvall TC-2 tissue chopper. After sectioning, the slices of medullary cones were rinsed overnight in cold buffer. After the overnight rinse, tissue was preincubated for 30 min at room temperature in Na+,K+ATPase reaction medium without substrate. This medium contained 45 mM KOH, 250 mM glycine, 3.8 mM lead citrate, and 2.5 mM levamisole (Sigma Chemical Co.), an inhibitor of nonNa+,K+-ATPasealkaline phosphatase activity. Following the preincubation,the tissue was incubated for 1hr at room temperature in the same medium but also containing the ATPase substrate p-nitrophenylphos hate (Sigma Chemical Co., Mg, p-NPP, Va?+ -free), pH 8.8. The control preincubationand completereaction media were the same as those described above, except these media contained the Na',K+ATPase inhibitor ouabain and NaOH (45 mM) rather than KOH. After the 1hr reaction period, the tissue slices were rinsed in 0.1 M cacodylate buffer with 0.25 M sucrose (pH 7.4) and were postfixed in 1% buffered OsO, for 1hr. The sections were then dehydrated in a graded ethanol series and embedded in Spurr's embedding medium. Sections were viewed either unstained or lightly stained with Reynold's lead citrate (Hayat, '81). Morphometry One medullary cone was serially cross sectioned so that five mammalian-type nephrons of different lengths could be studied. Sectionswere taken from each nephron a t the following points: thin descending limb close to the cortex, thin descending limb near the hairpin turn, transition region between thin descending limb and thick descending limb, hairpin turn, thick ascending limb close to the hairpin turn, and thick ascending limb close to the cortex. Using the methods of M'eibel and Bolender ('74), S7eibel ('791, Dhup ('85), and Mayhew ('79), mitochondrial density (the fraction of the cell occupied by mitochondria) and apical and basolateral membrane boundary lengths and areas were measured. In the case of membrane boundary lengths and areas, an anisotropic curvilinear test grid (after Merz, '67) was used to avoid biasing data because of isotropic (linear) organization of plasma membrane folds. Surface density (S,) of
NA, K-ATPase IN AN AVIAN LOOP OF HENLE
Fig. 1. Stained thin section taken from the middle region of a thin descending limb of the loop of He&. Within this tubular segment, the epithelium is composed of cells that resemble podocytes of the renal glomerulus. Cell bodies contain nuclei (N) and occasional large mitochondria (M). The
3
lumen (L) is bordered by thin extensionsof the cells that form simplejunctionalcomplexes with neighboringprocesses. The lateral intercellular spaces (IS) are dilated and contain microvillous processes of the cell bodies.
4
M. POST ET AL.
plasma membranes w8s calculated according to the following equation:
21 s" =-L,' where I is the number of intersections of the membrane with the lines of the test grid and Lt is the length of the projection of the test line within the volume of interest (Weibel, '79; Dhup, '85). RESULTS
Thin descending limb A complete cross section of a descending limb is shown in Figure 1.Cells of the thin descending limbs are noteworthy in two respects. The extracellular space at the basolateral margins of these cells often displaysnumerous cellular extensions
Fig. 2. Enlargementof the epitheliumof thethin descending limb. Neighboringprocesses form simplejunctional cornpiexes (arrowheads).The lateral intercellular space (IS) is
from cells outside of the plane of section. Also, the apical borders of these cells are adorned with loosely organized microvilli. An unusual feature of the thin descending limb is the structure of the border between the lumen and the interstitial space (Fig. 2). Although part of this border is composed of typical epithelial cells, other regions of the tubule border are made of thin regions of interdigitations of cellular processes that project from cells above and below the plane of section. The cellular processes are joined by simple occluding junctions, the typical junction of medullary cone cells. Thirteen percent of the volume of the cells in the thin descending limb (excluding thin basal and apical projections) was filled with mitochondria (Fig. 3). This value was independent of
expanded in regions where numerous processes form the luminal border. L, lumen;M, mitochondrion.
40
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5 Fig. 3. a:Schematicdrawing of the loop of Henle indicating the approximatesites at which the data were obtained for graphs in Figures 3b, 4, and 5. b: Volume of mitochondria expressed as a percentage of epithelial cell volume in the six regions as indicated in a. Values here and in following graphs are mean i standard error of the mean (SEM).
Fig. 4. Boundary length in micrometers per cell of apical (a)and basolateral (b) membranes from six regions of the loop of Henle. Fig. 5. Surface density (surface to volume ratio, fim-') of apical (a)and basolateral (b)membranes from six regions of the loop of Henle.
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M.POST ET AL.
