Cell specialization in collecting tubules of the guinea pig kidney: Carbonic anhydrase activity and...

THE ANATOMICAL RECORD 202431-443 (19821

Cell Specialization in Collecting Tubules of the Guinea Pig Kidney: Carbonic Anhydrase Activity and Glycosaminoglycan Production in Different Cells

AKIRA S A T 0 AND SAMUEL S. SPICER Department of Pathology, Medical University of South Carolina, Charleston, South Carolina 29425

ABSTRACT The distribution of complex carbohydrates has been investigated cytochemically at the light and electron microscope levels in collecting ducts of the guinea pig kidney. The dialyzed iron method demonstrated acidic complex carbohydrate ultrastructurally on the outer surface of the apical and the basolateral plasmalemma of the principal cells and in their maturing Golgi cisternae and secretory granules. Glycoconjugate in these sites stained for sulfate esters with the high iron diamine method but lacked reactivity toward the periodic acid-thiocarbohydrazide-silver proteinate (PA-T-SP) sequence for visualizing vic glycol-containing glycoprotein. Lability to testicular hyaluronidase and resis- tance to sialidase identified the Glycosaminoglycan (GAG) in principal cell granules and the plasmalemmae as a chondroitin sulfate. In contrast, intercalated cells of the collecting ducts failed to stain with the cationic reagents, but showed light PA-T-SP reactivity demonstrative of neutral glycoprotein in the glycocalyx of the apical plasmalemma. Immunostaining with the immunoglobulin-enzyme bridge procedure localized carbonic anhydrase selectively to the intercalated cells. The ultrastructural and cytochemical observations on the guinea pig collecting ducts implicate intercalated cells in fluid and electrolyte transport and principal cells in secretion of a chondroitin sulfate to the tubule lumen and intercellular space.

Physiologic evidence has accumulated con- cerning the role of the renal collecting duct in urine formation. The collecting duct under the influence of antidiuretic hormone (ADH), for example, functions in concentrating urine (see Jamison, 1976; Hays and Levine, 1976; Burg, 1981). Antidiuretic hormone acts by increasing the water permeability of the collecting duct and allowing water to leave the tubule lumen in response to the osmolar attraction of the interstitial fluid (Grantham and Burg, 1966; Burg et al, 1970; Schafer and Andreoli, 1972; Jamison et al, 1971; Sonnenberg, 1974; Mor- gan et al, 1968). The hyperosmolar interstitial fluid coincidentally contains metachromatic glycoconjugate which, presumably, plays a part in the attraction of water to the interstices in tandem with the ADH action. The in- terstital complex carbohydrate in the renal medulla has been identified as chondroitin sulfate (Spicer and Henson, 1967; Spicer et al, 1967a ,b) . The source of t h e renal metachromasia is investigated in this study by examining the guinea pig kidney with cytochemical methods for demonstrating com-

plex carbohydrates at the ultrastructural level. Collecting ducts further modify the ion con-

tent and pH of urine by mediating net resorp- tion of Na' from and secretion of K and H' into the lumen (see Burg, 1981; Grantham, 1976). Carbonic anhydrase activity relates to these transport processes by contributing protons for secretion or exchange with other cations and providing HCO; ions (Maren, 1967; Rec- tor, 1976). Evidence for the location of these transport activities thus can be derived from immunocytochemical demonstration of carbonic anhydrase. A recently described immunocytochemical procedure (Spicer et al., 1979) is, accordingly, employed here to localize carbonic anhydrase in the collecting duct of the guinea pig kidney as a means of obtaining evidence for proton or HCO; associated transport in this site. The interest in examining guinea pig kidney for glycoconjugate and carbonic anhydrase depended, in part, on the lack

llecrived March I9.ISXI. acccpted No\,rmbcr 10. 1981 Akiri, Sato's p r e t m t address IS I k p a r t m e n l \ of Anatomy and

Surgi,ry. S inshu University School of I l e d i r i n ~ . Matsurnoto. .Japau.

0003-276X18212024-0431$04.00 :C: 1982 ALAN R. LISS, INC.

432 A. SAT0 AND S.S. SPICER

of knowledge of the ultrastructure and cytochemistry of the nephron in this species.

