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Evidence Indicating that Renal Tubular Metabolism of Leptin Is Mediated by Megalin But Not by the Leptin Receptors

Division of Clinical Nephrology and Rheumatology (H.H., A.S., T.T., A.T., Y.X., K.S., J.J.K., F.G.) and Department of Applied Molecular Medicine (A.S., T.T.), Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan; and Division of Intensive Care Medicine (J.J.K.), Niigata University Medical Hospital, Niigata 951-8510, Japan

Address all correspondence and requests for reprints to: Akihiko Saito, M.D., Ph.D., Department of Applied Molecular Medicine, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata 951-8510, Japan. E-mail: akisaito{at}med.niigata-u.ac.jp.

	Abstract

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Leptin is secreted by adipocytes and is a circulating factor that regulates food intake and energy expenditure. Its serum level is elevated in patients with renal failure and has been suggested to be associated with malnutritional factors in these patients. Leptin has been suggested to be primarily metabolized by the kidneys, although the precise molecular mechanisms are not known. The purpose of this study was to determine the nephron segments and potential receptors involved in renal leptin metabolism. To determine the segment involved in leptin uptake, we performed histoautoradiography of kidney sections obtained from rats that had been injected iv with ¹²⁵I-leptin. The ability of megalin, a multiligand endocytic receptor in the proximal tubules, to bind and endocytose leptin was examined by ligand blotting analysis, quartz-crystal microbalance, and degradation assays using megalin-expressing rat yolk sac L2 cells. Immunohistochemistry was performed to localize leptin receptors (LEP-R) in the rat kidney using two antibodies that recognize different epitopes on the LEP-R proteins. Circulating ¹²⁵I-leptin was filtered by glomeruli and internalized by proximal convoluted tubules. Megalin bound leptin in the presence of Ca²⁺ and mediated its cellular internalization and degradation. On immunohistochemistry, LEP-R were localized in the proximal straight tubules, loops of Henle, distal tubules, and collecting ducts. In conclusion, circulating leptin was filtered by glomeruli and taken up by proximal convoluted tubules, where megalin likely mediates its binding and uptake. The localization of LEP-R suggests that they are not primarily involved in leptin metabolism in the proximal tubules.

	Introduction

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LEPTIN IS A 16-kDa protein product of the obese (ob) gene (Lep), which is mutated in the massively obese (ob/ob) mouse. Secreted mainly by white fat adipocytes (1), circulating leptin functions to lower body weight by reducing appetite and altering metabolic processes (2, 3, 4, 5). Leptin exerts these effects by binding with leptin receptors (LEP-R) (6) in the hypothalamus. The serum leptin concentration was found to be a good marker of body fat mass in obese patients (7) and in nonobese patients with chronic renal failure (CRF) (8). A number of studies have demonstrated that uremic patients have a markedly elevated serum leptin level or serum leptin to fat mass ratio (8, 9, 10, 11, 12), suggesting that the kidneys play an important role in the clearance of leptin and that hyperleptinemia may be a factor inducing anorexia and weight loss in patients with CRF. Experiments in which radiolabeled leptin was administered to rats also indicated that the kidneys are the main organ that metabolizes circulating leptin (13). Although the process was suggested to involve glomerular filtration followed by metabolic degradation in the renal tubules (14), the site of metabolic degradation of leptin in the tubules and the molecular mechanisms of these processes have not been determined.

To date, six isoforms of LEP-R have been identified. They are produced by alternative splicing of the LEP-R gene and include a long form (LEP-Rb) and five short forms (LEP-Ra, c, d, e, and f) (15, 16). In the murine kidney, in situ hybridization showed that the mRNA of LEP-Ra and LEP-Rb was expressed in the inner zone of the medulla (17). In contrast, the rat kidney was shown to express LEP-Ra and LEP-Rf but not LEP-Rb by RT-PCR (16). However, the precise immunohistochemical localization of LEP-R in the kidney and their role in leptin clearance have not been determined.

