Insulin and other regulatory factors modulate the growth and the phosphoenolpyruvate carboxykinase (PEPCK) activity of primary rabbit kidney proximal tubule cells in serum free medium

A high degree of functional polarity has been obtained in primary cultures of rabbit kidney proximal tubule cells grown on collagen IV-coated porous membranes. Tight confluency was attained 6 days after seeding and maintained for at least 6 more days, as shown by analysis of paracellular inulin diffusion. From day 6 onward, L-lactate, ammonia, and D-glucose concentration gradient and a pH difference of approximately 1 unit developed between the two nutrient medium compartments. Confluent monolayers expressed organic ion transport properties higher than those formerly reported for other cell models. Transcellular transport of 20 microM tetraethylammonium was directed from basal to apical compartment and was specifically inhibited by mepiperphenidol (1 mM). Unidirectional transport of 2.4 microM p-aminohippurate also occurred from basal to apical compartment, was saturable, and specifically inhibited by probenecid (1 mM). These results suggest that rabbit kidney proximal tubule cells, cultured under the experimental conditions described here, may be a useful model for the in vitro study of highly polarized renal transport processes.

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Regulation of Na(+)-K(+)-2Cl- cotransport activity in rabbit proximal tubule primary culture

AJP Renal Physiology ◽

10.1152/ajprenal.1994.267.1.f63 ◽

1994 ◽

Vol 267 (1) ◽

pp. F63-F69

Author(s):

N. J. Raat ◽

A. Hartog ◽

C. H. van Os ◽

R. J. Bindels

Keyword(s):

Proximal Tubule ◽

Primary Culture ◽

Pulse Method ◽

Rabbit Kidney ◽

Free Medium ◽

Hypertonic Medium ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Uptake Studies

The presence of Na(+)-K(+)-2Cl- cotransport in rabbit kidney proximal tubule cells in primary culture was demonstrated by bumetanide-sensitive, ouabain-insensitive 86Rb+ uptake studies. After addition of 10 microM bumetanide, 86RB+ uptake was inhibited from 11.1 +/- 0.8 to 1.1 +/- 0.1 nmol.mg protein-1.min-1 under isotonic (300 mosM) conditions. Na(+)-K(+)-2Cl- cotransport activities ranged from 2.2 +/- 0.3 to 13.2 +/- 1.0 nmol.mg protein-1.min-1 depending on the osmolarity of the medium (150-500 mosM). Decreasing extracellular pH to 6.5 inhibited, whereas increasing to 8.5 stimulated, transport at all osmolarities. Decreasing intracellular pH (pHi) by the NH4Cl pulse method showed similar results, suggesting a possible regulatory role of pHi on cotransport activity. Ca(2+)-free medium increased cotransport activity 35 and 20% at iso- and hypertonicity, respectively. At 300 mosM, ionomycin (5 microM) inhibited cotransport by 25%. The combination of forskolin (10 microM) and 3-isobutyl-1-methylxanthine (1 mM) resulted in inhibition of cotransport activity by 38% at hypertonic conditions. Calyculin (1 microM) increased cotransport activity 134 and 128% at 150 and 300 mosM, respectively. In hypertonic medium calyculin did not influence cotransport activity. Okadaic acid (1 microM) had no effect on cotransport activity at all three osmolarities. NaF (10 mM) increased cotransport at all osmolarities tested.

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