Albumin uptake and processing by the proximal tubule: physiological, pathological, and therapeutic implications.

For nearly 50 years the proximal tubule (PT) has been known to reabsorb, process, and either catabolize or transcytose albumin from the glomerular filtrate. Innovative techniques and approaches have provided insights into these processes. Several genetic diseases, nonselective PT cell defects, chronic kidney disease (CKD), and acute PT injury lead to significant albuminuria, reaching nephrotic range. Albumin is also known to stimulate PT injury cascades. Thus, the mechanisms of albumin reabsorption, catabolism, and transcytosis are being reexamined with the use of techniques that allow for novel molecular and cellular discoveries. Megalin, a scavenger receptor, cubilin, amnionless, and Dab2 form a nonselective multireceptor complex that mediates albumin binding and uptake and directs proteins for lysosomal degradation after endocytosis. Albumin transcytosis is mediated by a pH-dependent binding affinity to the neonatal Fc receptor (FcRn) in the endosomal compartments. This reclamation pathway rescues albumin from urinary losses and cellular catabolism, extending its serum half-life. Albumin that has been altered by oxidation, glycation, or carbamylation or because of other bound ligands that do not bind to FcRn traffics to the lysosome. This molecular sorting mechanism reclaims physiological albumin and eliminates potentially toxic albumin. The clinical importance of PT albumin metabolism has also increased as albumin is now being used to bind therapeutic agents to extend their half-life and minimize filtration and kidney injury. The purpose of this review is to update and integrate evolving information regarding the reabsorption and processing of albumin by proximal tubule cells including discussion of genetic disorders and therapeutic considerations.

Glycosylation of a key cubilin Asn residue results in reduced binding to albumin.

Kidney disease often manifests with an increase in proteinuria, which can result from both glomerular and/or proximal tubule injury. The proximal tubules are the major site of protein and peptide endocytosis of the glomerular filtrate, and cubilin is the proximal tubule brush border membrane glycoprotein receptor that binds filtered albumin and initiates its processing in proximal tubules. Albumin also undergoes multiple modifications depending upon the physiologic state. We previously documented that carbamylated albumin had reduced cubilin binding, but the effects of cubilin modifications on binding albumin remain unclear. Here, we investigate the cubilin-albumin binding interaction to define the impact of cubilin glycosylation and map the key glycosylation sites while also targeting specific changes in a rat model of proteinuria. We identified a key Asn residue, N1285, that when glycosylated reduced albumin binding. In addition, we found a pH-induced conformation change may contribute to ligand release. To further define the albumin-cubilin binding site, we determined the solution structure of cubilin's albumin-binding domain, CUB7,8, using small-angle X-ray scattering and molecular modeling. We combined this information with mass spectrometry crosslinking experiments of CUB7,8 and albumin that provides a model of the key amino acids required for cubilin-albumin binding. Together, our data supports an important role for glycosylation in regulating the cubilin interaction with albumin, which is altered in proteinuria and provides new insight into the binding interface necessary for the cubilin-albumin interaction.

Clinical and genetic characterization of children with cubilin variants.

BACKGROUND: Cubilin is one of the receptor proteins responsible for reabsorption of albumin in proximal tubules and is encoded by the CUBN gene. We aimed to evaluate clinical and genetic characterization of six patients with proteinuria who had CUBN mutations. METHODS: Patients' characteristics, serum creatinine, albumin, vitamin B12 levels, urine analysis, spot urine protein/creatinine, microalbumin/creatinine, beta-2 microglobulin/creatinine ratios, estimated glomerular filtration rates (eGFR), treatments, kidney biopsies, and genetic analyses were evaluated. RESULTS: Six patients (2 female, 4 male) with an incidental finding of proteinuria were evaluated. Mean admission age and follow-up time were 7.3 ± 2.9 and 6.5 ± 5.6 years, respectively. Serum albumin, creatinine, and eGFR were normal; urine analysis revealed no hematuria, and C3, C4, ANA, and anti-DNA were negative; kidney ultrasonography was normal for all patients. Urine protein/creatinine was 0.9 ± 0.3 mg/mg, and microalbumin was high in all patients. Serum vitamin B12 was low in two patients and normal in four. Kidney biopsy was performed in four patients, three demonstrated normal light microscopy, and there was one focal segmental glomerulosclerosis (FSGS). Genetic tests revealed four homozygous and two compound heterozygous mutations in the C-terminal part of cubilin. All patients had normal eGFR and still had non-nephrotic range proteinuria at last visit. CONCLUSIONS: CUBN gene mutations should be considered in patients with isolated non-nephrotic range proteinuria and normal kidney function. Diagnosing these patients, who are thought to have a better prognosis, is important in terms of avoiding unnecessary treatment and predicting prognosis. CUBN gene mutations may also present as FSGS which extends the spectrum of renal manifestation of these patients. A higher resolution version of the Graphical abstract is available as Supplementary information.

