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Renal clearance of absorbed intact GFP in the frog and rat intestine
Comparative Biochemistry and Physiology, Part A 147 (2007) 1067 – 1073 www.elsevier.com/locate/cbpa
Renal clearance of absorbed intact GFP in the frog and rat intestine E.V. Seliverstova, M.V. Burmakin, Yu.V. Natochin ⁎ Laboratory of Renal Physiology, Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia Received 26 January 2007; received in revised form 12 March 2007; accepted 13 March 2007 Available online 20 March 2007
Abstract Intestine absorption of intact green fluorescent protein (GFP) and its following accumulation in the renal proximal tubule cells after its intragastric administration have been established by confocal microscopy in the rat and frog. Reabsorbed GFP was revealed in the endosomes and lysosomes of the proximal tubule cells by the methods of GFP photooxidation and immunofluorescent microscopy. The GFP intestine absorption rate and GFP accumulation in the kidney were significantly higher in the frog than in the rat. No specific fluorescence was revealed in the liver and colon cells after the GFP intragastric administration. The data obtained indicate the ability of the small intestine in the frog and rat to absorb intact proteins and an important role of the kidney in exogenous protein metabolism. © 2007 Elsevier Inc. All rights reserved. Keywords: Green fluorescent protein (GFP); Protein absorption; Intestine; Kidney; Protein reabsorption; Rats; Frogs
1. Introduction According to the classic scheme of digestion, proteins are hydrolyzed in the intestine to amino acids, di- and tripeptides that subsequently are absorbed into the blood (Adibi, 1996; Daniel, 2004). However, there are data that some polypeptides (Borges et al., 2002; Roberts et al., 1999; Wheeler et al., 2002; Natochin et al., 2003) and proteins (Dickinson et al., 1999) also can be absorbed without destruction. Nowadays, there are a few data as to how widely the phenomenon of intact protein and polypeptide absorption is spread in the animal kingdom. Use of proteins with radioactive label or protein-bound fluorochrome does not provide a strong answer to the posed question, as there can be doubts whether protein structure is conserved during intestine absorption. To overcome these difficulties, we have suggested another approach — study of absorption in the small intestine of a fluorescent protein. An example of such a protein is green fluorescent protein (GFP), which is characterized by a fluorescence of molecules with only a preserved tertiary structure (Tsien, 1998) and by a high resistance to proteolytic enzymes (Crameri et al., 1996). Detection of such a protein not only in the intestine lumen, but also within enterocytes during ⁎ Corresponding author. E-mail address: [email protected] (Y.V. Natochin). 1095-6433/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2007.03.019
the absorption would allow us to claim that the molecule is absorbed without hydrolysis. In experiments on rats, we have shown that yellow fluorescent protein can be absorbed in the intestine without hydrolysis (Burmakin et al., 2005). It was of interest to use GFP to elucidate a possibility of its absorption from the rat and frog small intestine into the blood, to analyze its further fate in the body, and to find out whether it is accumulated in the liver or kidney after absorption. The study of this issue has become the goal of the present work. 2. Materials and methods GFP (27 kDa) was obtained from the Laboratory of Protein Synthesis Mechanisms (Institute of Protein, Russian Academy of Sciences, Pushchino-on-Oka, Russia). Experiments were carried out on 4–6-month old adult female Wistar rats Rattus norvegicus (150–200 g BW) and on winter male brown frogs Rana temporaria L. (40–50 g BW) kept in water reservoirs at 4 °C. In in vivo experiments, the rats and frogs were administered per os via a rubber probe with 0.1 ml of GFP solution (0.34 μg/ml in 0.01 M PBS-buffer, pH 7.3) per 150 g of BW. In control experiments, the same volume of PBS without GFP was administered to the animals. After 20, 120, and 300 min of the GFP administration, rats were decapitated under a light ether anesthesia, while in frogs, the spinal cord was
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destroyed with a thin needle; then the abdominal cavity of the animals was opened and a 2-cm long segment of the small intestine and a fragment of the renal tissue were dissected out. Specimens were fixed for 2–3 h in 4% paraformaldehyde in 0.05 M PBS (pH 7.3). After washing with PBS (2 × 15 min), the tissue pieces were cryoprotected overnight at 4 °C in PBS containing 30% sucrose. Specimens then were embedded in OCT compound (Bright, UK) and frozen. Frozen sections of 5– 10 μm thickness were cut using a Cryostat CM 1510 (Leica,
Germany), placed on glass slides and air-dried at RT. Dry sections were covered with cover glass slips using ImmunoFluore Mounting Medium (MP Biomedicals, Inc, Germany). Specimens were viewed and analyzed using a DM R confocal laser scanning unit coupled to a Leica TSC SL microscope (Leica, Germany). Immunohistochemistry was performed on frozen kidneys sections by indirect immunofluorescence using antibodies against GFP and lysosome membrane (LAMP1). After fixation
Fig. 1. Fluorescence in enterocytes (a, b) 3 min after GFP administration and in the proximal tubules cells (c, d) 2 h after GFP administration into the small intestine lumen. Higher fluorescence intensity is evident in intestine and kidney in frogs (Rana temporaria) (b, d) compared to rats (Rattus norvegicus) (a, c). No specific fluorescence is revealed in the goblet cells (a, asterisk), in the rat liver cells after GFP administration (e) and in the frog kidney epithelial cells without GFP administration (f). Scale bar — 10 μm.
