Antigenic expression of aminopeptidase M, dipeptidyl-peptidase IV and endopeptidase by primary cultures from rabbit kidney proximal tubule

Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occurred during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occurred only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.     

CHIP28 water channels are localized in constitutively water-permeable segments of the nephron

The sites of water transport along the nephron are well characterized, but the molecular basis of renal water transport remains poorly understood. CHIP28 is a 28-kD integral protein which was proposed to mediate transmembrane water movement in red cells and kidney (Preston, G. M., T. P. Carroll, W. B. Guggino, and P. Agre. 1992. Science [Wash. DC]. 256:385-387). To determine whether CHIP28 could account for renal epithelial water transport, we used specific polyclonal antibodies to quantitate and localize CHIP28 at cellular and subcellular levels in rat kidney using light and electron microscopy. CHIP28 comprised 3.8% of isolated proximal tubule brush border protein. Except for the first few cells of the S1 segment, CHIP28 was immunolocalized throughout the convoluted and straight proximal tubules where it was observed in the microvilli of the apical brush border and in basolateral membranes. Very little CHIP28 was detected in endocytic vesicles or other intracellular structures in proximal tubules. Uninterrupted, heavy immunostaining of CHIP28 was also observed over both apical and basolateral membranes of descending thin limbs, including both short and long loops of Henle. These nephron sites have constitutively high osmotic water permeabilities. CHIP28 was not detected in ascending thin limbs, thick ascending limbs, or distal tubules, which are highly impermeable to water. Moreover, CHIP28 was not detected in collecting duct epithelia, where water permeability is regulated by antidiuretic hormone. These determinations of abundance and structural organization provide evidence that the CHIP28 water channel is the predominant pathway for constitutive transepithelial water transport in the proximal tubule and descending limb of Henle's loop.     

Comparative study of ezrin phosphorylation among different tissues: more is good; too much is bad

In a comparison of three different tissues, the membrane cytoskeleton linker protein ezrin was found to assume high levels of phosphorylation on threonine-567 (T567) in the brush border membranes of renal proximal tubule cells and small intestine enterocytes, in contrast to the apical canalicular membrane of gastric parietal cells. Together with an earlier observation that increased T567 phosphorylation is associated with more elaborate microvilli in parietal cells, this comparative study suggested a higher phosphorylation level requirement for the denser and more uniform distribution of microvilli at brush border surfaces. Using a kinase inhibitor, staurosporin, and metabolic inhibitor, sodium azide, relatively high turnover of ezrin T567 phosphorylation was observed in all three epithelia. Aiming to understand the role of phosphorylation turnover in these tissues, detergent extraction analysis of gastric glands and proximal tubules revealed that an increased phosphorylation on ezrin T567 greatly enhanced its association with F-actin, while ezrin-membrane interaction persisted regardless of the changes of phosphorylation level on ezrin T567. Finally, expression of Thr567Asp mutant ezrin, which mimics the phospho-ezrin state but does not allow turnover, caused aberrant growth of membrane projections in cultured proximal tubule cells, consistent with what had previously been observed in several cell lines and gastric parietal cells. These results fit into a model of surface plasticity, which posits that the turnover of phosphorylation on T567 empowers ezrin to relax and reposition membrane to the underlying cytoskeleton under varying conditions of filament growth or rapid membrane expansion (or depletion).     

Polarized membrane expression of brush-border hydrolases in primary cultures of kidney proximal tubular cells depends on cell differentiation and is induced by dexamethasone

To analyze the influence of cell differentiation and the effects of hormones on the subcellular distribution of apical antigens in polarized epithelial cells, we have compared the localization of three brush border (BB) hydrolases [neutral endopeptidase (ENDO), aminopeptidase N (APN), and dipeptidylpeptidase IV (DPPIV)] in primary cultures of renal proximal tubule cells grown in various culture media. The degree of cell differentiation modulated by medium composition was estimated by measuring proximal functions, including glucose transport, specific enzymatic activities, and PTH responsiveness. In the dedifferentiated state observed in cells grown in 1% fetal calf serum (FCS)-supplemented medium, the three hydrolases are abnormally concentrated in a cytoplasmic vesicle compartment with weak expression on both membrane domains. By contrast, in serum-free hormonally defined medium (DM: insulin, 5 microgram/ml; dexamethasone, 5 x 10(-8) M), which markedly enhances morphological and functional cell differentiation, the distribution of hydrolases parallels that observed in the normal tubule. When added to the DM devoid of hormones, insulin has little polarizing effect, whereas dexamethasone dramatically increases the apical expression of the hydrolases, which then almost disappear from the basolateral membrane and cytoplasmic vesicular compartments. This glucocorticoid hormone augments the amount of immunoreactive antigen detectable on the apical domain in paraformaldehyde-fixed cells but does not change the total enzymatic activity. This suggests the presence in tubular cells of a dexamethasone-dependent polarizing machinery that requires de novo RNA and protein synthesis, and probably acts mainly by targeting a storage cytoplasmic pool of enzyme to the apical domain.     