MATERIALS AND METHODS Light microscopic complex carbohydrate

his tochemis try Kidneys obtained under nembutal

anesthesia from adult male albino guinea pigs were fixed 3 to 24 hours in 4% formalin buffered with 2% calcium acetate or 5 hours in Carnoy’s solution, prior to routine dehydration and embedment paraffin. The following histochemical procedures were applied to 5-pm tissue sections: (1) the periodic acid Schiff (PAS) method with and without prior diastase digestion (Lillie and Fullmer, 1976); (2) the Al- cian blue at pH 2.5 (AB)-PAS sequence (Mowry and Winkler, 1956); (3) the high iron diamine (HID)-AB sequence (Spicer, 1965); (4) the aldehyde fuchsin (AF) stain (Spicer et al., 1967a,b); and (5) testicular hyaluronidase (Spicer et al., 1967a,b) or sialidase (Spicer and Warren, 1960) digestion followed by the HID- AB sequence or AF stain.

Light microscopic immunohistochemistry Carbonic anhydrase was localized with the

immunoglobulin peroxidase bridge method (Mason et al., 1969) employing rabbit antihuman carbonic anhydrase I (CAI) or I1 (CAII) as the primary antiserum. These antisera were prepared and characterized for specificity as reported previously (Tashian et al., 1968; Osborne and Tashian, 1975), and employed ac- cording to a previously described postembed- ment, immunostaining procedure for demonstrating carbonic anhydrase (Spicer et al., 1979). In this procedure the paraffin sections from kidneys fixed 5 hours with Carnoy’s solution were deparaffinized, rehydrated, treated with 3% hydrogen peroxide to abolish the pseudoperoxidase activity of red blood cells, and then exposed 10 minutes to a 1:20 dilution in phosphate-buffered saline (PBS) of the primary antiserum to either CAI or CAII. This was followed by the sequential treatment with similarly diluted goat antirabbit immunoglobulin serum, then rabbit antihorseradish peroxidase serum prior to application of a 0.5 mg% horseradish peroxidase (Sigma Chemical, St. Louis, MO) solution in PBS. Con- trol sections were exposed to normal rabbit serum or rabbit antihuman ACTH serum as the first step of the bridge sequence, instead of anti-CAI or CAII sera. Sections thus treated were incubated 10 minutes in the Graham and

Karnovsky diaminobenzidine H,O, medium (Graham and Karnovsky, 1966), postfixed briefly with 1% osmium tetroxide, and then dehydrated and mounted. As a means of com- paring localization of complex carbohydrate- producing cells and carbonic anhydrase-rich cells, immunostained sections were counter- stained with Alcian blue or by other methods for demonstrating glycoconjugates. Ultrastructural carbohydrate cytochemistry Areas of renal cortex, medulla, and papilla

were minced finely and fixed for 2 hours in 2.5% glutaraldehyde buffered at pH 7.4 with 0.1 M sodium cacodylate. Cryostat sections cut at 40 pm from each specimen were stained overnight with either dialyzed iron (DI) (Wet- zel et al., 1966) to localize both carboxylated and sulfated glycoconjugate of the high iron diamine (HID) (Spicer et al., 1978) solution to visualize sulfated complex carbohydrate selectively. Control sections were exposed to a 10% MgC1, or 1.2% FeC1, solution in place of the DI or HID solution, respectively. These sections were postfixed 1 hour with 2% OsO, and rou- tinely dehydrated with ascending concentra- tions of ethanol. The remainder of each minced- specimen not utilized for cytochemistry was dehydrated with and without postosmication. All specimens were embedded in Epon 812. Ultrathin sections of the unosmicated specimens mounted on stainless steel grids were stained with the periodic acid-thiocarbohydrazide-silver proteinate (PA-T-SP) method (Thiery, 1967) for visualizing periodate reactive complex carbohydrate rich in hexoses with uicinal glycols. For a control, adjacent thin sections were stained with only the TCH- SP sequence. Thin sections from postosmi- cated specimens were stained conventionally with uranyl acetate and lead citrate for routine morphological demonstration. Ultrathin sections were examined in an Hitachi HS-8 electron microscope.

