Ultrastructura Organization of the Hamster Renal Pelvis ERIC R. LACY ' AND BODIL SCHMIDT-NIELSEN Mount Desert Zsland Biological Laboratory, Salsbury Cow, Maine 04672
ABSTRACT
The renal pelvis of the hamster has been studied by light microscopy (epo y resin sections), transmission electron microscopy, and morphometric analysis of electron micrographs. Three morphologically distinct epithelia line the pelvis, and each covers a different zone of the kidney. A thin epithelium covering the outer medulla (OM) consists of two cell types: (1) granular cells are most numerous and have apically positioned granules which stain intensely with toluidine blue, are membrane-bound, and contain a fine particulate matter that stains light grey to black in electron micrographs. (2) Basal cells do not have granules, are confined to the basal lamina region, and do not reach the mucosal epithelial surface. The inner medulla (IM) is covered by a pelvic epithelium morphologically similar to collecting duct epithelium of IM. Some cells in this portion of the pelvic epithelium (IM) stain intensely dark with toluidine blue, osmium tetroxide, lead, and uranyl acetate. Transitional epithelium, which separates cortex (C) from pelvic urine, has an asymmetric luminal plasma membrane and discoid vesicles, each of which is similar to those previously observed in mammalian ureter and urinary bladder epithelia. Based on morphological comparisons with other epithelia, the IM and OM pelvic epithelia would appear permeable to solutes and/or water, while the transitional epithelium covering the C appears relatively impermeable. It would also appear that the exchange of solutes and water between pelvic urine and OM would involve capillaries, primarily, since morphometric analysis showed t h a t both fenestrated and continuous capillaries of the OM were extremely abundant ( >60% of OM pelvic surface area) just under the thin pelvic epithelium.
Recent anatomical and physiological studies have presented evidence that the dynamics of urine formation may include exchange of water andlor solute between pelvic urine and renal parenchyma (Gertz et al., '66; Pfeiffer, '68; Schutz and Schnermann, '72; Khorshid and Moffat, '74; Silverblatt, '74; Kaissling et al., '75; Schmidt-Nielsen, '77; Bonventre et al., '78; Lacy and Schmidt-Nielsen, '79). Although this concept challenges the generally held belief that fluid exiting the Ductus Bellini is equivalent to the final urine, it provides a new perspective for understanding the maintenance of the medullary solute gradient and the production of hyper- and hypotonic urine. In a recently completed study of the hamster renal pelvis (Lacy and Schmidt-Nielsen, '791, it was shown that the outer medulla, as well as the inner medulla, may be an important site for solute and water exchange with pelvic urine. This conclusion was based parAM. J. ANAT. (1979)155: 403-424
tially on the fact that the outer medulla constituted about 50%, and the inner medulla about 25% of the total pelvic surface area. In addition, a thin epithelium separated pelvic urine from the outer medullary tubules and capillaries. The morphological differences between the outer and inner medullary pelvic epithelium further suggested that the permeability properties of these two epithelia are probably quite different. At the present time there have been only two reports published on the ultrastructure of the entire pelvic epithelium (Silverblatt, '74; Khorshid and Moffat, '74). Although both studies presented consistent results concerning the cortical and inner medullary epithelium, there was sharp disagreement over the ultrastructure of the outer medullary epitheReceived Sept. 18,'78. Accepted Mar 22, '79. I Present address: Electron Microscope Laboratories, Hannover Medical School, Karl-Wiechert-Allee 9. 3000 Hannover 61, Germany. 'To whom reprint requests should be addressed.
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lium. The paucity of ultrastructural studies of weighed five days per week including t h e two the pelvis and the controversy concerning the days prior to use. The weight of each animal at fine structure of t h e pelvic epithelium cover- the time of death was within 4% of their ing the outer medulla have thus made further weights at the beginning of the dietary study of this region of the kidney necessary. In regime. addition, Schmidt-Nielsen ('77) has suggested Surgical procedure and tissue fixation that t h e renal pelvis may also be the site of increased urea reabsorption in mammals mainFixation of pelvic tissue for electron microstained on a low-protein diet. copy was achieved by either (1) vascular perThe present study was undertaken to (1)de- fusion or (2) retrograde pelvic superfusion scribe t h e ultrastructure of t h e pelvic epithe- through the intact ureter. lium covering each major kidney zone, (2) quantify by morphometric techniques t h e Vascular perfusion spatial organization of outer medullary t u Twenty-four hamsters were used. Sevenbules and capillaries juxtaposed to t h e pelvic teen comprised group I, of which nine had epithelium, (3)determine the thickness of the water ad libitum, and eight were deprived of outer medullary pelvic epithelium over each water for 24 to 36 hours prior to perfusion. All type of tubule and capillary, and (4) determine seven hamsters from group I1 were deprived of whether a low-protein diet induced a n y drinking water for 24 to 36 hours prior to use, changes in the outer medullary pelvic epithe- in order to induce elevated urine osmolality, lium or !,he parenchyma just under it. during which urea conservation is maximal The results show t h a t the large outer med- (Schmidt-Nielsen, '58). Anesthesia, the techullary pelvic surface area is covered by a n epi- niques for surgery and vascular perfusion, as thelium which is ultrastructurally different well as the composition of the vascular buffer from that covering either t h e cortex or t h e in- rinse, were identical to those described previner medulla. The abundance of outer medul- ously (Lacy and Schmidt-Nielsen, '79). Fixalary capillaries, as well as their close prox- tive solutions used were either: (1)5 or 6%gluimity to the pelvic epithelium, strongly sug- taraldehyde in 0.2 M sodium cacodylate buffer gests that they play a n important role in or (2) 2%glutaraldehyde and 3%formaldehyde exchange of solutes and/or water with pelvic (Karnovsky, '65) diluted to the appropriate urine. A low-protein diet did not induce osmolality with 0.1 M sodium cacodylate changes in any of the measured parameters. buffer. The osmolality of fixative solutions was adjusted to approximately 800 mOsm/l MATERIALS AND METHODS for the outer medulla or 1,200 mOsm/l for the Right and left kidneys from 28 Syrian ham- inner medulla. sters (16 females, 12 males) were studied by light milcroscopy of 1-pm-thick epoxy-resin Pelvic superfusion sections and by transmission electron microsFour hamsters from group I were used. Two copy. All animals weighed between 90-115 had been deprived of drinking water for 24 grams and were obtained from a local supplier hours prior to surgery and two had water ad (Telaco, Inc., Bar Harbor, Maine) a t least six libitum. Anesthesia and preparation for surweeks prior to use. Twenty-one hamsters gery have been described previously (Lacy and (group I:l were used for an ultrastructural Schmidt-Nielsen, '79). In all four animals a study of t.he pelvic epithelium and seven ham- small midline incision was made near t h e sters (group 11) were used for morphometric pubis to allow access to the urinary bladder, analysis !of t h e fornix region of t h e pelvis. from which a urine sample was withdrawn. Urine osmolality was measured immediately, Diet using a vapor-pressure osmometer (Wescor, Group I: These animals were maintained on Inc.). The midline incision was then continued regular rat chow (Old Guilford J a x Lab 96) to t h e sternum and t h e right or left kidney exuntil used. Group 11: Four hamsters were posed by slightly retracting t h e viscera. The given a high-protein diet and three hamsters a viscera were then covered with surgical gauze low-protein diet for three weeks prior to use. and flooded with light mineral oil to avoid The diets, contained respectively 24% and 8% evaporative water loss. Ten to fifteen mm protein (39-235 or 39-234, Zeigler Bros., from the papilla tip, the ureter was nicked to Gardner, Pennsylvania). The animals were allow entry of a n appropriately-sized poly-
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ethylene catheter into its lumen. The catheter tip was inserted 2-3 mm into the ureter and urine was collected by capillary action for 5 to 10 minutes in two animals (1 had water ad libitum and 1 was water-deprived). It was not possible to gain a ureteral urine sample in the other two animals. At the end of this period the catheter was withdrawn and osmolality was determined on each urine sample (Wescor Inc.). A new polyethylene catheter, with a diameter sized to fit loosely into the cut ureter, was inserted and connected to a 30 ml syringe containing fixative. The osmolality of the fixative was adjusted to within 90 mOsm of the urine (ureteral urine in 2 animals, bladder urine in the other two) by either dilution with 0.1 M sodium cacodylate buffer or addition of NaCl to the stock fixative, which consisted of 2% glutaraldehyde and 3%formaldehyde in 0.1 M sodium cacodylate (approximately 1,900 mOsm/kg water) (Karnovsky, ’65). The appropriately adjusted fixative was then superfused into the pelvis by an infusion pump (Harvard Apparatus Co.) a t a rate of 100-150 pl/minute for 15 to 20 minutes, during which time fixative flowed freely out the ureter around the catheter.
