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Biochemical and Molecular Actions of Nutrients

Megalin-Mediated Reuptake of Retinol in the Kidneys of Mice Is Essential for Vitamin A Homeostasis^1,2

Institute of Nutritional Science, University of Potsdam, D-14558 Nuthetal (Bergholz-Rehbruecke), Germany and ^* Max-Delbrueck-Center for Molecular Medicine, D-13125 Berlin, Germany

³To whom correspondence should be addressed. E-mail: jraila{at}rz.uni-potsdam.de.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The reuptake of retinol (ROH) and retinol-binding protein (RBP) in the kidneys is mediated by the endocytic receptor megalin, suggesting an important role for this receptor in vitamin A (VA) metabolism. We examined the extent to which megalin deficiency may affect urinary ROH excretion, levels of ROH and RBP in plasma, as well as storage of VA in liver and kidney. For this purpose, mice with a kidney-specific megalin gene defect (megalin^lox/lox; apoE^Cre) and control mice (megalin^lox/lox) were fed either a basal diet containing 4500 retinol equivalents (RE)/kg diet or a diet without VA during experimental periods of 42 and 84 d. Urinary ROH excretion was observed only in megalin^lox/lox; apoE^Cre mice (P < 0.0001, 2-way ANOVA) and not in the controls. Plasma ROH and RBP differed only by diet (P < 0.05), but not genotype (P = 0.615). A major effect of megalin deficiency, however, was evident in retinyl ester levels in the liver (P < 0.05), which were 37% lower than those in megalin^lox/lox controls (P < 0.05, Student’s t test) during the 84-d period of dietary VA deprivation. Kidney levels of VA were not affected by the receptor gene defect. The findings demonstrate that urinary ROH excretion caused by megalin deficiency requires accelerated mobilization of hepatic VA stores to maintain normal plasma ROH levels, which suggests that megalin plays an essential role in systemic VA homeostasis.

KEY WORDS: • megalin • vitamin A homeostasis • mice

Vitamin A (VA)⁴ and its derived metabolites (retinoids) are required for the maintenance of many biological processes, including the visual cycle, reproduction, cellular growth and differentiation, embryonic development, and immune response (1). The central importance of VA is reflected in the homeostatic regulation of plasma retinol (ROH) transported by retinol-binding protein (RBP), which is synthesized mainly by the liver. The major physiologic role of RBP is to guarantee a constant and continuous supply of ROH to peripheral tissues despite fluctuations in dietary VA intake (2–4). As part of its role as the central organ for maintenance of VA homeostasis, the liver clears newly absorbed VA circulating in chylomicrons and chylomicron remnants and is also the main storage site for VA within the body (5,6). In addition, the kidneys also seem to play an important role in whole-body VA homeostasis. Kinetic studies showed that 50% of the circulating ROH pool originates in the kidneys and that the reabsorption of ROH from the glomerular filtrate may be important for this regulation (7,8). The reuptake of ROH in the kidney is mediated by the binding of its carrier RBP to the endocytic receptor megalin. The essential role of megalin-mediated uptake of ROH-RBP complexes in the kidney is supported by the documented loss of ROH and RBP in the urine of mice with megalin deficiency (9).

Megalin is a 600-kDa type 1 membrane protein belonging to the LDL receptor gene family (10). In the kidney, megalin expression is most abundant in the brush border, coated pits, endocytic vesicles, and in the membrane recycling compartment of renal proximal tubular cells (11). In addition to RBP, megalin is able to bind several unrelated ligands from the glomerular filtrate, notably carrier proteins for vitamins and hormones, such as transthyretin, vitamin D-binding protein, and transcobalamin II (12–14). An essential function for megalin in renal as well as systemic vitamin D metabolism was confirmed in mice with a receptor gene defect (13,15); however, the relevance of this receptor pathway for VA homeostasis awaits formal proof in vivo. Thus, it is unclear whether the urinary excretion of the ROH-RBP complex in megalin-deficient mice affects the mobilization of hepatic VA stores, the transport of ROH in plasma, and its delivery to tissues.

