Kidney cortex cells derived from SV40 transgenic mice retain intrinsic properties of polarized proximal tubule cells.
BACKGROUND
We have developed a nontransformed immortalized mice kidney cortex epithelial cell (MKCC) culture from a mouse transgenic for a recombinant plasmid adeno-SV40 (PK4). Methods and Results. After 12 months in culture, the immortalized cells had a stable homogeneous epithelial-like phenotype, expressed simian virus 40 (SV40) T-antigen, but failed to induce tumors after injection in nude mice. Epithelium exhibited polarity with an apical domain bearing many microvilli separated from lateral domains by junctional complexes with ZO1 protein. The transepithelial resistance was low. A Na-dependent glucose uptake sensitive to phlorizin and a Na-dependent phosphate uptake sensitive to arsenate were present. Western blot analysis of membrane fractions showed that anti-Na-Pi antiserum reacted with a 87 kD protein. The Na/H antiporters NHE-1, NHE-2, and NHE-3 mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). The corresponding proteins with molecular weights of 111, 81, and 75 kD, respectively, could be detected by Western blot and were shown to be functional. Parathyroid hormone (PTH) induced a tenfold increase in cAMP and reduced the Na-dependent phosphate uptake and NHE-3 activity, as observed in proximal tubule cells. Isoforms alpha, delta, epsilon, and zeta of protein kinase C (PKC) were present in the cells. Angiotensin II (Ang II) elicited a translocation of the PKC-alpha toward the basolateral and apical domains.


CONCLUSIONS
Thus, the MKCC culture retains the structural and functional properties of proximal tubular cells. To our knowledge, it is the first cell culture obtained from transgenic mice that exhibits the NHE-3 antiporter and type II Na-Pi cotransporter. MKCCs also display functional receptors for PTH and Ang II. Thus, MKCCs offer a powerful in vitro system to study the cellular mechanisms of ion transport regulation in proximal epithelium.
Chalumeau
C
C
INSERM Unité 356, Université Paris VI, Hôpital Broussais, Assistance Publique, Paris, France.
Lamblin
D
D
Bourgeois
S
S
Borensztein
P
P
Chambrey
R
R
Bruneval
P
P
Huyen
J P
JP
Froissart
M
M
Biber
J
J
Paillard
M
M
Kellermann
O
O
Poggioli
J
J
D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D016764:Cell Polarity / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology* / Q000648:ultrastructure; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D007672:Kidney Cortex / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D007687:Kidney Tubules, Proximal / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008854:Microscopy, Electron; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology; D010710:Phosphates / Q000493:pharmacokinetics; D010957:Plasmids; D017680:Potassium Compounds / Q000493:pharmacokinetics; D011493:Protein Kinase C / Q000032:analysis; D013539:Simian virus 40; D051269:Sodium-Glucose Transporter 1; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D010281:Parathyroid Hormone; D010710:Phosphates; D017680:Potassium Compounds; D051269:Sodium-Glucose Transporter 1; D017923:Sodium-Hydrogen Exchangers; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters; D000804:Angiotensin II; D001127:Arginine Vasopressin; C013216:potassium phosphate; D000242:Cyclic AMP; D011493:Protein Kinase C