RESULTS Light microscopy

Two cell types were distinguished with the Alcian blue, high iron diamine, or aldehyde fuchsin reagents in collecting ducts in the medullary rays of the cortical region and in the collecting ducts of the outer medulla. One type lacking basophilia was interpreted as the dark cell or intercalated cell described in other species (Rastegar et al, 1980; Myers et al, 1966; Ordonez and Spargo, 1976). The other type, the

CYMCHEMISTRY OF GUINEA PIG KIDNEY 433

light cell, or principal cell, stained blue with Al- cian blue, black with high iron diamine, and purple with AF in the apical portion of the cell (Fig. 1). Hyaluronidase digestion prior to the cationic dye largely eliminated or completely abolished the stainability of the principal cells (Fig. 2). but sialidase digestion failed to affect the basophilia (Fig. 3) . Intercalated cells were prevalent in the segment of the collecting duct in the outer cortex but became infrequent in the segment in the inner cortex and outer medulla (cf. Fig. 1 with Figs. 5 and 6). All the cells lining the collecting ducts in the inner medulla and papilla of the kidney revealed the same staining characteristics as the principal cells, although staining in the papillary collecting duct cells appeared more confined to the luminal surface.

As previously observed (Rovasio et al., 1974), the PAS procedure stained the basal lamina of all renal epithelial cells in the nephron and collecting ducts. The brush border of proximal convoluted and straight tubules also stained red with the PAS or the AB-PAS methods. The cells forming the collecting ducts were PAS negative (Fig. 4).

Immunostaining for carbonic anhydrase I or I1 disclosed intensely stained cells interspersed with unstained cells in the proximal portion of the collecting ducts in the cortex (Fig. 5). A sequence of the immunostain followed by cationic dye revealed that the im- munoreactive cells corresponded with the intercalated cells. Thus, the homogenous brown coloration demonstrative of carbonic anhydrase in the cytoplasm occurred in cells with no affinity for the cationic reagents in the apical cytoplasm (Fig. 6). Conversely, the cells identi- fiable as principal cells by their apical basophilia lacked immunoreactivity for carbonic anhydrase.

Ultrastructural morphology The presence of intercalated and principal

cells in the collecting ducts of the cortex and outer medulla of the guinea pig kidney was evident in routine morphological preparations (Figs. 7, 8). Intercalated cells were characterized by an electron-dense hyaloplasm, containing numerous mitochondria and a few small apical vesicles. A moderate number of fine processes, presumably representing microplicae, protruded vertically from the surface of these intercalated cells (Rhodin, 1958). In addition, intercalated cells characteristically revealed infoldings of the basal plasmalemma. A single

cilium often projected into the lumen from these cells (Fig. 7).

Intercalated cells were observed mainly in the proximal part of the collecting duct in the cortex, but even at this level, these cells were less prevalent than principal cells. The intercalated cells were infrequently en- countered in the outer medulla and virtually absent in the inner medulla and papilla.

The principal cell of the collecting ducts in the outer cortex displayed a lucent hyaloplasm which enclosed a relatively small number of mitochondria and infrequent apical vacuoles with low density content (Fig. 7). Sparse, short microvilli protruded from the principal cells in- to the lumen. The basal plasmalemma showed relatively little infolding. Well-developed Gol- gi complexes, mainly located above or beside the nucleus, comprised another distinctive feature of the principal cells (Fig. 9). The principal cells lining collecting ducts in the inner cortex and the outer medulla closely resembled mor- phologically those of the more proximal region of the collecting ducts except that the empty- looking, apical vacuoles were more numerous and larger (Fig. 8). The epithelium lining collecting ducts in the inner medulla and papilla consisted entirely of principal cells which resembled those in the cortex in content of empty-appearing vacuoles in the apical cytoplasm. Ultrastructural carbohydrate cytochemistry The periodic acid-thiocarbohydrazide-silver

proteinate (PA-T-SP) sequence failed to stain material in the apical vacuoles, the Golgi cisternae, and the apical or basolateral plasmalemma of the principal cells (Fig. 10). The apical plasmalemma of intercalated cells usually stained lightly but basolateral plasmalemma, Golgi cisternae, and small cytoplasmic vesicles of these cells lacked PA- TCH-SP reactivity (Fig. 10). This procedure densified abundant glycogen particles in both cell types, however. The basal lamina underlying all of the epithelial cells stained lightly in agreement with the PAS reactivity by light microscopy.

