Nephron specific ATP6AP2 knockout increases urinary excretion of fatty acids and decreases renal cortical megalin expression.

ATP6AP2 knockout in the renal nephron impairs receptor-mediated endocytosis, increasing urinary albumin and glucose excretion and impairing weight gain. Nonesterified fatty acids (NEFA) in urine are bound to albumin and reabsorbed in the proximal tubule through receptor-mediated endocytosis by the megalin-cubilin complex. We hypothesized that ATP6AP2 knockout increases urinary NEFA excretion through a reduction in megalin. Ten-week-old male C57BL/6 mice with nephron specific inducible ATP6AP2 knockout and noninduced controls were fed either normal diet (ND 12% fat) or high fat diet (HFD 45% fat) for 6 months. ATP6AP2 knockout significantly increased urine albumin:creatinine ratio in both ND and HFD fed mice while normalized urine NEFA concentration increased 489% and 259% in ND and HFD knockout mice compared to respective controls. Knockout decreased renal cortical megalin mRNA by 47% on ND and 49% on HFD while megalin protein expression decreased by 36% and 44% respectively. At the same time, markers of mTOR activity were increased while autophagy was impaired. Our results indicate that nephron specific ATP6AP2 knockout increases urinary NEFA excretion in the setting of impaired receptor-mediated endocytosis. Further investigation should determine whether ATP6AP2 contributes to obesity related ectopic lipid deposition in the proximal tubule.

Effect of Tissue-derived Angiotensinogen on Kidney Injury and Fibrosis in Obstructive Nephropathy.

BACKGROUND/AIM: Angiotensinogen (AGT), a precursor of angiotensin II (AngII), contributes to regulating (patho)physiological conditions, including blood pressure changes, inflammation, and kidney fibrosis. However, the precise role of tissue-specific AGT in kidney fibrosis independent of blood pressure remains to be fully understood. This study investigated the source of intrarenal AGT and its role in kidney injury and fibrosis during obstructive nephropathy. MATERIALS AND METHODS: Proximal tubule- (PT, major source secreting AGT in the kidney; PKO) or liver- (major source of circulating AGT; LKO) AGT knockout (KO) mice were subjected to unilateral ureteral obstruction (UUO), a blood pressure-independent fibrosis model. RESULTS: UUO increased AGT mRNA and protein levels in the kidneys. PKO decreased AGT mRNA, but LKO enhanced it in UUO kidneys compared with the control. In contrast, the intrarenal protein levels of AGT increased in PKO, but not in LKO in UUO kidneys, indicating that the liver is a major source of intrarenal AGT protein. Expression of megalin, a PT receptor involved in the uptake of circulating AGT, was down-regulated in UUO kidneys and was independent of PKO or LKO. However, none of these changes prevented UUO-induced tubular injury and kidney fibrosis. CONCLUSION: Hepatic and proximal tubule AGT play distinct roles in contributing to intrarenal AGT levels during UUO, and their genetic inhibitions fail to prevent kidney injury and fibrosis, suggesting a highly complicated signaling pathway of the renin-angiotensin system and an associated compensatory mechanism in obstructive nephropathy.

Megalin Knockout Reduces SGLT2 Expression and Sensitizes to Western Diet-induced Kidney Injury.

Megalin (Lrp2) is a multiligand receptor that drives endocytic flux in the kidney proximal tubule (PT) and is necessary for the recovery of albumin and other filtered proteins that escape the glomerular filtration barrier. Studies in our lab have shown that knockout (KO) of Lrp2 in opossum PT cells leads to a dramatic reduction in sodium-glucose co-transporter 2 (SGLT2) transcript and protein levels, as well as differential expression of genes involved in mitochondrial and metabolic function. SGLT2 transcript levels are reduced more modestly in Lrp2 KO mice. Here, we investigated the effects of Lrp2 KO on kidney function and health in mice fed regular chow (RC) or a Western-style diet (WD) high in fat and refined sugar. Despite a modest reduction in SGLT2 expression, Lrp2 KO mice on either diet showed increased glucose tolerance compared to control mice. Moreover, Lrp2 KO mice were protected against WD-induced fat gain. Surprisingly, renal function in male Lrp2 KO mice on WD was compromised, and the mice exhibited significant kidney injury compared with control mice on WD. Female Lrp2 KO mice were less susceptible to WD-induced kidney injury than male Lrp2 KO. Together, our findings reveal both positive and negative contributions of megalin expression to metabolic health, and highlight a megalin-mediated sex-dependent response to injury following WD.

The angiotensin II/type 1 angiotensin II receptor pathway is implicated in the dysfunction of albumin endocytosis in renal proximal tubule epithelial cells induced by high glucose levels.