To address these questions, we performed a feeding experiment in a mouse model with a kidney-specific megalin defect. In contrast to the complete receptor-knockout mice, which die perinatally due to holoprosencephaly (16), mice with this kidney-specific gene defect develop normally and therefore represent a useful model for studying the function of megalin in the metabolism of cholecalciferol as well as its role in the sodium phosphate cotransporter system (15,17).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and diets. Male conditional megalin knockout mice (megalin^lox/lox; apoE^Cre) and control mice (megalin^lox/lox) were used in the study. These mice were produced using the Cre-recombinase technology as described previously (15). After weaning (21 d of age), all groups had free access to a pelleted commercial standard rodent diet (18) containing 4500 retinol equivalents (RE)/kg diet until mice reached the age of 12 wk and were designated for the experiments. All mice were bred in-house according to the regulatory rules for animal welfare of the German Society for Laboratory Animal Science. Protocols were approved by the local Animal Welfare Committee Berlin, Germany.

    Expt. 1. In the first experiment, we investigated the effects of basal and VA-free diets on levels of ROH and retinyl esters in plasma, urine, liver, and kidneys of megalin-deficient and control mice. In this experiment, 12-wk-old megalin^lox/lox; apoE^Cre (n = 10), and 12-wk-old megalin^lox/lox (n = 10) mice were fed either the commercial rodent diet (18) containing 4500 RE/kg diet (basal) or the same diet without VA (–VA) for a period of 42 d. The experimental period of 42 d was chosen based on results of a feeding trial, which investigated the effects of a vitamin D–deficient diet on systemic cholecalciferol metabolism in megalin-deficient mice (15). The diets were consumed ad libitum and all mice had free access to water throughout the period. At the end of the feeding period, mice were placed in metabolic cages for a 16-h urine collection. Mice were then killed and blood, liver, and kidneys were removed. Serum was separated by centrifugation (1500 x g for 10 min at 4°C) and stored at –80°C until analysis, which occurred within 3 mo. Tissue samples from the livers and kidneys were either frozen in liquid nitrogen and kept at –80°C until analyzed or fixed in 4% buffered formaldehyde solution for immunohistochemistry (IHC).

    Expt. 2. Expanding on the initial results, a 2nd experiment was undertaken to determine the effects of an extended period of dietary VA deprivation on levels of ROH and retinyl esters in plasma, liver, and kidney of megalin-deficient mice. In this experiment, 12-wk-old megalin^lox/lox; apoE^Cre (n = 5), and 12-wk-old megalin^lox/lox (n = 5), mice were fed a VA-free diet for 84 d. At the end of this period, mice were killed and blood, liver, kidney, as well as spleen, brain, and eye samples were collected and processed as described for Expt. 1.

    Extraction and analytical determination of VA. ROH and retinyl ester concentrations in plasma and tissues were measured using a modified gradient reversed-phase HPLC-system (Waters). VA was extracted twice from plasma (100 µL) and urine (200 µL) using n-hexane (1 mL each time stabilized with 0.05% BHT) after deproteinization with ethanol (200 µL). Tissue samples were extracted 3 times in a 15-mL mixture of n-hexane and isopropanol (3:2; v:v, 0.05% BHT) (19). For separation of the compounds, a reversed-phase C30 column, (5 µm, 250 x 4.6 mm; YMC) in line with a C18 precolumn (Luna, Phenomenex) together with a solvent system consisting of solvent A, methanol:water (90:10; v:v, with 0.4 g/L ammonium acetate in H₂O) and solvent B, methanol:methyl-tert-butyl-ether:water (8:90:2; by vol, with 0.1 g/L ammonium acetate in H₂O) was applied (20). ROH and retinyl esters were quantified by measuring the absorption at 325 nm using external standards. Authentic standards of retinyl palmitate, retinyl oleate, retinyl stearate, retinyl linoleate, and retinyl myristate were a gift from Hoffmann-La Roche. The standard for ROH was purchased from Sigma. Results for ROH were compared with standard reference material 968a (National Institute of Standards Technology).