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ed solution of uranyl acetate (Watson, ’58) followed by counterstaining with lead citrate (Reynolds, ’63). Electron micrographs were made using a Hitachi HU-11C electron microscope. Morphometry Techniques One kidney from each of the seven hamsters in Group I1 (4 had a high-protein diet; 3 had a low-protein diet) was used for morphometric analysis of the fornix region of the pelvis. Following fixation of kidneys in situ, portions of the medial region of the upper pelvis, including one complete fornix from each kidney of each animal, were carefully excised. The tissue was trimmed by hand, perpendicular to the long axis of the interlobar artery and vein, until the fornix was exposed. (The orientation of the exposed fornix can be seen in Lacy and Schmidt-Nielsen, ’79: figs. 17, 18). Some tissue blocks exposed t h e fornix near t h e peripelvic columns, while in others the fornix was cut a t its upper extent. After the tissue was embedded in epoxy resin, one-micrometer thick sections were cut to verify the correct orientation of the fornix. For transmission electron microscopy, one Tissue preparation half of the fornix, which included the pelvic After the fixation of pelvic tissue in situ, space, the pelvic epithelium, the underlying the kidney was removed from each animal and parenchyma and one half of the interlobar appropriate areas of the pelvis excised and vein, was sectioned. These sections were colfixed in the original fixative for an additional lected on single-slotted formvar-coated copper 4 to 20 hours. All tissues, fixed by vascular grids which permitted an uninterrupted view perfusion or pelvic superfusion, were postfixed of each entire tissue section. Electron microin reduced osmium tetroxide (Karnovsky, ’71) graphs of the pelvic epithelium and underlyand embedded in epoxy resin, as previously de- ing tissue were then taken and printed a t a scribed (Lacy and Schmidt-Nielsen, ’791, for final magnification of 10,080 times. Microtransmission electron microscopy. graphs were arranged into a montage reconstructing the entire half of the fornix. (The avLight a n d electron microscopy erage length of the montage was approximateOne micrometer thick sections from each ly 14 meters.) Starting near the interlobar region of the pelvis were cut using an LKB U1- vein, the length of the mucosal membrane of tratome I11 and glass knives. The following re- the pelvic epithelium was divided into 40-mm gions of the pelvis (Lacy and Schmidt-Nielsen, units (fig. 1) which represented 3.97 p m on ’79) were sampled: (1)the entire papilla (in- the actual tissue. At the junction of each 40ner medulla) from its tip to its base, ( 2 ) each mm unit (termed “data-point’’ here) two disregion of the peripelvic columns (outer medul- tances were measured (fig. 1):(1)the shortest la), (3) the fornices (outer medulla) and (4) distance between the mucosal membrane of the outer wall of the lower pelvis (predomi- the pelvic epithelium and its basal lamina; nantly cortex). Sections were stained with to- this length represented the epithelial thickand sodium borate (1%) and ness; (b) the shortest distance between the luidine blue (1%) examined with the light microscope. Ap- point on the basal lamina from measurement propriate areas of pelvic tissue were sectioned (a) and the nearest tubule or capillary (termed for electron microscopy using a diamond “structure” here). This length was the inknife. Thin sections were stained in a saturat- terstitial distance between the pelvic epithe-
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Outer S t r i p e
1
Fig. 1 Diagram of half of fornix, which has been cut perpendicular to long axis of interlohar artery and vein. Dots on fixnix epithelium represent data-points where two measurements were made: (a) epithelial thickness and (b) interstitial distance. Tubules and capillaries were identified IJelow each data point. (PSp) Pelvic space.
lium and the underlying structure. The structure under each data-point was then identified as one of the following: (1) proximal tubule, (2) ascending thick limb of Henle, (3) descending thin limb of Henle, (4) collecting duct, ( 5 ) distal convoluted tubule, (6) fenestrated capillary, or ( 7 ) continuous capillary. In one hamster (high-protein diet) distinctions were not made between continuous and fenestrated capillaries. The morphology of the larger tubules was distinctive enough, as was that of the fenestrated capillaries, so t h a t positive identification of each structure could be made easily. Distinctions between the morphologically similar descending thin limbs of Henle and continuous capillaries were made by positive identification of these structures in tissue fixed by pelvic superfusion in which red blood cells were trapped in situ.
points, minus 1, and then dividing this value by t h e magnification of t h e micrograph (10,080); (2) t h e length and percentage of t h e fornix epithelium t h a t was underlain by the outer stripe of t h e outer medulla and by t h e inner stripe of the outer medulla; distinction between the two stripes of the outer medulla was made according to t h e criteria of Peter ('09) ; (3) mean epithelial thickness over each of the two stripes of the outer medulla as well as t h a t over each type of structure; (4)mean interstitial distance of each of the two stripes of the outer medulla as well a s that for each type of structure. Each of these values was then used to calculate t h e mean values for each dietary protein group (high and low) as well as for t h e entire population (n = 7 hamsters). The small interval between data-points assured t h a t t h e distribution of data-points accurately represented the smaller structures (capillaries, descending thin limbs of Henle) as well as t h e larger structures (proximal tubules, ascending thick limbs of Henle, etc.) The length of the interval between datapoints (3.97 p m on t h e tissue) was small enough t h a t each structure immediately under t h e pelvic epithelium was counted at least once and frequently twice or more. The distribution of data-points along the measured fornix lengths was then used to estimate the percentages of the fornix surface area underlain by each stripe of t h e outer medulla, as well as by each structure. Each fornix which was chosen for morphometric analysis resulted from random cuts (see above) and therefore the mean percentage values were considered typical of the entire fornix. RESULTS
There were no differences in the ultrastructure of t h e pelvis between: (1) male and female hamsters, (2) those on high- and lowprotein diets or (3) those with water ad libitum and those deprived of water. The following description of epithelial ultrastructure represents observations made from all groups of animals.
Outer medullary pelvic epithelium Data analysis The following values were calculated for each hamster: (1)the length of the fornix epithelium; these values were obtained from each hamster by multiplying t h e distance between adiacent data-Doints on the micrographs (40 mm) by the- total number of data-
Although a portion of the outer medulla is covered by transitional epithelium, t h e major surface area consists of what has previously been described as a simple epithelium with cells ranging from squamous to low cuboidal (Lacy and Schmidt-Nielsen, '79). Higher-magnification light microscopy (figs. 2-41, as w d l
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lar stripe of t h e outer medulla (inner stripe or outer stripe) ; instead, i t probably reflected t h e different stages of epithelial stretching during pelvic peristalsis as suggested previously (Lacy and Schmidt-Nielsen, ’79). Toluidine blue-staining granules were positioned in the apical cytoplasm of these cells (figs. 2-4) (termed “granular cells” here). Both t h e number and size of granules, however, were extremely variable from cell to cell, a s well as within different regions of the outer medulla. In some tissue sections, cells appeared devoid of granules; however, upon serial sectioning a t both t h e light and electron microscopic level, all cells were observed to contain some granules. Cells of t h e second type did not exhibit granules (figs. 2-41. These “basal cells” always appeared squamous and were most easily identified by their darkly staining ovoid nucleus, while the cytoplasm itself was difficult to visualize due to t h e attenuation of t h e cell as i t stretched along t h e basal lamina. Because basal cells were much fewer in number t h a n granular cells (particularly over the inner stripe, where we estimated t h a t basal cells were less t h a n 10%of the cell population) this epithelium can be considered simple. However t h e presence of basal cells makes t h e classification of pseudostratified epithelium more accurate. Ultrastructural observations of this epithelium confirmed the presence of the two cell types (fig. 5 ) described above. Granular cells Figs. 2-4 Light micrographs of outer medullary pelvic epithelium. Upper arrows (in pelvic space) indicate large consistently stained lighter t h a n did the basal darkly staining granules in apical region of granular cells, cells and had a rich complement of cellular orwhich are squamous (figs. 2, 4) or cuboidal (fig. 3). Lower ganelles, including mitochondria, Golgi comarrows (pointing towards top of page) indicate basal cells plex, rough endoplasmic reticulum, apically with darkly staining nuclei. Arrowhead inside tubule (fig. 2) indicates transition between pars recta segment of positioned granules, free ribonucleoprotein proximal tubule (PR) (outer stripe of outer medulla) and particles, glycogen and small vesicles (figs. 5, descending thin limb of Henle (DTL) (inner stripe of outer 6). The moderately abundant mitochondria medulla). (Ci Capillary. Magnifications: figure 2 x 618; were distributed evenly throughout t h e cytofigure 3 x 801; figure 4 x 2,250. plasm except in the apical region just below t h e mucosal membrane (figs. 5 , 6). The Golgi as electron microscopic observations (fig. 51, complex was most often positioned in t h e merevealed that the major portion of t h e outer dial third of the cell, sometimes in the lower medulla is covered by a n epithelium which third but never adjacent to t h e mucosal comprises two cell types: (1) cells which form plasma membrane (fig. 5 ) .Associated with t h e t h e mucosal epithelial surface and also con- Golgi complex were small membrane-bound tact t h e basal lamina and (2) cells which are spherical vesicles with either very lightly confined to the basal region of the epithelium staining or clear contents. Long strands of and do not reach t h e mucosal epithelial sur- rough endoplasmic reticulum were abundant face. throughout t h e cells, but were also rarely found adjacent to t h e mucosal membrane. The Cells of t h e first type ranged in shape from squamous to low cuboidal, but cellular shape number as well as the size of the granules indid not appear to be correlated with a particu- creased towards t h e mucosal plasma mem-
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Fig. 5 Electron micrograph of low cuboidal epithelium covering outer medulla. Darkly staining basal cell (dirwtly above 3 asterisks) is positioned under granular cells. Lateral cell margins (large arrows) are unconvoluted except in the region near basal cell. Asterisks lie on basal lamina. Membrane-bound granules (GR) positioned in mid- to apical region of cytoplasm. (N) Nucleus, (C) capillary with red blood cell, (RER) rough enda'plasmic reticulum. x 10,700.
