1Division of Nephrology, Department of Medicine, Indiana University School of Medicine, and The Indiana Center for Biological Microscopy, Indianapolis; 3Department of Chemistry, Purdue University, West Lafayette, Indiana 47907; and 2The Roudebush Veterans Affairs Medical Center, Indianapolis 46202
Submitted 6 January 2004 ; accepted in final form 1 April 2004
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ABSTRACT |
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proximal tubule cell
Two folate transport proteins have been identified in kidney tissue: the reduced folate carrier (RFC) and the folate receptor (FR) (27, 33, 49, 51). The RFC is found on the basolateral surface of kidney proximal tubule cells (55), while FRs are found on the apical brush-border membrane (3, 12, 22, 24, 49). Folate retention and transport back into blood vessels is known to be saturable (2), involving rapid uptake followed by slow transcytosis (50). Folate gold particles have been localized within coated pits and coated vesicles (2). FRs also have been colocalized to compartments of the clathrin-coated pit endocytic pathway (2, 3, 9, 10, 14, 22, 25, 32, 3437, 41, 48, 50, 52). Although these studies are illuminating, investigators have not yet been able to continuously track the intracellular path taken by folic acid after its initial binding to the apical brush border in living animals.
Recent advances in two-photon fluorescence microscopy have sparked an interest in intravital imaging (see references cited in Ref. 13). Briefly, the longer, far-red wavelengths used for double harmonic fluorescence excitation provide deeper optical penetration into biological specimens with less light scatter while illuminating only the sample plane in focus. This also results in markedly reduced phototoxicity when viewing a through-focus volume (13).
In the present studies, we used fluorescein and Texas red conjugates of folate (FF and FTR, respectively) in conjunction with two-photon microscopy to examine the uptake of folic acid into kidney proximal tubules. Our data demonstrate folate binding at the apical surface with subsequent uptake via endocytosis as well as the dynamic movement of FTR into and across the intact kidney.
Electron microscopic studies in which photoconversion techniques were used (17, 39, 45, 53, 54) revealed fusion of these small vesicles with the basolateral membrane domain, documenting transcytosis. Studies in which the microtubule depolymerizing agent colchicine was used revealed reduced apical uptake with no trafficking of endocytic vesicles below the subapical region of proximal tubules. In addition, long after the initial infusion of FTR and clearance from the proximal tubule lumen, staining persisted at the apical surface that was far too intense to be attributed solely to receptor binding. This observation may represent a novel mechanism by which proximal tubule cells sequester and retain folate for subsequent internalization, thereby preventing loss by excretion.
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MATERIALS AND METHODS |
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Synthesis of folate conjugates.
Standard Fmoc peptide chemistry was used to synthesize a folate peptide linked to Texas red attached to the -COOH terminal of folic acid. The sequence Gly-Lys-(
)Glu-pteroic acid was constructed by Fmoc chemistry with HBTU and N-hydroxybenzotriazole as the activating agents along with diisopropyethylamine as the base and 20% piperidine in dimethylformamide (DMF) for deprotection of the Fmoc groups. Fmoc-protected lysine containing a 4-methyltrityl protecting group on the
-amine was linked to Fmoc-protected glycine attached to a Wang resin. An
-t-Boc-protected N-
-Fmoc glutamic acid was then linked to the peptide to provide a
-linked conjugate on folate after N10-trifluoroacetylpteroic acid was attached to the peptide. The methoxytrityl protecting group on the
-amine of lysine was removed with 1% trifluoroacetic acid in dichloromethane to allow attachment of Texas red. Texas red N-hydroxysuccinimide (Molecular Probes, Eugene, OR) in DMF was reacted overnight with the peptide and then washed thoroughly from the peptide resin beads. The FTR peptide was then cleaved from the resin with 95% trifluoroacetic acid-2.5% water-2.5% triisopropylsilane solution. Diethyl ether was used to precipitate the product, and the precipitant was collected by centrifugation. The product was then washed twice with diethyl ether and dried under vacuum overnight. The product was then analyzed and confirmed by mass spectroscopic analysis ([M] calculated, 1,423; found, 1,422). To remove the N10-trifluoracetyl protecting group, the product was dissolved in 5 ml of water containing 0.5 ml of 10% ammonium hydroxide and stirred for 30 min at room temperature. The product was then precipitated with combined isopropanol and ether, and the precipitant was collected by centrifugation. The product was then added to a G-10 Sephadex gel filtration column (1.5 x 15 cm) with water used as the eluent. The product peaks were collected and lyophilized.