Megalin, which was originally identified as a target antigen of experimental Heymann nephritis (18), is a large ( 600 kDa) glycoprotein that belongs to the low-density lipoprotein (LDL) receptor family (19). It is abundantly expressed at the apical membranes of proximal tubule cells that reabsorb and metabolize proteins filtered by glomeruli (20). Megalin is located in clathrin-coated pits, it internalizes its ligands into endocytic compartments, and it is then recycled to the cell surface (20, 21). Ligands for megalin that are endocytosed include multiple low-molecular-weight proteins such as transcobalamin-B₁₂, vitamin D-binding protein, retinol-binding protein, PTH, insulin, ß₂-microglobulin, epidermal growth factor, prolactin, lysozyme, cytochrome c, ₁-microglobulin, pancreatitis-associated protein 1, odorant-binding protein, and transthyretin (22). Thus, it is very likely that megalin is also a leptin clearance receptor.

The present study demonstrates that glomerular-filtered leptin is primarily taken up by the apical domains of proximal convoluted tubule cells and that leptin is a novel ligand that is endocytosed by megalin. The immunohistochemical localization of LEP-R in the kidney is also demonstrated.

	Materials and Methods

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Reagents
Recombinant rat leptin, BSA (Fraction V), and chloroquine were purchased from Sigma-Aldrich (St. Louis, MO). Iodo-Beads were obtained from Pierce (Rockford, IL). Na¹²⁵I (IODINE-125, 3.7 GBq/ml), PD-10 columns prepacked with Sephadex G-25, and Hyperfilm MP were purchased from Amersham Biosciences (Piscataway, NJ). Autoradiography emulsion NTB-3 and developer D-19 were purchased from Eastman Kodak Company (Rochester, NY). 1-(3-dimethylaminopropyl)-3 ethylcarbodiimide hydrochloride was purchased from Sigma-Aldrich, and N-hydroxysuccinimide was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Normal rabbit serum (10%) in PBS with 0.1% sodium azide was purchased from Nichirei CO. (Tokyo, Japan). Fuchsin alkaline phosphatase substrate solution with an endogenous alkaline phosphatase inhibitor and 3,3'-diaminobenzidine substrate-chromogen solution were obtained from Dako (Carpinteria, CA), and normal goat serum was purchased from Chemicon (Temecula, CA). Ready Gel J and Immun-Blot polyvinylidine difluoride membranes were obtained from Bio-Rad Laboratories (Hercules, CA). DMEM and fetal calf serum were purchased from Life Technologies, Inc. (Rockville, MD).

Antibodies
Antirat megalin rabbit sera were raised as described previously (23), and protein A-purified IgG was prepared as described previously (24). Affinity-purified goat polyclonal IgG that had been raised against a peptide sequence (AA 877–894) at the intracellular carboxyl terminus of mouse LEP-Ra (M-18, sc-1834) and rabbit polyclonal IgG that had been raised against a recombinant protein corresponding to AA 541–840 in the extracellular domain of human LEP-R (H-300, sc-8325) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antigoat IgG conjugated to peroxidase-labeled amino acid polymers was purchased from Nichirei CO. Goat antirabbit IgG conjugated to alkaline phosphatase-labeled dextran polymers or to peroxidase-labeled dextran polymers were obtained from Dako.

Radioiodination
Recombinant rat leptin (100 µg) was radioiodinated using 1 mCi Na¹²⁵I and one Iodo-Bead according to the manufacturer’s instructions. Free Na¹²⁵I was removed from the labeled proteins using PD-10 columns. The specific activity of ¹²⁵I-leptin was 1.0–2.4 x 10⁶ cpm/µg.

