fine structure and staining of mucosubstances on \"intercalated cells\" from the rat...

Fine Structure and Staining of Mucosubstances on “Intercalated Cells“ from the Ra t Distal Convoluted Tubule and Collecting Duct

LYLE D. GRIFFITH, RUTH ELLEN BULGER AND BENJAMIN F. TRUMP University of Washington School of Medicine, Department of Pathology, Seattle, Washington, and Duke University Medical Center, Department of Pathology, Durham, North Carolina

ABSTRACT A second and previously unrecognized cell type which is identical to the intercalated cells of the collecting duct exists in the rat distal convoluted tubule. These cells are distinctly different from the principal cells which have long been recognized with light and electron microscopy.

The intercalated cells have numerous irregular apical microvilli, a high concentra- tion of nearly round mitochondria located in the apical cytoplasm, and a basal nucleus.

A variety of coated and uncoated vesicles in the apical cytoplasm were identified by electron microscopy. The cytoplasmic surface of the apical plasmalemma also was studded with club-shaped densities.

Light and electron microscopic histochemical techniques for acid mucosubstances were carried out on tissue prepared by rapid freezing techniques or intravascular perfusion hation. The luminal surfaces of the cells from both the distal convoluted tubule and collecting duct stained for acid mucosubstances. In some cases, the inter- calated cells stained more intensely with this technique than the adjacent principal cells. The apical cytoplasm of the intercalated cells was also more PAS positive than that of the principal cells.

A distinct cell type, variously called “intercalated cell,” “dark cell, ” ‘lost tribe of cells,” or “special cells” (Novikoff, ‘59; Oliver, ’44; von Mollendorfi?, ’30; Yoshi- mura and Nemoto, ’53) has been known to exist in the collecting duct of the mam- malian nephron since 1876 (Schachowa, 1876). Neither the origin nor the function of these cells has been defined conclusively but appear in the collecting ducts of many species of mammals, including man (Clark, ‘57; Flume et al., ’63; MacDonald et al., ’62; Myers et al., ’66; Novikoff, ’59; Yoshi- mura and Nemoto, ’53; Yoshimura and Sanuga, ’52).

We have found cells in the distal tubule of the rat nephron which are identical in morphology to those previously identified in the collecting duct by others. In this paper we will describe the structure and certain histochemical staining reactions of the intercalated cells of the distal tubule and collecting duct.

MATERIALS AND RlETHODS

The kidneys of female (200-250 gm) Sprague-Dawley rats were used throughout

ANAT. REC., 160: 643-662.

the experiments. The animals were given food and water ad libitum and studied in one of three states of water balance: (1) given water ad libitum; ( 2 ) dehydrated by water deprivation; or (3) put into a state of water diuresis by fluid administra- tion via stomach tube. The methods for inducing each of these states have been previously published (Griffith et al., ’67).

1. Tissue preparation for light and electron microscopy

The tissues were fured for light and electron microscopy by: (A) intravascular perfusion techniques (Griffith et al., ’67) utilizing formaldehyde, glutaraldehyde, osmium tetroxide, a combination of osmium tetroxide and glutaraldehyde (Trump and Bulger, ’66); or (B) im- mersion in a fixative containing a com- bination of glutaraldehyde and formalde- hyde diluted to approximately 1400 mOsm/ L (Karnovsky, ’65); or (C) a rapid-freeze technique (Waggoner and Collings, ’64). Tissues were dehydrated in a series of graded alcohols, embedded in Epon epoxy

643

644 LYLE D. GRIFFITH, RUTH ELLEN BULGER, AND BENJAMIN F. TRUMP

resin (Luft, '61), sectioned using glass or diamond knives, stained with uranyl acetate (Watson, '58) and lead tartrate (Millonig, '6 1 ), or lead citrate (Reynolds, ' 63 ) , and viewed with RCA EMU-3G, AEI- 6B, Hitachi HS-7, or Hitachi HU-11 elec- tron microscopes.

Sections 0.5-1 p thick of the Epon-em- bedded tissue were stained with toluidine blue (Trump et al., '61) for light micros- COPY.

11. Tissue preparation for electron microscopic histochemistry

Tissue was fixed by perfusion with either 4% formaldehyde buffered with phosphate (pH approximately 7.4) or 3% glutaralde- hyde buffered with 0.1 M cacodylate (Grif- fith et al., '67). The tissue was excised after three minutes of perfusion and slices approximately 2 mm in thickness, contain- ing tissue from the tip of the papilla to the cortex, were cut with a razor blade. These were allowed to remain in fixative (0-4°C) for one to two hours. The tissue was then briefly washed in 0.15 M NaCl, and handled in one of the following ways:

A. Diced with a razor blade into cubes less than 1 mm in diameter, and placed in a wet tea bag to facilitate transfer during the procedures described below.

