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1.
The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their complex foot process network. While deficiency of the polarity proteins Crumbs and aPKC result in impaired podocyte foot process architecture, the function of basolateral polarity proteins for podocyte differentiation and maintenance remained unclear. Here we report, that Scribble is expressed in developing podocytes, where it translocates from the lateral aspects of immature podocytes to the basal cell membrane and foot processes of mature podocytes. Immunogold electron microscopy reveals membrane associated localisation of Scribble predominantly at the basolateral site of foot processes. To further study the role of Scribble for podocyte differentiation Scribble(flox/flox) mice were generated by introducing loxP-sites into the Scribble introns 1 and 8 and these mice were crossed to NPHS2.Cre mice and Cre deleter mice. Podocyte-specific Scribble knockout mice develop normally and display no histological, ultrastructural or clinical abnormalities up to 12 months of age. In addition, no increased susceptibility to glomerular stress could be detected in these mice. In contrast, constitutive Scribble knockout animals die during embryonic development indicating the fundamental importance of Scribble for embryogenesis. Like in podocyte-specific Scribble knockout mice, the development of podocyte foot processes and the slit diaphragm was unaffected in kidney cultures from constitutive Scribble knockout animals. In summary these results indicate that basolateral polarity signaling via Scribble is dispensable for podocyte function, highlighting the unique feature of podocyte development with its significant apical membrane expansions being dominated by apical polarity complexes rather than by basolateral polarity signaling.  相似文献   

2.
Podocytes in glomerulus of rat kidney express a characteristic 44 KD protein   总被引:12,自引:0,他引:12  
We describe a new monoclonal antibody (MAb) directed against glomerular visceral epithelial cells (podocytes), generated by immunization with isolated rat kidney glomeruli. In immunoblotting experiments this MAb (IgG1 subclass) reacted with a 44 KD protein. In cryostat sections of normal rat kidney the MAb stained glomerular podocytes; therefore, we called the antigen pp44 (podocyte protein 44 KD). On 0.5-micron cryostat sections the signal could be more precisely ascribed to the podocyte foot processes, whereas the cell bodies appeared virtually unreactive. On ultra-thin frozen sections pp44 was found within the cytoplasm of podocyte foot processes at their origin from their parent processes. The podocyte cell membrane was not labeled. All other parts of the nephron were unreactive. An additional but weaker immunoreaction was found in the arterial endothelium; the endothelia of other vessels (peritubular capillaries, veins) were negative. In human kidney anti-pp44 revealed the same staining pattern as in rat kidney. The expression of pp44 was also studied in newborn rat kidney. The early stages of glomerular development (renal vesicle, S-shaped body) were negative. pp44 first appeared during the capillary loop stage, i.e., when formation of podocyte foot processes commences. In comparing the present results with published data, pp44 is clearly different from other antigens thus far described in podocytes. From the results of this investigation we conclude that pp44 represents a novel cytoplasmic protein of podocytes. Our data suggest a cytoskeletal role for pp44 in preserving the complex architecture of podocytes. This idea is confirmed by the simultaneous appearance of foot processes and anti-pp44 immunoreactivity during glomerular development.  相似文献   

3.
Chen HC  Chen CA  Guh JY  Chang JM  Shin SJ  Lai YH 《Life sciences》2000,67(19):2345-2353
The adhesion molecule integrin alpha3beta1 is the major receptor of podocyte to the glomerular capillary basement membrane (GBM). Since progressive alteration of the glomerular extracellular matrix (ECM) compartment leading to GBM thickening is common in diabetic nephropathy, we investigated the cellular distribution of alpha3beta1 integrin in podocytes of patients with diabetic nephropathy and streptozotocin-induced diabetic rats, and we evaluated the effects of high glucose on the cultured rat podocytes. Both human and rat kidneys were stained using the immunoelectron microscopy and immunoperoxidase technique with mouse monoclonal antibodies to human integrin alpha3 subunit. The results showed that both the number of immunogold particles and the staining of integrin alpha3 subunit on podocytes were weaker in patients with diabetic nephropathy than those of control kidneys. The staining of alpha3 on podocytes in the poorly-controlled diabetic rats was also weaker after one and three months of hyperglycemia. However, the staining was identical to controls in rats with only one week of hyperglycemia. High glucose (25 mM) but not streptozotocin in vitro suppressed the alpha3 expression of cultured rat podocytes. Our results demonstrated that the expression of integrin alpha3beta1 on podocytes was suppressed in both human and rats with diabetes, possibly due to the effects of hyperglycemia, and the suppression became more severe with the duration of diabetes.  相似文献   

