Curcumin Improves the Renal Autophagy in Rat Experimental Membranous Nephropathy via Regulating the PI3K/AKT/mTOR and Nrf2/HO-1 Signaling Pathways.

Membranous nephropathy (MN, also known as membranous glomerulopathy) is one of the many glomerular diseases causing nephrotic syndrome. The literature indicates that autophagy is associated with the homeostasis of podocytes in glomeruli. Curcumin, the main active component in turmeric, has drawn attention for its effective bioactivities against chronic kidney disease. The current study was aimed at assessing the effects of curcumin and exploring the underlying mechanism that mediates autophagy in an animal model of passive Heymann nephritis (PHN) in rats. Passive Heymann nephritis (PHN) was induced in male SD rats by intraperitoneal injection of anti-Fx1A serum. The rats were divided into 3 groups: control (n = 10, normal diet), model group (n = 10, 0.5% sodium carboxymethylcellulose), and curcumin (n = 10, 300 mg/kg/d). The kidney function and oxidative stress indicators were measured using commercial diagnostic kits, and the histomorphology of renal tissues was observed. The number of podocytes was measured by immunohistochemistry. Meanwhile, the autophagosomes in podocyte were analyzed by transmission electron microscopy and the immunofluorescence assay pointing to p62, an autophagic marker. Western blot analyzed the levels of apoptosis, autophagy, PI3K/AKT/mTOR, and Nrf2/HO-1 pathway-associated proteins. The total cholesterol (TC), triglycerides (TG), creatinine (Scr), blood urea nitrogen (BUN), urine volume, and urine albumin of PHN rats were significantly reduced by the administration of curcumin and attenuated renal histomorphological changes in model rats. Meanwhile, curcumin improved the oxidative stress response by decreasing MDA and increasing SOD, GSH, and CAT levels in the kidney of PHN rats. Furthermore, curcumin significantly ameliorated the podocyte loss, along with the fusion, and increased the autophagic vacuoles compared to the PHN control rats. In addition, curcumin downregulated the expression of Bax, Caspase-3, p62, PI3K, p-AKT, and p-mTOR proteins and upregulated the Bcl-2, beclin1, LC3, Nrf2, and HO-1 levels in this animal model. The results provide a scientific basis that curcumin could significantly alleviate the development of MN by inducing autophagy and alleviating renal oxidative stress through the PI3K/AKT/mTOR and Nrf2/HO-1 pathways.

A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity.

Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.

Betulinic acid, isolated from the leaves of Syzygium cumini (L.) Skeels, ameliorates the proteinuria in experimental membranous nephropathy through regulating Nrf2/NF-κB pathways.

Membranous nephropathy (MN) is associated with increased oxidative stress and inflammatory markers in the kidney. Betulinic acid (BA) is a potent antioxidant and anti-inflammatory compound isolated from the leaves of Syzygium cumini (L.) Skeels. In the present study, we investigated the effects of BA on experimental MN in rats and explored the mechanisms by which it enhances antioxidant activities and resolves inflammatory condition in experimental MN. Passive Heymann nephritis (PHN) was induced in Sprague-Dawley rats by a single tail vein injection of anti- Fx1A antiserum. The rats were orally administered BA (25 and 50 mg kg -1 d -1) or dexamethasone (DEX; 0.07 mg kg-1, reference compound) for 4 weeks after the induction of PHN. Blood, urine, and kidney tissue were collected for analysis at the end of the study. Treatment of PHN rats with BA or DEX significantly attenuated renal dysfunction, histopathological alterations and reduced immune complex deposition in the kidneys. Furthermore, BA ameliorated mRNA and protein expression of NF-κB, iNOS, TNF-α, Nrf2, HO-1 and NQO1 in the kidney. BA also restored malondialdehyde level and antioxidant enzyme activities in the kidney. In a nutshell, the protective effect of BA can be explained by its anti-inflammatory and anti-oxidant activities, which in turn is due to downregulation of NF-κB pathway and activation of Nrf2. The results indicated that BA can effectively suppress experimental PHN in rats by regulating Nrf2/NF-κB pathways.

Heymann nephritis in Lewis rats.

