Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines

Receptor-mediated endocytosis is a constitutive high capacity pathway for the reabsorption of proteins from the glomerular filtrate by the renal proximal tubule. ClC-5 is a voltage-gated chloride channel found in the proximal tubule where it has been shown to be essential for protein uptake, based on evidence from patients with Dent's disease and studies in ClC-5 knockout mice. To further delineate the role of ClC-5 in albumin uptake, we performed a yeast two-hybrid screen with the C-terminal tail of ClC-5 to identify any interactions of the channel with proteins involved in endocytosis. We found that the C-terminal tail of ClC-5 bound the actin depolymerizing protein, cofilin, a result that was confirmed by GST-fusion pulldown assays. In cultured proximal tubule cells, cofilin was distributed in nuclear, cytoplasmic, and microsomal fractions and co-localized with ClC-5. Phosphorylation of cofilin by overexpressing LIM kinase 1 resulted in a stabilization of the actin cytoskeleton. Phosphorylation of cofilin in two proximal tubule cell models (porcine renal proximal tubule and opossum kidney) was also accompanied by a pronounced inhibition of albumin uptake. This study identifies a novel interaction between the C-terminal tail of ClC-5 and cofilin, an actin-associated protein that is crucial in the regulation of albumin uptake by the proximal tubule.     

Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells

Diabetes mellitus (DM) has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA) has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt) in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160) and cytoplasmic tail of megalin. Mice with type 1 DM (T1D) displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications) study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN) at an earlier stage.     

Uptake of leptin and albumin via separate pathways in proximal tubule cells

The adipokine leptin and oncotic protein albumin are endocytosed in the proximal tubule via the scavenger receptor megalin. Leptin reduces megalin expression and activates cell signalling pathways that upregulate fibrotic protein expression. The aim of this study was to investigate if leptin uptake in proximal tubule cells was via the albumin-megalin endocytic complex. In immortalised proximal tubule Opossum kidney cells (OK) fluorescent leptin and albumin co-localised following 5 min exposure, however there was no co-localisation at 10, 20 and 30 min exposure. In OK cells, acute exposure to leptin for 2 h did not alter NHE3, ClC-5, NHERF1 and NHERF2 mRNA. However, acute leptin exposure increased NHERF2 protein expression in proximal tubule cells. In OK cells, immunoprecipitation experimentation indicated leptin did not bind to ClC-5. Leptin uptake in OK cells was enhanced by bafilomycin and ammonium chloride treatment, demonstrating that uptake was not dependent on lysosomal pH. Thus, it is likely that two pools of megalin exist in proximal tubule cells to facilitate separate uptake of leptin and albumin by endocytosis.     

The Clathrin Adaptor Proteins ARH,Dab2, and Numb Play Distinct Roles in Niemann-Pick C1-Like 1 Versus Low Density Lipoprotein Receptor-mediated Cholesterol Uptake

The uptake of circulating low density lipoproteins (LDL) is mediated by LDL receptor (LDLR) through clathrin-dependent endocytosis. At the early stage of this process, adaptor proteins ARH and Dab2 specifically bind the endocytic signal motif in LDLR and recruit clathrin/AP2 to initiate internalization. On the other hand, intestinal cholesterol is absorbed by Niemann-Pick C1-Like 1 (NPC1L1) through clathrin-dependent endocytosis. Another adaptor protein, Numb recognizes the endocytic motif in NPC1L1 C terminus and couples NPC1L1 to endocytic machinery. The ARH, Dab2, and Numb proteins contain a homogeneous phosphotyrosine binding (PTB) domain that directly binds endocytic motifs. Because ARH, Dab2, and Numb are all PTB domain family members, the emerging mystery is whether these adaptors act complementally in LDLR and NPC1L1 endocytosis. Here, we found that ARH and Dab2 did not bind NPC1L1 and were not required for NPC1L1 internalization. Similarly, Numb lacked the ability to interact with the LDLR C terminus and was dispensable for LDL uptake. Only the Numb isoforms with shorter PTB domain could facilitate NPC1L1 endocytosis. Besides the reported function in intestinal cholesterol absorption, Numb also mediated cholesterol reabsorption from bile in liver. We further identified a Numb variant with G595D substitution in humans of low blood LDL-cholesterol. The G595D substitution impaired NPC1L1 internalization and cholesterol reabsorption, due to attenuating affinity of Numb to clathrin/AP2. These results demonstrate that Numb specifically regulates NPC1L1-mediated cholesterol absorption both in human intestine and liver, distinct from ARH and Dab2, which selectively participate in LDLR-mediated LDL uptake.     