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(as described above), the tissue pieces were incubated overnight at 4 °C in 0.05 M PBS containing 0.15 M glycine, cryoprotected and frozen. Cryostat sections were placed on coverslips coated with poly-L-lysine (ISN Biomedicals, USA). Sections were rinsed in PBS and then blocked in blocking solution (3% BSA/ 0.2% Triton X-100 in 0.05 M PBS) to unmask antigenic sites for 30 min. The sections were incubated overnight at 4 °C or for 2 h at RT with primary antibody: mouse anti-GFP monoclonal antibody (Clone GFP-20) (Chemicon Int., USA) and mouse anti-Lamp1 monoclonal antibody (Calbiochem, USA). The antibodies were diluted 1:100 in incubation medium (0.1% BSA/0.2% Triton-X-100 in PBS). After this incubation, sections were washed three times with 0.1% BSA in PBS over the course of 1 h. For fluorescent visualization of bound primary antibodies (anti-LAMP1 and anti-GFP), sections were incubated with secondary Cy3-conjugated anti-mouse antibody (1:1000) (Sigma, USA). As the negative control, the primary antibody was either omitted from the incubation solution or used nonspecific antibody. Specimens were viewed and analyzed using the same confocal microscope. Electronic images were captured and edited in Adobe Photoshop (Adobe System Inc., Mountain View, California, USA). For the photooxidation procedure (Grabenbauer et al., 2005), specimens were sliced and fixed for 2–3 h in 2% paraformaldehyde in 0.05 M PBS (pH 7.3). After washing 3× with PBS, we blocked samples with 100 mM glycine and 100 mM potassium cyanide in PBS for 1 h. For photoconversion, we washed samples in PBS and then incubated them in a freshly prepared ice-cold solution of 1.5 mg/ml 3,3′-DAB in PBS (Sigma, St. Louis, USA). For bleaching, samples were illuminated with appropriate filter settings for the GFP (excitation filter BP) using a 100-W mercury lamp. Photobleaching (photooxidation) was stopped when the green fluorescence was faded and the cytosolic background staining (brownish reaction product) was occurred (in 20–30 min). After photoconversion, samples were treated in the electron microscopy procedure. Small pieces of rat intestine and kidney were fixed for electron microscopy by immersion in 2% paraformaldehyde/ 2.5% glutaraldehyde in 0.1 M Na cacodylate buffer, pH 7.3. Samples were washed for 30 min with the same buffer and further treated with 1% OsO4 in 0.1 M cacodylate. Specimens were dehydrated and embedded in an epoxy resin/araldite mixture according to routine procedures. Ultrathin sections were obtained using a Leica Ultracut UCT (Leica, Germany), collected on cupper grids and contrasted with uranyl acetate and lead citrate. Specimens were examined at 60 kV in a LEO 910 electron microscope (LEO Electron Microscopy Group, Germany). For the analysis of the images obtained using the confocal microscope, the computer program Image Tool III was used. The intensity of the fluorescence was measured in equal areas of the compared sites of the small intestine epithelium or proximal nephron segments. The intensity value was expressed in arbitrary units, their values being calculated as the ratio of fluorescent pixels to non-fluorescent pixels within this area (the scale was from 0 to 256). To study the distribution of
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fluorescence along the length of the gut villus, areas of epithelium (100 images in each experiment) from the upper third of the intestinal villi and the crypt zone were analyzed in various histological sections. To evaluate the intensity of fluorescence, the epithelial cell was measured as a whole, including the brush border. In all experiments, to determine the value of specific GFP fluorescence, the background fluorescence (autofluorescence) of control experiments without GFP administration was subtracted from the total fluorescence values in experiments with the administration of this protein. Unpaired t-test was used to estimate the statistical significance of differences; all data are presented as means ± SE. The threshold of significance was p b 0.05. 3. Results 3.1. Absorption of GFP in the small intestine After 3 min of GFP intragastric administration, specific fluorescence within the rat enterocytes was observed (Fig. 1a), which differed from the autofluorescence of epithelial cells in the control. The intensity of the GFP fluorescence rose depending on the time period after GFP administration. Fluorescence reached a maximum at 21 min and then decreased gradually to the background level (Fig. 2). Specific GFP fluorescence was revealed in enterocytes 3 min after GFP administration into the frog small intestine lumen, with its maximal fluorescence being observed 15 min after GFP administration (Fig. 1b); by 33 min, the fluorescence intensity of the epithelial layer of the frog small intestine decreased to the background level (Fig. 2). Distribution of the GFP within the enterocyte cytoplasm in rats and fogs was diffuse, but not level (Fig. 1a, b). Fluorescence in the supranuclear part of the enterocyte cytoplasm in rats was about 1.5 times higher than in the basal one. In addition, GFP fluorescence was located in the brush border. GFP fluorescence
Fig. 2. Intensity of the fluorescence of the epithelial layer of the upper third of the small intestinal villus depending on the time after GFP administration in rats (Rattus norvegicus) and in frogs (Rana temporaria). Abscissa — time (min), ordinate — fluorescence intensity (arbitrary units). ⁎ — p b 0.05, § — p b 0.01, $ — p b 0.001 compared with frog and rat.
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epithelium in frogs was similar to the rats, but fluorescence rose more intensively in frog tubule epithelium (Fig. 3). Subcellular distribution of GFP fluorescence in rat proximal tubule cells was irregular. A granular staining pattern of GFP was revealed by confocal microscopy. In the proximal cell cytoplasm of both rats and frogs, the most intensive specific fluorescence was concentrated predominantly in the round shape structures, 0.5–2.0 μm in diameter (Fig. 1c, d). Twenty minutes after GFP administration, these structures were located predominantly in the apical area of the cell cytoplasm, whereas 2 and 5 h later, they were revealed throughout the cytoplasm. No specific fluorescence was revealed in the liver (Fig. 1e) and colon cells (not shown) after the GFP intragastric administration. Fig. 3. Intensity of the fluorescence in the proximal tubule cells at various intervals after the intragastic GFP administration in rats and frogs. Abscissa — time (min), ordinate — fluorescence intensity (arbitrary units). ⁎ — p b 0.05, § — p b 0.001 compared with frog and rat.
