Intracellular compartmentation of organic anions within renal cells

1993 ◽  
Vol 264 (5) ◽  
pp. R882-R890 ◽  
Author(s):  
D. S. Miller ◽  
D. E. Stewart ◽  
J. B. Pritchard
Keyword(s):  
Organic Anion ◽  
Basolateral Membrane ◽  
Organic Anions ◽  
Epifluorescence Microscopy ◽  
Renal Cells ◽  
Bladder Epithelium ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Intracellular Compartments ◽  
Energy Dependent

Epifluorescence microscopy and video-image analysis were used to measure the distribution of the monovalent organic anion fluorescein (FL) within the cells of three organic anion-secreting renal epithelia: crab urinary bladder (a proximal tubule analogue), opossum kidney (OK) cells in culture, and intact teleost proximal tubules. In all three preparations the intracellular FL distribution was nonuniform. Two distinct intracellular compartments were detected, one being diffuse and cytoplasmic and the other punctate. With low FL concentrations in the medium (1 microM and below) dye accumulation in the punctate compartment exceeded that of the cytoplasm. In crab bladder epithelium FL uptake into both compartments was inhibited by external probenecid, p-aminohippurate (PAH), and LiCl and stimulated by 10-50 microM external glutarate, suggesting that the punctate compartment loaded by a two-step mechanism: transport into the cytoplasm at the basolateral membrane, followed by accumulation at specific intracellular sites. Experiments in which FL was microinjected into OK cells directly demonstrated movement of FL from the cytoplasmic to the punctate compartment. Accumulation in the latter was specific, i.e., inhibitable by coinjected PAH and probenecid, and energy dependent. Together, these findings indicate that during secretion organic anions are sequestered within renal cells. The role of sequestration in overall transport remains to be determined.

Differences between OK and LLC-PK1 cells: cystine handling

1991 ◽  
Vol 261 (1) ◽  
pp. C8-C16 ◽  
Author(s):  
B. States ◽  
D. Harris ◽  
S. Segal
Keyword(s):  
Cell Lines ◽  
Apical Membrane ◽  
Reduced Glutathione ◽  
Basolateral Membrane ◽  
Growth Period ◽  
Transport Systems ◽  
Amino Acid Transport System ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Dibasic Amino Acid

Cultured opossum kidney (OK) and porcine kidney (LLC-PK1) cells were compared for biochemical characteristics and cystine transport systems. The cell lines differ in amount of protein per cell, with OK cells having approximately one-half the amount found in LLC-PK1. Both cell lines contain 19 micrograms DNA/10(6) cells. As cells reach confluence, cystine uptake increases in OK and decreases in LLC-PK1 cells. Throughout the growth period, only lysine inhibits cystine uptake in OK, whereas glutamate is the inhibitor in LLC-PK1. The predominant site of cystine transport in OK cells is across the apical membrane, and the basolateral membrane is the corresponding site of transport in LLC-PK1 cells. Although the intracellular reduced glutathione pool is the same, the cysteine pool in OK cells is approximately one-fourth that found in LLC-PK1 cells. The ability of OK cells to reflect the shared cystine-dibasic amino acid transport system and LLC-PK1 to exhibit the cystine-glutamate antiporter system makes available two models for investigation of the development and structure of cystine transport systems.

Mechanism and regulation of vitamin B6 uptake by renal tubular epithelia: studies with cultured OK cells

2002 ◽  
Vol 282 (3) ◽  
pp. F465-F471 ◽  
Author(s):  
Hamid M. Said ◽  
Alvaro Ortiz ◽  
Nike D. Vaziri
Keyword(s):  
Vitamin B6 ◽  
Vitamin B ◽  
Maximal Velocity ◽  
Limited Information ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Intracellular Ca ◽  
Renal Tubular ◽  
Energy Dependent ◽  
Menten Constant

