Search

Donnai-Barrow syndrome (DBS) is an autosomal-recessive disorder characterized by multiple pathologies including malformation of forebrain and eyes, as well as resorption defects of the kidney proximal tubule. The underlying cause of DBS are mutations in LRP2, encoding the multifunctional endocytic receptor megalin. Here, we identified a unique missense mutation R3192Q of LRP2 in an affected family that may provide novel insights into the molecular causes of receptor dysfunction in the kidney proximal tubule and other tissues affected in DBS. Using patient-derived induced pluripotent stem cell lines we generated neuroepithelial and kidney cell types as models of the disease. Using these cell models, we documented the inability of megalin R3192Q to properly discharge ligand and ligand-induced receptor decay in lysosomes. Thus, mutant receptors are aberrantly targeted to lysosomes for catabolism, essentially depleting megalin in the presence of ligand in this affected family., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The tight junction proteins claudin-10 and -16 are crucial for the paracellular reabsorption of cations along the thick ascending limb of Henle's loop in the kidney. In patients, mutations in CLDN16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, while mutations in CLDN10 impair kidney function. Mice lacking claudin-16 display magnesium and calcium wasting, whereas absence of claudin-10 results in hypermagnesemia and interstitial nephrocalcinosis. In order to study the functional interdependence of claudin-10 and -16 we generated double-deficient mice. These mice had normal serum magnesium and urinary excretion of magnesium and calcium and showed polyuria and sodium retention at the expense of increased renal potassium excretion, but no nephrocalcinosis. Isolated thick ascending limb tubules of double mutants displayed a complete loss of paracellular cation selectivity and functionality. Mice lacking both claudin-10 and -16 in the thick ascending limb recruited downstream compensatory mechanisms and showed hypertrophic distal convoluted tubules with changes in gene expression and phosphorylation of ion transporters in this segment, presumably triggered by the mild decrease in serum potassium. Thus, severe individual phenotypes in claudin-10 and claudin-16 knockout mice are corrected by the additional deletion of the other claudin., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Studies in mice have suggested bone marrow transplantation as a strategy to correct the endocytic dysfunction of the proximal tubules in renal Fanconi syndrome, yet the mode of action has remained mysterious. Using a mouse model of Dent disease, Gabriel et al. now show that rescue of the resorptive capacity in the diseased kidney involves cell-to-cell contact between engrafted and host cells via nanotubes, cellular projections that enable transfer of wild-type activity into mutant cells of the proximal tubule., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

VPS10P domain receptors are a unique class of sorting receptors that direct intracellular transport of target proteins in neurons and that play central roles in neurodegenerative processes. Surprisingly, genome-wide association studies now implicate the very same receptors in cardiovascular and metabolic disturbances. In this review, we discuss current findings that uncovered some of the molecular mechanisms whereby sorting receptors, such as SORLA, sortilin, and SORCS1 control homeostasis in cardiovascular and metabolic tissues, and how they promote hypercholesterolemia, atherosclerosis, obesity, and diabetes, when being altered., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Megalin-mediated endocytic uptake constitutes the main pathway for clearance of plasma proteins from the glomerular filtrate in proximal tubules. Little is known, however, about mechanisms that control megalin expression and activity in the kidney. A widely discussed hypothesis states that upon ligand binding a regulated intramembrane proteolysis releases the cytosolic domain of megalin and this fragment subsequently modulates megalin gene transcription. Here, we tested this by generating a mouse model that co-expressed both the soluble intracellular domain and full-length megalin. Despite pronounced synthesis in the proximal tubules, the soluble intracellular domain failed to exert distinct effects on renal proximal tubular function, including megalin expression, endocytic retrieval of proteins, or global renal gene transcription. Hence, our study argues that the soluble intracellular domain does not have a role in regulating the activity of megalin in the kidney.

Megalin, a member of the LDL receptor family, is expressed on the apical membrane of proximal tubules and serves as an endocytic scavenger of filtered proteins and hence might contribute to the tubule injury as a consequence of glomerular disease. To study its role, we crossed megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte). Treatment of this transgenic mouse with the immunotoxin causes nephrotic syndrome, focal segmental glomerulosclerosis and tubule-interstitial injury. Following this treatment, the double transgenic mice had massive non-selective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells within each kidney showed that albumin, immunoglobulin light chain, IgA and IgG were preferentially accumulated in proximal tubule cells expressing megalin. Tubule injury markers such as heme-oxygenase-1, monocyte chemoattractant protein-1 and cellular apoptosis were also preferentially found in these megalin-expressing cells. These results show that megalin plays a pivotal role in the reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury.

Dysfunction of the proximal tubule (PT) is associated with variable degrees of solute wasting and low-molecular-weight proteinuria. We measured metabolic consequences and adaptation mechanisms in a model of inherited PT disorders using PT cells of ClC-5-deficient (Clcn5Y/-) mice, a well-established model of Dent's disease. Compared to cells taken from control mice, those from the mutant mice had increased expression of markers of proliferation (Ki67, proliferative cell nuclear antigen (PCNA), and cyclin E) and oxidative scavengers (superoxide dismutase I and thioredoxin). Transcriptome and protein analyses showed fourfold induction of type III carbonic anhydrase in a kidney-specific manner in the knockout mice located in scattered PT cells. Kidney-specific carbonic anhydrase type III (CAIII) upregulation was confirmed in other mice lacking the multiligand receptor megalin and in a patient with Dent's disease due to an inactivating CLCN5 mutation. The type III enzyme was specifically detected in the urine of mice lacking ClC-5 or megalin, patients with Dent's disease, and in PT cell lines exposed to oxidative stress. Our study shows that lack of PT ClC-5 in mice and men is associated with CAIII induction, increased cell proliferation, and oxidative stress.