Albuminuria-Related Genetic Biomarkers: Replication and Predictive Evaluation in Individuals with and without Diabetes from the UK Biobank.

Increased albuminuria indicates underlying glomerular pathology and is associated with worse renal disease outcomes, especially in diabetic kidney disease. Many single nucleotide polymorphisms (SNPs), associated with albuminuria, could be potentially useful to construct polygenic risk scores (PRSs) for kidney disease. We investigated the diagnostic accuracy of SNPs, previously associated with albuminuria-related traits, on albuminuria and renal injury in the UK Biobank population, with a particular interest in diabetes. Multivariable logistic regression was used to evaluate the influence of 91 SNPs on urine albumin-to-creatinine ratio (UACR)-related traits and kidney damage (any pathology indicating renal injury), stratifying by diabetes. Weighted PRSs for microalbuminuria and UACR from previous studies were used to calculate the area under the receiver operating characteristic curve (AUROC). CUBN-rs1801239 and DDR1-rs116772905 were associated with all the UACR-derived phenotypes, in both the overall and non-diabetic cohorts, but not with kidney damage. Several SNPs demonstrated different effects in individuals with diabetes compared to those without. SNPs did not improve the AUROC over currently used clinical variables. Many SNPs are associated with UACR or renal injury, suggesting a role in kidney dysfunction, dependent on the presence of diabetes in some cases. However, individual SNPs or PRSs did not improve the diagnostic accuracy for albuminuria or renal injury compared to standard clinical variables.

Modelling normal and nephrotic axial uptake of albumin and other filtered proteins along the proximal tubule.

Recent studies indicate that filtered albumin is retrieved in the proximal tubule (PT) via three pathways: receptor-mediated endocytosis via cubilin (high affinity) and megalin (low affinity), and fluid-phase uptake. Expression of megalin is required to maintain all three pathways, making it challenging to determine their respective contributions. Moreover, uptake of filtered molecules varies between the sub-segments (S1, S2 and S3) that make up the PT. Here we used new and published data to develop a mathematical model that predicts the rates of albumin uptake in mouse PT sub-segments in normal and nephrotic states, and partially accounts for competition by ß2 -microglobulin (ß2m) and immunoglobulin G (IgG). Our simulations indicate that receptor-mediated, rather than fluid-phase, uptake accounts for the vast majority of ligand recovery. Our model predicts that ∼75% of normally filtered albumin is reabsorbed via cubilin; however, megalin-mediated uptake predominates under nephrotic conditions. Our results also suggest that ∼80% of albumin is normally recovered in S1, whereas nephrotic conditions or knockout of cubilin shifts the bulk of albumin uptake to S2. The model predicts ß2m and IgG axial recovery profiles qualitatively similar to those of albumin under normal conditions. In contrast with albumin, however, the bulk of IgG and ß2m uptake still occurs in S1 under nephrotic conditions. Overall, our model provides a kinetic rationale for why tubular proteinuria can occur even though a large excess in potential PT uptake capacity exists, and suggests testable predictions to expand our understanding of the recovery profile of filtered proteins along the PT. KEY POINTS: We used new and published data to develop a mathematical model that predicts the profile of albumin uptake in the mouse proximal tubule in normal and nephrotic states, and partially accounts for competitive inhibition of uptake by normally filtered and pathological ligands. Three pathways, consisting of high-affinity uptake by cubilin receptors, low-affinity uptake by megalin receptors and fluid phase uptake, contribute to the overall retrieval of filtered proteins. The axial profile and efficiency of protein uptake depend on the initial filtrate composition and the individual protein affinities for megalin and cubilin. Under normal conditions, the majority of albumin is retrieved in sub-segment S1 but shifts to sub-segment S2 under nephrotic conditions. Other proteins exhibit different uptake profiles. Our model explains how tubular proteinuria can occur despite a large excess in potential proximal tubule uptake capacity.