Human membranous nephritis is a major cause of end-stage kidney disease. Active Heymann nephritis (HN) is an auto-immune model of membranous nephritis induced in Lewis rats by immunization with a crude renal tubular antigen (Fx1A) or megalin (gp330). The pathogenesis of HN is through the binding of anti-Fx1A autoantibodies to the auto-antigen expressed on glomerular epithelial cells, resulting in severe glomerular injury and proteinuria. The pathological features of HN include immune deposits in glomeruli and infiltration of glomeruli and the tubulointerstitium by macrophages and T cells. This unit describes the method of the preparation of Fx1A and the induction of HN in Lewis rats by immunization with Fx1A.

De novo expression of podocyte proteins in parietal epithelial cells during experimental glomerular disease.

Studies have shown that certain cells of the glomerular tuft begin to express proteins considered unique to other cell types upon injury. Little is known about the response of parietal epithelial cells (PEC) to injury. To determine whether PECs change their phenotype upon injury to also express proteins traditionally considered podocyte specific, the following four models of glomerular disease were studied: the transforming growth factor (TGF)-beta1 transgenic mouse model of global glomerulosclerosis, the adriamycin model of focal segmental glomerulosclerosis (FSGS), the anti-glomerular basement membrane (GBM) model of crescentic glomerulonephritis, and the passive Heymann nephritis model of membranous nephropathy. Double immunostaining was performed with antibodies to podocyte-specific proteins (synaptopodin and Wilms' tumor 1) and antibodies to PEC specific proteins (paired box gene 8 and claudin-1). No double staining was detected in normal mice. In contrast, the results showed a statistical increase in the number of cells attached to Bowman basement membrane that were double-positive for both podocyte/PEC proteins in TGF-beta1 transgenic, anti-GBM, and membranous animals. Double-positive cells for both podocyte and PEC proteins were also statistically increased in the glomerular tuft in TGF-beta1 transgenic, anti-GBM, and FSGS mice. These results are consistent with glomerular cells coexpressing podocyte and PEC proteins in experimental glomerular disease, but not under normal circumstances.

[The effect of splenic lymphocytes from oral immune tolerance rats on activity of NF-kappaB p65 in glomerular mesangial cells].

AIM: To explore the effect of splenic lymphocytes from oral immune tolerance rats on NF-kappaB p65 activity in glomerular mesangial cells. METHODS: 10 female Wistar rats with 6-8 weeks of age were divided randomly into two groups, 5 rats for each group. The rats of one group were given through intragastric feeding with 2 mL Fx1A antigen to induce oral immune tolerance, and the rats of the other group only with PBS (as control group). Two weeks later, the rats were sacrificed, and then their spleens were excised and lymphocytes were separated routinely for antigen-specific lymphocytes reaction. The culture supernatants of splenic lymphocytes from oral immune tolerance rats were prepared. The IL-10 level in the supernatants was detected by sandwich ELISA. After the cultured mesangial cells were stimulated with high dose of insulin, the culture supernatants of rat's splenic lymphocytes were added to insulin-treated mesangial cells. Twenty-four hours later, the NF-kappaB p65 activity in mesangial cells was detected by immunohistochemical staining and sandwich ELISA. RESULTS: As compared with control group, the proliferation reaction of splenic lymphocytes from oral immune tolerance rats was inhibited obviously, and IL-10 level in splenic lymphocyte culture supernatants from the tolerance rats rose markedly (P<0.01). NF-kappaB p65 activity induced by insulin in mesangial cells was inhibited by culture superatants of splenic lymphocytes from oral immune tolerance rats (P<0.05). CONCLUSION: NF-kappaB p65 activity in mesangial cells may be inhibited by IL-10 secreted by splenic lymphocytes from the oral immune tolerance rats.

[Change of activity of NF-kappaB p65 in lymphocytes from rats with oral tolerance induced by Fx1A].

