Polybrominated diphenyl ethers in dust, hair and urine: Exposure, excretion.

Polybrominated diphenyl ethers (PBDEs) have been detected in various environmental media and human tissues. PBDEs concentrations in dust from college buildings and homes and in paired hair and urine samples from students were determined. This is of great significance to explore the accumulation and excretion patterns of PBDEs in the human body. The median PBDEs concentrations in the dust (College: 84.59 ng/g; Home: 170.32 ng/g) and hair (undergraduate: 6.16 ng/g; Home: 3.25 ng/g) samples were generally lower than were found in the majority of previous studies. The PBDEs concentrations in the hair and urine samples were subjected to principal component analysis, and the results combined with the PBDEs detection rates confirmed that hair is a useful non-invasive sampling medium for assessing PBDEs exposure and the risks posed. Body mass indices (BMIs) were used to divide students who had not been exposed to large amounts of PBDEs into groups. Body fat percentage is an important factor affecting the accumulation of PBDE in the human body. Environmental factors were found to affect the PBDEs concentrations in the hair and urine samples less for normal-weight students (BMI≤24) than overweight students (BMI>24). Short-term environmental changes to more readily affect the PBDEs concentrations in the tissues of the normal-weight than overweight students. PBDEs with seven or more bromine substituents were found not to be readily excreted in urine. Performing molecular docking simulations of the binding of isomers BDE-99 and BDE-100 to megalin. The binding energy was higher for BDE-100 and megalin than for BDE-99 and megalin, meaning BDE-99 would be more readily excreted than BDE-100.

Low Density Lipoprotein Receptor-related Protein 2 Expression and Function in Cultured Astrocytes and Microglia.

Activation of glial cells, astrocytes and microglia, has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Amyloid ß (Aß), which is aggregated and the aggregation is detected as characteristic pathology in AD brain, is known to be produced by neurons and to activate glial cells. Clearance of Aß from the brain via active transport system is important to prevent the accumulation and aggregation. Low density lipoprotein receptor-related protein 2 (LRP2/megalin) is an Aß transporter. However, expression and contribution of LRP2 in astrocytes and microglia remain to be clarified. In the present study, we examined the expression of LRP2 and its roles in cultured astrocytes prepared from rat embryonic brain cortex and mouse microglial cell line BV-2. Both cultured rat astrocytes and BV-2 cells expressed LRP2 mRNA detected by RT-PCR. When lipopolysaccharide (LPS) or all-trans retinoic acid (ATRA) were added to BV-2 cells, LRP2 mRNA expression and uptake of microbeads, Aß and insulin were increased. On the other hand, LPS decreased LRP2 expression and uptake of Aß and insulin in cultured astrocytes. Knockdown of LRP2 using siRNA attenuated the LPS- or ATRA-increased uptake of microbeads, Aß and insulin in BV-2 cells. These results suggest that LRP2 was expressed in both astrocytes and microglia and might be involved in endocytosis activities. Adequate control of LRP2 expression and function in astrocytes and microglia might regulate Aß and insulin levels in brain and would be a potential target in AD pathology.

Filtration and tubular handling of EWE-hC3Nb1, a complement inhibitor nanobody, in wild type mice and a mouse model of proteinuric kidney disease.

Tubular activation and deposition of filtered complement proteins have been implicated in the progression of proteinuric kidney disease. The potent C3b-specific nanobody inhibitor of the alternative pathway, EWE-hC3Nb1, is likely freely filtered in the glomerulus to allow complement inhibition in the tubular lumen and may provide a novel treatment option to prevent tubulointerstitial injury. However, more information on the pharmacokinetic properties and renal tubular handling of EWE-hC3Nb1 nanobody is required for its pharmacological application in relation to kidney disease. Here, we examined the pharmacokinetic properties of free EWE-hC3Nb1 in mouse plasma and urine, following subcutaneous injection in wild-type control and podocin knock out (KO) mice with severe proteinuria. Tubular handling of filtered EWE-hC3Nb1 was assessed by immunohistochemistry (IHC) on kidney tissue from control, proteinuric mice, and KO mice deficient in the proximal tubule endocytic receptor megalin. Rapid plasma absorption and elimination of EWE-hC3Nb1 was observed in both control and proteinuric mice; however, urinary excretion of EWE-hC3Nb1 was markedly increased in proteinuric mice. Urinary EWE-hC3Nb1 excretion was amplified in megalin KO mice, and substantial accumulation of EWE-hC3Nb1 was observed in megalin-expressing renal proximal tubules by IHC. Moreover, free EWE-hC3Nb1 was found to be rapidly cleared from plasma. In conclusion, filtered EWE-hC3Nb1 is reabsorbed by a megalin-dependent process in the proximal tubules. Increased load of filtered proteins in the tubular fluid may inhibit the megalin-dependent uptake of EWE-hC3Nb1 in proteinuric mice. Treatment with EWE-hC3Nb1 may allow investigation of the effects of complement inhibition in the tubular fluid.