Monoclonal antibodies to rat renal tissue: an approach to the immunohistological analysis of the nephron-collecting duct system, and ultra-structural localization of antigens

Summary To obtain more accurate information on the nephron-collecting duct system, monoclonal antibodies against renal tissue were prepared. BALB/c mice were immunized every two weeks with rat renal tissue, either cortex or medulla. Spleen cells were collected and fused with myeloma cells sensitive to hypoxanthine-aminopterin-thymidine medium. Hybrids were selected for production of antibodies by indirect immunofluorescence and cloned by the limiting dilution method. Tissue reactivity of the antibodies obtained was defined by immunofluorescence. The intracellular localization of antigenic determinants was ascertained by immunoelectron microscopy. The antibodies were classified into four major groups: (1) antibodies against proximal tubules; (2) antibodies against distal tubules and the loop of Henle; (3) antibodies against collecting duct system; and (4) antibodies against glomeruli. Using immunoelectron microscopy, various intracellular antigenic determinants were recognized, such as brush border, apical canaliculi, vacuolar apparatus, luminal and basolateral plasma membranes. The results obtained indicated that electron microscopy is indispensable for the immunohistological study of the nephroncollecting duct system. The observations help to understand morphological and functional diversity of the nephron-collecting duct system.     

Evidence for receptor-mediated uptake of adenosine deaminase in rabbit kidney

We investigated the subcellular location of adenosine deaminase-complexing protein in the proximal renal tubules of rabbit kidney and its interaction with intravenously infused monomeric calf adenosine deaminase. Cortical tissue from non-infused animals, stained in suspension by the peroxidase-antiperoxidase method for complexing protein and embedded in resin, was examined by transmission electron microscopy. Positive staining indicated the presence of complexing protein on the surface of microvilli in the proximal tubules. Sections (1 micron) of resin-embedded cortex from infused rabbits, stained first for complexing protein and then for adenosine deaminase, were examined by light microscopy. After staining for complexing protein by indirect immunofluorescence, the sections were photographed and then immersed in buffer containing 6 M guanidine hydrochloride plus 2-mercaptoethanol for 3 hr at 60 degrees C to remove bound antibodies. The sections were then stained by the peroxidase-antiperoxidase method for infused enzyme. Vesicle-like apical structures, the basal membrane area and, as previously reported, the brush border of proximal tubule cells were positive for complexing protein. Vesicle-like structures and brush borders positive for complexing protein were also stained for adenosine deaminase. The basal membrane area did not stain. These results support the hypothesis that complexing protein can act as a receptor for adenosine deaminase.     

Changes in expression and subcellular localization of annexin IV in rabbit kidney proximal tubule cells during primary culture

In the present study, we investigated the polarized expression of annexin IV at various stages in the growth of rabbit kidney proximal tubule cells (PTC) in primary cultures. The results of immunoblotting analysis and indirect immunofluorescence studies using a specific anti-annexin IV monoclonal antibody, indicated that annexin IV is expressed in proximal tubule cultured cells, although it was not detected in the proximal tubules present in frozen sections of kidney cortex and freshly isolated proximal tubule cells. In either non-confluent or confluent cells which remained attached to the collagen-coated support, annexin IV was mainly concentrated around the nucleus, whereas in PTC forming the monolayer of domes, it was restricted to the basolateral membrane domain. This basolateral localization was identical to that observed in other polarized epithelial cell types such as enterocytes. When the domes burst, the cells returned to the collagen-coated support and the annexin IV was again localized around the nuclei. The fact that the change of localization was very rapid suggested the existence of a considerable difference between the differentiation states of dome forming and adherent confluent cells. Moreover, a transient association of annexin IV with the basal body of apically located cilia also seemed to be correlated with a particular polarization state and/or differentiation states of adherent cultured cells, corresponding to the beginning of the polarized expression of aminopeptidase N, a hydrolase located in the apical brush border membrane, and to the falling of cells onto the support, subsequent to the bursting of the domes. In conclusion, these results provide evidence that annexin IV may constitute a new marker of the basolateral membrane domain of polarized epithelial renal cells in primary cultures. © 1995 Wiley-Liss, Inc.     

