Urea transport in freshly isolated and cultured cells from rat inner medullary collecting duct

Summary Regulation of urea transport by vasopressin in inner medullary collecting duct (IMCD) cells is thought to be important for the urinary concentrating mechanism. Isolated tubule perfusion studies suggest the existence of a saturable urea carrier. We have measured¹⁴C-urea efflux in IMCD cells which were freshly isolated and grown in primary culture. Cells were isolated from rat papilla by collagenase digestion and hypotonic shock. In suspended cells,¹⁴C-urea efflux (J _urea from loaded cells was exponential with time constant 59±3 sec (sem,n=6, 23°C).J _urea had an activation energy of 4.1 kcal/mole and was inhibited 42±7% by 0.25mm phloretin and 30–40% by the high affinity urea analogues dimethylurea and phenylurea.J _urea was increased 40–60% by addition of vasopressin (10^–8 m) or 8-bromo-cAMP (1mm); stimulatedJ _urea was inhibited 55±8% by the kinase A inhibitor H-8. Phorbol esters and epidermal growth factor did not alterJ _urea. IMCD cells grown in primary culture were homogeneous in appearance with>fivefold stimulation of cAMP by vasopressin. The exponential time constant for urea efflux was 610±20 sec (n=3).J _urea was not altered by vasopressin, cAMP or phloretin. Another function of in vivo IMCD cells, vasopressin-dependent formation of endosomes containing water channels, was absent in the cultured cells. These results demonstrate presence of a urea transporter on suspended IMCD cells which is activated by cAMP and inhibited by phloretin and urea analogues. The urea transporter and its regulation by cAMP, and cAMP-dependent apical membrane endocytosis, are lost after growth in primary culture.     

Properties of a polarized primary culture from rat renal inner medullary collecting duct (IMCD) cells

Summary A primary culture from rat renal IMCD cells was established to investigate the permeability characteristics of the luminal and contraluminal plasma membranes of the papillary collecting duct in vitro. Freshly isolated IMCD cells were grown on filters in a special “epithelial cell” medium. Confluency was proved with an epithelial volt/ohm meter. After 7 d of culture the transepithelial resistance reached more than 1000 Ω×cm². A polarization of the cells with regard to a basolateral localization of a lactate efflux system, and an l-alanine transport system was achieved. The hypotonicity-activated release systems for the organic osmolytes sorbitol and betaine were also located basolaterally, whereas taurine, glycerophosphorylcholine, and myo-inositol left the cells at both cell poles but with different capacity. Morphological observations revealed also that the monolayer was well differentiated. Thus, a model of a renal collecting duct epithelium was established which can be used to analyze polarized and differentiated transport processes across the epithelial cells and their plasma membranes.     

Immortalized renal proximal and collecting duct cell lines derived from transgenic mice harboring L-type pyruvate kinase promoters as tools for pharmacological and toxicological studies

Targeted oncogenesis in transgenic mice, where an oncogene is placed under the control of the regulatory sequences of a cell-specific gene, has been used to derive lines of differentiated kidney epithelial cells derived from proximal or distal tubules or from the collecting duct. These renal cell lines were obtained from kidneys of transgenic mice harboring the large-T and little-t antigens placed under the control of regulatory sequences of the L-type pyruvate kinase gene. This review summarizes the main properties of these differentiated cell lines, which are usefulex vivo cell systems for pharmacological and toxicological studies. This revised version was published online in August 2006 with corrections to the Cover Date.     

