Megalin Function Research Articles

Influenza virus decreases albumin uptake and megalin expression in alveolar epithelial cells.

Acute respiratory distress syndrome (ARDS) is a common complication of influenza virus (IV) infection. During ARDS, alveolar protein concentrations often reach 40-90% of plasma levels, causing severe impairment of gas exchange and promoting deleterious alveolar remodeling. Protein clearance from the alveolar space is at least in part facilitated by the multi-ligand receptor megalin through clathrin-mediated endocytosis. To investigate whether IV infection impairs alveolar protein clearance, we examined albumin uptake and megalin expression in MLE-12 cells and alveolar epithelial cells (AEC) from murine precision-cut lung slices (PCLS) and in vivo, under IV infection conditions by flow cytometry and western blot. Transcriptional levels from AEC and broncho-alveolar lavage (BAL) cells were analyzed in an in-vivo mouse model by RNAseq. IV significantly downregulated albumin uptake, independently of activation of the TGF-β1/GSK3β axis that has been previously implicated in the regulation of megalin function. Decreased plasma membrane abundance, total protein levels, and mRNA expression of megalin were associated with this phenotype. In IV-infected mice, we identified a significant upregulation of matrix metalloproteinase (MMP)-14 in BAL fluid cells. Furthermore, the inhibition of this protease partially recovered total megalin levels and albumin uptake. Our results suggest that the previously described MMP-driven shedding mechanisms are potentially involved in downregulation of megalin cell surface abundance and clearance of excess alveolar protein. As lower alveolar edema protein concentrations are associated with better outcomes in respiratory failure, our findings highlight the therapeutic potential of a timely MMP inhibition in the treatment of IV-induced ARDS.

Megalin and Vitamin D Metabolism-Implications in Non-Renal Tissues and Kidney Disease.

Megalin is an endocytic receptor abundantly expressed in proximal tubular epithelial cells and other calciotropic extrarenal cells expressing vitamin D metabolizing enzymes, such as bone and parathyroid cells. The receptor functions in the uptake of the vitamin D-binding protein (DBP) complexed to 25 hydroxyvitamin D3 (25(OH)D3), facilitating the intracellular conversion of precursor 25(OH)D3 to the active 1,25 dihydroxyvitamin D3 (1,25(OH)2D3). The significance of renal megalin-mediated reabsorption of 25(OH)D3 and 1,25(OH)2D3 has been well established experimentally, and other studies have demonstrated relevant roles of extrarenal megalin in regulating vitamin D homeostasis in mammary cells, fat, muscle, bone, and mesenchymal stem cells. Parathyroid gland megalin may regulate calcium signaling, suggesting intriguing possibilities for megalin-mediated cross-talk between calcium and vitamin D regulation in the parathyroid; however, parathyroid megalin functionality has not been assessed in the context of vitamin D. Within various models of chronic kidney disease (CKD), megalin expression appears to be downregulated; however, contradictory results have been observed between human and rodent models. This review aims to provide an overview of the current knowledge of megalin function in the context of vitamin D metabolism, with an emphasis on extrarenal megalin, an area that clearly requires further investigation.

Sodium-glucose Cotransporter Type 2 Inhibitors: A New Insight into the Molecular Mechanisms of Diabetic Nephropathy.

Diabetic nephropathy is one of the chronic microvascular complications of diabetes and is a leading cause of end-stage renal disease. Fortunately, clinical trials have demonstrated that sodium-glucose cotransporter type 2 inhibitors could decrease proteinuria and improve renal endpoints and are promising agents for the treatment of diabetic nephropathy. The renoprotective effects of sodium-glucose cotransporter type 2 inhibitors cannot be simply attributed to their advantages in aspects of metabolic benefits, such as glycemic control, lowering blood pressure, and control of serum uric acid, or improving hemodynamics associated with decreased glomerular filtration pressure. Some preclinical evidence suggests that sodium-glucose cotransporter type 2 inhibitors exert their renoprotective effects by multiple mechanisms, including attenuation of oxidative and endoplasmic reticulum stresses, anti-fibrosis and anti-inflammation, protection of podocytes, suppression of megalin function, improvement of renal hypoxia, restored mitochondrial dysfunction and autophagy, as well as inhibition of sodium-hydrogen exchanger 3. In the present study, the detailed molecular mechanisms of sodium-glucose cotransporter type 2 inhibitors with the actions of diabetic nephropathy were reviewed, with the purpose of providing the basis for drug selection for the treatment of diabetic nephropathy.

