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.

Predicting the protein composition of human urine in normal and pathological states: Quantitative description based on Dent1 disease (CLCN5 mutation).

KEY POINTS: The presence of plasma proteins in urine is difficult to interpret quantitatively. It may be a result of impaired glomerular filtration or impaired proximal tubule (PT) reabsorption, or both. Dent1 disease (CLCN5 mutation) abolishes PT protein reabsorption leaving glomerular function intact. Using urine protein measurements from patients with Dent1 disease and normal individuals, we devised a mathematical model that incorporates two PT transport processes with distinct kinetics. This model predicts albumin, α1 -microglobulin (α1 -m), ß2 -microglobulin (ß2 -m) and retinol-binding protein 4 (RBP4) urine concentrations. Our results indicate that the urinary excretion of ß2 -m and RBP4 differs from that of albumin and α1 -m in their sensitivity to changes in the glomerular filtration rate, glomerular protein leak, tubular protein uptake via endocytosis and PT water reabsorption. The model predicts quantitatively how hyperfiltration and glomerular leak interact to promote albuminuria. Our model should contribute to improved understanding and interpretation of urine protein measurements in renal disease. ABSTRACT: To clarify the relative contributions of glomerular filtration and tubular uptake to urinary protein excretion, we developed a mathematical model of protein reabsorption in the human proximal tubule (PT) using Michaelis-Menten kinetics and molar urinary protein measurements taken from human Dent1 disease (CLCN5 loss-of-function mutation). ß2 -Microglobulin (ß2 -m) and retinol-binding protein 4 (RBP4) are normally reabsorbed with 'very high' efficiency uptake kinetics and fractional urinary excretion of 0.025%, whereas albumin and α1 -microglobulin (α1 -m) are reabsorbed by 'high' efficiency uptake kinetics and 50-fold higher fractional urinary excretion of 1.15%. Our model correctly predicts the urinary ß2 -m, RBP4 and α1 -m content in aristolochic acid nephropathy, and elevated ß2 -m excretion with increased single nephron glomerular filtration rate (SNGFR) following unilateral-nephrectomy. We explored how altered endocytic uptake, water reabsorption, SNGFR and glomerular protein filtration affect excretion. Our results help to explain why ß2 -m and RBP4 are more sensitive markers of PT dysfunction than albumin or α1 -m, and suggest that reduced PT sodium and water reabsorption in Fanconi syndrome may contribute to proteinuria. Transition of albumin excretion from normal to microalbuminuria, a 5-fold increase, corresponds to a 3.5-fold elevation in albumin glomerular filtration, supporting the use of microalbuminuria screening to detect glomerular leak in diabetes. In macroalbuminuria, small albumin permeability changes produce large changes in excretion. However, changes in SNGFR can alter protein excretion, and hyperfiltration with glomerular leak can combine to increase albuminuria. Our model provides a validated quantitative description of the transport processes underlying the protein composition of human urine in normal and pathophysiological states.

A well-developed endolysosomal system reflects protein reabsorption in segment 1 and 2 of rat proximal tubules.

Proteinuria is a well-established marker and predictor of kidney disease. The receptors megalin and cubilin reabsorb filtered proteins and thereby proteinuria is avoided. It is unknown if all segments of the proximal tubule are involved in clearing the filtrate or if there exists a reserve capacity in case of increased glomerular protein filtration. To determine this, we performed serial sectioning of rat kidney and used stereology to quantify the endolysosomal system of the three segments of cortical and juxtamedullary nephrons by electron microscopy. Immunohistochemistry was applied to analyze the adaptor protein Dab2, which assists in megalin mediated endocytosis, megalin, and endocytic uptake of two endogenous megalin ligands; retinol binding protein and ß2-microglobulin at exact tubular positions. Proteinuric rats (puromycin-treated) and mice (podocin knock-out) were analyzed to clarify the response of the tubule to increased protein filtration. We found that the endolysosomal system was most prominent in segment 1 and 2, whereas segment 3 was less developed. The depth of ligand uptake varied among nephrons, but it descended into segment 2 although uptake was lower than in segment 1 and it was never observed in segment 3. This was supported by prominent expression of Dab2 in segment 1 and 2. When protein filtration increased, segment 3 was included in the reabsorption process in proteinuric animals. Thus, segment 1 and 2 are responsible for clearing the filtrate for protein during normal physiological conditions, but the tubule exhibits plasticity and is able to include segment 3 under proteinuric stress.

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.

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

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

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions.

The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O-glycan sites. Moreover, we found that O-glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O-glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O-glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by ∼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.