was practically absent in the area of the junctional complex in the enterocytes and in goblet cells (Fig. 1a, b). Morphological examination demonstrated no alteration of the structural integrity of the epithelia after GFP introduction: the enterocytes stay intact with well-developed microvilli, and the cells remain joined by tightly closed junctions. 3.2. GFP accumulation in kidney cells after its administration into the gastrointestinal tract Specific GFP fluorescence was observed in rat proximal tubule epithelial cells 20 min after protein administration into the intestine lumen (Fig. 1c). No fluorescence was detected in epithelium cells of the other nephron segment. Specific fluorescence rose with an increase of time after the GFP administration and reached its maximal value 5 h later (Fig. 3). Specific fluorescence was also detected in proximal tubule cells in the frog kidney after the GFP administration (Fig. 1d). The dynamics of GFP accumulation in the proximal tubule
3.3. Ultrastructural localization of GFP in proximal tubule cells Ultrastructure examination of rat proximal tubule cells has shown the formation of large vesicles (up to 1–2 μm) in the apical cytoplasm after GFP administration (Fig. 4b) along with small numerous vesicles typical for this cell type in norm (Fig. 4a). Using the DAB photoconversion technique, the localization of GFP in proximal tubule cells was examined. GFP was photoconverted into an electron-dense reaction product during photobleaching. Most of the reaction product was observed in the vesicles after GFP administration (Fig. 4b), but not in control experiments (Fig. 4a). Heavy reaction product was confined to the lumen of large endocytic vesicles near the apical plasma membrane. 3.4. Immunofluorescent localization of GFP in the proximal tubule cells To determine the precise localization of GFP in rat epithelial cells, mouse anti-GFP (Clone GFP-20) monoclonal antibody was used. The immunoreactivity was detected by using immunofluorescence confocal microscopy with Cy3-conjugated anti-mouse antibody. The immunofluorescent Cy3 red signal
Fig. 4. Electron micrographs of proximal tubule cells of the rat kidney after GFP administration (b) and in control (a). Electron dense products (arrows) were confined to the lumen of large endocytic vesicles (ev) which formed after GFP introduction. Scale bar, 1 μm.
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Fig. 5. Immunocytochemical detection of GFP in rat proximal tubule cells 2 h after GFP administration into the small intestine lumen. Intense green fluorescence of GFP was detected within the cytoplasmic vesicles (a, d). The same localization (red) observed after incubation with anti-GFP antibody conjugated with Cy3 (b). The colocalization sites are seen yellow (c). Confocal laser scanning images showing the localization of GFP within lysosomes (d–f). The partial colocalization of GFP green fluorescence (d) and red anti-Lamp1 antibody fluorescence (e) is occur in some sites (f, arrows). Scale bar, 1 μm.
was localized within the intracellular granules (Fig. 5b). Green fluorescence (GFP) was detected within the same structures (Fig. 5a). The colocalization of both fluorescent signals confirms that administered GFP is located within the endocytic vesicles (Fig. 5c). Use of the anti-LAMP1 antibody as a marker for the late lysosome membrane has allowed for the detection of localization sites of accumulated GFP in the proximal tubule cells. Fig. 5(d)–(f) presents the results of immunochemical reactions with a partial colocalization of two labels in the same vesicular structures of epitheliocytes. Thus, GFP is taken up from the renal tubular fluid by endocytosis, transported into the endosome and then accumulated in the lysosomes of the proximal tubule cells.
4. Discussion The obtained results clearly demonstrate transepithelial transport and absorption of undegraded GFP in the frog and rat small intestine. The green fluorescence is seen within the cytoplasm of epithelial cells 3 min after GFP intragastric introduction (Fig. 1). Absorption of these proteins into the blood is documented by that 20 min after the GFP administration into the intestine, specific fluorescence is revealed in the cells of the renal proximal tubules (Fig. 3). The molecular mass of GFP is 27 kDa, hence, it is able to pass the glomerular filter and transfer with ultrafiltrate into the proximal tubule lumen. Cells of this nephron segment have been known as the major site of reabsorption of the endogenous proteins, which pass into
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coated vesicles, then into endosomes and lysosomes to be degraded (Maack and Park, 1990; Nielsen, 1994; Gburek et al., 2003; Christensen and Gburek, 2004). For the last decade, it has been established that the function of two multiligand receptors – megalin and cubilin – is required for reabsorption of proteins in the nephron proximal segment (Christensen et al., 1998). These proteins are strictly localized on epitheliocyte apical membranes, membranes of coated pits, apical tubular network, and endosomal membranes, i.e., in early endocytotic compartments (Verroust and Christensen, 2002). Expression of these proteins is also revealed in the small intestine (Yammani et al., 2001). Confocal microscopy examination revealed the granular character of GFP distribution in the proximal tubule cells. Our results, obtained using immunofluorescent confocal microscopy and electron microscopy, indicate the location of absorbed GFP in endosomes and lysosomes of the rat proximal tubule cells (Figs. 4, 5). Thus, GFP reabsorption can be effected by endocytosis, most likely by receptor-mediated endocytosis. On the contrary intracellular distribution of GFP fluorescence in the enterocyte cytosol was diffuse. There is a few data of the mechanism of intestinal macromolecular uptake. Horseradish peroxidase (HRP) are generally used as a marker for intestinal transcytosis study (Rombout et al., 1985; Berin et al., 1998; Kiliaan et al., 1998). However vesicular HRP transport within enterocyte cytoplasm is mediated by bulk-phase endocytosis (Heuser and Anderson, 1989; Cupers et al., 1994). The mechanism of GFP uptake in the enterocytes remains not clear and requires additional investigations. There are known facts of absorption of some intact proteins in the animal gastrointestinal tract. Albumin (Casartelli et al., 2005) and urease (Kurahashi et al., 2005) are absorbed in larva of the silkworm Bombyx mori L., casein and GFP in the plant bug Lygus hesperus Knight (Habibi et al., 2002), and amylase and lipase in rats (Cloutier et al., 2006). These proteins cross the epithelial barrier; however, the mechanism of the transepithelial transport is unknown. Mechanisms of protein transport are studied more completely in kidney proximal tubule cells of various taxonomic group representatives. In dog and mouse kidneys, transferrin