Albumin Endocytosis Research Articles

Monitoring the endocytosis of bovine serum albumin based on the fluorescence lifetime of small squaraine dye in living cells.

Bovine serum albumin (BSA) has a wide range of physiological functions involving the binding, transportation, and delivery of fatty acids, porphyrins, bilirubin, steroids, etc. In the present study, we prepared a small squaraine dye (SD), which can selectively detect BSA using fluorescence lifetime imaging microscopy (FLIM), to monitor the endocytosis of BSA in live cultured cells in real time. This approach revealed that BSA uptake is concentration-dependent in living cells. Furthermore, we used paclitaxel (PTX), a chemotherapeutic drug, to influence the endocytosis of BSA in living cells. The results demonstrated that the endocytic rate was clearly reduced after pretreatment with 0.4 µM PTX for 2 h. The present study demonstrates the potential value of using the fluorescence lifetime of SD to detect BSA concentration and study the physiological mechanism of chemotherapeutic drugs.

Mechanism Underlying Selective Albuminuria in Minimal Change Nephrotic Syndrome.

As water and solutes are filtered through the slit membrane, it is an a priori concept that a slit membrane is an essential filtration barrier for proteins, including albumin. However, in cases of minimal change nephrotic syndrome, the number of slit membranes is reduced by the foot process effacement and tight junction-like cell adhesion. Furthermore, albumin endocytosis is enhanced in the podocytes under condition of minimal change disease, and albumin is selectively transported by the albumin receptor FcRn. Suppressing the endocytosis of albumin with anti-FcRn antibody decreases the urinary protein level. The expression of motor molecules, such as cytoplasmic dynein 1 and myosin IX, is increased in the podocytes under conditions of minimal change nephrotic syndrome, suggesting the enhanced transport of vesicles containing albumin. Podocyte vesicle transport may play an important role in the pathology of selective albuminuria in cases of nephrotic syndrome.

Sertraline Reduces Albuminuria by Interfering with Caveolae-Mediated Endocytosis through Glomerular Endothelial and Epithelial Cells

Introduction: Previously, we reported the caveolae-mediated intracellular trafficking pathway of albumin through glomerular endothelial cells (GEnCs) as a new etiological hypothesis of urinary albumin excretion. The selective serotonin reuptake inhibitor, sertraline (Ser), inhibits dynamin, which plays a pivotal role in the fission of caveolae from the cell membrane during caveolae endocytosis. Objective: In this study, we evaluated whether Ser reduces albuminuria levels by interfering with albumin endocytosis through caveolae into GEnCs and podocytes as a novel treatment for glomerulonephritis. Methods: After treating the cells with Ser, albumin and caveolin-1 (Cav-1) expression levels were evaluated by immunofluorescence (IF) and western blot (WB) analyses. The albuminuria level was determined by histology in a puromycin aminonucleoside (PAN)-induced nephrotic syndrome mouse model (PAN mice) treated with or without Ser. Results: IF and WB analyses showed that the albumin expression level was significantly decreased by Ser treatment; however, Cav-1 expression was not decreased in GEnCs or podocytes based on the IF results. In PAN mice treated with or without Ser, Cav-1 expression increased, and the foot process effacement of podocytes and swelling of GEnCs were observed. However, proteinuria levels were not increased in PAN mice treated with Ser relative to that in normal control mice (p = 0.17), and a significant increase was observed in PAN mice without Ser treatment (p = 0.0027). Conclusions: Ser interfered with albumin internalization through the caveolae into GEnCs and podocytes and reduced albuminuria. Dynamin inhibitors may serve as a novel therapeutic option for reducing albuminuria in glomerulonephritis.

Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria

Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria.

