LRP2 controls sonic hedgehog-dependent differentiation of cardiac progenitor cells during outflow tract formation.

Conotruncal malformations are a major cause of congenital heart defects in newborn infants. Recently, genetic screens in humans and in mouse models have identified mutations in LRP2, a multi-ligand receptor, as a novel cause of a common arterial trunk, a severe form of outflow tract (OFT) defect. Yet, the underlying mechanism why the morphogen receptor LRP2 is essential for OFT development remained unexplained. Studying LRP2-deficient mouse models, we now show that LRP2 is expressed in the cardiac progenitor niche of the anterior second heart field (SHF) that contributes to the elongation of the OFT during separation into aorta and pulmonary trunk. Loss of LRP2 in mutant mice results in the depletion of a pool of sonic hedgehog-dependent progenitor cells in the anterior SHF due to premature differentiation into cardiomyocytes as they migrate into the OFT myocardium. Depletion of this cardiac progenitor cell pool results in aberrant shortening of the OFT, the likely cause of CAT formation in affected mice. Our findings identified the molecular mechanism whereby LRP2 controls the maintenance of progenitor cell fate in the anterior SHF essential for OFT separation, and why receptor dysfunction is a novel cause of conotruncal malformation.

Megalin: a Novel Determinant of Renin-Angiotensin System Activity in the Kidney?

PURPOSE OF REVIEW: Megalin is well known for its role in the reabsorption of proteins from the ultrafiltrate. Recent studies suggest that megalin also reabsorbs renin and angiotensinogen. Indeed, without megalin urinary renin and angiotensinogen levels massively increase, and even prorenin becomes detectable in urine. RECENT FINDINGS: Intriguingly, megalin might also contribute to renal angiotensin production, as evidenced from studies in megalin knockout mice. This review discusses these topics critically, concluding that urinary renin-angiotensin system components reflect diminished reabsorption rather than release from renal tissue sites and that alterations in renal renin levels or megalin-dependent signaling need to be ruled out before concluding that angiotensin production at renal tissue sites is truly megalin dependent. Future studies should evaluate megalin-mediated renin/angiotensinogen transcytosis (allowing interstitial angiotensin generation), and determine whether megalin prefers prorenin over renin, thus explaining why urine normally contains no prorenin.

Protection of Cystinotic Mice by Kidney-Specific Megalin Ablation Supports an Endocytosis-Based Mechanism for Nephropathic Cystinosis Progression.

BACKGROUND: Deletions or inactivating mutations of the cystinosin gene CTNS lead to cystine accumulation and crystals at acidic pH in patients with nephropathic cystinosis, a rare lysosomal storage disease and the main cause of hereditary renal Fanconi syndrome. Early use of oral cysteamine to prevent cystine accumulation slows progression of nephropathic cystinosis but it is a demanding treatment and not a cure. The source of cystine accumulating in kidney proximal tubular cells and cystine's role in disease progression are unknown. METHODS: To investigate whether receptor-mediated endocytosis by the megalin/LRP2 pathway of ultrafiltrated, disulfide-rich plasma proteins could be a source of cystine in proximal tubular cells, we used a mouse model of cystinosis in which conditional excision of floxed megalin/LRP2 alleles in proximal tubular cells of cystinotic mice was achieved by a Cre-LoxP strategy using Wnt4-CRE. We evaluated mice aged 6-9 months for kidney cystine levels and crystals; histopathology, with emphasis on swan-neck lesions and proximal-tubular-cell apoptosis and proliferation (turnover); and proximal-tubular-cell expression of the major apical transporters sodium-phosphate cotransporter 2A (NaPi-IIa) and sodium-glucose cotransporter-2 (SGLT-2). RESULTS: Wnt4-CRE-driven megalin/LRP2 ablation in cystinotic mice efficiently blocked kidney cystine accumulation, thereby preventing lysosomal deformations and crystal deposition in proximal tubular cells. Swan-neck lesions were largely prevented and proximal-tubular-cell turnover was normalized. Apical expression of the two cotransporters was also preserved. CONCLUSIONS: These observations support a key role of the megalin/LRP2 pathway in the progression of nephropathic cystinosis and provide a proof of concept for the pathway as a therapeutic target.