Dietary supplementation of cystinotic mice by lysine inhibits the megalin pathway and decreases kidney cystine content.

Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by L- and D-stereoisomers. In cystinotic mice, 2-month diets with 5x-L-lysine and 5x-L-arginine were overall well tolerated, while 5x-D-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-D-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by D-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo.

Megalin is involved in angiotensinogen-induced, angiotensin II-mediated ERK1/2 signaling to activate Na + -H + exchanger 3 in proximal tubules.

BACKGROUND: Kidney angiotensin (Ang) II is produced mainly from liver-derived, glomerular-filtered angiotensinogen (AGT). Podocyte injury has been reported to increase the kidney Ang II content and induce Na + retention depending on the function of megalin, a proximal tubular endocytosis receptor. However, how megalin regulates the renal content and action of Ang II remains elusive. METHODS: We used a mass spectrometry-based, parallel reaction-monitoring assay to quantitate Ang II in plasma, urine, and kidney homogenate of kidney-specific conditional megalin knockout (MegKO) and control (Ctl) mice. We also evaluated the pathophysiological changes in both mouse genotypes under the basal condition and under the condition of increased glomerular filtration of AGT induced by administration of recombinant mouse AGT (rec-mAGT). RESULTS: Under the basal condition, plasma and kidney Ang II levels were comparable in the two mouse groups. Ang II was detected abundantly in fresh spot urine in conditional MegKO mice. Megalin was also found to mediate the uptake of intravenously administered fluorescent Ang II by PTECs. Administration of rec-mAGT increased kidney Ang II, exerted renal extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, activated proximal tubular Na + -H + exchanger 3 (NHE3), and decreased urinary Na + excretion in Ctl mice, whereas these changes were suppressed but urinary Ang II was increased in conditional MegKO mice. CONCLUSION: Increased glomerular filtration of AGT is likely to augment Ang II production in the proximal tubular lumen. Thus, megalin-dependent Ang II uptake should be involved in the ERK1/2 signaling that activates proximal tubular NHE3 in vivo , thereby causing Na + retention.

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

Introduction: 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. Methods: 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. Results: IV significantly downregulated albumin uptake, independently of activation of the TGF-ß1/GSK3ß 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. Discussion: 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.

Severe kidney dysfunction in sialidosis mice reveals an essential role for neuraminidase 1 in reabsorption.

Sialidosis is an ultra-rare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe type II form of the disease manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology, and visceromegaly. A subset of these patients present with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in 2 NEU1-deficient mouse models, a constitutive Neu1-knockout, Neu1ΔEx3, and a conditional phagocyte-specific knockout, Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles, and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal distal tubules, and distal tubules was drastically increased, including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.

Role of the SLC22A17/lipocalin-2 receptor in renal endocytosis of proteins/metalloproteins: a focus on iron- and cadmium-binding proteins.

The transmembrane protein SLC22A17 [or the neutrophil gelatinase-associated lipocalin/lipocalin-2 (LCN2)/24p3 receptor] is an atypical member of the SLC22 family of organic anion and cation transporters: it does not carry typical substrates of SLC22 transporters but mediates receptor-mediated endocytosis (RME) of LCN2. One important task of the kidney is the prevention of urinary loss of proteins filtered by the glomerulus by bulk reabsorption of multiple ligands via megalin:cubilin:amnionless-mediated endocytosis in the proximal tubule (PT). Accordingly, overflow, glomerular, or PT damage, as in Fanconi syndrome, results in proteinuria. Strikingly, up to 20% of filtered proteins escape the PT under physiological conditions and are reabsorbed by the distal nephron. The renal distal tubule and collecting duct express SLC22A17, which mediates RME of filtered proteins that evade the PT but with limited capacity to prevent proteinuria under pathological conditions. The kidney also prevents excretion of filtered essential and nonessential transition metals, such as iron or cadmium, respectively, that are largely bound to proteins with high affinity, e.g., LCN2, transferrin, or metallothionein, or low affinity, e.g., microglobulins or albumin. Hence, increased uptake of transition metals may cause nephrotoxicity. Here, we assess the literature on SLC22A17 structure, topology, tissue distribution, regulation, and assumed functions, emphasizing renal SLC22A17, which has relevance for physiology, pathology, and nephrotoxicity due to the accumulation of proteins complexed with transition metals, e.g., cadmium or iron. Other putative renal functions of SLC22A17, such as its contribution to osmotic stress adaptation, protection against urinary tract infection, or renal carcinogenesis, are discussed.