The kidneys play an important role in regulating vitamin B6 body homeostasis, but limited information exists regarding the mechanism of pyridoxine uptake by renal epithelial cells, and no study exists on its regulation. To address these issues, we used the renal opossum-derived tubular epithelial (opossum kidney; OK) cells and found pyridoxine uptake to 1) be temperature and energy dependent, 2) be pH dependent, with a higher uptake at alkaline or neutral buffer pH compared with acidic pH, 3) be Na+independent, 4) involve a saturable component (apparent Michaelis- Menten constant of 2.40 ± 0.23 μM), 5) be inhibited by structural analogs, and 6) be amiloride sensitive. Maintaining OK cells in a vitamin B6-deficient growth medium (for 48 h) led to a significant upregulation of pyridoxine uptake. This upregulation was found to be specific for pyridoxine, inhibited by cyclohexamide and actinomycin D, reversible, and mediated via an increase in maximal velocity. Pretreating OK cells with modulates of a Ca2+/calmodulin-mediated pathway led to a significant downregulation in pyridoxine uptake via inhibition of maximal velocity. These results demonstrate that pyridoxine uptake by renal tubular epithelial OK cells is via a specialized pH-sensitive carrier-mediated mechanism. This mechanism appears to be regulated by extracellular vitamin B6 levels and an intracellular Ca2+/calmodulin-mediated pathway.

Indirect coupling of organic anion secretion to sodium in teleost (Paralichthys lethostigma) renal tubules

1991 ◽  
Vol 261 (6) ◽  
pp. R1470-R1477 ◽  
Author(s):  
D. S. Miller ◽  
J. B. Pritchard
Keyword(s):  
Steady State ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Epifluorescence Microscopy ◽  
Renal Tubules ◽  
Bathing Medium ◽  
Anion Secretion ◽  
Southern Flounder ◽  
Indirect Coupling ◽  
Tubular Lumen

Recent findings in both rat and crab indicate that renal accumulation of p-aminohippurate (PAH) across the basolateral membrane can be coupled indirectly to the Na gradient through PAH-glutarate exchange and Na/glutarate cotransport. However, the role of this mechanism in net transepithelial PAH secretion was not examined. Therefore, proximal tubules from Southern flounder kidney were used to assess both the presence of indirect coupling in the fish and its relationship to net secretion. [14C]glutarate uptake by proximal tubular masses was concentrative, Na dependent, and Li inhibitable. Glutarate efflux from preloaded masses was stimulated by addition of PAH to the medium. Thus flounder tubules exhibited both Na/glutarate uptake and glutarate-PAH exchange. Furthermore, steady-state [3H]PAH accumulation was increased 50% by 10-50 microM glutarate, and this increase was abolished by Li, indicating indirect coupling of PAH entry to Na. To determine whether indirect coupling affected net secretion as well as tissue accumulation, the steady-state accumulation of an anionic dye, fluorescein (FL), was measured in individual renal tubules by use of epifluorescence microscopy and video-image analysis. FL accumulated in tubules to levels that were 20-40 times higher than the medium. In most fish, luminal fluorescence was measurably higher than cellular, and uptake in both compartments was markedly reduced by PAH and Li. Moreover, FL accumulation in cells and lumina was increased by 70-100% when 50 microM glutarate was added to the bathing medium. Thus glutarate not only stimulated uphill FL entry into the cells, but also stimulated active secretion into the tubular lumen.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of NH2 and COOH termini in targeting, stability, and activity of sodium bicarbonate cotransporter 1

2006 ◽  
Vol 291 (3) ◽  
pp. F588-F596 ◽  
Author(s):  
Doris Joy D. Espiritu ◽  
Angelito A. Bernardo ◽  
Jose A. L. Arruda
Keyword(s):  
Cell Membrane ◽  
Sodium Bicarbonate ◽  
Basolateral Membrane ◽  
Membrane Stability ◽  
Hek293 Cells ◽  
Opossum Kidney ◽  
Cell Membrane Stability ◽  
Ok Cells ◽  
Sodium Bicarbonate Cotransporter

