Secretion Of Organic Anions Research Articles

Organic anion secretion in polycystic kidney disease.

This study examined whether organic anion secretion contributes to fluid accumulation in cysts in polycystic kidney disease. Clearance and micropuncture studies were done on young (7 to 16 wk old), mostly male, heterozygous Han:SPRD cystic rats and healthy control littermate rats. Heterozygous Han:SPRD rats manifest a slowly progressive autosomal dominant polycystic kidney disease that closely resembles the human disorder. Left kidney GFR (polyfructosan clearance), in microl/min per 100 g body wt, averaged 331 +/- 36 (SD) in seven healthy rats and 278 +/- 75 in seven cystic rats. The maximal rate of p-aminohippurate (PAH) secretion, in micromol/min per 100 g body wt, averaged 0.94 +/- 0.24 in healthy rats and 0.83 +/- 0.11 in cystic rats. In these young rats, there were no significant differences in GFR or the maximal rate of PAH secretion despite the presence of cystic disease. Using fluorescence microscopy, it was found that 27 of 29 proximal cysts secreted sulfonefluorescein, an organic anion transported by the PAH system. Transmission electron micrographs of superficial cysts that had secreted sulfonefluorescein demonstrated the presence of both normal-appearing and poorly differentiated proximal tubule cells. Segments of superficial proximal convoluted tubules or cysts, isolated by upstream and downstream wax blocks, failed to accumulate fluid when PAH was infused intravenously. With the stationary microperfusion technique, PAH secretion by both normal and cystic nephrons was demonstrated. It is concluded that most proximal cystic epithelia retain the ability to secrete organic anions. Secretion of organic anions, however, does not appear to contribute in any substantial way to fluid accumulation in cysts in the rat kidney.

Disruption of actin organization by cytochalasin D does not impair biliary secretion of organic anions in the rat.

The bile canaliculi of hepatocytes contract spontaneously, and it is hypothesized that this canalicular motility provides a propulsive force for normal intrahepatic bile flow. Cytochalasin disrupts actin polymerization, inhibits contraction, and decreases bile flow. We investigated whether this cholestasis was associated with impaired canalicular secretion. Isolated rat hepatocyte doublets, with and without incubation with 2 mumol/L cytochalasin D (cytD), were superfused, under first-order conditions, to steady state with fluorescein isothiocyanate-labeled glycocholic acid (FITC-GC) and carboxy-4',5'-dimethylfluorescein diacetate (CMFD), which are fluorescent substrates for the bile acid and the nonbile acid organic anion transport pathways, respectively. Fluorescent microscopic images were quantified and the data analyzed by noncompartmental and compartmental kinetic methods. cytD dilated the canalicular spaces fivefold but did not change the proportion of doublets that secreted either probe. Cytochalasin did not affect the mean cellular transit times of FITC-GC (2.8 and 2.5 minutes for control and cytochalasin-treated groups, respectively) and of carboxy-4',5'-dimethylfluorescein (3.8 and 3.7 minutes, respectively). Analysis with a three-compartment model gave estimates of the rate constants for canalicular secretion: 0.21 +/- 0.04 and 0.22 +/- 0.03 min-1 in control and treated cells, respectively, for FITC-GC, and 0.14 +/- 0.01 and 0.16 +/- 0.02 min-1, respectively, for carboxy-dimethylfluorescein. When kinetics are first-order, the canalicular secretion of organic anions is not altered by actin disruptive agents, suggesting that actin filaments do not modulate the function or distribution of these transporters. This suggests that impaired contractility rather than impaired canalicular secretion is the mechanism of cytD-induced cholestasis.

Multiple pathways of organic anion secretion in renal proximal tubule revealed by confocal microscopy.

