Inhibition of cyclooxygenases reduces complement-induced glomerular epithelial cell injury and proteinuria in passive Heymann nephritis

In the passive Heymann nephritis (PHN) model of rat membranous nephropathy, complement induces glomerular epithelial cell injury and proteinuria, which is partially mediated by eicosanoids. Glomerular cyclooxygenase (COX)-1 and -2 are up-regulated in PHN and contribute to prostanoid generation. In the current study, we address the role of COX isoforms in proteinuria, using the nonselective COX inhibitor indomethacin and the COX-2-selective inhibitor 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone (DFU). Four groups of rats with PHN were treated twice daily, from day 7 through 14 with vehicle, 1 mg/kg DFU, 10 mg/kg DFU, or 2 mg/kg indomethacin. Vehicle-treated rats with PHN showed significant proteinuria on day 14 (163 +/- 15 mg/d, n = 19), compared with normal rats (10 +/- 4 mg/d, n = 3, p < 0.001). Treatment with DFU (1 or 10 mg/kg) reduced proteinuria significantly (by ~33%), compared with vehicle, but to a lesser extent than indomethacin (56% reduction). Glomerular eicosanoid generation was reduced significantly in the DFU and indomethacin groups, compared with vehicle. There were no significant differences among vehicle- or DFU-treated groups in [(3)H]inulin clearance, or in glomerular expression of COX-1 and -2. DFU did not affect the autologous immune response. In cultured rat glomerular epithelial cells, COX inhibition reduced complement-induced cytotoxicity, and this reduction was reversed by the thromboxane A(2) analog 9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin F(2alpha) (U46619). Thus, in experimental membranous nephropathy, selective inhibition of COX-2 reduces proteinuria, without adversely affecting renal function. However, inhibition of both COX-1 and -2 is required to achieve a maximum cytoprotective and antiproteinuric effect.     

A rat hybridoma model of Heymann nephritis: production of a monoclonal anti GP330 from a nephritic rat

Autologous (Heymann) nephritis was induced by immunizing Sprague-Dawley rats with a crude membrane extract (Fx1A) prepared from renal cortical tubules. Urine protein excretion was monitored to determine the onset of nephritis and then the spleens of nephritic rats were fused with a non-secretor rat myeloma cell line. Supernatants from hybridoma cultures were first screened for production of anti-brush border membrane (BBM) antibody by immunodot blotting of highly purified rat BBM on nitrocellulose. Positive hybrids were then tested by indirect immunofluorescence for the presence of IgG, which binds to the brush border of rat renal proximal tubules. Those hybrids which were positive by both screening assays were subcloned twice. Two monoclonal antibodies (C5, D11) were studied in some detail. Both C5 and D11 immunoprecipitated a single polypeptide from BBM, labelled with 125I by the lactoperoxidase method. Radioautography of gradient 4-11% slab sodium dodecyl sulphate polyacrylamide gels revealed that the polypeptide against which C5 and D11 were directed co-migrated with the polypeptide immunoprecipitated by (i) IgG eluted from the renal cortex of nephritic rats, and by (ii) a mouse anti-rat monoclonal against gp 330 [a BBM constituent with proven pathogenicity [9]]. Supernatants which tested positive by immunodot blotting but negative by indirect immunofluorescence showed no detectable immunoprecipitate after reaction with BBM. Immunocytochemical staining by immunoperoxidase and immunogold methods localized C5 and D11 to the BBM of the renal proximal tubule and to the urine face of the glomerular epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Initial events in the formation of immune deposits in passive Heymann nephritis. gp330-anti-gp330 immune complexes form in epithelial coated pits and rapidly become attached to the glomerular basement membrane

