Successive histochemical differentiation steps during postnatal development of the collecting duct in rabbit kidney

Summary Immunohistochemical experiments with monoclonal antibodies (mabs) on the kidney of neonatal rabbits revealed that the primary expression of collecting duct typic structures does not occur in a continuous and parallel, but in a subsequent developmental process. Only mabs RCT-30 A, and CD 4-V revealed immunoreactivity at the ontogenetically oldest parts of the collecting duct, the ampullae, while the other used markers (CD 1–3, CD 5-V, RCT-30 and RMCX) did not. In contrast, all of the tested antibodies showed positive reactions at the medullary and cortical collecting duct of the neonatal kidney as well as of the adult kidney. Additional incubations with wheat-germ agglutinin (WGA) a marker of terminal-differentiated collecting duct cells demonstrated weak-labelled ampulla cells beside intensively labelled ampullary neck and medullary collecting duct cells. With peanut agglutinin (PNA) labelling a 3 step transition could be illuminated: weak-labelled ampulla cells were found beside continuously bright labelled ampullary neck cells and finally a punctuate pattern downwards to the papilla. If the ampullary neck is the zone of proliferation, our findings of WGA- and PNA-co-labelling in this zone indicate, that in early developmental stages characteristic structures of different mature cells, probably principal and intercalated cells, are co-expressed within one single cell type. Thus intercalated cells might derive from principal cells.     

Proliferation of acid-secretory cells in the kidney during adaptive remodelling of the collecting duct

The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H(+)-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H(+)-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH(4)Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH(4)Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions.     

Expression of binding sites for Dolichos biflorus agglutinin at the apical aspect of collecting duct cells in rat kidney

Summary To identify precisely the structural and functional cell type in the collecting duct of the rat kidney expressing binding sites for Dolichos biflorus agglutinin (DBA), we stained serial paraffin sections of kidney with horseradish peroxidase-labeled DBA and with immunocytochemical methods for localizing (Na⁺+K⁺)-ATPase and carbonic anhydrase II (CA II), enzymes found preferentially in principal and intercalated cells, respectively. Most principal cells expressing a strong basolateral staining for (Na⁺ + K⁺)-ATPase showed binding sites for DBA at their luminal surfaces. However, a minority of cells rich in CA II and showing morphologic characteristics of intercalated cells also expressed DBA binding sites at their luminal surface and apical cytoplasm. These data suggest that DBA cytochemistrycan provide a useful tool for studying the functional polarity of the main cell types of the collecting duct of the rat kidney.     

Immunohistochemical localization of atrial natriuretic peptide in the developing and adult mammalian kidney

The discovery, within the last decade, of atrial natriuretic peptide (ANP), a family of peptides with natriuretic/diuretic and vasorelaxant properties, has prompted much research into the mechanisms and sites of action of ANP within the kidney. In the present study, ANP was localized in the kidneys of several mammalian species by immunohistochemical techniques 1) to identify possible sites of synthesis; 2) to compare the localization of ANP to known physiological effects; 3) to determine species differences, if any, in ANP localization; and 4) to study the development of ANP immunoreactivity in the fetal and neonatal rat kidney. Using an antibody against rat ANP, IV, ANP was localized exclusively on the proximal convoluted tubule (PCT) brush border and within intercalated cells of the outer medullary and cortical collecting tubules and ducts of adult mouse, rat, pig, monkey, and human kidneys. The development of ANP immunoreactivity paralleled the differentiation and maturation of collecting duct epithelium in rat fetal kidney. Atrial natriuretic peptide found within intercalated cells of the cortical and outer medullary collecting ducts may be the result of endogenous synthesis and, following secretion, may be available to receptors in the inner medullary collecting ducts.     

Mechanisms and regulation of ion transport in the renal collecting duct

1. In the present paper the ion transport function of the renal mammalian collecting duct and its regulation is briefly reviewed. 2. The epithelium is characterized by different cell types: principal cells, intercalated cells, type A, and intercalated cells, type B. 3. Using microelectrodes and various microscopic techniques active Na+ absorption as well as K+ secretion has been localized to the principal cells, while Cl- absorption was found to proceed largely, though not exclusively, through the tight junctions between cells. 4. Intercalated cells of type A, which prevail in the outer medullary collecting duct, secrete H+ and intercalated cells of type B, which are most frequent in the late cortical collecting duct, secrete HCO3-. 5. This specialization of different cells in transporting individual ions provides the basis for the efficient adaptive regulation of urinary ion excretion.     