Megalin mediates 25-hydroxyvitamin D3 actions in human mesenchymal stem cells.

Osteoblast differentiation of human mesenchymal stem cells (hMSCs) is stimulated by 1α,25-dihydroxycholecalciferol [1α,25(OH)2D3] and 25-hydroxycholecalciferol [25(OH)D3]; the latter's effects require intracellular hydroxylation to 1α,25(OH)2D3. Thus, hMSCs are both a source of and target for 1α,25(OH)2D3. Megalin is a transmembrane receptor for serum d-binding protein (DBP) in kidney cells and is required for uptake of the 25(OH)D3-DBP complex. We tested the hypothesis that megalin is required for D actions in hMSCs with cells from surgically discarded marrow for RT-PCR, for effects of 25(OH)D3 and 1α,25(OH)2D3, for 1α,25(OH)2D3 biosynthesis, for osteoblastogenesis, and for small interfering RNA for megalin (si-Meg) and control (si-Ctr). In hMSCs with high constitutive megalin expression, both 1α,25(OH)2D3 and 25(OH)D3 stimulated osteoblastogenesis (P < 0.05), but only 1α,25(OH)2D3 did so in hMSCs with lower megalin (lo-Meg, P < 0.001) or in si-Meg cells (P < 0.05). In addition, 1α,25(OH)2D3 biosynthesis was significantly lower in lo-Meg (46%, P = 0.034) and in si-Meg (23%, P < 0.001) than each control. Leptin significantly stimulated megalin expression 2.1-fold in lo-Meg cells (P < 0.01). These studies show that megalin is expressed in hMSCs and is required for the biosynthesis of 1α,25(OH)2D3 and for the 25(OH)D3/DBP complex to stimulate vitamin D receptor targets and osteoblastogenesis.-Gao, Y., Zhou, S., Luu, S., Glowacki, J. Megalin mediates 25-hydroxyvitamin D3 actions in human mesenchymal stem cells.

Angiotensinogen and Megalin Interactions Contribute to Atherosclerosis-Brief Report.

Objective- AGT (Angiotensinogen) is the unique precursor of the renin-angiotensin system that is sequentially cleaved by renin and ACE (angiotensin-converting enzyme) to produce Ang II (angiotensin II). In this study, we determined how these renin-angiotensin components interact with megalin in kidney to promote atherosclerosis. Approach and Results- AGT, renin, ACE, and megalin were present in the renal proximal convoluted tubules of wild-type mice. Hepatocyte-specific AGT deficiency abolished AGT protein accumulation in proximal tubules and diminished Ang II concentrations in kidney, while renin was increased. Megalin was most abundant in kidney and exclusively present on the apical side of proximal tubules. Inhibition of megalin by antisense oligonucleotides (ASOs) led to ablation of AGT and renin proteins in proximal tubules, while leading to striking increases of urine AGT and renin concentrations, and 70% reduction of renal Ang II concentrations. However, plasma Ang II concentrations were unaffected. To determine whether AGT and megalin interaction contributes to atherosclerosis, we used both male and female low-density lipoprotein receptor-/- mice fed a saturated fat-enriched diet and administered vehicles (PBS or control ASO) or megalin ASO. Inhibition of megalin did not affect plasma cholesterol concentrations, but profoundly reduced atherosclerotic lesion size in both male and female mice. Conclusions- These results reveal a regulatory role of megalin in the intrarenal renin-angiotensin homeostasis and atherogenesis, positing renal Ang II to be an important contributor to atherosclerosis that is mediated through AGT and megalin interactions.

Circulating FABP4 is eliminated by the kidney via glomerular filtration followed by megalin-mediated reabsorption.