From pollakiuria to Donnai-Barrow syndrome diagnosis in pediatric age.

We report the case of two siblings with incomplete Donnai-Barrow syndrome (DBS) phenotype carrying three LRP2 variants never associated before with DBS phenotype.

Human parietal epithelial cells (PECs) and proteinuria in lupus nephritis: a role for ClC-5, megalin, and cubilin?

BACKGROUND: Parietal epithelial cells are a heterogeneous population of cells located on Bowman's capsule. These cells are known to internalize albumin with a still undetermined mechanism, although albumin has been shown to induce phenotypic changes in parietal epithelial cells. Proximal tubular cells are the main actors in albumin handling via the macromolecular complex composed by ClC-5, megalin, and cubilin. This study investigated the role of ClC-5, megalin, and cubilin in the parietal epithelial cells of kidney biopsies from proteinuric lupus nephritis patients and control subjects and identified phenotypical changes occurring in the pathological milieu. METHODS: Immunohistochemistry and immunofluorescence analyses for ClC-5, megalin, cubilin, ANXA3, podocalyxin, CD24, CD44, HSA, and LTA marker were performed on 23 kidney biopsies from patients with Lupus Nephritis and 9 control biopsies (obtained from nephrectomies for renal cancer). RESULTS: Two sub-populations of hypertrophic parietal epithelial cells ANXA3+/Podocalyxin-/CD44-, both expressing ClC-5, megalin, and cubilin and located at the tubular pole, were identified and characterized: the first one, CD24+/HSA-/LTA- had characteristics of human adult parietal epithelial multipotent progenitors, the second one, CD24-/LTA+/HSA+ committed to become phenotypically proximal tubular cells. The number of glomeruli presenting hypertrophic parietal epithelial cells positive for ClC-5, megalin, and cubilin were significantly higher in lupus nephritis patients than in controls. CONCLUSIONS: Our results may provide further insight into the role of hypertrophic parietal epithelial cells located at the tubular pole and their possible involvement in protein endocytosis in lupus nephritis patients. These data also suggest that the presence of hypertrophic parietal epithelial cells in Bowman's capsule represents a potential resource for responding to protein overload observed in other glomerulonephritis.

Differentiation and Subculturing of Renal Proximal Tubular-like Cells Derived from Human iPSC.

Recently, we have developed a protocol to differentiate human induced pluripotent stem cells (iPSC) into proximal tubular-like cells (PTL) (Chandrasekaran et al., 2021). These cells express proximal tubular-specific markers, including megalin, and form a polarized monolayer expressing tight junction proteins, including ZO-3 and occludin. Furthermore, PTL display functional properties, including megalin-facilitated endocytosis, P-glycoprotein (ABCB1) efflux, and respond to parathyroid hormone. Here, we report step-by-step protocols to culture iPSC prior to differentiation (Basic Protocol 1), to differentiate PTL from iPSC (Basic Protocol 2), and to passage and freeze-thaw PTL (Basic Protocol 3). Additionally, we provide a protocol (Basic Protocol 4) to culture PTL on microporous growth supports (transwells). Immunofluorescence stainings for characteristic markers, including megalin, are shown for unpassaged (Basic Protocol 2) and passaged (Basic Protocol 3) PTL. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: iPSC culture Basic Protocol 2: iPSC-derived PTL differentiation Basic Protocol 3: PTL passaging, culturing, and freezing Basic Protocol 4: PTL culturing on transwells Support Protocol 1: Preparation of Geltrex-coated cell culture plates Support Protocol 2: Preparation of RPTEC/TERT1 or fHDF/TERT166-ECM-coated cell culture plates Support Protocol 3: Preparation of human collagen IV-coated cell culture plates Support Protocol 4: Immunofluorescence staining.

Receptor-associated protein impairs ligand binding to megalin and megalin-dependent endocytic flux in proximal tubule cells.