Morphology of rabbit collecting duct.

Recently the assumed structural and functional homogeneity of the collecting duct (CD) has been questioned. The objective of this study was to determine if heterogeneity occurs in luminal surface membrane structure or in cytoplasmic configuration of cells in the collecting duct or both. Straight segments of cortical and medullary CD were examined in perfusion-fixed rabbit kidneys with scanning electron microscopy (SEM), light (LM) and transmission electron microscopy (TEM). Principal cells were the most abundant cells in all CD regions; intercalated cells comprised 37% of the cell population on the cortex, 18% in the outer medulla, and less than 1% in the inner medulla. SEM revealed two surface patterns among the ciliated principal cells: 1, located in the cortex and outer medulla, with few surface microvilli, and 2, located in the inner medulla, with abundant microvilli. Intercalated cells exhibited four distinctive luminal surface configurations: I, numerous short microvilli; II, both short and elongate microvilli; III, microplicae alone; and IV, both microvilli and microplicae. Intercalated cells with patterns I and II were predominant in the cortex, while cells with patterns III and IV were most common at the corticomedullary junction. TEM confirmed that marked variation existed in cytoplasmic structures of both principal and intercalated cells. These findings may either indicate the presence of several specific types of principal and intercalated cells or reflect different functional states of the principal and intercalated cells. Regardless of their significance, their presence must be considered in studies seeking to establish precise structural-functional relationships in this region of the rabbit renal tubule.     

Structural links between the renal stem/progenitor cell niche and the organ capsule

A special feature of the renal stem/progenitor cell niche is its always close neighborhood to the capsule during organ development. To explore this link, neonatal kidney was investigated by histochemistry and transmission electron microscopy. For adequate contrasting, fixation of specimens was performed by glutaraldehyde including tannic acid. The immunohistochemical data illustrate that renal stem/progenitor cells are not distributed randomly but are positioned specially to the capsule. Epithelial stem/progenitor cells are found to be enclosed by the basal lamina at a collecting duct (CD) ampulla tip. Only few layers of mesenchymal cells are detected between epithelial cells and the capsule. Most impressive, numerous microfibers reacting with soybean agglutinin, anti-collagen I and III originate from the basal lamina at a CD ampulla tip and line between mesenchymal stem/progenitor cells to the inner side of the capsule. This specific arrangement holds together both types of stem/progenitor cells in a cage and fastens the niche as a whole at the capsule. Electron microscopy further illustrates that the stem/progenitor cell niche is in contact with a tunnel system widely spreading between atypical smooth muscle cells at the inner side of the capsule. It seems probable that stem/progenitor cells are supplied here by interstitial fluid.     

Immunolocalization of C1-tetrahydrofolate synthase in the rat kidney

The distribution of the trifunctional enzyme C1-tetrahydrofolate synthase (C1-THF synthase) was examined in the rat kidney by immunolocalization with anti-C1-THF synthase serum using the peroxidase-antiperoxidase method. C1-THF synthase immunoreactivity was detected in both distal and proximal epithelial cells. Staining of the distal tubule epithelia was more intense and granular whereas staining of the proximal tubule epithelia was diffuse. All cells of the cortical collecting duct showed positive granular staining. In the outer medullary collecting duct, the intercalated cells showed intense granular cytoplasmic staining and the principal cells were either negative or weakly positive. The ascending thick limb of Henle's loop was also positive. Glomeruli and the inner medulla showed no staining for C1-THF synthase.     