A cellular model of albumin endocytosis uncovers a link between membrane and nuclear proteins.

Cubilin (CUBN) and amnionless (AMN), expressed in kidney and intestine, form a multiligand receptor complex called CUBAM that plays a crucial role in albumin absorption. To date, the mechanism of albumin endocytosis mediated by CUBAM remains to be elucidated. Here, we describe a quantitative assay to evaluate albumin uptake by CUBAM using cells expressing full-length CUBN and elucidate the crucial roles of the C-terminal part of CUBN and the endocytosis signal motifs of AMN in albumin endocytosis. We also demonstrate that nuclear valosin-containing protein-like 2 (NVL2), an interacting protein of AMN, is involved in this process. Although NVL2 was mainly localized in the nucleolus in cells without AMN expression, it was translocated to the extranuclear compartment when coexpressed with AMN. NVL2 knockdown significantly impaired internalization of the CUBN-albumin complex in cultured cells, demonstrating an involvement of NVL2 in endocytic regulation. These findings uncover a link between membrane and nucleolar proteins that is involved in endocytic processes.

Clinical and genetic characterization of a cohort of proteinuric patients with biallelic CUBN variants.

BACKGROUND: Proteinuria is a well-known risk factor for progressive kidney impairment. Recently, C-terminal cubilin (CUBN) variants have been associated with isolated proteinuria without progression of kidney disease. METHODS: Genetic testing of 347 families with proteinuria of suspected monogenic cause was performed by next-generation sequencing of a custom-designed kidney disease gene panel. Families with CUBN biallelic proteinuria-causing variants were studied at the clinical, genetic, laboratory and pathologic levels. RESULTS: Twelve families (15 patients) bearing homozygous or compound heterozygous proteinuria-causing variants in the C-terminal CUBN gene were identified, representing 3.5% of the total cohort. We identified 14 different sequence variants, five of which were novel. The median age at diagnosis of proteinuria was 4 years (range 9 months to 44 years), and in most cases proteinuria was detected incidentally. Thirteen patients had moderate to severe proteinuria at diagnosis without nephrotic syndrome. These patients showed lack of response to angiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blocker (ARB) treatment, normal kidney biopsy and preservation of normal kidney function over time. The two remaining patients presented a more severe phenotype, likely caused by associated comorbidities. CONCLUSIONS: Identification of C-terminal pathogenic CUBN variants is diagnostic of an entity characterized by glomerular proteinuria, normal kidney histology and lack of response to ACEi/ARB treatment. This study adds evidence and increases awareness about albuminuria caused by C-terminal variants in the CUBN gene, which is a benign condition usually diagnosed in childhood with preserved renal function until adulthood.

Renal Expression of CLC-5 and Megalin/Cubilin in Dent-1 Disease With Nonsense Mutations of CLCN5 Gene.

