Transport and metabolism of glucose by renal proximal tubular cells in primary culture

Chronic acidosis in vivo leads to an increase in proximal tubule Na/H antiporter activity that persists when the transporter is studied out of the acidotic environment. It is presently not clear whether a decrease in extracellular fluid pH alone is sufficient to elicit this adaptation. The present studies examined the effect of acid preincubation on Na/H antiporter activity in cultured proximal tubule cells. Antiporter activity was examined after a 2-day preincubation in control or acid medium, 1 hr after removal from the preincubation fluid.Na/H antiporter activity was assayed as the initial rate of (vol/vol) to the culture medium for the first 3 days to allow better cell attachment. After achieving confluence (8-12 days), cells were rendered quiescent by removal of insulin and hydrocortisone for 48 hr prior to and during preincubation (10).Cultured fibroblasts were derived from human foreskin and used in passages 5-7 (11). Fibroblasts were grown on glass coverslips in DMEM supplemented with penicillin (100 units/ ml), streptomycin (100 ,ug/ml), and 10% fetal bovine serum. Fibroblasts were incubated with a reduced concentration of fetal bovine serum (1%) for 24 hr prior to and during preincubation.Measurement of Intracellular pH and Na/H Antiporter Activity. Continuous measurement of cytoplasmic pH (pHi) was accomplished using the intracellularly trapped pHsensitive dye 2',7'-bis(2-carboxyethyl)-5-(and -6)carboxyfluorescein (BCECF acid). Cells were loaded with the acetoxymethyl ester of BCECF (10 ,uM)

mentioning

confidence: 99%

Preincubation in acid medium increases Na/H antiporter activity in cultured renal proximal tubule cells.

Horie

Moe

Tejedor

et al. 1990

“…Although an increase in the activity of the Na+/H+ antiporter has been associated with mitogenesis (7,(17)(18)(19)(20)(21)(22)(23), this is not invariable (8,25), and to our knowledge it has not been specifically associated with hypertrophy in the absence of cell proliferation. MATERIALS Primary cultures of rabbit proximal tubular cells were prepared as described previously by this laboratory (9), with a serum-free medium [50:50 (vol/vol) mixture of Dulbecco's modified Eagle's medium (DME medium) and Ham's F- 12 medium] supplemented with bovine insulin (5 pug/ml), human transferrin (1 ,ug/ml), and hydrocortisone (50 nM). Confluent monolayers were exposed to fresh basic medium (i.e., 50:50 DME/F-12 media containing transferrin) for 48 hr, which resulted in arrest in Go/G1.…”

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confidence: 99%

Stimulation of Na+/H+ antiport is an early event in hypertrophy of renal proximal tubular cells.

Fine¹,

Badie-Dezfooly²,

Lowe³

et al. 1985

101

Renal hypertrophy in vivo is achieved by an increase in protein content per cell and an increase in cell size with minimal hyperplasia. Hypertrophied renal tubular cells remain quiescent and demonstrate an increase in transcellular transport rates. This situation was simulated in vitro by exposing a confluent, quiescent primary culture of rabbit renal proximal tubular cells to either insulin, prostaglandin E1, or hypertonic NaCl for 24 or 48 hr. Protein per cell increased by 20-30% with little or no increase in [3H~thymidine incorporation into DNA. Mean cell volume was also increased in insulin-and hypertonic NaCl-treated but not in prostaglandin Eltreated cells. The lag period required to initiate DNA synthesis by a combination of insulin and hydrocortisone was the same in control and hypertrophied cells, indicating a quiescent state of the latter. Two hours of exposure to the growth stimuli increased amiloride-sensitive Na+ uptake, Na-dependent H+ efflux, and ouabain-sensitive Rb+ uptake, indicating that stimulation of Na+/H' antiport (exchange) occurs as an early event in their action. Hypertrophied cells continued to demonstrate enhanced Na+/H+ antiport after the growth stimuli were removed for 3 hr, by which time their acute effects are reversed.Removal of part of the kidney mass results in hypertrophy of the remaining renal tissue. The ensuing increase in kidney size is largely due to hypertrophy-i.e., an increase in protein per cell or protein per DNA, rather than hyperplasia (i.e., an increase in cell number or DNA content) (1). Although attempts have been made to identify kidney-specific growth factors or renotropins (2), an alternative explanation for the hypertrophy is that one or more local factors could sensitize renal cells to a permissive balance of circulating growth factors and that the local stimulus determines the organ specificity of the response. Since fluid transport across the proximal tubule is increased within 24 hr of uninephrectomy (3) and remains elevated in the hypertrophied proximal tubule (4), and since the activity of the Na+/H+ exchanger ("antiporter") in isolated brush border membranes is increased after subtotal nephrectomy (5,6), in the present study we examined whether an increased rate of Na+/H+ antiport is an early event in cell hypertrophy. Although an increase in the activity of the Na+/H+ antiporter has been associated with mitogenesis (7,(17)(18)(19)(20)(21)(22)(23) medium] supplemented with bovine insulin (5 pug/ml), human transferrin (1 ,ug/ml), and hydrocortisone (50 nM). Confluent monolayers were exposed to fresh basic medium (i.e., 50:50 DME/F-12 media containing transferrin) for 48 hr, which resulted in arrest in Go/G1. Experiments were performed by adding specific growth stimuli to this basic medium for 24 and 48 hr. Cells exposed to the basic medium alone acted as controls.Three stimuli to cell hypertrophy (denoted "growth stimuli") were added to the basic medium and studied: (i) insulin at 10 ,ug/ml; (it) PGE1 at 1 ,4M, and (iii) NaCl at 20 mM. The dose...

“…Primary cultures of rabbit proximal tubular cells (6,17) were grown to confluence in DME/Ham's F-12 media (1:1)…”

mentioning

confidence: 99%

BSC-1 growth inhibitor transforms a mitogenic stimulus into a hypertrophic stimulus for renal proximal tubular cells: relationship to Na+/H+ antiport activity.

Fine¹,

Holley²,

Nasri³

et al. 1985

Renal hypertrophy is characterized by an increase in cell size and protein content with minimal hyperplasia. The mechanisms of control of this pattern of cell growth have not been determined. The present studies examined whether the growth inhibitor elaborated by BSC-1 kidney epithelial cells (GI), which has nearly identical biological properties to transforming growth factor /3 (TGF-fi), could transform a mitogenic stimulus into a hypertrophic stimulus for rabbit renal proximal tubular cells in primary culture.Insulin (10 ,ug/ml) plus hydrocortisone (50 nM