Sodium bicarbonate cotransporter 1 (NBC1) mediates 80% of bicarbonate reabsorption by the kidney, but the molecular determinants for activity, targeting, and cell membrane stability are poorly understood. We generated truncation mutants involving the entire NH2 (ΔN424) or the entire COOH (ΔC92) terminus and examined the effects of these truncations on targeting, cell membrane stability, and NBC1 activity. ΔN424 and ΔC92 targeted to the plasma membrane of HEK293 cells or to the basolateral membrane of opossum kidney (OK) cells at 24 h but did not display NBC1 activity. Unlike the NBC1 wild-type and the ΔN424, ΔC92 expression was significantly decreased in the basolateral membrane at 48 h and yet the total ΔC92 expression in the cell was constant. We found that decreased ΔC92 expression in the basolateral membrane was due to increased endocytosis and mistargeting to the apical membrane. Increased endocytosis was prevented when both ΔN424 and ΔC92 were cotransfected together and more stable expression of ΔC92 was observed. Immunoprecipitation studies using NBC1 antibody specific for the COOH epitope were able to detect the COOH truncated NBC1 when probed with NH2 epitope-specific antibody or vice versa. Similar findings were observed with Ni-NTA pull-down assay. Cotransfection of both mutants partially restored NBC1 activity. In summary, NBC1 targets to the basolateral membrane of OK cells by a default mechanism and the COOH terminus plays a role on NBC1 stability in the basolateral membrane.

Nocodazole inhibition of organic anion secretion in teleost renal proximal tubules

1994 ◽  
Vol 267 (3) ◽  
pp. R695-R704 ◽  
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.

scholarly journals Immunohistochemical Localization of Multispecific Renal Organic Anion Transporter 1 in Rat Kidney

1999 ◽  
Vol 10 (3) ◽  
pp. 464-471 ◽  
Author(s):  
AKIHIRO TOJO ◽  
TAKASHI SEKINE ◽  
NORIKO NAKAJIMA ◽  
MAKOTO HOSOYAMADA ◽  
YOSHIKATSU KANAI ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Organic Anions ◽  
Proximal Tubules ◽  
Organic Anion Transporter ◽  
Electron Microscopic ◽  
Renal Vasculature ◽  
Initial Portion ◽  
Anion Transporter

Abstract. Renal proximal convoluted tubules have an important role, i.e., to excrete organic anions, including numerous drugs and endogenous substances. Recently, multispecific organic anion transporter 1 (OAT1) was isolated from rat kidney. In this study, the cellular and subcellular localization of OAT1 in rat kidney was investigated. Kidneys from normal rats were perfused and fixed with periodate-lysine-paraformaldehyde solution and were then processed for immunohistochemical analysis using the labeled streptavidin-biotin method, preembedding horseradish peroxidase method, and immunogold method. Light microscopic examination revealed immunostaining for OAT1 in the middle portion of the proximal tubule (S2 segment), but not in the initial portion of the proximal convoluted tubule, next to the glomerulus. Nephron segments other than the S2 segment and the renal vasculature were not stained with antibody to OAT1. Electron-microscopic observation using a preembedding method revealed that OAT1 was exclusively expressed in the basolateral membrane of S2 segments of proximal tubules. The immunogold method showed no labeling for OAT1 in the cytoplasmic vesicles, suggesting that OAT1 may not move together with organic anions into the cells. These results are consistent with previous physiologic data showing that organic anions, including para-aminohippurate, are taken up by the basolateral Na+-independent organic anion/dicarboxylate exchanger and excreted at S2 segments. In conclusion, OAT1 was localized to the basolateral membrane of S2 segments of proximal tubules in rat kidneys.