brane, although isolated small granules were occasionally observed in the medial to lower one-third of the cytoplasm. These granules, which were irregularly spherical in shape (regardless of their position in the cell), consisted of a single unit membrane which enclosed a matrix of homogeneously fine particles ranging from light gray to black (figs. 5 , 6). The mucosal plasma membrane consisted of two equally thick, electron-dense units separated by an electron-lucent core, all of which formed a tripartite unit measuring 7.5-8.0nm thick (fig. 7 ) .The lateral as well as the basal margins of these cells were formed by membranes which appeared identical to t h e mucosal plasma membrane. Plasma membranes of adjacent granular cells formed either long lines of fusion or multiple points of fusion (zonulae occludentes) near the apical
epithelial surface (fig. 8). Desmosomes could occasionally be found between adjacent granular cells, but were more common between granular and basal cells. Lateral cell margins were generally straight, without interdigitations, for much of the cells' depth (figs. 5 , 6). Nearer t h e basal lamina these margins became interdigitated with those of adjacent granular cells and with basal cells, when they were present (fig. 5 ) . Dilated intercellular spaces between adjacent granular cells (as well as between granular cells and basal cells) were erratically present in kidneys fixed by vascular perfusion or by pelvic superfusion. Extending away from the nuclear region, pseudopod-like processes of b a s a l cells stretched along the basal lamina, both under and between granular cells (fig. 5 ) . The smaller mitochondria, which were not as
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Fig. 6 Higher-magnification electron micrograph of apical region of two granular cells which are a t tached a t mucosal margin by zonula occludens (ZO).Lateral intercellular spaces (IS) are slightly dilated. Membrane-bound granules (GR) contain flocculent matrix. (N) Nucleus. x 46,935.
abundant a s in granular cells, were evenly distributed in all parts of t h e cytoplasm. The Golgi apparatus was in t h e lateral perinuclear position, while t h e rough endoplasmic reticulum was less dense in these cells, but free ribonucleoprotein particles were extremely abundant. Some microfilaments were observed, but not in bundles. Granules were not identified in any part of these cells. The upper plasma membrane (that nearer t h e mucosal epithelial surface) was not as highly interdigitated as were t h e lateral borders of t h e cell (fig. 5).The basal cell border was flat without broad interdigitations. The basal lamina was a darkly staining material which was distinctly separated from both t h e epithelial plasma mem-
brane and t h e interstitial matrix by a more lightly staining region (fig. 5 ) . Inner medullary pelvic epithelium
Light microscopy revealed t h a t a single layer of tall columnar cells, which surrounded t h e papilla tip (fig. 91, became progressively shorter towards t h e inner-outer medullary junction (fig. 10). Along t h e same axis, there were also differences in lateral cell borders, mucosal membranes, nuclear shape and the affinity for toluidine blue stain. Dilated lateral intercellular spaces were consistently observed only a t the papilla tip (figs. 9, 10) when t h e pelvis was fixed by superfusion. Kidneys fixed by vascular perfu-
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Fig. 9 Light micrograph of columnar pelvic epithelium a t papilla tip. Lateral intercellular spaces are dilated (arrows). Cells stain from light gray to intensely dark. Nuclei (N) are irregularly shaped. (C) Capillary. x 1,360.
Fig 7 High-magnification electron micrograph of mucosal membrane of granular cell. Tripartite structure of membrane (between arrows) is the same over microvilli (MV) as well as between them. Membrane measures approximately 7-8 nm thick. X 225,800. Fig. 8 High-power electron micrograph of zonula occludens (ZO) of two adjacent granular cells. Outer leaflets of adjacent. membranes fuse along a line. x 117,100.
sion, however, revealed a n inconsistent distribution of these dilated spaces. The mucosal membranes of cells covering t h e lower onehalf to one-fourth of the papilla were often slightly convex toward t h e pelvic space. Nearer the outer medulla, t h e mucosal membranes were flatter and more irregular. Although nuclear shape was extremely variable among hamsters, the epithelium nearer t h e
area cribrosa contained cells with irregularly rounded nuclei as well as nearly spherical nuclei. Nearer the outer medulla the nuclei were more consistently irregular but not as darkly staining. A distinctive feature of the inner medullary pelvic epithelium was the variability in staining among individual cells. There was a gradation of toluidine blue-staining from those cells which were very faint to those t h a t were so intensely blue t h a t little cellular detail could be observed (figs. 9, 10). This variability in staining generally decreased from the papilla tip to the outer-inner medullary junction, where darkly staining cells were not observed. There was, however, a great deal of variability among hamsters in the number and distribution Of darker along t h e Papilla. In Some hamsters only the papilla tip had deeply staining cells such as those shown in figure 9, while in other hamsters these extended to the regions of t h e papilla (fig. 10). Likewise, t h e incidence of these cells ranged from very numerous, as seen in figures 9 and 10, to nearly absent in some other animals. Both the mucosal membrane and the nucleus of the darker cells were more irregularly shaped than those of the lighter cells. Ultrastructural observations of the inner medullary pelvic epithelium (fig. 11) confirmed the presence of more darkly staining cells, as had been observed by light microscopy. Although these darker cells had some dis-
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Fig. 10 Low-power light micrograph of pelvic epithelium covering inner medulla midway between papilla tip and outer medulla. Darker staining cells (arrows) are interspersed among more lightly staining cells. Note darkly staining cells (DK) of collecting ducts (CD). X 450.
tinctive morphological features, their subcellular organization appeared to be basically the same as that of the adjacent lightly stained cells. Ultrastructural similarities between these two forms of cells will be reported first, followed by the morphological characteristics that distinguished the darker cells from the lighter ones. Mitochondria were generally found throughout the cells with the exception of the area just under the mucosal membrane and around the nucleus (figs. 11, 12). The Golgi complex was present in the perinuclear region but not uncommonly in the upper half of the cytoplasm. The nuclear contents of both light and dark cells were extremely homogenous in contrast to what was observed in the outer medullary and cortical pelvic epithelium. Small membrane-bound vesicles were abundant (fig. 12) throughout the cytoplasm, being particularly concentrated in the apical region. They were generally free of large inclusions, but had lightly flocculent contents (fig. 12). They were formed by a 7.5-8.0 nm-thick membrane with the typical symmetrical tripartite structure. Scattered filaments in the cytoplasm did not appear in bundles. Although free ribosomes were present in the perinuclear cytoplasm there was a conspicuous paucity of long strands of rough endoplasmic reticulum, as seen in the outer medullary pelvic epithelium. Small sacs of endoplasmic reticulum were present and had a more densely staining matrix than that of the cytoplasm. The mucosal membrane, which was formed into
numerous small microvilli (fig. 12) had the same trilaminar structure (fig. 13) as that of the outer medullary pelvic epithelial cells. Adjacent cells were attached by three to four points of membrane fusion, forming zonulae occludentes near their apical borders (fig. 13). Electron microscopy revealed that the lateral cell margins, which appeared straight or slightly curved with light microscopy (figs. 9, 101, were in fact highly interdigitated from the basal lamina region to the apical junctional complex (figs. 11-14).The basal plasma membrane was without interdigitation, being generally parallel with a slightly undulating basal lamina (fig. 14). This basal lamina did not appear as a distinct darkly staining layer underlying this epithelium, as i t did under the two other types of pelvic epithelia, but, instead, consisted of lightly staining homogeneously fine particles forming a broad band which abutted the basal plasma membrane and graded gently into the more flocculent but denser interstitial matrix (fig. 14). Basal laminar material appeared to extend into the lateral intercellular region between adjacent cells (fig. 14). In each case the following characteristics, which distinguished darker cells from lighter cells, were more prominent the darker the cell stained (fig. 11): (1) the contour of the mucosal membrane was more irregular in darker cells. (2) The cytoplasmic matrix of darker cells consisted of homogeneously fine particles throughout, while the cytoplasmic matrix of the lighter cells was flocculent and
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Fig. 11 Electron micrograph of inner medullary pelvic epithelium showing two darker staining cells between two lighter staining cells. Note extremely irregularly shaped nuclei and mucosal membrane of darker cells. Arrow indicates myelin-like figure within cytoplasm of darker cell. Asterisks indicate position of indistinct basal lamina. (N) Nucleus. X 6,237.