Previous studies with p-aminohippurate (PAH) and fluorescein (FL) have shown that cellular uptake and tubular secretion of organic anions is driven by indirect coupling to sodium. Here we used killifish proximal tubules and laser-scanning confocal microscopy to study the transport of a larger organic anion, fluorescein-methotrexate (FL-MTX, mol mass 923 Da). When tubules were incubated in medium containing 2 microM FL-MTX, dye accumulated in both cells and tubular lumens. At steady state, luminal fluorescence was 4-5 times higher than cellular fluorescence. Ouabain (0.1 mM) did not affect cellular or luminal fluorescence, and replacement of medium sodium by N-methylglucamine had only a modest effect; preincubation with glutarate had no effect. KCN did not affect cellular uptake but abolished secretion into the lumen. Uptake and secretion of FL-MTX were inhibited by micromolar concentrations of other organic anions (MTX, folate, probenecid, bromocresol green, bromosulfophthalein), but 1 mM PAH had a relatively small effect. FL-MTX secretion into the lumen was inhibited by leukotriene C4, cyclosporine A, and verapamil, none of which affected FL transport. Thus a substantial component of FL-MTX secretion is Na independent and ouabain insensitive. Both the basolateral and luminal steps in the Na-independent pathway differ from those usually associated with FL and PAH secretion.

Renal organic anion secretion: evidence for dopaminergic and adrenergic regulation.

To examine possible regulatory control of renal proximal tubule organic anion secretion, winter flounder (Pleuronectes americanus) proximal tubule primary cultures were mounted in Ussing chambers. Unidirectional fluxes of [2,4-(14)C]dichlorophenoxyacetic acid were determined under short-circuited conditions. Phorbol 12-myristate 13-acetate (1 microM) caused a significant (P < 0.01) inhibition of net 2,4-dichlorophenoxyacetic acid secretion. Preincubation with staurosporine (1 microM) blocked the phorbol 12-myristate 13-acetate-induced decrease in secretion. Neither forskolin (10 microM) nor W-7 (20 microM) had any effect on net transport. Elevation of intracellular calcium activity with either A-23187 or thapsigargin produced a slight, transient decrease in transport. Addition of dopamine (1 microM) to the peritubular side, but not the luminal side, caused a significant (P < 0.01) decrease in net secretion. Both the alpha-adrenergic agonist oxymetazoline (10 microM) and depletion of intracellular Na+ transiently, but significantly (P < 0.05), increased net transport. The data indicate that renal organic anion excretion may be regulated through dopaminergic inhibition and alpha-adrenergic stimulation of net transepithelial secretion.

Renal transport of drugs: specificity and molecular mechanisms.

1. The kidney is principally an excretory organ for drugs and their metabolites and has developed high capacity transport systems to rapidly eliminate the large quantities of foreign compounds delivered to it. 2. There are specific and selective transporters for organic cations and organic anions at the contraluminal and luminal membranes of epithelial cells lining the proximal tubule. 3. For organic anions, the transporter (termed PAH-transporter) is located at the contraluminal cell membrane and uptake is a tertiary active transport process. Important physico-chemical properties for interaction with this transporter are hydrophobicity, ionic charge strength and electron-attracting side groups. 4. For organic cations, there is evidence for one common transport system at the contraluminal membrane and substrate requirements are the degree of hydrophobicity, ionic charge strength and hydrogen bond formation. At the luminal membrane, an electroneutral H+/organic cation exchanger in which the degree of substrate hydrophobicity is critical for interaction and a choline transport system have been described. 5. P-Glycoprotein present in renal brush border membranes transports organic cationic drugs but is dissimilar to the luminal electroneutral H+/organic cation exchanger. 6. Clinically, several drugs have been implicated in causing interactions via these transport systems. For organic anions, probenecid blocks the tubular secretion of other organic anions and this has been used to prolong the duration of action of penicillin-like compounds. For organic cations, cimetidine and trimethoprim are potent inhibitors of the secretion of a number of organic cations, particularly procainamide, which can result in significant clinical toxicity. 7. An understanding of the mechanisms of renal tubular secretion of drugs and their metabolites will allow for the prediction of drug interactions involving renal excretory mechanisms.

