Cilia are organelles that play diverse roles, from fluid movement to sensory reception. Polaris, a protein associated with cystic kidney disease in Tg737°rpk mice, functions in a ciliogenic pathway. Here, we explore the role of polaris in primary cilia on Madin-Darby canine kidney cells. The results indicate that polaris localization and solubility change dramatically during cilia formation. These changes correlate with the formation of basal bodies and large protein rafts at the apical surface of the epithelia. A cortical collecting duct cell line has been derived from mice with a mutation in the Tg737 gene. These cells do not develop normal cilia, which can be corrected by reexpression of the wild-type Tg737 gene. These data suggest that the primary cilia are important for normal renal function and/or development and that the ciliary defect may be a contributing factor to the cystic disease in Tg737°rpk mice. Further characterization of these cells will be important in elucidating the physiological role of renal cilia and in determining their relationship to cystic disease.

The results indicate that polyductin is part of the group of polycystic kidney disease (PKD)-related proteins expressed in primary apical cilia. Our data also suggest that, in addition to its likely involvement in cilia function, polyductin probably serves in other subcellular functional roles. The detection of three different products using two antisera, with evidence for distinct subcellular localizations, suggests that PKHD1 encodes membrane-bound and soluble isoforms.

We describe a simplified method for the isolation of large numbers of nephron segments from rat and rabbit kidneys. In contrast to most previous protocols, the kidneys are not perfused. After removal from the animal, the kidney is sliced and torn in pieces that are subsequently digested in culture medium containing 0.5 mg/ml of collagenase at 37°C. If the preparation is agitated only very gently and infrequently, then the tissue gradually falls apart into a suspension containing long nephron fragments, often consisting of multiple connected segments. These are easily sorted into homogeneous segment populations that can be used for enzyme assays, protein extraction for immunoblotting, and RNA extraction for reverse transcription-polymerase chain reaction, all of which have been done successfully in our laboratory. For comparison, we have also examined cortical collecting tubule segments and cells prepared by the more rigorous protocol described previously (E. Schlatter, U. Fröbe, and R. Greger. Pflügers Arch. 421: 381–387, 1992). Even after the isolation of single cells in a Ca2+-free medium, the cells maintain their normal architecture and a distinct separation of apical and basolateral membranes.

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Unidirectional fluxes of L-35S-cystine and intracellular 35S activity were measured in isolated perfused segments of rabbit proximal straight tubule. The absorptive (lumen-to-both) flux of L-35S-cysteine showed a tendency toward saturation within the concentration limits imposed by the low solubility of cystine (0.3 mmol . l-1). In contrast, for the bath-to-lumen fluxes, there was a linear relation between the bathing solution concentration of L-35S-cystine and the rate of 35S appearance in the lumen. Nonlinear fitting of both sets of unidirectional flux data gave a maximal cystine transport rate (Jmax) of 1.45 +/- 0.27 (SEM) pmol min-1 mm-1, a Michaelis constant (Km) of 0.20 +/- 0.07 mmol . l-1, and an apparent permeability coefficient of 0.27 +/- 0.11 pmol min-1 mm-1 (mmol . l-1)-1 (approximately 0.06 micrometer/s). The 35S concentration in the cell exceeded that in the lumen by almost 60-fold during the lumen-to-bath flux, and exceeded the bathing solution concentration by 4.7-fold during the bath-to-lumen flux. Thus cystine was accumulated by the cells across either membrane, but over 77% of the intracellular activity was in the form of cysteine. Although the presence of luminal L-lysine or cycloleucine inhibited the absorptive flux of cystine, neither amino acid affected the bath-to-lumen flux.