patchy. (3) Both t h e cytoplasm and nucleus were stained darker. (4)The darker cells had a more irregularly shaped nucleus. (5) There were numerous light areas within the darker cells which were not membrane-bound and appeared devoid of cytoplasmic matrix (figs. 11, 12). Within these lighter regions were intensely dark inclusions which appeared somewhat similar to myelin figures. Cortical pelvic epithelium
Light microscopy of t h e transitional epithe-
lium revealed striking differences in epithelial thickness and cell shape, which was presumably due to fixation of the tissue during various stages of pelvic peristalsis (figs. 15, 16). We observed only two cell types in this epithelium: (1) superficial cells which formed a single continuous sheet adjacent to the pelvic space; (2) basal cells, which formed t h e remainder of the epithelial thickness, were two to three cell layers deep near the outer medulla. All cell boundaries were extremely difficult to discern at t h e light microscopic
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Fig. 1 2 Electron micrograph of upper one-third of darker cells. Note the paucity of cellular organelles just under mucosal membrane. Arrowheads point to irregularly shaped vesicles with darker staining matrix. Arrows point to myelin-like figures. Lateral cell borders are highly interdigitated below tight junction (TJ), (M)mitochondria, (G) Golgi complex. X 20,115.
level. Superficial cells generally stained lighter than did basal cells, being so pale in some cases that determination of cellular detail was not possible. Both superficial and basal cells were squamous in the thin epithelium (fig. 15). In some tissue sections, however, the individual cells were not as attenuated and t h e epithelium was generally thicker. In these cases the superficial cells
were not as elongated but still remained squamous (fig. 16). The basal cells changed from a polyhedral or ovoid form in the thicker, “relaxed’ epithelium (fig. 16) to a thin, elongated shape in the thinner “stretched” epithelium (fig. 15). Ultrastructural observations of the transitional epithelium revealed that superficial cells and basal cells were morphologically dif-
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Fig. 15 Light micrograph of transitional epithelium in “stretched’ position. Tissue sample taken midway between pelvic septum and ureter. Arrows mark approximate position of basal lamina above smooth muscle (SM). Epithelial cells are squamous, as are their nuclei. X 297. Fig. 16 Light micrograph of transitional epithelium in “relaxed” position. Tissue sample-site same as figure 15. Arrows mark approximate position of basal lamina above smooth muscle (SM). Basal cells are more cuboidal as are their nuclei. Superficial cells line the mucosal epithelial surface and are somewhat squamous. x 552.
Fig. 13 High-magnification electron micrograph showing zonula occludens (ZO) of two adjacent lighter cells. Outer leaflets of adjacent plasma membranes fuse at several points (arrowheads). Mucosal membrane between and over microvilli has tripartite structure (arrows) measuring about 7-8 nm thick. X 157,573. Fig. 14 Electron micrograph of basal region of inner medullary pelvic epithelium. Asterisks mark position of basal lamina. Border between basal lamina and flocculent interstitial matrix (IMX) is not distinct. Material similar to that of basal lamina appears along lateral cell borders (arrows). x 20,666.
ferent (fig. 17). Individual superficial cells were extremely long with a spindle-shaped nucleus occupying the central region of the cytoplasm. The lower three-fourths of these cells had more mitochondria and more rough endoplasmic reticulum than did t h e apical region of the cytoplasm (fig. 17). Golgi complexes and bundles of filaments were most often located in the lateral perinuclear position, but occasionally were found throughout the cytoplasm with the exception of the region just under the mucosal membrane (figs. 17, 18). The filamentous bundles were more numerous and larger in the medial regions of the cell. The upper 15-20%of each superficial cell was predominantly occupied by discoid vesicles with their long axis generally parallel with that of the mucosal plasma membrane (fig. 17). The core of each vesicle, which appeared free of stained material, was surrounded by a tripartite unit membrane measuring about 12.012.5 nm in thickness (fig. 18). Two osmiophilic leaflets (outer and inner) were separated by
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Fig. 17 Low power electron micrograph of transitional epithelium and underlying smooth muscle (SM). Asterisks mark the approximate position of the basal lamina. Arrows indicate the border between t h e superficial cell (SC) and basal cells (BC). Mucosal epithelial surface is scalloped and immediately underlain by discoid vesicles (DV). Large bundles of filaments (F) are present in the cytoplasm of superficial cells, but not basal cells. (M) Mitochondria. X 12,483.
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Fig. 18 Higher-power electron micrograph of apical region of superficial cell. Mucosal membrane (top of photo) is tripartite in structure (between arrows) with outer leaflet thicker than inner leaflet. Discoid vesicles (DV) have identical structure (arrows), except that inner leaflet is thicker than outer. Both membranes measure about 12 nm thick. x 184,860. Fig. 19 Higher-power electron micrograph of junctional complex of adjacent transitional epithelial cells. Outer leaflets of adjacent membranes fuse along several points to form zonula occludens (ZO). (DV) Discoid vesicle with asymmetric unit membrane. X 53,334.
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a n electron-lucent interior. The inner leaflet, which appeared denser than t h e outer leaflet, measured about 6 nm in thickness while t h e outer leaflet measured about 3 nm in thickness (fig. 18). The tripartite structure of t h e mucosal plasma membrane was identical to t h a t of t h e discoid vesicles, with t h e exception t h a t in t h e discoid vesicles the inner leaflet was thicker than t h e outer leaflet (fig. 18). This asymmetric unit membrane was confined to t h e discoid vesicles and the mucosal plasma membrane; t h e lateral and basal plasma membranes were approximately 7-8 nm thick, with both membrane leaflets of uniform thickness. Adjacent superficial cells were attached by typical zonulae occludentes at their mucosal margins (fig. 19), where t h e fusion of adjacent outer membrane leaflets occurred along a relative long line or many points of fusion. There
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was moderate but wide interdigitation of both lateral and basal cell borders. Some basal cells had extremely long contacts with the basal lamina, while others rested on the basal lamina along a short distance, with the major portion of the cell projecting deeper into t h e medial regions of t h e epithelium. There was a uniform distribution of cell organelles throughout t h e cytoplasm, in contrast t o t h e superficial cells (fig. 20). Rough endoplasmic reticulum was slightly less abundant but more dilated, while the number and size of mitochondria were about t h e same as in the superficial cells. The Golgi complex, although prominent, was also less abundant in these cells. Discoid vesicles or asymmetric unit membrane were not present in basal cells. Very small, uniformly-round vesicles were present, being most abundant
Fig. 20 Electron micrograph of basal region of transitional epithelium. Distinct basal lamina (asterisks) separates epithelial cells from underlying smooth muscle (SM). Hemidesmosomes (HD) are present along basal region of cell, as are small invaginations of the plasma membrane (arrows). (G) Golgi complex. (BC) Basal cell, (SC) superficial cell. X 26,730.
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near the Golgi complex. Filaments were often present in small groups or scattered singly throughout the cell, but not in large bundles. Common borders between basal cells appeared convoluted, with large pseudopod-like extensions interdigitating widely between adjacent cells, in contrast to the highly interdigitated cell borders of t h e inner medullary pelvic epithelium. The basal border was generally flat, but some small involutions of this membrane were observed (fig. 20). Normal 7-8-nm-thick plasma membrane, consisting of two symmetrically thick leaflets, surrounded the cytoplasm on all sides. Contacts between adjacent basal cells, as well as between basal cells and superficial cells, was by desmosomes. Morp hometry
In each of the estimated parameters of the fornix: (1) surface area, (2) epithelial thickness and (3) interstitial distance, for each outer medullary stripe and each structure, there were no statistically significant differences between hamsters on high- and lowprotein diets (table 1).Therefore, statistical tests included the values from all hamsters (both high- and low-protein diets) as one population (tables 2, 3). The entire fornix in t h e medial region of the kidney was approximately double the length given for the mean “total” measured fornix length (1,423.2 pm, table 2) since only one-half of each fornix was measured here. The entire medial fornix had a
circumference of about 3 mm (2,845.5 p m ) , which consisted of approximately 30.5% outer stripe of the outer medulla and 69.5% inner stripe of t h e outer medulla. Surface area Although the actual fornix surface area itself cannot be determined from the measurements made here, the relative surface area has been estimated. The most striking result of these surfacearea estimations was t h a t more than 60% of t h e fornix epithelium was underlain by capillaries, in both outer and inner stripes of the outer medulla (table 3). In both stripes, the fenestrated capillaries immediately underlay about 1.5times more surface area than did t h e continuous capillaries. In each outer medullary stripe, over 90% of t h e estimated surface area was underlain by capillaries plus one additional structure. This structure was the proximal tubule (pars recta segment) in the outer stripe, while in the inner stripe i t was the ascending thick limb of Henle. An extremely small percentage (1.0%) of outer stripe epithelium was underlain by distal convoluted tubules, which are normally present in the cortex. Epithelial thickness The mean pelvic epithelial thickness (table 3) was 2.3 p m greater over the inner stripe than over the outer stripe (paired t-test, p
ABSTRACT
The renal pelvis of the hamster has been studied by light microscopy (epo y resin sections), transmission electron microscopy, and morphometric analysis of electron micrographs. Three morphologically distinct epithelia line the pelvis, and each covers a different zone of the kidney. A thin epithelium covering the outer medulla (OM) consists of two cell types: (1) granular cells are most numerous and have apically positioned granules which stain intensely with toluidine blue, are membrane-bound, and contain a fine particulate matter that stains light grey to black in electron micrographs. (2) Basal cells do not have granules, are confined to the basal lamina region, and do not reach the mucosal epithelial surface. The inner medulla (IM) is covered by a pelvic epithelium morphologically similar to collecting duct epithelium of IM. Some cells in this portion of the pelvic epithelium (IM) stain intensely dark with toluidine blue, osmium tetroxide, lead, and uranyl acetate. Transitional epithelium, which separates cortex (C) from pelvic urine, has an asymmetric luminal plasma membrane and discoid vesicles, each of which is similar to those previously observed in mammalian ureter and urinary bladder epithelia. Based on morphological comparisons with other epithelia, the IM and OM pelvic epithelia would appear permeable to solutes and/or water, while the transitional epithelium covering the C appears relatively impermeable. It would also appear that the exchange of solutes and water between pelvic urine and OM would involve capillaries, primarily, since morphometric analysis showed t h a t both fenestrated and continuous capillaries of the OM were extremely abundant ( >60% of OM pelvic surface area) just under the thin pelvic epithelium.