SUN-003 MECHANISM OF SELECTIVE ALBUMINURIA IN MINIMAL CHANGE NEPHROTIC SYNDROME

Water and electrolytes are filtered through slit membranes, however in minimal change nephrotic syndrome with foot process effacement it is not well understood whether albumin molecules will be filtered through the slit membrane. We reported that albumin endocytosis by FcRn was enhanced in the podocyte of puromycin aminonucleoside (PAN)-induced minimal change nephrotic syndrome rat, and treatment with anti-FcRn antibody decreased albumin endocytosis and urinary protein (Kidney Int 2011). Minimal change nephrotic syndrome model was induced by puromycin amino nucleoside, and podocytes were observed by electron microscope and low vacuum scanning electron microscope. Electron microscopy demonstrated many vesicles were observed in the podocytes of PAN rat with 3D observation. SDS-polyacrylamide electrophoresis of glomerular proteins isolated from PAN rats showed that several protein bands were increased than those from control rats, and those increased protein bands were identified as dynein -1, myosin 7 and myosin 9 by mass spectrometry. These motor molecules are capable of transporting vesicles containing albumin in the podocytes of PAN nephrotic rat. In renal biopsy, electron microscopic examination of human minimal change disease cases showed numerous vesicles and microtubules in podocytes, and a number of exocytotic holes were found on the surface of podocytes by low vacuum scanning electron microscopy. In conclusion, podocyte vesicle transport could play an important role in the selective albuminuria in the minimal change nephrotic syndrome.

Elafibranor Inhibits Chronic Kidney Disease Progression in NASH Mice.

Identification of new pharmacological approaches to inhibit the excessive fat intake-induced steatohepatitis and chronic kidney disease (CKD) is important. High-fat diet (HFD)-induced steatohepatitis and CKD share common pathogenesis involving peroxisome proliferator-activated receptor (PPAR)-α and -δ. Elafibranor, a dual PPARα/δ agonist, can ameliorate the HFD-induced steatohepatitis. Nonetheless, the effects of HFD-induced CKD had not yet explored. This study investigated the effects of elafibranor (elaf) on the progression of HFD-induced CKD in mice. In vivo and in vitro renal effects were evaluated in HFD-elaf mice receiving 12 weeks of elafibranor (from 13th to 24th week of HFD feeding) treatment. In elafibranor-treated HFD mice, increased insulin sensitivity, reduced obesity and body fat mass, decreased severity of steatohepatitis, increased renal expression of PPARα, PPARδ, SIRT1, and autophagy (Beclin-1 and LC3-II) as well as glomerular/renal tubular barrier markers [synaptopodin (podocyte marker), zona occludin-1, and cubulin], reduced renal oxidative stress and caspase-3, and less urinary 8-isoprostanes excretion were observed. Aforementioned benefits of elafibranor were associated with low renal tubular injury and tubulointerstitial fibrosis scores, less albuminuria, low urinary albumin-to-creatinine ratio, and preserved glomerular filtration rate. Acute incubation of podocytes and HK-2 cells with elafibranor or recombinant SIRT1 reversed the HFD-sera-induced oxidative stress, autophagy dysfunction, cell apoptosis, barrier marker loss, albumin endocytosis, and reuptake reduction. Besides hepatoprotective and metabolic beneficial effects, current study showed that elafibranor inhibited the progression of HFD-induced CKD through activation of renal PPARα, PPARδ, SIRT1, autophagy, reduction of oxidative stress, and apoptosis in mice with steatohepatitis.

Lithium ameliorates tubule-interstitial injury through activation of the mTORC2/protein kinase B pathway.