is reabsorbed by megalin-dependent cubilin-mediated endocytosis (Kozyraki et al., 2001). The megalin-cubilin receptor system has been revealed in pronephros cells in zebrafish fries (Anzenberger et al., 2006). By receptor-mediated endocytosis, yeast invertase is absorbed in kidney epithelia of the fish Salmo alpinus L. (Smedsrud et al., 1984). Reabsorption of intraperitoneally injected fibronectin and TGF-beta has been found in proximal tubule cells of the axolotl nephron (Gross et al., 2002). The mechanism of interaction of immunoglobulins with receptors (pIgRs) during endocytosis in various vertebrate groups is suggested to be universal, as functional homology and conservatism of sequences of their receptors are revealed in mammals, birds, and amphibians (Wieland et al., 2004). Comparison of the results of our experiments on rats and frogs indicates that absorption in the gut and accumulation in the kidney proceed faster in the amphibian. In the frog kidney, unlike the rat kidney, blood supply is provided from two sources — renal arteries and the renoportal vein. The similarity
of the fluorescence character in the proximal tubule cells in rats and frogs after GFP administration into the intestine lumen allows us to think that GFP comes into the nephron lumen only during ultrafiltration. The high rate of GFP absorption can be a manifestation of the species-peculiar specificity as well as a reflection of the functional state of the organism's protein metabolism. No specific fluorescence has been revealed after GFP administration in the rat and frog liver (Fig. 3). Since blood from the small intestine reaches the liver via the portal system and then comes via the systemic circulation to the heart and renal vessels, the primary accumulation might have been expected in liver cells; however, this was not found. We can suggest that the liver does not get involved in the metabolism of the absorbed protein, while the kidney plays a dominant role in their metabolism. We tried to measure the absorbed GFP amount from the small intestine and the GFP renal excretion, if any. On the GFP administration at the used concentration, fluorescence of rat and frog blood serum was not detected; it was lower than the sensitivity of the RF-1501 spectrophotometer (Shimadzu, Japan). To reveal the plasma fluorescence, we increased the concentration of GFP administered into the gut in special experiments, that however, induced an alteration of the enterocyte ultrastructure seen by the enlargement of intercellular spaces and swelling of the Golgi apparatus cisterns. Thus, the performed study indicates that GFP can serve as a marker of intact protein absorption in the vertebrate body by the examples of frog and rat. Since GFP has fluorescence only with a preserved tertiary structure (Tsien, 1998), the obtained results certainly argue for intact GFP uptake into the enterocytes and transfer into the blood. Detection of GFP in cells of the kidney, but not the liver, shows an important function of renal proximal tubules in the metabolism of exogenous proteins entering the body environment. A comparative study of GFP absorption and accumulation in the kidney indicates a higher activity of these processes in amphibians compared to mammals. Acknowledgments The study was supported by the Russian Foundation for the Basic Research (05-04-49836) and the Leading Scientific School Program (NSh. 6576.206.4). References Adibi, S.A., 1996. Intestinal oligopeptide transporter: from hypothesis to cloning. News Physiol. Sci. 11, 133–137. Anzenberger, U., Bit-Avragim, N., Rohr, S., Rudolph, F., Dehmel, B., Willnow, T.E., Abdellilah-Seyfried, S., 2006. Elucidation of megalin/LRP2-dependent endocytic transport processes in the larval zebrafish pronephros. J. Cell Sci. 119, 2127–2137. Berin, M.C., Kiliaan, A.J., Yang, P.-C., Groot, J.A., Kitamura, Y., Perdue, M.H., 1998. The influence of mast cells on pathway of transepithelial antigen transport in rat intestine. J. Immunol. 161, 2561–2566. Borges, E.L., de Fatima Leite, M., Barbosa, A.J., Alves, J.B., 2002. Route of jejunal mucosa absorption of trypsin demonstrated by immunofluorescence. Histochem. J. 34, 525–528.
E.V. Seliverstova et al. / Comparative Biochemistry and Physiology, Part A 147 (2007) 1067–1073 Burmakin, M.V., Seliverstova, E.V., Natochin, Yu.V., 2005. Accumulation of yellow fluorescent protein in kidney after its absorption in rat intestine. Ross. Fiziol. Z. Im. I.M. Secenova 91, 1195–1204. Casartelli, M., Corti, P., Giovanna Leonardi, M., Fiandra, L., Burlini, N., Pennacchio, F., Giordana, B., 2005. Absorption of albumin by the midgut of a lepidopteran larva. J. Insect Physiol. 51, 933–940. Christensen, E.I., Gburek, J., 2004. Protein reabsorption in renal proximal tubule — function and dysfunction in kidney pathophysiology. Pediatr. Nephrol. 19, 714–721. Christensen, E.I., Birn, H., Verroust, P., Moestrup, S.K., 1998. Membrane receptors for endocytosis in the renal proximal tubule. Int. Rev. Cyt. 180, 237–284. Cloutier, M., Gingras, D., Bendayan, M., 2006. Internalization and transcytosis of pancreatic enzymes by the intestinal mucosa. J. Histochem. Cytochem. 54, 781–794. Crameri, A., Whitehorn, E.A., Tate, E., Stemmer, W.P., 1996. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14, 315–319. Cupers, P., Veithen, A., Kiss, A., Baudhuin, P., Courtoy, J., 1994. Clathrin polymerization is not required for bulk-phase endocytosis in rat fetal fibroblasts. J. Cell Biol. 127, 725–735. Daniel, H., 2004. Molecular and integrative physiology of intestinal peptide transport. Annu. Rev. Physiol. 66, 361–384. Dickinson, B.L., Badizadegan, K., Wu, Z., Ahouse, J.C., Zhu, X., Simister, N.E., Blumberg, R.S., Lencer, W.I., 1999. Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line. J. Clin. Invest. 104, 903–911. Gburek, J., Birn, H., Verroust, P.J., Goj, B., Jacobsen, C., Moestrup, S.K., Willnow, T.E., Christensen, E.I., 2003. Renal uptake of myoglobin is mediated by the endocytic receptors megalin and cubilin. Am. J. Physiol. Renal. Physiol. 285, F451–F458. Grabenbauer, M., Geerts, W.J., Fernandes-Rodriguez, J., Hoenger, A., Koster, A.J., Nilsson, T., 2005. Correlative microscopy and electron tomography of GFP through photooxidation. Nat. Methods 2, 857–862. Gross, M.L., Hanke, W., Koch, A., Ziebart, H., Amann, K., Ritz, E., 2002. Intraperitoneal protein injection in the axolotl: the amphibian kidney as a novel model to study tubulointerstitial activation. Kidney Int. 62, 51–59. Habibi, J., Brandt, S.L., Coudron, T.A., Wagner, R.M., Wright, M.K., Backus, E.A., Huesing, J.I., 2002. Uptake, flow, and digestion of casein and green fluorescent protein in the digestive system of Lygus hesperus Knight. Arch. Insect Biochem. Physiol. 50, 62–74. Heuser, J.E., Anderson, R.G.W., 1989. Hypertonic media inhibit receptormediated endocytosis by blocking clathrin-coated pit formation. J. Cell Biol. 108, 389–400.