AIM: To explore the activity change and significance of NF-kappaB p65 in lymphocytes from rats with oral tolerance induced by Fx1A. METHODS: 30 female Wistar rats were divided into two groups (oral tolerance and control groups), which stomachs were perfused with Fx1A and PBS, respectively. The delayed-type hypersensitivity (DTH) was evaluated by skin test, the immune function was detected by splenic lymphocyte proliferation test, the activity of NF-kappaB p65 and expression of TGF-beta1 in lymphoid tissues were examined by immunohistochemical staining and Sandwich ELISA. RESULTS: In comparison with control group, the DTH and antigen-specific lymphocyte proliferation reaction in oral tolerance group were suppressed significantly. The activity of NF-kappaB p65 and expression of TGF-beta1 in Peyer's patch were increased notably. However, the activity of NF-kappaB p65 was decreased, but expression of TGF-beta1 was increased markedly in splenic lymphocytes. CONCLUSION: Information of oral tolerance induced by Fx1A may be related to the change of NF-kappaB p65 activity in lymphocytes of body's different places.

A secreted soluble form of ApoE receptor 2 acts as a dominant-negative receptor and inhibits Reelin signaling.

Specialized neurons throughout the developing central nervous system secrete Reelin, which binds to ApoE receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR), triggering a signal cascade that guides neurons to their correct position. Binding of Reelin to ApoER2 and VLDLR induces phosphorylation of Dab1, which binds to the intracellular domains of both receptors. Due to differential splicing, several isoforms of ApoER2 differing in their ligand-binding and intracellular domains exist. One isoform harbors four binding repeats plus an adjacent short 13 amino acid insertion containing a furin cleavage site. It is not known whether furin processing of this ApoER2 variant actually takes place and, if so, whether the produced fragment is secreted. Here we demonstrate that cleavage of this ApoER2 variant does indeed take place, and that the resulting receptor fragment consisting of the entire ligand-binding domain is secreted as soluble polypeptide. This receptor fragment inhibits Reelin signaling in primary neurons, indicating that it can act in a dominant-negative fashion in the regulation of Reelin signaling during embryonic brain development.

Suppression of experimental membranous glomerulonephritis in rats by an anti-MHC class II antibody.

BACKGROUND: We previously reported that idiopathic membranous nephropathy (IMN) strongly correlated with HLA-DRB1*1501-DRB5*0101-DQAI*0102-DQB1* 0602, a specific haplotype of human major histocompatibility complex (MHC), in Japanese patients. To investigate the role of MHC in the development of rat Heymann nephritis (HN), an animal model of membranous nephropathy, a monoclonal antibody (mAb) specific to rat MHC class II antigen (RT1B) was administered, and its effectiveness in inhibiting HN was assessed. METHODS: Active HN was induced in HN-sensitive Lewis rats by administering brush border proteins of rat proximal uriniferous tubules (FX1A). Rats were divided into four groups: rats treated with 1,000 micorg anti-rat MHC class II mAb, rats treated with 100 microg anti-rat MHC class II mAb, rats treated with murine myeloma IgG, and rats that did not receive either FX1A or any other mAb. We examined the differences in 24-hour urinary protein excretion and serum alloantibody titers against FX1A between groups at different time intervals, and the histologic features of kidneys at the end of the study. RESULTS: HN was induced in Lewis rats by inoculation with FX1A antigen. Administration of anti-MHC class II mAb successfully lowered urinary proteins, production of anti-FX1A alloantibodies, and the development of glomerular lesions in a dose-dependent manner. CONCLUSION: The present results demonstrated that the MHC class II molecule itself is directly involved in the pathogenesis of HN, and suggest that this therapy would be any better (or less toxic) than nonselective immunosuppressants in the treatment of IMN.

In vitro and in vivo expression of a nephritogenic Ig heavy chain determinant: pathogenic autoreactivity requires permissive light chains.

