Action of aldosterone on citrate synthase in cultured renal collecting duct cells

Cultured renal collecting duct cells from neonatal rabbit kidney were used to examine the influence of aldosterone on enzymatic activity of citrate synthase during increase in Na+ transport. Control epithelia showed citrate synthase activity of 71 +/- 3 mU/mg protein (n = 28), while after aldosterone treatment citrate synthase activity was significantly increased to 79 +/- 6 mU/mg at 1 h (n = 5), to 88 +/- 6 mU/mg at 2 h (n = 6) and to 93 +/- 8 mU/mg protein at 3 h (n = 5). Citrate synthase activity subsequently decreased to basal values. Spironolactone fully blocked the aldosterone-induced increase in citrate synthase activity. The time course of enzyme stimulation after aldosterone administration indicates that the hormone activates citrate synthase during the physiological early response phase.     

Action of aldosterone on protein expression in cultured collecting duct cells from neonatal rabbit kidney

To investigate whether 'aldosterone-induced proteins' could be detected in mammalian species, cultured renal collecting duct epithelia from neonatal rabbit kidneys were labelled under aldosterone administration with radioactive methionine and subsequently fractionated into cytosolic and coarse membrane protein fractions. Newly synthesized proteins were then analyzed by SDS-PAGE, isoelectric focussing and two-dimensional electrophoresis. Quantitative estimates of individual newly synthesized proteins were performed utilizing gel slicing, scintillation counting and autoradiography. The labelling experiments demonstrated that, in comparison to controls, aldosterone (1 X 10(-6) M) generally increased the amount of radioactive protein. No qualitative changes in the pattern of newly synthesized proteins and, therefore, no classical aldosterone-induced proteins were observed. The increase of radioactive protein was already seen after 1, 6, and 18 h of hormone treatment. The effect could be blocked partially by spironolactone (1.5 X 10(-4) M), and totally by amiloride (1 X 10(-6) M), g-strophantin (5 X 10(-4) M), and cycloheximide (1 X 10(-6) M. Thus, the interference of aldosterone action at the receptor level, the Na+ channels and the Na+/K(+)-ATPase pump demonstrate that the expression of proteins in cultured renal collecting duct cells is a sensitive system and seems to be controlled by aldosterone at the receptor level, but also counter-controlled by specific plasma membrane sites.     

Dual influence of aldosterone on AQP2 expression in cultured renal collecting duct principal cells

In the renal collecting duct (CD) the major physiological role of aldosterone is to promote Na+ reabsorption. In addition, aldosterone may also influence CD water permeability elicited by vasopressin (AVP). We have previously shown that endogenous expression of the aquaporin-2 (AQP2) water channel in immortalized mouse cortical CD principal cells (mpkCCDC14) grown on filters is dramatically increased by administration of physiological concentrations of AVP. In the present study, we investigated the influence of aldosterone on AQP2 expression in mpkCCDC14 cells by RNase protection assay and Western blot analysis. Aldosterone reduced AQP2 mRNA and protein expression when administered together with AVP for short periods of time (< or =24 h). For longer periods of time, however, aldosterone increased AQP2 protein expression despite sustained low expression levels of AQP2 mRNA. Both events were dependent on mineralocorticoid receptor occupancy because they were both induced by a low concentration of aldosterone (10-9 m) and were abolished by the mineralocorticoid receptor antagonist canrenoate. Inhibition of lysosomal AQP2 protein degradation increased AQP2 protein expression in AVP-treated cells, an effect that was potentiated by aldosterone. Finally, both aldosterone and actinomycin D delayed AQP2 protein decay following AVP washout, but in a non-cumulative manner. Taken together, our data suggest that aldosterone tightly modulates AQP2 protein expression in cultured mpkCCDC14 cells by increasing AQP2 protein turnover while maintaining low levels of AQP2 mRNA expression.     