Abstract 10460: Urinary Fatty Acid Binding Protein 1 is a Biomarker for Impaired Proximal Tubular Protein Reabsorption and is Synergistically Enhanced by Concurrent Liver Injury

Introduction: Urinary fatty acid binding protein 1 (FABP1, also known as liver-type FABP) has been implicated as a biomarker of acute kidney injury (AKI) in humans. However, the precise biological mechanisms underlying its elevation remain elusive. Here, we show that urinary FABP1 primarily reflects impaired protein reabsorption in proximal tubule epithelial cells (PTECs). Methods and Results: Bilateral nephrectomy resulted in a marked increase in serum FABP1 levels, suggesting that the kidney is an essential organ for removing serum FABP1. Injected recombinant FABP1 was filtered through the glomeruli and robustly reabsorbed via the apical membrane of PTECs. Urinary FABP1 was significantly elevated in mice devoid of megalin, a giant endocytic receptor for protein reabsorption. Elevation of urinary FABP1 was also observed in patients with Dent disease, a rare genetic disease characterized by defective megalin function in PTECs. Urinary FABP1 levels were exponentially increased following acetaminophen overdose, with both nephrotoxicity and hepatotoxicity observed. FABP1-deficient mice with liver-specific overexpression of FABP1 showed a massive increase in urinary FABP1 levels upon acetaminophen injection, indicating that urinary FABP1 is liver-derived. Lastly, we employed transgenic mice expressing diphtheria toxin receptor (DT-R) either in a hepatocyte- or PTEC-specific manner, or both. Upon diphtheria toxin (DT) administration, massive excretion of urinary FABP1 was induced in mice with both kidney and liver injury, while mice with either injury type showed marginal excretion. Conclusions: Collectively, our data demonstrated that intact PTECs have a considerable capacity to reabsorb liver-derived FABP1 through a megalin-mediated mechanism. Thus, urinary FABP1, which is synergistically enhanced by concurrent liver injury, is a biomarker for impaired protein reabsorption in AKI. These findings address the use of urinary FABP1 as a biomarker of histologically injured PTECs that secrete FABP1 into primary urine, and suggest the use of this biomarker to simultaneously monitor impaired tubular reabsorption and liver function.

Urinary FABP1 is a biomarker for impaired proximal tubular protein reabsorption and is synergistically enhanced by concurrent liver injury.

Urinary fatty acid binding protein 1 (FABP1, also known as liver‐type FABP) has been implicated as a biomarker of acute kidney injury (AKI) in humans. However, the precise biological mechanisms underlying its elevation remain elusive. Here, we show that urinary FABP1 primarily reflects impaired protein reabsorption in proximal tubule epithelial cells (PTECs). Bilateral nephrectomy resulted in a marked increase in serum FABP1 levels, suggesting that the kidney is an essential organ for removing serum FABP1. Injected recombinant FABP1 was filtered through the glomeruli and robustly reabsorbed via the apical membrane of PTECs. Urinary FABP1 was significantly elevated in mice devoid of megalin, a giant endocytic receptor for protein reabsorption. Elevation of urinary FABP1 was also observed in patients with Dent disease, a rare genetic disease characterized by defective megalin function in PTECs. Urinary FABP1 levels were exponentially increased following acetaminophen overdose, with both nephrotoxicity and hepatotoxicity observed. FABP1‐deficient mice with liver‐specific overexpression of FABP1 showed a massive increase in urinary FABP1 levels upon acetaminophen injection, indicating that urinary FABP1 is liver‐derived. Lastly, we employed transgenic mice expressing diphtheria toxin receptor (DT‐R) either in a hepatocyte‐ or in a PTEC‐specific manner, or both. Upon administration of diphtheria toxin (DT), massive excretion of urinary FABP1 was induced in mice with both kidney and liver injury, while mice with either injury type showed marginal excretion. Collectively, our data demonstrated that intact PTECs have a considerable capacity to reabsorb liver‐derived FABP1 through a megalin‐mediated mechanism. Thus, urinary FABP1, which is synergistically enhanced by concurrent liver injury, is a biomarker for impaired protein reabsorption in AKI. These findings address the use of urinary FABP1 as a biomarker of histologically injured PTECs that secrete FABP1 into primary urine, and suggest the use of this biomarker to simultaneously monitor impaired tubular reabsorption and liver function. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Sodium-glucose cotransporter 2 inhibition attenuates protein overload in renal proximal tubule via suppression of megalin O-GlcNacylation in progressive diabetic nephropathy