    RBP immunoblot analysis. To assess the presence of RBP, we performed SDS-PAGE immunoblot analyses. Aliquots of 0.3 mg of plasma protein were assayed on 12% reducing SDS-PAGE. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% defatted milk was used to block nonspecific binding sites on the blot. The membrane was then incubated with a peroxidase-coupled rabbit anti-human RBP IgG (DakoCytomation; 1:300 diluted in TBS containing 0.1% Tween 20) for 1 h at room temperature. Antibody binding was visualized using the Luminol reaction (BM Chemiluminescence Blotting Substrate, Roche Diagnostics). The band intensity of RBP was read with an imager (Bio-Rad) and analyzed with the Bio-Rad Multi-Analyst software 1.1 (21).

    Immunohistochemistry (IHC). Polyclonal goat anti-rabbit megalin was prepared as described previously (22) and was used for IHC at a determined optimal dilution of 1:50 000 in 1% bovine serum albumin (BSA) in TBS. Polyclonal rabbit anti-human RBP (DakoCytomation) was used 1:400 diluted in 1% BSA in TBS. Peroxidase conjugated rabbit anti-sheep IgG and peroxidase-conjugated swine anti-rabbit IgG were purchased from DakoCytomation and both were used at a 1:100 dilution in 1% BSA in TBS. IHC was carried out with the indirect peroxidase immunostaining technique as previously described (23). The deparaffinized and rehydrated sections were incubated overnight in a humidified chamber at 4°C with the primary anti-megalin or anti-RBP antibody. The sections were then exposed to the secondary peroxidase-conjugated antibody for 30 min. Immunoreactivity was visualized using diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in 0.1 mol/L imidazole buffer (pH 7.1), producing a brown-colored stain. Counterstaining was performed with Papanicolaou hematoxylin. Negative controls, which included the omission of the primary antibodies, had no significant labeling. All incubations except for the primary antibody were performed at room temperature. The sections were examined and photographed with an Olympus BX-50 microscope (Olympus) equipped with a ColorView 12 CCD video camera (SIS). Images were processed using analySIS^TM 3.0 software (SIS).

    Statistical analysis. Results are presented as means ± SD. Results for Expt. 1 were processed by a 2-factor ANOVA to analyze the effect of megalin genotype, diet, or any interaction between megalin genotype and diet (SPSS statistical package, version 10.0). With the occurrence of a significant effect, a 1-way ANOVA was performed on groups segregated according to both genotype and diet. This was followed by a modified least-squared difference post hoc test of significance. If the data were not normally distributed or the variance was not equal, then log10-transformed data were analyzed. In the case of 2-group comparison in Expt. 2, differences between group means were analyzed by Student’s t test (SPSS). Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Evaluation of the mouse model by IHC. Initially, we examined the distribution of the megalin receptor and its ligand RBP in kidneys of megalin^lox/lox; apoE^Cre and megalin^lox/lox mice using IHC. Megalin was highly expressed at the apical surface of the epithelial cells in the proximal tubular cells of megalin^lox/lox controls (Fig. 1a). By contrast, receptor expression was not detectable in most proximal tubular cells of megalin^lox/lox; apoE^Cre mice (Fig. 1b). The residual megalin expression in a few proximal tubular cells were in line with previous observations that suggested incomplete inactivation of the receptor gene in 10–15% of the renal cells due to insufficient Cre-recombinase activity. IHC for RBP in megalin^lox/lox mice revealed a supranuclear granular staining in the epithelial cells of the proximal convoluted tubules (Fig. 1c) that colocalized with immunoreactive megalin. By contrast, RBP was absent in proximal tubular cells of megalin^lox/lox; apoE^Cre mice lacking the receptor (Fig. 1d).

View larger version (113K): [in this window] [in a new window]   FIGURE 1 Immunohistochemical localization of megalin (a, b) and RBP (c, d) in the renal proximal tubules of megalinlox/lox and megalinlox/lox; apoECre mice. Arrows indicate that immunoreactivity of both megalin and RBP is lower in mice with a kidney-specific inactivation of the megalin gene.

View larger version (11K): [in this window] [in a new window]   FIGURE 2 Semiquantitative detection of RBP in plasma of megalinlox/lox and megalinlox/lox; apoECre mice after consumption of a basal or VA-free (–VA) diet for 42 d. Values are expressed in arbitrary intensity units and are means ± SD, n = 5. There was a significant effect of VA (P < 0.05, 2-way ANOVA). Groups not sharing a letter differ, P < 0.05.