Nedd4-2 functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl(-) channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.     

Binding of 125I-labelled albumin by isolated rat renal brush-border membrane vesicles. Evidence for uptake and internalization process

1. In the kidney, filtered proteins are rapidly reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with the protein binding to the luminal brush-border membrane. 2. The binding of 125I-labelled albumin to rat renal brush-border membrane vesicles and the effect of a low molecular weight protein lysozyme on that binding was assessed by the filtration method. 3. The Scatchard plot revealed a one-component binding-type curve with a dissociation constant Kd of 430.9 nM and 39.6 pmol/mg membrane protein for the number of binding sites. 4. Albumin binding was saturable and reversible, time and temperature dependent and the initial rate enhanced by increasing amounts of lysozyme. 5. The fact that association of albumin with the brush-border membrane vesicles was dependent upon the intravesicular space suggested a double process, binding of the ligand to the membrane surface and its internalization. These data suggest that albumin has a different binding site than that of a low-molecular weight protein lysozyme, with a constant affinity value near physiological loads. That specificity may confer selectivity upon the endocytic uptake process.     

Regulation of albumin endocytosis by PSD95/Dlg/ZO-1 (PDZ) scaffolds. Interaction of Na+-H+ exchange regulatory factor-2 with ClC-5

The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl- channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney (OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.     

FCH domain only-2 organizes clathrin-coated structures and interacts with Disabled-2 for low-density lipoprotein receptor endocytosis

Clathrin-mediated endocytosis regulates the internalization of many nutrient and signaling receptors. Clathrin and endocytic accessory proteins are recruited to receptors by specific adaptors. The adaptor Disabled-2 (Dab2) recruits its cargoes, including the low-density lipoprotein receptor (LDLR), and mediates endocytosis, even when the major adaptor protein AP2 is depleted. We hypothesized that the accessory proteins normally recruited by AP2 may be recruited by Dab2 if AP2 is absent. We identified one such accessory protein, the F-BAR protein FCH domain only-2 (FCHO2), as a major Dab2-interacting protein. The μ-homology domain (μHD) of FCHO2 binds directly to DPF sequences in Dab2 that also bind AP2. Disrupting the Dab2-FCHO2 interaction inhibited Dab2-mediated LDLR endocytosis in AP2-depleted cells. Depleting FCHO2 reduced the number but increased the size of clathrin structures on the adherent surface of HeLa cells and inhibited LDLR and transferrin receptor clustering. However, LDLR was internalized efficiently by FCHO2-deficient cells when additional time was provided for LDLR to enter the enlarged structures before budding, suggesting that later steps of endocytosis are normal under these conditions. These results indicate FCHO2 regulates the size of clathrin structures, and its interaction with Dab2 is needed for LDLR endocytosis under conditions of low AP2.     

AMN Directs Endocytosis of the Intrinsic Factor‐Vitamin B12 Receptor Cubam by Engaging ARH or Dab2

Cubam is a multi‐ligand receptor involved in dietary uptake of intrinsic factor‐vitamin B₁₂ in the small intestine and reabsorption of various low‐molecular‐weight proteins (such as albumin, transferrin, apolipoprotein A‐I and vitamin D‐binding protein) in the kidney. Cubam is composed of two proteins: cubilin and amnionless. Cubilin harbors ligand binding capabilities, while amnionless provides membrane anchorage and potential endocytic capacity via two FXNPXF signals within the cytosolic domain. These signals are similar to the FXNPXY signals found in members of the low‐density lipoprotein receptor superfamily, which associate with clathrin‐associated sorting proteins, including Disabled‐2 (Dab2) and autosomal recessive hypercholesterolemia (ARH), during endocytosis. We therefore investigated the functionality of each amnionless FXNPXF signal and their respective interaction with sorting proteins. By sequential mutation and expression of a panel of amnionless mutants combined with yeast two‐hybrid analyses, we demonstrate that the signals are functionally redundant and both are able to mediate endocytosis of cubam through interaction with Dab2 and ARH.     

