MORPHOLOGICAL AND BIOCHEMICAL-CHARACTERIZATION OF PRIMARY CULTURE OF RABBIT PROXIMAL KIDNEY TUBULE CELLS GROWN ON COLLAGEN-IV COATED MILLICELL-CM

The aim of this study was to better characterize rabbit proximal kidne y tubule cells cultured on collagen IV-coated porous inserts, as compa red to the same cells seeded in standard plastic wells. Total protein contents in confluent monolayers on permeable membranes were about two fold higher than those measured in confluent cultures in plastic wells . Microscopy examinations suggested that such a difference was probabl y due to a higher cell density and to an impressive development of the apical brush-border membrane. Moreover, measurement of unidirectional transport of p-aminohippuric acid and tetraethylammonium bromide conf irmed the high polarization level of cultures on porous inserts. Resul ts of methyl(alpha-D[U-C-14]glyco)pyranoside uptake suggested that cel l phenotype was probably influenced by culture conditions. Analysis of different markers as a function of time in culture showed decreases o f alkaline phosphatase (AP), gamma-glutamyltranspeptidase (GGT), and N a+-K+-ATPase activities as well as increases in LDH ATP, and glutathio ne levels, similar to those formerly reported for cells cultured in st andard plastic plates. However, comparative data from 6-d-old monolaye rs have shown that AP, GGT, Na+-K+-ATPase, glutathione reductase (GRED ), and selenium-dependent glutathione peroxidase (Se-GPX) activities w ere 2.8-, 2.6-, 1.6-, 1.2-, and 2.1-fold, respectively, better preserv ed on precoated permeable membranes. On the other hand, this paper rep orts for the first time in the literature that GRED and Se-GPX, two ph ase II detoxification enzymes, were well maintained in cultures of rab bit proximal kidney tubule cells. Our results show that culturing rabb it proximal kidney tubule cells on collagen IV-coated porous membranes was accompanied by an improvement of both morphological and biochemic al properties of the cells.