4.
Nephrin is a transmembrane molecule essential for morphology and function of kidney podocytes. We and others reported previously that the cytoplasmic domain of human and mouse nephrin interacts with the adaptor protein, Nck, in a tyrosine phosphorylation-dependent manner. In the current study, we characterized the interaction of rat nephrin with Nck and further addressed its impact on cell morphology. Rat nephrin expressed in Cos-1 cells co-immunoprecipitated with Nck in a manner dependent on the phosphorylation of Y1204 and Y1228. Nephrin from normal rat glomeruli was also tyrosine phosphorylated and associated with Nck. Overexpression of rat nephrin in HEK293T cells induced morphological changes resembling process formation, which became more distinct when the extracellular domain of nephrin was cross-linked by antibodies. The morphological changes were attenuated by expression of dominant negative constructs of Nck. In the rat model of podocyte injury and proteinuria, nephrin tyrosine phosphorylation and nephrin-Nck interaction were both reduced significantly. Taken together, we propose that Nck couples nephrin to the actin cytoskeleton in glomerular podocytes and contributes to the maintenance of normal morphology and function of podocytes.  相似文献   

5.
6.
The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm’s tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.  相似文献   

7.

Background

α-Dystroglycan is a negatively charged glycoprotein that covers the apical and basolateral membrane of the podocyte. Its transmembrane binding to the cytoskeleton is regulated via tyrosine phosphorylation (pY892) of β-dystroglycan. At the basolateral side α-dystroglycan binds the glomerular basement membrane. At the apical membrane, it plays a role in the maintenance of the filtration slit. In this study, we evaluated whether ligation of α-dystroglycan with specific antibodies or natural ligands induces intracellular signaling, and whether there is an effect on podocyte architecture.

Methodology/Principal Findings

Conditionally immortalized podocytes were exposed in vitro to antibodies to α-dystroglycan, and to fibronectin, biglycan, laminin and agrin. Intracellular calcium fluxes, phosphorylation of β-dystroglycan and podocyte architecture were studied. Antibodies to α-dystroglycan could specifically induce calcium signaling. Fibronectin also induced calcium signaling, and led to dephosphorylation of pY892 in β-dystroglycan. Ligation of α-dystroglycan resulted in an altered actin architecture, a decreased number of podocyte pedicles and a more flattened appearance of the podocyte.

Conclusions/Significance

We conclude that ligation of α-dystroglycan on podocytes induces intracellular calcium signaling, which leads to an altered cytoskeleton architecture akin to the situation of foot process effacement. In particular the ability of fibronectin to induce intracellular signaling events is of interest, since the expression and excretion of this protein is upregulated in several proteinuric diseases. Therefore, fibronectin-induced signaling via dystroglycan may be a novel mechanism for foot process effacement in proteinuric diseases.  相似文献   