Histoautoradiography
This study was approved by the Animal Committee of Niigata University Graduate School of Medical and Dental Sciences. Six-wk-old male Sprague Dawley rats (Charles River Japan, Yokohama, Japan), weighing between 140 and 160 g, were anesthetized with ether and injected with ¹²⁵I-leptin (10 µg in 0.5 ml) into the femoral vein. After 10 min, the rats were killed by systemic perfusion with saline. The kidneys were removed and fixed with 4% paraformaldehyde in PBS overnight. They were then dehydrated through graded ethanol, embedded in paraffin, and sectioned at a thickness of 3 µm. The sections were deparaffinized with xylene, rehydrated with ethanol and distilled water, and coated with NTB-3 photoemulsion. Autoradiography was performed at 4 C for 10 d. Slides were developed with developer D-19 and counterstained with hematoxylin and eosin. Some sections were subsequently used for immunohistochemical analysis; slides were treated with normal goat serum at room temperature for 30 min, followed by incubation with rabbit antimegalin IgG (200 µg/ml) at 4 C for 12 h. The sections were then incubated with goat antirabbit IgG conjugated with alkaline phosphatase-labeled dextran polymers at room temperature for 30 min. After washing with PBS, the sections were incubated with a Fuchsin alkaline phosphatase substrate solution with an endogenous alkaline phosphatase inhibitor.

Protein purification
Recombinant rat receptor-associated protein (RAP) was prepared using a prokaryotic expression system as a fusion protein with glutathione-S-transferase (GST), as described previously (25). Rat megalin was prepared from renal microvillar membranes by affinity chromatography using monoclonal antibody 20B, as described previously (26).

Ligand blotting analysis
Ligand blotting analysis was carried out as described previously (27). Rat megalin (5 µg/lane) was prepared in a Laemmli sample buffer without ß-mercaptoethanol and subjected to 4% SDS-PAGE. The protein was electrophoretically transferred to polyvinylidine difluoride membranes. Nonspecific sites on the membranes were blocked by incubation in buffer A (10 mM HEPES, pH 7.4; 150 mM NaCl, 2 mM CaCl₂, and 0.2% Tween 20) containing 3% BSA at room temperature for 2 h. The membranes were then incubated in a blocking buffer containing ¹²⁵I-leptin (1 x 10⁶ cpm/ml) in the presence or absence of unlabeled leptin (100-fold excess of radiolabeled leptin) or EDTA (20 mM) at room temperature for 3 h and washed four times with buffer A (15 min each). Autoradiography using Hyperfilm MP was performed at –80 C with an intensifying screen for 3 d.

Quartz-crystal microbalance (QCM)
The binding of leptin to megalin was also examined using a highly sensitive 27-MHz QCM (Affinix Q; Initium, Tokyo, Japan) as described previously (28, 29). Briefly, the QCM sensor tips were activated with a 1:1 mixture of 100 mg/ml 1-(3-dimethylaminopropyl)-3 ethylcarbodiimide hydrochloride and 100 mg/ml N-hydroxysuccinimide in water. Rat megalin was then immobilized at a concentration of 10 µg/ml in buffer B (10 mM HEPES, pH 7.4; 150 mM NaCl, and 2 mM CaCl₂) on the sensor tips. The sensor tips were soaked at 23 C in 8 ml of buffer B or C (10 mM HEPES, pH 7.4; 150 mM NaCl, and 10 mM EDTA) in the incubation chamber. Leptin, diluted in the same buffer, was added into the chamber for binding to megalin. The resonance frequency of the QCM was defined as the 0 position after equilibrium. The stability and drift of the 27-MHz QCM frequency in the solution were ± 3 Hz. The binding affinity was determined from the frequency changes upon cumulative injection of a small volume (8 µl) of leptin solution (30).

Cellular internalization and degradation assays
Megalin-expressing rat yolk sac tumor-derived L2 cells (31) were grown (37 C, 5% CO₂) to confluence (1 x 10⁵ cells/well) in DMEM supplemented with 10% fetal calf serum on 12-well tissue culture plates coated with 1% gelatin. The cells were washed with DMEM and incubated in DMEM containing 0.1% BSA with ¹²⁵I-leptin (1.0 µg/ml) in the absence or presence of a competitor. Incubation was also performed in the presence of chloroquine (100 µM), an inhibitor of lysosomal enzyme activity (32). After incubation, the culture media were precipitated with 20% trichloroacetic acid (TCA), and the radioactivity level of the TCA-soluble degradation products was quantified by counting. To correct for liberation of iodine from ¹²⁵I-labeled ligands, the level of TCA-soluble radioactivity in medium incubated without cells was subtracted from that found in the samples. Statistical analyses were carried out using the unpaired Student’s t test.