Recent anatomical and physiological studies have presented evidence that the dynamics of urine formation may include exchange of water andlor solute between pelvic urine and renal parenchyma (Gertz et al., '66; Pfeiffer, '68; Schutz and Schnermann, '72; Khorshid and Moffat, '74; Silverblatt, '74; Kaissling et al., '75; Schmidt-Nielsen, '77; Bonventre et al., '78; Lacy and Schmidt-Nielsen, '79). Although this concept challenges the generally held belief that fluid exiting the Ductus Bellini is equivalent to the final urine, it provides a new perspective for understanding the maintenance of the medullary solute gradient and the production of hyper- and hypotonic urine. In a recently completed study of the hamster renal pelvis (Lacy and Schmidt-Nielsen, '791, it was shown that the outer medulla, as well as the inner medulla, may be an important site for solute and water exchange with pelvic urine. This conclusion was based parAM. J. ANAT. (1979)155: 403-424
tially on the fact that the outer medulla constituted about 50%, and the inner medulla about 25% of the total pelvic surface area. In addition, a thin epithelium separated pelvic urine from the outer medullary tubules and capillaries. The morphological differences between the outer and inner medullary pelvic epithelium further suggested that the permeability properties of these two epithelia are probably quite different. At the present time there have been only two reports published on the ultrastructure of the entire pelvic epithelium (Silverblatt, '74; Khorshid and Moffat, '74). Although both studies presented consistent results concerning the cortical and inner medullary epithelium, there was sharp disagreement over the ultrastructure of the outer medullary epitheReceived Sept. 18,'78. Accepted Mar 22, '79. I Present address: Electron Microscope Laboratories, Hannover Medical School, Karl-Wiechert-Allee 9. 3000 Hannover 61, Germany. 'To whom reprint requests should be addressed.
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lium. The paucity of ultrastructural studies of weighed five days per week including t h e two the pelvis and the controversy concerning the days prior to use. The weight of each animal at fine structure of t h e pelvic epithelium cover- the time of death was within 4% of their ing the outer medulla have thus made further weights at the beginning of the dietary study of this region of the kidney necessary. In regime. addition, Schmidt-Nielsen ('77) has suggested Surgical procedure and tissue fixation that t h e renal pelvis may also be the site of increased urea reabsorption in mammals mainFixation of pelvic tissue for electron microstained on a low-protein diet. copy was achieved by either (1) vascular perThe present study was undertaken to (1)de- fusion or (2) retrograde pelvic superfusion scribe t h e ultrastructure of t h e pelvic epithe- through the intact ureter. lium covering each major kidney zone, (2) quantify by morphometric techniques t h e Vascular perfusion spatial organization of outer medullary t u Twenty-four hamsters were used. Sevenbules and capillaries juxtaposed to t h e pelvic teen comprised group I, of which nine had epithelium, (3)determine the thickness of the water ad libitum, and eight were deprived of outer medullary pelvic epithelium over each water for 24 to 36 hours prior to perfusion. All type of tubule and capillary, and (4) determine seven hamsters from group I1 were deprived of whether a low-protein diet induced a n y drinking water for 24 to 36 hours prior to use, changes in the outer medullary pelvic epithe- in order to induce elevated urine osmolality, lium or !,he parenchyma just under it. during which urea conservation is maximal The results show t h a t the large outer med- (Schmidt-Nielsen, '58). Anesthesia, the techullary pelvic surface area is covered by a n epi- niques for surgery and vascular perfusion, as thelium which is ultrastructurally different well as the composition of the vascular buffer from that covering either t h e cortex or t h e in- rinse, were identical to those described previner medulla. The abundance of outer medul- ously (Lacy and Schmidt-Nielsen, '79). Fixalary capillaries, as well as their close prox- tive solutions used were either: (1)5 or 6%gluimity to the pelvic epithelium, strongly sug- taraldehyde in 0.2 M sodium cacodylate buffer gests that they play a n important role in or (2) 2%glutaraldehyde and 3%formaldehyde exchange of solutes and/or water with pelvic (Karnovsky, '65) diluted to the appropriate urine. A low-protein diet did not induce osmolality with 0.1 M sodium cacodylate changes in any of the measured parameters. buffer. The osmolality of fixative solutions was adjusted to approximately 800 mOsm/l MATERIALS AND METHODS for the outer medulla or 1,200 mOsm/l for the Right and left kidneys from 28 Syrian ham- inner medulla. sters (16 females, 12 males) were studied by light milcroscopy of 1-pm-thick epoxy-resin Pelvic superfusion sections and by transmission electron microsFour hamsters from group I were used. Two copy. All animals weighed between 90-115 had been deprived of drinking water for 24 grams and were obtained from a local supplier hours prior to surgery and two had water ad (Telaco, Inc., Bar Harbor, Maine) a t least six libitum. Anesthesia and preparation for surweeks prior to use. Twenty-one hamsters gery have been described previously (Lacy and (group I:l were used for an ultrastructural Schmidt-Nielsen, '79). In all four animals a study of t.he pelvic epithelium and seven ham- small midline incision was made near t h e sters (group 11) were used for morphometric pubis to allow access to the urinary bladder, analysis !of t h e fornix region of t h e pelvis. from which a urine sample was withdrawn. Urine osmolality was measured immediately, Diet using a vapor-pressure osmometer (Wescor, Group I: These animals were maintained on Inc.). The midline incision was then continued regular rat chow (Old Guilford J a x Lab 96) to t h e sternum and t h e right or left kidney exuntil used. Group 11: Four hamsters were posed by slightly retracting t h e viscera. The given a high-protein diet and three hamsters a viscera were then covered with surgical gauze low-protein diet for three weeks prior to use. and flooded with light mineral oil to avoid The diets, contained respectively 24% and 8% evaporative water loss. Ten to fifteen mm protein (39-235 or 39-234, Zeigler Bros., from the papilla tip, the ureter was nicked to Gardner, Pennsylvania). The animals were allow entry of a n appropriately-sized poly-
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ethylene catheter into its lumen. The catheter tip was inserted 2-3 mm into the ureter and urine was collected by capillary action for 5 to 10 minutes in two animals (1 had water ad libitum and 1 was water-deprived). It was not possible to gain a ureteral urine sample in the other two animals. At the end of this period the catheter was withdrawn and osmolality was determined on each urine sample (Wescor Inc.). A new polyethylene catheter, with a diameter sized to fit loosely into the cut ureter, was inserted and connected to a 30 ml syringe containing fixative. The osmolality of the fixative was adjusted to within 90 mOsm of the urine (ureteral urine in 2 animals, bladder urine in the other two) by either dilution with 0.1 M sodium cacodylate buffer or addition of NaCl to the stock fixative, which consisted of 2% glutaraldehyde and 3%formaldehyde in 0.1 M sodium cacodylate (approximately 1,900 mOsm/kg water) (Karnovsky, ’65). The appropriately adjusted fixative was then superfused into the pelvis by an infusion pump (Harvard Apparatus Co.) a t a rate of 100-150 pl/minute for 15 to 20 minutes, during which time fixative flowed freely out the ureter around the catheter.
