Transcytosis Of Albumin Research Articles

Albumin influences leucocyte FcRn expression in the early days of kidney transplantation.

FcRn, a receptor originally known for its involvement in IgG and albumin transcytosis and recycling, is also important in the establishment of the innate and adaptive immune response. Dysregulation of the immune response has been associated with variations in FcRn expression, as observed in cancer. Recently, a link between autophagy and FcRn expression has been demonstrated. Knowing that autophagy is strongly involved in the development of reperfusion injury in kidney transplantation and that albuminemia is transiently decreased in the first 2 weeks after transplantation, we investigated variations in FcRn expression after kidney transplantation. We monitored FcRn levels by flow cytometry in leukocytes from 25 renal transplant patients and considered parameters such as albumin concentrations, estimated glomerular filtration rate, serum creatinine, serum IgG levels, and ischaemia/reperfusion time. Two groups of patients could be distinguished according to their increased or non-increased FcRn expression levels between days 2 and 6 (d2-d6) post-transplantation. Leukocyte FcRn expression at d2-d6 was correlated with albumin concentrations at d0-d2. These results suggest that albumin concentrations at d0-d2 influence FcRn expression at d2-d6, raising new questions about the mechanisms underlying these original observations.

Positive association between circulating Caveolin-1 and microalbuminuria in overt diabetes mellitus in pregnancy.

Mounting evidence has shown that caveolin-1 plays a pathological role in the progression of albuminuria. Our study aimed to provide clinical evidence showing whether circulating caveolin-1 levels were associated with microalbuminuria (MAU) in women with overt diabetes mellitus in pregnancy (ODMIP). A total of 150 pregnant women were enrolled in different groups, including 40 women with ODMIP and MAU (ODMIP + MAU), 40 women with ODMIP, and 70 women without ODMIP (Non-ODMIP). Plasma caveolin-1 levels were determined by ELISA. The presence of caveolin-1 in the human umbilical vein vascular wall was evaluated by immunohistochemical and western blot analysis, respectively. Albumin transcytosis across endothelial cells was measured using an established nonradioactive in vitro approach. Significantly increased levels of plasma caveolin-1 were detected in ODMIP + MAU women. The Pearson's correlation analysis revealed a positive correlation between plasma caveolin-1 levels and Hemoglobin A1c (HbA1c %) as well as with MAU in the ODMIP + MAU group. Simultaneously, experimental knockdown or overexpression of caveolin-1 significantly decreased or increased the level of albumin transcytosis across both human and mouse glomerular endothelial cells (GECs), respectively. Our data showed a positive association between plasma caveolin-1 levels and microalbuminuria in ODMIP + MAU.

Preserved blood-brain barrier and neurovascular coupling in female 5xFAD model of Alzheimer's disease.

Dysfunction of the cerebral vasculature is considered one of the key components of Alzheimer's disease (AD), but the mechanisms affecting individual brain vessels are poorly understood. Here, using in vivo two-photon microscopy in superficial cortical layers and ex vivo imaging across brain regions, we characterized blood-brain barrier (BBB) function and neurovascular coupling (NVC) at the level of individual brain vessels in adult female 5xFAD mice, an aggressive amyloid-β (Aβ) model of AD. We report a lack of abnormal increase in adsorptive-mediated transcytosis of albumin and preserved paracellular barrier for fibrinogen and small molecules despite an extensive load of Aβ. Likewise, the NVC responses to somatosensory stimulation were preserved at all regulatory segments of the microvasculature: penetrating arterioles, precapillary sphincters, and capillaries. Lastly, the Aβ plaques did not affect the density of capillary pericytes. Our findings provide direct evidence of preserved microvascular function in the 5xFAD mice and highlight the critical dependence of the experimental outcomes on the choice of preclinical models of AD. We propose that the presence of parenchymal Aβ does not warrant BBB and NVC dysfunction and that the generalized view that microvascular impairment is inherent to Aβ aggregation may need to be revised.