The study aims to explore the clinicopathological features and whether the nonsense mutations of CLCN5 gene have effect on the renal expression of CLC-5 protein and megalin/cubilin complex in children with Dent-1 disease. The clinicopathological features and genetic examination of three patients with Dent-1 disease were investigated. The expression of CLC-5 and megalin/cubilin complex in renal tissues was detected by using immunohistochemistry method. Urinary albumin, α1-microglobulin, ß2-microglobulin, retinol binding protein, and calcium levels were measured by immunonephelometry. Urinary calcium and low molecular weight proteinuria (LMWP) were enhanced in three patients, and two presented with nephrotic range proteinuria. Focal glomerular obsolescence, minor tubulointerstitial injury, and focal calcification in corticomedullary junction were found in one patient. Nonsense mutations of CLCN5 gene from their mothers were identified in all three patients with Dent-1 disease; however, the expression of CLC-5 protein was not decreased in renal tubular cells. As the receptor complex of albumin and LMWP reabsorption, the expression of megalin/cubilin in the brush border of proximal tubules was decreased in Dent-1 patients. Even if the renal CLC-5 protein is expressed normally, the reduced expression of megalin/cubilin in the brush border of renal proximal tubules may be helpful to understand the physiopathology of Dent-1 disease with nonsense mutations of CLCN5 gene.

Endocytic adaptation to functional demand by the kidney proximal tubule.

The kidney proximal tubule (PT) efficiently recovers the low level of albumin and other proteins that normally escape the glomerular filtration barrier. Two large receptors, megalin and cubilin/amnionless (CUBAM), bind to and efficiently retrieve these predominantly low molecular-weight proteins via clathrin-mediated endocytosis. Studies in cell culture models suggest that PT cells may sense changes in shear stress to modulate recovery of filtered proteins in response to normal variations in filtration rate. Impairments in PT endocytic function lead to the excretion of filtered proteins into the urine (tubular proteinuria). Remarkably, when the glomerular filtration barrier is breached, the PT is able to recover excess albumin with a capacity that is orders of magnitude higher than normal. What mediates this excess capacity for albumin uptake under nephrotic conditions, and why doesn't it compensate to prevent tubular proteinuria? Here we propose an integrated new working model to describe the PT recovery of filtered proteins under normal and nephrotic states. We hypothesize that uptake via the fluid phase provides excess capacity to recover high concentrations of filtered proteins under nephrotic conditions. Further, concentration of tubular fluid along the tubule axis will enhance the efficiency of uptake in more distal regions of the PT. By contrast to cells where fluid phase and receptor-mediated uptake are independent pathways, expression of megalin is required to maintain apical endocytic pathway integrity and is essential for both uptake mechanisms. This model accounts for both the high-affinity and the high-capacity responses to filtration load in physiological and pathological states.

Mechanism of how carbamylation reduces albumin binding to FcRn contributing to increased vascular clearance.

Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin's reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.

Cubam receptor-mediated endocytosis in hindgut-derived pseudoplacenta of a viviparous teleost (Xenotoca eiseni).

Nutrient transfer from mother to embryo is essential for reproduction in viviparous animals. In the viviparous teleost Xenotoca eiseni (family Goodeidae), the intraovarian embryo intakes the maternal component secreted into the ovarian fluid via the trophotaenia. Our previous study reported that the epithelial layer cells of the trophotaenia incorporate a maternal protein via vesicle trafficking. However, the molecules responsible for the absorption were still elusive. Here, we focused on Cubam (Cubilin-Amnionless) as a receptor involved in the absorption, and cathepsin L as a functional protease in the vesicles. Our results indicated that the Cubam receptor is distributed in the apical surface of the trophotaenia epithelium and then is taken into the intracellular vesicles. The trophotaenia possesses acidic organelles in epithelial layer cells and cathepsin L-dependent proteolysis activity. This evidence does not conflict with our hypothesis that receptor-mediated endocytosis and proteolysis play roles in maternal macromolecule absorption via the trophotaenia in viviparous teleosts. Such nutrient absorption involving endocytosis is not a specific trait in viviparous fish. Similar processes have been reported in the larval stage of oviparous fish or the suckling stage of viviparous mammals. Our findings suggest that the viviparous teleost acquired trophotaenia-based viviparity from a modification of the intestinal absorption system common in vertebrates. This is a fundamental study to understand the strategic variation of the reproductive system in vertebrates.

Receptor-Mediated Endocytosis in the Proximal Tubule.