Tubule-interstitial injury (TII) is a critical step in the progression of renal disease. It has been proposed that changes in proximal tubule (PT) albumin endocytosis plays an important role in the development of TII. Some reports have shown protective effects of lithium on kidney injury animal models that was correlated to proteinuria. We tested the hypothesis that lithium treatment ameliorates the development of TII due to changes in albumin endocytosis. Two experimental models were used: (1) TII induced by albumin overload in an animal model; (2) LLC-PK1 cells, a PT cell line. Lithium treatment ameliorates TII induced by albumin overload measured by (1) proteinuria; (2) collagen deposition; (3) area of tubule-interstitial space, and (4) macrophage infiltration. Lithium treatment increased mTORC2 activity leading to the phosphorylation of protein kinase B (PKB) at Ser473 and its activation. This mechanism enhanced albumin endocytosis in PT cells, which decreased the proteinuria observed in TII induced by albumin overload. This effect did not involve changes in the expression of megalin, a PT albumin receptor. In addition, activation of this pathway decreased apoptosis in LLC-PK1 cells, a PT cell line, induced by higher albumin concentration, similar to that found in pathophysiologic conditions. Our results indicate that the protective role of lithium treatment on TII induced by albumin overload involves an increase in PT albumin endocytosis due to activation of the mTORC2/PKB pathway. These results open new possibilities in understanding the effects of lithium on the progression of renal disease.

O-GlcNAcylation reduces proximal tubule protein reabsorption and promotes proteinuria in spontaneously hypertensive rats

Hypertensive individuals are at greater risk for developing chronic kidney disease (CKD). Reducing proteinuria has been suggested as a possible therapeutic approach to treat CKD. However, the mechanisms underlying the development of proteinuria in hypertensive conditions are incompletely understood. Cardiac and vascular dysfunction is associated with changes in the O-GlcNAcylation pathway in hypertensive models. We hypothesized that O-GlcNAcylation is also involved in renal damage, especially development of proteinuria, associated with hypertension. Using the spontaneously hypertensive rat (SHR) model, we observed higher renal cortex O-GlcNAcylation, glutamine-fructose aminotransferase (GFAT), and O-GlcNAc transferase (OGT) protein expression, which positively correlated with proteinuria. Interestingly, this was observed in hypertensive, but not pre-hypertensive, rats. Pharmacological inhibition of GFAT decreased renal cortex O-GlcNAcylation, proteinuria, and albuminuria in SHR. Using a proximal tubule cell line, we observed that increased O-GlcNAcylation reduced megalin surface expression and albumin endocytosis in vitro, and the effects were correlated in vivo Moreover, megalin is O-GlcNAcylated both in vitro and in vivo In conclusion, our results demonstrate a new mechanism involved in hypertension-associated proteinuria.

High glucose reduces megalin-mediated albumin endocytosis in renal proximal tubule cells through protein kinase B O-GlcNAcylation

The role of albumin reabsorption in proximal tubule (PT) cells has emerged as an important factor in the genesis of albuminuria observed in the early stages of diabetes. Evidence has shown that a decrease in megalin expression could be the key mechanism in this process. In the present work, we investigated the molecular mechanism underlying the modulation of albumin endocytosis in LLC-PK1 cells, a model of PT cells. High glucose concentrations (HG) inhibited megalin expression and albumin endocytosis after 48 h of incubation. This inhibitory effect involves the entrance of glucose into PT cells through SGLT located at the luminal membrane. Once inside PT cells, glucose is diverted to the hexosamine biosynthetic pathway (HBP) increasing O-GlcNAcylation of several intracellular proteins, including PKB. This process promotes the inhibition of PKB activity measured by its phosphorylation at Thr-308 and Ser-473 and phosphorylation of specific substrates, glycogen synthase kinase 3β (GSK3β) and tuberous sclerosis complex 2. The decrease in PKB activity led to a decrease in megalin expression and, consequently, reducing albumin endocytosis in LLC-PK1 cells. HG did not change mammalian target of rapamycin (mTOR) C2 activity, responsible for phosphorylated PKB at Ser-473. In addition, HG activated the mTORC1/S6K pathway, but this effect was not correlated to the decrease in megalin expression or albumin endocytosis. Taken together, our data help to clarify the current understanding underlying the genesis of tubular albuminuria induced by hyperglycemia in the early stage of diabetes pathogenesis.

Increased megalin expression in early type 2 diabetes: role of insulin-signaling pathways.