The influence of moderate hypoalbuminaemia on the renal metabolism and dynamics of furosemide in the rabbit.

1. The present study aimed to investigate the influence of hypoalbuminaemia on the pharmacokinetics and pharmacodynamics of furosemide. Hypoalbuminaemia was produced by repeated plasmapheresis, to attain plasma albumin concentrations of 21.6 +/- 0.9 g l-1, compared with 33.0 +/- 0.6 g l-1 in controls (P < 0.001). The per cent of bound furosemide in hypoalbuminaemic rabbits (90.8 +/- 0.7%) was lower than that in control rabbits (97.4 +/- 0.5%, P < 0.001). The kinetics of intravenous furosemide (2.5 mg kg-1) were studied in control (n = 6) and hypoalbuminaemic rabbits (n = 6). 2. To assess the effect of hypoalbuminaemia on extrarenal clearance of furosemide, functional anephria was induced by ligating the renal pedicles of 12 rabbits, which were segregated in two groups, with and without hypoalbuminaemia. 3. In the control group, total, urinary and metabolic clearances of furosemide were 11.8 +/- 1.0, 5.0 +/- 0.4 and 6.8 +/- 0.6 ml min-1 kg-1, respectively. Compared with control rabbits, in hypoalbuminaemic rabbits, total clearance of furosemide increased by 40% (P < 0.001), result of the enhancement of furosemide metabolic clearance (C1m) from 5 to 10 ml min-1 kg-1 (P < 0.01). In hypoalbuminaemic rabbits, urinary excretion of furosemide was reduced by 26% (2451 +/- 115 vs 1818 +/- 134 micrograms h-1, P < 0.01). In anephric rabbits, furosemide total clearance was 1.77 +/- 0.12 ml min-1 kg-1, value not affected by hypoalbuminaemia, confirming that the increase in C1m induced by hypoalbuminaemia occurs in the kidneys. 4. Compared with controls, in hypoalbuminaemic rabbits, the rate of urinary excretion (142 +/- 9 vs 74 +/- 8 ml h-1, P < 0.001) and the rate of excretion of sodium (18.6 +/- 0.9 vs 9.9 +/- 0.9 mmol h-1, P < 0.001) were very much reduced. However, the dose-response curves were not different. 5. In conclusion, hypoalbuminaemia is associated with an increase in renal metabolic clearance of furosemide, possibly because of the increase in furosemide unbound concentration, and a decrease in the diuretic/natriuretic effect of furosemide, secondary to a reduction in furosemide tubular secretion. Thus, albumin facilitates the renal secretion of organic anions but not their metabolism.

Renal secretion of organic anions and cations

The renal proximal tubule actively transports charged, potentially toxic xenobiotics from blood to lumen. Basolateral uptake of organic anions is indirectly coupled to the sodium gradient through Na-dicarboxylate cotransport and dicarboxylate-organic anion exchange. Upon entry, a significant fraction of intracellular organic anion is sequestered within vesicles. Disruption of the cellular microtubular network can lead to both diminished vesicular movement and reduced transepithelial secretion. Luminal efflux of organic anions is energetically downhill, but carrier mediated. Both anion exchange and potential driven transport are present, but neither completely accounts for transport from cell to lumen. For organic cations, basolateral entry is downhill via potential driven facilitated diffusion. Intracellular sequestration of organic cations in vesicles is substantial, but its role in secretion is uncertain. Multiple carriers are available to drive organic cations uphill into the tubular lumen. The classical system indirectly taps the energy of the luminal Na gradient to drive organic cation efflux via Na(+)-H+ and proton-organic cation exchange. In addition, the multidrug resistance ATPase can pump organic cations into the tubular lumen. Thus, although much detailed information has been added over the last 50 years, it is not yet possible to provide a detailed, quantitative understanding of these important excretory systems.