Circulating fatty acid binding protein 4 (FABP4), secreted from adipocytes, is a potential biomarker for metabolic and cardiovascular diseases. Circulating FABP4 levels are positively associated with adiposity and adrenergic stimulation, but negatively with renal function. In this study, we addressed the issue of how the kidney regulates clearance of circulating FABP4. Tracing study revealed remarkable accumulation of 125I-labeled FABP4 in the kidney. Exogenous FABP4 was exclusively detected in the apical membrane of proximal tubule epithelial cells (PTECs). Bilateral nephrectomy resulted in marked elevation of circulating FABP4 levels. Accelerated lipolysis by ß-3 adrenergic stimulation led to a marked elevation in circulating FABP4 in mice with severe renal dysfunction. Megalin, an endocytic receptor expressed in PTECs, plays a major role in reabsorption of proteins filtered through glomeruli. Quartz-crystal microbalance study revealed that FABP4 binds to megalin. In kidney-specific megalin knockout mice, a large amount of FABP4 was excreted in urine while circulating FABP4 levels were significantly reduced. Our data suggest that circulating FABP4 is processed by the kidney via the glomerular filtration followed by megalin-mediated reabsorption. Thus, it is likely that circulating FABP4 levels are determined mainly by balance between secretion rate of FABP4 from adipocytes and clearance rate of the kidney.

Megalin dependent urinary cystatin C excretion in ischemic kidney injury in rats.

BACKGROUND: Cystatin C, a marker of kidney injury, is freely filtered in the glomeruli and reabsorbed by the proximal tubules. Megalin and cubilin are endocytic receptors essential for reabsorption of most filtered proteins. This study examines the role of these receptors for the uptake and excretion of cystatin C and explores the effect of renal ischemia/reperfusion injury on renal cystatin C uptake and excretion in a rat model. METHODS: Binding of cystatin C to megalin and cubilin was analyzed by surface plasmon resonance analysis. ELISA and/or immunoblotting and immunohistochemistry were used to study the urinary excretion and tubular uptake of endogenous cystatin C in mice. Furthermore, renal uptake and urinary excretion of cystatin C was investigated in rats exposed to ischemia/reperfusion injury. RESULTS: A high affinity binding of cystatin C to megalin and cubilin was identified. Megalin deficient mice revealed an increased urinary excretion of cystatin C associated with defective uptake by endocytosis. In rats exposed to ischemia/reperfusion injury urinary cystatin C excretion was increased and associated with a focal decrease in proximal tubule endocytosis with no apparent change in megalin expression. CONCLUSIONS: Megalin is essential for the normal tubular recovery of endogenous cystatin C. The increase in urinary cystatin C excretion after ischemia/reperfusion injury is associated with decreased tubular uptake but not with reduced megalin expression.

Recent Updates on the Proximal Tubule Renin-Angiotensin System in Angiotensin II-Dependent Hypertension.

It is well recognized that the renin-angiotensin system (RAS) exists not only as circulating, paracrine (cell to cell), but also intracrine (intracellular) system. In the kidney, however, it is difficult to dissect the respective contributions of circulating RAS versus intrarenal RAS to the physiological regulation of proximal tubular Na(+) reabsorption and hypertension. Here, we review recent studies to provide an update in this research field with a focus on the proximal tubular RAS in angiotensin II (ANG II)-induced hypertension. Careful analysis of available evidence supports the hypothesis that both local synthesis or formation and AT1 (AT1a) receptor- and/or megalin-mediated uptake of angiotensinogen (AGT), ANG I and ANG II contribute to high levels of ANG II in the proximal tubules of the kidney. Under physiological conditions, nearly all major components of the RAS including AGT, prorenin, renin, ANG I, and ANG II would be filtered by the glomerulus and taken up by the proximal tubules. In ANG II-dependent hypertension, the expression of AGT, prorenin, and (pro)renin receptors, and angiotensin-converting enzyme (ACE) is upregulated rather than downregulated in the kidney. Furthermore, hypertension damages the glomerular filtration barrier, which augments the filtration of circulating AGT, prorenin, renin, ANG I, and ANG II and their uptake in the proximal tubules. Together, increased local ANG II formation and augmented uptake of circulating ANG II in the proximal tubules, via activation of AT1 (AT1a) receptors and Na(+)/H(+) exchanger 3, may provide a powerful feedforward mechanism for promoting Na(+) retention and the development of ANG II-induced hypertension.

LRP2, an auxiliary receptor that controls sonic hedgehog signaling in development and disease.