The nephritogenic antigen of Heymann's nephritis (HN), gp330, was previously demonstrated (4-9) to be a resident glycoprotein of coated pits in the glomerular and proximal tubule epithelium of rats, and anti-gp330 IgG given intravenously was found to form IDs in glomeruli (passive HN). The purpose of this study was to investigate the detailed events that occur in the formation of IDs in passive HN. HN was induced by the injection of either 125I-labeled or unlabeled anti-gp330 IgG. At various times after injection (15 min to 8 d) the kidneys of some of the injected rats were fixed by perfusion, and the distribution of the rabbit IgG was determined by immunofluorescence and by immunoelectron microscopy. Glomeruli were isolated from the kidneys of injected rats and used for isolation of GBM fractions or for elution of the bound IgG. At 15 min to 1 h after injection, the rabbit IgG was localized by immunocytochemistry exclusively in coated pits along the podocyte plasmalemma facing the GBM. By 1-8 d, anti-gp330 IgG was detected in larger electron-dense IDs often located under the slit diaphragms. Serial sectioning revealed that each of the IDs maintained contact with a coated pit at some level. When GBMs isolated from rats given radiolabeled anti-gp330 IgG were examined by electron microscopy, the IDs were found to remain attached to the GBMs as early as 15 min after injection and coisolated with them at all time points. By double-immunolabeling of the isolated GBMs with two sizes of gold particles, both the antigen (gp330) and the anti-gp330 IgG could be demonstrated in IDs at all time points. When the amount of radiolabeled anti-gp330 bound to GBM fractions was compared with that of isolated glomeruli, it was found that 20% of the radiolabel remained bound to the purified GBMs at 15 min after injection, and 90% at 3 d. The bound IgG was released only by treatments that disrupt antibody-antigen complexes (high and low pH), but not by the other treatments we tried (detergent, high salt, heparinase, or collagenase digestion). When the IgG bound to glomeruli was eluted with acid citrate buffer 3 d after injection, it was found to specifically immunoprecipitate only gp330 from detergent-solubilized 125I-labeled kidney microvillar vesicles. By isoelectric focusing the eluate was found to be enriched in IgGs with acidic isoelectric points.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pathogenic antibodies inhibit the binding of apolipoproteins to megalin/gp330 in passive Heymann nephritis.

Megalin/gp330 is an endocytic receptor that internalizes multiple ligands including apolipoproteins E (apo E) and B100 (apo B). Megalin is the main antigenic target in passive Heymann nephritis (pHN), where it binds circulating autoantibodies leading to the formation of subepithelial immune deposits (ID)-the hallmark of pHN. Apo E and apo B were found recently to accumulate within these IDs, and evidence was provided that their lipids may undergo peroxidation, causing glomerular basement membrane damage and proteinuria. Here we investigated if ID-forming antimegalin IgG can inhibit the binding and internalization of apo E-betaVLDL (very low density lipoprotein) by megalin, and lead to their accumulation within IDs. By immunoelectron microscopy, apo E and apo B were detected in clathrin-coated pits and multivesicular bodies of podocytes in control rats, suggesting that the uptake of lipoproteins is a constitutive function of the glomerular epithelium. When pHN was induced by intravenous injection of antimegalin IgG, apo E and apo B were found within IDs by immunofluorescence and immunoelectron microscopy. Bound antibodies eluted from glomeruli of rats with pHN were found to inhibit the binding and internalization of apo E-enriched betaVLDL by megalin. These results indicate that pHN-inducing antimegalin IgG is capable of interfering with the uptake of lipoproteins by megalin in vivo during the formation of IDs.     

Immunocytochemical characterization of cells in rat glomerular culture

Identification and characterization of cells in glomerular tissue culture has been an ongoing concern of experimental renal biologists. We examined the immunocytochemical staining profiles of primary and passaged rat glomerular cultures using a battery of commercially available antisera. In all culture systems examined, two distinct cell populations could be distinguished. One cell type stained positively for desmin and had ultrastructurally smooth muscle features typical of contractile mesangial cells. The second cell type proliferated in a manner classically described for "epithelial" cells but, intriguingly, did not express any of the numerous epithelial markers tested. The second cell population may represent an undifferentiated reserve cell type. Immunocytochemical methods offer an independent modality to the evaluation of cells which proliferate in glomerular cell culture.     