Cells lining the proximal tubule (PT) of the kidney are highly specialized for apical endocytosis of filtered proteins and small bioactive molecules from the glomerular ultrafiltrate to maintain essentially protein-free urine. Compromise of this pathway results in low molecular weight (LMW) proteinuria that can progress to end-stage kidney disease. This review describes our current understanding of the endocytic pathway and the multiligand receptors that mediate LMW protein uptake in PT cells, how these are regulated in response to physiologic cues, and the molecular basis of inherited diseases characterized by LMW proteinuria.

Renal Handling of Albumin-From Early Findings to Current Concepts.

Albumin is the main protein of blood plasma, lymph, cerebrospinal and interstitial fluid. The protein participates in a variety of important biological functions, such as maintenance of proper colloidal osmotic pressure, transport of important metabolites and antioxidant action. Synthesis of albumin takes place mainly in the liver, and its catabolism occurs mostly in vascular endothelium of muscle, skin and liver, as well as in the kidney tubular epithelium. Long-lasting investigation in this area has delineated the principal route of its catabolism involving glomerular filtration, tubular endocytic uptake via the multiligand scavenger receptor tandem-megalin and cubilin-amnionless complex, as well as lysosomal degradation to amino acids. However, the research of the last few decades indicates that also additional mechanisms may operate in this process to some extent. Direct uptake of albumin in glomerular podocytes via receptor for crystallizable region of immunoglobulins (neonatal FC receptor) was demonstrated. Additionally, luminal recycling of short peptides into the bloodstream and/or back into tubular lumen or transcytosis of whole molecules was suggested. The article discusses the molecular aspects of these processes and presents the major findings and controversies arising in the light of the research concerning the last decade. Their better characterization is essential for further research into pathophysiology of proteinuric renal failure and development of effective therapeutic strategies.

Novel Cardiovascular Risk Factors in Patients with Diabetic Kidney Disease.

Patients with diabetic kidney disease (DKD) are at very high risk for cardiovascular events. Only part of this increased risk can be attributed to the presence of diabetes mellitus (DM) and to other DM-related comorbidities, including hypertension and obesity. The identification of novel risk factors that underpin the association between DKD and cardiovascular disease (CVD) is essential for risk stratification, for individualization of treatment and for identification of novel treatment targets.In the present review, we summarize the current knowledge regarding the role of emerging cardiovascular risk markers in patients with DKD. Among these biomarkers, fibroblast growth factor-23 and copeptin were studied more extensively and consistently predicted cardiovascular events in this population. Therefore, it might be useful to incorporate them in risk stratification strategies in patients with DKD to identify those who would possibly benefit from more aggressive management of cardiovascular risk factors.

Proximal tubule transferrin uptake is modulated by cellular iron and mediated by apical membrane megalin-cubilin complex and transferrin receptor 1.

Receptor-mediated endocytosis is responsible for reabsorption of transferrin (Tf) in renal proximal tubules (PTs). Although the role of the megalin-cubilin receptor complex (MCRC) in this process is unequivocal, modalities independent of this complex are evident but as yet undefined. Here, using immunostaining and Tf-flux assays, FACS analysis, and fluorescence imaging, we report localization of Tf receptor 1 (TfR1), the cognate Tf receptor mediating cellular holo-Tf (hTf) acquisition, to the apical brush border of the PT, with expression gradually declining along the PT in mouse and rat kidneys. In functional studies, hTf uptake across the apical membrane of cultured PT epithelial cell (PTEC) monolayers increased in response to decreased cellular iron after desferrioxamine (DFO) treatment. We also found that apical hTf uptake under basal conditions is receptor-associated protein (RAP)-sensitive and therefore mediated by the MCRC but becomes RAP-insensitive under DFO treatment, with concomitantly decreased megalin and cubilin expression levels and increased TfR1 expression. Thus, as well as the MCRC, TfR1 mediates hTf uptake across the PT apical brush border, but in conditions of decreased cellular iron, hTf uptake is predominated by augmented apical TfR1. In conclusion, both the MCRC and TfR1 mediate hTf uptake across apical brush border membranes of PTECs and reciprocally respond to decreased cellular iron. Our findings have implications for renal health, whole-body iron homeostasis, and pathologies arising from disrupted iron balance.

