The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium.

The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.

Loss and rearrangement of glomerular basement membrane laminin during acute nephrotoxic nephritis in the rat.

Many earlier studies have shown that the intravenous injection into rats of sheep antibodies against rat glomerular basement membrane (GBM) induces a rapid influx of neutrophils and proteinuria (nephrotoxic nephritis or NTN). The GBM antigens recognized by nephrotoxic antibodies (NTAbs) have not been identified conclusively. Our experiments presented here, however, showed that NTAbs did not significantly reduce binding of anti-laminin IgGs to laminin-coated enzyme-linked immunosorbent assay (ELISA) plates or to the GBM in vivo, indicating little cross-reactivity between the NTAbs and laminin. To evaluate possible changes in GBM architecture during acute stages of NTN, the ultrastructural distribution of laminin was determined by postfixation, postembedding immunogold labeling, and compared between normal and nephritic rats. The density of immunoreactive GBM laminin was significantly reduced in rats with acute NTN. In addition, conjugates of anti-laminin IgG and horseradish peroxidase were intravenously injected into rats that then received injections of NTAbs. Anti-laminin peroxidase conjugates were also injected after administering NTAbs. In both cases, an overall decrease in anti-laminin peroxidase reaction product was observed as compared to normal controls. The densest labeling was seen in the lamina rara interna, especially in areas of endothelial cell detachment. Some immunoperoxidase reaction product was also bound to basal surfaces of detaching endothelial cells, demonstrating the removal of at least some laminin from the GBM. A decrease in GBM binding of intravenously injected anti-laminin IgG, both before and after injection of rats with NTAbs, was also confirmed by postembedding immunogold labeling. Furthermore, morphometry showed that the GBM was significantly wider in nephritic rats than in controls, indicating a redistribution of laminin over a greatly increased area. These immunoultrastructural findings show, therefore, that GBM architecture is altered in the early phase of NTN.

Glomerular basement membrane: evidence for collagenous domain of the alpha 3 and alpha 4 chains of collagen IV.

A collagenous component(s) of Mr = 60K was extracted from glomerular basement membrane with urea and was purified. Upon digestion, it yielded a collagenase-resistant fragment(s) of Mr = 23.5K. Both component and fragment showed immunochemical identity with the noncollagenous domains of the new alpha 3 & alpha 4 chains of collagen IV. The component is characterized by a collagenous domain of about 280 residues and a noncollagenous domain of about 250 residues. These findings further establish these new chains as distinct entities of collagen IV.

Identification of disulfide-linked transforming growth factor-beta 1-specific binding proteins in rat glomeruli.

We have identified two distinct classes of transforming growth factor-beta (TGF-beta)-binding proteins by affinity labeling rat glomeruli with 125I-TGF-beta 1 and 125I-TGF-beta 2. The first type consists of a group of proteins that bind TGF-beta 1 but do not bind TGF-beta 2. When 125I-TGF-beta 1 affinity-labeled glomeruli were separated under nonreducing conditions, four prominent bands with Mr values of 320,000, 260,000, 170,000, and 90,000 were observed. Following reduction, the 320,000 and 170,000 bands yielded only a 100,000 band, the 260,000 complex yielded bands of 200,000, 100,000, and 85,000, and the 90,000 band migrated with an Mr of 85,000. Binding of 125I-TGF-beta 1 to these proteins was unaffected by the addition of as much as a 1,000-fold excess of TGF-beta 2. The second type of glomerular TGF-beta-binding protein consists of Mr 160,000-200,000 and 280,000 proteins that bind both TGF-beta 1 and beta 2. Digestion of these affinity-labeled proteins with heparitinase and chondroitinase resulted in a decrease of approximately 40,000 in their apparent molecular weights. Glomerular TGF-beta 1-binding proteins are distinct from previously described TGF-beta-binding proteins in their specificity for TGF-beta 1 and their formation of disulfide-linked multimers. The TGF-beta 1/beta 2-binding proteins share some properties of the previously described type III TGF-beta receptor.

Heparan sulfate proteoglycan from human tubular basement membrane. Comparison with this component from the glomerular basement membrane.

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.

Ultrastructural immunogold studies of heparan sulphate proteoglycan in normal human glomeruli and glomerulonephritis.

