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

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.

Download Full-text

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

Journal of the American Society of Nephrology ◽

10.1681/asn.v96960 ◽

1998 ◽

Vol 9 (6) ◽

pp. 960-968 ◽

Cited By ~ 1

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.

Download Full-text

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

Pflügers Archiv - European Journal of Physiology ◽

10.1007/bf00550876 ◽

1991 ◽

Vol 418 (4) ◽

pp. 383-392 ◽

Cited By ~ 46

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

Download Full-text

Nocodazole inhibition of organic anion secretion in teleost renal proximal tubules

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1994.267.3.r695 ◽

1994 ◽

Vol 267 (3) ◽

pp. R695-R704 ◽

Cited By ~ 7

Author(s):

D. S. Miller ◽

J. B. Pritchard

Keyword(s):

Organic Anion ◽

Basolateral Membrane ◽

Organic Anions ◽

Tubular Secretion ◽

Anion Secretion ◽

Proximal Tubular ◽

Renal Tubular ◽

The Impact ◽

Organic Anion Secretion

The impact of the microtubule-disrupting drug nocodazole on renal tubular secretion of organic anions was examined in vitro using proximal tubular masses from teleost fish. Nocodazole reversibly inhibited 20-30% of the tubular accumulation of two model organic anions, p-aminohippurate and fluorescein (FL), by winter flounder tubular masses. However, the drug had no effect on the initial rate of organic anion uptake. Thus it did not reduce transport into the cells at the basolateral membrane, either directly by affecting basolateral organic anion transport proteins or indirectly by altering metabolism or ion gradients. Instead, epifluorescence video microscopy and digital image analysis of killifish tubules showed that nocodazole greatly reduced luminal accumulation of FL and had a smaller effect on cellular dye accumulation. Luminal FL accumulation returned to control levels when tubules were incubated in drug-free medium. Confocal fluorescence microscopy confirmed the marked reduction in luminal FL concentration and demonstrated that intracellular punctate FL accumulation was also markedly reduced. Finally, immunohistochemistry with an anti-tubulin antibody showed that the concentrations of nocodazole used in the above experiments reversibly disrupted microtubules within renal epithelial cells. These data indicate that a component of organic anion secretion in teleost proximal tubule is dependent on an intact microtubular network.

Download Full-text

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

AJP Renal Physiology ◽

10.1152/ajprenal.1993.264.3.f404 ◽

1993 ◽

Vol 264 (3) ◽

pp. F404-F410 ◽

Cited By ~ 2

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)

Download Full-text

Action of EGF and PGE2 on basolateral organic anion uptake in rabbit proximal renal tubules and hOAT1 expressed in human kidney epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00326.2003 ◽

2004 ◽

Vol 286 (4) ◽

pp. F774-F783 ◽

Cited By ~ 33

Author(s):

C. Sauvant ◽

D. Hesse ◽

H. Holzinger ◽

K. K. Evans ◽

W. H. Dantzler ◽

...

Keyword(s):

Cell Line ◽

Ex Vivo ◽

Organic Anion ◽

Renal Tubules ◽

Proximal Tubule Cell ◽

Kidney Cells ◽

Opossum Kidney ◽

Opossum Kidney Cells ◽

Anion Uptake ◽

Stimulation Of

We recently showed that, in a proximal tubule cell line (opossum kidney cells), epithelial growth factor (EGF) stimulates basolateral organic anion transport (OAT) via ERK1/2, arachidonic acid, phospholipase A2, and generation of prostaglandins. PGE2 binds the prostanoid receptor and, thus, activates adenylate cyclase and PKA, which stimulate basolateral organic anion uptake. In the present study, we investigated whether this regulatory cascade is also true 1) for ex vivo conditions in isolated renal proximal (S2) tubules from rabbit and 2) in a human renal epithelial cell line stably expressing human OAT1 (IHKE-hOAT1). EGF activated ERK1/2 in S2 tubules and IHKE-hOAT1, and, in both cases, inhibition of ERK activation (by U-0126) abolished this stimulation. In S2 tubules and IHKE-hOAT1, EGF led to an increase of organic anion uptake, which again was inhibited by U-0126. PGE2 stimulated basolateral organic anion uptake in rabbit S2 tubules and IHKE-hOAT1. EGF- and PGE2-mediated stimulation of organic anion uptake was abolished by inhibition of PKA in rabbit S2 tubules and IHKE-hOAT1, respectively. We conclude that 1) stimulation of basolateral organic anion uptake by EGF or PGE2 is a widespread (if not general) regulatory mechanism, 2) the signal transduction pathway involved seems to be general, 3) stimulation of basolateral organic anion uptake by EGF or PGE2 is also present under ex vivo conditions and, thus, is not a cell culture artifact, 4) activation of OAT1 is sufficient to explain the stimulatory effects of EGF and PGE2 in opossum kidney cells and rabbit S2 segments, and 5) stimulation of basolateral OAT1 by EGF or PGE2 is also important in humans and, thus, may have clinical implications.

Download Full-text