To fulfill their multiple roles in organ development and adult tissue homeostasis, hedgehog (HH) morphogens act through their receptor Patched (PTCH) on target cells. However, HH actions also require HH binding proteins, auxiliary cell surface receptors that agonize or antagonize morphogen signaling in a context-dependent manner. Here, we discuss recent findings on the LDL receptor-related protein 2 (LRP2), an exemplary HH binding protein that modulates sonic hedgehog activities in stem and progenitor cell niches in embryonic and adult tissues. LRP2 functions are crucial for developmental processes in a number of tissues, including the brain, the eye, and the heart, and defects in this receptor pathway are the cause of devastating congenital diseases in humans. Developmental Dynamics 245:569-579, 2016. © 2016 Wiley Periodicals, Inc.

Megalin and cubilin in proximal tubule protein reabsorption: from experimental models to human disease.

Proximal tubule protein uptake is mediated by 2 receptors, megalin and cubilin. These receptors rescue a variety of filtered ligands, including biomarkers, essential vitamins, and hormones. Receptor gene knockout animal models have identified important functions of the receptors and have established their essential role in modulating urinary protein excretion. Rare genetic syndromes associated with dysfunction of these receptors have been identified and characterized, providing additional information on the importance of these receptors in humans. Using various disease models in combination with receptor gene knockout, the implications of receptor dysfunction in acute and chronic kidney injury have been explored and have pointed to potential new roles of these receptors. Based on data from animal models, this paper will review current knowledge on proximal tubule endocytic receptor function and regulation, and their role in renal development, protein reabsorption, albumin uptake, and normal renal physiology. These findings have implications for the pathophysiology and diagnosis of proteinuric renal diseases. We will examine the limitations of the different models and compare the findings to phenotypic observations in inherited human disorders associated with receptor dysfunction. Furthermore, evidence from receptor knockout mouse models as well as human observations suggesting a role of protein receptors for renal disease will be discussed in light of conditions such as chronic kidney disease, diabetes, and hypertension.

Renal Handling of Circulating and Renal-Synthesized Hepcidin and Its Protective Effects against Hemoglobin-Mediated Kidney Injury.

Urinary hepcidin may have protective effects against AKI. However, renal handling and the potential protective mechanisms of hepcidin are not fully understood. By measuring hepcidin levels in plasma and urine using mass spectrometry and the kidney using immunohistochemistry after intraperitoneal administration of human hepcidin-25 (hhep25) in C57Bl/6N mice, we showed that circulating hepcidin is filtered by the glomerulus and degraded to smaller isoforms detected in urine but not plasma. Moreover, hepcidin colocalized with the endocytic receptor megalin in proximal tubules, and compared with wild-type mice, megalin-deficient mice showed higher urinary excretion of injected hhep25 and no hepcidin staining in proximal tubules that lack megalin. This indicates that hepcidin is reaborbed in the proximal tubules by megalin dependent endocytosis. Administration of hhep25 concomitant with or 4 hours after a single intravenous dose of hemoglobin abolished hemoglobin-induced upregulation of urinary kidney injury markers (NGAL and KIM-1) and renal Interleukin-6 and Ngal mRNA observed 24 hours after administration but did not affect renal ferroportin expression at this point. Notably, coadministration of hhep25 and hemoglobin but not administration of either alone greatly increased renal mRNA expression of hepcidin-encoding Hamp1 and hepcidin staining in distal tubules. These findings suggest a role for locally synthesized hepcidin in renal protection. Our observations did not support a role for ferroportin in hhep25-mediated protection against hemoglobin-induced early injury, but other mechanisms of cellular iron handling may be involved. In conclusion, our data suggest that both systemically delivered and locally produced hepcidin protect against hemoglobin-induced AKI.

Megalin-Mediated Tubuloglomerular Alterations in High-Fat Diet-Induced Kidney Disease.