The distribution of heparan sulphate proteoglycans (HSPG) has been investigated in normal human glomeruli, membranous glomerulonephritis, mesangial IgA disease, and anti-glomerular basement membrane disease. HSPG was localized using anti-bovine HSPG antibody and 10 nm gold-labelled secondary antibody on paraformaldehyde-fixed, Lowicryl K4M resin-embedded kidneys. HSPG was present in all glomeruli and there was a zonation of its distribution in that it was predominantly on the epithelial aspect of the glomerular basement membrane (GBM) and mesangium with little in the central regions of the mesangial matrix. In the cases of immune complex glomerulonephritis, no HSPG was found in the electron-dense deposits. These findings contrast with our previous studies using the same technique in which type IV collagen and fibronectin were found predominantly on the endothelial aspect of the GBM.

The pathogenesis of Plasmodium falciparum-associated tissue lesions--what role for C3b receptors?

Human tissue sections from cerebral and cerebellar cortices, the liver, the stomach pylorus, the kidney and the placenta, were examined for the presence of C3b receptors. This was carried out in order to determine whether these receptors play any role in the pathological effects of malarial infections on these tissues. The sheep erythrocyte/rabbit antibody/complement system was used. C3b receptors were only detected in the kidney glomeruli, but not in the other tissues. It is suggested that during malarial infections complement may be indirectly involved in the pathogenesis of tissue damage in the organs examined by triggering the activation and participation of other body systems.

Autoradiographic localization of putative arginine vasotocin receptors in the kidney of a urodele amphibian.

The distribution and characteristics of putative arginine vasotocin (AVT) receptors in the urodele amphibian kidney were investigated using in vitro quantitative autoradiography. Specific binding sites for [3H]arginine vasopressin (AVP) in the kidney of the rough-skinned newt (Taricha granulosa) were located over the glomeruli. Scatchard analysis showed that, in the range of concentrations tested (0.2 to 22 nM), [3H]AVP bound to a single class of receptors with a dissociation constant of 1.4 nM and a binding site concentration of 36.5 fmol/mg protein. Binding displacement studies showed that both AVT and AVP were potent ligands for newt kidney receptors. Two specific antagonist peptides with anti-vasopressor (V1) activity, but not anti-antidiuretic (V2) activity, in rat tissues were tested as well. Both antagonists effectively displaced [3H]AVP from receptor sites in newt kidney slices, indicating that the binding sites in this amphibian resemble the V1 subtype of mammals in ligand specificity. Localization of AVT receptors over kidney glomeruli and ligand specificity of these sites is consistent with the hypothesis that AVT may cause antidiuresis in urodele amphibians at least in part via a glomerular vasoconstricting action.

Adhesions, focal sclerosis, protein crescents, and capsular lesions in membranous nephropathy.

All biopsies of membranous glomerulopathy collected between 1965 and 1988 (n = 198) were examined for the presence of adhesions, focal sclerosis, protein crescents, capsular drops, and other capsular lesions. These were observed with a frequency related to the number of glomeruli in the biopsy and the number of sections examined. A peculiar interrelation between these lesions was found. In the 63 cases with sufficient clinical follow-up, the capsular lesions were associated with a lower incidence of remission of the disease, and with more proteinuria and a higher serum creatinine at the last follow-up. It is hypothesized that focal detachment of podocytes may result in the formation of a protein crescent if a plasma-like filtrate is 'injected' between the epithelium of Bowman's capsule and its basement membrane. The detachment of the epithelium may lead to focal sclerosis and the formation of adhesions. The capsular drop is thought to be a final stage of inspissation of the protein crescent.

Detection of specific mRNAs in single nephron segments by use of the polymerase chain reaction.

We have developed a procedure to detect specific mRNAs in single renal nephron segments. This approach combines microdissection, reverse transcription (RT) of the target mRNA, and amplification of the resulting cDNA using the polymerase chain reaction (PCR). After microdissection, the sample is placed in a tube where it is permeabilized and where all reactions are performed directly without the need for isolation of the RNA. Our model target was the mRNA for aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol. Its expression is modulated by changes in extracellular osmolality in the renal medulla. RT-PCR of inner medullary collecting duct (1 mm) and glomeruli (6-10) yielded a product of the predicted length (670 base pairs) defined by the PCR primers. Its identity was confirmed by a specific oligonucleotide probe that differed from the primers. RT-PCR of proximal tubules (1 mm) resulted in no aldose reductase-specific amplification product. RT-PCR is generally applicable for measuring specific gene expression in single nephron segments or small numbers of cultured cells. Utility, limitations, and refinements of this approach are discussed.