B. Transferred to a model CTD Inter- national Harris Cryostat (International Equipment Co., Needham Heights, Mass.), frozen with Freon 22 and sectioned at 40 1.1 and 100 p.

The 40 1.1 and 100 1.1 sections, as well as the 1 mm cubes in tea bags, were then sub- jected to a colloidal iron method for acid mucosubstances (Wetzel et al., '66). The tissue was not treated with potassium fer- rocyanide. Following three rinses, in 2% acetic acid, the tissue was rinsed in an 0.1 M sodium acetate buffer (pH 5), post- fixed in 1% s-collidine buffered osmium tetroxide, dehydrated in a series of graded alcohols, embedded in Epon epoxy resin (Griffith et al., '67), and sectioned on a Porter Blum Ultramicrotome, using glass knives. Sections were mounted on carbon coated grids, and viewed either unstained, or stained with lead citrate (Reynolds, '63).

111. Tissue preparation for light microscopic histochemistry

Tissue was fixed by perfusion and freez- ing as described. The rapidly frozen tis- sue was either sectioned in a cryostat, or prepared by freeze-drying with vapor-phase formaldehyde fixation (Lagunoff et al., '61). All tissue, with the exception of the fresh frozen and frozen-dried, was dehydrated with ethanol and embedded in paraffin. All embedded tissue was sectioned at 2 p, stained, and examined as follows:

A. Toluidine blue, pH 2.5; viewed while wet (Pearse, '60).

B. Toluidine blue, pH 0.9, viewed while wet (Spicer, '60).

C. Alcian blue (Steedman, '50). D. Alcian blue, counter-stained with

nuclear fast red (Spicer et al., '65). E. Alcian blue, counter-stained with the

periodic acid Schfi (PAS) technique after blocking the sections with an aniline solu- tion in gacial acetic acid (Yanoff et al., '65).

F. Colloidal iron (Mowry, '58). Fresh frozen sections were cut at 6 CI

and stained for succinic dehydragenase (Nachlas et al., '57).

OBSERVATIONS

The rat distal convoluted tubule con- tained two distinct cell types: (1) A prin- cipal cell (Rhodin, '58, '63), and (2) a second cell type morphologically similar to the intercalated cell of the collecting duct but heretofore unreported in the dis- tal tubule of the mammalian nephron.

I . Light microscqy of intercalated cells of the distal tubule (f igs. I , 2,

10, 11, 14) The intercalated cell in the distal con-

voluted tubule could be easily recognized at the light microscopic level in toluidine blue stained sections embedded in Epon (figs. 1, 2). The intercalated cell ap- peared as a more darkly staining cell, in- terdigitated between the more lightly stain- ing principal cells. Microscopically, the principal cells were characterized by the apically located nucleus and parallel, long mitochondria in a wide subnuclear cyto- plasmic zone (Rhodin, '58, '63; Trump and

THE INTERCALATED CELL OF THE DISTAL NEPHRON 645

Bulger, In Press) (figs. 1, 2). The inter- calated cells had a densely staining cyto- plasm, more apical microvilli than seen on principal cells (figs. 1,2). The intercalated cells had a high concentration of nearly round to ellipsoidal mitochondria, many of which were seen between the nucleus and the apical cell membrane, and a basally-placed nucleus.

11. Ultrastructure of intercalated cells in

A. General Appearance The electron-dense cytoplasm contained

round to ellipsoidal mitochondrial profiles throughout the cytoplasm, but more on the luminal side of the nucleus. The nucleus appeared to be large and basally to cen- trally located (fig. 5).

B. Cytoplasmic Membrane Numerous large and irregularly-shaped

microvilli or flaps were found on the Ium- inal surface (figs. 4, 6, 7). At the base of these microvilli, many round membranous profiles were seen in close relationship to, or continuous with, the apical membranes (figs. 4-6). At higher magnifications, the apical cytoplasmic membrane (figs. 7, 8) was more complex than the membrane of adjacent principal cells. The outer surface of the membrane was covered with a dense “fuzz” (antennulae microvillares, Yamada, ’55) coat. The filaments of the fuzz were usually longer and more numerous than those found on the principal cells. The cell membrane frequently demonstrated a trilaminar structure. Small regularly- spaced, club-shaped densities were at- tached to the cytoplasmic side of the inner leaflet of the cell membrane by the handle of the club (figs. 6, 8).

The apical region of the lateral cell mem- brane was marked by a junctional com- plex (Farquhar and Palade, ’63), com- posed of short tight junctions an intermedi- ate junction of considerably longer length, and often a desmosome with a poorly or- ganized plaque. Fine cytoplasmic filaments in the apex were associated with the in- termediate junction. A second class of filaments of larger diameter was seen to converge upon the desmosomes from nearly

the distal tubule (figs. 3-6, 17, 18)

all directions in the cytoplasm. The lateral cell membrane profiles tended to be straight for long distances. However, there were often interlocking processes at some points along the lateral border of these cells (figs. 4, 5).