Leukotriene D4 is a mediator of proteinuria and glomerular hemodynamic abnormalities in passive Heymann nephritis.

We assessed the role of leukotrienes (LTs) in Munich-Wistar rats with passive Heymann nephritis (PHN), an animal model of human membranous nephropathy. 10 d after injection of anti-Fx1A antibody, urinary protein excretion rate (Upr) in PHN was significantly higher than that of control. Micropuncture studies demonstrated reduced single nephron plasma flow and glomerular filtration rates, increased transcapillary hydraulic pressure difference, pre- and postglomerular resistances, and decreased ultrafiltration coefficient in PHN rats. Glomerular LTB4 generation from PHN rats was increased. Administration of the 5-LO activating protein inhibitor MK886 for 10 d markedly blunted proteinuria and normalized glomerular hemodynamic abnormalities in PHN rats. An LTD4 receptor antagonist SK&F 104353 led to an immediate reduction in Upr and to reversal of glomerular hemodynamic impairment. Ia(+) cells/glomerulus were increased in PHN rats. In x-irradiated PHN rats, which developed glomerular macrophage depletion, augmented glomerular LT synthesis was abolished. Thus, in the autologous phase of PHN, LTD4 mediates glomerular hemodynamic abnormalities and a hemodynamic component of the accompanying proteinuria. The synthesis of LTD4 likely occurs directly from macrophages or from macrophage-derived LTA4, through LTC4 synthase in glomerular cells.     

Cellular pathology of Heyman nephritis: monoclonal antibodies against the pathogenic antigen Gp 330

Heymann nephritis is an experimental auto-immune glomerulonephritis, closely resembling human epimembranous nephritis, which is induced by identified antigens in the brush border membranes of kidney proximal tubules. The hallmark of the disease is the accumulation of immune deposits in a granular pattern in the lamina rara externa of the glomerular basement membrane. We have established that a large membrane glycoprotein with an apparent molecular weight of 330 kDal (pg 330) is the pathogenic antigen. By means of immunohistochemistry, using mono- and polyclonal anti-pg 330 IgG we found that gp 330 is present in the cell membranes of glomerular epithelial cells and that it is concentrated there in coated pits (specialized areas off the cell membrane which play a key role in receptor mediated endocytosis for ligands such as insulin and others). On the basis of these findings we propose the following mechanism of formation of an immune deposit: (1) "In-situ" formation of immune complexes of gp 330 and anti-gp 330 IgG; (2) shedding of the immune complexes into the basement membrane and (3) crosslinking of the immune complexes into the basement membrane. This scheme could be a general mechanism by which immune deposits are formed also in various other auto-immune diseases.     

正常人皮肤表皮干细胞定位特征对修复皮肤创伤的意义

目的:研究正常人皮肤中表皮干细胞的定位特征,探讨表皮干细胞与烧伤创面愈合的关系。方法:在需手术治疗的患者身上获取正常皮肤组织共24例,用α2、β1整合素和K10角蛋白作为第一抗体标记表皮干细胞,采用LSAB免疫组化方法研究皮肤中的表皮干细胞的表达位置。结果:分化高的K10阳性细胞分布于除表皮基底层以外的所有基底上层包括角质层、透明层、颗粒细胞层和棘细胞层的细胞中,而阴性染色区域则集中于表皮基底层。说明表皮基底层细胞不表达K10角蛋白;而α2、β1整合素阳性细胞位于基底膜上、邻接真皮的基底层,呈波浪状,起伏不平分布,同时在头皮的真皮网状层、毛囊末端底部及汗腺周围也发现有阳性细胞。而阴性染色细胞则位于基底层以上的表皮细胞。结论:表皮基底层、毛囊及汗腺周围中存在的表皮干细胞可能与皮肤创伤的修复有关,不同创面中残存的表皮干细胞可能是创伤修复过程中再上皮化的细胞来源,这提示对残存的表皮干细胞的保护、激活、促进其增殖和分化是研究促进创面愈合的关键,从而可以部分阐明创伤愈合的机制。     