The dialyzed iron (DI) technique clearly distinguished the two cell types in the collecting ducts of the cortex and outer medulla (Fig. 11). The principal cells revealed intense DI affinity in apical cytoplasmic granules in contrast to the lack of staining in intercalated cells (Fig. 12). The DI-reactive granules corresponded with the empty-looking apical vacuoles in rou-

CYTOCHEMISTRY OF GUINEA PIG KIDNEY 435

Fig. 1.-4. Areas in paraffin sections of formalin-fixed guinea pig kidney.

Fig. 1. A portion of the inner cortex reveals two cell types in the collecting duct (CD). Principal cells are more numerous than intercalated cells and disclose heavy (black) staining for sulfated complex carbohydrate in their luminal aspect and light reactivity in deeper cytoplasm. Intercalated cells (arrows) lack basophilia as do cells of the thick ascending limb of Henle’s loop (upper left) and the proximal convoluted tubule (lower right). High iron diamine-Alcian blue pH 2.5 (HID-AB) sequence. x 400.

Fig. 2. As in Figure 1 except the section was treated with testicular hyaluronidase prior to the staining. The enzyme digestion has eliminated staining of the principal cells in the proximal collecting ducts (cd) and, thus, identified the HID-positive material shown in Figure 1 as a chondroitin sulfate. The glomerulus at the left reveals light (blue) staining demonstrative of the presence of hyaluronidase-resistant glycoconjugate with carboxyl groups but no sulfates. x 200.

Fig. 3. Similar to Figure 1 except the section was treated with sialidase prior to the staining. The intense (black) HID staining in the luminal aspect of the principal cells resists sialidase; but the digestion has eliminated (blue) staining of the glomerulus a t the upper left identifying the alcianophilic groups as sialic acid. x 200.

Fig. 4. An area from the outer cortex reveals strong (red) coloration demonstrative of glycoprotein in the basement membrane underlying the proximal and distal convoluted tubules and the collecting ducts. The glomerulus and Bowman’s cap- sule at the lower left also appear reactive. Staining in the brush border of the proximal convoluted tubules (pct) contrasts with the lack of reactivity in the luminal aspect of the collecting ducts (cd). Periodic acid-Schiff (PAS) stain after diastase digestion. x 200.

Fig. 5. Cells with strong staining demonstrative of carbonic anhydrase lie interspersed between unstained cells in proximal collecting ducts of the outer cortex. The (brown) staining appears distributed throughout the cytoplasm of the cells and spares the nuclei. Immunoglobulin-peroxidase bridge procedure employing anticarbonic anhydrase I as the primary antiserum. x 200.

Fig, 6a. Intermittent cells distributed in proximal collecting ducts like the stained intercalated cells in Figure 5 show brown coloration and, accordingly, contain carbonic anhydrase. Intervening cells interpreted as principal cells lack the brown color but show apical blue staining which is obscure in this photograph taken with a blue filter (cf. Fig. 6b). The brush border of the proximal convoluted tubule a t the bottom shows (red) staining. Immunostain for carbonic anhydrase I followed by the Alcian-blue-PAS sequence. Carnoy-fixed kidney. x 400.

Fig. 6b. Another micrograph of the field shown in Figure 6a taken with a red filter. The strong (blue) staining evidencing acidic glycoconjugate in the luminal aspect of many cells (arrows) is now prominent. The lack of (brown) immunoreactivity for carbonic anhydrase serves to identify these as principal cells. Carbonic anhydrase-rich intercalated cells, thus, differ cytochemically from the acid glycoconjugate-containing principal cells. Carnoy-fixed kidney. x 400.


tinely processed specimens (cf. Fig. 8). The granules often revealed an unstained central area. Some developing granules near the Golgi zone also possessed an unstained core sur- rounded by reactive glycoconjugate (Fig. 13). Golgi cisternae at the maturing face of the complex contained intensely stained material and sometimes appeared continuous with nascent, DI-reactive granules. DI-positive mucosubstance also coated the luminal surface of the apical plasmalemma and adhered, in addition, at intervals to the outer surface of the basolateral plasmalemma. Notably, the intercalated cells lacked both surface staining and reactive organelles. The basal lamina underlying all of the collecting duct epithelium displayed light DI affinity. Dense particles were evident, scattered lightly between collagen bundles in the underlying interstitium and

Fig. 7.-9. Electron micrographs of a morphologic preparation of gluteraldehyde-fixed guinea pig kidney.