AimsThe crosstalk between sodium-glucose cotransporter 2 (SGLT2) inhibition and a membrane-associated endocytic receptor megalin function involved in renal proximal tubular protein overload in progressive diabetic nephropathy (DN) is uncertain. Here, we determined whether SGLT2 inhibition affects megalin endocytic function through suppressing its O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) and protects the diabetic kidney from protein overload. Materials and methodWe treated 8-week-old male non-obese and hypoinsulinemic KK/Ta-Ins2Akita (KK/Ta-Akita) mice which develop progressive DN with an SGLT2 inhibitor ipragliflozin or insulin for 6 weeks, and investigated the endocytic function (proximal tubular protein reabsorption), renal expression and O-GlcNAcylation of megalin along with their effects on renal phenotypes including histology and biochemical markers. ResultsThe treatment with ipragliflozin, but not insulin, suppressed megalin O-GlcNAcylation and accelerated its internalization, resulting in reduction in proximal tubular reabsorption of the highly filtered plasma proteins such as albumin and neutrophil gelatinase-associated lipocalin. These alterations following the ipragliflozin treatment contributed to amelioration of proximal tubular protein overload, mitochondrial morphological abnormality, and renal oxidative stress and tubulointerstitial fibrosis. ConclusionsThe present study provides a novel crosstalk mechanism between SGLT2 inhibition and megalin underlying the potential renal benefits of SGLT2 inhibition in DN.

Abstract A089: Challenging the free hormone hypothesis for vitamin D in the prostate has implications for the prostate cancer disparity in African American men

Abstract African American (AA) men are disproportionately at risk for both prostate cancer (PCa) and vitamin D (vitD) deficiency compared to European American (EA) men. Based on the numerous chemopreventative properties of vitD and its status with PCa aggressiveness and mortality, vitD deficiency has been hypothesized as a biologic contributor to the PCa disparity in AA men. We recently reported that despite having deficient levels of vitD in the serum, AAs have higher levels of vitD in their prostate tissue compared to EAs. This suggests a mechanism of active vitD transport across the membrane and challenges the longstanding free hormone hypothesis, which asserts that the activity of a hormone is due to the bioavailable fraction and its passive diffusion across the membrane. The majority of VitD circulates bound to D binding protein (DBP) and is sequestered in the serum, rendering it unavailable for passive diffusion. A mechanism of active DBP-vitD endocytosis has been well characterized in the kidney by the extracellular receptor megalin. We hypothesized that megalin is functional in prostate epithelium and facilitates adequate prostatic vitD levels in AAs with vitD deficiency. Here, we report megalin protein expression in prostate tissue, LNCaP, and primary human prostate cells. Megalin function was further assessed in primary human-derived organoids treated with 25D in the presence or absence of DBP. Surprisingly, vitD import and metabolism occurred faster in +DBP conditions and suggests that DBP-vitD active transport is more efficient than passive diffusion of vitD alone. VDR activation was diminished in a dose-responsive manner by addition of a megalin antagonist. Additionally, megalin protein levels in PCa were significantly lower compared to benign (p<0.05) on a tissue microarray quantified by immunofluorescent staining. In summary, our data support a model of megalin-mediated active transport for vitD in the prostate and demonstrate ancestry-related and PCa differences in megalin regulation. These findings challenge the free hormone hypothesis and postulate that vitD deficiency is more complex than previously thought. Citation Format: Zachary Richards, Larisa Nonn. Challenging the free hormone hypothesis for vitamin D in the prostate has implications for the prostate cancer disparity in African American men [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A089.