Figures 6-7
NA, K-ATPM IN AN AVIAN LOOP OF HENLE
7
Fig. 8. Micrograph of an unstained section showing the distribution of Na+,K+-ATPaseactivity in the basolateral region of cells at the hairpin turn of a loop (bottom half of figure) and in another portion of thick limb (top half). Enzy-
matic deposition has filled most of the cytoplasmic space in basolateral folds (e.g., between arrowheads). M, mitochondrion.
whether the measurement was made close to the cortex or close to the hairpin turn. A typical cell of the thin limb has an apical membrane length of 9.5 Km (Fig. 4a);the basolateral length was 3.1-fold greater, or 29.7 gm (Fig. 4b). Expressed as membrane area per volume of cell, these values correspond to 0.36gm2/~m3 for apical membrane and 1.28 pm2/gm3for basolateral membrane (Fig. 5a,b).No attempt was made
to account for the presence of the interdigitating processes in these morphometric calculations. Thin descending limbs from tissue demonstrating Na+,K+-ATPase activity showed extremely low levels of that activity, too low to yield reaction product (Fig.6). Some lead deposition (which is likely artifact) can be seen around the nuclei of these cells. For comparison, a small segment of a thick limb cell can be seen at the
Fig. 6. Unstained thin section of thin limb treated to reveal Na+,K' -ATPaseactivity.No accumulationof reaction product is visible in association with plasma membranes in this tubule segment. Some precipitate has accumulated in the nuclear envelopes of these epithelial cells, and particularly dense deposits are apparent in the adjacent thick limb at the top edge of the figure. IS, lateral intercellularspace; L, lumen; N, nucleus.
Fig. 7. Electron micrograph of a stained thin section showing a cell from the transition from descending thin limb to descendingthicklimb. The cell appears unspecialized, with simple contours of the apical and basolateral membranes. A centriole at the apical surface (arrowhead)probably is a basal body to a single, central cilium. RL, basal lamina: L, lumen; M, mitochondrion; N, nucleus.
Figurea 9-10
NA, K-ATF'ase IN AN AVIAN LOOP OF HENLE
extreme top of the micrograph. Heavy lead deposition is apparent a t the basolateral border of the cell. The thin limb in this section also shows basolateral and apical projections as well as regions of interdigitating cellular processes forming a border between the lumen and the interstitial space.
Thin-thick transition cells The thin descending limb thickens prior to the hairpin turn. A cell from such a region is shown in Figure 7. The cell possesses neither apical nor basolateral surface membrane amplifications. Basal bodies are often seen. Descending limbs just prior to the hairpin turn never show the narrow border of interdigitating processes seen in the thin limb of the same nephron closer to the cortex. The hairpin turn Between the thin descending limb and the hairpin turn, the cells of the avian nephron change in terms of basolateral membrane amplification and mitochondrial density. Cells a t the tip of the haupin turn display an extensive network of infoldings of the basolateral cell membrane surrounding large numbers of mitochondria. Approximately 50 5% of the volume of a cell at the hairpin turn is composed of mitochondria lying between basolateral membrane infoldings (Fig. 3). The degree of membrane amplification can be seen in Figure 4a,b, which shows that each of these cells has 25 times as much basolateral as apical membrane (3.26 pm2of basolateral membrane/pm3 of cell volume and 0.13 pm2of apical membrane/pm3of cell). Figure 8 shows the tip of a hairpin turn cell (the lumen is beyond the plane of the section) from a medullary cone slice reacted for demonstration of Na' ,Kf -ATPase activity. Numerous mitochondria are apparent between basolateral membrane infoldings. Along their cytoplasmic surfaces, those membranes show dense deposits of reaction product, indicating a high level of Na+,K+-ATPaseactivity. Thick ascending limb Figure 9 shows a cell from the thick ascending limb. These cells are typified by having numer-
Fig. 9. Stained cross sedion of cells from the ascending thick limb in the center of a medullary cone. Mitochondria (M) within basolateral folds occupy a large fraction of the cytoplasm. L, lumen; N, nucleus.
9
ous mitochondria surrounded by infoldings of the basolateral membrane. Cells in any part of the ascending limb, from the hairpin turn to about halfway up the nephron, had mitochondrial volumes on the order of 40% of the cell volume (Fig. 3). These cells, like others in the mammalian-type nephrons, are joined by simple occludingjunctions. In all segments of the ascending limbs that we measured, the surface area density of basolateral membrane (per unit of cell volume) greatly exceeded the apical membrane area density. The same was true for the boundary length of the cell membranes, with basolateral length far exceeding apical length. There was a tendency for the degree of basolateral membrane amplification (compared to apical membrane length or area) to decrease toward the cortex (Figs. 4,5). The basolateral membranes of ascending limb cells show intense Na+,K+-ATPase reaction product deposition (Fig. 10). Apical membranes showed little if any deposition, although there was some diffusion of reaction product onto mitochondrial membranes. Tissue that normally showed intense reactivity showed little reaction product when the incubation was run in the presence of ouabain and in the absence of potassium ions (Fig. 11). DISCUSSION
The major difference in osmoticallyactivemolecules between avian and mammalian urine is the lack of urea in avian urine. The major difference between the classical countercurrent multiplier model and the more recent two-solutecountercurrent multiplier model is the lack of a function for urea in the classical model. This parallelism suggests that, whereas the twosolute countercurrent system (discussed below) is thought to operate in the mammalian kidney, the classical countercurrent system is a more appropriate model for the avian kidney. In the two-solute countercurrent multiplier model of the mammalian nephron, NaCl (the first solute) is reabsorbed along the entire ascending limb of the loop of Henle. NaCl transport from the thick ascending limb is active, but the thin ascending limb of the loop secretes NaCl by a passive process. This passive process is driven by a urea gradient (urea being the second solute)
Fig. 10. Electron micrographof thick ascendinglimb that demonstrates dense deposition of Na+,K+-ATPasereaction product within basolateral folds. Lighter deposition is seen within the intracristal space of mitochondria (arrowhead), whereas no deposition is seen at the luminal border. L, lumen; M, mitochondrion.
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M. POST ET AL.