Localization of aquaporin-7 in rat and mouse kidney using RT-PCR, immunoblotting, and immunocytochemistry

To establish the segmental, cellular, and subcellular localization of AQP7 in rat and mouse kidney, we used RT-PCR, immunocytochemical, and immunoblotting approaches. RT-PCR of rat and mouse kidney zones revealed AQP7 mRNA in cortex and outer stripe of the outer medulla. RT-PCR on microdissected nephron segments revealed AQP7 mRNA in proximal convoluted and straight tubules. Immunoblotting using peptide-derived rabbit antibodies to either rat or mouse AQP7 revealed a 28-kDa band in kidney and testes from rat and mouse, respectively. Immunocytochemistry revealed strong AQP7 labeling of segment 3 proximal tubules and weaker labeling of proximal convoluted tubules in both rat and mouse kidneys. The labeling was almost exclusively confined to the brush border with no basolateral labeling. No labeling was observed of thin descending limbs or collecting duct. Immunolabeling controls were negative. The presence of AQP7 in the proximal tubule brush border indicates a role of AQP7 in proximal tubule water reabsorption.     

Differential expression pattern of protein ARVCF in nephron segments of human and mouse kidney

The protein ARVCF is a member of the p120 subfamily of armadillo proteins whose members have been described to occur in junction-bound and non-junction-bound forms. Studies on ARVCF were constrained because the endogenous protein was difficult to detect with the available reagents. We have generated novel monoclonal and polyclonal antibodies usable for biochemical and localization studies. By systematic immunohistochemical analysis of various tissues protein ARVCF is prominently detected in mouse, bovine and human kidney. Using antibodies against specific markers of nephron segments protein ARVCF is localized in proximal tubules according to double label immunofluorescence. Besides its occurrence in proximal tubules of adult kidney and in renal cell carcinoma derived from proximal tubules ARVCF is also detected in maturing nephrons in early mouse developmental stages such as, for example, 15 days of gestation (E15). Immunoblotting of total extracts of cultured cells of renal origin showed that ARVCF is detected in all human and murine cultured cells analyzed. Upon immunolocalization ARVCF is mostly detected in the cytoplasm occurring in a fine granular form. This prominent cytoplasmic localization of ARVCF in cultured cells and its occurrence in proximal tubules implies an involvement of ARVCF in specific functional processes of proximal tubules of kidney. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence for a decreased membrane recycling in the cells of renal proximal tubules exposed to high concentrations of ferritin

Summary The present study was performed to investigate whether membrane recycling via the dense apical tubules in cells of renal proximal tubules could be modified after exposure to large amounts of cationized ferritin. Proximal tubules in the rat kidney were microinfused in vivo with cationized ferritin for 10 or 30 min and then fixed with glutaraldehyde by microinfusion, or proximal tubules were microinfused with ferritin for 30 min and then fixed 2 h thereafter. The tubules were processed for electron microscopy, and the surface density and the volume density of the different cell organelles involved in endocytosis were determined by morphometry. The morphometric analyses showed that after loading of the endocytic vesicles with ferritin the surface density of dense apical tubules decreased to about 50% of the original value. However, 2 h later when ferritin had accumulated in the lysosomes the surface density of dense apical tubules had returned to control values. Furthermore, cationized ferritin was virtually absent from the Golgi region, indicating that the Golgi apparatus in these cells does not participate in membrane recycling. In conclusion, the present study shows that membrane recycling in renal proximal tubule cells can in part be inhibited by loading the endocytic vacuoles with ferritin.     