The megalin/cubilin complex is responsible for the majority of serum protein reclamation in the proximal tubules. The current study examined if decreases in their renal expression, along with the albumin recycling protein neonatal Fc receptor (FcRn) could account for proteinuria/albuminuria in the Zucker diabetic fatty rat model of type 2 diabetes. Immunoblots of renal cortex samples obtained at worsening disease stages demonstrated no loss in megalin, cubilin, or FcRn, even when proteinuria was measured. Additionally, early diabetic rats exhibited significantly increased renal megalin expression when compared with controls (adjusted P < 0.01). Based on these results, the ability of insulin to increase megalin was examined in a clonal subpopulation of the opossum kidney proximal tubule cell line. Insulin treatments (24 h, 100 nM) under high glucose conditions significantly increased megalin protein ( P < 0.0001), mRNA ( P < 0.0001), and albumin endocytosis. The effect on megalin expression was prevented with inhibitors against key effectors of insulin intracellular signaling, phosphatidylinositide 3-kinase and Akt. Studies using rapamycin to inhibit the mechanistic target of rapamycin complex 1 (mTORC1) resulted in a loss of insulin-induced megalin expression. However, subsequent evaluation demonstrated these effects were independent of initial mTORC1 suppression. The presented results provide insight into the expression of megalin, cubilin, and FcRn in type 2 diabetes, which may be impacted by elevated insulin and glucose. Furthermore, proximal tubule endocytic activity in early diabetics may be enhanced, a process that could have a significant role in proteinuria-induced renal damage.

The presence of human mesenchymal stem cells of renal origin in amniotic fluid increases with gestational time

BackgroundEstablished therapies for managing kidney dysfunction such as kidney dialysis and transplantation are limited due to the shortage of compatible donated organs and high costs. Stem cell-based therapies are currently under investigation as an alternative treatment option. As amniotic fluid is composed of fetal urine harboring mesenchymal stem cells (AF-MSCs), we hypothesized that third-trimester amniotic fluid could be a novel source of renal progenitor and differentiated cells.MethodsHuman third-trimester amniotic fluid cells (AFCs) were isolated and cultured in distinct media. These cells were characterized as renal progenitor cells with respect to cell morphology, cell surface marker expression, transcriptome and differentiation into chondrocytes, osteoblasts and adipocytes. To test for renal function, a comparative albumin endocytosis assay was performed using AF-MSCs and commercially available renal cells derived from kidney biopsies. Comparative transcriptome analyses of first, second and third trimester-derived AF-MSCs were conducted to monitor expression of renal-related genes.ResultsRegardless of the media used, AFCs showed expression of pluripotency-associated markers such as SSEA4, TRA-1-60, TRA-1-81 and C-Kit. They also express the mesenchymal marker Vimentin. Immunophenotyping confirmed that third-trimester AFCs are bona fide MSCs. AF-MSCs expressed the master renal progenitor markers SIX2 and CITED1, in addition to typical renal proteins such as PODXL, LHX1, BRN1 and PAX8. Albumin endocytosis assays demonstrated the functionality of AF-MSCs as renal cells. Additionally, upregulated expression of BMP7 and downregulation of WT1, CD133, SIX2 and C-Kit were observed upon activation of WNT signaling by treatment with the GSK-3 inhibitor CHIR99201. Transcriptome analysis and semiquantitative PCR revealed increasing expression levels of renal-specific genes (e.g., SALL1, HNF4B, SIX2) with gestational time. Moreover, AF-MSCs shared more genes with human kidney cells than with native MSCs and gene ontology terms revealed involvement of biological processes associated with kidney morphogenesis.ConclusionsThird-trimester amniotic fluid contains AF-MSCs of renal origin and this novel source of kidney progenitors may have enormous future potentials for disease modeling, renal repair and drug screening.

Reciprocal regulation of eNOS and caveolin-1 functions in endothelial cells.