Relationship between biliary lipid and protoporphyrin secretion; potential role of mdr2 P-glycoprotein in hepatobiliary organic anion transport

Background/Aims: Erythropoietic protoporphyria, caused by ferrochelatase deficiency, leads to protoporphyrin accumulation in the liver. Therapeutic attempts to increase the secretion of ths hydrophobic organic anion into bile are hampered by a lack of understanding of the secretory mechanism(s) involved. We have investigated biliary secretion of protoporphyrin in rats and mice, primarily targeted on the role of biliary lipids in this process. Methods: Gel permeation chromatography was applied to investigate the association of porphyrins with lipid fractions in bile. Secretion of endogenous porphyrins was studied in (GY mutant) rats and mdr2 P-glycoprotein deficient mice, under conditions of widely varying biliary lipid secretion rates. Results: Gel permeation chromatography revealed that, in native human and rat bile, protoporphyrin associated with cholesterol/phospholipid vesicles upon elution with bile salt-free buffer. In contrast, the more hydrophilic corporpyrin isomers I and III were found only in bile salt/organic anion hybrid particles and smaller aggregates. Interruption of the enterohepatic circulation in normal Wistar rats resulted in a parallel decrease of endogenous protoporphyrin-, lipid-, and bile salt secretion, but did not alter the secretion of coproporphyrin I and III. Uncoupling of lipid- from bile salt secretion by sulfated taurolithocholate resulted in imparied secretion into bile of protoporpyrin only. Conversely, secretion of coproporphyrin I and III, but not that of protoporphyrin, was impaired in mutant Gronigen Yellow rats with defective ATP-dependent hepatobiliary organic anions transport. In mice homozygous for a disruption of the mdr2 P-glycoprotein gene, resulting in complete absence of phospholipids in bile and strongly reduced cholesterol output, secretion of protoporphyrin was reduced by 90%, whereas that of coproporpyrin I and III was affected to a much lesser extent. Conclusions: Our data demonstrate a close association between protoporphyrin and lipid secretion into bile, indicating that these processes are, at least functionally coupled. This finding implicates a role of mdr2 P-glycoprotein activity in hepatobiliary removal of the hydrophobic organic anion protoporphyrin. Hence, it may be speculated that protoporphyrin secretion can be influenced by drugs, diet or other means that affect biliary lipid secretion.

Effect of diethylmaleate on bile secretion and ultrastructural appearance of hepatocytes in normal rats and mutant rats with defective organic anion secretion.

Diethylmaleate is an organic anion secreted into bile as a glutathione conjugate. Its transport by the hepatocyte is associated with dilatation of the Golgi apparatus and the appearance of small vesicles in the pericanalicular area. It has been speculated that the Golgi apparatus could play a role in the intracellular transport and/or the biliary canalicular secretion of diethylmaleate. The purpose of this work was to determine whether the alterations in the Golgi apparatus and the pericanalicular vesicles could mediate the canalicular secretion of diethylmaleate. Diethylmaleate biliary secretion and diethylmaleate-induced bile flow were measured in Sprague-Dawley rats, and in TR- rats which have an inherited defect in the excretion into bile of organic anions, including glutathione conjugates. Livers of both Sprague-Dawley and TR-rats were examined by electron microscopy, to characterize the changes in intracellular organelles. In Sprague-Dawley rats, as previously described, diethylmaleate administration was associated with an increase in bile flow, which was parallel in time to the secretion into bile of diethylmaleate conjugates. Electron microscopic examination of the liver after diethylmaleate administration showed dilatation of the Golgi saccules. In contrast, in TR- rats, the increase in bile flow and the secretion of diethylmaleate conjugated were nearly absent. Nevertheless, electron microscopic examination showed a dilatation of the Golgi saccules similar to that observed in Sprague-Dawley rats. TR- rats, in addition to the changes in the Golgi apparatus, had marked dilatation of the endoplasmic reticulum. These results show that biliary secretion of diethylmaleate conjugates was severely impaired in TR- rats, in spite of a dilatation of the Golgi apparatus and of the endoplasmic reticulum. We conclude that it is unlikely that the alterations in the Golgi apparatus (and the endoplasmic reticulum) induced by diethylmaleate play a role in the canalicular secretion of diethylmaleate. We do not exclude the possibility that these organelles could play a role in intracellular transport of this compound. Alternatively, these alterations could be due to a "toxic" effect of diethylmaleate accumulation in hepatocytes.