Fig. 11. Unstained section of thick ascending limb showing a control reaction for Na-,K+-ATPaseactivity. In this case, the enzyme reaction mixture was free of K' and contained ouabain. Only a very light dusting of precipitate is apparent on the external surfaces of the basohteral membranes (e.g., between arrowheads) and within mitochondria
(MI.
established through active mechanisms in the thick ascending limb (reviewed in Jamison and M d y , '76). The structure of the mammalianloop of Henle is consonant with the two-solute model. The cells of the thick ascending limb of the loop are specialized for active salt transport in that these cells have numerous mitochondria surrounded by infoldings of the basolateral cell membrane. These membranes have been shown to be rich in the sodium transport enzyme Na+,K'-ATPase (Ernst and Schreiber, '81). The cells of the thin ascendinglimb lack obvioustransport specialiations; they have few mitochondria and few, if any, basolateral membrane infoldings. The urea that accumulates in the interstitium drives the
movement of salt and water from the thin limbs of the loop (which are themselves not capable of active transport) to the interstitium. Because the fluid in the avian nephron does not contain urea, the two-solute countercurrent system based on urea and NaCl clearly could not function in a bird's kidney. Indeed, NaCl is the predominant osmotically active solute in avian urine, and there is no other solute in avian urine that could take the place of urea (Skadhauge and Schmidt-Nielsen,'67). The simplesthypothesis accounting for countercurrent multiplication in the medullary cones of avian kidney is that NaCl is actively reabsorbed along the entire length of the avian ascending limb. Thus, in the avian mammalian-type nephron, as is the case in the true mammalian long-loopednephron, there is an excellent match between structure and function. In the avian case, there appears to be a functional requirement for active salt transport along the entire length of the ascending limb and, indeed, from the hairpin turn to the distal convolution the cells of the ascending limb show transport specialization, namely, mitochondria surrounded by Na+,K+-ATPase-rich infoldings of the basolateral plasma membrane. Although the structure-function relationships of the avian ascending limb conform with the requirementsof the classicalcountercurrentmultiplier, the apparent transport capability of the thick descending limb just prior to the hairpin turn is diflicult to explain. Braun and Reimer ('88) suggest that "this additional transport capacity should augment the diluting capacity of the ascending limb . . . [and/or] would tend to facilitate the build up of the gradient toward the papilla tip." The thin (descending) limb of the avian loop has a structure that seems to indicate a high permeability to solutes and/or water. Cells in the midregion of the thin limb showed numerous, narrow, interdigitating cell processes connected by extremely shallow occluding junctions. Simple cell junctions with shallow areas of adhesion have been related to high ion rather than water permeability in the mammalian thin ascending limb (Bachmann and Kriz, '82). In the thin descending limb, however, one would expect a higher osmotic than ionic permeability of the junctions, because, in the classical countercurrent system, the NaCl transported by the ascending limb causes an increase in concentration of the luminal contents of the descending limb by water abstraction from that nephron segment.
NA, K-ATPase IN AN AVIAN LOOP OF HENLE
Similar interdigitating regions have also been seen by Braun and Reimer ('88) in thin limbs of Gambel's quail, by Schwartz and Venkatachalam ('74) in the hairpin turn region in rats, and by Bachmann and Kriz ('82) in the ascending and descending thin limbs of hamsters. In summary, this study, along with that of Braun and Reimer ('88),supports the conclusion that the avian mammalian-type nephron achieves concentration of the urine by mechanisms more closely related to the classical countercurrent system than to the two-solute system or any of its more recent variants (e.g., Imai et al.,'87). Still to be explained in more detail is the significance of morphological transport specializations in the descending limb prior to the hairpin turn. Also, as was shown by Braun and Reimer ('88)' the thin descending limb has different structural characteristics between the abrupt origin of the thin limb close to the cortex and its abrupt end at the beginning of the thick descending limb. The functional significance of these differences also is yet to be explained. ACKNOWLEDGMENT
This work was supported by NSF grant DMB8417744 to L.G. LITERATURE CITED Bachmann, S., and W. Kriz (1982) Histotopography and ultrastructure of the thin limbs of the long loop of Henle in the hamster. Cell Tissue Res. 225r111-127. Braun, E.J., and W.H. Dantzler (1972) Function of mammalian-type and reptilian-type nephrons in kidney of desert quail. Am. J. Physiol. 222.617429. Braun, E.J., and P.R. Reimer (1988) Structure of avian loop of Henle as related to counkcurrent multiplier system. Am. J. Physiol. 255:F5WF512. de Rouffignac, C., and R.L. Jamison (1987) The urinary concentratingmechanism. Kidney Int. 31501472. D@rup,J. (1985) Ultrastructure of distal nephron cells in rat renal cortex. J. Ultrastruct. Res. 92r101-118.