Immunohistochemistry for LEP-R
Six-week-old male Sprague Dawley rats were anesthetized with ether and killed by systemic perfusion with PBS containing 4% paraformaldehyde and 0.1% glutaraldehyde. The kidneys were removed and fixed with the same fixative at 4 C overnight. They were then dehydrated through graded ethanol, embedded in paraffin, and sectioned at a thickness of 3 µm. The sections were deparaffinized with xylene and rehydrated with ethanol and distilled water. Endogenous peroxidase activity was quenched by incubation in 0.6% H₂O₂ in methanol for 30 min. The slides were rinsed with PBS, and incubated with normal rabbit serum (10%) in PBS at room temperature for 30 min. Subsequently, they were incubated with goat anti-LEP-R IgG (M-18, 2 µg/ml) at 4 C for 24 h. After washing with PBS, the sections were incubated with rabbit antigoat IgG conjugated with peroxidase-labeled amino acid polymers at room temperature for 30 min. After washing with PBS, the sections were incubated with the 3,3'-diaminobenzidine substrate-chromogen solution. Immunohistochemistry was also carried out using the same procedure with rabbit anti-LEP-R IgG (H-300, 4 µg/ml) as the primary antibody and goat antirabbit IgG conjugated with peroxidase-labeled dextran polymers as the secondary antibody.

	Results

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Circulating leptin was filtered by glomeruli and taken up by the apical portion of proximal convoluted tubules
Circulating leptin was reported to be eliminated primarily by the kidneys (13). The process was suggested to involve glomerular filtration followed by tubular uptake and degradation (14). To confirm this process and further define the tubular segments that participate in leptin uptake, histoautoradiography was performed on kidney sections from rats that had been iv-administered ¹²⁵I-leptin. As shown in Fig. 1A, radioactivity was exclusively detected in the renal cortex in structures histologically suggested to be proximal convoluted tubules. No apparent uptake was detected in the glomeruli, vasculatures, or other tubular segments.

View larger version (145K): [in this window] [in a new window]   FIG. 1. Glomerular-filtered 125I-labeled leptin is primarily taken up by the apical membrane of proximal convoluted tubules. (A) 125I-labeled leptin was administered iv to rats, and the renal uptake was investigated using histoautoradiography, which showed that leptin was taken up by the cortical tubules. The sections were then stained with hematoxylin and eosin. The renal cortex (C), outer stripe (OS), and inner stripe (IS) are indicated. Original magnifications, x20. (B) Alkaline-phosphatase-based immunostaining with an antibody against megalin, a proximal tubular marker, of kidney sections that had been subjected to histoautoradiography. Leptin molecules were primarily taken up by the apical membrane of proximal convoluted tubules (PCT) in the early stages of their passage through megalin-expressing tubular regions. No leptin uptake was found in the glomeruli (G) or distal tubules (DT). Original magnifications, x100.

Leptin bound to megalin in a Ca²⁺-dependent manner
Megalin has been implicated in playing a scavenger function and mediating the reabsorption of proteins from the glomerular filtrate and is known to bind a number of ligands in a Ca²⁺-dependent manner. To determine whether megalin has the potential to mediate leptin absorption, immunoaffinity-purified megalin was assayed for leptin binding activity by ligand blotting analysis. ¹²⁵I-leptin bound to immobilized megalin in buffer containing Ca², but the binding was suppressed by the addition of 100-fold unlabeled leptin and was abolished in the presence of EDTA (Fig. 2). These results indicate that megalin is capable of specifically interacting with leptin and that the binding is Ca²⁺ dependent. To confirm the results and evaluate the binding affinity, QCM analysis was carried out (Fig. 3). In the analysis, leptin bound to megalin in a Ca²⁺-dependent manner (Fig. 3, A and B), and the binding was inhibited by RAP, a competitor for ligand binding to megalin (Fig. 3C) (26). On the affinity analysis, the maximum binding quantity and equilibrium dissociation constant for the binding of leptin to megalin were found to be 3.0 x 10² Hz and 0.20 µmol/liter, respectively (Fig. 3D). This equilibrim dissociation constant value is comparable to those of other low-molecular-weight protein ligands that bind to megalin (33).