ANGPT2/CAV1 regulates albumin transcytosis of glomerular endothelial cells under high glucose exposure and is impaired by losartan

BackgroundMicroalbuminuria is a common clinical symptom that manifests in the early stages of diabetic kidney disease (DKD) and is also the main feature of glomerular endothelial cells (GECs) injury. There is increasing evidence that the transcytosis of albumin across GECs is closely related to the formation of albuminuria. Our previous studies have shown that angiopoietin 2 (ANGPT2) can inhibit albumin transcytosis across renal tubular epithelial cells by activating caveolin 1 (CAV1) phosphorylation during high glucose (HG) exposure. The role of ANGPT2 in albumin transcytosis across GECs remains unclear. Losartan significantly reduces albuminuria, but the mechanism has not been clarified. MethodsWe established an in vitro albumin transcytosis model to investigate the change in albumin transcytosis across human renal glomerular endothelial cells (hrGECs) under normal glucose (NG), high glucose (HG) and losartan intervention. We knocked down ANGPT2 and CAV1 to evaluate their roles in albumin transcytosis across hrGECs and verified the relationship between them. In vivo, DKD mouse models were established and treated with different doses of losartan. Immunohistochemistry and Western blot were used to detect the expression of ANGPT2 and CAV1. ResultsIn vitro, the transcytosis of albumin across hrGECs was significantly increased under high glucose stimulation, and losartan inhibited this process. The expression of ANGPT2 and CAV1 were both increased in hrGECs under HG conditions and losartan intervention reduced the expression of them. Moreover, ANGPT2 downregulation reduced albumin transcytosis in hrGECs by regulating CAV1 expression. In vivo, the expression of ANGPT2 and CAV1 in the glomerulus was both increased significantly in DKD mice. Compared with DKD mice, losartan treatment reduced albuminuria and decreased the expression of ANGPT2 and CAV1 in a dose-dependent manner. ConclusionsANGPT2 exacerbated albumin transcytosis across GECs by increasing CAV1 expression during HG exposure, thereby increasing albuminuria. Losartan reduces albumin transcytosis and albuminuria formation in DKD by inhibiting the upregulation of ANGPT2 under HG conditions. Our findings suggest that ANGPT2 and CAV1 may be novel therapeutic targets for diabetic albuminuria. In addition, we provide new evidence to elaborate on the mechanism of losartan in the development of DKD.

ANGPT2/CAV1 regulates albumin transcytosis of glomerular endothelial cells under high glucose exposure and is impaired by losartan

BackgroundMicroalbuminuria is a common clinical symptom that manifests in the early stages of diabetic kidney disease (DKD) and is also the main feature of glomerular endothelial cells (GECs) injury. There is increasing evidence that the transcytosis of albumin across GECs is closely related to the formation of albuminuria. Our previous studies have shown that angiopoietin 2 (ANGPT2) can inhibit albumin transcytosis across renal tubular epithelial cells by activating caveolin 1 (CAV1) phosphorylation during high glucose (HG) exposure. The role of ANGPT2 in albumin transcytosis across GECs remains unclear. Losartan significantly reduces albuminuria, but the mechanism has not been clarified. MethodsWe established an in vitro albumin transcytosis model to investigate the change in albumin transcytosis across human renal glomerular endothelial cells (hrGECs) under normal glucose (NG), high glucose (HG) and losartan intervention. We knocked down ANGPT2 and CAV1 to evaluate their roles in albumin transcytosis across hrGECs and verified the relationship between them. In vivo, DKD mouse models were established and treated with different doses of losartan. Immunohistochemistry and Western blot were used to detect the expression of ANGPT2 and CAV1. ResultsIn vitro, the transcytosis of albumin across hrGECs was significantly increased under high glucose stimulation, and losartan inhibited this process. The expression of ANGPT2 and CAV1 were both increased in hrGECs under HG conditions and losartan intervention reduced the expression of them. Moreover, ANGPT2 downregulation reduced albumin transcytosis in hrGECs by regulating CAV1 expression. In vivo, the expression of ANGPT2 and CAV1 in the glomerulus was both increased significantly in DKD mice. Compared with DKD mice, losartan treatment reduced albuminuria and decreased the expression of ANGPT2 and CAV1 in a dose-dependent manner. ConclusionsANGPT2 exacerbated albumin transcytosis across GECs by increasing CAV1 expression during HG exposure, thereby increasing albuminuria. Losartan reduces albumin transcytosis and albuminuria formation in DKD by inhibiting the upregulation of ANGPT2 under HG conditions. Our findings suggest that ANGPT2 and CAV1 may be novel therapeutic targets for diabetic albuminuria. In addition, we provide new evidence to elaborate on the mechanism of losartan in the development of DKD.