Effect of SITS on organic anion transport in the rabbit kidney cortical slice

1978 ◽  
Vol 234 (4) ◽  
pp. F302-F307
Author(s):  
S. K. Hong ◽  
J. M. Goldinger ◽  
Y. K. Song ◽  
F. J. Koschier ◽  
S. H. Lee
Keyword(s):  
Organic Cation ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Organic Anions ◽  
Rabbit Kidney ◽  
Cortical Slice ◽  
Tissue Water Content ◽  
Tissue Water ◽  
Inulin Space ◽  
Disulfonic Stilbene

The effect of SITS (4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene) on the transport of organic ions in the rabbit kidney cortical slice was studied. SITS at a concentration of 10(-4) to 10(-3) M significantly decreased the slice-to-medium (S/M) concentration ratio of the organic anions p-aminohippurate (PAH), and 2,4,5-trichlorophenoxyacetate, but had no significant effect on that of the organic cation tetraethylammonium. The S/M ratio of PAH decreased to 0.52 +/- 0.03 (SE) in the presence of 10(-3) M SITS. The inhibition of PAH uptake caused by SITS was reversed in the presence of 0.5% bovine serum albumin in the medium. SITS at a concentration of 10(-4) M had no significant effect on the efflux of PAH. However, there was a small increase in PAH efflux at a concentration of 10(-3) M SITS. A Lineweaver-Burk analysis of the data indicates that SITS competitively inhibits PAH uptake and that SITS has a Ki value of 2.3 X 10(-4) M. SITS had no effect on the tissue water content, [14C]inulin space, or intracellular Na and K concentrations. It is suggested that the primary effect of SITS is to inhibit the entry of organic anions from the medium into the cell across the basolateral membrane.

scholarly journals Immunolocalization of Multispecific Organic Anion Transporters, OAT1, OAT2, and OAT3, in Rat Kidney

2002 ◽  
Vol 13 (4) ◽  
pp. 848-857 ◽  
Author(s):  
Ryoji Kojima ◽  
Takashi Sekine ◽  
Masanao Kawachi ◽  
Seok Ho Cha ◽  
Yoshio Suzuki ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Organic Anions ◽  
Proximal Tubules ◽  
Transepithelial Transport ◽  
Thick Ascending Limb ◽  
Apical Surface ◽  
Organic Anion Transporters ◽  
Anion Transporters

ABSTRACT. Recently, a family of multispecific organic anion transporters has been identified, and several isoforms have been reported. However, the physiologic and pharmacologic roles of each isoform, except OAT1, in the transepithelial transport of organic anions in the kidney remain to be elucidated. To address this issue, it is essential to determine the intrarenal distribution and membrane localization of each OAT isoform along the nephron. In this study, the intrarenal distributions of rOAT1, rOAT2, and rOAT3 were investigated by an immunofluorescence method that used frozen rat serial kidney sections. Confocal microscopic analysis showed that immunoreactivity for rOAT1 was detected exclusively in the proximal tubules (S1, S2, S3) in the cortex with basolateral membrane staining. rOAT2 was detected in the apical surface of the tubules in the medullary thick ascending limb of Henle’s loop (MTAL) and cortical and medullary collecting ducts (CD). rOAT3 was localized in the basolateral digitation of the cell membrane in all the segments (S1, S2, and S3) of the proximal tubules, MTAL, cortical TAL, connecting tubules, and cortical and medullary CD. These results on the distribution of each OAT isoform will facilitate the understanding of the role of OATs in the renal processing of organic anions.