Reevaluating the role of megalin in renal vitamin D homeostasis using a human cell-derived microphysiological system

The role of megalin in the regulation of renal vitamin D homeostasis has previously been evaluated in megalin-knockout mice and rat proximal tubule epithelial cells. We revisited these hypotheses that were previously tested solely in rodent models, this time using a 3-dimensional proximal tubule microphysiological system incorporating primary human proximal tubule epithelial cells. Using this human cell-derived model, we confirmed that 25OHD3 is transported into the human proximal tubule epithelium via megalin-mediated endocytosis while bound to vitamin D binding protein. Building upon these findings, we then evaluated the role of megalin in modulating the cellular uptake and biological activity of 1α,25(OH)2D3. Inhibition of megalin function decreased the 1α,25(OH)2D3-mediated induction of both cytochrome P450 24A1 protein levels and 24-hydroxylation activity following perfusion with vitamin D binding protein and 1α,25(OH)2D3. The potential for reciprocal effects from 1α,25(OH)2D3 on megalin expression were also tested. Contrary to previously published observations from rat proximal tubule epithelial cells, 1α,25(OH)2D3 did not induce megalin gene expression, thus highlighting the potential for meaningful interspecies differences in the homeostatic regulation of megalin in rodents and humans. These findings challenge a recently promoted hypothesis, predicated on the rodent cell data, that attempts to connect 1α,25(OH)2D3-mediated regulation of renal megalin expression and the pathology of chronic kidney disease in humans. In addition to providing specific insights related to the importance of renal megalin in vitamin D homeostasis, these results constitute a proof-of-concept that human-derived microphysiological systems are a suitable replacement for animal models for quantitative pharmacology and physiology research.

MicroRNA-148b regulates megalin expression and is associated with receptor downregulation in mice with unilateral ureteral obstruction.

Megalin is a multiligand, endocytic receptor that is important for the normal, proximal tubule reabsorption of filtered proteins, hormones, enzymes, essential nutrients, and nephrotoxins. Megalin dysfunction has been associated with acute, as well as chronic kidney diseases. Tubular proteinuria has been observed following unilateral ureteral obstruction (UUO), suggesting megalin dysfunction; however, the pathophysiological mechanism has not been determined. To identify potential regulators of megalin expression, we examined renal microRNAs (miRNAs) expression and observed an upregulation of microRNA-148b (miR-148b) in obstructed mouse kidneys 7 days after UUO, which was associated with a significant reduction in proximal tubule megalin expression and accumulation of megalin ligands. By in silico miRNA target prediction analysis, we identified megalin messenger RNA (mRNA) as a potential target of miR-148b and confirmed using a dual-luciferase reporter assay that miR-148b targeted the 3'-untranslated region of the megalin gene. Transfection of LLC-PK1 cells with miR-148b mimic reduced endogenous megalin mRNA and protein levels in a concentration-dependent manner, while transfection with miR-148b inhibitor resulted in an increase. Our findings suggest that miR-148b directly downregulates megalin expression and that miR-148b negatively regulates megalin expression in UUO-induced kidney injury. Furthermore, the identification of a miRNA regulating megalin expression may allow for targeted interventions to modulate megalin function and proximal tubule uptake of proteins, as well as other ligands.