The B1 subunit of the H+ATPase is a PDZ domain-binding protein. Colocalization with NHE-RF in renal B-intercalated cells

The 56-kDa B1 subunit of the vacuolar H(+)ATPase has a C-terminal DTAL amino acid motif typical of PDZ-binding proteins that associate with the PDZ protein, NHE-RF (Na(+)/H(+) exchanger regulatory factor). This B1 isoform is amplified in renal intercalated cells, which play a role in distal urinary acid-base transport. In contrast, proximal tubules express the B2 isoform that lacks the C-terminal PDZ-binding motif. Both the B1 56-kDa subunit and the 31-kDa (E) subunit of the H(+)ATPase are pulled down by glutathione S-transferase NHE-RF bound to GSH-Sepharose beads. These subunits associate in vivo as part of the cytoplasmic V1 portion of the H(+)ATPase, and the E subunit was co-immunoprecipitated from rat kidney cytosol with NHE-RF antibodies. The interaction of H(+)ATPase subunits with NHE-RF was inhibited by a peptide derived from the C terminus of the B1 but not the B2 isoform. NHE-RF colocalized with H(+)ATPase in either the apical or the basolateral region of B-type intercalated cells, whereas NHE-RF staining was undetectable in A-intercalated cells. In proximal tubules, NHE-RF was located in the apical brush border. In contrast, H(+)ATPase was concentrated in a distinct membrane domain at the base of the brush border, from which NHE-RF was absent, consistent with the expression of the truncated B2 subunit isoform in this tubule segment. The colocalization of NHE-RF and H(+)ATPase in B- but not A-intercalated cells suggests a role in generating, maintaining, or modulating the variable H(+)ATPase polarity that characterizes the B-cell phenotype.     

Passive Heymann nephritis induced by rabbit antiserum to membrane antigens isolated from rat visceral yolk-sac microvilli

Passive Heymann nephritis (PHN) is an animal model of immune-complex-induced renal disease resembling human membranous glomerulonephritis. It was induced in rats by injecting rabbit antiserum directed against glycoprotein antigens isolated from rat embryonic visceral yolk-sac microvilli (VYS-MV). The glycoprotein antigens were isolated by extracting the VYS-MV with detergent Nonidet P-40 followed by gel filtration in Sephacryl S-300 and finally by lectin affinity chromatography with Ricinus communis agglutinin I. In vitro immunofluorescent localization studies demonstrated that the nephritogenic antibodies were localized along the apical region of the visceral yolk-sac endodermal cells and the brush border of the proximal tubular cells of the kidney. Rats injected with a single dose of the antiserum manifested proteinuria. Indirect immunofluorescent studies showed that the injected rabbit IgG was localized in vivo along the capillary walls of the glomerulus in a granular fashion. Electron microscopic examination of the same kidney glomeruli revealed numerous electron-dense deposits along the lamina rara externa of the glomerular basement membrane. Fusion of the epithelial foot processes was also present. These findings represent the typical immunopathological characteristics of Heymann nephritis. Furthermore, with the aid of Ouchterlony analysis, the antiserum against the isolated VYS antigens exhibited an immunoprecipitin band which was in common with that formed by the antiserum against the homogeneous nephritogenic antigen (gp330) of renal brush border origin. Thus, the nephritogenic antigens which have been found to be associated with the brush border of the renal proximal tubules may also be present or cross-reacted in the microvilli of the rat embryonic visceral yolk-sac.     

Enhancement of gentamicin-induced nephrotoxicity by Mg deficiency in non-pregnant rats. Morphological, enzyme histochemical and immunohistochemical studies

Non-pregnant rats fed an Mg-deficient diet showed some degenerated and calcified proximal tubules in the inner region of the medullary rays accompanied by reduced or absent enzyme activities. After gentamicin treatment some damaged convoluted proximal tubules occurred. Enzyme histochemistry revealed decreased activities for brush border and lysosomal hydrolases; using immunohistochemistry lesions were detectable for the cytoskeletal proteins keratin and vimentin. Administration of gentamicin to Mg-deficient rats led to a further decrease of hydrolase activities in obviously intact proximal tubules and drastic structural and enzymatic defects as well as alterations of the cytoskeletal proteins in the convoluted and straight segments of other proximal tubules and to a lesser degree also in glomeruli and further portions of the tubular apparatus including the collecting ducts.     

Intracellular localization and endocytosis of brush border enzymes in the enterocyte-like cell line Caco-2.