The expression of specific proteins in cultured renal collecting duct cells

It is still uncertain whether cell cultures attain the functional maturity of corresponding in vivo cells. The degree of differentiation of cultured collecting-duct (CD) epithelium cells was therefore examined using immunohistochemical procedures. Three monoclonal antibodies (mabs CD1, CD2, and CD3) were raised against proteins (PCD) isolated from the renal papilla. At Western-blot analysis, each of these antibodies reacted with a specific protein that was distinguishable according to its molecular weight [PCD1, 190 kilodaltons (kDa); PCD2, 210 kDa; PCD3, 50 kDa]. Using immunofluorescence, these proteins were found to be localized exclusively in the renal CD system. Other renal structures, such as the proximal or distal tubular portions, the glomeruli and the interstitial network, were not reactive. The mabs, CD2 and CD3, labeled both the cortical and medullary CD in a uniform way, whereas mab CD1 produced heterogeneous immunolabeling along the length of the cortical, medullary, and papillary CD. As revealed by immunohistochemistry, the mabs revealed differences with respect to the expression of the specific renal proteins in cultured CD cells. In polar-differentiated epithelium cultured for 5 days on a specific renal support, mab CD1 was unreactive, whereas mabs CD2 and CD3 were positive. This demonstrated the biochemical immaturity of this cultured epithelium with respect to CD1 reactivity. In morphologically dedifferentiated CD monolayer cells grown on the bottom of a culture dish, only a weak reaction for mab CD3 was observed. The loss of epithelial polarization in CD monolayer cells obviously coincides with the absence of the renal proteins PCD1 and PCD2.(ABSTRACT TRUNCATED AT 250 WORDS)     

The expression of specific proteins in cultured renal collecting duct cells

Summary It is still uncertain whether cell cultures attain the functional maturity of corresponding in vivo cells. The degree of differentiation of cultured collecting-duct (CD) epithelium cells was therefore examined using immunohistochemical procedures. Three monoclonal antibodies (mabs CD 1, CD 2, and CD 3) were raised against proteins (P_CD) isolated from the renal papilla. At Western-blot analysis, each of these antibodies reacted with a specific protein that was distinguishable according to its molecular weight [P_CD1, 190 kilodaltons (kDa); P_CD2, 210 kDa; P_CD3, 50 kDa]. Using immunofluorescence, these proteins were found to be localized exclusively in the renal CD system. Other renal structures, such as the proximal or distal tubular portions, the glomeruli and the interstitial network, were not reactive. The mabs, CD 2 and CD 3, labeled both the cortical and medullary CD in a uniform way, whereas mab CD 1 produced heterogeneous immunolabeling along the length of the cortical, medullary, and papillary CD. As revealed by immunohistochemistry, the mabs revealed differences with respect to the expression of the specific renal proteins in cultured CD cells. In polar-differentiated epithelium cultured for 5 days on a specific renal support, mab CD 1 was unreactive, whereas mabs CD 2 and CD 3 were positive. This demonstrated the biochemical immaturity of this cultured epithelium with respect to CD 1 reactivity. In morphologically dedifferentiated CD monolayer cells grown on the bottom of a culture dish, only a weak reaction for mab CD 3 was observed. The loss of epithelial polarization in CD monolayer cells obviously coincides with the absence of the renal proteins P_CD1 and P_CD2. Thus, our mabs proved to be valuable tools for the investigation of the differentiation and dedifferentiation of renal CD cells in culture.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Methylation of cytosolic proteins may be a possible biochemical pathway of early aldosterone action in cultured renal collecting duct cells

The common model of aldosterone-dependent sodium transport is that the hormone increases sodium transport during the "early" and "late" response phases by inducing specific proteins (AIPs). However, in actual biochemical studies, AIPs were mostly detected 6-24 h after aldosterone application. Regarding the physiological early response phase, this implies temporal dissociation of the physiological and biochemical events. The discrepancy raises the question as to whether other biochemical events, such as protein modifications, may be involved in addition to the novo protein synthesis. Labelling of cultured renal collecting duct epithelia for 1-5 h with a radioactive methylgroup donor, S-adenosyl methionine (SAM), following tissue fractionation, resulted in progressive methylations of specific cytosolic proteins. Aldosterone-dependent methylations increased consistently with time, and accounted for a 60% increase in total cytosolic protein content as compared to controls after 5 h labelling. The different methylated proteins showed a molecular weight of 220, 97 and 75 kd and comprised groups of proteins with an isoelectric point of 5.1-5.7 and 6.0-7.5. Methylation of identical proteins was obtained by incubation of the epithelia with unlabelled SAM instead of aldosterone. SAM-induced as well as aldosterone-induced methylation of proteins with an isoelectric point of 6.0-7.5 could be inhibited by the methylation inhibitor S-adenosylhomocysteine. The results indicate that aldosterone may influence the SAM cycle in cultured collecting-duct epithelia during increase of the Na+-transport.     