Secretion of Organic Anions by Hepatocytes: Involvement of Homologues of the Multidrug Resistance Protein

The canalicular multispecific organic anion transporter (cMOAT) is one of at least four ATP-dependent transport systems identified so far in the canalicular membrane domain. Using mutant rat strains that lack organic anion secretion, the substrate specificity of cMOAT has been characterized. cMOAT appears to be responsible for the elimination of amphipathic anionic conjugates formed by phase II conjugation. Very recently it has been shown that the hepatocyte-specific homologue (MRP2) of the multidrug resistance protein (MRP1) functions as cMOAT in the canalicular membrane, whereas MRP1 is present in the lateral membrane only at very low levels in quiescent cells. The defective excretion of organic anions in TR- rate livers is caused by the total absence of mrp2 due to a deletion of one nucleotide in the mrp2 gene. In conclusion, the liver-specific mrp2/MRP2, a new member of the ATP-binding cassette superfamily, appears to function as cMOAT in the hepatobiliary secretion of organic anions.

Hepatocellular transport: role of ATP-binding cassette proteins.

The interest of mammalian biologists in ATP-binding cassette (ABC) proteins is relatively recent. However, ABC proteins are widespread in distribution and have long been known to play an important transport role in prokaryotes. The review includes a brief overview of the structure, regulation, and varied functions of ABC proteins in different cell types as well as a synopsis of the emerging role of ABC proteins in human biology and disease. The review then focuses on the established (canalicular secretion of organic cations by the multidrug resistance, or MDR 1, gene product; ductular secretion of fluid and electrolytes mediated by CFTR), probable (biliary phospholipid secretion by the MDR 2 gene product; secretion of non-bile acid organic anions by the multidrug resistance protein, or MRP), and possible (bile acid secretion; biliary secretion of the signaling molecule, ATP; hormone transport) roles of known and novel ABC proteins in hepatobiliary secretion.

Intracellular Compartmentation of Organic Anions and Cations during Renal Secretion

Although plasma membrane events mediating organic anion and cation secretion have been intensively investigated, intracellular events associated with transcellular fluxes of secreted solutes have rece

Inhibition of renal xenobiotic excretion by tetrachloroguaiacol: Mechanism and possible consequences

AbstractThe weak acid tetrachloroguaiacol (teCG; pKa 6.0) is the major chlorinated phenol produced during chlorine bleaching of wood pulp. Upon its release into the environment, it is persistent and markedly bioaccumulated (100‐ to 1,000‐fold). Within the organism (pH ∼ 7.4), it should be &gt;95% ionized, making it a likely inhibitor of renal organic anion transport. Its effects on both passive membrane permeability and mediated transport processes were determined in basolateral (BLM) and luminal membrane (BBM) vesicles isolated from the rat kidney cortex. Vesicular solute uptake was measured by the rapid filtration technique at pH 7.5. Passive membrane permeability (assessed by mannitol uptake) was altered only at very high concentrations of teCG (≥ 1,000 μM). In contrast, transport of the model organic anion p‐aminohippurate (PAH) by BLM vesicles was significantly inhibited by as little as 5 μM (1.3 mg/L) teCG and totally abolished by 200 μM. Inhibition was competitive (K1 = 31.3 μM). Decreasing assay pH diminished inhibition of the PAH uptake, reflecting the reduced concentration of ionized teCG. Other membrane effects were seen at higher teCG concentrations (&gt; 100 μM). The BLM sodium/glutarate cotransport was modestly, but significantly, stimulated by 100 to 500 μM teCG, whereas BBM sodium/glucose cotransport was inhibited by similar teCG concentrations. Thus, teCG has multiple effects on renal membrane function. However, at environmental levels (0.1 to 30 μM in plasma), its principal membrane effects would be related to changes in organic anion secretion.