8.
The appearance and distribution of podocalyxin on the glomerular epithelium (podocytes) during glomerular development was determined in the newborn rat kidney using specific monoclonal and affinity-purified polyclonal antibodies. Kidneys from 2-day-old rats were perfusion-fixed and processed for immunofluorescence or immunoperoxidase localization or immunogold labeling on ultrathin frozen sections. Podocalyxin first appeared on the apical surfaces of the presumptive podocytes of the S-shaped body above the level of the junctional complexes that connect the cells at this stage. The latter consist of a shallow occluding zonule and a deeper adhering zonule. Early in the capillary loop stage, when the urinary spaces open and the junctional complexes migrate from the apex to the base of the cells, labeling for podocalyxin extended along the lateral plasmalemma above the migrating junctions. In the maturing glomerulus when the foot processes form and the occluding and adhering junctions give way to developing slit diaphragms, podocalyxin was found along all newly-opened surfaces above the occluding junctions or slit membranes. No labeling was found below the latter. Podocalyxin was also detected intracellularly throughout the entire exocytotic pathway--i.e., in the rough endoplasmic reticulum and perinuclear cisternae, in Golgi cisternae and associated vesicles, and in carrier vesicles presumably en route to the cell surface. It is concluded that 1) podocalyxin is synthesized at a high rate in the differentiating podocyte; 2) its distribution is restricted to the apical plus lateral plasmalemmal domain facing the urinary spaces above the migrating junctions; 3) its time of appearance and distribution during glomerular development are identical to that reported earlier for epithelial polyanion; and 4) its synthesis and insertion into the podocyte plasmalemma is closely coupled to the development of the foot processes and filtration slits.  相似文献   

9.
IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were determined in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (α-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties.  相似文献   

10.
The human LARGE gene encodes a protein with two putative glycosyltransferase domains and is required for the generation of functional alpha-dystroglycan (alpha-DG). Monoclonal antibodies IIH6 and VIA4-1 recognize the functional glycan epitopes of alpha-DG that are necessary for binding to laminin and other ligands. Overexpression of full-length mouse Large generated functionally glycosylated alpha-DG in Pro(-5) Chinese hamster ovary (CHO) cells, and the amount was increased by co-expression of protein:O-mannosyl N-acetylglucosaminyltransferase 1. However, functional alpha-DG represented only a small fraction of the alpha-DG synthesized by CHO cells or expressed from an alpha-DG construct. To identify features of the glycan epitopes induced by Large, the production of functionally glycosylated alpha-DG was investigated in several CHO glycosylation mutants. Mutants with defective transfer of sialic acid (Lec2), galactose (Lec8), or fucose (Lec13) to glycoconjugates, and the Lec15 mutant that cannot synthesize O-mannose glycans, all produced functionally glycosylated alpha-DG upon overexpression of Large. Laminin binding and the alpha-DG glycan epitopes were enhanced in Lec2 and Lec8 cells. In Lec15 cells, functional alpha-DG was increased by co-expression of core 2 N-acetylglucosaminyltransferase 1 with Large. Treatment with N-glycanase markedly reduced functionally glycosylated alpha-DG in Lec2 and Lec8 cells. The combined data provide evidence that Large does not transfer to Gal, Fuc, or sialic acid on alpha-DG nor induce the transfer of these sugars to alpha-DG. In addition, the data suggest that human LARGE may restore functional alpha-DG to muscle cells from patients with defective synthesis of O-mannose glycans via the modification of N-glycans and/or mucin O-glycans on alpha-DG.  相似文献   

11.
Podocytes are specialized cells of the kidney that form the blood filtration barrier in the kidney glomerulus. The barrier function of podocytes depends upon the development of specialized cell-cell adhesion complexes called slit-diaphragms that form between podocyte foot processes surrounding glomerular blood vessels. Failure of the slit-diaphragm to form results in leakage of high molecular weight proteins into the blood filtrate and urine, a condition called proteinuria. In this work, we test whether the zebrafish pronephros can be used as an assay system for the development of glomerular function with the goal of identifying novel components of the slit-diaphragm. We first characterized the function of the zebrafish homolog of Nephrin, the disease gene associated with the congenital nephritic syndrome of the Finnish type, and Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome. Zebrafish nephrin and podocin were specifically expressed in pronephric podocytes and required for the development of pronephric podocyte cell structure. Ultrastructurally, disruption of nephrin or podocin expression resulted in a loss of slit-diaphragms at 72 and 96 h post-fertilization and failure to form normal podocyte foot processes. We also find that expression of the band 4.1/FERM domain gene mosaic eyes in podocytes is required for proper formation of slit-diaphragm cell-cell junctions. A functional assay of glomerular filtration barrier revealed that absence of normal nephrin, podocin or mosaic eyes expression results in loss of glomerular filtration discrimination and aberrant passage of high molecular weight substances into the glomerular filtrate.  相似文献   