Host-dependent Lewis (Le) antigen expression in Helicobacter pylori cells recovered from Leb-transgenic mice

Variation of surface antigen expression is a mechanism used by microbes to adapt to and persist within their host habitats. Helicobacter pylori, a persistent bacterial colonizer of the human stomach, can alter its surface Lewis (Le) antigen expression. We examined H. pylori colonization in mice to test the hypothesis that host phenotype selects for H. pylori (Le) phenotypes. When wild-type and Le^b-expressing transgenic FVB/N mice were challenged with H. pylori strain HP1, expressing Le^x and Le^y, we found that bacterial populations recovered after 8 mo from Le^b-transgenic, but not wild-type, mice expressed Le^b. Changes in Le phenotype were linked to variation of a putative galactosyltransferase gene (β-(1,3)galT); mutagenesis and complementation revealed its essential role in type I antigen expression. These studies indicate that H. pylori evolves to resemble the host''s gastric Le phenotype, and reveal a bacterial genetic locus that is subject to host-driven selection pressure.For microbes that are obligatory parasites of outbred host species, an important challenge is to adapt to each new individual host (Moxon et al., 1994; Falk et al., 2000; Bayliss et al., 2004; Blaser and Kirschner, 2007). Such coevolved bacteria use multiple strategies, including stealth, variation, and antidefense (Monack et al., 2004; Blaser and Kirschner, 2007). One mechanism to generate variation is the use of contingency genes to change expression of bacterial cell-surface structures relevant to the hosts being colonized (Moxon et al., 1994; Bayliss et al., 2001; Bayliss et al., 2004).Humans are polymorphic for the expression of the fucosylated Lewis (Le) antigens on erythrocytes and in other body compartments, including the gastric epithelium (Sakamoto et al., 1989). Helicobacter pylori, the dominant human gastric bacteria (Bik et al., 2006; Andersson et al., 2008), are also polymorphic for expression of Le antigens (Fig. S1; Wang et al., 2000). Most strains predominately express Le^x and Le^y (type II antigens), which are major human gastric antigens (Simoons-Smit et al., 1996), whereas <5% express Le^a and Le^b (type I antigens; Wirth et al., 1996), which are also expressed in the stomach (Sakamoto et al., 1989). H. pylori may vary type II Le expression using a variety of genetic mechanisms (Appelmelk et al., 1998; Wang et al., 1999; Wirth et al., 2006; Sanabria-Valentín et al., 2007; Nilsson et al., 2008).We have hypothesized that H. pylori Le expression reflects host selection operating on a population of stochastically varying strains that have differential fitness in particular hosts (Webb and Blaser, 2002). Observations in humans naturally colonized with H. pylori (Wirth et al., 1997) and in rhesus monkeys experimentally infected with H. pylori (Wirth et al., 2006) support this hypothesis. However, these studies are not conclusive, because the human studies were correlative, and the monkey studies were an experimental challenge with multiple strains and a small number of study animals (Wirth et al., 1997; Wirth et al., 2006).Wild-type mice do not express Le^b in their stomach. The creation of transgenic mice that express a human α-1,3/4 fucosyltransferase (accession no. EC 2.4.1.65 from the IntEnz database, available at http://www.ebi.ac.uk/intenz/index.jsp) in their mucus-producing gastric pit cells led to Le^b expression (Falk et al., 1995; Guruge et al., 1998). The presence of Le^b in the gastric mucosa of these mice and its absence in their nontransgenic littermates presented an opportunity to examine whether host phenotype selects for H. pylori phenotypic (Le antigen) expression. We hypothesized that among H. pylori strains introduced into “humanized” Le^b-transgenic mice but not their isogenic Le^b-negative (wild-type) littermates, there would be selection for bacterial Le^b expression. In the present study, we verify this hypothesis, and characterize the genetic loci and mechanisms responsible for the changed H. pylori phenotype.     