Culture of human main pancreatic duct epithelial cells

Summary Attempts to grow human pancreatic duct epithelial cells in long-term culture have proven difficult. We have developed a system of growing these cells for several passages by adapting methods used to culture dog pancreatic duct cells. Epithelial cells were enzymatically dissociated from the main pancreatic duct and plated onto collagen-coated culture inserts suspended above a human fibroblast feeder layer. After primary culture, the cells were either passaged onto new inserts or plastic tissue culture plates in the absence of collagen. Cells grown on the latter plates were maintained in a serum-free medium. Primary pancreatic duct epithelial cells grow steadily to confluence as a monolayer in the feeder layer system. After primary culture, cells passaged onto new inserts with fresh feeder layer or plastic plates and fed with serum-free medium continued to develop into confluent monolayers for up to four passages. The cells were columnar with prominent apical microvilli, sub-apical secretory vesicles, and lateral intercellular junctions resembling the morphology of normal in vivo epithelial cells. These cells were also positive for cytokeratin 19, 7, and 8 and carbonic anhydrase II, as measured by immunohistochemistry. Metabolically, these cells synthesized and secreted mucin, as measured by incorporation of tritiated N-acetyl-d-glucosamine. In conclusion, we demonstrated that human pancreatic epithelial cells from the main duct can be successfully grown in culture and repeatedly passaged using a feeder layer system, with serum-free medium, and in organotypic cultures.     

Apico-basal osmotic gradient induces transcytosis in cultured renal collecting duct epithelium

Summary The present experiments report the existence of an apico-basal plasma membrane shuttle in cultured renal collecting duct principal cell epithelium. Apical and basal perfusion under isotonic conditions, 290 mosm phosphate-buffered saline (PBS), has no effect on the shape of the epithelium. In contrast, gradient perfusion bf the epithelium with 75 mosm PBS on the apical side and 290 mosm PBS on the basal side for 10 min alters the morphology of the epithelium by causing the originally columnar epithelial cells to become lower, the intercellular spaces to dilate, and the intracellular vesicles to enlarge. Perfusion of the epithelium with isotonic PBS in the presence of electron-dense cellular markers such as gold-coupled GP_CDI antibody, recognizing a glycoprotein in the plasma membrane of collecting duct cells (W.W. Minuth, G. Lauer, S. Bachman and W. Kriz,Histochemistry 80:171–182, 1984), cationized ferritin (CF), horseradish peroxidase (HRP) and native ferritin (NF) for 10 min reveals their binding at the apical plasma membrane. Little endocytosis is observable. However, after labeling the luminal side by the cellular markers and following exposure to apical hypotonicity, 75 mosm PBS for 10 min, endocytosis of all markers is enhanced to a high degree. Furthermore, the gold-coupled GP_CDI antibody and cationized ferritin are transported within vesicles unidirectionally through the epithelium and are exocytosed at the basolateral aspect, indicating the retrieval and possible translocation of apical plasma membrane. In contrast, volume markers such as NF and HRP are also endocytosed under osmotic gradient exposure, but are not seen to be transcytosed. Therefore, the function of this membrane pathway seems not to be related to water reabsorption, but may be part of a cellular response as protection against the osmotic gradient.     

Novel cellular mechanism that mediates the collecting duct formation during postnatal renal development

We have previously demonstrated that kidney embryonic structures are present in rats, and are still developing until postnatal Day 20. Consequently, at postnatal Day 10, the rat renal papilla contains newly formed collecting duct (CD) cells and others in a more mature stage. Performing primary cultures, combined with immunocytochemical and time-lapse analysis, we investigate the cellular mechanisms that mediate the postnatal CD formation. CD cells acquired a greater degree of differentiation, as we observed that they gradually lose the ability to bind BSL-I lectin, and acquire the capacity to bind Dolichos biflorus. Because CD cells retain the same behavior in culture than in vivo, and by using DBA and BSL-I as markers of cellular lineage besides specific markers of epithelial/mesenchymal phenotype, the experimental results strongly suggest the existence of mesenchymal cell insertion into the epithelial CD sheet. We propose such a mechanism as an alternative strategy for CD growing and development.     

Modulation of renin angiotensin system components by high glucose levels in the culture of collecting duct cells

