Receptor-mediated endocytosis of albumin in cultured opossum kidney cells: a model for proximal tubular protein reabsorption

1991 ◽  
Vol 418 (4) ◽  
pp. 383-392 ◽  
Author(s):  
Johann S. Schwegler ◽  
Bernd Heppelmann ◽  
Sigrid Mildenberger ◽  
Stefan Silbernagl
Keyword(s):  
Kidney Cells ◽  
Opossum Kidney ◽  
Receptor Mediated Endocytosis ◽  
Opossum Kidney Cells ◽  
Proximal Tubular ◽  
Protein Reabsorption

scholarly journals Long-term protein exposure reduces albumin binding and uptake in proximal tubule-derived opossum kidney cells.

1998 ◽  
Vol 9 (6) ◽  
pp. 960-968 ◽  
Author(s):  
M Gekle ◽  
S Mildenberger ◽  
R Freudinger ◽  
S Silbernagl
Keyword(s):  
Amino Acids ◽  
Proximal Tubule ◽  
Uptake Rate ◽  
Kidney Cells ◽  
Proximal Tubular Cells ◽  
Opossum Kidney ◽  
Tubular Cells ◽  
Opossum Kidney Cells ◽  
Proximal Tubular ◽  
Protein Reabsorption

To avoid renal loss of large amounts of proteins, filtered proteins are reabsorbed by endocytosis along the proximal tubule. However, although protein reabsorption is a task of proximal tubular cells, it is also a threat because it may cause cell injury. This study determines whether exposure to bovine serum albumin (BSA) leads to regulatory changes in endocytosis of FITC-BSA in proximal tubule-derived opossum kidney cells. Preincubation with BSA led to a decrease of FITC-BSA endocytosis with an IC50 value of 0.58 g/L. Specific binding of FITC-BSA to the apical membrane was also reduced (IC50 = 0.69 g/L). Kinetic analyses revealed that maximal uptake rate and maximal binding capacity were decreased with no change in affinity. Similar effects were observed after preincubation with equimolar amounts of other proteins (lactalbumin, transferrin, and conalbumin), but not after preincubation with dextran. The effect of preincubation with BSA could be mimicked by preincubation with some amino acids. Preincubation with L-Ala, L-Gln, or NH4Cl, but not with L-Leu, L-Glu, or L-Asp, reduced FITC-BSA endocytosis and binding. Preincubation with BSA, but not with dextran, reduced protein degradation and increased ammonia production, vesicular pH, as well as the rate of lactate dehydrogenase release. Apical fluid-phase endocytosis and apical uptake of neutral amino acids were not reduced. It is concluded that proximal tubular cells reduce the uptake rate for proteins, but not for other substrates, in response to increased protein load. This reduction is achieved by reducing the number of apical binding sites, partially in response to increased ammoniagenesis with deranged vesicular pH and enzyme activities. Thus, increased protein filtration could result in reduced protein reabsorption, thereby enhancing proteinuria.

scholarly journals Na+/Pi co-transport alters rapidly cytoskeletal protein polymerization dynamics in opossum kidney cells

1996 ◽  
Vol 315 (1) ◽  
pp. 241-247 ◽  
Author(s):  
Evangelia A. PAPAKONSTANTI ◽  
Dimitrios S. EMMANOUEL ◽  
Achille GRAVANIS ◽  
Christos STOURNARAS
Keyword(s):  
Immunoblot Analysis ◽  
Kidney Cells ◽  
Opossum Kidney ◽  
Protein Polymerization ◽  
Phosphonoformic Acid ◽  
Opossum Kidney Cells ◽  
Proximal Tubular ◽  
Microtubule Stabilizer ◽  
Dynamic Equilibria ◽  
Contrast Measurement