Proximal tubule (PT) cells retrieve albumin and a broad array of other ligands from the glomerular ultrafiltrate. Efficient uptake of albumin requires PT expression of both megalin and cubilin receptors. Although most proteins engage cubilin selectively, megalin is required to maintain robust flux through the apical endocytic pathway. Receptor-associated protein (RAP) is a chaperone that directs megalin to the cell surface, and recombinant RAP dramatically inhibits the uptake of numerous megalin and cubilin ligands. The mechanism by which this occurs has been suggested to involve competitive inhibition of ligand binding and/or conformational changes in megalin that prevent interaction with ligands and/or with cubilin. To discriminate between these possibilities, we determined the effect of RAP on endocytosis of albumin, which binds to cubilin and megalin receptors with high and low affinity, respectively. Uptake was quantified in opossum kidney (OK) cells and in megalin or cubilin (Cubn) knockout (KO) clones. Surprisingly, RAP inhibited fluid-phase uptake in addition to receptor-mediated uptake in OK cells and Cubn KO cells but had no effect on endocytosis when megalin was absent. The apparent Ki for RAP inhibition of albumin uptake was 10-fold higher in Cubn KO cells compared with parental OK cells. We conclude that in addition to its predicted high-affinity competition for ligand binding to megalin, the primary effect of RAP on PT cell endocytosis is to globally dampen megalin-dependent endocytic flux. Our data explain the complex effects of RAP on binding and uptake of filtered proteins and reveal a novel role in modulating endocytosis in PT cells.NEW & NOTEWORTHY Receptor-associated protein inhibits binding and uptake of all known endogenous ligands by megalin and cubilin receptors via unknown mechanism(s). Here, we took advantage of recently generated knockout cell lines to dissect the effect of this protein on megalin- and cubilin-mediated endocytosis. Our study reveals a novel role for receptor-associated protein in blocking megalin-stimulated endocytic uptake of fluid-phase markers and receptor-bound ligands in proximal tubule cells in addition to its direct effect on ligand binding to megalin receptors.

Association between urinary C-megalin levels and progressive kidney dysfunction: a cohort study based on the diabetes distress and care registry at Tenri (DDCRT 24).

AIMS: The aim of this cohort study was to evaluate the association between urinary levels of C-megalin, a full-length form of megalin, and kidney dysfunction progression and its dependence on the urinary albumin-creatinine ratio (UACR) in individuals with diabetes. METHODS: We enrolled 1,547 individuals with diabetes who visited the ambulatory clinic at Tenri Hospital, a regional tertiary-care hospital in Tenri City, Nara Prefecture, Japan, with an estimated glomerular filtration (eGFR) of ≥ 30 mL/min/1.73 m2. The hazard ratio (HR) and 95% confidence interval (CI) were estimated using Cox proportional hazard models to examine the association between urinary C-megalin levels and eGFR decline by ≥ 40% from baseline. RESULTS: Urinary C-megalin level was not associated with ≥ 40% eGFR decline in an age-, sex-, eGFR-, systolic blood pressure-, hemoglobin-, and UACR-adjusted model in the 1,547 patients enrolled in the study. However, urinary C-megalin levels were associated with a ≥ 40% decline in eGFR when accounting for the relationship between urinary C-megalin levels and UACR in the model. This association was UACR-dependent. CONCLUSIONS: High urinary C-megalin levels were associated with progressive kidney dysfunction in individuals with diabetes, and this association was attenuated by high UACRs.

Proprotein convertase subtilisin/kexin type 9 targets megalin in the kidney proximal tubule and aggravates proteinuria in nephrotic syndrome.

Proteinuria is a prominent feature of chronic kidney disease. Interventions that reduce proteinuria slow the progression of chronic kidney disease and the associated risk of cardiovascular disease. Here, we propose a mechanistic coupling between proteinuria and proprotein convertase subtilisin/kexin type 9 (PCSK9), a regulator of cholesterol and a therapeutic target in cardiovascular disease. PCSK9 undergoes glomerular filtration and is captured by megalin, the receptor responsible for driving protein reabsorption in the proximal tubule. Accordingly, megalin-deficient mice and patients carrying megalin pathogenic variants (Donnai Barrow syndrome) were characterized by elevated urinary PCSK9 excretion. Interestingly, PCSK9 knockout mice displayed increased kidney megalin while PCSK9 overexpression resulted in its reduction. Furthermore, PCSK9 promoted trafficking of megalin to lysosomes in cultured proximal tubule cells, suggesting that PCSK9 is a negative regulator of megalin. This effect can be accelerated under disease conditions since either genetic destruction of the glomerular filtration barrier in podocin knockout mice or minimal change disease (a common cause of nephrotic syndrome) in patients resulted in enhanced tubular PCSK9 uptake and urinary PCSK9 excretion. Pharmacological PCSK9 inhibition increased kidney megalin while reducing urinary albumin excretion in nephrotic mice. Thus, glomerular damage increases filtration of PCSK9 and concomitantly megalin degradation, resulting in escalated proteinuria.