Lymphocyte antigen receptors are promising targets for immune intervention strategies in disorders marked by repertoire skewing or expansion of lymphocyte subsets. Appropriate application of immune receptor modulation is predicated on understanding the role of a particular receptor in pathogenesis and disease regulation. The VHB/W16 gene, restricted to mice carrying the j haplotype for the J558 family, is overexpressed by murine lupus anti-DNA Ig. This gene is also expressed recurrently among nephritogenic anti-DNA Ig recovered from several autoimmune strains, suggesting that cells expressing this pathogenic receptor are positively selected during disease progression. To explore the extent and mechanisms by which Ig H chains expressing this gene contribute to autoimmunity, an Ig H chain gene was engineered for in vitro and in vivo recombination studies. Site-directed mutagenesis generated unique restriction sites to link PCR-amplified V region (VDJ) cDNA to previously isolated genomic fragments containing Ig regulatory and signal sequences. The new 3 kb VDJ gene was then ligated to a 9 kb fragment encoding the IgM constant region. Transfection of H chain loss variant myeloma with the complete 12 kb construct, termed 238H-Cmicro, resulted in secretion of intact Ig pairing 238H-Cmicro, with a lambda L chain; however, transfectant Ig lacked autoreactivity and pathogenicity. Introduction of the 238H-Cmicro H chain as a transgene onto the non-autoimmune C57BL/6 background resulted in abundant B cell surface expression of 238H-Cmicro, however, four transgenic Ig recovered by fusion of LPS-stimulated splenocytes and formed by combination of 238H-Cmicro, with endogenous kappa chains do not bind DNA or laminin. These results indicate that the antigen binding sites encoded by this disease-associated gene and/or H chain must associate with permissive L chains to specify autoimmunity. The 238H-Cmicro, transgenic model should prove useful in dissecting the in vivo fate of 238H-Cmicro, L combinations that produce pathogenic autoreactive receptors and in evaluating receptor-targeted interventions.

Functional characterization of rat gp600/megalin promoter: combination of proximal Sp1 site and JCV repeat is important in rat gp600/megalin promoter activation.

Gp600/megalin is an endocytic receptor belonging to the low-density lipoprotein receptor family. Up or down regulation of this protein were observed in certain disease states. To understand the mechanisms that control gp600/megalin gene expression, we cloned and functionally characterized a 738-bp fragment of the 5'-flanking region of rat gp600/megalin gene. A transcription start site was mapped to 33 bp downstream of TAGAAA sequence (TATA-like box). Multiple transcription factor binding sites were identified. Serial 5' deletions and transient transfection assays showed that the deletion fragment containing the Sp1 site proximal to the TATA-like box and a JCV repeat retained 80% of the promoter activity. Individual mutations of the proximal Sp1 site and JCV repeat reduced the promoter activity by 60 and 34% respectively. Double mutations of the proximal Sp1 site and JCV repeat produced a dramatic 80% reduction in the promoter activity. However, deletions and mutations or double mutations of other transcription factor binding sites in the promoter region had a minor effect on the promoter activity. These results indicate that the combination of proximal Sp1 site and the JCV repeat are necessary for activation of gp600/megalin expression. Moreover, Sp1 and Sp3 proteins interacted with the proximal and the distal Sp1 sites in the nuclear extracts of gp600/megalin expressing cell lines. TCF site seems to be involved in negative regulation of this promoter but no nuclear protein(s) were found to bind to this site. In addition, Ap2 site responsible for 28% promoter activity is able to bind two dominant unknown nuclear proteins. This functional characterization of the regulation of gp600/megalin gene is likely to advance the knowledge of the regulation of this gene in health and disease.

A two-receptor pathway for catabolism of Clara cell secretory protein in the kidney.

Clara cell secretory protein (CCSP) is a transport protein for lipophilic substances in bronchio-alveolar fluid, plasma, and uterine secretion. It acts as a carrier for steroid hormones and polychlorinated biphenyl metabolites. Previously, the existence of receptors for uptake of CCSP.ligand complexes into the renal proximal tubules had been suggested. Using surface plasmon resonance analysis, we demonstrate that CCSP binds to cubilin, a peripheral membrane protein on the surface of proximal tubular cells. Binding to cubilin results in uptake and lysosomal degradation of CCSP in cultured cells. Surprisingly, internalization of CCSP is blocked not only by cubilin antagonists but also by antibodies directed against megalin, an endocytic receptor that does not bind CCSP but associates with cubilin. Consistent with a role of both receptors in renal uptake of CCSP in vivo, patients deficient for cubilin or mice lacking megalin exhibit a defect in tubular uptake of the protein and excrete CCSP into the urine. These findings identify a cellular pathway consisting of a CCSP-binding protein (cubilin) and an endocytic coreceptor (megalin) responsible for tissue-specific uptake of CCSP and associated ligands.

Internalization of transthyretin. Evidence of a novel yet unidentified receptor-associated protein (RAP)-sensitive receptor.