SARS-CoV-2 spike protein inhibits megalin-mediated albumin endocytosis in proximal tubule epithelial cells

Patients with COVID-19 have high prevalence of albuminuria which is used as a marker of progression of renal disease and is associated with severe COVID-19. We hypothesized that SARS-CoV-2 spike protein (S protein) could modulate albumin handling in proximal tubule epithelial cells (PTECs) and, consequently contribute to the albuminuria observed in patients with COVID-19. In this context, the possible effect of S protein on albumin endocytosis in PTECs was investigated. Two PTEC lines were used: HEK-293A and LLC-PK1. Incubation of both cell types with S protein for 16 h inhibited albumin uptake at the same magnitude. This effect was associated with canonical megalin-mediated albumin endocytosis because: (1) DQ-albumin uptake, a marker of the lysosomal degradation pathway, was reduced at a similar level compared with fluorescein isothiocyanate (FITC)-albumin uptake; (2) dextran-FITC uptake, a marker of fluid-phase endocytosis, was not changed; (3) cell viability and proliferation were not changed. The inhibitory effect of S protein on albumin uptake was only observed when it was added at the luminal membrane, and it did not involve the ACE2/Ang II/AT1R axis. Although both cells uptake S protein, it does not seem to be required for modulation of albumin endocytosis. The mechanism underlying the inhibition of albumin uptake by S protein encompasses a decrease in megalin expression without changes in megalin trafficking and stability. These results reveal a possible mechanism to explain the albuminuria observed in patients with COVID-19.     

Dual roles for the Dab2 adaptor protein in embryonic development and kidney transport

The Disabled-2 (Dab2) gene has been proposed to act as a tumor suppressor. Cell culture studies have implicated Dab2 in signal transduction by mitogens, TGFbeta and endocytosis of lipoprotein receptors. To identify in vivo functions of Dab2, targeted mutations were made in the mouse. In the absence of Dab2, embryos arrest prior to gastrulation with a phenotype reminiscent of those caused by deletion of some TGFbeta signal transduction molecules involved in Nodal signaling. Dab2 is expressed in the extra-embryonic visceral endoderm but not in the epiblast. Dab2 could be conditionally deleted from the embryo without affecting normal development, showing that Dab2 is required in the visceral endoderm but dispensable in the embryo proper. Conditionally mutant Dab2(-/-) mice are overtly normal, but have reduced clathrin-coated pits in kidney proximal tubule cells and excrete specific plasma proteins in the urine, consistent with reduced transport by a lipoprotein receptor, megalin/gp330, in the proximal tubule. This evidence indicates that Dab2 is pleiotropic and regulates both visceral endoderm function and lipoprotein receptor trafficking in vivo.     

Signaling cascade of insulin-induced stimulation of L-dopa uptake in renal proximal tubule cells

The inward l-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of l-dopa transport into isolated rat PTCs. Insulin (50-500 microU/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 microU/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 microM). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 microM) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 microM), but not maximal (5 microM), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 microM LY-294002) and protein kinase C (PKC; 1 microM RO-318220). Insulin-induced activation of PKC-zeta was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-zeta, and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs.     

Tubular proteinuria in mice and humans lacking the intrinsic lysosomal protein SCARB2/Limp-2

Deficiency of the intrinsic lysosomal protein human scavenger receptor class B, member 2 (SCARB2; Limp-2 in mice) causes collapsing focal and segmental glomerular sclerosis (FSGS) and myoclonic epilepsy in humans, but patients with no apparent kidney damage have recently been described. We now demonstrate that these patients can develop tubular proteinuria. To determine the mechanism, mice deficient in Limp-2, the murine homolog of SCARB2, were studied. Most low-molecular-weight proteins filtered by the glomerulus are removed in the proximal convoluted tubule (PCT) by megalin/cubilin-dependent receptor-mediated endocytosis. Expression of megalin and cubilin was unchanged in Limp-2(-/-) mice, however, and the initial uptake of injected Alexa Fluor 555-conjugated bovine serum albumin (Alexa-BSA) was similar to wild-type mice, indicating that megalin/cubilin-dependent, receptor-mediated endocytosis was unaffected. There was a defect in proteolysis of reabsorbed proteins in the Limp-2(-/-) mice, demonstrated by the persistence of Alexa-BSA in the PCT compared with controls. This was associated with the failure of the lysosomal protease cathepsin B to colocalize with Alexa-BSA and endogenous retinol-binding protein in kidneys from Limp-2(-/-) mice. The data suggest that tubular proteinuria in Limp-2(-/-) mice is due to failure of endosomes containing reabsorbed proteins to fuse with lysosomes in the proximal tubule of the kidney. Failure of proteolysis is a novel mechanism for tubular proteinuria.     