Immunocytochemical localization of a renal glycoprotein (gpCDI) synthesized by cultured collecting duct cells

A sulfated, proline-rich glycoprotein (gpCDI, apparent molecular weight 200,000 in column chromatography and 150,000 in SDS-PAGE) was isolated from cultured renal collecting duct epithelium by centrifugation. Triton X100 extraction and DEAE-cellulose ion exchange chromatography. A DEAE-cellulose ion exchange chromatography fraction with the enriched gpCDI was used for immunization of guinea pigs. The antiserum was prepared for antigen localization by indirect immunofluorescence in collecting duct cell cultures and in tissue sections of neonatal and adult rabbit kidneys. In the cultured collecting duct epithelium, antibody staining of the epithelium and structures of the extracellular matrix was age dependent. Cultures of dedifferentiated collecting duct monolayers revealed positive reaction in the cytoplasm. In neonatal and adult rabbit kidneys, the antibody was localized in the entire collecting duct system but not in the collecting duct ampullae of the newborn kidney. Staining of the cytoplasm was found only in medullary collecting ducts of the neonatal kidney; other portions revealed staining mostly at the basal circumference of the tubule and at the luminal cell borders. Apart from collecting ducts, no other tubular segments were reactive. The cortical and the medullary interstitium contained fluorescent fibres which were concentrated around vascular structures. A possible relation between gpCDI and collagenous compounds is discussed. Bowman's capsule reacted positively, whereas staining of the mesangial matrix was weak. The localization of the antigen, as revealed by indirect immunofluorescence, suggests that gpCDI occurs both in intracellular and extracellular (interstitial) location. Two main points are emphasized: Firstly gpCDI is considered an important constituent in different stages of collecting duct development, and secondly, the staining pattern of the antibody varies with the different portions of both young and adult kidney collecting ducts; this staining heterogeneity may correspond with the known regional differences of collecting duct functions.     

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.     

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

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 of 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 GPCDI 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 GPCDI 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.     

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.     

Immunocytochemical localization of a renal glycoprotein (gp CD I) synthesized by cultured collecting duct cells

Summary A sulfated, proline-rich glycoprotein (gp_CDI, apparent molecular weight 200,000 in column chromatography and 150,000 in SDS-PAGE) was isolated from cultured renal collecting duct epithelium by centrifugation, Triton X100 extraction and DEAE-cellulose ion exchange chromatography. A DEAE-cellulose ion exchange chromatography fraction with the enriched gp_CDI was used for immunization of guinea pigs. The antiserum was prepared for antigen localization by indirect immunofluorescence in collecting duct cell cultures and in tissue sections of neonatal and adult rabbit kidneys. In the cultured collecting duct epithelium, antibody staining of the epithelium and structures of the extracellular matrix was age dependent. Cultures of dedifferentiated collecting duct monolayers revealed positive reaction in the cytoplasm. In neonatal and adult rabbit kidneys, the antibody was localized in the entire collecting duct system but not in the collecting duct ampullae of the newborn kidney. Staining of the cytoplasm was found only in medullary collecting ducts of the neonatal kidney; other portions revealed staining mostly at the basal circumference of the tubule and at the luminal cell borders. Apart from collecting ducts, no other tubular segments were reactive. The cortical and the medullary interstitium contained fluorescent fibres which were concentrated around vascular structures. A possible relation between gp_CDI and collagenous compounds is discussed. Bowman's capsule reacted positively, whereas staining of the mesangial matrix was weak. The localization of the antigen, as revealed by indirect immunofluorescence, suggests that gp_CDI occurs both in intracellular and extracellular (interstitial) location. Two main points are emphasized: Firstly, gp_CDI is considered an important constituent in different stages of collecting duct development, and secondly, the staining pattern of the antibody varies with the different portions of both young and adult kidney collecting ducts; this staining heterogeneity may correspond with the known regional differences of collecting duct functions.     