Distinct functions of megalin and cubilin receptors in recovery of normal and nephrotic levels of filtered albumin.

Proximal tubule (PT) cells express a single saturable albumin-binding site whose affinity matches the estimated tubular concentration of albumin; however, albumin uptake capacity is greatly increased under nephrotic conditions. Deciphering the individual contributions of megalin and cubilin to the uptake of normal and nephrotic levels of albumin is impossible in vivo, as knockout of megalin in mice globally disrupts PT endocytic uptake. We quantified concentration-dependent albumin uptake in an optimized opossum kidney cell culture model and fit the kinetic profiles to identify albumin-binding affinities and uptake capacities. Mathematical deconvolution fit best to a three-component model that included saturable high- and low-affinity uptake sites for albumin and underlying nonsaturable uptake consistent with passive uptake of albumin in the fluid phase. Knockdown of cubilin or its chaperone amnionless selectively reduced the binding capacity of the high-affinity site, whereas knockdown of megalin impacted the low-affinity site. Knockdown of disabled-2 decreased the capacities of both binding sites. Additionally, knockdown of megalin or disabled-2 profoundly inhibited the uptake of a fluid phase marker, with cubilin knockdown having a more modest effect. We propose a novel model for albumin retrieval along the PT in which cubilin and megalin receptors have different functions in recovering filtered albumin in proximal tubule cells. Cubilin binding to albumin is tuned to capture normally filtered levels of the protein. In contrast, megalin binding to albumin is of lower affinity, and its expression is also essential for enabling the recovery of high concentrations of albumin in the fluid phase.

Megalin-mediated albumin endocytosis in cultured murine mesangial cells.

Endocytosis by podocytes is gaining increased attention as a biologic means of removing large proteins such as serum albumin from the glomerular barrier. Some of this function has been attributed to the megalin/cubilin (Lrp2/Cubn) receptor complex and the albumin recycling protein FcRn (Fcgrt). However, whether other glomerular cells possess the potential to perform this same phenomenon or express these proteins remains uncharacterized. Mesangial cells are uniquely positioned in glomeruli and represent a cell type capable of performing several diverse functions. Here, the expression of megalin and FcRn in murine mesangial cells along with the megalin adaptor protein Dab-2 (Dab2) was shown for the first time. Cubilin mRNA expression was detected, but the absence of the cubilin partner amnionless (Amn) suggested that cubilin is minimally functional, if at all, in these cells. Mesangial cell endocytosis of albumin was characterized and shown to involve a receptor-mediated process. Albumin endocytosis was significantly impaired (p < 0.01) under inducible megalin knockdown conditions in stably transduced mesangial cells. The current work provides both the novel identification of megalin and FcRn in mesangial cells and the functional demonstration of megalin-mediated albumin endocytosis.

Genetic Analyses in Dent Disease and Characterization of CLCN5 Mutations in Kidney Biopsies.

Dent disease (DD), an X-linked renal tubulopathy, is mainly caused by loss-of-function mutations in CLCN5 (DD1) and OCRL genes. CLCN5 encodes the ClC-5 antiporter that in proximal tubules (PT) participates in the receptor-mediated endocytosis of low molecular weight proteins. Few studies have analyzed the PT expression of ClC-5 and of megalin and cubilin receptors in DD1 kidney biopsies. About 25% of DD cases lack mutations in either CLCN5 or OCRL genes (DD3), and no other disease genes have been discovered so far. Sanger sequencing was used for CLCN5 gene analysis in 158 unrelated males clinically suspected of having DD. The tubular expression of ClC-5, megalin, and cubilin was assessed by immunolabeling in 10 DD1 kidney biopsies. Whole exome sequencing (WES) was performed in eight DD3 patients. Twenty-three novel CLCN5 mutations were identified. ClC-5, megalin, and cubilin were significantly lower in DD1 than in control biopsies. The tubular expression of ClC-5 when detected was irrespective of the type of mutation. In four DD3 patients, WES revealed 12 potentially pathogenic variants in three novel genes (SLC17A1, SLC9A3, and PDZK1), and in three genes known to be associated with monogenic forms of renal proximal tubulopathies (SLC3A, LRP2, and CUBN). The supposed third Dent disease-causing gene was not discovered.