Fig. 7. Proximal collecting duct from the guinea pig kidney processed for routine morphologic examination reveals an intercalated cell (I) sandwiched by principal cells (P). An electron-dense hyaloplasm in the intercalated cell encloses a few small apical vesicles and numerous mitochondria. A moderate number of long microvilli and cilium protrude into the tubular lumen (TL) from the intercalated cells. The intercalated cell also discloses elaborate basal infoldings in contrast to the relatively flat basal plasmalemma of the neighboring principal cell (cf. also Fig. 8). Principal cells display a less dense hyaloplasm, contain fewer mitochondria and have sparse short microvilli a t the apex. Neighboring cells appear loosely interlocked and show empty-appearing slits between the cells. Thin section stained with uranyl acetate-lead citrate (UA-LC). x 8,200.

Fig. 8. An area from the outer medulla of the guinea pig kidney shows a collecting duct with adjacent principal (P) and intercalated (I) cells and the basal portion of a distal tubule (DT) cell. The principal and intercalated cells differ in structure of the plasmalemma. electron opacity of the hyaloplasm, and the abundance of mitochondria. A number of empty-looking vacuoles located mainly in the apical cytoplasm and near the well-developed Golgi complex (G) charac- terize the principal cell a t this more distal level in the collecting duct. UA-LC stain. x 7,175.

Fig. 9. A Golgi complex from a principal cell in the proximal collecting duct consists of stacked parallel lamellae. small vesicles, and vacuoles presumed to be nascent granules. Some developing vacuoles appear continuous with Golgi lamellae (arrows). The content of the vacuoles varies but often comprises a finely granular rim (arrowheads) around an empty-looking core. x 19,950.

spaced at periodic intervals in or on collagen fibrils.

The high iron diamine (HID) method yielded results comparable to those obtained with the DI stain (Figs. 14-20). Thus, the principal cells disclosed intense HID affinity in the apical secretory granules that corresponded with the empty-appearing vacuoles in morphologic preparations. Trans cisternae of the Golgi complex stained similarly (Fig. 19). Material attached to both the luminal glycocalyx and the outer surface of the basolateral plasmalemma revealed strong HID binding. That the principal cell extended cytoplasmic processes beneath intercalated cells was evident from prominent HID staining of plasmalemma of these processes underlying intercalated cells. The HID-reactive mucosubstance lay aligned on the plasmalemma of principal cells in dis- crete globular deposits (Figs. 16-18). The basal lamina also revealed light punctate staining and underlying collagen fibrils showed periodic densification (Fig. 18). In contrast, intercalated cells totally lacked affinity for the HID reagent.

DISCUSSION

The collecting duct of the kidney is thought to play an important role in modifying the urine content of electrolytes, functioning in secretion of ammonia, and transport of K and H' to and of Na' from the solution in the duct lumen (Jamison, 1976; Hays and Levine, 1976; Burg, 1981). A problem remains, however, in assigning functions to segments of the duct and cell types within the segment. The present immunohistochemical localization of carbonic anhydrase in intercalated but not in principal cells in the proximal collecting ducts indicates a role of intercalated cells in contributing protons for secretion or exchange with Na in ion transport (Rector, 1976).

Recent evidence (Rastegar et al., 1980) which has further documented an important role of collecting ducts in countering excess potassium intake (Grantham, 1976) depends on secretion of K by the principal cells. Cells interpreted as principal cells in the guinea pig collecting duct, however, lack the basolateral plasmalemma amplification that presumably contains abundant NalK ATPase in the rat principal cells (Rastegar et al., 1980). The guinea pig collecting ducts have apparently not been examined ultrastructurally before and a question arises whether these collecting duct cells function comparably to absorb Na'


and secrete K' in both guinea pigs and rats. Presumably the guinea pig principal cells function like those of the rat in ADH-stimulated water transport and their production of chondroitin sulfate does not seem inconsistent with such activity. Fine structural changes in intercalated cells of collecting ducts in potassium depletion states implicates these cells in reabsorption of K from the duct lumen (Rastegar et al., 1980; Stetson et al., 1981; Hansen et al., 1978). The intercalated cells observed here resemble those of rat collecting ducts (Tisher, 1981), in fine structural features, including abundance of mitochondria, and ap-