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 approximately 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.

Background: The endocytic receptor megalin constitutes the major pathway for clearance of low-molecular weight plasma proteins from the glomerular filtrate into the renal proximal tubules. Furthermore, the receptor has been implicated in a number of other functions in the kidney including uptake and activation of 25-(OH) vitamin D3, calcium and sodium reabsorption as well as signal transduction., Methods: We used genome-wide expression profiling by microarray technology to detect changes in the gene expression pattern in megalin knockout mouse kidneys and to uncover some of the renal pathways affected by megalin deficiency., Results: Alterations were identified in several (patho)physiologic processes in megalin-deficient kidneys including the renal vitamin D metabolism, transforming growth factor (TGF)-beta1 signal transduction, lipid transport and heavy metal detoxification. Most importantly, changes were detected in the mRNA levels of 25-(OH) vitamin D-24-hydroxylase and 25-(OH) vitamin D-1alpha-hydroxylase as well as strong up-regulation of TGF-beta1 target genes. Both findings indicate plasma vitamin D deficiency and lack of vitamin D signaling in renal tissues., Conclusions: Expression profiling confirms a crucial role for megalin in renal vitamin D metabolism.

Megalin is an endocytic receptor expressed on the luminal surface of the renal proximal tubules. The receptor is believed to play an important role in the tubular uptake of macromolecules filtered through the glomerulus. To elucidate the role of megalin in vivo and to identify its endogenous ligands, we analyzed the proximal tubular function in mice genetically deficient for the receptor. We demonstrate that megalin-deficient mice exhibit a tubular resorption deficiency and excrete low molecular weight plasma proteins in the urine (low molecular weight proteinuria). Proteins excreted include small plasma proteins that carry lipophilic compounds including vitamin D-binding protein, retinol-binding protein, alpha(1)-microglobulin and odorant-binding protein. Megalin binds these proteins and mediates their cellular uptake. Urinary loss of carrier proteins in megalin-deficient mice results in concomitant loss of lipophilic vitamins bound to the carriers. Similar to megalin knockout mice, patients with low molecular weight proteinuria as in Fanconi syndrome are also shown to excrete vitamin/carrier complexes. Thus, these results identify a crucial role of the proximal tubule in retrieval of filtered vitamin/carrier complexes and the central role played by megalin in this process.

The sites and precise mechanisms of the catabolism of the atherogenic lipoprotein[a] (Lp[a]) are unknown. It has been proposed that the low density lipoprotein receptor (LDL-R) and the low density lipoprotein receptor-related protein (LRP) are involved in the catabolism of Lp[a]. To address the question whether and to what extent the LDL-R and/or LRP are involved in the catabolism of Lp[a], we studied the cellular uptake of Lp[a] via those two receptors using mouse embryonic fibroblast (MEF) cell lines lacking either the LDL-R, the LRP, or both receptors due to disruption of the respective mouse genes. 125I-labeled LDL and 125I-labeled Lp[a] uptake by wild-type fibroblasts (MEF1) was compared with that by fibroblasts homozygous for the disrupted LRP allele (MEF2), fibroblasts with two defective alleles for the LDL-R (MEF3), and fibroblasts homozygous for defects both in the LDL-R and LRP gene (MEF4). Compared with MEF1, 125I-labeled LDL uptake by MEF2 was 77%, by MEF3 30%, and by MEF4 24% of that by MEF1. However, no significant differences in the specific 125I-labeled Lp[a] uptake by the four mouse embryonic cell lines was observed. In comparison with MEF1, the 125I-labeled Lp[a] uptake by MEF2 was 98%, by MEF3 111%, and 73% by MEF4. Approximately 50% of the total cellular uptake of 125I-labeled Lp[a] was nonspecific. In conclusion, our results suggest that Lp[a] is a poor ligand for the LDL receptor and the LRP. The data of the displacement studies, however, indicated that the nonspecific uptake of Lp[a] constitutes a major route for the cellular Lp[a] catabolism in this study.

Recent technological advances have enabled us to manipulate specific genes in laboratory animals in a specific predetermined manner. This has opened new areas of research on physiological processes not previously accessible to such precise experimental manipulation. Over-expression of genes by traditional transgenic techniques has recently been complemented by methods that allow the efficient transfer of exogenous genes into various somatic tissues of adult animals. The development of homologous recombination technology in embryonic stem cells (ESC) and the application of this technology to specifically disrupt a given gene of interest in the germline of a mouse has been particularly useful to determine the physiologically relevant processes in which these genes participate in vivo. Rather than introducing random mutations into the genome by chemical mutagenesis or by retroviral insertion, techniques that have been employed in the past, gene targeting not only allows us to disrupt any cloned gene, but also to specifically introduce single nucleotide changes into its genomic sequence. The past few years have witnessed an explosion of research reports in all areas of biological research that have employed these ground-breaking tools of modern genetics to study the physiological roles of a plethora of different genes in neurobiology immunology, endocrinology, development, etc. Our laboratory has also extensively used these new approaches to study the function of several genes that are involved in the metabolism of lipoproteins on the systemic as well as on the cellular level. In this article, we will review the various approaches we have used to define the roles of the low density lipoprotein (LDL) receptor, the LDL receptor-related protein (LRP) and the receptor-associated protein (RAP) in hepatic lipoprotein metabolism.