It is well-established that dysfunction of megalin-mediated albumin endocytosis by proximal tubule epithelial cells (PTECs) and the activation of the Renin-Angiotensin System (RAS) play significant roles in the development of Diabetic Kidney Disease (DKD). However, the precise correlation between these factors still requires further investigation. In this study, we aimed to elucidate the potential role of angiotensin II (Ang II), a known effector of RAS, as the mediator of albumin endocytosis dysfunction induced by high glucose (HG) in PTECs. To achieve this, we utilized LLC-PK1 and HK-2 cells, which are well-established in vitro models of PTECs. Using albumin-FITC or DQ-albumin as tracers, we observed that incubation of LLC-PK1 and HK-2 cells with HG (25 mM for 48 h) significantly reduced canonical receptor-mediated albumin endocytosis, primarily due to the decrease in megalin expression. HG increased the concentration of Ang II in the LLC-PK1 cell supernatant, a phenomenon associated with an increase in angiotensin-converting enzyme (ACE) expression and a decrease in prolyl carboxypeptidase (PRCP) expression. ACE type 2 (ACE2) expression remained unchanged. To investigate the potential impact of Ang II on HG effects, the cells were co-incubated with angiotensin receptor inhibitors. Only co-incubation with 10-7 M losartan (an antagonist for type 1 angiotensin receptor, AT1R) attenuated the inhibitory effect of HG on albumin endocytosis, as well as megalin expression. Our findings contribute to understanding the genesis of tubular albuminuria observed in the early stages of DKD, which involves the activation of the Ang II/AT1R axis by HG.

Effects of High-Dose Vitamin D Supplementation on Placental Vitamin D Metabolism and Neonatal Vitamin D Status.

Vitamin D (vitD) deficiency (25-hydroxy-vitamin D < 50 nmol/L) is common in pregnancy and associated with an increased risk of adverse pregnancy outcomes. High-dose vitD supplementation is suggested to improve pregnancy health, but there is limited knowledge about the effects on placental vitD transport and metabolism and the vitD status of newborns. Comparing the current standard maternal supplementation, 10 µg/day to a 90 µg vitD supplement, we investigated placental gene expression, maternal vitD transport and neonatal vitD status. Biological material was obtained from pregnant women randomized to 10 µg or 90 µg vitD supplements from week 11-16 onwards. Possible associations between maternal exposure, neonatal vitD status and placental expression of the vitD receptor (VDR), the transporters (Cubilin, CUBN and Megalin, LRP2) and the vitD-activating and -degrading enzymes (CYP24A1, CYP27B1) were investigated. Maternal vitD-binding protein (VDBP) was determined before and after supplementation. Overall, 51% of neonates in the 10 µg vitD group were vitD-deficient in contrast to 11% in the 90 µg group. High-dose vitD supplementation did not significantly affect VDBP or placental gene expression. However, the descriptive analyses indicate that maternal obesity may lead to the differential expression of CUBN, CYP24A1 and CYP27B1 and a changed VDBP response. High-dose vitD improves neonatal vitD status without affecting placental vitD regulation.

Nanotherapeutic kidney cell-specific targeting to ameliorate acute kidney injury.

Acute kidney injury (AKI) increases the risk of in-hospital death, adds to expense of care, and risk of early chronic kidney disease. AKI often follows an acute event such that timely treatment could ameliorate AKI and potentially reduce the risk of additional disease. Despite therapeutic success of dexamethasone in animal models, clinical trials have not demonstrated broad success. To improve the safety and efficacy of dexamethasone for AKI, we developed and characterized a novel, kidney-specific nanoparticle enabling specific within-kidney targeting to proximal tubular epithelial cells provided by the megalin ligand cilastatin. Cilastatin and dexamethasone were complexed to H-Dot nanoparticles, which were constructed from generally recognized as safe components. Cilastatin/Dexamethasone/H-Dot nanotherapeutics were found to be stable at plasma pH and demonstrated salutary release kinetics at urine pH. In vivo, they were specifically biodistributed to the kidney and bladder, with 75% recovery in the urine and with reduced systemic toxicity compared to native dexamethasone. Cilastatin complexation conferred proximal tubular epithelial cell specificity within the kidney in vivo and enabled dexamethasone delivery to the proximal tubular epithelial cell nucleus in vitro. The Cilastatin/Dexamethasone/H-Dot nanotherapeutic improved kidney function and reduced kidney cellular injury when administered to male C57BL/6 mice in two translational models of AKI (rhabdomyolysis and bilateral ischemia reperfusion). Thus, our design-based targeting and therapeutic loading of a kidney-specific nanoparticle resulted in preservation of the efficacy of dexamethasone, combined with reduced off-target disposition and toxic effects. Hence, our study illustrates a potential strategy to target AKI and other diseases of the kidney.