Diabetes mellitus and its complications have become a major health concern in Western countries. Increased activity of the intrarenal renin–angiotensin system (RAS) contributes to diabetic nephropathy (DN). We previously reported that in mesangial cells, the high glucose concentration (HG) leads to upregulation of angiotensin-converting enzyme (ACE) messenger RNA, suggesting that ACE was modulated by angiotensin II (Ang II) release. However, this relation in the collecting duct has not yet been studied. We, therefore, aimed to evaluate RAS modulation in inner medullary collecting duct cells (IMCD) exposed to HG. The IMCD were divided into normal glucose (5 mM D -glucose, NG), high glucose (30 mM, HG), and mannitol (30 mM, M) groups. The cells were cultured 48 hr in their respective media. The intracellular and extracellular ACE activity was measured using hippuryl-His-Leu as substrate via a fluorimetric assay and expression was analyzed using western blot analysis. ACE activity, intracellular (27%) and extracellular (22%), was significantly lower in the HG group than in NG and M. ACE2 activity and Ang 1–7 levels were higher in the intracellular compartment. Our data suggest that the HG cannot modify ACE synthesis in IMCD cells but can modulate its activity. The decrease in ACE activity may result in decreased levels of Ang II to protect the IMCD against proliferative and inflammatory deleterious effects of this peptide. Conversely, the increase of ACE2 generating high levels of Ang 1–7, a vasodilator peptide, suggesting that this peptide can induce glucose uptake and protect cells against oxidative stress, which can elicit insulin resistance.     

Heterogeneity of tight junctions along the collecting duct in the renal medulla

Summary The tight junctions along the medullary collecting duct in the kidneys of the rat and the rabbit were studied with freeze-fracture electron microscopy and quantitated according to the number of strands and the apico-basal depth (nm) of the junctions.The most elaborate tight junctions were found in the inner stripe of the outer medulla; rat: 10.6±0.8 strands and 205±24nm; rabbit: 11.6±2.4 strands and 291±55 nm.The elaboration of the tight junctions decreased continuously towards the papillary tip. Inner zone I; rat: 9.3±2.6 strands and 186±38nm, rabbit: 9.5±2.3 strands and 247±59nm. Inner zone II; rat: 7.1±2.2 strands and 129±32nm, rabbit: 8.5±1.4 strands and 199±26nm. Inner zone III; rat: 6.0±1.6 strands and 111 + 19 nm, rabbit: 7.0±1.5 strands and 183±43 nm. In the inner zone III comprising the papillary tip tight junctions with only 1–3 strands were not infrequently seen. Preliminary findings in the kidney of the golden hamster indicate a similar decline of junctional tightness along the collecting duct.These morphological observations suggest that the permeability of the paracellular pathway of the medullary collecting duct increases towards the tip of the papilla, especially in the rat. The functional implications for the medullary recycling of urea and electrolytes, and for the urinary concentrating mechanism are discussed.In addition, the tight junctions of the papillary epithelium are described.     

Cell type-specific glycoconjugates of collecting duct cells during maturation of the rat kidney

Summary The ontogeny of lectin-positive epithelial cell types and the maturation of polarized expression of the glycocalyx of the collecting ducts (CD) of the rat kidney were studied from samples of 18th-day fetal and neonatal kidneys of various ages. Lectins from Dolichos biflorus (DBA) and Vicia villosa (VVA), with preferential affinity to principal cells, stained virtually all CD cells of the fetal kidneys. However, within two days postnatally, the number of cells positive for DBA and VVA decreased to amounts found in the adult kidneys. Moreover, a characteristic change occurred rapidly after birth in the intracellular polarization of the reactive glycoconjugates, from a uniform plasmalemmal to a preferentially apical staining. In contrast, lectins from Arachis hypogaea (PNA), Maclura pomifera (MPA) and Lotus tetragonolobus (LTA), reacting indiscriminatively with principal and intercalated cells of adult kidneys, stained most CD cells in the fetal kidneys, and failed to show any postnatal change in the amount of positive cells or in the intracellular polarization.The immunocytochemical tests for (Na + K)-ATPase and carbonic anhydrase (CA II) revealed the characteristic postnatal decrease in the amount of principal cells and simultaneous increase in the amount of CA II rich intercalated cells. DBA and VVA reactive cells also decreased postnatally, paralleling the changes observed in the (Na + K)-ATPase positive principal cells. The present results suggest that the expression of the cell type-specific glycocalyx of principal and intercalated cells is developmentally regulated, undergoes profound changes during maturation, and is most likely associated with electrolyte transport phenomena.     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Cancer specific mortality in patients with collecting duct vs. clear cell renal carcinoma