View larger version (34K): [in this window] [in a new window]   FIG. 2. Ligand blotting analysis for the binding of leptin to megalin. Rat megalin (5 µg/lane) was loaded on 4% sodium dodecyl sulfate-polyacrylamide gels, transferred to polyvinylidine difluoride membranes, and incubated with 125I-labeled leptin in a buffer containing 2 mM Ca2+ in the absence (lane 1) or presence of 100-fold unlabeled leptin (lane 2) or 20 mM EDTA (lane 3). Radiolabeled leptin directly bound to megalin, and the binding was inhibited by the addition of unlabeled leptin and by the addition of EDTA, indicating that leptin specifically binds to megalin in a Ca2+-dependent manner.

View larger version (20K): [in this window] [in a new window]   FIG. 3. QCM analysis for the binding of leptin to megalin. Leptin was added to the QCM chamber in which megalin had been immobilized on the sensor tip. The binding of leptin to megalin was shown by the presence of a change in the frequency (F). A, Leptin (0.5 µg/ml) was added to the QCM chamber in a buffer containing Ca2+ (2 mM). Leptin was added at the time point indicated by the arrow. B, Leptin (0.5 µg/ml) was added to the QCM chamber in a buffer containing EDTA (20 mM). Leptin was added at the time point indicated by the arrow. Leptin did not bind to megalin in this condition, indicating that leptin binds to megalin in a Ca2+-dependent manner. C, RAP (0.5 µg/ml) was added to the QCM chamber and allowed to reach the steady state of binding megalin. Then, leptin (0.5 µg/ml) was added to the chamber at the time point indicated by the arrow. The binding of leptin to megalin was inhibited by RAP. D, To evaluate the affinity of binding of leptin to megalin, the frequency changes upon cumulative addition of leptin to the QCM chamber were analyzed by fitting to a Michaelis-Menten plot, with an inset Lineweaver-Burk plot.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Megalin mediated the cellular internalization and degradation of leptin. Cultured L2 cells were incubated with 125I-labeled leptin (1 µg/ml) in DMEM containing 0.1% BSA for 2 h for cellular degradation assays in the absence (control) or presence of the indicated competitors. The addition of unlabeled leptin to the culture media significantly inhibited the degradation of 125I-labeled leptin, indicating the presence of a specific pathway. The addition of chloroquine (100 µM) suppressed the degradation of 125I-labeled leptin, suggesting that the process was due to receptor-mediated endocytosis. The addition of antimegalin IgG (200 µg/ml) or GST-RAP (100 µg/ml) inhibited the degradation of 125I-labeled leptin compared with the addition of nonimmune IgG (200 µg/ml) or GST (100 µg/ml), respectively, indicating that megalin mediates the cellular internalization and degradation of leptin. Values (mean ± SD, n = 4) are expressed relative to the control. *, P < 0.01.

View larger version (179K): [in this window] [in a new window]   FIG. 5. Immunohistochemical localization of LEP-R in the rat kidney. Immunohistochemical expression of LEP-R is shown by using two anti-LEP-R antibodies, M-18 (A, C, and E) and H-300 (B, D, and F), in the renal cortex (A and B), the outer stripe (C and D), and the inner stripe (E and F). In the renal cortex, the distal tubules (DT) are positively stained, but the glomeruli (G) and proximal convoluted tubules (PCT) are not. In the outer stripe, the proximal straight tubules (PST) and descending thin limbs (DL) are positively stained. In the inner stripe, the ascending thick limbs (AL) and collecting ducts (CD) are also positively stained. Vascular bundles (VB) are all negative for LEP-R. Original magnifications, x100.