Animal model.
Male Sprague-Dawley or Munich-Wistar rats initially weighing between 200 and 250 g were placed on a folate-deficient diet for 24 wk before the studies. For some studies, adult male balb/c mice weighing 20 g were placed on a folate-deficient diet 24 wk before imaging. The rats and mice were anesthetized with pentobarbital sodium (55 mg/kg body wt; Besse Scientific, Louisville, KY) (13) before surgery.
Surgical procedures. The anesthetized animal's midsection was shaved completely, and a small, 10- to 15-mm lateral incision was made dorsally. The kidney was gently externalized, and fluorescent probes were infused by one of three methods: 1) a femoral venous line inserted into the left leg, 2) a butterfly catheter inserted into the tail vein, or 3) intraperitoneal (IP) injection. Regardless of delivery method, the eventual intracellular localization was identical; however, IP injection provided a more protracted delivery of the probes with greater clearance times, because absorption into the bloodstream was significantly prolonged.
Fluorescent probes.
Fluorescent probes were injected in a bolus with normal saline used as a carrier as described in Surgical procedures in a total combined volume not exceeding 1 ml. In rat studies, 600 µg (200 µg for mice) of the dye Hoechst 33342 (Molecular Probes) was used for nuclear staining (13) and was injected 510 min before FTR injection to allow time for incorporation. Approximately 2 mg of a small, 3,000 mol wt fluorescent dextran conjugated to the pH-insensitive dye Alexa 488 (Molecular Probes) was sometimes injected to localize the lysosomes (13). A 10,000 mol wt rhodamine dextran and a 500,000 mol wt amino dextran conjugated to rhodamine (Molecular Probes) were used to label the proximal tubules and microvasculature, respectively, in the FF studies. In rat studies,
200 µg (80 µg for mice) of FTR or FF were injected.
Lysosomal localization, colchicine treatment, and nonfluorescent folate competition experiments.
The 3,000 mol wt Alexa 488 dextran mentioned above was injected 1.5 h before FTR infusion to fluorescently label the lysosomes (13). This was done to ensure that the early endosomal compartment would be free of all traces of the dextran before FTR was introduced. To further characterize the abundance of FTR localization to the apical brush border in proximal tubule cells, rats were first labeled with dextran as described and then infused with a 10-fold excess of folic acid (Sigma) in normal saline, pH 7.0, followed by FTR infusion 10 min later. For colchicine treatment studies, 3.2 mg/kg body wt of colchicine was injected intraperitoneally into balb/c mice 12 h before imaging (44).
Microscopy. A Bio-Rad MRC-1024 two-photon laser scanning microscope (Hercules, CA) mounted on a Nikon Diaphot inverted stage platform (Fryer, Huntley, IL) with a Ti:Sapphire laser (Spectra-Physics, Franklin, MA) was used. Acquisition parameters and placement of the rat on the microscope stage were performed as previously described (13). All images were collected with the use of a x60 water-immersion objective with a numerical aperture of 1.2. A wavelength of 800 nm was used to excite the mixture of fluorescent probes.
Image processing and volume rendering. Images were processed, and three-dimensional projections were generated from through-focus data sets using Voxx rendering software (a proprietary software program developed at the Indiana Center for Biological Microscopy facility, available as freeware) or Metamorph (Universal Imaging, West Chester, PA) as previously described (13).