In contrast to the principal cells, inter- digitations of the basal cell membrane did not generally contain elongate mitochon- dria (fig. 5). The intercalated cell did, however, have numerous recesses into the basilar cytoplasm (figs. 4, 5) similar to those seen in the principal cell of the col- lecting duct. These recesses occasionally contained basement membrane and other interstitial material. The cellular projec- tions formed by the infolded membranes were often composed of further subdivi- sions which then interdigitated with sub- divisions of adjacent projections from the same cell.

C. Cytoplasmic Organelles 1. Mitochondria. The numerous api-

cally located, nearly round mitochondrial profiles were one of the most distinguish- ing characteristics (figs. 4, 5). The mito- chondria had plate-like cristae and fre- quent dense matrical granules. Within fairly narrow limits, all were of a similar size and shape as described by Myers et al. (’66) for the human collecting duct inter- calated cell. Only rarely did the mitochon- dria in the intercalated cell have a pleo- morphic shape but some were shaped like short rods.

2. Vesicles. Several types of vesicles were seen in the apical cytoplasm (figs. 3-6,9). The most numerous were bounded by a trilaminar membrane and studded with a thick cytoplasmic layer of radially arranged club-shaped densities (fig. 9). The structure and thickness of these mem- branes were similar to that described for the apical cell membrane. Although the majority of these vesicles were relatively large, there was also a population of smaller vesicles (fig. 6) of similar morphol- ogy. Some of the large, coated vesicles contained spherical dense bodies. Another class of round or irregular vesicle was bounded by a single membrane varied in diameter (fig. 6). This class lacked the well-organized cytoplasmic coating and

646 LYLE D. GRIFFITH, RUTH ELLEN BULGER, AND BENJAMIN F. TRUMP

sometimes contained amorphous material. These apical vesicles might have arisen by fragmentation of the larger smooth-sur- faced elements.

3. Other cytoplasmic organelles. Mul- tivesicular bodies were found occasionally (fig. 4); the Golgi apparatus, located in the apical or lateral cytoplasm, was often extensive (figs. 4, 5). Small amounts of smooth and rough endoplasmic reticulum were seen. Cytosomes and cytosegresomes were seen frequently (fig. 5) and a group of smaller dense bodies were present. As discussed in the electron microscopic his- tochemical results, these cytosomes were frequently iron-positive (fig. 17).

D. Cytoplasmic Sap The following structures contributed to

the density of the cytoplasm: numerous free ribosomes, both single and in clusters; numerous randomly distributed fine dense filaments; and the club-shaped densities which surrounded many of the vesicles

111. Light microscopic histochemical characteristics of intercalated cells from distal tubule and collecting

duct (figs. 10-1 5) Tissue which had been prepared by

rapid-freezing techniques or intravascular perfusion fixation stained more intensely with alcian blue, toluidine blue, and col- loidal iron than tissue fixed by immersion techniques. Although the frozen tissue re- tained considerable acid mucosubstances, the ice crystal artifacts and lack of resolu- tion made it difficult to make specific in- terpretations. Perfusion fixation with formaldehyde or glutaraldehyde was per- formed in an attempt to achieve better tis- sue preservation while preserving the his- tochemically active materials. The length of the glutaraldehyde fixation was usually short, i.e., three to four minutes, but this did not markedly affect the histochemical response as compared to that observed after freeze-dry techniques. Results ob- tained on tissue allowed to remain in glu- taraldehyde for three to four weeks were similar to those on tissue fixed in glutaral- dehyde for short periods, which were in

(fig. 6).

turn similar to those on the formaldehyde- fixed tissue.

The luminal surfaces of the cells from the distal convoluted tubule and the col- lecting duct were positive for toluidine blue (pH. 2.5), toluidine blue (pH 0.9), alcian blue, alcian blue-PAS (figs. 12, 13), and colloidal iron (figs. 10, 11, 14, 15). In some cases, the intercalated cells had a more distinct coating.

IV. Electron microscopic histochemical characteristics of the intercalated cell The general distribution of the electron-

dense particles was similar to the localiza- tion of colloidal iron reported in light mi- croscopic studies by Rambourg et al. ('66) and Mowry ('58). However, many fine distinctions were made by us using the electron microscope that could not be made at the light microscopic level.

