Growth characteristics and measurement of confluency of rabbit primary proximal tubule kidney cells grown on millicell-HA membrane filters

Palmoski, Marshall J.; Masters, Barbara A.; Ford, Sue; Flint, Oliver

doi:10.1007/bf02388121

Cited by 3 publications

(3 citation statements)

References 18 publications

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“…Thus, these cultures maintain tight junctions and active transepithelial transport function for extended periods without the use of a collagen substrate. In contrast, Ford et al (20) and Palmoski et al (21) reported that primary cultures of rabbit RPT cells seeded at 4 mg/membrane, a density approximately fourfold higher than that used in this study, and grown on 30-mm Millicell-HA filters without collagen required 10 to 12 d to reach confluence. The basal-to-apical transepithelial flux of PAH in these cultures was reduced only 55% during a 20-min coincubation with probenecid (21).…”

Section: Discussioncontrasting

confidence: 68%

“…In contrast, Ford et al (20) and Palmoski et al (21) reported that primary cultures of rabbit RPT cells seeded at 4 mg/membrane, a density approximately fourfold higher than that used in this study, and grown on 30-mm Millicell-HA filters without collagen required 10 to 12 d to reach confluence. The basal-to-apical transepithelial flux of PAH in these cultures was reduced only 55% during a 20-min coincubation with probenecid (21). Thus, in contrast to previous studies, the cultures grown under the conditions described in the present study reached confluence more quickly and maintained carrier-mediated organic anion transport for extended periods.…”

Section: Discussioncontrasting

confidence: 68%

“…Primary cultures of rabbit RPT cells grown on permeable membranes were used as the in vitro model to study transepithelial flux of OTA. Compared with cultures grown on noncoated Millicell HA membranes (20,21), primary cultures of rabbit RPT cells grown on collagen IV-coated membranes reached confluence after 5 d and maintained tight monolayers for up to 15 d in culture (22). However, in the current study, monolayers grown under improved culture conditions on noncoated Transwell membranes with 1 mM AscP added to the media reached confluence within 4 to 5 d, had improved The apical-to-basal ratio of 0.01 for fluorescein-inulin diffusion across primary cultures after 6 h was significantly less than the ratio of 0.06 for inulin diffusion previously reported after 4 h in primary cultures of rabbit RPT cells grown on collagen type IV (22).…”

Section: Discussionmentioning

confidence: 99%

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Ochratoxin A Secretion in Primary Cultures of Rabbit Renal Proximal Tubule Cells

Groves

Nowak

Morales

1999

Journal of the American Society of Nephrology

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Abstract. Primary cultures of rabbit renal proximal tubule cells grown under improved culture conditions were used to study the transepithelial transport of the nephrotoxic mycotoxin ochratoxin A. The basal-to-apical transepithelial flux, i.e., secretion, of this fluorescence organic acid was measured in primary cultures of rabbit renal proximal tubule cells. The basal-to-apical flux of ochratoxin A increased with time and reached a steady state after 12 h. On the other hand, the apical-to-basal flux, i.e., reabsorption, of ochratoxin A was minimal over time. The secretory flux of ochratoxin A was as much as eightfold greater than the reabsorptive flux, indicating that net secretion is the primary mechanism for ochratoxin A clearance by the proximal tubule. The kinetic analysis of ochratoxin A flux revealed secretion to be a saturable and very high-affinity process with an apparent K50 of 0.33 ± 0.21 mM. A saturating concentration of the prototypical organic anion substrate para-aminohippurate (PAH) reduced ochratoxin A secretion by approximately 75%. The kinetic analysis of PAH inhibition of ochratoxin A secretion revealed an IC50 of 195 mM, which is similar to the IC50 for PAH inhibition of peritubular ochratoxin A uptake in tubule suspensions and the Km values for peritubular PAH uptake. The organic anions probenecid, octanoate, and α-ketoglutarate reduced ochratoxin A excretion to the same degree as PAH, whereas the amino acid phenylalanine had a minimal effect on ochratoxin A secretion. Thus, collectively, these observations indicate that the secretion of ochratoxin A in primary cultures of rabbit renal proximal tubules is limited to the organic anion secretory pathway. The high affinity measured for the basal-to-apical flux of ochratoxin A suggests that at concentrations typical of naturally occurring exposures, transepithelial secretion by the organic anion transport pathway represents a significant avenue for excretion of this mycotoxin by the renal proximal tubule.

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Section: Discussioncontrasting

confidence: 68%