Tumoral cubilin is a predictive marker for treatment of renal cancer patients with sunitinib and sorafenib

PurposeTyrosine kinase inhibitors like sunitinib and sorafenib are commonly used to treat metastatic renal cell cancer patients. Cubilin is a membrane protein expressed in the proximal renal tubule. Cubilin and megalin function together as endocytic receptors mediating uptake of many proteins. There is no established predictive marker for metastatic renal cell cancer patients and the purpose of the present study was to assess if cubilin can predict response to treatment with tyrosine kinase inhibitors.MethodsCubilin protein expression was analyzsed in tumor tissue from a cohort of patients with metastatic renal cell cancer (n = 139) using immunohistochemistry. One hundred and thirty six of the patients were treated with sunitinib or sorafenib in the first- or second-line setting. Thirty of these were censored because of toxicity leading to the termination of treatment and the remaining (n = 106) were selected for the current study.ResultsFifty-three (50%) of the tumors expressed cubilin in the membrane. The median progression-free survival was 8 months in patients with cubilin expressing tumors and 4 months in the cubilin negative group. In addition, the overall survival was better for patients with cubilin positive tumors. We also found that the fraction of cubilin negative patients was significantly higher in the non-responding group (PFS ≤3 months) compared to responding patients (PFS >3 months).ConclusionsWe show for the first time that tumoral expression of cubilin is a positive predictive marker for treatment of metastatic renal cell cancer patients with sunitinib and sorafenib.

Megalin-deficiency causes high myopia, retinal pigment epithelium-macromelanosomes and abnormal development of the ciliary body in mice.

In man, mutations of the megalin-encoding gene causes the rare Donnai-Barrow/Facio-Oculo-Acoustico-Renal Syndrome, which is partially characterized by high-grade myopia. Previous studies of renal megalin function have established that megalin is crucial for conservation of renal filtered nutrients including vitamin A; however, the role of megalin in ocular physiology and development is presently unknown. Therefore, we investigate ocular megalin expression and the ocular phenotype of megalin-deficient mice. Topographical and subcellular localization of megalin as well as the ocular phenotype of megalin-deficient mice were examined with immunological techniques using light, confocal and electron microscopy. We identified megalin in the retinal pigment epithelium (RPE) and non-pigmented ciliary body epithelium (NPCBE) in normal mouse eyes. Immunocytochemical investigations furthermore showed that megalin localizes to vesicular structures in the RPE and NPCBE cells. Histological investigations of ocular mouse tissue also identified a severe myopia phenotype as well as enlarged RPE melanosomes and abnormal ciliary body development in the megalin-deficient mice. In conclusion, the complex ocular phenotype observed in the megalin-deficient mice suggests that megalin-mediated developmental abnormalities may contribute to the high myopia phenotype observed in the Donnai-Barrow Syndrome patients and, thus, that megalin harbors important roles in ocular development and physiology. Finally, our data show that megalin-deficient mice may provide a valuable model for future studies of megalin in ocular physiology and pathology.

Transforming growth factor-β inhibits alveolar epithelial protein transport by glycogen synthase kinase 3-β-mediated downregulation of megalin

A hallmark of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is the accumulation of protein-rich edema fluid in the distal airspaces removal of which is critical for patient survival. We have previously shown that albumin transport across the alveolar epithelial barrier is an active process mediated by megalin. Here we set out to identify the mechanisms that may impair megalin function upon ALI/ARDS. Particularly, we investigated the role of glycogen synthase kinase 3-β (GSK3-β), a serine-threonine kinase that is involved in various inflammatory disorders and that has been proposed to phosphorylate megalin at the PPPSP motif thereby causing its downregulation.

Role of megalin and the soluble form of its ligand RAP in Cd-metallothionein endocytosis and Cd-metallothionein-induced nephrotoxicity in vivo