Renal handling of albumin in rats with early stage diabetes: A theoretical analysis.

In early diabetic nephropathy (DN), recent studies have shown that albuminuria stems mostly from alterations in tubular function rather than from glomerular damage. Several factors in DN, including hyperfiltration, hypertrophy and reduced abundance of the albumin receptors megalin and cubilin, affect albumin endocytosis in the proximal tubule (PT). To assess their respective contribution, we developed a model of albumin handling in the rat PT that couples the transport of albumin to that of water and solutes. Our simulations suggest that, under basal conditions, ∼75% of albumin is retrieved in the S1 segment. The model predicts negligible uptake in S3, as observed experimentally. It also accurately predicts the impact of acute hyperglycaemia on urinary albumin excretion. Simulations reproduce observed increases in albumin excretion in early DN by considering the combined effects of increased glomerular filtration rate (GFR), osmotic diuresis, hypertrophy, and megalin and cubilin downregulation, without stipulating changes in glomerular permselectivity. The results indicate that in isolation, glucose-elicited osmotic diuresis and glucose transporter upregulation raise albumin excretion only slightly. Enlargement of PT diameter not only augments uptake via surface area expansion, but also reduces fluid velocity and thus shear stress-induced stimulation of endocytosis. Overall, our model predicts that downregulation of megalin and cubilin and hyperfiltration both contribute significantly to increasing albumin excretion in rats with early-stage diabetes. The results also suggest that acute sodium-glucose cotransporter 2 inhibition lowers albumin excretion only if GFR decreases sufficiently, and that angiotensin II receptor blockers mitigate urinary albumin loss in early DN in large part by upregulating albumin receptor abundance. KEY POINTS: The urinary excretion of albumin is increased in early diabetic nephropathy (DN). It is difficult to experimentally disentangle the multiple factors that affect the renal handling of albumin in DN. We developed a mathematical model of albumin transport in the rat proximal tubule (PT) to examine the impact of elevated plasma glucose, hyperfiltration, PT hypertrophy and reduced abundance of albumin receptors on albumin uptake and excretion in DN. Our model predicts that glucose-elicited osmotic diuresis per se raises albumin excretion only slightly. Conversely, increases in PT diameter and length favour reduced albumin excretion. Our results suggest that downregulation of the receptors megalin and cubilin in PT cells and hyperfiltration both contribute significantly to increasing albumin excretion in DN. The model helps to better understand the mechanisms underlying urinary loss of albumin in early-stage diabetes, and the impact of specific treatments thereupon.

Megalin-related mechanism of hemolysis-induced acute kidney injury and the therapeutic strategy.

Hemolysis-induced acute kidney injury (AKI) is attributed to heme-mediated proximal tubule epithelial cell (PTEC) injury and tubular cast formation due to intratubular protein condensation. Megalin is a multiligand endocytic receptor for proteins, peptides, and drugs in PTECs and mediates the uptake of free hemoglobin and the heme-scavenging protein α1-microglobulin. However, understanding of how megalin is involved in the development of hemolysis-induced AKI remains elusive. Here, we investigated the megalin-related pathogenesis of hemolysis-induced AKI and a therapeutic strategy using cilastatin, a megalin blocker. A phenylhydrazine-induced hemolysis model developed in kidney-specific mosaic megalin knockout (MegKO) mice confirmed megalin-dependent PTEC injury revealed by the co-expression of kidney injury molecule-1 (KIM-1). In the hemolysis model in kidney-specific conditional MegKO mice, the uptake of hemoglobin and α1-microglobulin as well as KIM-1 expression in PTECs was suppressed, but tubular cast formation was augmented, likely due to the nonselective inhibition of protein reabsorption in PTECs. Quartz crystal microbalance analysis revealed that cilastatin suppressed the binding of megalin with hemoglobin and α1-microglobulin. Cilastatin also inhibited the specific uptake of fluorescent hemoglobin by megalin-expressing rat yolk sac tumor-derived L2 cells. In a mouse model of hemolysis-induced AKI, repeated cilastatin administration suppressed PTEC injury by inhibiting the uptake of hemoglobin and α1-microglobulin and also prevented cast formation. Hemopexin, another heme-scavenging protein, was also found to be a novel ligand of megalin, and its binding to megalin and uptake by PTECs in the hemolysis model were suppressed by cilastatin. Mass spectrometry-based semiquantitative analysis of urinary proteins in cilastatin-treated C57BL/6J mice indicated that cilastatin suppressed the reabsorption of a limited number of megalin ligands in PTECs, including α1-microglobulin and hemopexin. Collectively, cilastatin-mediated selective megalin blockade is an effective therapeutic strategy to prevent both heme-mediated PTEC injury and cast formation in hemolysis-induced AKI. © 2024 The Pathological Society of Great Britain and Ireland.

