24-25-dihydroxyvitamin-d-3 and methylglucoside

We have described and compared culture systems for proximal tubule cell (PTC) preparations from dog and rat kidney. Cells were prepared from kidney cortex by enzyme digestion and purified on Percoll density gradient. The dog PTC and rat PTC differed in their growth characteristics in culture. Although the dog PTC tended to overgrow the contaminating fibroblastic cells, the rat PTC tended to be overgrown by the latter cells when cultured in medium containing 15% fetal calf serum (FCS). Cultures of rat PTC in serum-free medium or medium containing only 2% FCS yielded only epithelium-like cells exhibiting characteristics of cells that are proximal tubular in origin. These properties include protrusion of microvilli, high alkaline phosphatase activity, ability to transport sugar, and responsiveness to parathyroid hormone (PTH) in terms of cAMP production. The time course and dose-response curves of PTH-stimulated cAMP accumulation were studied in dog and rat PTC. The estimated half-maximal concentrations (Kact) for PTH in dog and rat PTC were 1.2 and 100 nM, respectively. Both values are within the range reported in the literature for the respective renal membrane preparations. In addition to our previously reported data on dog PTC, this study revealed the presence of PTH-inhibitable 25-hydroxyvitamin D3-24-hydroxylase in dog PTC. These two model cell culture systems should prove useful in studying PTH action in renal cells.

Topics: 24,25-Dihydroxyvitamin D 3; Alkaline Phosphatase; Animals; Calcifediol; Cells, Cultured; Cyclic AMP; Dihydroxycholecalciferols; Dogs; Glucose; Kidney Tubules, Proximal; Methylglucosides; Microscopy, Electron; Microscopy, Phase-Contrast; Parathyroid Hormone; Rats

Primary cultures of murine renal epithelial cells were established from a preparation of proximal tubule fragments. Confluent cultures exhibited multiple dome formation, indicating the presence of tight junctions and an intact transcellular transport process. Ultrastructural analysis revealed a monolayer of polarized cells, with a sparse but clearly defined microvillar surface facing the growth medium and a basolateral surface attached to the substratum. Cultures grown on collagen gels did not show domes. The epithelial monolayer exhibited several differentiated functions of the proximal tubule: a) parathyroid hormone (PTH)-stimulated cAMP synthesis; b) production of 24,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3; c) high alkaline phosphatase activity; and d) Na+-dependent transport of phosphate (Pi) and alpha-methylglucoside (alpha-MG). The sugar uptake was selectively inhibited by phlorizin, a competitive inhibitor of glucose uptake at the luminal membrane. Kinetic analysis revealed independent transport systems for Pi and alpha-MG, with Km values corresponding to the high affinity systems identified in brush border membrane vesicles derived from the proximal tubule. Pi uptake by the epithelial monolayers was regulated by the concentration of Pi in the growth medium. Phorbol esters and PTH did not exert an effect on Pi and alpha-MG transport in mouse primary cultures. The present study demonstrates that primary cultures provide a useful in vitro preparation to investigate renal proximal tubular function.

Topics: 24,25-Dihydroxyvitamin D 3; Alkaline Phosphatase; Animals; Biological Transport; Calcifediol; Cells, Cultured; Cyclic AMP; Dihydroxycholecalciferols; Epithelial Cells; Epithelium; Histocytochemistry; Kidney; Kidney Tubules, Proximal; Kinetics; Methylglucosides; Mice; Microvilli; Parathyroid Hormone; Phlorhizin; Phosphates; Sodium