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Kiliaan, A.J., Saunders, P.R., Bijlsma, P.B., Berin, M.C., Taminiau, J.A., Groot, J.A., Perdue, M.H., 1998. Stress stimulates transepithelial macromolecular uptake in the rat jejunum. Am. J. Physiol. 275, G1037–G1044. Kozyraki, R., Fyfe, J., Verroust, P.J., Jacobsen, C., Dautry-Varsat, A., Gburek, J., Willnow, T.E., Christensen, E.I., Moestrup, S.K., 2001. Megalin-dependent cubilin-mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia. Proc. Natl. Acad. Sci. 98, 12491–12496. Kurahashi, H., Atiwetin, P., Nagaoka, S., Miyata, S., Kitajima, S., Sugimura, Y., 2005. Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori. J. Insect. Physiol. 51, 1055–1061. Maack, T., Park, C.H., 1990. Endocytosis and lysosomal hydrolysis of proteins in proximal tubules. Methods Enzymol. 191, 340–354. Natochin, Yu.V., Grigoriev, A.I., Buravkova, L.B., Larina, I.M., Prutskova, N.P., Shakhmatova, E.I., 2003. Antidiuretic reaction of the human and rat kidney to peroral administration of arginine–vasopressin and desmopressin. Ross. Fiziol. Z. Im. I.M. Secenova 89, 184–192. Nielsen, R., 1994. Endocytosis in renal proximal tubules. Experimental electron microscopical studies of protein absorption and membrane traffic in isolated, in vitro perfused proximal tubules. J. Dance Med. Sci. 41, 243–263. Roberts, P.R., Burney, J.D., Black, K.W., Zaloga, G.P., 1999. Effect of chain length on absorption of biologically active peptides from the gastrointestinal tract. Digestion 60, 332–337. Rombout, J.H.W.M., Lamers, C.H.J., Helfrich, M.H., Dekker, A., TaverneThiele, J.J., 1985. Uptake and transport of intact macromolecules in the intestinal epithelium of carp (Cyprinus carpio L.) and the possible immunological implications. Cell Tissue Res. 239, 519–530. Smedsrud, T., Dannevig, B.H., Tollenshaug, H., Berg, T., 1984. Endocytosis of a mannose-terminated glycoprotein and formaldehyde-treated human serum albumin in liver and kidney cells from fish (Salmo alpinus L.). Dev. Comp. Immunol. 8, 579–588. Tsien, R.Y., 1998. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544. Verroust, P.J., Christensen, E.I., 2002. Megalin and cubilin — the story of two multipurpose receptors unfolds. Nephrol. Dial. Transplant 17, 1867–1871. Wheeler, S., McGinn, B.J., Lucas, M.L., Morrison, J.D., 2002. Absorption of biologically active peptide hormones from the small intestine of rat. Acta Physiol. Scand. 176, 203–213. Wieland, W.H., Orzaez, D., Lammers, A., Parmentier, H.K., Verstegen, M.W.A., Schots, A., 2004. A functional polymeric immunoglobulin receptor in chicken (Gallus gallus) indicates ancient role of secretory IgA in mucosal immunity. Biochem. J. 380, 669–676. Yammani, R.R., Seetharam, S., Seetharam, B., 2001. Cubilin and megalin expression and their interaction in the rat intestine: effect of thyroidectomy. Am. J. Physiol. Endocrinol. Metab. 281, E900–E907.
Renal clearance of absorbed intact GFP in the frog and rat intestine E.V. Seliverstova, M.V. Burmakin, Yu.V. Natochin ⁎ Laboratory of Renal Physiology, Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia Received 26 January 2007; received in revised form 12 March 2007; accepted 13 March 2007 Available online 20 March 2007
Abstract Intestine absorption of intact green fluorescent protein (GFP) and its following accumulation in the renal proximal tubule cells after its intragastric administration have been established by confocal microscopy in the rat and frog. Reabsorbed GFP was revealed in the endosomes and lysosomes of the proximal tubule cells by the methods of GFP photooxidation and immunofluorescent microscopy. The GFP intestine absorption rate and GFP accumulation in the kidney were significantly higher in the frog than in the rat. No specific fluorescence was revealed in the liver and colon cells after the GFP intragastric administration. The data obtained indicate the ability of the small intestine in the frog and rat to absorb intact proteins and an important role of the kidney in exogenous protein metabolism. © 2007 Elsevier Inc. All rights reserved. Keywords: Green fluorescent protein (GFP); Protein absorption; Intestine; Kidney; Protein reabsorption; Rats; Frogs
1. Introduction According to the classic scheme of digestion, proteins are hydrolyzed in the intestine to amino acids, di- and tripeptides that subsequently are absorbed into the blood (Adibi, 1996; Daniel, 2004). However, there are data that some polypeptides (Borges et al., 2002; Roberts et al., 1999; Wheeler et al., 2002; Natochin et al., 2003) and proteins (Dickinson et al., 1999) also can be absorbed without destruction. Nowadays, there are a few data as to how widely the phenomenon of intact protein and polypeptide absorption is spread in the animal kingdom. Use of proteins with radioactive label or protein-bound fluorochrome does not provide a strong answer to the posed question, as there can be doubts whether protein structure is conserved during intestine absorption. To overcome these difficulties, we have suggested another approach — study of absorption in the small intestine of a fluorescent protein. An example of such a protein is green fluorescent protein (GFP), which is characterized by a fluorescence of molecules with only a preserved tertiary structure (Tsien, 1998) and by a high resistance to proteolytic enzymes (Crameri et al., 1996). Detection of such a protein not only in the intestine lumen, but also within enterocytes during ⁎ Corresponding author. E-mail address: [email protected] (Y.V. Natochin). 1095-6433/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2007.03.019
the absorption would allow us to claim that the molecule is absorbed without hydrolysis. In experiments on rats, we have shown that yellow fluorescent protein can be absorbed in the intestine without hydrolysis (Burmakin et al., 2005). It was of interest to use GFP to elucidate a possibility of its absorption from the rat and frog small intestine into the blood, to analyze its further fate in the body, and to find out whether it is accumulated in the liver or kidney after absorption. The study of this issue has become the goal of the present work. 2. Materials and methods GFP (27 kDa) was obtained from the Laboratory of Protein Synthesis Mechanisms (Institute of Protein, Russian Academy of Sciences, Pushchino-on-Oka, Russia). Experiments were carried out on 4–6-month old adult female Wistar rats Rattus norvegicus (150–200 g BW) and on winter male brown frogs Rana temporaria L. (40–50 g BW) kept in water reservoirs at 4 °C. In in vivo experiments, the rats and frogs were administered per os via a rubber probe with 0.1 ml of GFP solution (0.34 μg/ml in 0.01 M PBS-buffer, pH 7.3) per 150 g of BW. In control experiments, the same volume of PBS without GFP was administered to the animals. After 20, 120, and 300 min of the GFP administration, rats were decapitated under a light ether anesthesia, while in frogs, the spinal cord was