Fig. 10. This figure and those to follow show guinea pig kidney stained by ultrastructural methods for localizing glycoconjugates. The apical plasmalemma of a principal cell on the right lacks reactivity, contrasting with the light staining demonstrative of glycoprotein on the microvillar surface of the neighboring intercalated cell. The apical vacuoles of the principal cell (arrows) and small vesicles in the intercalated cell fail to stain. Stained glycogen particles in the principal cell exceed those in the intercalated cell. Inset shows a cilium of an intercalated cell with light punctuate densification on the luminal surface. Periodic acid-thiocarbohydrazide-silver proteinate (PA-T-SP) sequence. x 12,635. Inset. x 12,000.

Fig. 11. The luminal surface of principal cells (PI in the proximal collecting duct stains heavily, in contrast to the lack of basophilia on the luminal surface of the neighboring intercalated cell (I). The strong reactivity of the principal cell surface contrasts also with the absence of PA-T-SP staining in this site (cf. Fig. 10). Dialyzed iron (DI) stain. x 19.000.

Fig. 12. The luminal aspect of a collecting duct principal cell (P) in the outer medulla of the kidney is covered with a surface mucous coat with intense reactivity demonstrative of acidic glycoconjugate. Heavily densified material also adheres to the outer surface of the infoldings of the basal plasmalemma (arrows; cf. Figs. 17,18). Secretory granules in the apical cytoplasm and neighboring the paranuclear Golgi area display strong staining as well. The basal lamina reveals light punctate staining as does the interstitium (I) to a lesser extent. A nearby distal tubule cell (DT) lacks basophilia. DI stain x 10,500.

Fig. 13. The maturing or trans cisternae of a Golgi complex in a principal cell contain material with basophilia demonstrative of acidic mucosubstance. Vesicles (arrowheads) bordering the Golgi lamellae contain material stained like that in forming granules nearby (arrows). Mitochondria (M) lack basophilia. DI stain. x 25.270.

pear capable of comparable activity. The carbonic anhydrase activity in intercalated cells observed in guinea pig kidney with immunostaining and by Lonnerholm (1971) in rat kidney with the cobalt technique (Lon- nerholm, 1971) indicate this enzyme con- tributes to the K' adsorption process. Exchange of K' for protons accruing from carbonic anhydrase activity would account for both the K adsorption and the H' secretion (Rector, 1976; Burg, 1981) characteristic of collecting duct function. Secretion of H' seems less compatible with the K secreting activity of the principal cells. The neutral glycoprotein compromising the glycocalyx of the apical plasmalemma of the intercalated cells differs from the acidic glycocalyx of neighboring principal cells and most other epithelia (Spicer et al., 1981). Notably, gastric parietal cells share this unusual feature which, accordingly, could be considered a possible property of a plasmalemma secreting protons.

Principal cells in the guinea pig have been thought from light microscopic examination to possess acidic glycosaminoglycans bound ex- clusively to their apical plasma membrane (Rovasioet al., 1974; Lis and Monis, 1978). Evi- dence for this derived from the staining of the apical plasmalemma of principal cells by Al- cian blue and ruthenium red (Rovasio et al., 1974). The present study extends previous in- vestigations on the histochemical character- ization of these cells by revealing sulfated glycoconjugate not only on the luminal surface but also on the basolateral surface, in Golgi cisternae and in granules of presumed secretory nature as well. The finding that the trans cisternae of the Golgi complex, mature and im- mature cytoplasmic granules, and apical and basolateral plasmalemmae share the property of binding HID and DI, while lacking PA-T-SP reactivity, provides strong evidence that the surface coats represent Golgi material pack- aged into secretory granules and secreted and absorbed to the cell surface. Thus, the granules arising from the Golgi lamellae migrate mainly apically and to a lesser extent basolaterally, and discharge their content into both the tubular lumen and the basolateral extracellular space. This interpretation could explain the oc- curence of glycoconjugate of intrarenal origin in the urine and the source of acidic glycosaminoglycans in the stroma of kidney.