Receptor-Mediated Endocytosis of Lysozyme in Renal Proximal Tubules of the Frog Rana Temporaria

The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endo-somes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intra-cellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.Key words: Endocytic receptor, frog, kidney, lysozyme, protein uptake, proximal tubule     

Disabled-2 (Dab2) inhibits Wnt/β-catenin signalling by binding LRP6 and promoting its internalization through clathrin

Canonical Wnt signalling requires caveolin-dependent internalization of low-density lipoprotein receptor-related protein 6 (LRP6). Here we report that the tumour suppressor and endocytic adaptor disabled-2 (Dab2), previously described as an inhibitor of Wnt/β-catenin signalling, selectively recruits LRP6 to the clathrin-dependent endocytic route, thereby sequestering it from caveolin-mediated endocytosis. Wnt stimulation induces the casein kinase 2 (CK2)-dependent phosphorylation of LRP6 at S1579, promoting its binding to Dab2 and internalization with clathrin. LRP6 receptor mutant (S1579A), deficient in CK2-mediated phosphorylation and Dab2 binding, fails to associate with clathrin, and thus escapes the inhibitory effects of Dab2 on Wnt/β-catenin signalling. Our data suggest that the S1579 site of LRP6 is a negative regulatory point during LRP6-mediated dorsoventral patterning in zebrafish and in allograft mouse tumour models. We conclude that the tumour suppressor functions of Dab2 involve modulation of canonical Wnt signalling by regulating the endocytic fate of the LRP6 receptor.     

The activation of Akt/PKB signaling pathway and cell survival

Akt/PKB is a serine/threonine protein kinase that functions as a critical regulator of cell survival and proliferation. Akt/PKB family comprises three highly homologous members known as PKBalpha/Akt1, PKBbeta/Akt2 and PKBgamma/Akt3 in mammalian cells. Similar to many other protein kinases, Akt/PKB contains a conserved domain structure including a specific PH domain, a central kinase domain and a carboxyl-terminal regulatory domain that mediates the interaction between signaling molecules. Akt/PKB plays important roles in the signaling pathways in response to growth factors and other extracellular stimuli to regulate several cellular functions including nutrient metabolism, cell growth, apoptosis and survival. This review surveys recent developments in understanding the molecular mechanisms of Akt/PKB activation and its roles in cell survival in normal and cancer cells.     

Akt/PKB localisation and 3' phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction

Protein kinase B (PKB or Akt) is a mitogen-regulated protein kinase involved in the protection of cells from apoptosis, the promotion of cell proliferation and diverse metabolic responses [1]. Its activation is initiated by the binding of 3' phosphorylated phosphoinositide lipids to its pleckstrin homology (PH) domain, resulting in the induction of activating phosphorylation at residues Thr308 and Ser473 by upstream kinases such as phosphoinositide-dependent protein kinase-1 (PDK1) [2]. Adhesion of epithelial cells to extracellular matrix leads to protection from apoptosis via the activation of phosphoinositide (PI) 3-kinase and Akt/PKB through an unknown mechanism [3] [4]. Here, we use the localisation of Akt/PKB within the cell to probe the sites of induction of PI 3-kinase activity. In fibroblasts, immunofluorescence microscopy showed that endogenous Akt/PKB localised to membrane ruffles at the outer edge of the cell following mitogen treatment as did green fluorescent protein (GFP) fusions with full-length Akt/PKB or its PH domain alone. In epithelial cells, the PH domain of Akt/PKB localised to sites of cell-cell and cell-matrix contact, distinct from focal contacts, even in the absence of serum. As this localisation was disrupted by PI 3-kinase inhibitory drugs and by mutations that inhibit interaction with phosphoinositides, it is likely to represent the sites of constitutive 3' phosphoinositide generation that provide a cellular survival signal. We propose that the attachment-induced, PI-3-kinase-mediated survival signal in epithelial cells is generated not only by cell-matrix interaction but also by cell-cell interaction.     