Appearance of specific proteins in the apical plasma membrane of cultured renal collecting duct principal cell epithelium after chronic administration of aldosterone and arginine vasopressin

Principal cell epithelium of renal collecting duct from neonatal rabbit kidney was cultured in the presence of aldosterone (1 x 10(-6) M) and arginine vasopressin (AVP; 1 x 10(-6) M) for 10 days to investigate, by immunohistochemical methods using specific monoclonal antibodies, whether the hormones influence the expression and insertion of plasma membrane proteins. The experiments demonstrated that aldosterone alone or aldosterone plus AVP significantly increased the number of epithelial cells reacting at the luminal and lateral plasma membrane with the antibodies CD 2 and 3, specific for renal collecting duct, as we have shown in the kidney. In cultures treated with aldosterone and aldosterone plus AVP, nearly all epithelial cells were labelled by the antibodies, while controls or AVP treatment showed 41% and 24% unreactive cells, respectively. These findings were complemented with electrophysiological experiments, in which epithelia pretreated by aldosterone or aldosterone plus AVP showed significantly hyperpolarized transepithelial voltage (Vte) and higher resistance (Rte) than controls or AVP-treated specimens. The experiments demonstrated that chronic administration of aldosterone or of aldosterone plus AVP to increase Na+-transport was paralleled by the appearance of collecting-duct-specific proteins in the epithelium. Consequently, this result indicates that aldosterone influences the functional maturity of the cultured epithelium.     

Effects of glycoprotein and basement membrane synthesis inhibitors on the growth of cultured renal collecting duct epithelium

Summary The biochemical and morphological extent of glycoprotein synthesis inhibition of cellular and extracellular proteins was studied on cultured renal collecting duct (CD) epithelium. We found that tunicamycin (4 g/ml) inhibits the glycosylation of a 150,000 d glycoprotein (gp_CDI). A 85,000 d glycoprotein (gp_CDII) was not affected. The inhibition by tunicamycin demonstrates that gp_CDI has characteristics of a N-glycan, whereas gp_CDII seems to be an O-glycan. 6-diazo-5-oxo-norleucine (4×10^–5M) which was used as glutamine analogue, did not show a comparable inhibitory effect as seen with tunicamycin. The lack of effect of norleucine demonstrates that glutamine is not the locus of glycosylation in both proteins. However, because of the tunicamycin inhibition it points to asparagine as the site of glycosylation in the gp_CDI. Long term cultures of the tissue up to 15 days in the presence of tunicamycin and norleucine and of substances usually used as basement membrane inhibitors, such as hydroxy-d-proline (1 mM), l-azetidine-2-carboxylic acid (1 mM) and o- and p-nitrophenyl-xylopyranoside (1 mM), revealed that it is possible to eliminate completely the fibroblasts beneath the cultured epithelium and within the degenerating corematerial. Experiments with hydroxy-d-proline showed the most striking effect. Experiments with l-azetidine-2-carboxylic acid and nitrophenyl-xylopyranoside resulted in the elimination of fibroblasts and dedifferentiation of the collecting duct epithelium.     

Effects of glycoprotein and basement membrane synthesis inhibitors on the growth of cultured renal collecting duct epithelium

The biochemical and morphological extent of glycoprotein synthesis inhibition of cellular and extracellular proteins was studied on cultured renal collecting duct (CD) epithelium. We found that tunicamycin (4 micrograms/ml) inhibits the glycosylation of a 150,000 d glycoprotein (gpCDI). A 85,000 d glycoprotein (gpCDII) was not affected. The inhibition by tunicamycin demonstrates that gpCDI has characteristics of a N-glycan, whereas gpCDII seems to be an O-glycan. 6-diazo-5-oxo-norleucine (4 X 10(-5) M) which was used as glutamine analogue, did not show a comparable inhibitory effect as seen with tunicamycin. The lack of effect of norleucine demonstrates that glutamine is not the locus of glycosylation in both proteins. However, because of the tunicamycin inhibition it points to asparagine as the site of glycosylation in the gpCDI. Long term cultures of the tissue up to 15 days in the presence of tunicamycin and norleucine and of substances usually used as basement membrane inhibitors, such as hydroxy-D-proline (1 mM), L-azetidine-2-carboxylic acid (1 mM) and o- and p-nitrophenyl-xylopyranoside 1 mM), revealed that it is possible to eliminate completely the fibroblasts beneath the cultured epithelium and within the degenerating corematerial. Experiments with hydroxy-D-proline showed the most striking effect. Experiments with L-azetidine-2-carboxylic acid and nitrophenyl-xylopyranoside resulted in the elimination of fibroblasts and dedifferentiation of the collecting duct epithelium.     