In both the distal tubule and the collect- ing duct, colloidal iron particles were bound to the apical cytoplasmic membrane (figs. 16-18). The apical cell membrane of some intercalated cells showed greater affinity for the iron particles than did the apical cell membranes of the adjacent principal cells of the collecting duct, or the principal cells of the distal convoluted tubule. The apical vesicles which appeared to be simi- lar in structure to the apical cytoplasmic membrane, were generally negative (figs. 16-18), even on the 40 p sections, in which cell membranes were presumably disrupted before the colloidal iron reaction was per- formed. Only in a few instances (fig. 17) did vesicular structures in the cytoplasm appear to be iron positive. In these cases the structures most likely represent sec- tions through the base of apical cytoplas- mic in-pocketings. The cytosomes, how- ever, often contained large dense aggre- gates of electron-dense particles (see fig. 17). All other cytoplasmic elements, the nucleus, the lateral intercellular space, and the extracellular space at the base of these cells were negative in unstained material.

DISCUSSION

Although intercalated cells have been described in collecting ducts by numerous workers, the observation of cells with iden-

THE INTERCALATED CELL OF THE DISTAL NEPHRON 647

tical morphology lining the distal tubule appears to be a new observation. Rhodin (’63) reported dark cells in the collecting duct of mice, commented on their similar- ity to cells of the distal convoluted tubule, but did not distinguish two distinct types of cells in the distal tubule of this species. Clark (’57) and Hancox and Komender (’63) discussed intercalated cells only within the collecting duct. Oliver (’44) has postulated that the intercalated cells of the collecting ducts originate from the distal convoluted tubule. Flume et al. (’63) briefly commented that “compact dark cells” occur in the distal convoluted tubule of man. Biava et al. (’66) comments that intercalated cells were present in distal tubules of human.

The pattern of distribution of intercal- ated cells in the distal tubule has not been studied. Although they may be confined to the region near the transition to the collecting ducts, cells with intense sur- face staining with alcian blue are seen near the macula densa.

Since the first observation of intercalated cells in mammalian collecting ducts, there has been much speculation as to their function; no convincing evidence for a discrete function, however, has been pre- sented. For example, they have been con- sidered as possible sites of antidiuretic hormone action (Hancox and Komender, ’63). Hancox and Komender suggested that the secretory product might be hyalu- ronidase, a concept compatible with Ginet- zinsky’s (’58) hyaluronidase theory of uri- nary concentration and they noted that the intercalated cells occupy the proper loca- tion at the “head” of the collecting duct. However for this idea to be consistent with what is known about urinary concentration in the rat, a hormone sensitive cell would have to be found in the distal convoluted tubule also. Physiologic studies concerning the sites of action of antidiuretic hormone implicate the distal convoluted tubule as well as the collecting duct since antidiure- tic hormone has been shown to alter its permeability to water (Gottschalk, ’64; Ullrich et al., ’64).

Histochemical localization of succinic dehydrogenase has been of value in deter- mining the distribution of intercalated cells

in the collecting duct (Hancox and Ko- mender, ’63; Oliver, ’44). This technique was not useful in identifying intercalated cells in the distal convoluted tubule, be- cause the principal cells in this region are as rich in mitochondria as the inter- calated cells. Oliver (’44) also showed that intercalated cells of the collecting duct could be well demonstrated by their metachromasia after toluidine blue stain- ing. Our results on tissues preserved by perfusion with glutaraldehyde are consis- tent with those seen by Oliver; the inter- calated cells were positively stained in the distal tubule as well as the collecting duct. It thus appears that acid mucosubstance staining may provide a means of identify- ing intercalated cells in the distal con- voluted tubule for they reveal a distinguish- ing characteristic which is not based on the occurrence of mitochondria, as is the case with succinic dehydrogenase reaction. It appears, however, that the best method of distinguishing the intercalated cells is by electron microscopy.

In certain instances the electron micro- scopic histochemical procedure showed an increased reactivity of the apical surface of the intercalated cell (figs. 16-18). This was true not only in the collecting duct where the increased binding was reason- ably apparent even at the light microscopic level, but also in the distal tubule where the distinction was not as easily appreci- ated at that level. The stained layer seen over the intercalated cells, at the light mi- croscopic level, appeared to result from two different characteristics :

1. A more complex apical surface with many long microvilli which bind colloidal iron.

2. A greater colloidal iron-binding ca- pacity per unit of surface area in some cases. It also appeared that the “cap” of acid mucosubstance, seen within the apical cyto- plasm, is not due to staining of the nu- merous vesicles in this region, as they are colloidal iron negative at the electron mi- croscopic level. In addition, the blue gran- ules seen at the light microscopic level are cytosomes and not vesicles. The ultrastruc- tural location of the PAS positive material was not delineated.

648 LYLE D. GRIFFITH, RUTH ELLEN BULGER, AND BENJAMIN F. TRUMP

Contrary to the results of Ito ('56) and Curran and Clark ('63), the colloidal iron reaction in these experiments was precise and certainly not random in distribution. The iron particles were rarely, if ever, seen in tubular lumina.