Urinary Podocyte Excretion Predicts Urinary Protein Selectivity and Renal Prognosis.

Background Urinary podocyte excretion is related to a reduction in glomerular podocyte numbers, glomerulosclerosis, and urinary protein selectivity. To elucidate the role of urinary podocytes in proteinuria and renal prognosis and to identify the factors that cause podocyte detachment, we examined urinary podocytes in 120 renal biopsy patients. Methods Podocytes were identified in urinary sediments stained with fluorescent-labeled anti-podocalyxin antibodies in ten high power fields. The amounts of protein bands, separated by SDS-polyacrylamide gel electrophoresis, were calculated using an image software program and the correlation with urinary podocytes was analyzed. Podocyte surface pores were observed using a low-vacuum scanning electron microscope. The renal prognosis, including induction of hemodialysis or 30% reduction in eGFR, was investigated. Results Urinary podocyte excretion showed a higher positive correlation with albumin excretion compared to IgG, prealbumin, and transferrin. There were no significant correlations between urinary podocyte count and low molecular weight proteins, including β2-microglobulin and α1-microglobulin. The number of podocyte surface pores was positively correlated with proteinuria, suggesting enhanced albumin transcytosis. The hemodynamic pressure on the glomerular capillary wall, including products of pulse pressure and pulse rate (water hammer pressure), was positively correlated with urinary podocyte excretion. Urinary podocyte excretion and Tamm–Horsfall protein (THP) were independent risk factors for renal prognosis but were not related to response to treatment. Conclusion Urinary podocyte excretion was correlated with urinary albumin excretion, indicating specific albumin transport by podocytes. Podocytes were detached from the glomerular capillaries by water hammer pressure and THP was involved in the renal prognosis.

Angiopoietin2-mediated caveolin1 phosphorylation regulating transcytosis of renal tubular epithelial cell contributes to the occurrence of albuminuria under high glucose exposure

BackgroundMicrolbuminuria is the earliest clinical evidence of diabetic kidney disease (DKD) and contributes to the induction and/or progression of DKD. Previous studies have shown that increased expression of angiopoietin2 (ANGPT2) is correlated with an increase in albuminuria. However, the critical role of ANGPT2 in albuminuria development remains unclear. Some studies have shown the significance of transcytosis in the occurrence of albuminuria, but it is unknown whether it takes place in albumin recycling in renal tubular cells of patients with DKD. Furthermore, the potential mechanism of this association also remains unclear.MethodsIn this study, human renal tubular epithelial cells (HK-2) were cultured with high glucose in a Transwell plate to establish a transcytosis model, while C57BL/6 mice were intraperitoneally injected with streptozotocin to establish a DKD model. The expression of ANGPT2 and caveolin1 (CAV1) phosphorylation was dectected through immunohistochemistry and western blot analysis.ResultsTranscytosis of albumin in renal tubular epithelial cells was downregulated after high glucose exposure, and increased expression of ANGPT2 and CAV1 phosphorylation both in vivo and in vitro was observed. Inhibition of ANGPT2 and CAV1 independently promoted transcytosis. Furthermore, ANGPT2 downregulation inhibited CAV1 phosphorylation, whereas CAV1 phosphorylation had no effect on the expression of ANGPT2.ConclusionsANGPT2 reduces albumin transcytosis across renal tubular epithelial cells under high glucose conditions by activating CAV1 phosphorylation, thus increasing albuminuria in DKD. These findings suggested that ANGPT2 and CAV1 may be promising therapeutic targets for albuminuria in DKD.

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

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

Autophagy gene ATG7 regulates albumin transcytosis in renal tubule epithelial cells.