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destroyed with a thin needle; then the abdominal cavity of the animals was opened and a 2-cm long segment of the small intestine and a fragment of the renal tissue were dissected out. Specimens were fixed for 2–3 h in 4% paraformaldehyde in 0.05 M PBS (pH 7.3). After washing with PBS (2 × 15 min), the tissue pieces were cryoprotected overnight at 4 °C in PBS containing 30% sucrose. Specimens then were embedded in OCT compound (Bright, UK) and frozen. Frozen sections of 5– 10 μm thickness were cut using a Cryostat CM 1510 (Leica,
Germany), placed on glass slides and air-dried at RT. Dry sections were covered with cover glass slips using ImmunoFluore Mounting Medium (MP Biomedicals, Inc, Germany). Specimens were viewed and analyzed using a DM R confocal laser scanning unit coupled to a Leica TSC SL microscope (Leica, Germany). Immunohistochemistry was performed on frozen kidneys sections by indirect immunofluorescence using antibodies against GFP and lysosome membrane (LAMP1). After fixation
Fig. 1. Fluorescence in enterocytes (a, b) 3 min after GFP administration and in the proximal tubules cells (c, d) 2 h after GFP administration into the small intestine lumen. Higher fluorescence intensity is evident in intestine and kidney in frogs (Rana temporaria) (b, d) compared to rats (Rattus norvegicus) (a, c). No specific fluorescence is revealed in the goblet cells (a, asterisk), in the rat liver cells after GFP administration (e) and in the frog kidney epithelial cells without GFP administration (f). Scale bar — 10 μm.
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(as described above), the tissue pieces were incubated overnight at 4 °C in 0.05 M PBS containing 0.15 M glycine, cryoprotected and frozen. Cryostat sections were placed on coverslips coated with poly-L-lysine (ISN Biomedicals, USA). Sections were rinsed in PBS and then blocked in blocking solution (3% BSA/ 0.2% Triton X-100 in 0.05 M PBS) to unmask antigenic sites for 30 min. The sections were incubated overnight at 4 °C or for 2 h at RT with primary antibody: mouse anti-GFP monoclonal antibody (Clone GFP-20) (Chemicon Int., USA) and mouse anti-Lamp1 monoclonal antibody (Calbiochem, USA). The antibodies were diluted 1:100 in incubation medium (0.1% BSA/0.2% Triton-X-100 in PBS). After this incubation, sections were washed three times with 0.1% BSA in PBS over the course of 1 h. For fluorescent visualization of bound primary antibodies (anti-LAMP1 and anti-GFP), sections were incubated with secondary Cy3-conjugated anti-mouse antibody (1:1000) (Sigma, USA). As the negative control, the primary antibody was either omitted from the incubation solution or used nonspecific antibody. Specimens were viewed and analyzed using the same confocal microscope. Electronic images were captured and edited in Adobe Photoshop (Adobe System Inc., Mountain View, California, USA). For the photooxidation procedure (Grabenbauer et al., 2005), specimens were sliced and fixed for 2–3 h in 2% paraformaldehyde in 0.05 M PBS (pH 7.3). After washing 3× with PBS, we blocked samples with 100 mM glycine and 100 mM potassium cyanide in PBS for 1 h. For photoconversion, we washed samples in PBS and then incubated them in a freshly prepared ice-cold solution of 1.5 mg/ml 3,3′-DAB in PBS (Sigma, St. Louis, USA). For bleaching, samples were illuminated with appropriate filter settings for the GFP (excitation filter BP) using a 100-W mercury lamp. Photobleaching (photooxidation) was stopped when the green fluorescence was faded and the cytosolic background staining (brownish reaction product) was occurred (in 20–30 min). After photoconversion, samples were treated in the electron microscopy procedure. Small pieces of rat intestine and kidney were fixed for electron microscopy by immersion in 2% paraformaldehyde/ 2.5% glutaraldehyde in 0.1 M Na cacodylate buffer, pH 7.3. Samples were washed for 30 min with the same buffer and further treated with 1% OsO4 in 0.1 M cacodylate. Specimens were dehydrated and embedded in an epoxy resin/araldite mixture according to routine procedures. Ultrathin sections were obtained using a Leica Ultracut UCT (Leica, Germany), collected on cupper grids and contrasted with uranyl acetate and lead citrate. Specimens were examined at 60 kV in a LEO 910 electron microscope (LEO Electron Microscopy Group, Germany). For the analysis of the images obtained using the confocal microscope, the computer program Image Tool III was used. The intensity of the fluorescence was measured in equal areas of the compared sites of the small intestine epithelium or proximal nephron segments. The intensity value was expressed in arbitrary units, their values being calculated as the ratio of fluorescent pixels to non-fluorescent pixels within this area (the scale was from 0 to 256). To study the distribution of
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fluorescence along the length of the gut villus, areas of epithelium (100 images in each experiment) from the upper third of the intestinal villi and the crypt zone were analyzed in various histological sections. To evaluate the intensity of fluorescence, the epithelial cell was measured as a whole, including the brush border. In all experiments, to determine the value of specific GFP fluorescence, the background fluorescence (autofluorescence) of control experiments without GFP administration was subtracted from the total fluorescence values in experiments with the administration of this protein. Unpaired t-test was used to estimate the statistical significance of differences; all data are presented as means ± SE. The threshold of significance was p b 0.05. 3. Results 3.1. Absorption of GFP in the small intestine After 3 min of GFP intragastric administration, specific fluorescence within the rat enterocytes was observed (Fig. 1a), which differed from the autofluorescence of epithelial cells in the control. The intensity of the GFP fluorescence rose depending on the time period after GFP administration. Fluorescence reached a maximum at 21 min and then decreased gradually to the background level (Fig. 2). Specific GFP fluorescence was revealed in enterocytes 3 min after GFP administration into the frog small intestine lumen, with its maximal fluorescence being observed 15 min after GFP administration (Fig. 1b); by 33 min, the fluorescence intensity of the epithelial layer of the frog small intestine decreased to the background level (Fig. 2). Distribution of the GFP within the enterocyte cytoplasm in rats and fogs was diffuse, but not level (Fig. 1a, b). Fluorescence in the supranuclear part of the enterocyte cytoplasm in rats was about 1.5 times higher than in the basal one. In addition, GFP fluorescence was located in the brush border. GFP fluorescence
Fig. 2. Intensity of the fluorescence of the epithelial layer of the upper third of the small intestinal villus depending on the time after GFP administration in rats (Rattus norvegicus) and in frogs (Rana temporaria). Abscissa — time (min), ordinate — fluorescence intensity (arbitrary units). ⁎ — p b 0.05, § — p b 0.01, $ — p b 0.001 compared with frog and rat.