Orally administered Cd is predominantly distributed to the intestine, and the majority of this mucosal Cd is bound to metallothionein (MT). MT attenuates heavy metal-induced cytotoxicity by sequestering these metals and lowering their intracellular concentrations. In addition, MT acts as an extracellular transporter of orally administered Cd to the kidney. Because of its low molecular weight, the Cd-MT complex is freely filtered at the glomerulus, and the filtered Cd-MT is then incorporated into renal proximal tubular cells. Megalin, a multiligand endocytic receptor (also known as low-density lipoprotein receptor-related protein 2 or Lrp2), acts as the receptor for Cd-MT in a renal proximal tubular cell model. Here, we used the soluble form of 39-kDa receptor-associated protein (sRAP; also known as Lrpap1), a ligand of megalin, to inhibit megalin function, and then analyzed the effect of megalin loss on Cd-MT distribution and Cd-MT-induced nephrotoxicity in an animal model. Administration of sRAP to mice caused acute loss of megalin function by removing megalin in the brush border membrane. The pre-injection of sRAP decreased renal Cd content and decreased Cd-MT-induced kidney damage. Our results demonstrate that sRAP reduces Cd-MT-induced kidney toxicity in vivo.

Megalin mediates the transport of leptin across the blood-CSF barrier

Leptin, a peptide hormone secreted by adipose tissue, exhibits a large range of central and peripheral actions. It has been proposed that the participation of leptin in diseases such as obesity is due to, at least in part, its impaired transport across the blood–brain barrier (BBB). Since, the mechanisms by which brain takes up leptin remain unclear, we set out to study how leptin may cross the BBB. We have used different immunoassays and lentiviral vectors to analyze the role of megalin in the transport of leptin in rodents and humans. We demonstrate that circulating leptin is transported into the brain by binding to megalin at the choroid plexus epithelium. Indeed, the downregulation of megalin expression in physiological and pathological situations such as aging and Alzheimer's disease was correlated with poor entry of leptin into the brain. Moreover, amyloid beta (Aβ) deposits of choroid plexus could be disturbing megalin function. The present data indicate that leptin represents a novel megalin ligand of importance in the levels and therapeutic actions of leptin into the brain.

Proximal tubule endocytic apparatus as the specific renal uptake mechanism for vitamin D‐binding protein/25‐(OH)D3 complex (Review Article)

The renal proximal tubule exhibits a very extensive apical endocytic apparatus that is involved in the reabsorption of molecules filtered in the glomeruli. Several key receptors appear to be involved in this function, which serves not only to conserve protein but also to reabsorb different vitamins in complex with their binding proteins. Recent research has established megalin as probably the most important receptor in this endocytosis process. Cubilin is another receptor identified in the proximal tubule endocytic apparatus. Because cubilin lacks transmembrane or cytoplasmic domains required for endocytosis, this receptor associates with megalin to recycle and internalize its ligands. Recent studies have shown that vitamin D-binding protein (DBP)/25-(OH)D3 complex is one of the megalin/cubilin ligands. Megalin knockout mice develop vitamin D deficiency and bone disease owing to an inability of the proximal tubules to capture the DBP/25-(OH)D3 complexes from the glomerular filtrate. In the same way, kidney-specific megalin knockout mice have severe plasma vitamin D deficiency, hypocalcaemia and serious bone disease, like the complete megalin knockout mice. Anti-cubilin antibodies inhibit cellular uptake of DBP/25-(OH)D3 by up to 70%. Anti-megalin antibodies produced a similar reduction in DBP/25-(OH)D3 endocytosis. When both antibodies were applied, impairment of DBP/25-(OH)D3 was only slightly more impaired (around 80%), suggesting that cubilin and megalin function through the same endocytic pathway. Specific forms of renal Fanconi syndrome are associated with endocytic pathway dysfunction with disruption of megalin-mediated uptake DBP/25-(OH)D3 complex, producing metabolic bone disease in affected individuals as a prominent clinical finding.