BackgroundCollecting duct carcinoma (CDC) is biologically more aggressive than clear cell renal cell carcinoma (ccRCC). We tested for differences in cancer specific mortality (CSM) rates according to CDC vs. ISUP (International Society of Urological Pathology) 4 ccRCC histological subtype. We hypothesized that the survival disadvantage still applies, even after most detailed adjustments.MethodsWithin Surveillance, Epidemiology, and End Results database (2004–2018), we identified 380 CDC vs. 6273 ISUP 4 ccRCC patients of all stages. Propensity score matching (age, sex, race/ethnicity, T, N, and M stages, nephrectomy, and systemic therapy status), Kaplan-Meier plots and multivariable Cox regression models were used.ResultsAll 380 CDC were matched (1:2) with 760 ISUP4 ccRCC patients. Prior to matching CDC patients exhibited higher rates of lymph node invasion (37.6 % vs. 14.7 %, p < 0.001), and of distant metastases (40.8 % vs. 30.4 %, p < 0.001). Systemic therapy rates were higher in CDC (29.5 % vs. 20.5 %, p < 0.001). However, nephrectomy rates were higher in ISUP4 ccRCC patients (97.5 % vs. 84.7 %, p < 0.001). After matching, in multivariable Cox regression models addressing CSM, CDC was associated with a HR of 1.5 (p < 0.001) in the overall population vs. 1.9 (p = 0.014) in stage I-II vs. 1.4 (p = 0.022) in stage III vs. 1.6 in stage IV (p < 0.001), relative to ISUP4 ccRCC.ConclusionCDC patients exhibited 40–90 % higher CSM than their ISUP4 ccRCC counterparts in the overall analysis, as well as in stage specific analyses. The CSM disadvantage applies despite higher rates of systemic therapy in CDC patients.     

A Calcium-activated and nucleotide-sensitive nonselective cation channel in M-1 mouse cortical collecting duct cells

We recently reported that M-1 mouse cortical collecting duct cells show nonselective cation (NSC) channel activity (Proc. Natl. Acad. Sci. USA 89:10262–10266, 1992). In this study, we further characterize the M-1 NSC channel using single-channel current recordings in excised inside-out patches. The M-1 NSC channel does not discriminate between Na⁺, K⁺, Rb⁺, Cs⁺, and Li⁺. It has a linear I-V relation with a conductance of 22.7±0.5 pS (n=78) at room temperature. The P_cation/ P_anion ratio is about 60 and there is no measurable conductance for NMDG, Ca²⁺, Ba²⁺, and Mn²⁺. Cytoplasmic calcium activates the M-1 NSC channel at a threshold of 10^–6 m and depolarization increases channel activity (NP _o ). Cytoplasmic application of adenine nucleotides inhibits the M-1 NSC channel. At doses of 10^–4 m and 10^–3 m, ATP reduces NP _o by 23% and 69%, respectively.Furthermore, since ADP (10^–3 m) reduces NP _o by 93%, the inhibitory effect of adenine nucleotides is not dependent on the presence of a -phosphoryl group and therefore does not involve protein phosphorylation. The channel is not significantly affected by 8-Br-cGMP (10^–4 m) or by cGMP-dependent protein kinase (10^–7 m) in the presence of 8-Br-cGMP (10^–5 m) and ATP (10^–4 m). The NSC channel is not sensitive to amiloride (10^–4 m cytoplasmic and/or extracellular) but flufenamic acid (10^–4 m) produces a voltage-dependent block, reducing NP _o by 35% at depolarizing voltages and by 80% at hyperpolarizing voltages.We conclude that the NSC channel of M-1 mouse cortical collecting duct cells belongs to an emerging family of calcium-activated and nucleotide-sensitive nonselective cation channels. It does not contribute to amiloride-sensitive sodium absorption and is unlikely to be a major route for calcium entry. The channel is normally quiescent but may be activated under special physiological conditions, e.g., during volume regulation.The expert technical assistance of U. Fink and I. Doering-Hirsch is gratefully acknowledged. We thank A. Rabe and Dr. J. Disser for programming the computer software.This work was supported by a grant from the Deutsche Forschungsge-meinschaft (DFG grant Fr 233/9-1) and a grant from the National Institutes of Health (NIH grant DK-17433).     