12.
B1 and B2 are two highly homologous isoforms of the vacuolar H+-ATPase (V-ATPase) 56-kDa B subunit. We investigated whether the B2 subunit is expressed alongside B1 in proton-secreting cells of the rodent kidney collecting duct (intercalated cells, IC) and epididymis (clear cells) by using antibodies against distinct COOH-terminal peptides from the two B isoforms. B2 was detected not only in the kidney proximal tubule, thick ascending limb, distal convoluted tubule, and connecting segment but also in A- and B-type IC of collecting ducts (CD) in both rat and mouse. B2 had a predominant cytoplasmic localization in most IC but was clearly located in a tighter apical band together with the V-ATPase 31-kDa E subunit in some A-IC, especially in the medulla. Apical membrane staining was confirmed by immunogold electron microscopy. B2 was very weakly expressed on the basolateral membranes of B-IC in control kidney CD, but some connecting segment B-IC had more distinct basolateral staining. In response to chronic carbonic anhydrase inhibition by acetazolamide, many A-IC showed a strong apical membrane localization of B2, where it colocalized with E and B1. In rat and mouse epididymis, B2 isoform expression was detected in clear cells, where it was concentrated in subapical vesicles. Unlike B1, B2 did not colocalize with the E subunit in the apical microvilli. These findings indicate that in addition to its role in the acidification of intracellular organelles, the B2 isoform could also contribute to transepithelial proton secretion and the maintenance of acid-base homeostasis. vacuolar H+-ATPase B subunit; intercalated cells; clear cells; urogenital tract; immunofluorescence  相似文献   

13.
Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprin beta in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprin beta in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprin beta expression. The glomerular meprin beta expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprin beta staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprin beta is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprin beta in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprin beta in this form of glomerulonephritis.  相似文献   

14.

Background

Podocytes are highly specialized epithelial cells on the visceral side of the glomerulus. Their interdigitating primary and secondary foot processes contain an actin based contractile apparatus that can adjust to changes in the glomerular perfusion pressure. Thus, the dynamic regulation of actin bundles in the foot processes is critical for maintenance of a well functioning glomerular filtration barrier. Since the actin binding protein, cofilin-1, plays a significant role in the regulation of actin dynamics, we examined its role in podocytes to determine the impact of cofilin-1 dysfunction on glomerular filtration.

Methods and Findings

We evaluated zebrafish pronephros function by dextran clearance and structure by TEM in cofilin-1 morphant and mutant zebrafish and we found that cofilin-1 deficiency led to foot process effacement and proteinuria. In vitro studies in murine and human podocytes revealed that PMA stimulation induced activation of cofilin-1, whereas treatment with TGF-β resulted in cofilin-1 inactivation. Silencing of cofilin-1 led to an accumulation of F-actin fibers and significantly decreased podocyte migration ability. When we analyzed normal and diseased murine and human glomerular tissues to determine cofilin-1 localization and activity in podocytes, we found that in normal kidney tissues unphosphorylated, active cofilin-1 was distributed throughout the cell. However, in glomerular diseases that affect podocytes, cofilin-1 was inactivated by phosphorylation and observed in the nucleus.

Conclusions

Based on these in vitro and in vivo studies we concluded cofilin-1 is an essential regulator for actin filament recycling that is required for the dynamic nature of podocyte foot processes. Therefore, we describe a novel pathomechanism of proteinuria development.  相似文献   