CYTOCHEMISTRY OF GUINEA PIG KIDNEY 44 1

Notably, the surface of distal convoluted tubule cells in rat kidney differs from that of guinea pig collecting ducts in that only intercalated cells disclosed evidence for a surface coat rich in carbohydrate and this had acidic properties in the rat (Griffith et al., 1968). Such surface differences between principal and intercalated cells of rat distal convoluted tubule, compared with guinea pig collecting duct, suggest a difference in function, presumably in transport of fluid or ion a t the two sites.

Fig. 14. Mature apical granules and forming graules near the Golgi zone stain heavily in principal cells of a collecting duct (CD) in the outer medulla. Similar basophilia demonstrative of sulfated mucosubstance is localized also on the luminal surface of the apical plasmalemma, on the basolateral plasmalemma, and in the intercellular space. In contrast, the cells lining a distal tubule (DT) and a segment of Henle’s loop (HL) appear unstained. HID stain. x 14,250.

Fig. 15. A principal cell (P) from a relatively proximal part of the collecting duct exhibits strong staining of apical plasmalemma and cytoplasmic granules, presumably secretory in nature. The apical microvilli and small vesicles of an adjacent intercalated cell lack reactivity. High iron diamine (HID) stain. x 3,150.

Fig. 16. An apical area of a principal cell displays hetero- geneous secretory granules with intense staining. Some granules appear homogeneously stained throughout where- as others consist of two zones including a stained cortex and unreactive core. The densified sulfated glycoconjugate aligns at intervals on the apical plasmalemma in globular deposits. HID stain. x 14,000.

Fig. 17. Principal cells (P) with heavy staining in the apical granules and plasmalemma sandwich an unreactive intercalated cell (I). The principal cells appear to extend between the lightly stained basal lamina and the basalplasma-

Glycosaminoglycans derive principally from connective tissue cells and occupy ground substance or matrix of intercellular spaces and the fibers of connective tissue (Spicer et al., 1967a). Epithelial cells, on the other hand, as a rule produce and secrete glycoproteins. Human and canine gastric chief cells constitute the known exception to this generalization in stor- ing chondroitin sulfatelike material in their secretory granules (Spicer et al., 1967b; Cathcart et al., 1974). The DI and HID binding demonstrative of sulfate esters and the lack of

lemma of the intercalated cell. Reactive material adheres to the outer surface of the interposed basolateral plasmalemma of the principal cells. This sulfated glycoconjugate unique in the guinea pig renal epithelium to collecting duct principal cells affirms the evidence from morphologic ultrastructural examination that principal cell processes extend widely under neighboring intercalated cells. x 8,400.

Fig. 18. Stained material lies aggregated in globular deposits along the outer surface of the lateral plasmalemma of principal cells. Small granules lying near the basal plasmalemma (arrows) appear densified and could provide a transport mechanism for mucosubstance secreted hasolaterally. The basal lamina (arrowhead) displays light punctate staining as do the collagen fibrils in the interstitium (I) x 14,250.

Fig. 19. Cisternae at the maturing face of a Golgi complex (G) in a principal cell stain lightly in contrast to the lack of reactivity in cisternae at the forming face above. Three nascent granules (arrows) near the Golgi complex contain sulfated glycoconjugate. Two of these enclose unstained, carbohydrate-free foci. x 38,000.

Fig. 20. Secretory granules (arrows), the Golgi complex (G), and the plasmalemma lack appreciable densification in a control specimen exposed to 1.2% FeC4 solution in place of the HID reagent. x 7,450.


PA-T-SP reactivity of the principal cell secre- tions observed in this investigation provide evidence for identifying the complex carbohydrate produced by the intercalated cells as a glycosaminoglycan. This substance is further identified as a chrondroitin sulfate by its hyaluronidase lability. Although the physiologic significance of the chondroitin sulfate of the interstitial matrix is not explained, the hydrophilic properties of this mucosubstance could be viewed as contributing to the antidiuretic hormone-mediated water transport from collecting ducts.

ACKNOWLEDGMENTS

The authors are grateful for the efficient technical assistance of Ms. Lori Anderson, B.J. Sturcken, and Zandra McCormack and editorial, secretarial, and laboratory aide assistance of Ms. Fran Cameron, Dot Smith, and Pamela Nuckolls.

This research was supported by NIH grants AM-10956 and AM-11028.

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Cell specialization in collecting tubules of the guinea pig kidney: Carbonic anhydrase activity and...

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