Megalin, cubilin, and Dab2 drive endocytic flux in kidney proximal tubule cells.

The kidney proximal tubule (PT) elaborates a uniquely high-capacity apical endocytic pathway to retrieve albumin and other proteins that escape the glomerular filtration barrier. Megalin and cubilin/amnionless (CUBAM) receptors engage Dab2 in these cells to mediate clathrin-dependent uptake of filtered ligands. Knockout of megalin or Dab2 profoundly inhibits apical endocytosis and is believed to atrophy the endocytic pathway. We generated CRISPR/Cas9 knockout (KO) clones lacking cubilin, megalin, or Dab2 expression in highly differentiated PT cells and determined the impact on albumin internalization and endocytic pathway function. KO of each component had different effects on the concentration dependence of albumin uptake as well its distribution within PT cells. Reduced uptake of a fluid phase marker was also observed, with megalin KO cells having the most dramatic decline. Surprisingly, protein levels and distribution of key endocytic proteins were preserved in KO PT cell lines and in megalin KO mice, despite the reduced endocytic activity. Our data highlight specific functions of megalin, cubilin, and Dab2 in apical endocytosis and demonstrate that these proteins drive endocytic flux without compromising the physical integrity of the apical endocytic pathway. Our studies suggest a novel model to explain how these components coordinate endocytic uptake in PT cells.

Expression of leptin receptor in renal tubules is sparse but implicated in leptin-dependent kidney gene expression and function.

Leptin regulates energy balance via leptin receptors expressed in central and peripheral tissues, but little is known about leptin-sensitive kidney genes and the role of the tubular leptin receptor (Lepr) in response to a high-fat diet (HFD). Quantitative RT-PCR analysis of Lepr splice variants A, B, and C revealed a ratio of ∼100:10:1 in the mouse kidney cortex and medulla, with medullary levels being ∼10 times higher. Leptin replacement in ob/ob mice for 6 days reduced hyperphagia, hyperglycemia, and albuminuria, associated with normalization of kidney mRNA expression of molecular markers of glycolysis, gluconeogenesis, amino acid synthesis, and megalin. Normalization of leptin for 7 h in ob/ob mice did not normalize hyperglycemia or albuminuria. Tubular knockdown of Lepr [Pax8-Lepr knockout (KO)] and in situ hybridization revealed a minor fraction of Lepr mRNA in tubular cells compared with endothelial cells. Nevertheless, Pax8-Lepr KO mice had lower kidney weight. Moreover, while HFD-induced hyperleptinemia, increases in kidney weight and glomerular filtration rate, and a modest blood pressure lowering effect were similar compared with controls, they showed a blunted rise in albuminuria. Use of Pax8-Lepr KO and leptin replacement in ob/ob mice identified acetoacetyl-CoA synthetase and gremlin 1 as tubular Lepr-sensitive genes that are increased and reduced by leptin, respectively. In conclusion, leptin deficiency may increase albuminuria via systemic metabolic effects that impinge on kidney megalin expression, whereas hyperleptinemia may induce albuminuria by direct tubular Lepr effects. Implications of Lepr variants and the novel tubular Lepr/acetoacetyl-CoA synthetase/gremlin 1 axis remain to be determined.NEW & NOTEWORTHY This study provides new insights into kidney gene expression of leptin receptor splice variants, leptin-sensitive kidney gene expression, and the role of the leptin receptor in renal tubular cells for the response to diet-induced hyperleptinemia and obesity including albuminuria.

Angiotensin II type 2 receptor activation preserves megalin in the kidney and prevents proteinuria in high salt diet fed rats.