Effects of a new immunosuppressive agent, FK506, in rats with active Heymann nephritis.

FK506 is a recently-developed immunosuppressive drug. The aim of the present work was to investigate the effects of FK506 in experimental autoimmune glomerulonephritis (active Heymann nephritis) in rats. Active Heymann nephritis was induced in female Lewis rats by two immunizations with the homologous brush border vesicles (BBVs) at day 0 and day 28 (groups I, II, V, and VI). Rats of groups III and IV received the third immunization at day 56. In rats of groups I and III, FK506 was injected (1 mg/kg/day IM) from day 0 for 14 days. In rats of groups II and IV, significant proteinuria was observed (group II, 112.8 mg/16 hours; group IV, 55.4 mg/16 hours) at the time the rats were killed (day 84). Coarse subepithelial immune deposits (IDs) were found in these rats. In contrast, urinary protein excretion remained within normal range (less than 3.0 mg/16 hours) in groups I and III rats, and tiny subepithelial IDs were only occasionally seen. Circulating anti-BBV antibody levels were markedly lower in group I and III rats than in those of groups II and IV during the period between day 14 and day 56. To investigate the effects of FK506 on the proteinuric rats, FK506 (1 mg/kg/day, IM) was administered every day for 2 weeks beginning on day 56 (group V).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthetic peptides of Goodpasture's antigen in antiglomerular basement membrane nephritis in rats

Goodpasture's syndrome (GPS) is an autoimmune disease characterized by pulmonary hemorrhage, glomerulonephritis and anti-glomerular basement membrane (GBM) antibodies. The alpha(3) noncollagenous domain (NC1) of type IV collagen [alpha(3)(IV)] is the pathogen. The disease is T-cell-dependent; thus linear peptides initiate the autoimmune process. Studies in a rat model of GPS, experimental autoimmune glomerulonephritis (EAG), have shown that the carboxy-terminal 36 amino acids (purportedly the pathogenic epitope) are not responsible for disease induction. More recent studies implicate the amino terminus of alpha(3)(IV)NC1. Finding the nephritogenic epitope(s) is crucial in the understanding of the disease and for treatment. Because alpha(3)(IV)NC1 contains the antigens that induce GN in rats and human beings, we hypothesized that regions of the alpha(3)(IV)NC1 other than the carboxy terminus were responsible for disease. We investigated overlapping peptides spanning the entire NC1 domain of the alpha(3)(IV) chain N-terminal to the 36-mer (Goodpasture epitope) using the EAG rat model. Most peptides elicited antibody responses exclusively to themselves but not to native GBM. T-cells from GBM-immunized rats proliferated in vitro after stimulation with peptides 6, 8, 14, and 15, 24-mer and 23-mer. Fifteen percent of peptide 8 and peptide 14 rats had mild glomerulonephritis. In none of the animals immunized with other peptides did glomerulonephritis develop. These data suggest that conformation-dependent sites, posttranslational modification, multiple epitopes, concomitant antibody formation, or other disturbances are important in the ability of alpha(3)(IV)NC1 to induce EAG in rats and may also be important in the induction of GPS in human beings.     