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Emery, N., T.L. Poulson,and W.B. Kinter (1972)Production of concentrated urine by avian kidneys. Am. J. Physiol. 223:18&187. Ernst, S.A., and J.H. Schreiher (1981)Ultrastructural localiiration of Na+,K+-ATPasein rat and rabbit kidney medulla. J. Cell Biol. 91t803413. Greenwald, L., W.B. Stone, and T.J. Cade (1967) Physiologicaladjustments of the budgerygah (Melopsittacus undulatus) to dehydrating conditions. Comp. Biochem. Physiol. 22.91-100. Hayat, M.A. (1981) Principles and Techniques of Electron Microscopy. Vol 1. Baltimore: University Park Press, pp. 1-324. Imai, M., J. Taniguchi, and K. Tabei (1987) Function of thin loops of Henle. Kidney Int. 315654579. Jamison, R.L., and W. Kriz (1982) Urinary Concentrating Mechanism: Structure and Function. New York Oxford University Press, pp. 1340. Jamison,R.L.,andR.H. MafFly(1976)Theurinaryconcentrating mechanism. N. Engl. J. Med. 29.51059-1067. Mayhew, T.M. (1979) Basic stereological relationships for quantitative microscopical anatomy-A simple systematic approach.J. Anat. 129:9!+105. Merz, W.A. (1967) Die Streckemnessung an gerichteten Struckturen im Mikroskop und ihre Anwendung zur Bestimmung von Oberflachen-Volumen-Relationen in Knochengewebe. Mikrcskopie 22:132-142. Podson, T. (1965) Countercurrent multipliers in avian kidneys. Science 14&:389-391. Schwartz, M.M., and M.A. Venkatachalam (1974)Structural differences in thin limbs of Henle: Physiological implications. Kidney Int. 6r193-208. Skadhauge, E., and B. Schmidt-Nielsen(1967)Renal medullary electrolyte and urea gradient in chickens and turkeys. Am. J. Physiol. 212r131.1-1318. S p m , A.R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26:3143. Ueno, S., H. Mayahara, M. Ueck, I Tsukkahara, and K. Ogawa (1983) Ultracytochemical localization of ouabainsensitive, potassium dependent p-nitrophenylphcsphatase activity in the lacrimal gland of the rat. Cell Tissue Res. 234t497-518. Weibel, E.R. (1979) Stereological Methods, Vol. 1. Practical Methods for Biological Morphometry. New York Academic Press. Weibel, E.R., and R.P. Bolender (1974) Stereological techniques for electron microscopic morphometry. In M.A. Hayat (4): Principles and Techniques of Electron Microscopy. New York: Van Nostrand hinhold Co., pp. 237-296.
Cellular Basis of an Avian Countercurrent Multiplier System MARC POST, LEWIS GREENWALD, AND DAVID STETSON Department of Zoology, The Ohio State Uniuersity Columbus, Ohio 43210
ABSTRACT A kidney from the budgerigar (budgie,parakeet;Melopsittacus undulutus) is composed of cortical reptilian-type nephrons (without loops of Henle) and mammalian-type nephrons (with loops) grouped together in medullary cones. The loop of the mammalian-type nephrons has a descending segment composed of thin and highly interdigitated cells. These thin limb cells have few mitochondria (15% of cell volume), undetectable Na+,K+-ATPase activity, and virtually no basolateral surface amplification. Prior to the hairpin turn, the descending limb thickens, but the cells continue to lack basolateral amplification. Cells just prior to and within the hairpin turn resemble cells of the entire ascending limb. These cells are thick (there is no thin ascending segment in the avian loop), with extensive infoldings of the basolateral membrane surrounding numerous mitochondria (45?& of cell volume). The area of basolateral membrane is 25 times that of the apical membrane. The basolateral membrane (but not the apical membrane) is enriched in Na ,K+-ATPase activity. The structure of the avian mammalian-type nephron (as epitomized by the budgie nephron) and the fact that NaCl accounts for over 90 % of the osmotic activity of avian urine leads to the conclusion that the countercurrent multiplier of the avian kidney functions by active NaCl transport from the entire ascending limb. No explanation is offered for the transport specializationsfound in the thick descending segment of the loop,just prior to the hairpin turn. +
Among the vertebrates, only mammals and birds are able to form a urine that is more osmotically concentrated than the blood. The mammalian urinary-concentratingmechanism, the countercurrent multiplier system, has been the subject of numerous studies (reviewed in Jamison and Kriz, ’82; de Rouffignac and Jamison, ’87). Although the avian concentrating mechanism has been studied less, and is less well understood, there is little doubt that the bird kidney also uses some form of countercurrent multiplier system to form a concentrated urine. The evidence for this conclusion takes three forms. First, in addition to having nephrons lacking loops of Henle (reptilian-typenephrons),avian kidneys also possess nephrons with loops of Henle, the so-called mammalian-type nephrons, organized in groups c d e d medullary cones. These medullary cones are analogousto the mammalian medulla (Braun and Dantzler, ’72). Second, Poulson (’65) found that, as the number of mammalian-type nephions decreased in various birds, so did the urine concentration. At the extrapolated value of zero mammalian-type nephrons, the extrapolated urine osmotic concentration was the same as that of the blood, suggesting that the mammalian-type nephrons (those with loops of Henle) 0
1990 WILEY-LISS, INC.
are responsible for urine to plasma osmotic ratios ( U p ) ofgreater than one. Finally, the microcryoscopic analysis by Emery et al. (’72) showed that filtrate in the hairpin turns of mammaliantype nephrons had a greater osmolality than filtrate in tubules closer to the cortex, where the osmolality approached that of plasma. Osmotic concentration increased along most of the length of the collecting duct. These observationsare all consistent with a countercurrent concentrating mechanism. Although the above evidence suggeststhat the avian kidney uses a countercurrent multiplier system, as Braun and Reimer (’88) note, there are differences between the avian and mammalian systems. These differences may derive from the virtual lack of urea in avian urine (Skadhauge and Schmidt-Nielsen,’67).As is discussed below, urea is an important solute in the mammalian countercurrent system, and its absence in bird urine must have profound functional and perhaps structural consequences. Given that the avian and the mammal kidneys both use “mammalian-type nephrons” and countercurrent multiplication to form a concentrated urine, we sought to examine the extent to which the mechanisms are indeed similar, using