Uromodulin is expressed in the distal convoluted tubule, where it is critical for regulation of the sodium chloride cotransporter NCC.

Uromodulin, the most abundant protein in normal urine, is essentially produced by the cells lining the thick ascending limb. There it regulates the activity of the cotransporter NKCC2 and is involved in sodium chloride handling and blood pressure regulation. Conflicting reports suggested that uromodulin may also be expressed in the distal convoluted tubule (DCT) where its role remains unknown. Using microdissection studies combined with fluorescent in situ hybridization and co-immunostaining analyses, we found a significant expression of uromodulin in mouse and human DCT at approximately 10% of thick ascending limb expression levels, but restricted to the early part of the DCT (DCT1). Genetic deletion of Umod in mouse was reflected by a major shift in NCC activity from the DCT1 to the downstream DCT2 segment, paralleled by a compensatory expansion of DCT2. By increasing the distal sodium chloride and calcium ion load with chronic furosemide administration, an intrinsic compensatory defect in the DCT from Umod-/- compared to wild type mice was found manifested as sodium wasting and hypercalciuria. In line, co-expression studies in HEK cells suggested a facilitating role for uromodulin in NCC phosphorylation, possibly via SPAK-OSR1 modulation. These experiments demonstrate a significant expression of uromodulin in the early part of mouse and human DCT. Thus, biosynthesis of uromodulin in the DCT1 is critical for its function, structure and plasticity, suggesting novel links between uromodulin, blood pressure control and risk of kidney stones.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice.

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

Apoptotic properties of the type 1 interferon induced family of human mitochondrial membrane ISG12 proteins.

BACKGROUND INFORMATION: Interferons are a family of cytokines with growth inhibitory and antiviral functions, which exert their biological actions through the expression of interferon-stimulated genes (ISGs). The human ISG12 family of proteins comprises ISG12A, ISG12B, ISG12C and ISG6-16. Due to differential splicing and a gene variation, the human ISG12A protein exists as a full-length ISG12A form and three ISG12A variants. ISG12 genes have been found transcriptionally dysregulated in many disorders. High levels of ISG12A mRNA have been found in breast and ovarian cancers. Loss of heterozygosity at the position of the ISG12 genes often occurs in ovarian carcinomas and lymphoblastic leukemias. Both ISG12A and ISG6-16 are up-regulated in psoriasis. RESULTS: We demonstrate here that expression of the human full-length ISG12A protein sensitises cells for TNFα and the BH3 mimetic gossypol induced apoptosis, and the other ISG12A variants as well as ISG12B and ISG12C can induce apoptosis directly in HEK293 cells. Also ISG6-16 sensitises HEK293 cells for gossypol-induced apoptosis. In the ISG12 motif, two putative Bcl-2 homology (BH)3 like motifs were found, which may be decisive for the apoptotic properties of the ISG12 proteins. A series of BH3 mutants was made in ISG12AΔ-S, the smallest apoptosis-inducing ISG12A variant and our results indicate that ISG12AΔ-S indeed possesses features resembling those of BH3-only proteins. Supporting this notion are our findings that the full-length ISG12A co-immunoprecipitates with the Bcl-2 protein, and the apoptotic properties of the ISG12A variants are reduced in Bcl-2 expressing HEK293 cells. In addition, full-length ISG12A is able to form homodimers, which suggests a possible involvement in pore formation during apoptosis. The full-length ISG12A, the three ISG12A variants and the ISG12B proteins were found to be localised in the mitochondria. CONCLUSIONS: Our results suggest that the ISG12 family of proteins has an important role for the apoptotic properties induced by type 1 interferon. SIGNIFICANCE: The ISG12 family constitute small hydrophic proteins involved in apoptosis. This is the first comparison of the apoptotic potentials of the full-length ISG12A protein and the three ISG12A variants. The differential apoptotic potentials of these proteins might have an impact on the strategies to monitor and interpret their dysregulation associated with many disorders.

Nephron morphometry in mice and rats using tomographic microscopy.