Disabled-2 protein facilitates assembly polypeptide-2-independent recruitment of cystic fibrosis transmembrane conductance regulator to endocytic vesicles in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical plasma membrane of fluid-transporting epithelia, where the plasma membrane abundance of CFTR is in part controlled by clathrin-mediated endocytosis. The protein networks that control CFTR endocytosis in epithelial cells have only been partially explored. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor critical for optimal clathrin coat formation. AP-2 is essential for recruitment of cargo proteins bearing the YXXΦ motif. Although AP-2 interacts directly with CFTR in vitro and facilitates CFTR endocytosis in some cell types, it remains unknown whether it is critical for CFTR uptake into clathrin-coated vesicles (CCVs). Disabled-2 (Dab2) is a clathrin-associated sorting protein (CLASP) that contributes to clathrin recruitment, vesicle formation, and cargo selection. In intestinal epithelial cells Dab2 was not found to play a direct role in CFTR endocytosis. By contrast, AP-2 and Dab2 were shown to facilitate CFTR endocytosis in human airway epithelial cells, although the specific mechanism remains unknown. Our data demonstrate that Dab2 mediates AP-2 independent recruitment of CFTR to CCVs in polarized human airway epithelial cells. As a result, it facilitates CFTR endocytosis and reduces CFTR abundance and stability in the plasma membrane. These effects are mediated by the DAB homology domain. Moreover, we show that in human airway epithelial cells AP-2 is not essential for CFTR recruitment to CCVs.     

Albumin handling by renal tubular epithelial cells in a microfluidic bioreactor

Epithelial cells in the proximal tubule of the kidney reclaim and metabolize protein from the glomerular filtrate. Proteinuria, an overabundance of protein in the urine, affects tubular cell function and is a major factor in the progression of chronic kidney disease. By developing experimental systems to study tubular protein handling in a setting that simulates some of the environmental conditions of the kidney tubule in vivo, we can better understand how microenviromental conditions affect cellular protein handling to determine if these conditions are relevant in disease. To this end, we used two in vitro microfluidic models to evaluate albumin handling by renal proximal tubule cells. For the first system, cells were grown in a microfluidic channel and perfused with physiological levels of shear stress to evaluate the effect of mechanical stress on protein uptake. In the second system, a porous membrane was used to separate an apical and basolateral compartment to evaluate the fate of protein following cellular metabolism. Opossum kidney (OK) epithelial cells were exposed to fluorescently labeled albumin, and cellular uptake was determined by measuring the fluorescence of cell lysates. Confocal fluorescence microscopy was used to compare uptake in cells grown under flow and static conditions. Albumin processed by the cells was examined by size exclusion chromatography (SEC) and SDS-PAGE. Results showed that cellular uptake and/or degradation was significantly increased in cells exposed to flow compared to static conditions. This was confirmed by confocal microscopy. Size exclusion chromatography and SDS-PAGE showed that albumin was broken down into small molecular weight fragments and excreted by the cells. No trace of intact albumin was detectable by either SEC or SDS-PAGE. These results indicate that fluid shear stress is an important factor mediating cellular protein handling, and the microfluidic bioreactor provides a novel tool to investigate this process.     

Effect of ACE inhibition on glomerular permselectivity and tubular albumin concentration in the renal ablation model

Despite the central role of tubular plasma proteins that characterize progressive kidney diseases, protein concentrations along the nephron in pathological conditions have not been quantified so far. We combined experimental techniques and theoretical analysis to estimate glomerular and tubular levels of albumin in the experimental model of 5/6 nephrectomy (Nx) in the rat, with or without angiotensin-converting enzyme (ACE) inhibition. We measured glomerular permselectivity by clearance of fluorescent Ficoll and albumin and used theoretical analysis to estimate tubular albumin. As expected, 5/6 Nx induced an elevation of the fractional clearance of the largest Ficoll molecules (radii >56 ?, P < 0.05), increasing the importance of the shunt pathway of the glomerular membrane and the albumin excretion rate (119 ± 41 vs. 0.6 ± 0.2 mg/24 h, P < 0.01). ACE inhibition normalized glomerular permselectivity and urinary albumin (0.5 ± 0.3 mg/24 h). Theoretical analysis indicates that with 5/6 Nx, an increased albumin filtration overcomes proximal tubule reabsorption, with a massive increase in average albumin concentration along the tubule, reaching the highest value of >2,500 μg/ml at the end of the collecting duct. ACE inhibition improved glomerular permselectivity, limiting albumin filtration under proximal tubule reabsorption capacity, with low albumin concentration along the entire nephron, averaging <13 μg/ml at the end of the collecting duct. These results reinforce our understanding of the mechanisms of renal disease progression and the effects of angiotensin II antagonism. They also suggest that evaluation of tubular protein concentration levels could help to identify patients at risk of kidney disease progression and to improve clinical management.