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epithelium in frogs was similar to the rats, but fluorescence rose more intensively in frog tubule epithelium (Fig. 3). Subcellular distribution of GFP fluorescence in rat proximal tubule cells was irregular. A granular staining pattern of GFP was revealed by confocal microscopy. In the proximal cell cytoplasm of both rats and frogs, the most intensive specific fluorescence was concentrated predominantly in the round shape structures, 0.5–2.0 μm in diameter (Fig. 1c, d). Twenty minutes after GFP administration, these structures were located predominantly in the apical area of the cell cytoplasm, whereas 2 and 5 h later, they were revealed throughout the cytoplasm. No specific fluorescence was revealed in the liver (Fig. 1e) and colon cells (not shown) after the GFP intragastric administration. Fig. 3. Intensity of the fluorescence in the proximal tubule cells at various intervals after the intragastic GFP administration in rats and frogs. Abscissa — time (min), ordinate — fluorescence intensity (arbitrary units). ⁎ — p b 0.05, § — p b 0.001 compared with frog and rat.
was practically absent in the area of the junctional complex in the enterocytes and in goblet cells (Fig. 1a, b). Morphological examination demonstrated no alteration of the structural integrity of the epithelia after GFP introduction: the enterocytes stay intact with well-developed microvilli, and the cells remain joined by tightly closed junctions. 3.2. GFP accumulation in kidney cells after its administration into the gastrointestinal tract Specific GFP fluorescence was observed in rat proximal tubule epithelial cells 20 min after protein administration into the intestine lumen (Fig. 1c). No fluorescence was detected in epithelium cells of the other nephron segment. Specific fluorescence rose with an increase of time after the GFP administration and reached its maximal value 5 h later (Fig. 3). Specific fluorescence was also detected in proximal tubule cells in the frog kidney after the GFP administration (Fig. 1d). The dynamics of GFP accumulation in the proximal tubule
3.3. Ultrastructural localization of GFP in proximal tubule cells Ultrastructure examination of rat proximal tubule cells has shown the formation of large vesicles (up to 1–2 μm) in the apical cytoplasm after GFP administration (Fig. 4b) along with small numerous vesicles typical for this cell type in norm (Fig. 4a). Using the DAB photoconversion technique, the localization of GFP in proximal tubule cells was examined. GFP was photoconverted into an electron-dense reaction product during photobleaching. Most of the reaction product was observed in the vesicles after GFP administration (Fig. 4b), but not in control experiments (Fig. 4a). Heavy reaction product was confined to the lumen of large endocytic vesicles near the apical plasma membrane. 3.4. Immunofluorescent localization of GFP in the proximal tubule cells To determine the precise localization of GFP in rat epithelial cells, mouse anti-GFP (Clone GFP-20) monoclonal antibody was used. The immunoreactivity was detected by using immunofluorescence confocal microscopy with Cy3-conjugated anti-mouse antibody. The immunofluorescent Cy3 red signal
Fig. 4. Electron micrographs of proximal tubule cells of the rat kidney after GFP administration (b) and in control (a). Electron dense products (arrows) were confined to the lumen of large endocytic vesicles (ev) which formed after GFP introduction. Scale bar, 1 μm.
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Fig. 5. Immunocytochemical detection of GFP in rat proximal tubule cells 2 h after GFP administration into the small intestine lumen. Intense green fluorescence of GFP was detected within the cytoplasmic vesicles (a, d). The same localization (red) observed after incubation with anti-GFP antibody conjugated with Cy3 (b). The colocalization sites are seen yellow (c). Confocal laser scanning images showing the localization of GFP within lysosomes (d–f). The partial colocalization of GFP green fluorescence (d) and red anti-Lamp1 antibody fluorescence (e) is occur in some sites (f, arrows). Scale bar, 1 μm.
was localized within the intracellular granules (Fig. 5b). Green fluorescence (GFP) was detected within the same structures (Fig. 5a). The colocalization of both fluorescent signals confirms that administered GFP is located within the endocytic vesicles (Fig. 5c). Use of the anti-LAMP1 antibody as a marker for the late lysosome membrane has allowed for the detection of localization sites of accumulated GFP in the proximal tubule cells. Fig. 5(d)–(f) presents the results of immunochemical reactions with a partial colocalization of two labels in the same vesicular structures of epitheliocytes. Thus, GFP is taken up from the renal tubular fluid by endocytosis, transported into the endosome and then accumulated in the lysosomes of the proximal tubule cells.