We studied with biochemical and immunofluorescent techniques the interactions between the actin microfilament and tubulin microtubule cytoskeleton and Na+/Pi co-transport in opossum kidney cells, a line with proximal tubular characteristics. On brief (5 min) incubation of the cells with a low (0.1 mM) concentration of Pi, a rapid F-actin depolymerization takes place, which fails to occur in cells incubated under similar conditions with 1 mM Pi. The disassembly of actin microfilaments could be quantitatively expressed as a 33% increase in the ratio of monomeric G-actin to polymerized F-actin (G/F-actin ratio from 0.80±0.03 to 1.06±0.06, n = 28, P < 0.01), owing to a significant decrease in the latter. Under these conditions microfilaments were also markedly destabilized, as shown by their diminished resistance to graded cytochalasin B concentrations. In addition, incubation of opossum kidney cells with low Pi concentrations (0.1 mM) resulted within 5 min in a substantial depolymerization of microtubules, shown by immunofluorescence microscopy and measured as a 70.9±6.9% (n = 11, P < 0.01) decrement by immunoblot analysis. These changes, which occur only when extracellular Pi concentrations are kept low, seem to be related to a significant increase within 5 min in the rate of cellular Pi uptake by 25.5% under these conditions. The shifts in the dynamic equilibria between monomeric and polymerized actin and tubulin in response to cellular Pi uptake were transient, being fully reversible within 30 min. Moreover, the effect of Pi seemed to be specific because inhibition of its uptake by phosphonoformic acid blunted microtubular disassembly markedly. In contrast, measurement of Pi uptake in the presence of agents known to stabilize cytoskeletal structures showed a substantial decrease with phallacidin, which stabilizes microfilaments, whereas the microtubule stabilizer taxol had no apparent effect. These results indicate that acute alterations in the polymerization dynamics and stability of both microfilaments and microtubules are involved in the modulation of Na+/Pi co-transport and suggest important cytoskeletal participation in proximal tubular transport functions.

Parathyroid hormone degradation by opossum kidney cells via receptor-mediated endocytosis and lysosomal hydrolysis

1992 ◽  
Vol 127 (3) ◽  
pp. 267-270 ◽  
Author(s):  
Toru Yamaguchi ◽  
Makoto Arao ◽  
Masaaki Fukase
Keyword(s):  
Parathyroid Hormone ◽  
Biologically Active ◽  
Main Role ◽  
Kidney Cells ◽  
Binding Studies ◽  
Opossum Kidney ◽  
Receptor Mediated Endocytosis ◽  
Opossum Kidney Cell ◽  
Opossum Kidney Cells

The mechanisms involved in parathyroid hormone (PTH) degradation by proximal renal tubule cells were studied using an opossum kidney cell line possessing PTH receptors as an in vitro model system. One hour incubation of 5 nmol/l human (h) PTH-(1-84) with intact opossum kidney cells (4.0× 106 cells) resulted in about 70% degradation and disappearance of hPTH-(1-84) from the medium, as determined by a two-site immunoradiometric assay. Preincubation with 100 nmol/l h[Nle8, Nle18, Tyr34]PTH-(1-34)amide for 6, 24, 48 and 72 h caused a 26, 47, 62 and 73% decrease, respectively, in PTH degradation by opossum kidney cells. Binding studies with 125I-labeled h[Nle8, Nle18, Tyr34]PTH-(1-34)amide as a radioligand showed that PTH receptor binding decreased with the time of pretreatment with the agonist. Pretreatments of the cells with monensin, an inhibitor of endocytosis, and the lysosomotropic agents such as chloroquine, ammonium chloride and leupeptin, inhibited degradation of hPTH-(1-84) by 87, 71, 76 and 72%, respectively. Concentrations of 5 nmol/l hPTH-(39-84) and hPTH-(39-68), which are known not to bind to PTH receptors appreciably, were not degraded by opossum kidney cells during 1 h incubations. Thus intact, biologically active PTH, but not its inactive fragments, is degraded by opossum kidney cells, by receptor-mediated endocytosis and lysosomal hydrolysis. A mechanism resembling the peritubular uptake of intact PTH by perfused kidneys reported previously appears to play a main role in PTH metabolism by cultured renal cells.

scholarly journals Modulation of the Basolateral and Apical Step of Transepithelial Organic Anion Secretion in Proximal Tubular Opossum Kidney Cells

2001 ◽  
Vol 276 (18) ◽  
pp. 14695-14703 ◽  
Author(s):  
Christoph Sauvant ◽  
Hildegard Holzinger ◽  
Michael Gekle
Keyword(s):  
Organic Anion ◽  
Kidney Cells ◽  
Opossum Kidney ◽  
Anion Secretion ◽  
Opossum Kidney Cells ◽  
Proximal Tubular ◽  
Organic Anion Secretion