Obesity, an important risk factor for metabolic syndrome (MetS) and cardiovascular disease, is often complicated by CKD, which further increases cardiovascular risk and causes ESRD. To elucidate the mechanism underlying this relationship, we investigated the role of the endocytic receptor megalin in proximal tubule epithelial cells (PTECs). We studied a high-fat diet (HFD)-induced obesity/MetS model using kidney-specific mosaic megalin knockout (KO) mice. Compared with control littermates fed a normal-fat diet, control littermates fed an HFD for 12 weeks showed autolysosomal dysfunction with autophagy impairment and increased expression of hypertrophy, lipid peroxidation, and senescence markers in PTECs of the S2 segment, peritubular capillary rarefaction with localized interstitial fibrosis, and glomerular hypertrophy with mesangial expansion. These were ameliorated in HFD-fed megalin KO mice, even though these mice had the same levels of obesity, dyslipidemia, and hyperglycemia as HFD-fed control mice. Intravital renal imaging of HFD-fed wild-type mice also demonstrated the accumulation of autofluorescent lipofuscin-like substances in PTECs of the S2 segment, accompanied by focal narrowing of tubular lumens and peritubular capillaries. In cultured PTECs, fatty acid-rich albumin induced the increased expression of genes encoding PDGF-B and monocyte chemoattractant protein-1 via megalin, with large (auto)lysosome formation, compared with fatty acid-depleted albumin. Collectively, the megalin-mediated endocytic handling of glomerular-filtered (lipo)toxic substances appears to be involved primarily in hypertrophic and senescent PTEC injury with autophagy impairment, causing peritubular capillary damage and retrograde glomerular alterations in HFD-induced kidney disease. Megalin could be a therapeutic target for obesity/MetS-related CKD, independently of weight, dyslipidemia, and hyperglycemia modification.

LRP2 Acts as SHH Clearance Receptor to Protect the Retinal Margin from Mitogenic Stimuli.

During forebrain development, LRP2 promotes morphogen signaling as an auxiliary SHH receptor. However, in the developing retina, LRP2 assumes the opposing function, mediating endocytic clearance of SHH and antagonizing morphogen action. LRP2-mediated clearance prevents spread of SHH activity from the central retina into the retinal margin to protect quiescent progenitor cells in this niche from mitogenic stimuli. Loss of LRP2 in mice increases the sensitivity of the retinal margin for SHH, causing expansion of the retinal progenitor cell pool and hyperproliferation of this tissue. Our findings document the ability of LRP2 to act, in a context-dependent manner, as activator or inhibitor of the SHH pathway. Our current findings uncovered LRP2 activity as the molecular mechanism imposing quiescence of the retinal margin in the mammalian eye and suggest SHH-induced proliferation of the retinal margin as cause of the large eye phenotype observed in mouse models and patients with LRP2 defects.

Regulation of Selenium Metabolism and Transport.

Selenium is regulated in the body to maintain vital selenoproteins and to avoid toxicity. When selenium is limiting, cells utilize it to synthesize the selenoproteins most important to them, creating a selenoprotein hierarchy in the cell. The liver is the central organ for selenium regulation and produces excretory selenium forms to regulate whole-body selenium. It responds to selenium deficiency by curtailing excretion and secreting selenoprotein P (Sepp1) into the plasma at the expense of its intracellular selenoproteins. Plasma Sepp1 is distributed to tissues in relation to their expression of the Sepp1 receptor apolipoprotein E receptor-2, creating a tissue selenium hierarchy. N-terminal Sepp1 forms are taken up in the renal proximal tubule by another receptor, megalin. Thus, the regulated whole-body pool of selenium is shifted to needy cells and then to vital selenoproteins in them to supply selenium where it is needed, creating a whole-body selenoprotein hierarchy.

Megalin-mediated specific uptake of chitosan/siRNA nanoparticles in mouse kidney proximal tubule epithelial cells enables AQP1 gene silencing.

RNAi-based strategies provide a great therapeutic potential for treatment of various human diseases including kidney disorders, but face the challenge of in vivo delivery and specific targeting. The chitosan delivery system has previously been shown to target siRNA specifically to the kidneys in mice when administered intravenously. Here we confirm by 2D and 3D bioimaging that chitosan formulated siRNA is retained in the kidney for more than 48 hours where it accumulates in proximal tubule epithelial cells (PTECs), a process that was strongly dependent on the molecular weight of chitosan. Chitosan/siRNA nanoparticles, administered to chimeric mice with conditional knockout of the megalin gene, distributed almost exclusively in cells that expressed megalin, implying that the chitosan/siRNA particle uptake was mediated by a megalin-dependent endocytotic pathway. Knockdown of the water channel aquaporin 1 (AQP1) by up to 50% in PTECs was achieved utilizing the systemic i.v. delivery of chitosan/AQP1 siRNA in mice. In conclusion, specific targeting PTECs with the chitosan nanoparticle system may prove to be a useful strategy for knockdown of specific genes in PTECs, and provides a potential therapeutic strategy for treating various kidney diseases.