Hypertonic sodium chloride and mannitol induces COX-2 via different signaling pathways in mouse cortical collecting duct M-1 cells

The kidney cortical collecting duct is an important site for the maintenance of sodium balance. Previous studies have shown that, in renal medullary cells, hypertonic stress induces expression of cyclooxygenase-2 (COX-2) via NF-kappaB activation, but little is known about COX-2 expression in response to hypertonicity in the cortical collecting duct. Therefore, we examined the mechanism of hypertonic induction of COX-2 in M-1 cells derived from mouse cortical collecting duct. Induction of COX-2 protein was detected within 6 h of treatment with hypertonic sodium chloride. The treatment also increased COX-2 mRNA accumulation in a cycloheximide-independent manner, suggesting that ongoing protein synthesis is not required for COX-2 induction. Using reporter plasmids containing 0.2-, 0.3-, and 1.5-kb fragments of the COX-2 promoter, we found that hypertonic induction of COX-2 was due to an increase in promoter activity. The COX-2-inductive effect of hypertonicity was inhibited by SB203580, indicating that the effect is mediated by p38 MAPK. Since p38 MAPK can activate NF-kappaB, we made point mutations in the NF-kappaB binding site within the COX-2 promoter. The mutations did not block the induction of COX-2 promoter activity by hypertonic sodium chloride, and hypertonic sodium chloride failed to activate NF-kappaB binding site-driven reporter gene constructs. In contrast, hypertonic mannitol activated NF-kappaB, indicating that hypertonic mannitol and hypertonic sodium chloride activate COX-2 by different mechanisms. Thus, induction of COX-2 expression in M-1 cells by hypertonic sodium chloride does not involve activation of NF-kappaB. Furthermore, the signal transduction pathways that respond to hypertonic stress vary for different osmolytes in cortical collecting duct cells.     

Effect of dDAVP on basolateral cell surface water permeability in the outer medullary collecting duct

We report a novel approach for assessing the volume of living cells which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The aim of this study was to evaluate the short-term effect of vasopressin on basolateral cell surface water permeability in the outer medullary collecting duct (OMCD). The permeability of the basolateral cell membrane was determined in the tubules where the apical membrane was blocked with oil injected into the lumen. The apparent coefficient of water permeability (P _f) was evaluated by measuring the cell swelling after the step from hypertonic to isotonic medium (600 mosm to 300 mosm). Desmopressin (dDAVP) induced an increase of the basolateral P _f from 113.7±8.5 μm/s in control cells to 186.6±11.4 μm/s in micro-dissected fragments of the OMCD incubated in vitro (10⁻⁷ M dDAVP, 30 min at 37 °C) (P<0.05). Mercury caused pronounced inhibition of basolateral water permeability (26.0±6.9 μm/s; P<0.05). The effect of mercury (1.0 mM HgCl₂) was reversible: after washing the fragments with PBS for 20 min, P _f values were restored to the control levels (125.0±9.5 μm/s). The results of the study indicate the existence of a mechanism controlling the osmotic water permeability of the basolateral cell membrane in the OMCD epithelium.     

Isolation and characterization of epithelial cells from bovine pancreatic duct

Summary Epithelial cells derived from bovine pancreatic duct have been grown continuously in culture for 30 weeks (approximately 90 doublings of the cell population). The cells were grown in Eagle's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, and antibiotics. In confluent cultures, the cells are multilayered and form circular structures. When tested at various passages, the cells neither formed colonies in soft agar nor produced tumors after inoculation into athymic, nude mice. Hydrocortisone (1 and 5 μg per ml) and insulin (1,5 and 10 μg per ml) had no effect on the growth of the cells. β-Retinyl acetate inhibited growth rate and cell yield at a concentration of 5 μg per ml but was not growth-inhibitory at lower concentrations. By electron microscopy the cells have numerous mitochondria, Golgi and microvilli. Mucous droplets were observed in a small proportion of the cells. Desmosome-like structures and occluding junctions were observed more frequently between cells that had been transferred as aggregates than between cells transferred as single cells. Cytochemical studies indicated that some cells produce PAS positive granules that were not removed after treatment of the cultures with diastase. Eleven cell clones were isolated from the mass culture. The growth rates of the clones are different as well as the period of time in which the clones can be propagated in vitro. This work was supported in part by Y01 CP 60204 and N01 CP 43237.     

