Growth, immortalization, and differentiation potential of normal adult human proximal tubule cells.
Human proximal tubule epithelial cell lines are potentially useful models to elucidate the complex cellular and molecular details of water and electrolyte homeostasis in the kidney. Samples of normal adult human kidney tissue were obtained from surgical specimens, and S1 segments of proximal convoluted tubules were microdissected, placed on collagen-coated culture plate inserts, and cocultured with lethally irradiated 3T3 fibroblasts. Primary cultures of proximal tubule epithelial cells were infected with a replication-defective retroviral construct encoding either wild-type or temperature-sensitive simian virus 40 large T-antigen. Cells forming electrically resistive monolayers were selected and expanded in culture. Three cell lines (HPCT-03-ts, HPCT-05-wt, and HPCT-06-wt) were characterized for proximal tubule phenotype by electron microscopy, electrophysiology, immunofluorescence, Southern hybridization, and reverse transcriptase-polymerase chain reaction. Each of the three formed polarized, resistive epithelial monolayers with apical microvilli, tight junctional complexes, numerous mitochondria, well-developed Golgi complexes, extensive endoplasmic reticulum, convolutions of the basolateral plasma membrane, and a primary cilium. Each exhibited succinate, phosphate, and Na,K- adenosine triphosphatase (ATPase) transport activity, as well as acidic dipeptide- and adenosine triphosphate-regulated mechanisms of ion transport. Transcripts for Na(+)-bicarbonate cotransporter, Na(+)-H(+) exchanger isoform 3, Na,K-ATPase, parathyroid hormone receptor, epidermal growth factor receptor, and vasopressin V2 receptor were identified. Furthermore, immunoreactive sodium phosphate cotransporter type II, vasopressin receptor V1a, and CLIC-1 (NCC27) were also identified. These well-differentiated, transport-competent cell lines demonstrated the growth, immortalization, and differentiation potential of normal, adult, human proximal tubule cells and consequently have wide applicability in cell biology and renal physiology.
Orosz
David E
DE
Department of Physiology, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Woost
Philip G
PG
Kolb
Robert J
RJ
Finesilver
Margaret B
MB
Jin
Wenwu
W
Frisa
Phyllis S
PS
Choo
Chee-Keong
CK
Yau
Chung-Fai
CF
Chan
Kwok-Wah
KW
Resnick
Martin I
MI
Douglas
Janice G
JG
Edwards
John C
JC
Jacobberger
James W
JW
Hopfer
Ulrich
U
D000818:Animals; D001692:Biological Transport; D015415:Biomarkers; D002454:Cell Differentiation; D002460:Cell Line; D016764:Cell Polarity; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007477:Ions / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D010641:Phenotype; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; D007477:Ions; D008565:Membrane Proteins