The glomerular mesangium. II. Studies of macromolecular uptake in nephrotoxic nephritis in rats

These studies were designed to explore the effects of nephrotoxic serum (NTS) in rats on the uptake and processing by the glomerular mesangium of intravenously administered protein macromolecules (radiolabeled aggregated human IgG, [125I]AHIgG). Measurements of [125I]AHIgG levels in preparations of isolated glomeruli correlated well with qualitative estimates of glomerular IgG deposition seen by immunofluorescent microscopy. Rats given 2 ml NTS received 25 mg/100 g body wt [125I]AHIgG 48 h later and were sacrificed at varying time intervals thereafter. NTS-treated animals demonstrated a marked increase in glomerular uptake of [125I]AHIgG as compared with concurrent controls but a normal ability to clear [125I]AHIgG from the mesangium over 72 hr. Animals given 1.0, 0.5, and 0.25 ml NTS had neither proteinuria nor significant light microscopic changes, yet had increased uptake of [125I]AHIgG of 4.4, 3.0, and 2.1 times control values, respectively at 8 h after the infusion of aggregates. Rats given 1 ml NTS and 12.5, 6.25, and 3.125 mg [125I]AHIgG/100 g body wt had increased glomerular uptake at 8 h, but there was, within both NTS and control groups, a disproportionate decrease in uptake at lower [125I]AHIgG doses. Rats given cobra venom factor (CoF) followed by a NTS shown to be complement dependent (proteinuria reduced by prior complement depletion with CoF) had less mesangial [125I]AHIgG uptake than NTS controls but greater uptake compared with normal controls. CoF itself had no effect on mesangial or reticuloendothelial system [125I]AHIgG uptake. These studies demonstrate a striking change in glomerular mesangial activity after fixation of nephrotoxic antibodies to the glomerular basement membrane, even in the absence of proteinuria and suggest that subtle alterations of the filtration surface can influence mesangial function. The effect of NTS on the mesangium is due, in large part, to the glomerular injury and proteinuria which nephrotoxic antibodies produce.     

Effect of antimacrophage serum on the proliferation of glomerular cells in nephrotoxic serum nephritis in the rat

To evaluate the effect of a rabbit anti-rat macrophage serum (AMS) on glomerular cells in vivo, glomeruli were isolated from an accelerated autologous form of nephrotoxic serum nephritis (NTSN) in rats and grown in tissue culture. The prominent feature of the glomerular outgrowth of the glomeruli in the NTSN was the presence of large numbers of type III (macrophages) cells, which were not present in cultured normal glomeruli. In addition, there were significantly greater numbers of type II (mesangial) cells in culture from the NTSN rats as compared with glomeruli from normal rats though the numbers of type I (epithelial) cells were the same. The administration of AMS prevented the outgrowth of macrophages and reduced the number of mesangial cells in the outgrowth of glomeruli from the NTSN rats. The AMS-treated rats showed profound reduction in proteinuria. Light micrographs showed only minor histologic lesion in the AMS-treated rats. These findings suggest that AMS may be applicable to the modulation of the proliferative response seen in NTSN.     

Platelets and neutrophils are critical to the enhanced glomerular arachidonate metabolism in acute nephrotoxic nephritis in rats.

Nephrotoxic nephritis (NTN) is characterized by a marked increase in glomerular eicosanoid synthesis, which appears to play an important role in the pathophysiology of this disease model. In this study, we investigated the biochemical and cellular basis of this metabolic change. By examining the enzymatic conversion of exogenous substrates by intact glomeruli, we found that cyclooxygenase, TX synthase, and 5-lipoxygenase activities increased 4-, 8-, and 100-fold, respectively, in acute NTN. PGH2-PGE2 isomerase and leukotriene A4 hydrolase activities did not change. The cellular basis of these changes was examined using dissociated glomerular cells in vitro and by depleting platelets in vivo. Dissociated glomerular cells from nephritic glomeruli (largely mesangial cells and leukocytes) exhibited an enhanced arachidonate metabolism similar to intact nephritic glomeruli. Depletion of neutrophils (PMNs) from these cell preparations by 90% commensurately decreased 5-lipoxygenase and cyclooxygenase activity but had little effect on TX synthase activity. The recovered PMN fraction, however, did exhibit TX synthase activity. Immunocytochemical analysis of dissociated cells using an antiplatelet antibody demonstrated the presence of platelets, both adherent to cells and noncell associated. Depletion of platelets in vivo using this antibody substantially attenuated the increase in glomerular eicosanoid synthesis that accompanied NTN. Platelet depletion also decreased the influx of PMNs into the glomerulus by 50%. These data show that PMNs and platelets colocalize to the glomerulus in acute NTN and are coordinately essential to the increase in glomerular arachidonate metabolism.     