The present study suggests that the intercalated cell may be more actively in- volved with renal handling of acid muco- stances than the adjacent principal cells, although there is no way on the basis of our data to say that this represents in- creased synthesis, increased binding, or increased reabsorption. Other data con- sistent with this possibility include:

1. Acid mucopolysaccharides are found in urine (Oliver, '44).

2. Distal convoluted tubule and collect- ing duct are known to be coated with acid mucopolysaccharides (Mowry, '58; Ram- bourg et al., '66).

3. Collecting duct cells have been shown to produce acid mucopolysaccha- rides in tissue culture (Morard and Riedel, '65) and intercalated cells are known to be more resistant to manipulation for ob- taining tissue cultures (Clark, '57; Oliver, '44). Therefore these cells might be more likely to survive the procedures which are used to culture kidney tissue.

4. The morphology is consistent with a secretory function showing an extensive Golgi apparatus, large numbers of mito- chondria, and numerous apical vesicles. However, the lack of positive staining of the Golgi apparatus and the apical vesicles with colloidal iron is negative evidence for the secretion involving acid mucopoly- saccharides (Wetzel et al., '66) whereas the positive staining of cytosomes favors the possibility that the intercalated cells may be involved in reabsorption of acid mucosubstances.

ACKNOWLEDGMENTS The authors wish to express their

appreciation to Dr. David Lagunoff, De- partment of Pathology, University of Washington, for his assistance with the histochemical procedures and to Jessie Calder and Robert Pendergrass for tech- nical assistance.

This investigation was supported in part by United States Public Health Service

research grants AM-07919, AM-10698, AM- 10922, 5-TI-GM-726, and 5-TI-GM-00100.

LITERATURE CITED Biava, C., A. Grossman, and M. West 1966

Ultrastructural observations on renal glycogen in normal and pathologic human kidneys. Lab. Invest., 15: 330-356.

Clark, S. L., Jr. 1957 Cellular differentiation in the kidneys of newborn mice studied with the electron microscope. J. Biophysic. and Bio- chem. Cytol., 3: 349-361.

Curran, R. C., and A. E. Clark 1963 The use of the colloidal-iron method for acid muco- polysaccharides in electron microscopy. Bio- chem. J., 90: 2 P.

Farquhar, M. C., and G. E. Palade 1963 Junc- tional complexes in various epithelia. J. Cell Biol., 17: 375-412.

Flume, J. B., C. T. Ashworth, and J. A. James 1963 An electron microscopic study of tubu- lar lesions in human kidney biopsy specimens. Am. J. Path., 43: 1067-1087.

Ginetzinsky, A. G. 1958 Role of hyaluronidase in the re-absorption of water in renal tubules: The mechanism of action of the antidiuretic hormone. Nature, London, 182: 1218-1219.

Gottschalk, C. W. 1964 Osmotic concentration and dilution of the urine. Am. J. Med., 36: 670-685.

Griflith, L. D., R. E. Bulger, and B. F. Trump 1967 The ultrastructure of the functioning kidney. Lab. Invest., 16: 220-246.

Hancox, N. M. and J. Komender 1963 Quanti- tative and qualitative changes in the "dark" cells of the renal collecting tubules in rats deprived of water. Quart. J. Exp. Physiol., 48:

Ito, S. 1956 The enteric surface coat on the intestinal microvilli. J. Cell Biol., 27: 475-491.

Karnovsky, M. J. 1965 A formaldehyde-glu- taraldehyde k a t i v e of high osmolarity for use in electron microscopy. J. Cell Biol., 27: 137A- 138A.

1961 The histochemical demonstration of histamine in mast cells. J. Histochem. and Cytochem., 9: 534-54 1.

Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414.

MacDonald, M. K., M. S. Sabour, A. T. Lambie, and J. S. Robson 1962 The nephropathy of experimental potassium deficiency. An electron microscopic study. Quart. J. Exp. Physiol., 47: 262-272.

Millonig, G. A. 1961 A modified procedure for lead staining of thin sections. J. Biophysic. Biochem. Cytol., 11: 736-739.

Morard, J. C., and B. Riedel 1965 Sur I'origine des mucopolysaccharides acides du n'ephron CtudiCe in vitro. Experientia, 21: 621-622.

Mowry, R. W. 1958 Improved procedure for the staining of acidic polysaccharides by Miiller's colloidal (hydrous) ferric oxide and

346-354.

Lagunoff, D., M. Phillips, and E. P. Benditt

THE INTERCALATED CELL OF THE DISTAL NEPHRON 649

its combination with the feulgen and the periodic acid-Schiff reactions. Lab. Invest., 7:

Myers, C. E., R. E. Bulger, C. C. Tisher, and B. F. Trump 1966 Human Renal Ultrastructure. IV. Collecting duct of healthy individuals. Lab. Invest., 15: 1921-1950.