We hypothesized that the maintenance of vascular homeostasis is critically dependent on the expression and reciprocal regulation of caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs). Skeletal muscle biopsies from subjects with type 2 diabetes showed 50% less Cav-1 and eNOS than those from lean healthy controls. The Cav-1:eNOS expression ratio was 200:1 in primary culture human ECs. Cav-1 small interfering RNA (siRNA) reduced eNOS protein and gene expression in association with a twofold increase in eNOS phosphorylation and nitrate production per molecule of eNOS, which was reversed in cells overexpressing Adv-Cav-1-GFP. Upon addition of the Ca2+ ionophore A23187 to activate eNOS, we observed eNOS Ser1177 phosphorylation, its translocation to β-catenin-positive cell–cell junctions, and increased colocalization of eNOS and Cav-1 within 5 min. We also observed Cav-1 S-nitrosylation and destabilization of Cav-1 oligomers in cells treated with A23187 as well as insulin or albumin, and this could be blocked by L-NAME, PP2, or eNOS siRNA. Finally, caveola-mediated endocytosis of albumin or insulin was reduced by Cav-1 or eNOS siRNA, and the effect of Cav-1 siRNA was rescued by Adv-Cav-1-GFP. Thus, Cav-1 stabilizes eNOS expression and regulates its activity, whereas eNOS-derived NO promotes caveola-mediated endocytosis.

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.

ORAI channels are critical for receptor-mediated endocytosis of albumin

Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. Here we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and diethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. The albumin endocytosis is Ca2+-dependent and accompanied by ORAI1 internalization. Amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs, which determines clathrin-mediated endocytosis. These findings provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria.

Albumin uptake in human podocytes: a possible role for the cubilin-amnionless (CUBAM) complex

Albumin re-uptake is a receptor-mediated pathway located in renal proximal tubuli. There is increasing evidence of glomerular protein handling by podocytes, but little is known about the mechanism behind this process. In this study, we found that human podocytes in vitro are committed to internalizing albumin through a receptor-mediated mechanism even after exposure to low doses of albumin. We show that these cells express cubilin, megalin, ClC-5, amnionless and Dab2, which are partners in the tubular machinery. Exposing human podocytes to albumin overload prompted an increase in CUBILIN, AMNIONLESS and CLCN5 gene expression. Inhibiting cubilin led to a reduction in albumin uptake, highlighting its importance in this mechanism. We demonstrated that human podocytes are committed to performing endocytosis via a receptor-mediated mechanism even in the presence of low doses of albumin. We also disclosed that protein overload first acts on the expression of the cubilin-amnionless (CUBAM) complex in these cells, then involves the ClC-5 channel, providing the first evidence for a possible role of the CUBAM complex in albumin endocytosis in human podocytes.

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.

Endocytosis of Albumin Induces Matrix Metalloproteinase-9 by Activating the ERK Signaling Pathway in Renal Tubule Epithelial Cells.

Matrix metalloproteinase-9 (MMP-9) is dysregulated in chronic kidney diseases including diabetic nephropathy. This study was performed to examine the expression of MMP-9 in renal tubule epithelial cells (TECs) under diabetic conditions and its regulatory mechanisms. We characterized MMP-9 protein in diabetic animals and primary cultured rat TECs exposed to exogenous albumin and high glucose. We also used specific inhibitors to determine if internalization of albumin and/or extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation were required for MMP-9 secretion. Immunostaining of kidney sections revealed enhanced MMP-9 signal in the damaged proximal tubules in Zucker diabetic fatty (ZDF) rats. ZDF rats also exhibited an albuminuria-related and age-dependent increase in MMP-9 excretion, which was prevented by rosiglitazone. In primary cultured rat TECs, high glucose exposure did not increase MMP-9 secretion. In contrast, administration of rat serum albumin (RSA, 0.1–0.5 mg/mL) resulted in a dose-dependent increase in MMP-9 expression and secretion by TECs, which was abolished in the presence of an ERK1/2-specific inhibitor, U0126. Simvastatin, an inhibitor of albumin endocytosis, also prevented MMP-9 secretion. Taken together, these results demonstrate that endocytosis of albumin stimulates MMP-9 secretion by TECs through the ERK signaling pathway.