15.
The kidney filtration barrier consists of the capillary endothelium, the glomerular basement membrane and the slit diaphragm localized between foot processes of neighbouring podocytes. We report that collagen XVII, a transmembrane molecule known to be required for epithelial adhesion, is expressed in podocytes of normal human and mouse kidneys and in endothelial cells of the glomerular filtration barrier. Immunoelectron microscopy has revealed that collagen XVII is localized in foot processes of podocytes and in the glomerular basement membrane. Its role in kidney has been analysed in knockout mice, which survive to birth but have high neonatal mortality and skin blistering and structural abnormalities in their glomeruli. Morphometric analysis has shown increases in glomerular volume fraction and surface densities of knockout kidneys, indicating an increased glomerular amount in the cortex. Collagen XVII deficiency causes effacement of podocyte foot processes; however, major slit diaphragm disruptions have not been detected. The glomerular basement membrane is split in areas in which glomerular and endothelial basement membranes meet. Differences in the expression of collagen IV, integrins α3 or β1, laminin α5 and nephrin have not been observed in mutant mice compared with controls. We propose that collagen XVII has a function in the attachment of podocyte foot processes to the glomerular basement membrane. It probably contributes to podocyte maturation and might have a role in glomerular filtration.  相似文献   

16.
S-adenosylhomocysteine (SAH) hydrolase is a cytosolic enzyme present in the kidney. Enzyme activities of SAH hydrolase were measured in the kidney in isolated glomeruli and tubules. SAH hydrolase activity was 0.62 +/- 0.02 mU/mg in the kidney, 0.32 +/- 0.03 mU/mg in the glomeruli, and 0.50 +/- 0.02 mU/mg in isolated tubules. Using immunohistochemical methods, we describe the localization of the enzyme SAH hydrolase in rat kidney with a highly specific antibody raised in rabbits against purified SAH hydrolase from bovine kidney. This antibody crossreacts to almost the same extent with the SAH hydrolase from different species such as rat, pig, and human. Using light microscopy, SAH hydrolase was visualized by the biotin-streptavidin-alkaline phosphatase immunohistochemical procedure. SAH hydrolase immunostaining was observed in glomeruli and in the epithelium of the proximal and distal tubules. The collecting ducts of the cortex and medulla were homogeneously stained. By using double immunofluorescence staining and two-channel immunofluorescence confocal laser scanning microscopy, we differentiated the glomerular cells (endothelium, mesangium, podocytes) and found intensive staining of podocytes. Our results show that the enzyme SAH hydrolase is found ubiquitously in the rat kidney. The prominent staining of SAH hydrolase in the podocytes may reflect high rates of transmethylation. (J Histochem Cytochem 48:211-218, 2000)  相似文献   

17.
In an attempt to understand the mechanism underlying the tissue-dependent function, the expression of NHE-1 protein and its sub cellular localization was examined in the rat GI-tract and other tissues. Rat NHE-1 polyclonal antibodies were raised in rabbits using a NHE-1 fusion protein antigen. The antibodies recognized a 110 kD protein in rats and mice, but not in human or rabbit RBCs. Colon, stomach, brain, spleen and kidney expressed NHE-1 protein abundantly, whereas the skeletal muscle the least abundant. Ouabain-sensitive-K+-stimulated p-nitrophenylphosphatase (PNPPase), the partial activity of the sodium pump and alkaline phosphatase (Apase) were used as the markers of the basolateral and apical membranes. NHE-1 was detected predominantly in the PNPPase enriched membrane fractions, but was also detected in the apical membrane enriched fractions in the kidney cortex, jejunum and colon at a lower level. NHE-1 was detected in the plasma membrane enriched fractions from the skeletal muscle and ventricle. Immunofluorescence data showed a similar localization pattern of NHE-1 in the colon and kidney sections. These findings suggest that NHE-1 is localized both on the apical and basolateral membrane. In view of its similar sub cellular localization in the GI-tract and kidney, but a different level of expression, might suggest that the level of protein, but not the sub cellular distribution is important to regulate its tissue-dependent function.  相似文献   