Differentiation properties of renal collecting duct cells in culture

In the present study, we were particularly interested in distinguishing specific patterns of structural and functional proteins in the collecting duct system of neonatal and adult kidneys and in cultured renal collecting duct epithelia in order to ascertain the degree of differentiation in the cultures. We studied the distribution of specific renal collecting duct cell markers using morphological, immunohistochemical and biochemical procedures. Cultured renal collecting duct epithelium undergoes maturation in vitro. Examples of morphological differentiation include the appearance of cilia and microvilli at the apical cell pole, and a basement membrane at the basal aspect of the epithelium. Tight junctions with five to seven strands separate the wide intercellular spaces from the apical cell surface. Physiological maturation from a 'leaky' to a 'tight' epithelium is evident from the acquisition of the alpha-subunit of Na/K-ATPase and the development of a high transepithelial potential difference and resistance. Biochemical differentiation is revealed by the expression of specific proteins. The simple-epithelium cytokeratins, PKK1 and PKK2, which are typical intracellular-matrix proteins of mature collecting duct epithelium, maintain the same distribution in cell culture as in neonatal and adult kidneys. An indicator of maturation in vitro is the expression of the collecting duct-specific proteins, PCD2 and PCD3. Newly developed monoclonal antibodies against these antigens reacted similarly with cultured cells and cells of the mature collecting duct system, but they did not label the embryonic ampullae in the cortex of neonatal rabbit kidneys. In contrast, a third collecting duct-specific protein, PCD1, is not expressed by the cultured cells, which indicates the retention of an embryonic characteristic in vitro. Embryonic collecting duct ampullae of the neonatal kidney in situ contain laminin during their development. Laminin is, however, absent in cultured collecting duct epithelium. Biochemical stimulation of the adenylate cyclase system by arginine vasopressin resulted in a twofold stimulation of the enzyme activity. This degree of stimulation is similar to that found in maturing kidneys of neonatal rabbits and indicates another embryonic feature of the cultures.     

Differentiation properties of renal collecting duct cells in culture

In the present study, we were particularly interested in distinguishing specific patterns of structural and functional proteins in the collecting duct system of neonatal and adult kidneys and in cultured renal collecting duct epithelia in order to ascertain the degree of differentiation in the cultures. We studied the distribution of specific renal collecting duct cell markers using morphological, immuno-histochemical and biochemical procedures. Cultured renal collecting duct epithelium undergoes maturation in vitro. Examples of morphological differentiation include the appearance of cilia and microvilli at the apical cell pole, and a basement membrane at the basal aspect of the epithelium. Tight junctions with five to seven strands separate the wide intercellular spaces from the apical cell surface. Physiological maturation from a ‘leaky’ to a ‘tight’ epithelium is evident from the acquisition of the α-subunit of Na/K-ATPase and the development of a high transepithelial potential difference and resistance. Biochemical differentiation is revealed by the expression of specific proteins. The simple-epithelium cytokeratins. PKK1 and PKK2, which are typical intracellular-matrix proteins of mature collecting duct epithelium, maintain the same distribution in cell culture as in neonatal and adult kidneys. An indicator of maturation in vitro is the expression of the collecting duct-specific proteins, P^CD2 and P^CD3. Newly developed monoclonal antibodies against these antigens reacted similarly with cultured cells and cells of the mature collecting duct system, but they did not label the embryonic ampullae in the cortex of neonatal rabbit kidneys. In contrast, a third collecting duct-specific protein, PcDl, is not expressed by the cultured cells, which indicates the retention of an embryonic characteristic in vitro. Embryonic collecting duct ampullae of the neonatal kidney in situ contain laminin during their development. Laminin is. however, absent in cultured collecting duct epithelium. Biochemical stimulation of the adenylate cyclase system by arginine vasopressin resulted in a twofold stimulation of the enzyme activity. This degree of stimulation is similar to that found in maturing kidneys of neonatal rabbits and indicates another embryonic feature of the cultures.