Immunoelectron microscopy was used to localize the brush border hydrolases sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPPIV) in the human colon carcinoma cell line Caco-2. Both enzymes were detected at the microvillar membrane, in small vesicles and multivesicular bodies (MVBs), and in lysosomal bodies. In addition, DPPIV was found in the Golgi apparatus, a variety of apical vesicles and tubules, and at the basolateral membrane. To investigate whether the hydrolases present in the lysosomal bodies were endocytosed from the apical membrane, endocytic compartments were marked with the endocytic tracer cationized ferritin (CF). After internalization from the apical membrane through coated pits, CF was first recovered in apical vesicles and tubules, and larger electronlucent vesicles (early endosomes), and later accumulated in MVBs (late endosomes) and lysosomal bodies. DPPIV was localized in a subpopulation of both early and late endocytic vesicles, which contained CF after 3 and 15 min of uptake, respectively. Also, internalization of the specific antibody against DPPIV and gold labeling on cryosections showed endocytosed DPPIV in both early and late endosomes. However, unlike CF, no accumulation of DPPIV was seen in MVBs or lysosomal bodies after longer chase times. The results indicate that in Caco-2 cells the majority of brush border hydrolases present in lysosomal bodies are not endocytosed from the brush border membrane. Furthermore, the labeling patterns obtained, suggest that late endosomes may be involved in the recycling of endocytosed DPPIV to the microvilli.     

Apolipoprotein A-II is catabolized in the kidney as a function of its plasma concentration

We investigated in vivo catabolism of apolipoprotein A-II (apo A-II), a major determinant of plasma HDL levels. Like apoA-I, murine apoA-II (mapoA-II) and human apoA-II (hapoA-II) were reabsorbed in the first segment of kidney proximal tubules of control and hapoA-II-transgenic mice, respectively. ApoA-II colocalized in brush border membranes with cubilin and megalin (the apoA-I receptor and coreceptor, respectively), with mapoA-I in intracellular vesicles of tubular epithelial cells, and was targeted to lysosomes, suggestive of degradation. By use of three transgenic lines with plasma hapoA-II concentrations ranging from normal to three times higher, we established an association between plasma concentration and renal catabolism of hapoA-II. HapoA-II was rapidly internalized in yolk sac epithelial cells expressing high levels of cubilin and megalin, colocalized with cubilin and megalin on the cell surface, and effectively competed with apoA-I for uptake, which was inhibitable by anti-cubilin antibodies. Kidney cortical cells that only express megalin internalized LDL but not apoA-II, apoA-I, or HDL, suggesting that megalin is not an apoA-II receptor. We show that apoA-II is efficiently reabsorbed in kidney proximal tubules in relation to its plasma concentration.     

Enhancement of gentamicin-induced nephrotoxicity by Mg deficiency in non-pregnant rats

Summary Non-pregnant rats fed an Mg-deficient diet showed some degenerated and calcified proximal tubules in the inner region of the medullary rays accompanied by reduced or absent enzyme activities. After gentamicin treatment some damaged convoluted proximal tubules occurred. Enzyme histochemistry revealed decreased activities for brush border and lysosomal hydrolases; using immunohis-tochemistry lesions were detectable for the cytoskeletal proteins keratin and vimentin. Administration of gentamicin to Mg-deficient rats led to a further decrease of hydrolase activities in obviously intact proximal tubules and drastic structural and enzymatic defects as well as alterations of the cytoskeletal proteins in the convoluted and straight segments of other proximal tubules and to a lesser degree also in glomeruli and further portions of the tubular apparatus including the collecting ducts.Supported by the Deutsche Forschungsgemeinschaft (Sfb 174)     

Membrane traffic after inhibition of endocytosis in renal proximal tubules

This study was performed to examine quantitatively the cellular organelles involved in membrane recycling after inhibition of luminal endocytosis in renal proximal tubules. Paraffin oil was microinfused into rat renal proximal convoluted tubules to prevent luminal endocytosis. After 1-2 hr the kidneys were fixed by perfusion and prepared for electron microscopy. Segment 1 proximal tubules infused with paraffin oil and control tubules from the same kidney were studied. In addition we examined proximal tubules from kidneys fixed by immersion 30 sec after removal of the kidney. In the oil-infused tubules the large endocytic vacuoles (greater than 0.5 micron) disappeared, the amount of small endocytic vacuoles (less than 0.5 micron) was reduced to about 10%, and the amount of dense apical tubules was significantly increased. The dense apical tubules were very seldom seen connected to the apical plasma membrane in controls but this was occasionally observed in tubules fixed by immersion and relatively often in oil-infused tubules. An ultrastructural morphometric analysis substantiated and extended the qualitative observations and provided quantitative estimates of volumes and surface areas for large endocytic vacuoles, lysosomes, mitochondria, small endocytic vacuoles, and dense apical tubules in control and experimental tubules. The results strongly support the suggestion that the dense apical tubules located in the apical cytoplasm represent the vehicle for the recycling of membrane from endocytic vacuoles back to the plasma membrane, and show that in renal proximal tubule cells small and large endocytic vacuoles are transformed into dense apical tubules when endocytosis is stopped.     