NHE3 Na+/H+ exchanger supports proximal tubular protein reabsorption in vivo

2004 ◽  
Vol 287 (3) ◽  
pp. F469-F473 ◽  
Author(s):  
Michael Gekle ◽  
Katharina Völker ◽  
Sigrid Mildenberger ◽  
Ruth Freudinger ◽  
Gary E. Shull ◽  
...  
Keyword(s):  
Ph Homeostasis ◽  
Genetic Studies ◽  
Opossum Kidney ◽  
Protein Excretion ◽  
Opossum Kidney Cells ◽  
Sds Page ◽  
Proximal Tubular ◽  
Protein Reabsorption ◽  
Endosomal Ph

Proximal tubular receptor-mediated endocytosis (RME) of filtered proteins prevents proteinuria. Pharmacological and genetic studies in cultured opossum kidney cells have shown that the apical Na+/H+ exchanger isoform 3 (NHE3) supports RME by interference with endosomal pH homeostasis and endocytic fusion events. However, it is not known whether NHE3 also supports proximal tubular RME in vivo. We analyzed proximal tubular protein reabsorption by microinfusion experiments in rats and investigated renal protein excretion in NHE3 knockout (Nhe3 −/−) mice. Inhibition of NHE3 by EIPA or S-3226 reduced the fractional reabsorption of [14C]cytochrome c by ∼50% during early proximal microinfusion. During early distal microinfusion, no protein reabsorption could be detected. Urinary protein excretion of Nhe3 −/− or heterozygous mutant mice was significantly higher compared with wild-type mice. SDS-PAGE analysis of urinary proteins revealed that Nhe3 −/− animals excreted proteins the size of albumin or smaller. Thus a reduction in NHE3 activity or abundance causes tubular proteinuria. These data show that NHE3 supports proximal tubular RME of filtered proteins in vivo.

Kinetics of receptor-mediated endocytosis of albumin in cells derived from the proximal tubule of the kidney (opossum kidney cells): influence of Ca2+ and cAMP

1995 ◽  
Vol 430 (3) ◽  
pp. 374-380 ◽  
Author(s):  
Ruth Freudinger ◽  
Stefen Silbernagl ◽  
Michel Gekle ◽  
Sigrid Mildenberger
Keyword(s):  
Proximal Tubule ◽  
Kidney Cells ◽  
Opossum Kidney ◽  
Receptor Mediated Endocytosis ◽  
Opossum Kidney Cells ◽  
Kinetics Of ◽  
In Cells

Calcitriol stimulates Na(+)-Pi cotransport in a subclone of opossum kidney cells (OK-7A) by a genomic mechanism

1993 ◽  
Vol 264 (3) ◽  
pp. F404-F410 ◽  
Author(s):  
M. Allon ◽  
M. Parris
Keyword(s):  
Kidney Cells ◽  
Dihydroxyvitamin D3 ◽  
Prolonged Incubation ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Opossum Kidney Cells ◽  
Proximal Tubular ◽  
Renal Proximal Tubular Cells ◽  
Renal Proximal Tubular ◽  
Contrast Ct

Calcitriol (CT) stimulates Na(+)-Pi cotransport in a subclone of opossum kidney cells (OK-7A) by a genomic mechanism. An experimental model of renal proximal tubular cells in which CT affects Na(+)-Pi cotransport would be useful for examining the mechanisms of this effect. This study evaluated the effect of CT on Na(+)-Pi cotransport in opossum kidney (OK) cells. CT had no effect on Na(+)-Pi cotransport in wild-type OK cells and in the OK-B, OK-H, and OK-P subclones. In contrast, CT at physiological concentrations stimulated Na(+)-Pi cotransport in the OK-7A subclone; the effect was dose related with a 52% increase at 10(-11) M CT, as well as a maximal twofold stimulation at 10(-9) M. CT (10(-11) M) increased the maximum uptake for Na(+)-Pi cotransport (Vmax = 3.55 +/- 0.16 vs. 2.51 +/- 0.17 nmol.mg protein-1.5 min-1, P < 0.01), without affecting the apparent Michaelis constant (Km = 30.6 +/- 1.0 vs. 30.8 +/- 0.7 microM). The stimulatory effect on Na(+)-Pi cotransport was specific for CT and did not occur with 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, or 1 beta,25-dihydroxyvitamin D3. At 10(-11) M CT, the stimulation of Na(+)-Pi uptake in OK-7A cells was maximal at 3 h; it was completely abolished by preincubation with actinomycin D or cycloheximide. Both calphostin C, an inhibitor of protein kinase C (PKC), or prolonged incubation with phorbol 12-myristate 13-acetate, to downregulate the PKC pathway, partially inhibited the stimulatory effect of CT on Na(+)-Pi cotransport in OK-7A cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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