Proteinuria is a risk factor for and consequence of kidney injury. Angiotensin II type 2 receptor (AT2R) is an emerging reno-protective target and is anti-proteinuric under pathological conditions, including high salt-fed obese animals. However, the mechanisms remain unknown, particularly whether the anti-proteinuric activity of AT2R is independent of its anti-hypertensive and anti-inflammatory effects. In the present study, obese Zucker rats were fed high sodium (4%) diet (HSD) for 48 h, a time in which blood pressure does not change. HSD caused proteinuria without affecting glomerular slit diaphragm proteins (nephrin and podocin), glomerular filtration rate, inflammatory and fibrotic markers (TNFα, IL-6, and TGF-ß), ruling out glomerular injury, inflammation and fibrosis but indicating tubular mechanisms of proteinuria. At cellular and molecular levels, we observed a glycogen synthase kinase (GSK)-3ß-mediated megalin phosphorylation, and its subsequent endocytosis and lysosomal degradation in HSD-fed rat kidneys. Megalin is a major proximal tubular endocytic protein transporter. The AT2R agonist C21 (0.3 mg/kg/day, i.p.) administration prevented proteinuria and rescued megalin surface expression potentially by activating Akt-mediated phosphorylation and inactivation of GSK-3ß in HSD-fed rat kidneys. Overall, AT2R has a direct anti-proteinuric activity, potentially via megalin regulation, and is suggested as a novel target to limit kidney injury.

Regulation of Prostate Androgens by Megalin and 25-hydroxyvitamin D Status: Mechanism for High Prostate Androgens in African American Men.

Vitamin D deficiency is associated with an increased risk of prostate cancer mortality and is hypothesized to contribute to prostate cancer aggressiveness and disparities in African American populations. The prostate epithelium was recently shown to express megalin, an endocytic receptor that internalizes circulating globulin-bound hormones, which suggests regulation of intracellular prostate hormone levels. This contrasts with passive diffusion of hormones that is posited by the free hormone hypothesis. Here, we demonstrate that megalin imports testosterone bound to sex hormone-binding globulin into prostate cells. Prostatic loss of Lrp2 (megalin) in a mouse model resulted in reduced prostate testosterone and dihydrotestosterone levels. Megalin expression was regulated and suppressed by 25-hydroxyvitamin D (25D) in cell lines, patient-derived prostate epithelial cells, and prostate tissue explants. In patients, the relationships between hormones support this regulatory mechanism, as prostatic DHT levels are higher in African American men and are inversely correlated with serum 25D status. Megalin levels are reduced in localized prostate cancer by Gleason grade. Our findings suggest that the free hormone hypothesis should be revisited for testosterone and highlight the impact of vitamin D deficiency on prostate androgen levels, which is a known driver of prostate cancer. Thus, we revealed a mechanistic link between vitamin D and prostate cancer disparities observed in African Americans. Significance: These findings link vitamin D deficiency and the megalin protein to increased levels of prostate androgens, which may underpin the disparity in lethal prostate cancer in African America men.

The endocytosis receptor megalin: From bench to bedside.

The large (∼600 kDa) endocytosis receptor megalin/low-density lipoprotein receptor-related protein 2 is highly expressed at the apical membrane of proximal tubular epithelial cells (PTECs). Megalin plays an important role in the endocytosis of various ligands via interactions with intracellular adaptor proteins, which mediate the trafficking of megalin in PTECs. Megalin mediates the retrieval of essential substances, including carrier-bound vitamins and elements, and impairment of the endocytic process may result in the loss of those substances. In addition, megalin reabsorbs nephrotoxic substances such as antimicrobial (colistin, vancomycin, and gentamicin) or anticancer (cisplatin) drugs and advanced glycation end product-modified or fatty acid-containing albumin. The megalin-mediated uptake of these nephrotoxic ligands causes metabolic overload in PTECs and leads to kidney injury. Blockade or suppression of the megalin-mediated endocytosis of nephrotoxic substances may represent a novel therapeutic strategy for drug-induced nephrotoxicity or metabolic kidney disease. Megalin reabsorbs urinary biomarker proteins such as albumin, α1-microglobulin, ß2-microglobulin, and liver-type fatty acid-binding protein; thus, the above-mentioned megalin-targeted therapy may have an effect on the urinary excretion of these biomarkers. We have previously established a sandwich enzyme-linked immunosorbent assay to measure the ectodomain (A-megalin) and full-length (C-megalin) forms of urinary megalin using monoclonal antibodies against the amino- and carboxyl-terminals of megalin, respectively, and reported their clinical usefulness. In addition, there have been reports of patients with novel pathological anti-brush border autoantibodies targeting megalin in the kidney. Even with these breakthroughs in the characterization of megalin, a large number of issues remain to be addressed in future research.