The mitogenic effect of epidermal growth factor (EGF) and the characteristics of EGF binding were studied on primary cultures of rabbit proximal tubular cells. EGF was found to be a potent mitogen and stimulated DNA synthesis 18-fold above the level observed in quiescent cells. Using 125I-EGF as a ligand, two classes of specific EGF receptors were identified on the proximal tubular cell in culture, i.e., a high-affinity receptor with a dissociation constant (Kd) of 0.3 nM and maximal binding (Bmax) of 1.7 X 10(4) receptors/cell and a low-affinity receptor with a Kd of 1.9 nM and Bmax of 5.3 X 10(4) receptors/cell. Because angiotensin II (ANG II) appeared to possess many properties common to growth factors, we also examined the interaction of ANG II and EGF on these cells. ANG II was not mitogenic, but it potentiated the mitogenic effect of EGF with a maximal effect at 10(-9) M. The dose-response curve of EGF-induced mitogenesis was shifted to the left in the presence of 10(-9) M ANG II, decreasing the approximate half-maximal stimulatory concentration from 3 X 10(-8) to 5 X 10(-9) M. ANG II also stimulated prostaglandin E2 (PGE2) release, but inhibition of basal and ANG II-stimulated PGE2 synthesis had no effect on mitogenesis. ANG II had no effect on the binding of EGF to the high-affinity receptor from 1 to 20 h and did not alter receptor downregulation. ANG II (10(-9) M) had no effect on cell protein content, RNA and protein synthesis, Na+-H+ antiport, and intracellular free Ca2+ concentration. Higher concentrations of ANG II (5 X 10(-8) to 5 X 10(-6) M) led to a rapid and transient dose-dependent rise in cytosolic free Ca2+ concentration. These studies demonstrate that ANG II potentiates EGF-induced mitogenesis at one or more postreceptor steps that may include small changes in cytosolic Ca2+ concentration.

A highly purified suspension of rabbit proximal tubules was cultured in a hormone-supplemented serum-free medium. This primary culture yielded a homogeneous population of cells that demonstrated functional and morphological polarity in mono-layers. The characteristics of the Na-dependent glucose transporter in the luminal membrane were studied by measuring the uptake of alpha-methylglucoside (AMG). The kinetics of Na-dependent AMG uptake were consistent with a single saturable system with an apparent Km of 0.8 mM and Jmax of 0.14 nmol X mg-1 X min-1. AMG permeability was 0.10 microliter X mg-1 X min-1. Uptake was inhibited 95% by 0.1 mM phlorizin and by removal of sodium. The stoichiometry of Na/glucose interaction with the carrier was 2:1. These characteristics are typical of the characteristics described for the late proximal tubule. To examine whether the glucose that enters the cell across the luminal membrane is incorporated into the metabolic pool of the cell, we studied the oxidation of [14C]glucose to 14CO2 in the absence and presence of phlorizin. Significant decarboxylation of [1-14C]glucose and [6-14C]glucose was observed, consistent with the existence of aerobic metabolism and a hexose monophosphate shunt. In the presence of 0.1 mM phlorizin, uptake and oxidation of D-glucose were inhibited to an identical degree, suggesting that luminal uptake is a rate-limiting step in the oxidation of glucose by these proximal tubular cells. These studies indicate that proximal tubular cells in primary culture utilize glucose as an energy source and that the glucose derived from transport across the luminal membrane is incorporated into the metabolic pool of the cell.

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...

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