Outcome of the acute glomerular injury in proliferative lupus nephritis.

Treatment with total lymphoid irradiation (TLI) and corticosteroids markedly reduced activity of systemic lupus erythematosis in 10 patients with diffuse proliferative lupus nephritis (DPLN) complicated by a nephrotic syndrome. Physiologic and morphometric techniques were used serially before, and 12 and 36 mo post-TLI to characterize the course of glomerular injury. Judged by a progressive reduction in the density of glomerular cells and immune deposits, glomerular inflammation subsided. A sustained reduction in the fractional clearance of albumin, IgG and uncharged dextrans of radius greater than 50 A, pointed to a parallel improvement in glomerular barrier size-selectivity. Corresponding changes in GFR were modest, however. A trend towards higher GFR at 12 mo was associated with a marked increase in the fraction of glomerular tuft area occupied by patent capillary loops as inflammatory changes receded. A late trend toward declining GFR beyond 12 mo was associated with progressive glomerulosclerosis, which affected 57% of all glomeruli globally by 36 mo post-TLI. Judged by a parallel increase in volume by 59%, remaining, patent glomeruli had undergone a process of adaptive enlargement. We propose that an increasing fraction of glomeruli continues to undergo progressive sclerosis after DPLN has become quiescent, and that the prevailing GFR depends on the extent to which hypertrophied remnant glomeruli can compensate for the ensuing loss of filtration surface area.     

Identification of a major sialoprotein in the glycocalyx of human visceral glomerular epithelial cells.

Glomerular visceral epithelial cells are endowed with a sialic acid-rich surface coat (the "glomerular epithelial polyanion"), which in rat tissue contains the sialoprotein podocalyxin. We have identified a major membrane sialoprotein in human glomeruli that is similar to rat podocalyxin in its sialic acid-dependent binding of wheat germ agglutinin and in its localization on the surface of glomerular epithelial and endothelial cells, as shown by immunoelectron microscopy, using the monoclonal antibody PHM5. Differences in the sialoproteins of the two species are indicated by the discrepancy of their apparent molecular weights in sodium dodecyl sulfate gels, by the lack of cross reactivity of their specific antibodies, and by the lack of homology of their proteolytic peptide maps. It is therefore possible that the human glomerular sialoprotein and rat podocalyxin are evolutionarily distinct, but have similar functions.     

Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells.

In reticulocytes, the enzyme 15-lipoxygenase (15-LO) is believed to contribute to cellular differentiation, and in leukocytes and airway cells 15-LO generates inflammatory mediators. The recent availability of antibodies to 15-LO now allows us to determine which specific cells contain the enzyme, to characterize its subcellular localization, and to determine its expression at the translational level. A polyclonal antibody to recombinant human reticulocyte 15-LO was used with a standard immunofluorescent technique. In rabbit red blood cells, fluorescence appeared during the course of anemia. Early reticulocytes did not fluoresce, but more mature reticulocytes showed increased fluorescent intensity. Late reticulocytes contained little fluorescence. Among human leukocytes, only eosinophils fluoresced. In human trachea, 15-LO immunofluorescence was localized to epithelial cells, and both basal and ciliated cells fluoresced. In all cells studied, fluorescence was localized to the cytoplasm and was variable in degree among cells in each preparation. We conclude that the 15-LO of airway cells and eosinophils is immunologically related to the reticulocyte 15-LO. Furthermore, the variable fluorescence among cells (e.g., in epithelium) and during development (e.g., reticulocytes) suggests a role of 15-LO in cell growth and development.