Nachlas, M. M., K. Tsou, E. De Souza, C. Cheng and A. M. Seligman 1957 Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem., 5: 420- 436.

Novikoff, A. B. 1959 The rat kidney: cyto- chemical and electron microscopic studies. In: Henry Ford Hospital, International Symposium on Pylonephritis. E. Kass and E. Quinn, eds. Little, Brown & Co., Boston, p. 113-144.

Oliver, J. 1944 New directions in renal mor- phology: A method, its results and its future. The Harvey Lectures, Ser XL: 102-155.

Pearse, A. G. E. 1960 Histochemistry, Theo- retical and Applied, second ed., Little, Brown and Co., Boston.

Rambourg, A., M. Neutra, and C. P. Leblond 1966 Presence of a ‘cell coat’ rich in carbohy- drate a t the surface of cells in the rat. Anat. Rec., 154: 41-72.

Reynolds, E. S. 1963 The use of Iead citrate in high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208-212.

Rhodin, J. 1958 Anatomy of kidney tubules. In: International Rev. Cytol., VII. G. H. Bourne and J. F. Danielli, eds. Academic Press, Inc., New York, pp. 485-534.

1963 Structure of the kidney. In: Diseases of the Kidney, chapter 1, p. 21. M. B. Strauss and L. G. Welt, eds. Little, Brown and Co., Boston.

Schachowa, S. 1876 “Undersuchungen iiber die Niere.” Med. Diss. Bern. Quoted by Mollendorff, 1930.

Spicer, S. S. 1960 A correlative study of the histochemical properties of rodent acid muco- polysaccharides. J. Histochem. Cytochem., 8:

1965 Outline of Mucosaccharide Histochemistry. Section on Biophysical Histology, Laboratory of Experimental Pathology, National Institute of

566-576.

18-35. Spicer, S. S., T. J. Leppi, and P. J. Stoward

Arthritis and Metabolic Diseases, National Insti- tutes of Health, Bethesda, Md.

Steedman, H. F. 1950 Alcian blue 8 GS. A new stain for mucin. Quart. J. Micr. Sci., 91: 477-479.

Trump, B. F., and R, E. Bulger 1966 New ultrastructural characteristics of cells fixed in a glutaraldehyde-osmium tetroxide mixture. Lab. Invest., 15: 368-379.

The morphology of the kidney. In: The Structural Basis of Renal Disease. E. L. Becker, ed. Hoeber, New York. In press.

Trump, B. F., E. A. Smuckler and E. P. Benditt 1961 A method for staining epoxy sections for light microscopy, J. Ultrastruct. Res., 5: 343-348.

1964 Water permeability and transtubular water flow in the cortical nephron segments under various diuretic states. Arch. Gesamte Physiol., Mens Tiere (Pfluegers) 280: 99-119.

von Mollendoe, W. 1930 Handbuch der Mikroskopischen Anatomie des Menschen. Siebenter Band. Harn-und Geschlechtsapparat. Julius Springer, Berlin.

Waggoner, W. C., and W. D. Collings 1964 Microanatomy of quick-frozen kidneys after acute experimental renal disturbances. Texas Rep. Biol. Med., 22: 628-639.

Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophysic. Biochem. Cytol., 4: 475-478.

Wetzel, M. G., B. K. Wetzel, and S. S. Spicer 1966 Ultrastructural localization of acid mucosubstances in the mouse colon with iron containing stains. J. Cell Biol., 30: 299-315.

Yamada, E. 1955 The h e structure of the gall bladder epithelium of the mouse. J. Biophysic. Biochem. Cytol., 1: 445-458.

Yanoff, M., L. E. Zimmerman, and B. S. Fine 1965 Glutaraldehyde fixation of whole eyes. Am. J. Clin. Path., 44: 167-171.

Yoshimura, F., and M. Nemoto 1953 Cytologi- cal studies on the special cells in the epithelium of the junctional and collecting segments in the mammalian renal tubules. Gunma .T. med.

Ullrich, K. J., G. Rumrich, and G. Fuchs

Sci., 2: 315-329. Yoshimura, F., and Y. Sanuga 1952 Endo-

crinological studies on renin. Arch. Hist., Jap., 3: 255-269.

PLATE 1

EXPLANATION OF FIGURES

1 Light micrograph of a cross-section of a distal convoluted tubule (DCT) from the cortical region from an animal deprived of water for three days. Note the intercalated cells (IC) which are found adjacent to the principal cells of the distal tubule. The basally located nucleus, the apical mitochondria, and the increased number of microvilli along the luminal surface are illustrated. The tubular lumina are open, free of cellular debris, and lack distortion of apical cytoplasm along the luminal surface. This tissue was fixed by intravascular perfusion with 2% osmium tetroxide buffered in s-collidine, dehydrated in alcohol, and embedded in Epon epoxy resin, and sectioned at 0.5 p , stained with toluidine blue. X 1,800.