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ed solution of uranyl acetate (Watson, ’58) followed by counterstaining with lead citrate (Reynolds, ’63). Electron micrographs were made using a Hitachi HU-11C electron microscope. Morphometry Techniques One kidney from each of the seven hamsters in Group I1 (4 had a high-protein diet; 3 had a low-protein diet) was used for morphometric analysis of the fornix region of the pelvis. Following fixation of kidneys in situ, portions of the medial region of the upper pelvis, including one complete fornix from each kidney of each animal, were carefully excised. The tissue was trimmed by hand, perpendicular to the long axis of the interlobar artery and vein, until the fornix was exposed. (The orientation of the exposed fornix can be seen in Lacy and Schmidt-Nielsen, ’79: figs. 17, 18). Some tissue blocks exposed t h e fornix near t h e peripelvic columns, while in others the fornix was cut a t its upper extent. After the tissue was embedded in epoxy resin, one-micrometer thick sections were cut to verify the correct orientation of the fornix. For transmission electron microscopy, one Tissue preparation half of the fornix, which included the pelvic After the fixation of pelvic tissue in situ, space, the pelvic epithelium, the underlying the kidney was removed from each animal and parenchyma and one half of the interlobar appropriate areas of the pelvis excised and vein, was sectioned. These sections were colfixed in the original fixative for an additional lected on single-slotted formvar-coated copper 4 to 20 hours. All tissues, fixed by vascular grids which permitted an uninterrupted view perfusion or pelvic superfusion, were postfixed of each entire tissue section. Electron microin reduced osmium tetroxide (Karnovsky, ’71) graphs of the pelvic epithelium and underlyand embedded in epoxy resin, as previously de- ing tissue were then taken and printed a t a scribed (Lacy and Schmidt-Nielsen, ’791, for final magnification of 10,080 times. Microtransmission electron microscopy. graphs were arranged into a montage reconstructing the entire half of the fornix. (The avLight a n d electron microscopy erage length of the montage was approximateOne micrometer thick sections from each ly 14 meters.) Starting near the interlobar region of the pelvis were cut using an LKB U1- vein, the length of the mucosal membrane of tratome I11 and glass knives. The following re- the pelvic epithelium was divided into 40-mm gions of the pelvis (Lacy and Schmidt-Nielsen, units (fig. 1) which represented 3.97 p m on ’79) were sampled: (1)the entire papilla (in- the actual tissue. At the junction of each 40ner medulla) from its tip to its base, ( 2 ) each mm unit (termed “data-point’’ here) two disregion of the peripelvic columns (outer medul- tances were measured (fig. 1):(1)the shortest la), (3) the fornices (outer medulla) and (4) distance between the mucosal membrane of the outer wall of the lower pelvis (predomi- the pelvic epithelium and its basal lamina; nantly cortex). Sections were stained with to- this length represented the epithelial thickand sodium borate (1%) and ness; (b) the shortest distance between the luidine blue (1%) examined with the light microscope. Ap- point on the basal lamina from measurement propriate areas of pelvic tissue were sectioned (a) and the nearest tubule or capillary (termed for electron microscopy using a diamond “structure” here). This length was the inknife. Thin sections were stained in a saturat- terstitial distance between the pelvic epithe-
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Outer S t r i p e
1
Fig. 1 Diagram of half of fornix, which has been cut perpendicular to long axis of interlohar artery and vein. Dots on fixnix epithelium represent data-points where two measurements were made: (a) epithelial thickness and (b) interstitial distance. Tubules and capillaries were identified IJelow each data point. (PSp) Pelvic space.
lium and the underlying structure. The structure under each data-point was then identified as one of the following: (1) proximal tubule, (2) ascending thick limb of Henle, (3) descending thin limb of Henle, (4) collecting duct, ( 5 ) distal convoluted tubule, (6) fenestrated capillary, or ( 7 ) continuous capillary. In one hamster (high-protein diet) distinctions were not made between continuous and fenestrated capillaries. The morphology of the larger tubules was distinctive enough, as was that of the fenestrated capillaries, so t h a t positive identification of each structure could be made easily. Distinctions between the morphologically similar descending thin limbs of Henle and continuous capillaries were made by positive identification of these structures in tissue fixed by pelvic superfusion in which red blood cells were trapped in situ.
points, minus 1, and then dividing this value by t h e magnification of t h e micrograph (10,080); (2) t h e length and percentage of t h e fornix epithelium t h a t was underlain by the outer stripe of t h e outer medulla and by t h e inner stripe of the outer medulla; distinction between the two stripes of the outer medulla was made according to t h e criteria of Peter ('09) ; (3) mean epithelial thickness over each of the two stripes of the outer medulla as well as t h a t over each type of structure; (4)mean interstitial distance of each of the two stripes of the outer medulla as well a s that for each type of structure. Each of these values was then used to calculate t h e mean values for each dietary protein group (high and low) as well as for t h e entire population (n = 7 hamsters). The small interval between data-points assured t h a t t h e distribution of data-points accurately represented the smaller structures (capillaries, descending thin limbs of Henle) as well as t h e larger structures (proximal tubules, ascending thick limbs of Henle, etc.) The length of the interval between datapoints (3.97 p m on t h e tissue) was small enough t h a t each structure immediately under t h e pelvic epithelium was counted at least once and frequently twice or more. The distribution of data-points along the measured fornix lengths was then used to estimate the percentages of the fornix surface area underlain by each stripe of t h e outer medulla, as well as by each structure. Each fornix which was chosen for morphometric analysis resulted from random cuts (see above) and therefore the mean percentage values were considered typical of the entire fornix. RESULTS
There were no differences in the ultrastructure of t h e pelvis between: (1) male and female hamsters, (2) those on high- and lowprotein diets or (3) those with water ad libitum and those deprived of water. The following description of epithelial ultrastructure represents observations made from all groups of animals.
Outer medullary pelvic epithelium Data analysis The following values were calculated for each hamster: (1)the length of the fornix epithelium; these values were obtained from each hamster by multiplying t h e distance between adiacent data-Doints on the micrographs (40 mm) by the- total number of data-
Although a portion of the outer medulla is covered by transitional epithelium, t h e major surface area consists of what has previously been described as a simple epithelium with cells ranging from squamous to low cuboidal (Lacy and Schmidt-Nielsen, '79). Higher-magnification light microscopy (figs. 2-41, as w d l
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lar stripe of t h e outer medulla (inner stripe or outer stripe) ; instead, i t probably reflected t h e different stages of epithelial stretching during pelvic peristalsis as suggested previously (Lacy and Schmidt-Nielsen, ’79). Toluidine blue-staining granules were positioned in the apical cytoplasm of these cells (figs. 2-4) (termed “granular cells” here). Both t h e number and size of granules, however, were extremely variable from cell to cell, a s well as within different regions of the outer medulla. In some tissue sections, cells appeared devoid of granules; however, upon serial sectioning a t both t h e light and electron microscopic level, all cells were observed to contain some granules. Cells of t h e second type did not exhibit granules (figs. 2-41. These “basal cells” always appeared squamous and were most easily identified by their darkly staining ovoid nucleus, while the cytoplasm itself was difficult to visualize due to t h e attenuation of t h e cell as i t stretched along t h e basal lamina. Because basal cells were much fewer in number t h a n granular cells (particularly over the inner stripe, where we estimated t h a t basal cells were less t h a n 10%of the cell population) this epithelium can be considered simple. However t h e presence of basal cells makes t h e classification of pseudostratified epithelium more accurate. Ultrastructural observations of this epithelium confirmed the presence of the two cell types (fig. 5 ) described above. Granular cells Figs. 2-4 Light micrographs of outer medullary pelvic epithelium. Upper arrows (in pelvic space) indicate large consistently stained lighter t h a n did the basal darkly staining granules in apical region of granular cells, cells and had a rich complement of cellular orwhich are squamous (figs. 2, 4) or cuboidal (fig. 3). Lower ganelles, including mitochondria, Golgi comarrows (pointing towards top of page) indicate basal cells plex, rough endoplasmic reticulum, apically with darkly staining nuclei. Arrowhead inside tubule (fig. 2) indicates transition between pars recta segment of positioned granules, free ribonucleoprotein proximal tubule (PR) (outer stripe of outer medulla) and particles, glycogen and small vesicles (figs. 5, descending thin limb of Henle (DTL) (inner stripe of outer 6). The moderately abundant mitochondria medulla). (Ci Capillary. Magnifications: figure 2 x 618; were distributed evenly throughout t h e cytofigure 3 x 801; figure 4 x 2,250. plasm except in the apical region just below t h e mucosal membrane (figs. 5 , 6). The Golgi as electron microscopic observations (fig. 51, complex was most often positioned in t h e merevealed that the major portion of t h e outer dial third of the cell, sometimes in the lower medulla is covered by a n epithelium which third but never adjacent to t h e mucosal comprises two cell types: (1) cells which form plasma membrane (fig. 5 ) .Associated with t h e t h e mucosal epithelial surface and also con- Golgi complex were small membrane-bound tact t h e basal lamina and (2) cells which are spherical vesicles with either very lightly confined to the basal region of the epithelium staining or clear contents. Long strands of and do not reach t h e mucosal epithelial sur- rough endoplasmic reticulum were abundant face. throughout t h e cells, but were also rarely found adjacent to t h e mucosal membrane. The Cells of t h e first type ranged in shape from squamous to low cuboidal, but cellular shape number as well as the size of the granules indid not appear to be correlated with a particu- creased towards t h e mucosal plasma mem-
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Fig. 5 Electron micrograph of low cuboidal epithelium covering outer medulla. Darkly staining basal cell (dirwtly above 3 asterisks) is positioned under granular cells. Lateral cell margins (large arrows) are unconvoluted except in the region near basal cell. Asterisks lie on basal lamina. Membrane-bound granules (GR) positioned in mid- to apical region of cytoplasm. (N) Nucleus, (C) capillary with red blood cell, (RER) rough enda'plasmic reticulum. x 10,700.