Electron microscopy. Approximately 1 h after FTR infusion, the kidney was excised and small pieces were cut and fixed in 4% freshly thawed paraformaldehyde in PBS overnight. The specimens were then washed briefly in PBS, and 100-µm-thick vibratome sections were cut from the outer cortex. The thin sections were photoconverted and processed routinely for electron microscopy as previously described (46, 53).
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RESULTS |
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DISCUSSION |
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While these studies are very illuminating, the previously available technology did not permit continuous observation of the pathway under in vivo physiological conditions. Because folate gold nanoparticles and anti-folate receptor antibodies are multivalent, and because multimerization of folate receptors by antibodies has been shown to affect the internalization itinerary (34), data from these studies may not reflect the intracellular pathway taken by monomeric folate conjugates after internalization (2, 3). Although studies with [3H]folate uptake into kidney proximal tubules in vivo followed by radiography allow a more accurate assessment of the internalization pathway for folic acid (22), they do not allow continuous tracking of folic acid binding, endocytosis, and intracellular transport, which is possible with in vivo two-photon microscopy.
In the present study, several important aspects of intracellular folate handling are described. First, internalization occurs rapidly via endocytosis, with the bulk of the compound directed to the lysosomal pool. Second, strong evidence from electron and fluorescence microscopic data supports a mechanism for transcytosis directly from the apical to the basal membrane. Accumulation of FTR-containing vesicles at the subapical region without subsequent migration to the basal pole after colchicine treatment further supports evidence for transcytosis. Although vesicles derived from both clathrin-coated pits and caveolae use microtubules for intracellular movement, the lack of caveolin expression at the apical surface of proximal tubules (7) suggests that this phenomenon occurs via the former.
Lack of colocalization between dextrans and FTR in these discrete basal vesicles suggests that they did not originate from lysosomes. Also, during the initial infusion of FTR, movement into the peritubular space was seen. FTR quickly returned to the plasma volume, because no trace of residual FTR was seen in these areas after 4 min. The appearance of the small basal vesicles occurred too long after FTR infusion to be accounted for by the transient phenomenon of leakage into the peritubular space.
During these experiments, at no time was there evidence of FTR freely released into the cytosol of proximal tubule cells. Previous publications have noted this with the use of radiolabeled forms of folate when different cellular fractions were examined (22). When using such procedures, the risk of rupturing compartments containing the marker and contaminating other fractions raises concerns. Lack of cytosolic localization further bolsters the observation that internalization occurs solely though endocytosis and not likely through a surface channel. One limitation of this study is the inability to attain quantitative information, particularly for the fraction that is transcytosed. Continued accumulation of FTR within the lysosomes quickly creates a highly fluorescent intracellular compartment. Vesicles destined for transcytosis near the basal membranes conversely remain relatively dim. In addition, defining a transcytotic vesicle simply by close proximity to the basal membrane presents another pitfall that could lead to an overestimation, because not all vesicles at the basal pole will fuse. Finally, once transcytosis occurs, the dim, compartmentalized fluorescence is immediately lost as the contents are diluted with fluid in the peritubular space. Studies in which [3H]folate was used in isolated, microperfused rabbit proximal tubules reported transcytosis of 5% of the perfused amount (2). Barring species differences between rabbit and rat, this established value would likely be similar because those studies also used an intact tubule.
In conclusion, this study exploits newly developed techniques in live animal imaging to study the fate of a fluorescent analog of folate after internalization by proximal tubule cells. Taking our present results together with previous data, we surmise that the bulk of folate endocytosed by the cells trafficks directly to the lysosomes, where it is retained, while the receptor recycles back to the surface membrane. A small pool of folate is transcytosed directly across the cell to the basal membrane, with no evidence of cytosolic distribution throughout the length of the study. This result is contraindicative of the fate of folate in neoplastic cells, where cytosolic release in tumor cells is readily observed (40, 54). This underlying difference may play a pivotal role in the toxicity of folate drug conjugates to tumor cells and the absence of toxicity of the same conjugates to the kidney.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supplementary Material to this article (Movies 1 and 2) is available online at http://ajpcell.physiology.org/cgi/content/full/00006.2004/DC1.
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