The phenotype of cystic fibrosis (CF) includes abnormalities in transepithelial transport of Cl- (refs 1-5), decreased sialylation and increased sulphation and fucosylation of glycoproteins, and lung colonization with Pseudomonas. It is not apparent how these abnormalities are interrelated, nor how they result from loss of function of the CF gene-encoded transmembrane regulator (CFTR). We have previously shown that that the pH of a secretory granule is regulated by the vesicular conductance for Cl- (ref. 11). Here we find defective acidification in CF cells of the trans-Golgi/trans-Golgi network, of prelysosomes and of endosomes as a result of diminished Cl- conductance. Sialytation of proteins and lipids is reduced and ligand traffic altered. These abnormalities can result from defective acidification because vacuolar pH regulates glycoprotein processing and ligand transport. The CF phenotype is similar to that of alkalinized cells and acidification-defective mutatants.

Many adult organs contain stem cells, which are pluripotent and are involved in organ maintenance and repair after injury. In situ, these cells often have a low cycling rate and locate in specialized regions (niches). To detect such cells in the kidney, we administered a pulse of the nucleotide bromodeoxyuridine (BrdU) to rat and mouse pups and, after a long (more than 2-month) chase, examined whether the kidney contained a population of low-cycling cells. We found that in the adult kidney, BrdU-retaining cells were very sparse except in the renal papilla, where they were numerous. During the repair phase of transient renal ischemia, these cells entered the cell cycle and the BrdU signal quickly disappeared from the papilla, despite the absence of apoptosis in this part of the kidney. In vitro isolation of renal papillary cells showed them to have a plastic phenotype that could be modulated by oxygen tension and that when injected into the renal cortex, they incorporated into the renal parenchyma. In addition, like other stem cells, papillary cells spontaneously formed spheres. Single-cell clones of these cells coexpressed mesenchymal and epithelial proteins and gave rise to myofibroblasts, cells expressing neuronal markers, and cells of uncharacterized phenotype. These data indicate that the renal papilla is a niche for adult kidney stem cells

The fundamental characteristics that allow vectorial transport across an epithelial cell are the differential sorting and insertion of transport proteins either in the apical or the basolateral plasma membrane, and the preferential association of endocytosis and exocytosis with one or the other pole of the cell. Asymmetrical cellular structure and function, being manifestations of terminal differentiation, might be expected to be predetermined and invariant. Here we show that the polarity of transepithelial H+ transport, endocytosis and exocytosis in kidney can be reversed by environmental stimuli. The HCO3- secreting cell in the cortical collecting tubule is found to be an intercalated cell possessing a Cl-/HCO3- exchanger in the apical membrane and proton pumps in endocytic vesicles that fuse with the basolateral membrane; the H+-secreting cell in the medullary collecting tubule has these transport functions on the opposite membranes. Further, the HCO3- -secreting cell can be induced to change its functional polarity to that of the H+-secreting cell by acid-loading the animal.

Many adult organs contain stem cells, which are pluripotent and are involved in organ maintenance and repair after injury. In situ, these cells often have a low cycling rate and locate in specialized regions (niches). To detect such cells in the kidney, we administered a pulse of the nucleotide bromodeoxyuridine (BrdU) to rat and mouse pups and, after a long (more than 2-month) chase, examined whether the kidney contained a population of low-cycling cells. We found that in the adult kidney, BrdU-retaining cells were very sparse except in the renal papilla, where they were numerous. During the repair phase of transient renal ischemia, these cells entered the cell cycle and the BrdU signal quickly disappeared from the papilla, despite the absence of apoptosis in this part of the kidney. In vitro isolation of renal papillary cells showed them to have a plastic phenotype that could be modulated by oxygen tension and that when injected into the renal cortex, they incorporated into the renal parenchyma. In addition, like other stem cells, papillary cells spontaneously formed spheres. Single-cell clones of these cells coexpressed mesenchymal and epithelial proteins and gave rise to myofibroblasts, cells expressing neuronal markers, and cells of uncharacterized phenotype. These data indicate that the renal papilla is a niche for adult kidney stem cells.

Chronic colonization and infection of the lung with Pseudomonas aeruginosa is the major cause of morbidity and mortality in cystic fibrosis (CF) patients. We found that polarized CF bronchial and pancreatic epithelia bound P. aeruginosa in a reversible and dose-dependent manner. There was significantly greater binding to CF bronchial and pancreatic cells than to their matched pairs rescued with the wild-type CF transmembrane conductance regulator. Bound P. aeruginosa were easily displaced by unlabeled P. aeruginosa but not by Escherichia coli, an organism that does not cause significant pulmonary disease in CF. In contrast, Staphylococcus aureus, a frequent pathogen in CF, could effectively displace bound P. aeruginosa from its receptor. We found undersialylation of apical proteins and a higher concentration of asialoganglioside 1 (aGM1) in apical membranes of CF compared with rescued epithelia. Colonization of the respiratory tract by Pseudomonas is present in most patients with cystic fibrosis (CF) (1). This infection occurs in infancy before significant inflammatory damage to the lung generates bronchiectasis (2). Younger patients often have antecedent pulmonary infections with Staphylococcus aureus, and they are frequently colonized by both S. aureus and Pseudomonas aeruginosa. Loss of function of the CF gene product [CF transmembrane conductance regulator (CFTR)] is responsible for chronic infection with Pseudomonas (3, 4) because the severity of the defect in CFTR function correlates both with the age at colonization (5) and with the extent of binding of Pseudomonas to respiratory cells taken from CF patients (6). However, the identification of CFTR as an apical membrane Cl-channel has not suggested why CF children are so readily infected with Pseudomonas.Adherence of bacteria to specific cellular receptors is the initial event in many infectious diseases (7-9). In an in vitro assay, P. aeruginosa bound to asialoganglioside 1 (aGMi) and aGM2 but not to the sialylated homologues GM1 and GM2 (10). Respiratory cells from CF patients had more aGM1 than normal cells (11). In fact, undersialylation is also found in mucus glycoproteins of clinical specimens and in secretions from immortalized CF cell lines (12). We recently proposed that loss of the CFTR Cl-channel from intracellular glycosylation compartments impairs sialyltransferase activity (13,14). Thus, we hypothesized that defective sialylation of gangliosides generates Pseudomonas receptors (13, 14), and we show here that there are a larger number of these receptors on the apical surfaces of CF epithelia.MATERIALS AND METHODS Cells and Bacteria. CF bronchial epithelial cells, IB3 (genotype AF508/W1282X), and a matched rescued control cell line, C38, were from P. Zeitlin, Johns Hopkins University (15). These cells were maintained in LCH-8 medium (Biofluids, Rockville, MD) at 37°C in 5% C02/95% air. The second CF epithelial cell line, CFPAC (genotype AF508/AF508), and its matched rescued control cell line, CFPAC/CFTR, were from M. Drumm, University ...