ClC-5 Does Not Affect Megalin Expression and Function in the Thyroid

Megalin is an endocytic receptor responsible for thyroglobulin (Tg) transcytosis, a process that favors hormone release. Accordingly, megalin KO mice have primary hypothyroidism. In the kidney, megalin expression is reduced when the gene encoding the chloride transporter ClC-5 is mutated. We investigated whether megalin expression and function in the thyroid are affected by ClC-5 using a ClC-5 KO mouse model. By Western blotting, ClC-5 was found in thyroid tissue extracts of WT, but not of ClC-5 KO mice. In addition, ClC-5 was found to be expressed by cultured thyroid cells (FRTL-5). The thyroid size, weight, and histology were similar in ClC- 5 KO and WT mice, as were the amounts of megalin in thyroid extracts. Accordingly, serum Tg, a measure of megalin-mediated transcytosis, was similar in WT and ClC-5 KO mice, suggesting that megalin function was unaffected. Thus, unlike in megalin KO mice, in ClC-5 KO mice thyroid function was unchanged, as indicated by the normal serum FT4 and TSH. We concluded that in the thyroid, unlike in the kidney, ClC-5 does not affect megalin expression and function, suggesting that megalin is differentially regulated in these two organs.

Intraperitoneal Administration of Recombinant Receptor-Associated Protein Causes Phosphaturia via an Alteration in Subcellular Distribution of the Renal Sodium Phosphate Co-Transporter

Megalin is a multifunctional endocytic receptor that is expressed in renal proximal tubules and plays critical roles in the renal uptake of various proteins. It was hypothesized that megalin-dependent endocytosis might play a role in renal phosphate reabsorption. For addressing the short-term effects of altered megalin function, a recombinant protein for the soluble form of 39-kD receptor-associated protein (RAP) was administered intraperitoneally to 7-wk-old mice. Histidine (His)-tagged soluble RAP (amino acids 39 to 356) lacking the amino-terminal signal peptide and the carboxy-terminal endoplasmic reticulum retention signal was prepared by bacterial expression (designated His-sRAP). After the direct interaction between His-sRAP and megalin was confirmed, mice were given a single intraperitoneal administration of His-sRAP (3.5 mg/dose). Immunostaining and Western blot analyses demonstrated the uptake of His-sRAP and the accelerated internalization of megalin in proximal tubular cells 1 h after administration. In addition, internalization of the type II sodium/phosphate co-transporter (NaPi-II) was observed. The effects of three sequential administrations of His-sRAP (3.5 mg/dose, three doses at 4-h intervals) then were examined, and increased urinary excretion of low molecular weight proteins, including vitamin D-binding protein, was found, which is consistent with findings reported for megalin-deficient mice. It is interesting that urinary excretion of phosphate was also increased, and the protein level of NaPi-II in the brush border membrane was decreased. Serum concentration of 25-hydroxyvitamin D was decreased, whereas the plasma level of intact parathyroid hormone was not altered by the administration of His-sRAP. The results suggest that the His-sRAP-induced acceleration of megalin-mediated endocytosis caused phosphaturia via altered subcellular distribution of NaPi-II.

Characterization of ANKRA, a novel ankyrin repeat protein that interacts with the cytoplasmic domain of megalin.

Ankyrin-repeat family A protein (ANKRA) is a novel protein that interacts directly and specifically with the cytoplasmic tail of megalin in the yeast two-hybrid system and glutathione-S-transferase pull-down assays. ANKRA has three ankyrin repeats and shows 61% overall homology to regulatory factor X, ankyrin repeat-containing protein. Mapping studies show that the three ankyrin repeats and C-terminus of ANKRA are required for binding to a unique juxtamembrane, 19-amino acid sequence on the megalin tail. Point mutational analysis reveals that a proline-rich motif (PXXPXXP) within this region is the site of ANKRA binding. ANKRA interacts with megalin but not with low-density lipoprotein receptor related protein, in keeping with the fact that the sequence of the megalin tail is unique. By cell fractionation, ANKRA is found both in the cytosol and associated with membranes enriched in megalin in L2 cells and proximal tubule cells. By immunofluorescence, ANKRA is concentrated near megalin along the plasma membrane of L2 cells and in the kidney cortex is expressed in glomerular and proximal tubule epithelia which also express megalin. These observations suggest that ANKRA may play a unique role in megalin's function as a clearance receptor in the kidney and L2 cells. In addition, ANKRA may have other partners because northern blot analysis reveals that ANKRA is more broadly expressed than megalin, and by immunofluorescence ANKRA is also expressed in connecting tubule cells and principal cells of collecting ducts.