brane, although isolated small granules were occasionally observed in the medial to lower one-third of the cytoplasm. These granules, which were irregularly spherical in shape (regardless of their position in the cell), consisted of a single unit membrane which enclosed a matrix of homogeneously fine particles ranging from light gray to black (figs. 5 , 6). The mucosal plasma membrane consisted of two equally thick, electron-dense units separated by an electron-lucent core, all of which formed a tripartite unit measuring 7.5-8.0nm thick (fig. 7 ) .The lateral as well as the basal margins of these cells were formed by membranes which appeared identical to t h e mucosal plasma membrane. Plasma membranes of adjacent granular cells formed either long lines of fusion or multiple points of fusion (zonulae occludentes) near the apical
epithelial surface (fig. 8). Desmosomes could occasionally be found between adjacent granular cells, but were more common between granular and basal cells. Lateral cell margins were generally straight, without interdigitations, for much of the cells' depth (figs. 5 , 6). Nearer t h e basal lamina these margins became interdigitated with those of adjacent granular cells and with basal cells, when they were present (fig. 5 ) . Dilated intercellular spaces between adjacent granular cells (as well as between granular cells and basal cells) were erratically present in kidneys fixed by vascular perfusion or by pelvic superfusion. Extending away from the nuclear region, pseudopod-like processes of b a s a l cells stretched along the basal lamina, both under and between granular cells (fig. 5 ) . The smaller mitochondria, which were not as
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Fig. 6 Higher-magnification electron micrograph of apical region of two granular cells which are a t tached a t mucosal margin by zonula occludens (ZO).Lateral intercellular spaces (IS) are slightly dilated. Membrane-bound granules (GR) contain flocculent matrix. (N) Nucleus. x 46,935.
abundant a s in granular cells, were evenly distributed in all parts of t h e cytoplasm. The Golgi apparatus was in t h e lateral perinuclear position, while t h e rough endoplasmic reticulum was less dense in these cells, but free ribonucleoprotein particles were extremely abundant. Some microfilaments were observed, but not in bundles. Granules were not identified in any part of these cells. The upper plasma membrane (that nearer t h e mucosal epithelial surface) was not as highly interdigitated as were t h e lateral borders of t h e cell (fig. 5).The basal cell border was flat without broad interdigitations. The basal lamina was a darkly staining material which was distinctly separated from both t h e epithelial plasma mem-
brane and t h e interstitial matrix by a more lightly staining region (fig. 5 ) . Inner medullary pelvic epithelium
Light microscopy revealed t h a t a single layer of tall columnar cells, which surrounded t h e papilla tip (fig. 91, became progressively shorter towards t h e inner-outer medullary junction (fig. 10). Along t h e same axis, there were also differences in lateral cell borders, mucosal membranes, nuclear shape and the affinity for toluidine blue stain. Dilated lateral intercellular spaces were consistently observed only a t the papilla tip (figs. 9, 10) when t h e pelvis was fixed by superfusion. Kidneys fixed by vascular perfu-
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Fig. 9 Light micrograph of columnar pelvic epithelium a t papilla tip. Lateral intercellular spaces are dilated (arrows). Cells stain from light gray to intensely dark. Nuclei (N) are irregularly shaped. (C) Capillary. x 1,360.
Fig 7 High-magnification electron micrograph of mucosal membrane of granular cell. Tripartite structure of membrane (between arrows) is the same over microvilli (MV) as well as between them. Membrane measures approximately 7-8 nm thick. X 225,800. Fig. 8 High-power electron micrograph of zonula occludens (ZO) of two adjacent granular cells. Outer leaflets of adjacent. membranes fuse along a line. x 117,100.
sion, however, revealed a n inconsistent distribution of these dilated spaces. The mucosal membranes of cells covering t h e lower onehalf to one-fourth of the papilla were often slightly convex toward t h e pelvic space. Nearer the outer medulla, t h e mucosal membranes were flatter and more irregular. Although nuclear shape was extremely variable among hamsters, the epithelium nearer t h e
area cribrosa contained cells with irregularly rounded nuclei as well as nearly spherical nuclei. Nearer the outer medulla the nuclei were more consistently irregular but not as darkly staining. A distinctive feature of the inner medullary pelvic epithelium was the variability in staining among individual cells. There was a gradation of toluidine blue-staining from those cells which were very faint to those t h a t were so intensely blue t h a t little cellular detail could be observed (figs. 9, 10). This variability in staining generally decreased from the papilla tip to the outer-inner medullary junction, where darkly staining cells were not observed. There was, however, a great deal of variability among hamsters in the number and distribution Of darker along t h e Papilla. In Some hamsters only the papilla tip had deeply staining cells such as those shown in figure 9, while in other hamsters these extended to the regions of t h e papilla (fig. 10). Likewise, t h e incidence of these cells ranged from very numerous, as seen in figures 9 and 10, to nearly absent in some other animals. Both the mucosal membrane and the nucleus of the darker cells were more irregularly shaped than those of the lighter cells. Ultrastructural observations of the inner medullary pelvic epithelium (fig. 11) confirmed the presence of more darkly staining cells, as had been observed by light microscopy. Although these darker cells had some dis-
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Fig. 10 Low-power light micrograph of pelvic epithelium covering inner medulla midway between papilla tip and outer medulla. Darker staining cells (arrows) are interspersed among more lightly staining cells. Note darkly staining cells (DK) of collecting ducts (CD). X 450.
tinctive morphological features, their subcellular organization appeared to be basically the same as that of the adjacent lightly stained cells. Ultrastructural similarities between these two forms of cells will be reported first, followed by the morphological characteristics that distinguished the darker cells from the lighter ones. Mitochondria were generally found throughout the cells with the exception of the area just under the mucosal membrane and around the nucleus (figs. 11, 12). The Golgi complex was present in the perinuclear region but not uncommonly in the upper half of the cytoplasm. The nuclear contents of both light and dark cells were extremely homogenous in contrast to what was observed in the outer medullary and cortical pelvic epithelium. Small membrane-bound vesicles were abundant (fig. 12) throughout the cytoplasm, being particularly concentrated in the apical region. They were generally free of large inclusions, but had lightly flocculent contents (fig. 12). They were formed by a 7.5-8.0 nm-thick membrane with the typical symmetrical tripartite structure. Scattered filaments in the cytoplasm did not appear in bundles. Although free ribosomes were present in the perinuclear cytoplasm there was a conspicuous paucity of long strands of rough endoplasmic reticulum, as seen in the outer medullary pelvic epithelium. Small sacs of endoplasmic reticulum were present and had a more densely staining matrix than that of the cytoplasm. The mucosal membrane, which was formed into
numerous small microvilli (fig. 12) had the same trilaminar structure (fig. 13) as that of the outer medullary pelvic epithelial cells. Adjacent cells were attached by three to four points of membrane fusion, forming zonulae occludentes near their apical borders (fig. 13). Electron microscopy revealed that the lateral cell margins, which appeared straight or slightly curved with light microscopy (figs. 9, 101, were in fact highly interdigitated from the basal lamina region to the apical junctional complex (figs. 11-14).The basal plasma membrane was without interdigitation, being generally parallel with a slightly undulating basal lamina (fig. 14). This basal lamina did not appear as a distinct darkly staining layer underlying this epithelium, as i t did under the two other types of pelvic epithelia, but, instead, consisted of lightly staining homogeneously fine particles forming a broad band which abutted the basal plasma membrane and graded gently into the more flocculent but denser interstitial matrix (fig. 14). Basal laminar material appeared to extend into the lateral intercellular region between adjacent cells (fig. 14). In each case the following characteristics, which distinguished darker cells from lighter cells, were more prominent the darker the cell stained (fig. 11): (1) the contour of the mucosal membrane was more irregular in darker cells. (2) The cytoplasmic matrix of darker cells consisted of homogeneously fine particles throughout, while the cytoplasmic matrix of the lighter cells was flocculent and
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Fig. 11 Electron micrograph of inner medullary pelvic epithelium showing two darker staining cells between two lighter staining cells. Note extremely irregularly shaped nuclei and mucosal membrane of darker cells. Arrow indicates myelin-like figure within cytoplasm of darker cell. Asterisks indicate position of indistinct basal lamina. (N) Nucleus. X 6,237.