Selenoprotein P and apolipoprotein E receptor-2 interact at the blood-brain barrier and also within the brain to maintain an essential selenium pool that protects against neurodegeneration.

Selenoprotein P (Sepp1) and its receptor, apolipoprotein E receptor 2 (apoER2), account for brain retaining selenium better than other tissues. The primary sources of Sepp1 in plasma and brain are hepatocytes and astrocytes, respectively. ApoER2 is expressed in varying amounts by tissues; within the brain it is expressed primarily by neurons. Knockout of Sepp1 or apoER2 lowers brain selenium from ∼120 to ∼50 ng/g and leads to severe neurodegeneration and death in mild selenium deficiency. Interactions of Sepp1 and apoER2 that protect against this injury have not been characterized. We studied Sepp1, apoER2, and brain selenium in knockout mice. Immunocytochemistry showed that apoER2 mediates Sepp1 uptake at the blood-brain barrier. When Sepp1(-/-) or apoER2(-/-) mice developed severe neurodegeneration caused by mild selenium deficiency, brain selenium was ∼35 ng/g. In extreme selenium deficiency, however, brain selenium of ∼12 ng/g was tolerated when both Sepp1 and apoER2 were intact in the brain. These findings indicate that tandem Sepp1-apoER2 interactions supply selenium for maintenance of brain neurons. One interaction is at the blood-brain barrier, and the other is within the brain. We postulate that Sepp1 inside the blood-brain barrier is taken up by neurons via apoER2, concentrating brain selenium in them.

Megalin in acute kidney injury: foe and friend.

The kidney proximal tubule is a key target in many forms of acute kidney injury (AKI). The multiligand receptor megalin is responsible for the normal proximal tubule uptake of filtered molecules, including nephrotoxins, cytokines, and markers of AKI. By mediating the uptake of nephrotoxins, megalin plays an essential role in the development of some types of AKI. However, megalin also mediates the tubular uptake of molecules implicated in the protection against AKI, and changes in megalin expression have been demonstrated in AKI in animal models. Thus, modulation of megalin expression in response to AKI may be an important part of the tubule cell adaption to cellular protection and regeneration and should be further investigated as a potential target of intervention. This review explores current evidence linking megalin expression and function to the development, diagnosis, and progression of AKI as well as renal protection against AKI.

Renal uptake of the antiapoptotic protein survivin is mediated by megalin at the apical membrane of the proximal tubule.

The inhibitor of apoptosis protein survivin is a bifunctional molecule that regulates cellular division and survival. We have previously shown that survivin protein can be found at high concentrations in the adult kidney, particularly in the proximal tubules. Here, survivin is localized primarily at the apical membrane, a pattern that may indicate absorption of the protein. Several proteins in primary urine are internalized by megalin, an endocytosis receptor, which is in principle found in the same localization as survivin. Immunolabeling for survivin in different species confirmed survivin signal localizing to the apical membrane of the proximal tubule. Immunoelectron microscopy also showed apical localization of survivin in human kidneys. Furthermore, in polarized human primary tubular cells endogenous as well as external recombinant survivin is stored in the apical region of the cells. Costaining of survivin and megalin by immunohistochemistry and immunoelectron microscopy confirmed colocalization. Finally, by surface plasmon resonance we were able to demonstrate that survivin binds megalin and cubilin and that megalin knockout mice lose survivin through the urine. Survivin accumulates at the apical membrane of the renal tubule by reuptake, which is achieved by the endocytic receptor megalin, collaborating with cubilin. For this to occur, survivin will have to circulate in the blood and be filtered into the primary urine. It is not known at this stage what the functional role of tubular survivin is. However, a small number of experimental and clinical reports implicate that renal survivin is important for functional integrity of the kidney.

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.