	Discussion

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In the present study, immunohistochemistry of kidney sections showed for the first time that LEP-R were localized in the proximal straight tubules, loops of Henle, distal tubules, and collecting ducts in rats. The distribution of LEP-R was confirmed by using two antibodies that recognize different epitopes on LEP-R proteins; one antibody was expected to recognize all LEP-R isoforms except for LEP-Re, which does not have transmembrane and intracellular domains, and the second antibody was expected to recognize all LEP-R isoforms. A significant finding was that LEP-R immunoreactivity was absent at the major sites of accumulation of circulating leptin, suggesting that LEP-R are not involved in the clearance of leptin by the proximal convoluted tubules.

There are reports indicating that leptin acts on the rat kidney to increase diuresis (36) and natriuresis (37). However, another report suggested that the rat kidney did not express LEP-Rb (16), which is known to mediate leptininduced Janus kinase/signal transducer and activator of transcription 3 signal transduction (38). Therefore, the LEP-R expression detected in the rat kidney may have been derived from short isoforms such as LEP-Ra or LEP-Rf. Recently, LEP-Ra was suggested to mediate the transcellular transport of leptin across the blood-brain barrier (39). However, it is not known whether LEP-Ra is involved in leptin-induced signal transduction. To clarify the functions of LEP-R in the kidney, it is necessary to identify the particular isoforms expressed in the tubular segments using isoform-specific antibodies and to examine possible leptin transport activity and signal transduction. Because the proximal convoluted tubules appear to be the main site of accumulation of glomerular-filtered leptin, it should also be investigated whether this is the site of the action of leptin in the kidneys and, further, whether megalin plays a role in leptin-induced signal transduction. LDL receptor-related protein, another member of the LDL receptor family that is very similar to megalin in structure and function, is also recognized as a receptor that not only scavenges many ligands but also processes the signals that they evoke (40). Recent studies have identified intracellular proteins that bind to the cytoplasmic tail of megalin (41, 42, 43, 44), which might be involved in the intracellular signaling induced by the binding of leptin to megalin.

Anorexia and malnutrition are distinctive clinical features of uremic patients, and hyperleptinemia has been suggested as a contributing factor. Cross-sectional studies (10, 45, 46) as well as a recent longitudinal analysis (47) have indicated that an elevated serum leptin level or elevated ratio of serum leptin to fat mass is correlated with malnutritional markers in patients with CRF. Thus, leptin may be a uremic toxin protein like PTH and ß₂-microglobulin (48). Such proteins also include advanced glycation end products, which we have recently found are endocytosed by L2 cells via a megalin-dependent mechanism (27). The identification of leptin as a ligand endocytosed via megalin, like PTH (49) and ß₂-microglobulin (50), will aid in the development of megalin-mediated strategies to facilitate the removal of leptin in patients with uremia. We have recently developed a novel cell therapy model that involves subcutaneous implantation of megalin-expressing cells to metabolize ß₂-microglobulin in experimental renal failure (51). The cell therapy model may also be useful for reducing the markedly elevated serum leptin level in patients with CRF, thereby alleviating uremic malnutrition.

In conclusion, we confirmed that glomerular-filtered leptin is taken up primarily by proximal convoluted tubule cells, in which megalin likely functions in its binding and internalization. In contrast, immunohistochemical LEP-R expression was localized in the proximal straight tubules, loops of Henle, distal tubules, and collecting ducts in rats, suggesting that LEP-R are not involved in the metabolism of leptin in the proximal tubules. Further studies are required to elucidate the molecular mechanisms of the actions of leptin on the kidneys.

	Acknowledgments

 
The authors thank Dr. Akiyoshi Kakita for technical assistance and Jin Hayatsu for manuscript preparation.

	Footnotes

 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (14571018).

Abbreviations: CRF, Chronic renal failure; GST, glutathione-S-transferase; LDL, low-density lipoprotein; LEP-R, leptin receptors; QCR, quartz-crystal microbalance; RAP, receptor-associated protein; TCA, trichloroacetic acid.

Received January 22, 2004.

Accepted for publication April 29, 2004.

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