The aim was to quantify the glomerular capillary surface area, the segmental tubular radius, length, and area of single nephrons in mouse and rat kidneys. Multiple 2.5-µm-thick serial Epon sections were obtained from three mouse and three rat kidneys for three-dimensional reconstruction of the nephron tubules. Micrographs were aligned for each kidney, and 359 nephrons were traced and their segments localized. Thirty mouse and thirty rat nephrons were selected for further investigation. The luminal radius of each segment was determined by two methods. The luminal surface area was estimated from the radius and length of each segment. High-resolution micrographs were recorded for five rat glomeruli, and the capillary surface area determined. The capillary volume and surface area were corrected for glomerular shrinkage. A positive correlation was found between glomerular capillary area and proximal tubule area. The thickest part of the nephron, i.e., the proximal tubule, was followed by the thinnest part of the nephron, i.e., the descending thin limb, and the diameters of the seven identified nephron segments share the same rank in the two species. The radius and length measurements from mouse and rat nephrons generally share the same pattern; rat tubular radius-to-mouse tubular radius ratio ≈ 1.47, and rat tubular length-to-mouse tubular length ratio ≈ 2.29, suggesting relatively longer tubules in the rat. The detailed tables of mouse and rat glomerular capillary area and segmental radius, length, and area values may be used to enhance understanding of the associated physiology, including existing steady-state models of the urine-concentrating mechanism.

Untargeted Metabolomics Analysis of ABCC6-Deficient Mice Discloses an Altered Metabolic Liver Profile.

Loss-of-function mutations in the transmembrane ABCC6 transport protein cause pseudoxanthoma elasticum (PXE), an ectopic, metabolic mineralization disorder that affects the skin, eye, and vessels. ABCC6 is assumed to mediate efflux of one or several small molecule compounds from the liver cytosol to the circulation. Untargeted metabolomics using liquid chromatography-mass spectrometry was employed to inspect liver cytosolic extracts from mice with targeted disruption of the Abcc6 gene. Absence of the ABCC6 protein induced an altered profile of metabolites in the liver causing accumulation of compounds as more features were upregulated than downregulated in ABCC6-deficient mice. However, no differences of the identified metabolites in liver could be detected in plasma, whereas urine reflected some of the changes. Of note, N-acetylated amino acids and pantothenic acid (vitamin B5), which is involved in acetylation reactions, were accumulated in the liver. None of the identified metabolites seems to explain mineralization in extrahepatic tissues, but the present study now shows that abrogated ABCC6 function does cause alterations in the metabolic profile of the liver in accordance with PXE being a metabolic disease originating from liver disturbance. Further studies of these changes and the further identification of yet unknown metabolites may help to clarify the liver-related pathomechanism of PXE.

Tubular proteinuria in patients with HNF1α mutations: HNF1α drives endocytosis in the proximal tubule.

Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor expressed in the liver, pancreas, and proximal tubule of the kidney. Mutations of HNF1α cause an autosomal dominant form of diabetes mellitus (MODY-HNF1A) and tubular dysfunction. To gain insights into the role of HNF1α in the proximal tubule, we analyzed Hnf1a-deficient mice. Compared with wild-type littermates, Hnf1a knockout mice showed low-molecular-weight proteinuria and a 70% decrease in the uptake of ß2-microglobulin, indicating a major endocytic defect due to decreased expression of megalin/cubilin receptors. We identified several binding sites for HNF1α in promoters of Lrp2 and Cubn genes encoding megalin and cubilin, respectively. The functional interaction of HNF1α with these promoters was shown in C33 epithelial cells lacking endogenous HNF1α. Defective receptor-mediated endocytosis was confirmed in proximal tubule cells from these knockout mice and could be rescued by transfection of wild-type but not mutant HNF1α. Transfection of human proximal tubule HK2 cells with HNF1α was able to upregulate megalin and cubilin expression and to increase endocytosis of albumin. Low-molecular-weight proteinuria was consistently detected in individuals with HNF1A mutations compared with healthy controls and patients with non-MODY-HNF1A diabetes mellitus. Thus, HNF1α plays a key role in the constitutive expression of megalin and cubilin, hence regulating endocytosis in the proximal tubule of the kidney. These findings provide new insight into the renal phenotype of individuals with mutations of HNF1A.

In-depth phenotyping of a Donnai-Barrow patient helps clarify proximal tubule dysfunction.