Detection of glycosylated sites in embryonic rabbit kidney by lectin chemistry

The reciprocal cell biological interaction between mesenchymal and epithelial tissue plays a critical role during nephrogenesis. It is unknown to date whether the tissues interact during nephron induction by pure diffusion of substances or whether cellular contacts via gap junctions or focal adhesion molecules are involved. In neonatal rabbit kidney the interface between both tissues shows unique features. It consists of a distinct space, which is filled with specific extracellular matrix consisting of glycosylated proteins such as fibronectin, laminin, collagen, and proteoglycans. In the present experiments we tested by histochemistry whether it is possible to detect additional glycosylated proteins using Soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Ulex europaeus I agglutinin (UEA I), and Peanut agglutinin (PNA) as molecular markers. All tested lectins showed distinct labeling patterns in embryonic renal tissue. Within the collecting duct ampulla, DBA and UEA I revealed intensive cellular reaction. In contrast, PNA and SBA reacted at the basal aspect of the collecting duct ampulla tip in addition to a cellular reaction. To identify the individual molecules labeled by the lectins, embryonic tissue was fractionated and separated by electrophoretic methods. For the first time, we were able to show by two-dimensional electrophoresis and subsequent western blot experiments that lectins bind to a series of individual protein spots, which have not been identified to date.     

Ultrastructural organization of the transition from the distal nephron to the collecting duct in the desert rodent Psammomys obesus

Summary The transition from the nephron to the collecting duct is formed by three tubular segments (convoluted part of the distal tubule, connecting tubule, cortical collecting duct), which in the desert rodent, Psammomys obesus, transform gradually from one segment to the next, due to intermingling of their different cell types.The convoluted part of the distal tubule (DTC) starts abruptly, shortly beyond the macula densa and initially is homogeneously composed of characteristic DTC-cells. Subsequently, the DTC-cells intermingle with intercalated cells. The first appearance of the connecting-tubule cell, which gradually replaces the DTC-cell, is regarded as the beginning of the connecting tubule. The major portion of the connecting tubule is lined by connecting-tubule cells and intercalated cells. The first appearance of the principal cell between them defines the beginning of the cortical collecting duct, which in the medullary ray is lined by principal and intercalated cells only.Each cell type is described in detail and discussed in relation to the assumed function of the tubular segments.Interspecies differences in the cellular composition of the transitional zone from the nephron to the collecting duct are discussed in relation to the different organization of the collecting duct system.     

Cytochemical localization of carbohydrates in intercalated duct and acinar cells of mouse parotid gland

Summary The glycoconjugate composition of mouse intercalated duct and acinar cells of parotid gland has been compared. Mucins containing 1,2-glycols were demonstrated by the tannic acid-uranyl acetate technique. Hexose residues of glycoconjugates were identified using ferritin conjugated withCanavalia ensiformis agglutinin (Con A),Triticum vulgare or wheat germ agglutinin (WGA),Ricinus communis I agglutinin (RCA-I),Phaseolus vulgaris agglutinin (PHA-E) andArachis hypogaea agglutinin (PNA). Whereas qualitative and quantitative differences were observed in sugar residues of secretory granules in intercalated duct and acinar cells, apical plasmalemmae were labelled sparsely and similarly. This indicates that the glycocalyx composition of apical plasma minae in the parotid acinar and intercalated duct cells is little influenced by secretory granule composition.     

Ultrastructure of the kidney of a South American caecilian,Typhlonectes compressicaudus (Amphibia,Gymnophiona)

Summary The ultrastructure of the distal nephron, the collecting duct and the Wolffian duct was studied in a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) by transmission and scanning electron microscopy (TEM, SEM). The distal tubule (DT) is made up of one type of cell that has a well-developed membrane labyrinth established both by interdigitating processes and by interlocking ramifications. The processes contain large mitochondria, the ramifications do not. The tight junction is shallow and elongated by a meandering course. The connecting tubule (CNT) is composed of CNT cells proper and intercalated cells, both of which are cuboidal in shape. The CNT cells are characterized by many lateral interlocking folds. The intercalated cells have a dark cytoplasm densely filled with mitochondria. Their apical cell membrane is typically amplified by microplicae beneath which a layer of globular particles (studs) is found. The collecting duct (CD) is composed of principal cells and intercalated cells, again both cuboidal in shape. The CD epithelium is characterized by dilated intercellular spaces, which are often filled with lateral microfolds projecting from adjacent principal cells. The apical membrane is covered by a prominent glycocalyx. The intercalated cells in the CD are similar to those in the CNT. The Wolffian duct (WD) has a tall pseudostratified epithelium established by WD cells proper, intercalated cells and basal cells. The WD cells contain irregular-shaped dense granules located beneath the apical cell membrane. The intercalated cells of the WD have a dark cytoplasm with many mitochondria; their nuclei display a dense chromatin pattern.Research fellow of the Alexander von Humboldt Foundation     