4. Discussion The obtained results clearly demonstrate transepithelial transport and absorption of undegraded GFP in the frog and rat small intestine. The green fluorescence is seen within the cytoplasm of epithelial cells 3 min after GFP intragastric introduction (Fig. 1). Absorption of these proteins into the blood is documented by that 20 min after the GFP administration into the intestine, specific fluorescence is revealed in the cells of the renal proximal tubules (Fig. 3). The molecular mass of GFP is 27 kDa, hence, it is able to pass the glomerular filter and transfer with ultrafiltrate into the proximal tubule lumen. Cells of this nephron segment have been known as the major site of reabsorption of the endogenous proteins, which pass into
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coated vesicles, then into endosomes and lysosomes to be degraded (Maack and Park, 1990; Nielsen, 1994; Gburek et al., 2003; Christensen and Gburek, 2004). For the last decade, it has been established that the function of two multiligand receptors – megalin and cubilin – is required for reabsorption of proteins in the nephron proximal segment (Christensen et al., 1998). These proteins are strictly localized on epitheliocyte apical membranes, membranes of coated pits, apical tubular network, and endosomal membranes, i.e., in early endocytotic compartments (Verroust and Christensen, 2002). Expression of these proteins is also revealed in the small intestine (Yammani et al., 2001). Confocal microscopy examination revealed the granular character of GFP distribution in the proximal tubule cells. Our results, obtained using immunofluorescent confocal microscopy and electron microscopy, indicate the location of absorbed GFP in endosomes and lysosomes of the rat proximal tubule cells (Figs. 4, 5). Thus, GFP reabsorption can be effected by endocytosis, most likely by receptor-mediated endocytosis. On the contrary intracellular distribution of GFP fluorescence in the enterocyte cytosol was diffuse. There is a few data of the mechanism of intestinal macromolecular uptake. Horseradish peroxidase (HRP) are generally used as a marker for intestinal transcytosis study (Rombout et al., 1985; Berin et al., 1998; Kiliaan et al., 1998). However vesicular HRP transport within enterocyte cytoplasm is mediated by bulk-phase endocytosis (Heuser and Anderson, 1989; Cupers et al., 1994). The mechanism of GFP uptake in the enterocytes remains not clear and requires additional investigations. There are known facts of absorption of some intact proteins in the animal gastrointestinal tract. Albumin (Casartelli et al., 2005) and urease (Kurahashi et al., 2005) are absorbed in larva of the silkworm Bombyx mori L., casein and GFP in the plant bug Lygus hesperus Knight (Habibi et al., 2002), and amylase and lipase in rats (Cloutier et al., 2006). These proteins cross the epithelial barrier; however, the mechanism of the transepithelial transport is unknown. Mechanisms of protein transport are studied more completely in kidney proximal tubule cells of various taxonomic group representatives. In dog and mouse kidneys, transferrin is reabsorbed by megalin-dependent cubilin-mediated endocytosis (Kozyraki et al., 2001). The megalin-cubilin receptor system has been revealed in pronephros cells in zebrafish fries (Anzenberger et al., 2006). By receptor-mediated endocytosis, yeast invertase is absorbed in kidney epithelia of the fish Salmo alpinus L. (Smedsrud et al., 1984). Reabsorption of intraperitoneally injected fibronectin and TGF-beta has been found in proximal tubule cells of the axolotl nephron (Gross et al., 2002). The mechanism of interaction of immunoglobulins with receptors (pIgRs) during endocytosis in various vertebrate groups is suggested to be universal, as functional homology and conservatism of sequences of their receptors are revealed in mammals, birds, and amphibians (Wieland et al., 2004). Comparison of the results of our experiments on rats and frogs indicates that absorption in the gut and accumulation in the kidney proceed faster in the amphibian. In the frog kidney, unlike the rat kidney, blood supply is provided from two sources — renal arteries and the renoportal vein. The similarity
of the fluorescence character in the proximal tubule cells in rats and frogs after GFP administration into the intestine lumen allows us to think that GFP comes into the nephron lumen only during ultrafiltration. The high rate of GFP absorption can be a manifestation of the species-peculiar specificity as well as a reflection of the functional state of the organism's protein metabolism. No specific fluorescence has been revealed after GFP administration in the rat and frog liver (Fig. 3). Since blood from the small intestine reaches the liver via the portal system and then comes via the systemic circulation to the heart and renal vessels, the primary accumulation might have been expected in liver cells; however, this was not found. We can suggest that the liver does not get involved in the metabolism of the absorbed protein, while the kidney plays a dominant role in their metabolism. We tried to measure the absorbed GFP amount from the small intestine and the GFP renal excretion, if any. On the GFP administration at the used concentration, fluorescence of rat and frog blood serum was not detected; it was lower than the sensitivity of the RF-1501 spectrophotometer (Shimadzu, Japan). To reveal the plasma fluorescence, we increased the concentration of GFP administered into the gut in special experiments, that however, induced an alteration of the enterocyte ultrastructure seen by the enlargement of intercellular spaces and swelling of the Golgi apparatus cisterns. Thus, the performed study indicates that GFP can serve as a marker of intact protein absorption in the vertebrate body by the examples of frog and rat. Since GFP has fluorescence only with a preserved tertiary structure (Tsien, 1998), the obtained results certainly argue for intact GFP uptake into the enterocytes and transfer into the blood. Detection of GFP in cells of the kidney, but not the liver, shows an important function of renal proximal tubules in the metabolism of exogenous proteins entering the body environment. A comparative study of GFP absorption and accumulation in the kidney indicates a higher activity of these processes in amphibians compared to mammals. Acknowledgments The study was supported by the Russian Foundation for the Basic Research (05-04-49836) and the Leading Scientific School Program (NSh. 6576.206.4). References Adibi, S.A., 1996. Intestinal oligopeptide transporter: from hypothesis to cloning. News Physiol. Sci. 11, 133–137. Anzenberger, U., Bit-Avragim, N., Rohr, S., Rudolph, F., Dehmel, B., Willnow, T.E., Abdellilah-Seyfried, S., 2006. Elucidation of megalin/LRP2-dependent endocytic transport processes in the larval zebrafish pronephros. J. Cell Sci. 119, 2127–2137. Berin, M.C., Kiliaan, A.J., Yang, P.-C., Groot, J.A., Kitamura, Y., Perdue, M.H., 1998. The influence of mast cells on pathway of transepithelial antigen transport in rat intestine. J. Immunol. 161, 2561–2566. Borges, E.L., de Fatima Leite, M., Barbosa, A.J., Alves, J.B., 2002. Route of jejunal mucosa absorption of trypsin demonstrated by immunofluorescence. Histochem. J. 34, 525–528.
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