patchy. (3) Both t h e cytoplasm and nucleus were stained darker. (4)The darker cells had a more irregularly shaped nucleus. (5) There were numerous light areas within the darker cells which were not membrane-bound and appeared devoid of cytoplasmic matrix (figs. 11, 12). Within these lighter regions were intensely dark inclusions which appeared somewhat similar to myelin figures. Cortical pelvic epithelium
Light microscopy of t h e transitional epithe-
lium revealed striking differences in epithelial thickness and cell shape, which was presumably due to fixation of the tissue during various stages of pelvic peristalsis (figs. 15, 16). We observed only two cell types in this epithelium: (1) superficial cells which formed a single continuous sheet adjacent to the pelvic space; (2) basal cells, which formed t h e remainder of the epithelial thickness, were two to three cell layers deep near the outer medulla. All cell boundaries were extremely difficult to discern at t h e light microscopic
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Fig. 1 2 Electron micrograph of upper one-third of darker cells. Note the paucity of cellular organelles just under mucosal membrane. Arrowheads point to irregularly shaped vesicles with darker staining matrix. Arrows point to myelin-like figures. Lateral cell borders are highly interdigitated below tight junction (TJ), (M)mitochondria, (G) Golgi complex. X 20,115.
level. Superficial cells generally stained lighter than did basal cells, being so pale in some cases that determination of cellular detail was not possible. Both superficial and basal cells were squamous in the thin epithelium (fig. 15). In some tissue sections, however, the individual cells were not as attenuated and t h e epithelium was generally thicker. In these cases the superficial cells
were not as elongated but still remained squamous (fig. 16). The basal cells changed from a polyhedral or ovoid form in the thicker, “relaxed’ epithelium (fig. 16) to a thin, elongated shape in the thinner “stretched” epithelium (fig. 15). Ultrastructural observations of the transitional epithelium revealed that superficial cells and basal cells were morphologically dif-
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Fig. 15 Light micrograph of transitional epithelium in “stretched’ position. Tissue sample taken midway between pelvic septum and ureter. Arrows mark approximate position of basal lamina above smooth muscle (SM). Epithelial cells are squamous, as are their nuclei. X 297. Fig. 16 Light micrograph of transitional epithelium in “relaxed” position. Tissue sample-site same as figure 15. Arrows mark approximate position of basal lamina above smooth muscle (SM). Basal cells are more cuboidal as are their nuclei. Superficial cells line the mucosal epithelial surface and are somewhat squamous. x 552.
Fig. 13 High-magnification electron micrograph showing zonula occludens (ZO) of two adjacent lighter cells. Outer leaflets of adjacent plasma membranes fuse at several points (arrowheads). Mucosal membrane between and over microvilli has tripartite structure (arrows) measuring about 7-8 nm thick. X 157,573. Fig. 14 Electron micrograph of basal region of inner medullary pelvic epithelium. Asterisks mark position of basal lamina. Border between basal lamina and flocculent interstitial matrix (IMX) is not distinct. Material similar to that of basal lamina appears along lateral cell borders (arrows). x 20,666.
ferent (fig. 17). Individual superficial cells were extremely long with a spindle-shaped nucleus occupying the central region of the cytoplasm. The lower three-fourths of these cells had more mitochondria and more rough endoplasmic reticulum than did t h e apical region of the cytoplasm (fig. 17). Golgi complexes and bundles of filaments were most often located in the lateral perinuclear position, but occasionally were found throughout the cytoplasm with the exception of the region just under the mucosal membrane (figs. 17, 18). The filamentous bundles were more numerous and larger in the medial regions of the cell. The upper 15-20%of each superficial cell was predominantly occupied by discoid vesicles with their long axis generally parallel with that of the mucosal plasma membrane (fig. 17). The core of each vesicle, which appeared free of stained material, was surrounded by a tripartite unit membrane measuring about 12.012.5 nm in thickness (fig. 18). Two osmiophilic leaflets (outer and inner) were separated by
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Fig. 17 Low power electron micrograph of transitional epithelium and underlying smooth muscle (SM). Asterisks mark the approximate position of the basal lamina. Arrows indicate the border between t h e superficial cell (SC) and basal cells (BC). Mucosal epithelial surface is scalloped and immediately underlain by discoid vesicles (DV). Large bundles of filaments (F) are present in the cytoplasm of superficial cells, but not basal cells. (M) Mitochondria. X 12,483.
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Fig. 18 Higher-power electron micrograph of apical region of superficial cell. Mucosal membrane (top of photo) is tripartite in structure (between arrows) with outer leaflet thicker than inner leaflet. Discoid vesicles (DV) have identical structure (arrows), except that inner leaflet is thicker than outer. Both membranes measure about 12 nm thick. x 184,860. Fig. 19 Higher-power electron micrograph of junctional complex of adjacent transitional epithelial cells. Outer leaflets of adjacent membranes fuse along several points to form zonula occludens (ZO). (DV) Discoid vesicle with asymmetric unit membrane. X 53,334.
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a n electron-lucent interior. The inner leaflet, which appeared denser than t h e outer leaflet, measured about 6 nm in thickness while t h e outer leaflet measured about 3 nm in thickness (fig. 18). The tripartite structure of t h e mucosal plasma membrane was identical to t h a t of t h e discoid vesicles, with t h e exception t h a t in t h e discoid vesicles the inner leaflet was thicker than t h e outer leaflet (fig. 18). This asymmetric unit membrane was confined to t h e discoid vesicles and the mucosal plasma membrane; t h e lateral and basal plasma membranes were approximately 7-8 nm thick, with both membrane leaflets of uniform thickness. Adjacent superficial cells were attached by typical zonulae occludentes at their mucosal margins (fig. 19), where t h e fusion of adjacent outer membrane leaflets occurred along a relative long line or many points of fusion. There
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was moderate but wide interdigitation of both lateral and basal cell borders. Some basal cells had extremely long contacts with the basal lamina, while others rested on the basal lamina along a short distance, with the major portion of the cell projecting deeper into t h e medial regions of t h e epithelium. There was a uniform distribution of cell organelles throughout t h e cytoplasm, in contrast t o t h e superficial cells (fig. 20). Rough endoplasmic reticulum was slightly less abundant but more dilated, while the number and size of mitochondria were about t h e same as in the superficial cells. The Golgi complex, although prominent, was also less abundant in these cells. Discoid vesicles or asymmetric unit membrane were not present in basal cells. Very small, uniformly-round vesicles were present, being most abundant
Fig. 20 Electron micrograph of basal region of transitional epithelium. Distinct basal lamina (asterisks) separates epithelial cells from underlying smooth muscle (SM). Hemidesmosomes (HD) are present along basal region of cell, as are small invaginations of the plasma membrane (arrows). (G) Golgi complex. (BC) Basal cell, (SC) superficial cell. X 26,730.
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near the Golgi complex. Filaments were often present in small groups or scattered singly throughout the cell, but not in large bundles. Common borders between basal cells appeared convoluted, with large pseudopod-like extensions interdigitating widely between adjacent cells, in contrast to the highly interdigitated cell borders of t h e inner medullary pelvic epithelium. The basal border was generally flat, but some small involutions of this membrane were observed (fig. 20). Normal 7-8-nm-thick plasma membrane, consisting of two symmetrically thick leaflets, surrounded the cytoplasm on all sides. Contacts between adjacent basal cells, as well as between basal cells and superficial cells, was by desmosomes. Morp hometry
In each of the estimated parameters of the fornix: (1) surface area, (2) epithelial thickness and (3) interstitial distance, for each outer medullary stripe and each structure, there were no statistically significant differences between hamsters on high- and lowprotein diets (table 1).Therefore, statistical tests included the values from all hamsters (both high- and low-protein diets) as one population (tables 2, 3). The entire fornix in t h e medial region of the kidney was approximately double the length given for the mean “total” measured fornix length (1,423.2 pm, table 2) since only one-half of each fornix was measured here. The entire medial fornix had a
circumference of about 3 mm (2,845.5 p m ) , which consisted of approximately 30.5% outer stripe of the outer medulla and 69.5% inner stripe of t h e outer medulla. Surface area Although the actual fornix surface area itself cannot be determined from the measurements made here, the relative surface area has been estimated. The most striking result of these surfacearea estimations was t h a t more than 60% of t h e fornix epithelium was underlain by capillaries, in both outer and inner stripes of the outer medulla (table 3). In both stripes, the fenestrated capillaries immediately underlay about 1.5times more surface area than did t h e continuous capillaries. In each outer medullary stripe, over 90% of t h e estimated surface area was underlain by capillaries plus one additional structure. This structure was the proximal tubule (pars recta segment) in the outer stripe, while in the inner stripe i t was the ascending thick limb of Henle. An extremely small percentage (1.0%) of outer stripe epithelium was underlain by distal convoluted tubules, which are normally present in the cortex. Epithelial thickness The mean pelvic epithelial thickness (table 3) was 2.3 p m greater over the inner stripe than over the outer stripe (paired t-test, p