Receptor-mediated albumin transport in proximal tubule epithelial cells (PTECs) is important to control proteinuria. Autophagy is an evolutionarily conserved degradation pathway, and its role in intracellular trafficking through interactions with the endocytic pathway has recently been highlighted. Here, we determined whether autophagy regulates albumin transcytosis in PTECs and suppresses albumin-induced cytotoxicity using human proximal tubule (HK-2) cells. The neonatal Fc receptor (FcRn), a receptor for albumin transcytosis, is partially colocalized with autophagosomes. Recycling of FcRn was attenuated, and FcRn accumulated in autophagy-related 7 (ATG7) knockdown HK-2 cells. Colocalization of FcRn with RAB7-positive late endosomes and RAB11-positive recycling endosomes was reduced in ATG7 knockdown cells, which decreased recycling of FcRn to the plasma membrane. In ATG7 or autophagy-related 5 (ATG5) knockdown cells and Atg5 or Atg7 knockout mouse embryonic fibroblasts, albumin transcytosis was significantly reduced and intracellular albumin accumulation was increased. Finally, the release of kidney injury molecule-1, a marker of tubule injury, from ATG7 or ATG5 knockdown cells was increased in response to excess albumin. In conclusion, suppression of autophagy in tubules impairs FcRn transport, thereby inhibiting albumin transcytosis. The resulting accumulation of albumin induces cytotoxicity in tubules.NEW & NOTEWORTHY Albumin transport in proximal tubule epithelial cells (PTECs) is important to control proteinuria. The neonatal Fc receptor (FcRn), a receptor for albumin transcytosis, is partially colocalized with autophagosomes. Recycling of FcRn to the plasma membrane was decreased in autophagy-related 7 (ATG7) knockdown cells. In addition, albumin transcytosis was decreased in ATG7 or autophagy-related 5 (ATG5) knockdown PTECs. Finally, release of kidney injury molecule-1 from ATG7 or ATG5 knockdown cells was increased in response to excess albumin.

Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis.

LDL (low-density lipoprotein) transcytosis across the endothelium is performed by the SR-BI (scavenger receptor class B type 1) receptor and contributes to atherosclerosis. HMGB1 (high mobility group box 1) is a structural protein in the nucleus that is released by cells during inflammation; extracellular HMGB1 has been implicated in advanced disease. Whether intracellular HMGB1 regulates LDL transcytosis through its nuclear functions is unknown. Approach and Results: HMGB1 was depleted by siRNA in human coronary artery endothelial cells, and transcytosis of LDL was measured by total internal reflection fluorescence microscopy. Knockdown of HMGB1 attenuated LDL transcytosis without affecting albumin transcytosis. Loss of HMGB1 resulted in reduction in SR-BI levels and depletion of SREBP2 (sterol regulatory element-binding protein 2)-a transcription factor upstream of SR-BI. The effect of HMGB1 depletion on LDL transcytosis required SR-BI and SREBP2. Overexpression of HMGB1 caused an increase in LDL transcytosis that was unaffected by inhibition of extracellular HMGB1 or depletion of RAGE (receptor for advanced glycation endproducts)-a cell surface receptor for HMGB1. The effect of HMGB1 overexpression on LDL transcytosis was prevented by knockdown of SREBP2. Loss of HMGB1 caused a reduction in the half-life of SREBP2; incubation with LDL caused a significant increase in nuclear localization of HMGB1 that was dependent on SR-BI. Animals lacking endothelial HMGB1 exhibited less acute accumulation of LDL in the aorta 30 minutes after injection and when fed a high-fat diet developed fewer fatty streaks and less atherosclerosis. Endothelial HMGB1 regulates LDL transcytosis by prolonging the half-life of SREBP2, enhancing SR-BI expression. Translocation of HMGB1 to the nucleus in response to LDL requires SR-BI.

Appearances Can Be Deceiving - Viral-like Inclusions in COVID-19 Negative Renal Biopsies by Electron Microscopy.