Electron microscopic immunohistochemical localization of components of Na+-cotransporters along the rat nephron

The localization of Na+-cotransport proteins in cortex and outer medulla of rat kidney was investigated with five monoclonal antibodies. Recently, it was found that these antibodies altered Na+-D-glucose cotransport and/or Na+-dependent high affinity phlorizin binding in pig kidney cortex and that three of these antibodies interacted also with Na+-cotransporters for lactate, L-alanine and/or L-glutamate (Koepsell, H., K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, D. Ollig, J. Biol. Chem. 263, 18,419-18,429 (1988]. In pig and rat the monoclonal antibodies bind to two brush-border membrane polypeptides with identical molecular weights and isoelectric points of 75,000 and pI 5.5, and 47,000 and pI 5.4. These polypeptides have been previously identified as components of the porcine renal Na+-D-glucose cotransporter (Neeb, M., U. Kunz, H. Koepsell, J. Biol. Chem. 262, 10,718-10,727 (1987] and may also be part of other Na+-cotransporters. The electron microscopic localization of antibody binding was demonstrated by protein A-gold labeling on ultrathin plastic sections. Three antibodies bound to brush-border membranes of proximal convoluted and straight tubules. In the proximal convoluted tubules all antibodies reacted with apical endocytic vacuoles, apical dense tubules and lysosomes. Since dense tubules are supposed to originate from endocytic vacuoles and to fuse with brush-border membranes the data suggest recycling of Na+-cotransporters in the proximal convoluted tubule. In the outer medulla two antibodies bound to apical membranes of descending thin limbs (DTL) of short loops of Henle and to apical and basal membranes of DTL of long loops of Henle. Three antibodies bound to apical membranes of collecting ducts. These data indicate that Na+-cotransporters or homologous proteins exist beyond the proximal tubule.     

Histochemical localization of enzyme activity in the kidneys of male marmosets (Callithrix jacchus and Callithrix penicillata).

The distribution of several hydrolases and oxidoreductases was studied in the renal parenchyma of adult male marmosets (Callithrix jacchus and Callithrix penicillata). The oxidative enzymes showed a high reactivity in the proximal and distal tubules, whereas the hydrolases reacted strongly in the proximal tubules but only weakly or not at all in the thick limb of Henle's loop, distal tubules and collecting ducts. The NAD-dependent enzymes (except alpha-GPDH) showed a stronger reactivity in the proximal tubules, while the NADP-dependent ones were more reactive in the thick limb of Henle's loop and distal convoluted tubules. Two groups of interstitial cells were found in the medulla. A first group inside the outer medulla, showing cells rich in acid phosphatase and nonspecific esterases and a second group, close to the papilla, reactive to a certain number of oxidative enzymes. A different reactivity in cells of the distal convoluted tubules, thick limb of Henle's loops and collecting ducts (dark cells) was seen in the case of some enzymes like nonspecific esterase, alpha-GPDH and SDH.     

Brush cells of the mouse intestine possess a specialized glycocalyx as revealed by quantitative lectin histochemistry. Further evidence for a sensory function.

Brush cells occur in the epithelium of the small intestine and in various other epithelia of endodermal origin. Ultrastructural and histochemical characteristics suggest that they represent sensory cells. Because the apical membrane of brush cells might be involved in and specialized for (chemo-)receptive functions, we investigated the composition of the glycocalyx and compared it with that of enterocytes. Ultrathin sections of murine small intestine were labeled with a panel of eight lectins. Their binding sites in the brush border and on vesicles of the apical cytoplasm were detected by colloidal gold and quantified using image analysis. The glycocalyx of brush cells contained significantly higher amounts of l-fucose residues than that of enterocytes, as detected by the lectins UEA-I and LTA. In contrast, most of the other lectins bound more avidly to the glycocalyx of enterocytes. The cytoplasmic vesicles closely resembled the apical membrane in their labeling pattern. Quantitation of the brush cells' distribution revealed that the epithelia of the Peyer's patches contained 10-fold higher numbers of brush cells than the small intestinal mucosa distant from lymphoid tissue. We conclude that brush cells possess a glycocalyx with a specialized composition and differ significantly from enterocytes. Because similar peculiarities of the apical membrane have previously been described for sensory cells of the olfactory and gustatory organs, this study provides further evidence in favor of a sensory function of brush cells.