Comparative insights into the mechanisms of renal organic anion and cation secretion

1991 ◽  
Vol 261 (6) ◽  
pp. R1329-R1340 ◽  
Author(s):  
J. B. Pritchard ◽  
D. S. Miller
Keyword(s):  
Organic Cation ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Cation Transport ◽  
Organic Anions ◽  
Proton Exchange ◽  
Metabolic Energy ◽  
Organic Cations ◽  
Free Concentration ◽  
Indirect Coupling

Comparative models have played a major role in defining the mechanisms that enable vertebrate proximal tubules to transport organic anions and cations from the peritubular interstitium to the urine. The unique advantages of these models and their contributions to our understanding of organic anion and cation transport mechanisms are summarized here. Recent studies of the organic anion transport system suggest that transport is coupled to metabolic energy via indirect coupling to the sodium gradient. Organic anions enter the cell across the basolateral membrane in exchange for alpha-ketoglutarate (alpha-KG), and the alpha-KG is returned to the interior via Na-alpha-KG cotransport. Indirect coupling to Na has been demonstrated in both isolated membranes and intact renal epithelial cells of species ranging from marine crustaceans to mammals. This mechanism was shown to drive not only cellular accumulation but also secretory transepithelial fluxes of organic anions. Luminal exit of secreted organic anions appears to be carrier mediated but is, at present, poorly understood, with mediated potential-driven efflux and anion exchange-driven efflux implicated in some species. As for organic anions, the renal clearance of some organic cations approaches the renal plasma flow. Although there is considerable variation in the handling of specific substrates between species, the basic properties of organic cation transport include carrier-mediated potential-driven uptake at the basolateral membrane, intracellular sequestration that reduces the free concentration of the cation, and luminal exit by organic cation-proton exchange. Reabsorptive transport is also observed for some organic cations, but its mechanisms and driving forces are not well understood.

Expression of the angiotensinogen gene is synergistically stimulated by 8-BrcAMP and Dex in opossum kidney cells

1995 ◽  
Vol 268 (1) ◽  
pp. R105-R111 ◽  
Author(s):  
M. Ming ◽  
T. T. Wang ◽  
S. Lachance ◽  
A. Delalandre ◽  
S. Carriere ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Dependent Manner ◽  
Fusion Genes ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Angiotensinogen Gene ◽  
Opossum Kidney Cells ◽  
Dependent Protein ◽  
Flanking Region ◽  
Responsive Element

We transiently transfected fusion genes with the 5'-flanking region of the angiotensinogen gene linked to a bacterial chloramphenicol acetyltransferase (CAT) coding sequence as a reporter into opossum kidney (OK) cells. The addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) (10(-3)-10(-7) M) or forskolin (10(-9)-10(-5) M) stimulated the expression of the plasmid pOCAT [angiotensinogen nucleotide (N) -1498/+18] fusion gene in OK cells in a dose-dependent manner. The addition of dexamethasone (Dex) (10(-6) M) further enhanced the stimulatory effect of 8-BrcAMP or forskolin, whereas the addition of (R)-p-adenosine 3',5'-cyclic monophosphorothioate [(Rp)-cAMP[S], an inhibitor of cAMP-dependent protein kinase A, I and II] blocked the stimulatory effect of 8-BrcAMP. Furthermore, the addition of 8-BrcAMP (10(-3) M) or Dex (10(-6) M) or a combination of both stimulated the expression of pOCAT (angiotensinogen N -1138/+18), pOCAT (angiotensinogen N -960/+18), pOCAT (angiotensinogen N -814/+18), and pOCAT (angiotensinogen N -688/+18), but had no effect on the expression of pOCAT (angiotensinogen N -280/+18), pOCAT (angiotensinogen N -198/+18), pOCAT (angiotensinogen N -110/+18), pOCAT (angiotensinogen N -53/+18), and pOCAT (angiotensinogen N -35/+18). To further localize the putative cAMP-responsive element (CRE) in the angiotensinogen gene, we constructed fusion genes by inserting the DNA fragments angiotensinogen N -814 to N -689, angiotensinogen N -814 to N -761, and angiotensinogen N -760 to N -689 of the 5'-flanking region of the angiotensinogen gene upstream of the thymidine kinase (TK) promoter fused to a CAT gene and introduced them into OK cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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