Since the discovery of the causative agent for the novel severe acute respiratory syndrome (SARS)–like pneumonia syndrome pandemic that started in China in 2019 (1,2), a coronavirus named SARS coronavirus 2 (SARS-CoV-2), electron microscopy images have populated the medical literature (2) and media outlets alike displaying the characteristic 60–140 nm round particles surrounded by a “corona” of 9–12 nm distinctive spikes (2). Although many of these images were obtained after “ in vitro ” infection of cultured cells with SARS-CoV-2 (2) and are thus likely a true representation of viral particles, we have observed morphologically indistinguishable inclusions within podocytes and tubular epithelial cells both in patients negative for coronavirus disease 2019 (COVID-19) as well as in renal biopsies from the pre–COVID-19 era. Although direct infection of the kidney is theoretically possible, given the presence of angiotensin-converting enzyme 2 (the receptor used by SARS-CoV-2 to gain access to cells) within proximal tubular epithelium (3) and podocytes (4), the virus has not been detected by real-time RT-PCR in urine samples from patients with COVID-19 (5⇓–7). Additionally, for the virus to have access to kidney parenchyma, viremia should occur, and this has only been detected in a minority of patients (6⇓–8). We would, therefore, like to issue a note of caution for inferring viral tissue infection by morphology alone using electron microscopy images from tissues obtained from biopsies or autopsy material in patients with COVID-19. Moreover, caution should be used when interpreting immunohistochemical results, especially within proximal tubules, which are prone to nonspecific staining by a variety of antibodies due to their intense reabsorptive capacity. Additionally, more specific techniques such as immunoelectron microscopy using specific viral antigens (9), or in situ hybridization for viral RNA, are likely necessary to undoubtedly confirm tissue infection in …

Contribution of DNA methylation to the expression of FCGRT in human liver and myocardium

FcRn mediates recycling and transcytosis of IgG and albumin in various cell types. The MHC-class-I-like protein of the FcRn heterodimer is encoded by FCGRT. Few determinants of variable FCGRT expression in humans have been identified so far. In this study, we investigated the presence of DNA methylation in regulatory regions of FCGRT in samples of human liver and myocardium tissue, and we examined the impact of FCGRT methylation on FcRn expression in model cell lines. Quantitative DNA methylation analysis of the FCGRT locus revealed differentially methylated regions in DNA from liver and myocardium. Methylation status in individual CpG sites correlated with FCGRT mRNA expression. Data from model cell lines suggest that differential methylation in the −1058 to −587 bp regulatory region of FCGRT contributes to FcRn expression. Chromatin immunoprecipitation assays indicate that CpG site methylation impacts the binding of the methylation sensitive transcription factors Zbtb7a and Sp1. This study provides a foundation to further define the contribution of epigenetic factors during the control of FcRn expression and IgG traffic in human tissues.

CD36 mediates albumin transcytosis by dermal but not lung microvascular endothelial cells: role in fatty acid delivery.

In healthy blood vessels, albumin crosses the endothelium to leave the circulation by transcytosis. However, little is known about the regulation of albumin transcytosis or how it differs in different tissues; its physiological purpose is also unclear. Using total internal reflection fluorescence microscopy, we quantified transcytosis of albumin across primary human microvascular endothelial cells from both lung and skin. We then validated our in vitro findings using a tissue-specific knockout mouse model. We observed that albumin transcytosis was saturable in the skin but not the lung microvascular endothelial cells, implicating a receptor-mediated process. We identified the scavenger receptor CD36 as being both necessary and sufficient for albumin transcytosis across dermal microvascular endothelium, in contrast to the lung where macropinocytosis dominated. Mutations in the apical helical bundle of CD36 prevented albumin internalization by cells. Mice deficient in CD36 specifically in endothelial cells exhibited lower basal permeability to albumin and less basal tissue edema in the skin but not in the lung. Finally, these mice also exhibited a smaller subcutaneous fat layer despite having identical total body weights and circulating fatty acid levels as wild-type animals. In conclusion, CD36 mediates albumin transcytosis in the skin but not the lung. Albumin transcytosis may serve to regulate fatty acid delivery from the circulation to tissues.