M. POST ET AL.
2
ultrastructural analyses and cytochemical localization of Na+,K+-ATPase,the sodium P U P enzyme. The subject of our study was the common parakeet (budgerigar),Melopsittacus undu&us, a domesticated species originallyfrom arid regions of Australia. The budgie has been shown to possess a number of physiologicaladaptations to its arid habitat, all of which contribute to the budgie's ability to survive in the laboratory without drinking water (Greenwald,et al., '67). Avian mammalian-type nephrons differ from true mammalian long-looped nephrons in that the avian descending thin limb thickens before, rather than after, the hairpin turn. Thus, there is a thick descending segment just prior to the turn, and the entire ascending limb of the loop of Henle is thick (reviewed in Braun and Dantzler, '72). There is no thin ascendmg limb. This arrangement resembles that of mammalian shortlooped nephrons. In the Results section, we discuss the structure of the avian mammalian-type nephrons starting from the thin descendinglimb, progressing to the transition where the thin limb thickens, then to the hairpin turn, and finally to the ascendinglimb. h4ATEFWK.S AND METHODS
Animals Budgies were obtained from local pet stores, were fed (ad libitum) commercialparakeet seed, and were provided continuously with drinking water. Electron microscopy To fix renal tissue, we decapitated birds quickly and removed their kidneys to a dish of fixative. The kidney was then teased apart under fixative until medullary cones were found. The cones, which contain the mammalian-type nephrons, were removed, and fixation was continued for a total of 1hr. For studies involving general electron microscopy or morphometry, the fixative contained 2.5% glutaraldehyde, 1%(v/v) acrolein, 0.1 M N42-hydroxyethyl) piperazineN-(2-ethanesulfonic acid) (HEPES), 5 9% (v/v) dimethyl sulfoxide (DMSO),0.2 M sucrose, and 0.5% (v/v) H202 The pH was adjusted to 7.4. After fixation and buffer rinse, the tissue was osmicated (1%OsO, for 1hr), dehydrated in a graded ethanol series, and embedded in Spurr's medium (Spurr, '69). Sections were stained with Reynold's lead citrate (Hayat, '81). Na+,K+-ATPasecytochemistry Tissue to be used for localization of Na+,K' ATPase was processed according to the method of Ueno et al. ('83). The medullary cones were
fixed for 20 min in 2 76 paraformaldehyde,0.25 % glutaraldehyde,0.25 M sucrme,5 % (v/v)DMSO, and 0.1 M sodium cacodylate, pH 7.4,4"C. Following fixation, the tissue was rinsed twice in cold buffer [0.1 M sodium cacodylate, 0.25 M sucrose, 10% (v/v) DMSO, pH 7.41. After the second rinse, the cones were embedded in agar and chopped into 75-pm-thick cross sectionswith a Sorvall TC-2 tissue chopper. After sectioning, the slices of medullary cones were rinsed overnight in cold buffer. After the overnight rinse, tissue was preincubated for 30 min at room temperature in Na+,K+ATPase reaction medium without substrate. This medium contained 45 mM KOH, 250 mM glycine, 3.8 mM lead citrate, and 2.5 mM levamisole (Sigma Chemical Co.), an inhibitor of nonNa+,K+-ATPasealkaline phosphatase activity. Following the preincubation,the tissue was incubated for 1hr at room temperature in the same medium but also containing the ATPase substrate p-nitrophenylphos hate (Sigma Chemical Co., Mg, p-NPP, Va?+ -free), pH 8.8. The control preincubationand completereaction media were the same as those described above, except these media contained the Na',K+ATPase inhibitor ouabain and NaOH (45 mM) rather than KOH. After the 1hr reaction period, the tissue slices were rinsed in 0.1 M cacodylate buffer with 0.25 M sucrose (pH 7.4) and were postfixed in 1% buffered OsO, for 1hr. The sections were then dehydrated in a graded ethanol series and embedded in Spurr's embedding medium. Sections were viewed either unstained or lightly stained with Reynold's lead citrate (Hayat, '81). Morphometry One medullary cone was serially cross sectioned so that five mammalian-type nephrons of different lengths could be studied. Sectionswere taken from each nephron a t the following points: thin descending limb close to the cortex, thin descending limb near the hairpin turn, transition region between thin descending limb and thick descending limb, hairpin turn, thick ascending limb close to the hairpin turn, and thick ascending limb close to the cortex. Using the methods of M'eibel and Bolender ('74), S7eibel ('791, Dhup ('85), and Mayhew ('79), mitochondrial density (the fraction of the cell occupied by mitochondria) and apical and basolateral membrane boundary lengths and areas were measured. In the case of membrane boundary lengths and areas, an anisotropic curvilinear test grid (after Merz, '67) was used to avoid biasing data because of isotropic (linear) organization of plasma membrane folds. Surface density (S,) of
NA, K-ATPase IN AN AVIAN LOOP OF HENLE
Fig. 1. Stained thin section taken from the middle region of a thin descending limb of the loop of He&. Within this tubular segment, the epithelium is composed of cells that resemble podocytes of the renal glomerulus. Cell bodies contain nuclei (N) and occasional large mitochondria (M). The
3
lumen (L) is bordered by thin extensionsof the cells that form simplejunctionalcomplexes with neighboringprocesses. The lateral intercellular spaces (IS) are dilated and contain microvillous processes of the cell bodies.