Light micrograph of a cross-section of a distal convoluted tubule (DCT) from the cortical region of an animal given water ad libitum. Note the presence of an intercalated cell (IC) containing many spherical, dense mitochondria in a densely staining cytoplasm and an apical layer of small microvilli. The tubular lumina are open, but contain cytoplasmic projections, presumably artifacts result- ing from immersion hation. This tissue was fixed in combination-fixative (one part 50% glutaraldehyde, two parts 2% osmium tetroxide, and five parts s-colli- dine buffer), dehydrated in alcohol, and embedded in Epon epoxy resin, and sectioned at 0.5 p , stained with toluidine blue. x 1,100.

Electron micrograph of a cross-section of a distal convoluted tubule adjacent to a renal corpuscle from an animal deprived of water for two days. Note the patent tubular lumen (L), a large peritubular capillary (Cap), and widely patent Bowman’s space (B). The intercalated cells (IC) are seen to have an apical layer of microvilli; apically located, spherical mitochondria; a basally located nucleus; and numerous small apical vesicles. Cilium (Ci). Fixed by intravascular perfusion with osmium tetroxide buffered in s-collidine. X 2,000.

2

3

650

THE INTERCALATED CELL OF THE DISTAL NEPHRON Lyle D. Griffith, Ruth Ellen Bulger and Benjamin %. Trump

PLATE 1

PLATE 2

EXPLANATION OF FIGURE

4 Electron micrograph of a cross-section of a distal convoluted tubule showing a n intercalated cell ( IC) with increased apical microvilli (Mv) and numerous small vesicles ( V ) in the apical cytoplasm. Fixed by intravascular perfusion with osmium tetroxide buffered in s-collidine. x 11,500.

652

THE INTERCALATED CELL OF THE DISTAL NEPHRON Lyle D. Griffith, Ruth Ellen Bulger and Benjamin F. Trump

PLATE 2

PLATE 3

EXPLANATION OF FIGURE

5 Electron micrograph showing a n intercalated cell ( IC) between two typical distal tubular principal cells (PC). Note apically located nucleus and multiple rod-shaped mitochondria located in the basal infoldings of the principal distal tubule epithelial cells contrasted with the basally located nucleus and the more circular mitochondria1 profiles not seen in interdigitating processes of the intercalated cell. Numerous small vesicles ( V ) are found in the apex. X 12,900.

654

THE INTERCALATED CELL OF THE DISTAL NEPHRON Lyle D. Griffith, Ruth Ellen Bulger and Benjamin F. Trump

PLATE 3

655

PLATE 4

EXPLANATION O F FIGURES

6 Electron micrograph of a n apical region of a n intercalated cell (IC) and adjacent principal cell (PC) of the distal convoluted tubule illustrating the microvilli o€ the apical plasmalemma with a thin layer of fuzz on the luminal surface. The microvilli of the intercal- ated cell contain parallel fine filaments. Coated vesicles ( V ) are found in the apical cytoplasm of the intercalated cell with radially arranged club-shaped densities on the cytoplasmic surface (arrows). Some vesicles (V’) are surrounded by a single uncoated membrane and sometimes contains amorphous material. Fixed by immersion in Karnovsky’s fixative (Karnovsky, ’65), diluted to - 1400 mOsm. x 59,600.

7 Electron micrograph showing the apical plasmalemma of an inter- calated cell (IC) and a principal cell (PC). Note the densities on the cytoplasmic side of the apical membrane. x 50,200.

8 Electron micrograph showing the apical cytoplasmic membrane coated on the luminal surface with a thin layer of fuzz and on the cytoplasmic surface with small regularly spaced club-shaped densities (arrows) which are attached to the cytoplasmic side of the inner leaflet by the handle of the club. Fixed by immersion in Karnovsky’s fixative (Karnovsky, ’65, diluted to - 1400 mOsm. X 149,200.

Electron micrograph of the wall of a vesicle found in a n apical region of distal convoluted tubular intercalated cell demonstrating trilaminar membrane coated by dense cytoplasmic layer of radially arranged club-shaped densities (arrows), Fixed by immersion in Karnovsky’s fixative (Karnovsky, ’65), diluted to - 1400 mOsm. X 149,300.