Albumin nanoparticles conjugates binding with glycan - A strategic approach for targeted drug delivery

Understanding the transcytosis phenomenon through albumin binding receptor (glycoproteins) is an important step towards targeted drug delivery research. The transcytosis of bovine serum albumin (BSA) occurs through its uptake by binding to glycoprotein (gp60) present on the endothelial cell membrane. gp60 possess abundant glycans like N-acetylneuraminic acid (sialic acid, NANA), l-fucose (FUC), N-acetyl glucosamine (NAG), d-mannose (MAN) and d-galactose (GAL). Hence, the objective of the present study was to insight the binding between BSA-gold nanoparticles (GNPs) and BSA-silver nanoparticles (SNPs) conjugates with model glycans using several biophysical techniques. The fluorescence spectroscopy analysis established the strong affinity of BSA-NPs conjugates with glycan's as observed with the bimolecular quenching rate, the Stern-Volmer constant (Ksv), binding constant and binding site. The increase in hydrodynamic radii of conjugates exhibited strong association and zeta potential confirmed the significant stability of conjugates. Circular dichroism (CD) study showed that the substantial amount of secondary structure (α-helix) was retained in NPs bound BSA while interacting with glycans. Raman spectroscopy study of BSA-GNP and BSA-SNP with glycans depicted the intrusion in the spectral range of 400–1800 cm−1. The overall results showed that BSA-NPs conjugates can act as a virtuous system for targeted drug delivery in endothelial cells.

Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment.

The neonatal Fc receptor (FcRn) is responsible for the recycling and transcytosis of IgG and albumin. FcRn level was found altered in cancer tissues and implicated in tumor immunosurveillance and neoplastic cell growth. However, the consequences of FcRn down-regulation in the anti-tumor immune response are not fully elucidated. By using the B16F10 experimental lung metastasis model in an FcRn-deficient microenvironment (FcRn−/− mice), we found lung metastasis associated with an abnormal natural killer (NK) cell phenotype. In FcRn−/− mice, NK cells were immature, as shown by their surface marker profile and their decreased ability to degranulate and synthesize interferon γ after chemical and IL-2 or IL-12, IL-15 and IL-18 activation. These new findings support the critical role of FcRn downregulation in the tumor microenvironment in anti-tumor immunity, via NK cell maturation and activation.

Nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant results in albuminuria and mesangial expansion.

Intracellular trafficking processes play a key role for the establishment and maintenance of membrane surfaces in renal epithelia. Therefore, dysfunctions of these trafficking processes could be key events and important determinants in the onset and progression of diseases. The presence of cellular vacuoles-observed in many histologic analyses of renal diseases-is a macroscopic hint for disturbed intracellular trafficking processes. However, how vacuoles develop and which intracellular pathways are directly affected remain largely unknown. Previous studies showed that in some cases, vacuolization is linked to malfunction of the Vac14 complex. This complex, including the scaffold protein Vac14, the lipid kinase PIKfyve, and its counteracting lipid phosphatase Fig4, regulates intracellular phosphatidylinositol phosphate levels, which in turn, control the maturation of early-into-late endosomes, as well as the processing of autophagosomes into autophagolysosomes. Here, we analyzed the role of Vac14 in mice and observed that the nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant led to albuminuria, accompanied by mesangial expansion, increased glomerular size, and an elevated expression of several kidney injury markers. Overexpression of this Vac14 variant in podocytes did not reveal a strong in vivo phenotype, indicating that Vac14-dependent trafficking processes are more important for tubular than for glomerular processes in the kidney. In vitro overexpression of Vac14L156R in Madin-Darby canine kidney cells had no impact on apico-basal polarity defects but resulted in a faster reassembly of junctional structures after Ca2+ depletion and delayed endo- and transcytosis rates. Taken together, our data suggest that increased albuminuria of Vac14L156R-overexpressing mice is a consequence of a lowered endo- and transcytosis of albumin in renal tubules.

Intestinal uptake and transport of albumin nanoparticles: potential for oral delivery.