Microheterogeneity of the collecting duct system in rabbit kidney as revealed by monoclonal antibodies

Summary This report describes the immunolocalization of three monoclonal antibodies along the collecting duct system in rabbit kidney. The antibodies were raised against antigens derived from a membrane fraction of homogenized papillary tissue. Western Blot analysis demonstrated that each of the antibodies recognized a single band of about 190000 (P_CD1), 210000 (P_CD2) and 50000 (P_CD3) daltons. In renal tissue, the antibodies bound specifically to the epithelia of the connecting tubule (CNT), the collecting duct (CD) and the papillary surface epithelium. Differences in the binding patterns of the antisera were limited to the cortex. p_CD1 labeled only a few scattered cells in the CNT, and exhibited a heterogeneous binding along the cortical collecting duct (CCD). P_CD2 and P_CD3 binding patterns were similar. In the CNT, these antibodies bound to the intercalated cells (IC-cells) but not to the CNT-cells proper. In the CCD, both IC-cells and principal cells were labeled. The binding to the medullary collecting duct by all three antisera was identical. The ureter was labeled only by P_CD2 and P_CD3, and none of the antisera bound to the bladder epithelium.The antibody binding patterns provide information concerning tubular axial heterogeneity and embryogenetic aspects of the CNT and the CCD. These antibodies may be used as differentiation markers in studies of the developing kidney and of renal tissue culture systems.These studies were supported by Deutsche Forschungsgemeinschaft, Forschergruppe Niere, Kr 546/5-1     

The influence of culture media on embryonic renal collecting duct cell differentiation

Summary During kidney development the embryonic ampullar collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. Part of this development can be mimicked under in vitro conditions, when embryonic collecting duct epithelia are isolated from neonatal rabbit kidneys and kept under perfusion culture. The differentiation pattern is quite different when the embryonic collecting duct epithelia are cultured in standard Iscove’s modified Dulbecco’s medium as compared to medium supplemented with additional NaCl. Thus, the differentiation behavior of embryonic CD epithelia is unexpectedly sensitive. To obtain more information about how much influence the medium has on cell differentiation, we tested medium 199, basal medium Eagle, Williams’ medium E, McCoys 5A medium, and Dulbecco’s modified Eagle medium under serum-free conditions. The experiments show that in general, all of the tested media are suitable for culturing embryonic collecting duct epithelia. According to morphological criteria, there is no difference in morphological epithelial cell preservation. The immunohistochemical data reveal two groups of expressed antigens. Constitutively expressed antigens such as cytokeratin 19, P_CD 9, Na/K ATPase, and laminin are present in all cells of the epithelia independent of the culture media used. In contrast, a group of antigens detected by mab 703, mab 503, and PNA is found only in individual series. Thus, each culture medium produces epithelia with a very specific cell differentiation pattern.     

Temporal-spatial co-localisation of tissue transglutaminase (Tgase2) and matrix metalloproteinase-9 (MMP-9) with SBA-positive micro-fibres in the embryonic kidney cortex