9

656

THE INTERCALATED CELL OF THE DISTAL NEPHRON Lyle D. Griffith, Ruth Ellen Bulger and Benjamin F. Trump

PLATE 4

PLATE 5

EXPLANATION OF FIGURES

Note figures 10 through 15 are photomicrographs of renal tissue which have been fixed by intravascular perfusion with glutaraldehyde, dehydrated i n a series of graded alcohol, and embedded in paraffin. All were sectioned at 2 p, and stained as indicated under the figures.

10

11

12

13

14

15

Light micrograph showing renal corpuscle, arteriole (A) , and cross-section of distal convoluted tubule (in box) with a macula densa. This tissue section has been stained with Mowry colloidal iron technique and counter-stained with nuclear fast red. A no. 22 orange filter was employed to increase contrast. The box indicates the area seen in figure 11. x 460.

Higher magnification cf figure 10. Dark areas are produced by the colloidal iron staining which appears blue in the section, with the exception of the nuclei, which appear black in the black and white photograph because of the nuclear fast red stain. Note luminal surface of distal tubule to be intensely stained thus appearing dark in contrast to luminal surface of the proximal convoluted tubule. This picture indicates the possible existence of intercalated cells (arrows) in the distal convoluted tubule in the region of the macula densa. Note the cell with a basally located nucleus, a large region of apical cytoplasm and intensely colloidal iron positive apical cell membrane. The slight colloidal iron staining of the apical surface of the entire macula densa region exists but is hard to identify on this photograph. X 1,400.

Light micrograph of a section stained with alcian blue-PAS and photographed with a red-orange filter to accentuate the alcian blue staining reaction. The dense layer on the apical surface (arrow) is produced by the binding of alcian blue in this region. Note apparent lack of alcian blue binding by brush border of adjacent proximal convoluted tubule (PCT). Note a layer of alcian blue staining adjacent to both the distal convoluted tubule and proxinial convoluted tubule. x 1,900.

Light micrograph of the same section as figure 12 and photographed with a green filter to accentuate the PAS reaction of the tissue. The dark appearance of the cyto- plasm of the intercalated cell from a collecting duct results from the intense PAS positivity of the region. Also note PAS positive brush border to the adjacent proximal convoluted tubule (PCT) seen i n the bottom of the picture.

Light micrograph of tissue stained with colloidal iron and counter-stained with nuclear fast red. Cross-section of distal convoluted tubule showing a n intercalated cell (IC) with a n intensely staining colloidal iron positive apical cytoplasmic membrane. Al- though positive, the adjacent principal cell (PC) membrane shows a thinner layer of colloidal iron staining material. Note appearance of nucleus in the basilar region of the intercalated cell in contrast to apically located nucleus of principal cell. x 1,500.

Light micrograph illustrating intercalated cells (IC) of the collecting duct region of the nephron. Note intense staining of apical cytoplasmic region of intercalated cells (arrows). The nuclei are counter-stained with nuclear fast red.

X 1,900.

X 1,670.

658

THE INTERCALATED CELL OF THE DISTAL NEPHRON Lyle D. Griffith, Ruth Ellen Bulger and Benjamin F. Trump

PLATE 5

PLATE 6

EXPLANATION O F FIGURES

16 Electron micrograph of the apical cell membrane of the intercalated cell ( IC) found in the collecting duct of the rat reacted with colloidal iron solution to demonstrate thick layer of acid mucopolysaccharides. Note the lack of non-specific binding of colloidal iron substances and the sharp transition from the cell cytoplasmic membrane of the principal cell to the cytoplasmic membrane of the intercalated cell (arrows). Tissue was prepared with intravascular perfusion with formaldehyde ( l o % , NBF), frozer, and sectioned at 40 p with a cryostat, reacted with colloidal iron solution according to Spicer's method (Spicer, '60; Spicer et al., '65), all were post-osmicated, dehydrated through graded alcohols, embedded in Epon epoxy resin, sectioned for electron microscopy and viewed without staining. Note increased number of microvilli on the luminal surface of the inter- calated cell. x 8,500.

Electron micrograph of intercalated cell showing colloidal iron posi- tive surface layer and a positive staining reaction in the cytosomes ( C ) . Figures 17 and 18 are tissues fixed by intravascular perfusion with glutaraldehyde. Tissue cubes, less than 1 mm, were reacted with colloidal iron. These were post-fixed in s-collidine buffered osmium tetroxide for one hour, dehydrated in ethanol, embedded and viewed without staining. x 12,800.

Electron micrograph of the apical region of an intercalated cell from a distal convoluted tubule of rat reacted with colloidal iron solution. Note specificity of colloidal iron binding which sharply ends at the margins of the intercalated cell (arrows). The apical vesicles ( V ) do not show a positive staining reaction. x 11,000.

17

18

660

THE INTERCALATED CELL OF THE DISTAL NEPHRON Lyle D. Griffith, Ruth Ellen Bulger and Benjamin F. Trump

PLATE 6