BACKGROUND: The megalin/cubilin/amnionless complex is essential for albumin and low molecular weight (LMW) protein reabsorption by renal proximal tubules (PT). Mutations of the LRP2 gene encoding megalin cause autosomal recessive Donnai-Barrow/facio-oculo-acoustico-renal syndrome (DB/FOAR), which is characterized by LMW proteinuria. The pathophysiology of DB/FOAR-associated PT dysfunction remains unclear. CLINICAL CASE: A 3-year-old girl presented with growth retardation and proteinuria. Clinical examination was unremarkable, except for a still-opened anterior fontanel and myopia. Psychomotor development was delayed. At 6, she developed sensorineural hearing loss. Hypertelorism was noted when she turned 12. Blood analyses, including renal function parameters, were normal. Urine sediment was bland. Proteinuria was significant and included albumin and LMW proteins. Immunoblotting analyses detected cubilin and type 3 carbonic anhydrase (CA3) in the urine. Renal ultrasound was unremarkable. Optical examination of a renal biopsy did not disclose any tubular or glomerular abnormality. Electron microscopy revealed that PT apical endocytic apparatus was significantly less developed. Immunostaining for megalin showed a faint signal in PT cytosol contrasting with the distribution of cubilin at the apical membrane. The diagnostic procedure led to identifying two mutations of the LRP2 gene. CONCLUSIONS: The functional loss of megalin in DB/FOAR causes PT dysfunction characterized by increased urinary shedding of CA3 and cubilin.

Haploinsufficiency of the ammonia transporter Rhcg predisposes to chronic acidosis: Rhcg is critical for apical and basolateral ammonia transport in the mouse collecting duct.

Ammonia secretion by the collecting duct (CD) is critical for acid-base homeostasis and, when defective, causes distal renal tubular acidosis (dRTA). The Rhesus protein RhCG mediates NH(3) transport as evident from cell-free and cellular models as well as from Rhcg-null mice. Here, we investigated in a Rhcg mouse model the metabolic effects of Rhcg haploinsufficiency, the role of Rhcg in basolateral NH(3) transport, and the mechanisms of adaptation to the lack of Rhcg. Both Rhcg(+/+) and Rhcg(+/-) mice were able to handle an acute acid load, whereas Rhcg(-/-) mice developed severe metabolic acidosis with reduced ammonuria and high mortality. However, chronic acid loading revealed that Rhcg(+/-) mice did not fully recover, showing lower blood HCO(3)(-) concentration and more alkaline urine. Microperfusion studies demonstrated that transepithelial NH(3) permeability was reduced by 80 and 40%, respectively, in CDs from Rhcg(-/-) and Rhcg(+/-) mice compared with controls. Basolateral membrane permeability to NH(3) was reduced in CDs from Rhcg(-/-) mice consistent with basolateral Rhcg localization. Rhcg(-/-) responded to acid loading with normal expression of enzymes and transporters involved in proximal tubular ammoniagenesis but reduced abundance of the NKCC2 transporter responsible for medullary accumulation of ammonium. Consequently, tissue ammonium content was decreased. These data demonstrate a role for apical and basolateral Rhcg in transepithelial NH(3) transport and uncover an incomplete dRTA phenotype in Rhcg(+/-) mice. Haploinsufficiency or reduced expression of RhCG may underlie human forms of (in)complete dRTA.

Three-dimensional reconstruction of the rat nephron.

This study gives a three-dimensional (3D) structural analysis of rat nephrons and their connections to collecting ducts. Approximately 4,500 2.5-µm-thick serial sections from the renal surface to the papillary tip were obtained from each of 3 kidneys of Wistar rats. Digital images were recorded and aligned into three image stacks and traced from image to image. Short-loop nephrons (SLNs), long-loop nephrons (LLNs), and collecting ducts (CDs) were reconstructed in 3D. We identified a well-defined boundary between the outer stripe and the inner stripe of the outer medulla corresponding to the transition of descending thick limbs to descending thin limbs and between the inner stripe and the inner medulla, i.e., the transition of ascending thin limbs into ascending thick limbs of LLNs. In all nephrons, a mosaic pattern of proximal tubule (PT) cells and descending thin limb (DTL) cells was observed at the transition between the PT and the DTL. The course of the LLNs revealed tortuous proximal "straight" tubules and winding of the DTLs within the outer half of the inner stripe. The localization of loop bends of SLNs in the inner stripe of the outer medulla and the bends of LLNs in the inner medulla reflected the localization of their glomeruli; i.e., the deeper the glomerulus, the deeper the bend. Each CD drained approximately three to six nephrons with a different pattern than previously established in mice. This information will provide a basis for evaluation of structural changes within nephrons as a result of physiological or pharmaceutical intervention.

Spatial organization of the vascular bundle and the interbundle region: three-dimensional reconstruction at the inner stripe of the outer medulla in the mouse kidney.