Growth of the kidney is a complex process piloted by the collecting duct (CD) ampullae. The dichotomous arborisation and consecutive elongation of this tubular element determines the exact site and time for the induction of nephrons in the overlaying mesenchymal cap condensates. The mechanism by which the CD ampullae find the correct orientation is currently unknown. Recently, we have demonstrated micro-fibres that originate from the basal aspect of the CD ampullae and extend through the mesenchyme to the organ capsule. The micro-fibres are assumed to be involved in the growth and arborisation process of the CD ampulla. Therefore, we have investigated the specific distribution of the micro-fibres during branching morphogenesis. We have also analysed whether the micro-fibres co-localise with extracellular matrix (ECM)-modulating enzymes and whether the co-localisation pattern changes during CD ampulla arborisation. Micro-fibres were detected in all stages of CD ampulla arborisation. Tissue transglutaminase (Tgase2) co-localised with soybean agglutinin (SBA)-positive micro-fibres, whose presence depended upon the degree of CD branching. Matrix metalloproteinase-9 (MMP-9) also co-localised with micro-fibres, but its expression pattern was different from that for Tgase2. Western blotting experiments demonstrated that Tgase2 and MMP-9 co-migrated with SBA-labelled proteins. Thus, the micro-fibres are developmentally modulated by enzymes of the ECM in embryonic kidney cortex. These findings illustrate the importance of micro-fibres in directing CD ampulla growth.     

Heterogeneity of apical glycoconjugates in kidney collecting ducts: Further studies using simultaneous detection of lectin binding sites and immunocytochemical detection of key transport enzymes

Summary In the search for a functional role for the polarized glycoconjugates of rat collecting duct epithelial cells, the relation between binding of various lectins and expression of cellular transport enzyme profile of the cells was studied. For this purpose, principal and intercalated cells of rat kidney collecting duct were identified by morphological criteria and by their immunocytochemically determined content of (Na⁺+K⁺)-ATPase and carbonic anhydrase (CA II), respectively. VariousN-acetylgalactosamine-specific lectins such as those fromHelix pomatia andMaclura pomifera revealed heterogeneity among both principal and intercalated cells, whereas -N-acetylgalactosa nine-specific lectin fromDolichos biflorus andVicia villosa bound preferentially to principal cells. Still another lectin fromArachis hypogaea reacted with most collecting duct cells in the cortex and outer medulla, but only with a subpopulation of cells in the inner medulla. Interestingly, some lectins reacted exclusively with the apical aspect of the collecting duct epithelial cells, whereas others revealed both an apical and basolateral distribution of lectin reactive glycoconjugates. The results thus show subtle differences in the glycocalyx structure of principal and intercalated cells and differences in the intracellular polarization of glycoconjugates of these cells. Thus, lectins may be useful tools in the study of the molecular mechanisms which establish and maintain the polarized functions of principal and intercalated cells.     

Structure of the kidney in the crab-eating frog, Rana cancrivora

The structure of the nephron in the ranid frog, Rana cancrivora, was studied by light and electron microscopy. This frog is the only amphibian species to live in mangrove swamps of very high salinity. The nephron consists of the following parts: renal corpuscle, ciliated neck segment, proximal tubule, ciliated intermediate segment, distal tubule, connecting tubule, and collecting duct. The distal tubule is located in the ventromedial region of the kidney, and the other tubules are situated in the dorsolateral region. Renal corpuscles are found between the two regions. Some renal corpuscles have a wide Bowman's space because of the small glomerulus within them. The proximal tubules are composed of columnar cells with a dense luminal brush border of long microvilli and numerous apical vesicles and vacuoles. The initial part of the distal tubule consists of heavily interdigitated cells, characterized by a very regular palisade arrangement of mitochondria. In the terminal part of the distal tubule, shorter mitochondria of the infolding cells are situated irregularly around the nucleus. The connecting tubule consists of principal cells and canaliculus cells. The collecting duct consists of columnar or cuboidal cells; cytoplasmic organelles are relatively sparse. The canaliculus cells are intercalated between principal cells from the terminal distal tubule to the proximal part of the collecting duct. Our findings indicate that the kidney of R. cancrivora is structurally similar to kidneys of other amphibians. These findings are discussed with regard to probable correlations between ultrastructure and function in R. cancrivora.     