    VA in kidney and other tissues. After the 42-d feeding period, total kidney VA and retinyl ester concentrations were affected only by diet (both P < 0.05, 2-way ANOVA), and not by genotype (Table 1). ROH levels, which accounted for 67–78% of total VA, were not affected by either genotype or diet. After consumption of the VA-free diet for 84 d, concentrations of VA, ROH and retinyl esters in the kidneys did not differ between megalin^lox/lox; apoE^Cre and megalin^lox/lox mice (Table 2). In addition, levels of VA in spleen (megalin^lox/lox; apoE^Cre vs. megalin^lox/lox: 2.02 ± 0.83 vs. 2.15 ± 0.27 nmol/g), brain (0.50 ± 0.08 vs. 0.59 ± 0.17 nmol/g) and eye (22.3 ± 7.52 vs. 23.1 ± 7.74 nmol/g) did not differ among the groups.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Megalin is well recognized as an endocytic receptor essential for tubular uptake of vital substances filtered through the glomerulus. In particular, tubular reabsorption of vitamin-carrier proteins appears to be important both for maintaining vitamin homeostasis and in the metabolism of certain vitamins (24). Several vitamin-carrier proteins including RBP, vitamin D-binding protein, and transcobalamin were identified as ligands for megalin through the analysis of kidney and urine samples from mice with targeted disruption of the megalin gene. Thus, it is established that megalin-mediated uptake of these binding proteins prevents the loss of their ligands in urine (9,13,25).

For evaluation of the animal model, we performed IHC, showing that the receptor expression of megalin was absent in most but not all proximal tubular cells of megalin^lox/lox; apoE^Cre mice. The remnant megalin activity was not determined in this study, but it can be assumed to affect 10–15% of the nephrons as previously described (15,17). In agreement with this notion, some tubules stained positive for internalized RBP reflecting a small number of megalin-expressing cells in megalin^lox/lox; apoE^Cre kidneys. Thus, megalin^lox/lox; apoE^Cre mice represent a suitable model with which to evaluate the role of this receptor in renal and systemic VA metabolism. One should still keep in mind, however, that the residual megalin activity in these mice may partially obscure the actual extent of VA imbalance caused by megalin deficiency. In humans, urinary ROH excretion is observed in several pathophysiologic conditions, such as severe infections, sepsis, and pregnancy complications (26–28). Consequences of such urinary ROH loss may be a decrease in circulating ROH and impaired VA status (29). Moreover, urinary ROH excretion during infection addresses research questions to recommendations of dietary VA supply (30,31). Therefore, the model of kidney-specific megalin deficiency may be of particular interest in elucidating the underlining mechanisms and the effects of urinary ROH excretion on the whole-body VA turnover.

This study was designed to examine the effects of megalin deficiency on the mobilization of hepatic VA stores, the transport of ROH to tissues, and its excretion in urine of mice having a kidney-specific defect of the megalin gene. The results demonstrate that the kidney-specific megalin inactivation causes urinary ROH excretion and lower levels of hepatic retinyl ester, likely due to the accelerated depletion of hepatic VA stores necessary for the stabilization of otherwise homeostatic plasma ROH levels. Impairment of VA homeostasis is seen only in mice fed a VA-free diet, whereas megalin-deficient mice fed a VA-replete diet seem capable of compensating the uncontrolled urinary loss of ROH through constant dietary intake of VA.

