TEMPERATURE-SENSITIVE SV40 IMMORTALIZED RAT PROXIMAL TUBULE CELL-LINEHAS FUNCTIONAL RENIN-ANGIOTENSIN SYSTEM

Immortalized rat proximal tubule cell (IRPTC) lines should be useful f or investigation of proximal tubule (PT) regulation and function but p reviously have been unavailable. We now report the establishment and c haracterization of an immortalized transformed, temperature-sensitive IRPTC cell line containing renin-angiotensin system (RAS) components. Primary PT cells prepared from male Wistar rats (4-5 wk old) after col lagenase digestion, sieving, and Percoll gradient were cultured on col lagen-coated T-75 flasks in Dulbecco's modified Eagle's medium contain ing 5% fetal calf serum. Subconfluent PT cells were transfected with t he temperature-sensitive SV40 mutant viruses (tsA SV40) by direct expo sure. After 7-8 wk, several clones were obtained, from which one has b een characterized and designated as line 3-2. This cell line appears s table up to 45 passages. Clonal cells transformed with this virus exhi bit a transformed phenotype at a permissive temperature of 34 degrees C and grow in multiple layers. When the cells are subsequently placed at a nonpermissive temperature of 41 degrees C, they return to morphol ogy similar to that of untransformed cells of the same lineage. At eit her 34 degrees C or 41 degrees C, this cell line expresses a variety o f PT markers including alkaline phosphatase, cytokeratin, carbonic anh ydrase, and glucose transporter isoform 2 (GLUT2), while not expressin g factor VIII. Uniquely, these cells also appear to express PT protein s gp330 and CHIP28, markers which are usually lost in cultured cells. Furthermore, the cell line expresses protein and mRNA components of RA S, including angiotensinogen, angiotensin converting enzyme, and renin . The IRPTC cell line expresses few angiotensin II (ANG II) receptors at 34 degrees C, the permissive temperature. However, at the nonpermis sive temperature, 41 degrees C, IRPTC expresses ANG II receptor (disso ciation constant of 0.7 nM; maximum binding capacity of 265 fmol/mg pr otein). ANG II (10(-8) M) induced a transient rise in cytoplasmic Ca2 concentration, which was nearly abolished with losartan but not PD-12 3319, suggesting this finding is AT(1) receptor mediated. This cell li ne should provide an excellent model of PT and should make it possible to study the cell and molecular biology of the RAS, as well as other regulatory systems of the PT.