The effect of cholecystokinin-octapeptide on the hepatobiliary dysfunction caused by total parenteral nutrition

Purpose: Patients on total parenteral nutrition (TPN) commonly have hepatobiliary dysfunction. Interruption of the enterohepatic circulation (EHC) and gallbladder stasis are part of the pathogenesis. Cholecystokinin-octapeptide (CCK-OP), by emptying the gallbaldder, stimulates the EHC. This study was performed to determine whether daily CCK-OP infusions can ameliorate the hepatobiliary dysfunction caused by TPN. Methods: Rabbits maintained on a standard TPN for 12 days were divided into two groups. One group (n = 6) received daily intravenous doses of CCK-OP, and the other (n = 13) received TPN only. A lab-chow-fed (LCF) group (n = 8) served as controls. The authors studied bile flow and bile acid secretion rates, sulfobromophthalein (BSP) secretion, gallbladder emptying in response to CCK-OP, and liver histology. Results: The LCF group had a bile flow of 82.3 μL/kg/min; that for the TPN-only group was 45.7 μL/kg/min ( P < .001). The daily CCK-OP group did not improve more than the TPN-only group, with a bile flow of 45.8 μL/kg/min ( P = NS). Bile acid secretion was 0.64 μmol/kg/min for the LCF group, 0.46 for the TPN-only group ( P = NS), and 0.46 for the daily CCK-OP group ( P = NS). TPN impaired the ability of the gallbladder to empty, and this was restored with daily CCK-OP. In the LCF group, the mean BSP secretion was 81.7% of a 5-mg/kg bolus within 60 minutes, compared with 72.5% in the daily CCK-OP group ( P = NS) and 63.5% in the TPN-only group ( P < .01). Histological examination of the liver showed that daily CCK-OP produced less periportal inflammation and fibrosis, although all TPN groups had hepatocyte damage in the centrilobular area. Conclusion: Stimulation of the EHC with daily CCK-OP infusions during TPN decreased periportal inflammation and fibrosis, maintained gallbladder emptying capacity, and improved organic anion (BSP) secretion, although bile flow and bile acid secretion were not improved, and hepatocyte damage persisted.

Transport Mechanisms of Drugs in Renal Tubular Epithelial Cells.

Renal handling of drugs is a complex phenomenon involving glomerular filtration, tubular secretion and tubular reabsorption. Tubular secretion of organic cations and anions is viewed as a means of eliminating exogenous, toxic compounds such as drugs from the body. The sequence of movement of organic ions is transport across basolateral membrane, diffusion in the cells, followed by efflux from the cell across brush-border membrane. The luminal, intracellular and peritubular compartments are separated by two distinct membranes, luminal brush-border and contraluminal basolateral membrane. To characterize the specific membrane events underlying the transcellular transport of organic ions, we have used brush-border and basolateral membrane vesicles isolated from renal cortex. In addition, we have established the in vitro model system to study the secretory process of organic ions in intact cells using cultured renal cells.This article concerns the transport mechanisms of tetraethylammonium (organic cation) in renal brush-border and basolateral membranes, and characteristics of transcellular transport of p-aminohippurate (organic anion) in OK cells, an established cell line derived from the American opossum kidney. The studies with isolated plasma membrane vesicles and cultured renal cells would provide new insights into our understanding of renal tubular transport mechanisms of drugs.

Heat-shock-stimulated transepithelial daunomycin secretion by flounder renal proximal tubule primary cultures.