Intracellular transcytosis of albumin in glomerular endothelial cells after endocytosis through caveolae.

We previously described albumin endocytosis through caveolae in human renal glomerular endothelial cells (HRGECs). This suggested a new albumin transcytosis pathway, in addition to the fenestral pathway. As a next step, we investigated albumin transcytosis in HRGECs after caveolar endocytosis. HRGECs were incubated with Alexa Fluor 488-labeled bovine serum albumin from 0 to 360 min. Next, markers for endosomes, endoplasmic reticulum (ER), golgi apparatus (GA), lysosomes, and proteasomes and Fc receptors, microtubules, and actin were monitored by immunofluorescence. Labeled albumin co-localization with endosomes was gradually and significantly increased and it was significantly higher than with the other markers at any timepoint. Albumin, placed on inside of the Transwell membrane, diffused through HRGEC monolayers during a 360 min incubation period. This transportation of albumin through HRGECs was inhibited by methyl beta cyclodextrin (MBCD), a caveolae disrupting agent. MBCD also decreased albuminuria, causing decreased caveolin-1 (Cav-1) expression on glomerular capillaries, in puromycin aminonucleoside induced nephrotic mice. Albumin transcytosis depends on early endosomes, but not on other organelles, Fc receptors, or cytoskeletal components. Caveolae disruption prevented albumin transportation through HRGECs and decreased albuminuria in nephrotic mice. This newly described caveolae-dependent albumin pathway through glomerular endothelial cells is a potential pathogenetic mechanism for albuminuria, independent of the fenestrae.

Enhanced podocyte vesicle transport in the nephrotic rat.

Albumin endocytosis is enhanced in the podocytes of minimal change nephrotic syndrome. We investigated that the endocytic vesicle transport in the podocyte using three-dimensional observation in a rat model of puromycin aminonucleoside (PAN)-induced nephrotic syndrome. At day 7, Evans Blue-labeled albumin was intravenously injected in PAN rats, and one kidney was fixed for a morphological analysis; the other was used for the isolation of glomeruli through sieving and protein analyses. Evans Blue-labeled albumin was found to accumulate in an increased number of vesicles in the podocytes of PAN rat. Continuous sections and its three-dimensional observation demonstrated that vesicles may be transported from the cytoplasm to the apical membrane of the podocytes. The increased protein bands in the gel electrophoresis of the sieved glomeruli of nephrotic rats were analyzed by mass spectrometry in comparison to the control rats. The major proteins increased in the nephrotic rats were cytoplasmic dynein 1 heavy chain, myosin IX, and myosin VIIb. In conclusion, the podocyte endocytic vesicles carrying albumin increased with glomerular cytoplasmic dynein and myosin in minimal change nephrotic rats.

Novel routes of albumin passage across the glomerular filtration barrier

Albuminuria is a hallmark of kidney diseases of various aetiologies and an unambiguous symptom of the compromised integrity of the glomerular filtration barrier. Furthermore, there is increasing evidence that albuminuria per se aggravates the development and progression of chronic kidney disease. This review covers new aspects of the movement of large plasma proteins across the glomerular filtration barrier in health and disease. Specifically, this review focuses on the role of endocytosis and transcytosis of albumin by podocytes, which constitutes a new pathway of plasma proteins across the filtration barrier. Thus, we summarize what is known about the mechanisms of albumin endocytosis by podocytes and address the fate of the endocytosed albumin, which is directed to lysosomal degradation or transcellular movement with subsequent vesicular release into the urinary space. We also address the functional consequences of overt albumin endocytosis by podocytes, such as the formation of pro-inflammatory cytokines, which might eventually result in a deterioration of podocyte function. Finally, we consider the diagnostic potential of podocyte-derived albumin-containing vesicles in the urine as an early marker of a compromised glomerular barrier function. In terms of new technical approaches, the review covers how our knowledge of the movement of albumin across the glomerular filtration barrier has expanded by the use of new intravital imaging techniques.