4
M. POST ET AL.
plasma membranes w8s calculated according to the following equation:
21 s" =-L,' where I is the number of intersections of the membrane with the lines of the test grid and Lt is the length of the projection of the test line within the volume of interest (Weibel, '79; Dhup, '85). RESULTS
Thin descending limb A complete cross section of a descending limb is shown in Figure 1.Cells of the thin descending limbs are noteworthy in two respects. The extracellular space at the basolateral margins of these cells often displaysnumerous cellular extensions
Fig. 2. Enlargementof the epitheliumof thethin descending limb. Neighboringprocesses form simplejunctional cornpiexes (arrowheads).The lateral intercellular space (IS) is
from cells outside of the plane of section. Also, the apical borders of these cells are adorned with loosely organized microvilli. An unusual feature of the thin descending limb is the structure of the border between the lumen and the interstitial space (Fig. 2). Although part of this border is composed of typical epithelial cells, other regions of the tubule border are made of thin regions of interdigitations of cellular processes that project from cells above and below the plane of section. The cellular processes are joined by simple occluding junctions, the typical junction of medullary cone cells. Thirteen percent of the volume of the cells in the thin descending limb (excluding thin basal and apical projections) was filled with mitochondria (Fig. 3). This value was independent of
expanded in regions where numerous processes form the luminal border. L, lumen;M, mitochondrion.
40
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5 Fig. 3. a:Schematicdrawing of the loop of Henle indicating the approximatesites at which the data were obtained for graphs in Figures 3b, 4, and 5. b: Volume of mitochondria expressed as a percentage of epithelial cell volume in the six regions as indicated in a. Values here and in following graphs are mean i standard error of the mean (SEM).
Fig. 4. Boundary length in micrometers per cell of apical (a)and basolateral (b) membranes from six regions of the loop of Henle. Fig. 5. Surface density (surface to volume ratio, fim-') of apical (a)and basolateral (b)membranes from six regions of the loop of Henle.
6
M.POST ET AL.
Figures 6-7
NA, K-ATPM IN AN AVIAN LOOP OF HENLE
7
Fig. 8. Micrograph of an unstained section showing the distribution of Na+,K+-ATPaseactivity in the basolateral region of cells at the hairpin turn of a loop (bottom half of figure) and in another portion of thick limb (top half). Enzy-
matic deposition has filled most of the cytoplasmic space in basolateral folds (e.g., between arrowheads). M, mitochondrion.
whether the measurement was made close to the cortex or close to the hairpin turn. A typical cell of the thin limb has an apical membrane length of 9.5 Km (Fig. 4a);the basolateral length was 3.1-fold greater, or 29.7 gm (Fig. 4b). Expressed as membrane area per volume of cell, these values correspond to 0.36gm2/~m3 for apical membrane and 1.28 pm2/gm3for basolateral membrane (Fig. 5a,b).No attempt was made
to account for the presence of the interdigitating processes in these morphometric calculations. Thin descending limbs from tissue demonstrating Na+,K+-ATPase activity showed extremely low levels of that activity, too low to yield reaction product (Fig.6). Some lead deposition (which is likely artifact) can be seen around the nuclei of these cells. For comparison, a small segment of a thick limb cell can be seen at the
Fig. 6. Unstained thin section of thin limb treated to reveal Na+,K' -ATPaseactivity.No accumulationof reaction product is visible in association with plasma membranes in this tubule segment. Some precipitate has accumulated in the nuclear envelopes of these epithelial cells, and particularly dense deposits are apparent in the adjacent thick limb at the top edge of the figure. IS, lateral intercellularspace; L, lumen; N, nucleus.
Fig. 7. Electron micrograph of a stained thin section showing a cell from the transition from descending thin limb to descendingthicklimb. The cell appears unspecialized, with simple contours of the apical and basolateral membranes. A centriole at the apical surface (arrowhead)probably is a basal body to a single, central cilium. RL, basal lamina: L, lumen; M, mitochondrion; N, nucleus.
Figurea 9-10
NA, K-ATF'ase IN AN AVIAN LOOP OF HENLE
extreme top of the micrograph. Heavy lead deposition is apparent a t the basolateral border of the cell. The thin limb in this section also shows basolateral and apical projections as well as regions of interdigitating cellular processes forming a border between the lumen and the interstitial space.