Dexamethasone stimulates endothelin-1 gene expression in renal collecting duct cells

Aldosterone stimulates the endothelin-1 gene (Edn1) in renal collecting duct (CD) cells by a mechanism involving the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). The goal of the present study was to determine if the synthetic glucocorticoid dexamethasone affected Edn1 gene expression and to characterize GR binding patterns to an element in the Edn1 promoter. Dexamethasone (1μM) induced a 4-fold increase in Edn1 mRNA in mIMCD-3 inner medullary CD cells. Similar results were obtained from cortical collecting duct-derived mpkCCD(c14) cells. RU486 inhibition of GR completely blocked dexamethasone action on Edn1. Similarly, 24h transfection of siRNA against GR reduced Edn1 expression by approximately 50%. However, blockade of MR with either spironolactone or siRNA had little effect on dexamethasone induction of Edn1. Cotransfection of MR and GR siRNAs together had no additive effect compared to GR-siRNA alone. The results indicate that dexamethasone acts on Edn1 exclusively through GR and not MR. DNA affinity purification studies revealed that either dexamethasone or aldosterone resulted in GR binding to the same hormone response element in the Edn1Edn1 promoter. The Edn1 hormone response element contains three important sequence segments. Mutational analysis revealed that one of these segments is particularly important for modulating MR and GR binding to the Edn1 hormone response element.     

A role for collecting duct epithelial cells in renal antibacterial defences

Urinary tract infections (UTIs), which are mainly due to uropathogenic Escherichia coli (UPEC), occur via the retrograde ascent of the bacteria along the urinary tract system. The adhesion and invasion mechanisms of UPEC have been extensively studied in bladder epithelial cells, but less is known about the role of renal tubule epithelial cells (RTEC) in renal antibacterial defences. This review considers recent advances in the understanding of the role of RTECs in inducing an innate immune response mediated by Toll-like receptors (TLRs) in experimental UTI. Collecting duct cells are a preferential site of adhesion of UPEC colonizing the kidneys. Epithelial TLR4 activation induces an inflammatory response and the recruitment of lipid rafts to the plasma membrane, both of which facilitate the transcytosis of non-cytolytic UPEC strains across intact collecting duct cell layers to invade the renal interstitium. Arginine vasopressin, which regulates water absorption in the collecting duct, also acts as a potent modulator of the TLR4-mediated intrarenal innate response caused by UPEC. The role of epithelial TLR5 in renal host defences is also discussed. These findings highlight the role of RTECs in triggering the innate immune response in the context of ascending UTIs.     

Vasopressin modifies osmium impregnation of the endoplasmic reticulum in cultured kidney collecting duct cells

The ultrastructure of collecting duct epithelia was studied with the osmium impregnation technique in renal cortical explants grown in culture in the form of globular bodies. When this technique was applied to 7-day-old globular bodies, the endoplasmic reticulum (ER) of the superficial layer cells was faintly impregnated in the presence or absence of arginine-vasopressin (AVP) in the incubation medium; the ER of the cells located in the core of the globular bodies was densely impregnated with osmium. When these globular bodies were sectioned in 2 fragments and one was incubated in AVP for 30 min while the other was used as a control, a marked increase in osmium impregnation occurred: osmium black deposits were then noted in the lumen of the endoplasmic reticulum of two-thirds of the cells in the superficial layer. Various patterns of impregnation were observed. Cryptlike formations gave rise to mature epithelial cells showing the same pattern of osmium impregnation. When cyclic adenosine monophosphate (cAMP) was substituted for AVP in the incubation medium, the treated globular bodies revealed the same ultrastructural characteristics. Our data suggest that this primary culture of collecting duct epithelia is made up of heterogeneous cells with characteristics of principal and intercalated cells and that the AVP has a stimulatory effect on ER maturation, which is mediated by the adenylate cyclase system.