18.
Uptake of P(i) at the cellular membrane is essential for the maintenance of cell viability. However, phosphate overload is also stressful for cells and can result in cellular damage. In the present study, we investigated the effects of the transgenic overexpression of type III P(i) transporter Pit-1 to explore the role of extracellular P(i) in glomerular sclerosis during chronic renal disease. Pit-1 transgenic (TG) rats showed progressive proteinuria associated with hypoalbuminemia and dyslipidemia. Ultrastructural analysis of TG rat kidney by transmission electron microscopy showed a diffuse effacement of the foot processes of podocytes and a thickening of the glomerular basement membrane, which were progressively exhibited since 8 wk after birth. TG rats died at 32 wk of age due to cachexia. At this time, more thickening of the glomerular basement membrane and segmental sclerosis were observed in glomeruli of the TG rats. Immunohistochemical examination using anti-connexin 43 and anti-desmin antibodies suggested the progressive injury of podocytes in TG rats. TG rats showed higher P(i) uptake in podocytes than wild-type rats, especially under low P(i) concentration. When 8-wk-old wild-type and TG rats were fed a 0.6% normal phosphate (NP) or 1.2% phosphate (HP) diet for 12 wk, HP diet-treated TG rats showed more progressive proteinuria and higher serum creatinine levels than NP diet-treated TG rats. In conclusion, our findings suggest that overexpression of Pit-1 in rats induces phosphate-dependent podocyte injury and damage to the glomerular barrier, which result in the progression of glomerular sclerosis in the kidney.  相似文献   

19.
Copper is essential for human growth and survival. Enterocytes mediate the absorption of dietary copper from the intestinal lumen into blood as well as utilizing copper for their biosynthetic needs. Currently, the pathways for copper entry into enterocytes remain poorly understood. We demonstrate that the basolateral copper uptake into intestinal cells greatly exceeds the apical uptake. The basolateral but not apical transport is mediated by the high affinity copper transporter hCTR1. This unanticipated conclusion is supported by cell surface biotinylation and confocal microscopy of endogenous hCTR1 in Caco2 cells as well as copper influx measurements that show saturable high affinity uptake at the basolateral but not the apical membrane. Basolateral localization of hCTR1 and polarized copper uptake are also conserved in T84 cells, models for intestinal crypt cells. The lateral localization of hCTR1 seen in intestinal cell lines is recapitulated in immunohistochemical staining of mouse intestinal sections. Biochemical and functional assays reveal the basolateral localization of hCTR1 also in renal Madin-Darby canine kidney cells and opossum kidney cells. Overexpression of hCTR1 in Madin-Darby canine kidney cells results in both apical and basolateral delivery of the overexpressed protein and greatly enhanced copper uptake at both cell surfaces. We propose a model of intestinal copper uptake in which basolateral hCTR1 plays a key role in the physiologically important delivery of copper from blood to intracellular proteins, whereas its role in the initial apical uptake of dietary copper is indirect.  相似文献   

20.
A progressive decrease in podocyte number underlies the development of glomerulosclerosis and reduced kidney function in aging nephropathy. Recent data suggest that under certain disease states, parietal epithelial cells (PECs) begin to express proteins considered specific to podocytes. To determine whether this phenomenon increases in aging kidneys, 4-, 12-, and 20-mo ad libitum-fed and 20-mo calorie-restricted (CR) rats were studied. Single and double immunostaining were performed with antibodies to the PEC protein paired box gene 2 (PAX2) and tight junction protein claudin-1, the podocyte-specific protein Wilms' tumor 1 (WT-1), and the proliferating cell protein (Ki-67). ImageJ software measured Bowman's basement membrane (BBM) length and glomerular tuft area in individual glomeruli from each animal to assess glomerular size. The results showed that in aged ad libitum rats, the decrease in number of podocytes/glomerular tuft area was accompanied by an increase in the number of PECs/BBM length at 12 and 20 mo (P < 0.01 vs. 4 mo). The increase in PEC number was due to proliferation (increase in PAX2/Ki-67 double-positive cells). Aging was accompanied by a progressive increase in the number of glomerular cells double staining for PAX2 and WT-1. In contrast, the control 20-mo-old CR rats had no increase in glomerular size, and podocyte and PEC number were not altered. These results suggest that although the number of PECs and PECs expressing podocyte proteins increase in aging nephropathy, they are likely not sufficient to compensate for the decrease in podocyte number.  相似文献   

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