Carbonic anhydrase in the monkey kidney

The distribution of carbonic anhydrase in the kidney of the cynomolgus monkey was studied by the histochemical method of Hansson. Glomeruli and Bowman's capsule were inactive. Convoluted proximal tubules showed high enzyme activity at the brush border and the basolateral membranes and the cytoplasm. Straight proximal tubules were less intensely stained. In nephrons with long loops of Henle, the descending thin limb contained weak enzyme activity, whereas the ascending thin limb was inactive. The thick limb of Henle's loop displayed most enzyme activity at the luminal cell border. In distal convoluted tubules enzyme activity was restricted to the basal part of the cells. In the late distal tubule, intercalated cells appeared among the "ordinary" distal cells and contained abundant cytoplasmic enzyme. Many intensely stained intercalated cells were also found in the cortical and outer medullary segments of the collecting duct, intermingled with more weakly stained chief cells. In the inner medullary segment of the collecting duct, enzyme activity gradually disappeared. Many capillaries were clearly stained for enzyme activity. The capillary staining apparently varied with that of the kidney tubules; virtually all capillaries in the cortex, but very few in the inner medulla, were stained. The distribution of carbonic anhydrase in the kidney tubules of the monkey is very similar to that in man and in the rat, but the primate kidney differs from the rat kidney by the presence of capillary enzyme activity. The functional importance of this difference is not clear at present.     

Endogenous retinoic acid activity in principal cells and intercalated cells of mouse collecting duct system

Background

Retinoic acid is the bioactive derivative of vitamin A, which plays an indispensible role in kidney development by activating retinoic acid receptors. Although the location, concentration and roles of endogenous retinoic acid in post-natal kidneys are poorly defined, there is accumulating evidence linking post-natal vitamin A deficiency to impaired renal concentrating and acidifying capacity associated with increased susceptibility to urolithiasis, renal inflammation and scarring. The aim of this study is to examine the presence and the detailed localization of endogenous retinoic acid activity in neonatal, young and adult mouse kidneys, to establish a fundamental ground for further research into potential target genes, as well as physiological and pathophysiological roles of endogenous retinoic acid in the post-natal kidneys.

Methodology/Principal Findings

RARE-hsp68-lacZ transgenic mice were employed as a reporter for endogenous retinoic acid activity that was determined by X-gal assay and immunostaining of the reporter gene product, β-galactosidase. Double immunostaining was performed for β-galactosidase and markers of kidney tubules to localize retinoic acid activity. Distinct pattern of retinoic acid activity was observed in kidneys, which is higher in neonatal and 1- to 3-week-old mice than that in 5- and 8-week-old mice. The activity was present specifically in the principal cells and the intercalated cells of the collecting duct system in all age groups, but was absent from the glomeruli, proximal tubules, thin limbs of Henle''s loop and distal tubules.

Conclusions/Significance

Endogenous retinoic acid activity exists in principal cells and intercalated cells of the mouse collecting duct system after birth and persists into adulthood. This observation provides novel insights into potential roles for endogenous retinoic acid beyond nephrogenesis and warrants further studies to investigate target genes and functions of endogenous retinoic acid in the kidney after birth, particularly in the collecting duct system.     

Immunohistochemical localization of a protein fraction derived from rabbit renal papilla

Studies were carried out to define antigenic characteristics of the rabbit renal collecting duct. Renal papillae of adult rabbits were homogenized, centrifuged, and the 600 X g pellet was extracted with 0.5% Triton X-100 in the presence of 1 M NaCl. The crude extract was fractionated on an anion exchange column (DEAE cellulose). A fraction enriched in acidic proteins that co-purified with a radioactive 150 kd glycoprotein from cultured collecting duct cells (Minuth 1982), was used for immunization of guinea pigs. The antiserum shows the following characteristics as revealed by indirect immunofluorescence on the rabbit kidney: 1) Among all tubular epithelial cells only principal cells of the collecting duct and the connecting tubule cell show immunoreactivity. 2) The antiserum decorates the epithelial-interstitial interface of the whole collecting duct as well as of connecting tubule and thick ascending limb of Henle's loop. 3) There is immunoreactivity of interstitial fibers throughout the kidney. 4) Epithelial cells in a variety of other organs in rabbit did not react with the antiserum. Our data demonstrate an antigenic distinction of both, the connecting tubule cell and the principal cell, discriminating these cells from other tubular epithelial cells including the intercalated cells of the collecting duct system. Furthermore, our findings point to a heterogeneity along the distal nephron with respect to the constituents of the epithelial-interstitial interface.