To explore the potential of albumin nanoparticles for oral drug delivery. Sub-150nm human serum albumin nanoparticles were fabricated via a desolvation technique. Nanoparticle cell uptake and epithelial translocation were tested in Caco-2 monolayers, while comparing with albumin solution. Data suggest epithelial transcytosis of albumin, applied in solution form, via neonatal Fc receptor. Cell uptake of albumin nanoparticles demonstrated behaviors indicating a different cell uptake pathway compared with albumin solution. Importantly, application of equivalent concentrations of albumin solution or nanoparticles resulted in higher epithelial transport capacity of the latter, suggesting improvement of intestinal delivery via nanoformulation. This study highlights for the first time that simply fabricated, nontoxic human serum albumin nanoparticles may find application in oral drug delivery.

Abstract 452: Estrogen Inhibits LDL Transcytosis by Coronary Artery Endothelial Cells via GPER and SR-BI

Objectives: Premenopausal women have a significantly lower rate of atherosclerosis than men, an effect attributed to estrogen. The atheroprotective effects of estrogen are independent of circulating lipid levels and may be due to an effect on the vessel wall. Early on in atherosclerosis, circulating LDL is able to cross an intact endothelial monolayer by transcytosis and accumulate in the intima. Little is known about the mechanisms of transcytosis but using novel assays we recently discovered a role for the SR-BI and ALK1 receptors. We hypothesized that estrogen can attenuate LDL transcytosis by coronary artery endothelial cells. Approach and Results: Using a recently described assay based on total internal reflection fluorescence microscopy, we quantified transcytosis of LDL across human coronary artery endothelial cells treated with physiological concentrations of estrogen. Estrogen significantly attenuated LDL transcytosis by endothelial cells from male donors; the effect was ligand-specific as transcytosis of albumin was not affected. Estrogen caused down-regulation of endothelial SR-BI but not ALK1 and over-expression of SR-BI was sufficient to restore LDL transcytosis. Similarly, depletion of SR-BI by siRNA attenuated endothelial LDL transcytosis and prevented any further effect of estrogen. In contrast, treatment with estrogen had no effect on SR-BI expression by liver cells from a male donor. Inhibition of estrogen receptors α and β had no effect on estrogen-mediated attenuation of LDL transcytosis. However, estrogen’s effect was blocked by depletion of the G-protein coupled estrogen receptor (GPER) by siRNA. GPER was found to be enriched in endothelial cells compared to hepatocytes and is known to signal via transactivation of the epidermal growth factor receptor (EGFR); we observed that inhibition of EGFR also prevented the effect of estrogen on LDL transcytosis. Lastly, male mice demonstrated more vascular deposition of LDL than age-matched female mice after acute injection. Conclusions: Physiological concentrations of estrogen significantly inhibit LDL transcytosis by down-regulating endothelial SR-BI; this effect requires GPER. This may contribute to the lower rates of atherosclerosis in premenopausal women.

The Role of Caveolae on Albumin Passage through Glomerular Endothelial and Epithelial Cells: The New Etiology of Urinary Albumin Excretion.

In the traditional theory of albuminuria, small amounts of albumin pass through the fenestrae in glomerular endothelial cells, then through the slit membrane in the gaps between foot processes of glomerular epithelial cells. In the novel theory, large amounts of albumin pass through glomerular capillaries and are taken up by megalin and cubilin receptors on tubular epithelial cells. These etiologies of urinary albumin excretion are still controversial, and the details of albumin passage through the three layers of glomerular capillaries (glomerular endothelial cells, basement membrane, and epithelial cells) have never been entirely elucidated. Recent advances in basic research have shown that caveolae, which are cell invaginations located on the surface of both glomerular endothelial and epithelial cells, play pivotal roles in the endocytosis, transcytosis, and exocytosis of albumin. Albumin enters into glomerular endothelial and epithelial cells through caveolae; subsequent transcytosis of albumin is not actin- or microtubule-dependent in glomerular endothelial cells, but is actin-dependent in glomerular epithelial cells. Exocytosis of albumin in glomerular endothelial cells occurs via early endosomes through a process that bypasses other endosome-associated organelles. In contrast, exocytosis of albumin in glomerular epithelial cells occurs via early endosomes through a process that results in lysosomal degradation of some albumin particles. Key Messages: This caveolae-dependent pathway may provide a new pathophysiology for albumin passage through glomerular endothelial and epithelial cells, leading to a new etiology for urinary albumin excretion that connects both traditional and novel theories of albuminuria.

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.