The vascular bundle (VB) is a complex structure that resides in the inner stripe of the outer medulla. At present, the tubulovascular spatial organization of the VB, which is crucial for the formation of the osmolarity gradient and for solute transport, is still under debate. In this study, we used computer-assisted digital tracing combined with aquaporin-1 immunohistochemistry to reconstruct all tubules and vessels in the VB of the mouse kidney. We found, first, that the descending and ascending vasa recta travelled exclusively through the VB. The ascending vasa recta received no tributaries (no branches) along their entire path in the medulla and were not connected with the capillary plexus in the interbundle region. Second, a specific group of the descending vasa recta were closely accompanied by the longest ascending vasa recta, which connected only to the capillary plexus at the tip of the papilla. Third, the descending thin limbs of all short-looped nephrons travelled exclusively through the outer part of the VB. The loops of these nephrons (both descending and ascending parts) were distributed in a regular pattern based on their length. Finally, the thick ascending limbs of all long-looped nephrons were located at the margin of the VB (except a few within the VB), which formed a layer separating the VB from the interbundle region. In conclusion, our three-dimensional analysis of the VB strongly suggest a lateral osmolarity heterogeneity across the inner stripe of the outer medulla, which might work as a driving force for water and solute transport.

Megalin-deficiency causes high myopia, retinal pigment epithelium-macromelanosomes and abnormal development of the ciliary body in mice.

In man, mutations of the megalin-encoding gene causes the rare Donnai-Barrow/Facio-Oculo-Acoustico-Renal Syndrome, which is partially characterized by high-grade myopia. Previous studies of renal megalin function have established that megalin is crucial for conservation of renal filtered nutrients including vitamin A; however, the role of megalin in ocular physiology and development is presently unknown. Therefore, we investigate ocular megalin expression and the ocular phenotype of megalin-deficient mice. Topographical and subcellular localization of megalin as well as the ocular phenotype of megalin-deficient mice were examined with immunological techniques using light, confocal and electron microscopy. We identified megalin in the retinal pigment epithelium (RPE) and non-pigmented ciliary body epithelium (NPCBE) in normal mouse eyes. Immunocytochemical investigations furthermore showed that megalin localizes to vesicular structures in the RPE and NPCBE cells. Histological investigations of ocular mouse tissue also identified a severe myopia phenotype as well as enlarged RPE melanosomes and abnormal ciliary body development in the megalin-deficient mice. In conclusion, the complex ocular phenotype observed in the megalin-deficient mice suggests that megalin-mediated developmental abnormalities may contribute to the high myopia phenotype observed in the Donnai-Barrow Syndrome patients and, thus, that megalin harbors important roles in ocular development and physiology. Finally, our data show that megalin-deficient mice may provide a valuable model for future studies of megalin in ocular physiology and pathology.

Long-term enzyme replacement therapy is associated with reduced proteinuria and preserved proximal tubular function in women with Fabry disease.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene. Deficiency of α-galactosidase A (α-Gal A) causes intracellular accumulations of globotriaosylceramide (GL-3) and related glycosphingolipids in all organs, including the kidney, often leading to end-stage renal failure. In women with Fabry disease, accumulation of GL-3 in the glomerular podocytes and other renal cells induces progressive, proteinuric nephropathy, but not as severe as in men. Enzyme replacement therapy (ERT) with recombinant α-Gal A reduces cellular GL-3 deposits in podocytes and tubular epithelial cells. We have previously shown that α-Gal A is delivered to these cells by different pathways involving different receptors. This study investigated the long-term changes in albuminuria, estimated glomerular filtration rate (eGFR) and urinary markers of both glomerular and tubular dysfunction in women with Fabry disease treated with ERT. METHODS: A retrospective, single centre, cohort study evaluated the long-term association between ERT, albuminuria and eGFR in 13 women with Fabry disease and mild renal involvement. In particular, we analysed the changes in the proteinuric profile, including the glomerular marker IgG, the tubular markers α1-microglobulin and retinol-binding protein (RBP), and the shared tubular and glomerular markers albumin and transferrin. RESULTS: ERT was associated with a significant reduction in albuminuria and a relatively stable eGFR. The decrease in albuminuria was paralleled by a decrease in both glomerular and tubular urine protein markers. CONCLUSIONS: The data indicate that long-term ERT is associated with a reduction in albuminuria and glomerular and tubular urinary protein markers in women with Fabry disease and mild renal manifestations.

Endocytic receptors in the renal proximal tubule.

Protein reabsorption is a predominant feature of the renal proximal tubule. Animal studies show that the ability to rescue plasma proteins relies on the endocytic receptors megalin and cubilin. Recently, studies of patients with syndromes caused by dysfunctional receptors have supported the importance of these for protein clearance of human ultrafiltrate. This review focuses on the molecular biology and physiology of the receptors and their involvement in renal pathological conditions.