Preparation and Characterization of a Hormone-responsive Renal Plasma Membrane Fraction

Marx, Stephen J.; Fedak, Susan A.; Aurbach, G. D.

doi:10.1016/s0021-9258(19)44672-3

Cited by 163 publications

(3 citation statements)

References 32 publications

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“…Notably, the proximal tubule cultures are not similarly responsive to these hormones. In yet greater contrast, the renal medulla, which is not enriched with proximal tubules, exhibits dramatic responsiveness to arginine vasopressin (47).…”

Section: Discussionmentioning

confidence: 91%

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium.

Chung¹,

Alavi²,

Livingston³

et al. 1982

The Journal of Cell Biology

285

225

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Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serumfree medium (Medium RK-1). A hormone-deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and Ta. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and Ta, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors.Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated a-methylglucoside (a-MG) against a concentration gradient. However, little or no a-MG accumulation was observed in the absence of Na ÷. Metabolic inhibitor studies also indicated that a-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K ÷ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM c~-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na÷-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and y-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.Primary kidney-cell culture is valuable for studying renal functions in vitro (1). Although several established kidney-ceU lines have been used to study the mechanism of transepithelial solute transport by the kidney (2, 3,4,5,6), investigations using such cell lines have several limitations. First, the available cell lines may 0nly resemble particular kidney-cell types to a limited extent. Secondly, only a limited number of established kidney-ceU lines are available for study. Consequently, culture systems are also needed that closely resemble cells in a number of nephron segments (7). Primary kidney-cell culture provides an excellent means to resolve these problems (8).Previous studies with primary-kidney cultures have been, however, impeded by the presence of serum in the tissueculture medium (9). The transporting epithelial cells in primary-kidney cultures maintained in serum-supplemented medium, have been overgrown by fibrobl...

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Section: Discussionmentioning

confidence: 91%

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium.

Chung¹,

Alavi²,

Livingston³

et al. 1982

The Journal of Cell Biology

285

225

View full text Add to dashboard Cite

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“…(pH 6.8) containing 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.5 mmol/L EDTA, and 100 mmol/L dithio-Crude renal membrane (CM) preparations were cartreitol (final concentrations). Forty micrograms of BBM ried out by a modification of the method described by proteins per lane were electrophoresed on 10% sodium Marx, Fedak, and Aurbach [26]. One hundred to 150 mg dodecyl sulfate-polyacrylamide gel electrophoresis (SDSof frozen renal powder were homogenized in 1.5 mL iso-PAGE) gel according to the method of Laemmli [30], tonic medium containing 0.25 mol/L sucrose, 10 mmol/L and the samples were electrotransferred onto nitrocellu-Tris, and 1 mmol/L ethylenediaminetetraacetic acid lose membranes.…”

Section: Crude Renal Membrane Preparationsmentioning

confidence: 99%

Sulfate homeostasis, NaSi-1 cotransporter, and SAT-1 exchanger expression in chronic renal failure in rats

Fernandes¹,

Laouari²,

Tutt³

et al. 2001

Kidney International

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These results demonstrate that both NaSi-1 and Sat-1 total protein abundances are decreased in CRF, which may contribute to the increase in fractional sulfate excretion. Strikingly, NaSi-1 density was not decreased in CRF three weeks after Nx, and furthermore, increased six weeks after Nx, in contrast to NaPi-2 density, which was decreased at both times. The significance of this difference remains to be determined, but may explain why hypersulfatemia occurs earlier than hyperphosphatemia in CRF.

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“…Control incubations contained either 100 milliunits of PCPLC or no added phospholipase. At the indicated times, samples of the incubation mixtures were centrifuged (lOOOOOg, 60 min), and the supernatant and pellet fractions were analyzed for both [3H] folic acid counts (by scintillation counting) and alkaline phosphatase activity (with p-nitrophenyl phosphate substrate; Marx et al, 1972). One unit of FBP corresponds to binding of 1 pmol of [3H] folic acid.…”

Section: Methodsmentioning

confidence: 99%

Folate binding protein from kidney brush border membranes contains components characteristic of a glycoinositol phospholipid anchor

Lee¹,

Shoda²,

Krall³

et al. 1992

Biochemistry

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A number of cell surface proteins have been shown to be anchored to the plasma membrane by a covalently attached glycoinositol phospholipid (GPL) in amide linkage to the C-terminus of the mature protein. We applied several criteria to establish that folate binding protein (FBP) in brush border membranes of rat kidney contains a GPL anchor. Brush border membranes were isolated and labeled with [3H]folate, and the complex of FBP and [3H]folate was shown to be released to the supernatant by incubation with purified bacterial phosphatidylinositol-specific phospholipase C (PIPLC) but not by incubation with a purified bacterial phosphatidylcholine-specific phospholipase C. The FBP-[3H]folate complex both in crude extracts and after FBP purification by ligand-directed affinity chromatography interacted with Triton X-114 micelles, and prior incubation with PIPLC prevented this detergent interaction. Individual residues characteristic of GPL anchors were found to be covalently associated with FBP following polyacrylamide gel electrophoresis in sodium dodecyl sulfate. These included glucosamine and ethanolamine, which were radiolabeled by reductive methylation and identified by chromatography on an amino acid analyzer, and inositol phosphate, which was inferred by Western blotting with an anti-CRD antisera. This antisera gave positive immunostaining only after FBP had been cleaved by PIPLC, a reliable diagnostic of a GPL anchor. The relationship between GPL-anchored FBP in biological membranes and soluble FBP in biological fluids also is discussed.

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Preparation and Characterization of a Hormone-responsive Renal Plasma Membrane Fraction

Cited by 163 publications

References 32 publications

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium.

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium.

Sulfate homeostasis, NaSi-1 cotransporter, and SAT-1 exchanger expression in chronic renal failure in rats

Folate binding protein from kidney brush border membranes contains components characteristic of a glycoinositol phospholipid anchor

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