Cryo-EM structures elucidate the multiligand receptor nature of megalin.

Megalin (low-density lipoprotein receptor-related protein 2) is a giant glycoprotein of about 600 kDa, mediating the endocytosis of more than 60 ligands, including those of proteins, peptides, and drug compounds [S. Goto, M. Hosojima, H. Kabasawa, A. Saito, Int. J. Biochem. Cell Biol. 157, 106393 (2023)]. It is expressed predominantly in renal proximal tubule epithelial cells, as well as in the brain, lungs, eyes, inner ear, thyroid gland, and placenta. Megalin is also known to mediate the endocytosis of toxic compounds, particularly those that cause renal and hearing disorders [Y. Hori et al., J. Am. Soc. Nephrol. 28, 1783-1791 (2017)]. Genetic megalin deficiency causes Donnai-Barrow syndrome/facio-oculo-acoustico-renal syndrome in humans. However, it is not known how megalin interacts with such a wide variety of ligands and plays pathological roles in various organs. In this study, we elucidated the dimeric architecture of megalin, purified from rat kidneys, using cryoelectron microscopy. The maps revealed the densities of endogenous ligands bound to various regions throughout the dimer, elucidating the multiligand receptor nature of megalin. We also determined the structure of megalin in complex with receptor-associated protein, a molecular chaperone for megalin. The results will facilitate further studies on the pathophysiology of megalin-dependent multiligand endocytic pathways in multiple organs and will also be useful for the development of megalin-targeted drugs for renal and hearing disorders, Alzheimer's disease [B. V. Zlokovic et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4229-4234 (1996)], and other illnesses.

Mechanisms of gelofusine protection in an in vitro model of polymyxin B-associated renal injury.

Polymyxins are a last-resort treatment option for multidrug-resistant gram-negative bacterial infections, but they are associated with nephrotoxicity. Gelofusine was previously shown to reduce polymyxin-associated kidney injury in an animal model. However, the mechanism(s) of renal protection has not been fully elucidated. Here, we report the use of a cell culture model to provide insights into the mechanisms of renal protection. Murine epithelial proximal tubular cells were exposed to polymyxin B. Cell viability, lactate dehydrogenase (LDH) release, polymyxin B uptake, mitochondrial superoxide production, nuclear morphology, and apoptosis activation were evaluated with or without concomitant gelofusine. A megalin knockout cell line was used as an uptake inhibition control. Methionine was included in selected experiments as an antioxidant control. A polymyxin B concentration-dependent reduction in cell viability was observed. Increased viability was observed in megalin knockout cells following comparable polymyxin B exposures. Compared with polymyxin B exposure alone, concomitant gelofusine significantly increased cell viability as well as reduced LDH release, polymyxin B uptake, mitochondrial superoxide, and apoptosis. Gelofusine and methionine were more effective at reducing renal cell injury in combination than either agent alone. In conclusion, the mechanisms of renal protection by gelofusine involve decreasing cellular drug uptake, reducing subsequent oxidative stress and apoptosis activation. These findings would be valuable for translational research into clinical strategies to attenuate drug-associated acute kidney injury.NEW & NOTEWORTHY Gelofusine is a gelatinous saline solution with the potential to attenuate polymyxin-associated nephrotoxicity. We demonstrated that the mechanisms of gelofusine renal protection involve reducing polymyxin B uptake by proximal tubule cells, limiting subsequent oxidative stress and apoptosis activation. In addition, gelofusine was more effective at reducing cellular injury than a known antioxidant control, methionine, and a megalin knockout cell line, indicating that gelofusine likely has additional pharmacological properties besides only megalin inhibition.

Megalin-targeting and ROS-responsive elamipretide-conjugated polymeric prodrug for treatment of acute kidney injury.

Acute kidney injury (AKI) is associated with both kidney function loss and increased mortality. In the pathological progression of ischemia-reperfusion-induced AKI, the surge of reactive oxygen species (ROS) plays a crucial role. To combat this, mitochondrial-targeted antioxidant therapy shows great promise as mitochondria are the primary source of ROS in AKI. However, most strategies aiming to target mitochondria directly result in nanodrugs that are too large to pass through the glomerular system and reach the renal tubules, which are the main site of damage in AKI. This study focused on synthesizing a Megalin receptor-targeted polymeric prodrug, low molecular weight chitosan-thioketal-elamipretide (LMWC/TK/Ela), to mitigate excessive ROS in renal tubular epithelial cells for AKI. This soluble polymeric prodrug has the ability to successfully reach the tubular site by crossing the glomerular barrier. Once there, it can responsively release elamipretide, which possesses excellent antioxidative properties. Therefore, this research offers a novel approach to actively target renal tubular epithelial cells and intracellular mitochondria for the relief of AKI.

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.