Transthyretin (TTR) is a plasma carrier of thyroxine and retinol-binding protein (RBP). Though the liver is the major site of TTR degradation, its cellular uptake is poorly understood. We explored TTR uptake using hepatomas and primary hepatocytes and showed internalization by a specific receptor. RBP complexed with TTR led to a 70% decrease of TTR internalization, whereas TTR bound to thyroxine led to a 20% increase. Different TTR mutants showed differences in uptake, suggesting receptor recognition dependent on the structure of TTR. Cross-linking studies using hepatomas and (125)I-TTR revealed a approximately 90-kDa complex corresponding to (125)I-TTR bound to its receptor. Given previous evidence that a fraction of TTR is associated with high-density lipoproteins (HDL) and that in the kidney, megalin, a member of the low-density lipoprotein receptor family (LDLr) internalizes TTR, we hypothesized that TTR and lipoproteins could share related degradation pathways. Using lipid-deficient serum in uptake assays, no significant changes were observed showing that TTR uptake is not lipoprotein-dependent or due to TTR-lipoprotein complexes. However, competition studies showed that lipoproteins inhibit TTR internalization. The scavenger receptor SR-BI, a HDL receptor, and known LDLr family hepatic receptors did not mediate TTR uptake as assessed using different cellular systems. Interestingly, the receptor-associated protein (RAP), a ligand for all members of the LDLr, was able to inhibit TTR internalization. Moreover, the approximately 90-kDa TTR-receptor complex obtained by cross-linking was sensitive to the presence of RAP. To confirm that RAP sensitivity observed in hepatomas did not represent a mechanism absent in normal cells, primary hepatocytes were tested, and similar results were obtained. The RAP-sensitive TTR internalization together with displacement of TTR uptake by lipoproteins, further suggests that a common pathway might exist between TTR and lipoprotein metabolism and that an as yet unidentified RAP-sensitive receptor mediates TTR uptake.

Structural, biochemical and signaling properties of the low-density lipoprotein receptor gene family.

The low-density lipoprotein (LDL) receptor (LDL-R) family members (LDL-R, LRP, megalin, VLDL-R, apoER2) bind several extra-cellular structurally dissimilar ligands and internalize them for degradation by lysosomes by a process called receptor-mediated endocytosis. The receptor-mediated endocytosis involves immobilization of circulating ligands onto the cell-surface followed by their internalization and degradation. All the receptors can perform both of these functions. However, in the majority of the cases, other proteins immobilize ligands on to the cell-surface and subsequent internalization is mediated by these receptors. The LDL-R and LRP play important roles in plasma cholesterol homeostasis and fetal development. Megalin is an antigenic determinant for Heymann nephritis in rats and may be important for re-absorption of various molecules by the kidney. VLDL-R homologue in chicken is essential for female fertility. This receptor and apoER2 are critical for the proper development of the brain in mice. The members of the LDL-R gene family contain several complement-type and EGF precursor-like repeats, and single transmembrane and cytoplasmic domain. Cysteine-rich complement-type repeats containing DxSDE sequences at the C-termini constitute ligand-binding domains. In contrast to the ligand binding domains, receptor-binding domains in different ligands do not share sequence homology. It has been proposed that positive electrostatic surface potentials, not the primary sequences, in different ligands constitute receptor-binding domains. The EGF precursor homology repeats in receptors are important for the dissociation of ligands from receptors in endocytic vesicles. The transmembrane domain is necessary for anchoring to membranes and the cytoplasmic domain is required for their targeting to coated pits and subsequent internalization. The receptor-mediated endocytosis involves recognition of the NPXY motif by clathrin. Recently, this motif has also been implicated in signaling pathways that are crucial in brain development. The signaling process involves the recognition of the NPXY motif by Disabled-1 protein and possibly other proteins involved in intracellular signaling cascade. The LDL-R gene family has provided important insights into the mechanisms of ligand catabolism and may serve as new targets for the treatment of different cardiovascular and neuronal disorders. In the future, their role in signaling may provide novel insights into brain development and neuronal layering.

An endocytosis defect as a possible cause of proteinuria in polycystic kidney disease.