Primary monolayer cultures of winter flounder renal proximal tubule epithelium mounted in Ussing chambers were used to characterize transepithelial transport of daunomycin (Dau). Control tissues performed active net secretion of Dau (0.064 +/- 0.027 nmol.cm-2.h-1). Mild heat shock (5 degrees C elevation for 6-8 h followed by return to normal temperature) almost doubled Dau secretion (0.114 +/- 0.026 nmol.cm-2.h-1). This response was inhibited approximately 40% by addition of the protein synthesis inhibitor, cycloheximide. Dau secretion was inhibited by verapamil, vinblastine, cyclosporin A, and to a lesser degree by the organic cation, tetraethylammonium. In addition, tetraethylammonium secretion was inhibited by vinblastine. Dau secretion was not inhibited by the organic anion, p-aminohippurate, and p-aminohippurate secretion was not inhibited by vinblastine. The transepithelial reabsorptive flux of Dau and the electrical characteristics of the tissues, including rheogenic glucose transport, were unaffected by any of the above treatments. Reaction of tissues with a monoclonal antibody to P-glycoprotein (C219) revealed the presence of this transporter on only apical microvilli. The data indicate that flounder possess an active mechanism for the renal excretion of Dau that is stimulated by mild heat shock. This mechanism is distinct from organic anion, but not organic cation, transport and has characteristics consistent with transport by an apical P-glycoprotein.

Nocodazole inhibition of organic anion secretion in teleost renal proximal tubules

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.

Confocal microscopic analysis of fluorescein compartmentation within crab urinary bladder cells

Fluorescein (FL), a fluorescent organic anion, is compartmentalized in cells of organic anion-secreting epithelia, e.g., OK cells, teleost proximal tubule, and crab (Cancer borealis) urinary bladder, a proximal tubule analogue. To further examine the processes involved, FL uptake and distribution were studied in C. borealis urinary bladder cells using epifluorescence and laser confocal microscopy combined with video-image analysis. Intracellular FL was about evenly split between diffuse and punctate compartments after in vitro or in vivo loading. Treatments that affected FL transport into cells (incubation with p-aminohippuric acid or glutarate) altered the FL content of both compartments. However, nocodazole, a microtubule inhibitor, did not affect diffuse FL but significantly reduced punctate FL. Finally, confocal analysis indicated that individual sites of punctate FL accumulation moved in the secretory direction at 0.83 micron/min. Nocodazole nearly abolished this movement and significantly reduced transepithelial organic anion secretion. Thus, in crab urinary bladder, a substantial fraction of total cellular FL is sequestered in vesicles, and these vesicles move in the secretory direction, by a microtubule-dependent process.

Epithelial polarity and differentiation in polycystic kidney disease.

Renal cysts are central pathological features in a number of human congenital and acquired diseases, and produce significant morbidity and mortality. This review describes our laboratory's efforts to identify specific alterations in epithelial cell polarity and differentiation associated with renal tubular cyst formation and progressive enlargement. Studies in a murine model of human autosomal recessive polycystic kidney disease, the C57BL/6J cpk/cpk (CPK) mouse have demonstrated quantitative (increased activity) and qualitative (apical membrane distribution) alterations in Na+,K(+)-adenosine triphosphatase activity that mediate tubular cyst formation. Proximal tubular cyst formation in CPK kidneys is characterized by increased activity of a basolateral Na+,K(+)-ATPase, which drives organic anion secretion and consequent tubular fluid secretion. In contrast, collecting tubule cyst formation is characterized by increased apical membrane Na+,K(+)-ATPase expression, which may be a marker of the relatively undifferentiated phenotype of cyst lining cells. If such apically expressed enzyme is active, it may have pathogenic import in collecting tubule cyst formation and enlargement by mediating net basal to apical vectorial solute and fluid transport.

Mechanisms mediating renal secretion of organic anions and cations

Mechanisms mediating renal secretion of organic anions and cations