Thin-thick transition cells The thin descending limb thickens prior to the hairpin turn. A cell from such a region is shown in Figure 7. The cell possesses neither apical nor basolateral surface membrane amplifications. Basal bodies are often seen. Descending limbs just prior to the hairpin turn never show the narrow border of interdigitating processes seen in the thin limb of the same nephron closer to the cortex. The hairpin turn Between the thin descending limb and the hairpin turn, the cells of the avian nephron change in terms of basolateral membrane amplification and mitochondrial density. Cells a t the tip of the haupin turn display an extensive network of infoldings of the basolateral cell membrane surrounding large numbers of mitochondria. Approximately 50 5% of the volume of a cell at the hairpin turn is composed of mitochondria lying between basolateral membrane infoldings (Fig. 3). The degree of membrane amplification can be seen in Figure 4a,b, which shows that each of these cells has 25 times as much basolateral as apical membrane (3.26 pm2of basolateral membrane/pm3 of cell volume and 0.13 pm2of apical membrane/pm3of cell). Figure 8 shows the tip of a hairpin turn cell (the lumen is beyond the plane of the section) from a medullary cone slice reacted for demonstration of Na' ,Kf -ATPase activity. Numerous mitochondria are apparent between basolateral membrane infoldings. Along their cytoplasmic surfaces, those membranes show dense deposits of reaction product, indicating a high level of Na+,K+-ATPaseactivity. Thick ascending limb Figure 9 shows a cell from the thick ascending limb. These cells are typified by having numer-
Fig. 9. Stained cross sedion of cells from the ascending thick limb in the center of a medullary cone. Mitochondria (M) within basolateral folds occupy a large fraction of the cytoplasm. L, lumen; N, nucleus.
9
ous mitochondria surrounded by infoldings of the basolateral membrane. Cells in any part of the ascending limb, from the hairpin turn to about halfway up the nephron, had mitochondrial volumes on the order of 40% of the cell volume (Fig. 3). These cells, like others in the mammalian-type nephrons, are joined by simple occludingjunctions. In all segments of the ascending limbs that we measured, the surface area density of basolateral membrane (per unit of cell volume) greatly exceeded the apical membrane area density. The same was true for the boundary length of the cell membranes, with basolateral length far exceeding apical length. There was a tendency for the degree of basolateral membrane amplification (compared to apical membrane length or area) to decrease toward the cortex (Figs. 4,5). The basolateral membranes of ascending limb cells show intense Na+,K+-ATPase reaction product deposition (Fig. 10). Apical membranes showed little if any deposition, although there was some diffusion of reaction product onto mitochondrial membranes. Tissue that normally showed intense reactivity showed little reaction product when the incubation was run in the presence of ouabain and in the absence of potassium ions (Fig. 11). DISCUSSION
The major difference in osmoticallyactivemolecules between avian and mammalian urine is the lack of urea in avian urine. The major difference between the classical countercurrent multiplier model and the more recent two-solutecountercurrent multiplier model is the lack of a function for urea in the classical model. This parallelism suggests that, whereas the twosolute countercurrent system (discussed below) is thought to operate in the mammalian kidney, the classical countercurrent system is a more appropriate model for the avian kidney. In the two-solute countercurrent multiplier model of the mammalian nephron, NaCl (the first solute) is reabsorbed along the entire ascending limb of the loop of Henle. NaCl transport from the thick ascending limb is active, but the thin ascending limb of the loop secretes NaCl by a passive process. This passive process is driven by a urea gradient (urea being the second solute)
Fig. 10. Electron micrographof thick ascendinglimb that demonstrates dense deposition of Na+,K+-ATPasereaction product within basolateral folds. Lighter deposition is seen within the intracristal space of mitochondria (arrowhead), whereas no deposition is seen at the luminal border. L, lumen; M, mitochondrion.
10
M. POST ET AL.
Fig. 11. Unstained section of thick ascending limb showing a control reaction for Na-,K+-ATPaseactivity. In this case, the enzyme reaction mixture was free of K' and contained ouabain. Only a very light dusting of precipitate is apparent on the external surfaces of the basohteral membranes (e.g., between arrowheads) and within mitochondria
(MI.
established through active mechanisms in the thick ascending limb (reviewed in Jamison and M d y , '76). The structure of the mammalianloop of Henle is consonant with the two-solute model. The cells of the thick ascending limb of the loop are specialized for active salt transport in that these cells have numerous mitochondria surrounded by infoldings of the basolateral cell membrane. These membranes have been shown to be rich in the sodium transport enzyme Na+,K'-ATPase (Ernst and Schreiber, '81). The cells of the thin ascendinglimb lack obvioustransport specialiations; they have few mitochondria and few, if any, basolateral membrane infoldings. The urea that accumulates in the interstitium drives the
movement of salt and water from the thin limbs of the loop (which are themselves not capable of active transport) to the interstitium. Because the fluid in the avian nephron does not contain urea, the two-solute countercurrent system based on urea and NaCl clearly could not function in a bird's kidney. Indeed, NaCl is the predominant osmotically active solute in avian urine, and there is no other solute in avian urine that could take the place of urea (Skadhauge and Schmidt-Nielsen,'67). The simplesthypothesis accounting for countercurrent multiplication in the medullary cones of avian kidney is that NaCl is actively reabsorbed along the entire length of the avian ascending limb. Thus, in the avian mammalian-type nephron, as is the case in the true mammalian long-loopednephron, there is an excellent match between structure and function. In the avian case, there appears to be a functional requirement for active salt transport along the entire length of the ascending limb and, indeed, from the hairpin turn to the distal convolution the cells of the ascending limb show transport specialization, namely, mitochondria surrounded by Na+,K+-ATPase-rich infoldings of the basolateral plasma membrane. Although the structure-function relationships of the avian ascending limb conform with the requirementsof the classicalcountercurrentmultiplier, the apparent transport capability of the thick descending limb just prior to the hairpin turn is diflicult to explain. Braun and Reimer ('88) suggest that "this additional transport capacity should augment the diluting capacity of the ascending limb . . . [and/or] would tend to facilitate the build up of the gradient toward the papilla tip." The thin (descending) limb of the avian loop has a structure that seems to indicate a high permeability to solutes and/or water. Cells in the midregion of the thin limb showed numerous, narrow, interdigitating cell processes connected by extremely shallow occluding junctions. Simple cell junctions with shallow areas of adhesion have been related to high ion rather than water permeability in the mammalian thin ascending limb (Bachmann and Kriz, '82). In the thin descending limb, however, one would expect a higher osmotic than ionic permeability of the junctions, because, in the classical countercurrent system, the NaCl transported by the ascending limb causes an increase in concentration of the luminal contents of the descending limb by water abstraction from that nephron segment.