Because proteinuria has been demonstrated in patients with autosomal-dominant polycystic kidney disease (ADPKD), we have investigated whether proteinuria also occurs in the (cy/+) rat, a widely used model for ADPKD. Increased urinary excretion of proteins, in particular of albumin, can be found in 16-wk-old (cy/+) rats, with a gel electrophoresis pattern compatible with a tubular origin of proteinuria. Using FITC-labeled dextran as an in vivo tracer for renal tubular endosomal function, we could show that portions of cyst-lining epithelia from proximal tubules have lost the ability to endocytose, which is necessary for the reabsorption of low-molecular-weight proteins. By immunohistochemistry, the expression of other proteins implicated in endocytosis, such as the chloride channel ClC-5 and the albumin receptor megalin, correlated well with the presence and absence of FITC-dextran in cysts. As an example of growth factor systems possibly being affected by this endocytosis defect, we could detect increased urinary levels of insulin-like growth factor-I protein in (cy/+) animals. These data indicate that proteinuria and albuminuria in the aforementioned rat model for ADPKD are due to a loss of the endocytic machinery in epithelia of proximal tubular cysts. This may also affect the concentration of different growth factors and hormones in cyst fluids and thus modulate cyst development.

Evidence of macrophage foam cell formation by very low-density lipoprotein receptor: interferon-gamma inhibition of very low-density lipoprotein receptor expression and foam cell formation in macrophages.

BACKGROUND: Expression of the VLDL receptor, primarily in macrophages, has been confirmed in human and rabbit atherosclerotic lesions. The high binding affinity of the VLDL receptor for remnant particles implicates the VLDL receptor pathway in the foam cell formation mechanism in macrophages. This study investigates the effect of interferon (IFN)-gamma on VLDL receptor expression in phorbol-12-myristate-13-acetate (PMA)-treated THP-1, HL-60 macrophages, and human monocyte-derived macrophages. METHODS AND RESULTS: THP-1 cells were induced to differentiate into macrophages by PMA treatment. IFN-gamma was added to the medium, and expression of the VLDL receptor was determined. (125)I-beta-VLDL degradation study and oil red O staining were examined. In THP-1 macrophages, VLDL receptor protein expression decreased at 2 days after PMA treatment but increased at 3 days and increased up to 5 days. Scavenger receptor proteins, which were not originally present, appeared at 3 days after PMA treatment. IFN-gamma inhibited VLDL receptor expression in a dose-and time-dependent manner in macrophages. However, no inhibitory effect was observed in monocytes. Moreover, IFN-gamma receptor mRNA increased during differentiation to macrophages. (125)I-beta-VLDL degradation study and oil red O staining showed that IFN-gamma significantly inhibited foam cell formation after the uptake of beta-VLDL. LDL receptor-related protein (LRP) and LDL receptor mRNAs were not expressed in macrophages. In PMA-treated HL-60 macrophages and human monocyte-derived macrophages, IFN-gamma also inhibited VLDL receptor expression and foam cell formation by beta-VLDL. CONCLUSIONS: VLDL receptor expression is upregulated during monocyte-macrophage differentiation. IFN-gamma inhibits VLDL receptor expression and foam cell formation only in macrophages. Remnant particles induce macrophage foam cell formation through the VLDL receptor pathway.

Mechanisms of albumin uptake by proximal tubular cells.

The likely role of albumin in the induction tubulo-interstitial injury in proteinuria has stimulated considerable interest in the entry of albumin into the proximal tubule and its subsequent uptake by proximal tubular cells. Currently, there is considerable controversy over the degree of glomerular permeability to albumin. After filtration, however, albumin binds to megalin and cubulin, two giant receptors in the apical membrane of proximal tubular cells. Albumin is subsequently re-absorbed by proximal tubular cells by receptor-mediated endocytosis, a process subject to complex regulation. The interaction of albumin with proximal tubule cells also leads to the generation of intracellular signals. The understanding of these pathways may provide important insights into the pathogenesis of renal scarring in proteinuria.

Transcytosis of lipoprotein lipase across cultured endothelial cells requires both heparan sulfate proteoglycans and the very low density lipoprotein receptor.