The finding that hepatic VA concentrations in megalin^lox/lox; apoE^Cre mice were significantly lower compared with controls indicates that kidney-specific megalin inactivation affects hepatic VA storage, a result that provides new insights into the regulation of VA store mobilization. Furthermore, the absence of lower plasma ROH concentrations in megalin^lox/lox; apoE^Cre mice after consumption of a VA-free diet for 84 d demonstrates that plasma ROH does not decline until virtually all VA in the liver has been exhausted; this agrees with the general concept that the liver content of VA is used to regulate plasma ROH homeostasis (3). Although only 5–14% of RBP-ROH (holo-RBP) is normally not in complex with TTR and therefore free for filtration by the renal glomerula (32,33), the most likely cause for rapid hepatic VA mobilization in megalin^lox/lox; apoE^Cre mice is the uncontrolled urinary ROH excretion. The mechanism by which urinary ROH loss affects hepatic VA mobilization remains highly suggestive but not fully resolved. In acute renal failure, kidney malfunction leads to an increase in plasma ROH and RBP due to insufficient catabolism of apo-RBP (RBP without bound ROH), which was found to be a positive physiological feedback signal from peripheral tissues for hepatic ROH release (34,35). Recently, Quadro et al. (36) investigated the mechanisms of mobilization of hepatic retinoid stores by human RBP, which was expressed on a muscle-specific promoter in an RBP-deficient mouse model. The authors demonstrated that human RBP, unlike the murine form, was unable to mobilize hepatic VA stores. Although their work scrutinized the role of extrahepatically synthesized human RBP in the mobilization of hepatic VA, it leaves open the question of what role circulating murine RBP actually plays in the hepatic VA metabolism of mouse models.

In addition to the liver, the kidneys are also a major active site for VA metabolism. The second greatest abundance of newly absorbed VA is found in the kidneys, and kinetic studies revealed that the kidneys play an important role in the recycling and turnover of ROH (37). In rats, 50% of plasma ROH turnover is related to the kidneys, which is almost 5 times the disposal rate for VA (7,8), indicating that most of the ROH taken up by the kidneys has to be reabsorbed in the proximal tubules by megalin-mediated endocytosis. Our finding of high renal ROH concentrations, which accounted for 67–78% of total VA, reflects the important role of the kidneys in renal ROH metabolism. Surprisingly, identical renal ROH concentrations in normal and megalin-deficient mice suggest that megalin-deficiency does not affect renal VA accumulation. Thus, we conclude that the remnant megalin activity in megalin^lox/lox; apoE^Cre mice may be adequate to drive mechanisms that influence renal ROH accumulation. Moreover, it is unclear how much VA derived from circulation may contribute to the ROH pool in renal tissue. It was shown that ROH recycling in the kidney can be affected by VA status. During VA deficiency, ROH recycling through the kidneys is 5 times higher compared with the situation of normal VA status, in which the ROH recycling is highest within the liver (8). Higher levels of retinyl esters and total VA in the kidneys of megalin^lox/lox; apoE^Cre mice fed the basal diet indicate that megalin deficiency possibly influences ROH esterification activity in the kidneys, an aspect that requires further consideration and exploration.

When viewed in total, our results provide new insights into the intrinsic role of megalin in the regulation of systemic VA metabolism. The receptor is essential for ROH homeostasis due to its role in ROH-RBP reuptake in the kidney proximal tubules and through interaction with the mobilization of hepatic VA stores. We therefore propose that this animal model could be of use for future studies addressing the regulation of VA metabolism, especially for questions on the mechanisms involved in recycling and turnover of VA.

	ACKNOWLEDGMENTS

 
We thank A. Hurtienne, E. Pilz, E. Meyer, and U. Neumann for their skillful technical assistance.

	FOOTNOTES

 
¹ Presented in part at 40. Wissenschaftlicher Kongress der Deutschen Gesellschaft für Ernährung [Raila J, Leheste JR, Willnow TE, Schweigert FJ. Einfluss einer reduzierten Vitamin-A-Versorgung mit dem Futter auf den Transport und die Speicherung von Vitamin A bei Megalin-defizitären-Mäusen (abstract). Proc Germ Nutr Soc. 2003;5:27].

² Supported by grants from the Deutsche Forschungsgemeinschaft, DFG, and the Bundesministerium für Bildung und Forschung, BMBF (to T.E.W.).

⁴ Abbreviations used: BSA, bovine serum albumin; IHC, immunohistochemistry; megalin^lox/lox; apoE^Cre, conditional megalin knockout mice; megalin^lox/lox, control mice; RBP, retinol-binding protein; RE, retinol equivalent; ROH, retinol; TBS, Tris-buffered saline; VA, vitamin A; –VA, diet without vitamin A.

Manuscript received 19 May 2005. Initial review completed 8 June 2005. Revision accepted 20 July 2005.

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