NA, K-ATPase IN AN AVIAN LOOP OF HENLE
Similar interdigitating regions have also been seen by Braun and Reimer ('88) in thin limbs of Gambel's quail, by Schwartz and Venkatachalam ('74) in the hairpin turn region in rats, and by Bachmann and Kriz ('82) in the ascending and descending thin limbs of hamsters. In summary, this study, along with that of Braun and Reimer ('88),supports the conclusion that the avian mammalian-type nephron achieves concentration of the urine by mechanisms more closely related to the classical countercurrent system than to the two-solute system or any of its more recent variants (e.g., Imai et al.,'87). Still to be explained in more detail is the significance of morphological transport specializations in the descending limb prior to the hairpin turn. Also, as was shown by Braun and Reimer ('88)' the thin descending limb has different structural characteristics between the abrupt origin of the thin limb close to the cortex and its abrupt end at the beginning of the thick descending limb. The functional significance of these differences also is yet to be explained. ACKNOWLEDGMENT
This work was supported by NSF grant DMB8417744 to L.G. LITERATURE CITED Bachmann, S., and W. Kriz (1982) Histotopography and ultrastructure of the thin limbs of the long loop of Henle in the hamster. Cell Tissue Res. 225r111-127. Braun, E.J., and W.H. Dantzler (1972) Function of mammalian-type and reptilian-type nephrons in kidney of desert quail. Am. J. Physiol. 222.617429. Braun, E.J., and P.R. Reimer (1988) Structure of avian loop of Henle as related to counkcurrent multiplier system. Am. J. Physiol. 255:F5WF512. de Rouffignac, C., and R.L. Jamison (1987) The urinary concentratingmechanism. Kidney Int. 31501472. D@rup,J. (1985) Ultrastructure of distal nephron cells in rat renal cortex. J. Ultrastruct. Res. 92r101-118.
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Emery, N., T.L. Poulson,and W.B. Kinter (1972)Production of concentrated urine by avian kidneys. Am. J. Physiol. 223:18&187. Ernst, S.A., and J.H. Schreiher (1981)Ultrastructural localiiration of Na+,K+-ATPasein rat and rabbit kidney medulla. J. Cell Biol. 91t803413. Greenwald, L., W.B. Stone, and T.J. Cade (1967) Physiologicaladjustments of the budgerygah (Melopsittacus undulatus) to dehydrating conditions. Comp. Biochem. Physiol. 22.91-100. Hayat, M.A. (1981) Principles and Techniques of Electron Microscopy. Vol 1. Baltimore: University Park Press, pp. 1-324. Imai, M., J. Taniguchi, and K. Tabei (1987) Function of thin loops of Henle. Kidney Int. 315654579. Jamison, R.L., and W. Kriz (1982) Urinary Concentrating Mechanism: Structure and Function. New York Oxford University Press, pp. 1340. Jamison,R.L.,andR.H. MafFly(1976)Theurinaryconcentrating mechanism. N. Engl. J. Med. 29.51059-1067. Mayhew, T.M. (1979) Basic stereological relationships for quantitative microscopical anatomy-A simple systematic approach.J. Anat. 129:9!+105. Merz, W.A. (1967) Die Streckemnessung an gerichteten Struckturen im Mikroskop und ihre Anwendung zur Bestimmung von Oberflachen-Volumen-Relationen in Knochengewebe. Mikrcskopie 22:132-142. Podson, T. (1965) Countercurrent multipliers in avian kidneys. Science 14&:389-391. Schwartz, M.M., and M.A. Venkatachalam (1974)Structural differences in thin limbs of Henle: Physiological implications. Kidney Int. 6r193-208. Skadhauge, E., and B. Schmidt-Nielsen(1967)Renal medullary electrolyte and urea gradient in chickens and turkeys. Am. J. Physiol. 212r131.1-1318. S p m , A.R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26:3143. Ueno, S., H. Mayahara, M. Ueck, I Tsukkahara, and K. Ogawa (1983) Ultracytochemical localization of ouabainsensitive, potassium dependent p-nitrophenylphcsphatase activity in the lacrimal gland of the rat. Cell Tissue Res. 234t497-518. Weibel, E.R. (1979) Stereological Methods, Vol. 1. Practical Methods for Biological Morphometry. New York Academic Press. Weibel, E.R., and R.P. Bolender (1974) Stereological techniques for electron microscopic morphometry. In M.A. Hayat (4): Principles and Techniques of Electron Microscopy. New York: Van Nostrand hinhold Co., pp. 237-296.