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of circulating lipoprotein triglyceride molecules, is synthesized in myocytes and adipocytes but functions while bound to heparan sulfate proteoglycans (HSPGs) on the luminal surface of vascular endothelial cells. This requires transfer of LPL from the abluminal side to the luminal side of endothelial cells. Studies were performed to investigate the mechanisms of LPL transcytosis using cultured monolayers of bovine aortic endothelial cells. We tested whether HSPGs and members of the low density lipoprotein (LDL) receptor superfamily were involved in transfer of LPL from the basolateral to the apical side of cultured endothelial cells. Heparinase/heparinitase treatment of the basolateral cell surface or addition of heparin to the basolateral medium decreased the movement of LPL. This suggested a requirement for HSPGs. To assess the role of receptors, we used either receptor-associated protein, the 39-kDa inhibitor of ligand binding to the LDL receptor-related protein and the very low density lipoprotein (VLDL) receptor, or specific receptor antibodies. Receptor-associated protein reduced (125)I-LPL and LPL activity transfer across the monolayers. When the basolateral surface of the cells was treated with antibodies, only anti-VLDL receptor antibodies inhibited transcytosis. Moreover, overexpression of the VLDL receptor using adenoviral-mediated gene transfer increased LPL transcytosis. Thus, movement of active LPL across endothelial cells involves both HSPGs and VLDL receptor.

Neuronal apoptosis by apolipoprotein E4 through low-density lipoprotein receptor-related protein and heterotrimeric GTPases.

The epsilon4 genotype of apolipoprotein E (apoE4) is the most established predisposing factor in Alzheimer's disease (AD); however, it remains unclear how apoE4 contributes to the pathophysiology. Here, we report that the apoE4 protein (ApoE4) evokes apoptosis in neuronal cells through the low-density lipoprotein receptor-related protein (LRP) and heterotrimeric GTPases. We examined neuron/neuroblastoma hybrid F11 cells and found that these cells were killed by 30 microg/ml ApoE4, but not by 30 microg/ml ApoE3. ApoE4-induced death occurred with typical features for apoptosis in time- and dose-dependent manners, and was observed in SH-SY5Y neuroblastomas, but not in glioblastomas or non-neuronal Chinese hamster ovary cells. Activated, but not native, alpha2-macroglobulin suppressed this ApoE4 toxicity. Suppression by the antisense oligonucleotide to LRP and inhibition by low nanomolar concentrations of LRP-associated protein RAP provided evidence for the involvement of LRP. The involvement of heterotrimeric GTPases was demonstrated by the findings that (1) ApoE4-induced death was suppressed by pertussis toxin (PTX), but not by heat-inactivated PTX; and (2) transfection with PTX-resistant mutant cDNAs of Galpha(i) restored the toxicity of ApoE4 restricted by PTX. We thus conclude that one of the neurotoxic mechanisms triggered by ApoE4 is to activate a cell type-specific apoptogenic program involving LRP and the G(i) class of GTPases and that the apoE4 gene may play a direct role in the pathogenesis of AD and other forms of dementia.

The receptor-associated protein (RAP) interacts with several resident proteins of the endoplasmic reticulum including a glycoprotein related to actin.

The receptor-associated protein (RAP) is a chaperone found primarily in the endoplasmic reticulum (ER) that plays a necessary role in the folding and exocytic trafficking of members of the LDL receptor gene family including megalin and the LDL receptor-related protein (LRP). Recently, RAP has been shown to interact with a growing number of proteins including several that are unrelated to the LDL receptor family as well as new members of this rapidly expanding family. Based on these observations, we have applied chemical crosslinking procedures to identify additional novel RAP-interacting proteins, and thereby better characterize the scope of RAP's ER-related function. In this study, we have identified eight proteins with molecular weights of 32, 35, 46, 55, 70, 95, 170, and 200 kDa that interact with endogenous RAP. These proteins were found to associate with RAP in multiple cell types from different species, suggesting that their expression and interactions with RAP are ubiquitous. Results of pulse-chase experiments show that most of the proteins remain sensitive to endoglycosidase-H digestion, and also remain stably associated with RAP over an extended period, suggesting that they are ER resident proteins. All of the RAP-associated proteins appear to be largely soluble as they partition into the aqueous phase following TX-114 detergent extraction. Sequence analysis and immunoblotting of the 46-kDa RAP-associated glycoprotein (gp46) shows that it is structurally and immunologically related to actin. If gp46 is also functionally related to actin as an intracellular structural protein, it may represent a novel component of the putative ER matrix.