Imerslund-Gräsbeck Syndrome in an Infant with a Novel Intronic Variant in the AMN Gene: A Case Report.

Imerslund-Gräsbeck syndrome (IGS) is a rare autosomal recessive disorder clinically characterized by megaloblastic anemia, benign mild proteinuria, and other nonspecific symptoms. Several pathogenetic variants in the amnionless (AMN) or cubilin (CUBN) genes have been described in IGS. We describe a case of IGS with urinary tract infection and mild but persistent proteinuria at onset in an 11-month-old female child. With the appearance of macrocytic anemia, aphthous stomatitis, and neurological signs, IGS was clinically suspected, and vitamin B12 parenteral therapy was started. Sequence analysis showed the presence of a novel intronic variant c.513+5G>A of AMN, never before described in the literature, that was in compound heterozygosity with the known pathogenetic variant c.1006+34_1007-31del. Analysis extension to the parents revealed the presence of variant c.1006+34_1007-31 in the father and c.513+5G>A in the mother. In the present case with IGS, the novel intronic variant of AMN was identified in "trans" with a known pathogenic variant (c.1006-31 del) and the new variant was interpreted to be pathogenetic since it was not found in the public database of polymorphisms and because it was predicted to alter a donor splicing site. Our case underlines the relevance in detecting certain subtle symptoms, such as mild but persistent proteinuria associated with megaloblastic anemia, to reach a correct diagnosis of a rare but treatable disorder.

Cadmium Complexed with ß2-Microglubulin, Albumin and Lipocalin-2 rather than Metallothionein Cause Megalin:Cubilin Dependent Toxicity of the Renal Proximal Tubule.

Cadmium (Cd2+) in the environment is a significant health hazard. Chronic low Cd2+ exposure mainly results from food and tobacco smoking and causes kidney damage, predominantly in the proximal tubule. Blood Cd2+ binds to thiol-containing high (e.g., albumin, transferrin) and low molecular weight proteins (e.g., the high-affinity metal-binding protein metallothionein, ß2-microglobulin, α1-microglobulin and lipocalin-2). These plasma proteins reach the glomerular filtrate and are endocytosed at the proximal tubule via the multiligand receptor complex megalin:cubilin. The current dogma of chronic Cd2+ nephrotoxicity claims that Cd2+-metallothionein endocytosed via megalin:cubilin causes renal damage. However, a thorough study of the literature strongly argues for revision of this model for various reasons, mainly: (i) It relied on studies with unusually high Cd2+-metallothionein concentrations; (ii) the KD of megalin for metallothionein is ~105-times higher than (Cd2+)-metallothionein plasma concentrations. Here we investigated the uptake and toxicity of ultrafiltrated Cd2+-binding protein ligands that are endocytosed via megalin:cubilin in the proximal tubule. Metallothionein, ß2-microglobulin, α1-microglobulin, lipocalin-2, albumin and transferrin were investigated, both as apo- and Cd2+-protein complexes, in a rat proximal tubule cell line (WKPT-0293 Cl.2) expressing megalin:cubilin at low passage, but is lost at high passage. Uptake was determined by fluorescence microscopy and toxicity by MTT cell viability assay. Apo-proteins in low and high passage cells as well as Cd2+-protein complexes in megalin:cubilin deficient high passage cells did not affect cell viability. The data prove Cd2+-metallothionein is not toxic, even at >100-fold physiological metallothionein concentrations in the primary filtrate. Rather, Cd2+-ß2-microglobulin, Cd2+-albumin and Cd2+-lipocalin-2 at concentrations present in the primary filtrate are taken up by low passage proximal tubule cells and cause toxicity. They are therefore likely candidates of Cd2+-protein complexes damaging the proximal tubule via megalin:cubilin at concentrations found in the ultrafiltrate.