Distribution of beta 1 and beta 3 integrins in human fetal and adult kidney.

The distribution of beta 1 and beta 3 integrins was studied in fetal and adult human kidneys by indirect immunofluorescence microscopy. In the developing kidney, the cells of the undifferentiated metanephric blastema displayed strong cell surface-confined beta 1 integrin immunoreactivity, whereas the cells of primary vesicles and comma- and S-shaped bodies reacted more weakly. In mature fetal as well as adult glomeruli, beta 1 integrins were distinctly localized, apparently confining to the basal cell surfaces of endothelial cells and podocytes abutting the glomerular basement membrane. In adult proximal tubules, beta 1 integrin immunoreactivity was strictly confined to the basal aspect of the epithelial cells, being absent laterally, which is unusual for membrane proteins of polarized epithelial cells. A more diffuse overall immunoreactivity was seen in distal tubules and collecting ducts. The epithelial cells of developing proximal and distal tubules displayed an overall distribution of beta 1 integrins. In each case, talin immunoreactivity followed that of beta 1 integrins. Compared with beta 1 integrins, beta 3 integrins showed a more restricted distribution, and differences were seen in the reactions of mono- and polyclonal antibodies. In developing glomeruli, beta 3 integrin immunoreactivity was prominently seen in the cells of Bowman's capsule, possibly revealing the presence of vitronectin receptor. Solitary cells, that reacted also with antibodies to the platelet glycoprotein IIb, were consistently detected in fetal glomeruli, suggesting the presence of megakaryocytes. The results show that during nephrogenesis, beta 1 integrins become distinctly polarized both in glomerular endothelial cells and podocytes, as well as in the epithelial cells of proximal tubules.

Glomerular basement membrane. Identification of a fourth chain, alpha 4, of type IV collagen.

The noncollagenous domain hexamer of collagen IV from bovine glomerular basement membrane was further investigated to determine the types of collagen chain from which subunits M2*b and M3 are derived. M2*b was shown to be a shorter form, containing 9 fewer residues, of M2*a which was previously established as the noncollagenous domain of a third chain, alpha 3, of collagen IV (Saus, J., Wieslander, J., Langeveld, J.P.M., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). M3 was identified as the noncollagenous domain of a fourth chain, alpha 4, of type IV collagen, on the basis of additional sequence data together with previous findings. A comparison of the collagenous-noncollagenous junction regions of alpha 3(IV) and alpha 4(IV) chains with those of classical alpha 1(IV) and alpha 2(IV) chains reveals structural information which provides a potential strategy for molecular cloning of these novel chains. The results further reveal the complexity of electrophoresis patterns of the hexamer and potential ambiguities in using one-dimensional patterns to determine whether molecular defects of collagen IV occur in pathological processes affecting basement membranes.

Urticaria, nephritis, and pseudotumor cerebri.

The spectrum of chronic urticarial disease ranges from chronic urticarial skin lesions alone to well-characterized systemic lupus erythematosus with urticarial vasculitis as the major skin manifestation. Within this spectrum is the syndrome of urticarial vasculitis associated with systemic disease manifestations. There have been six previously recorded cases of urticarial vasculitis associated with pseudotumor cerebri. At least two of these have included membranoproliferative glomerulonephritis. The authors report a case of chronic urticarial disease associated with pseudotumor cerebri and membranoproliferative glomerulonephritis, but without demonstrable vasculitis. It is possible that this represents a distinct entity within the spectrum of chronic urticarial disease and cca be easily screened for in clinical practice.

Affinity cytochemical labeling of glomerular basement membrane anionic sites using specific biotinylation and colloidal gold probes.

To label specific anionic sites on glomerular capillary wall structures, biotin was covalently linked to sialic acid residues by sequential treatment with mild peroxidation and biotin hydrazide, while carboxyl groups were biotinylated by exposure to the combination of biotin hydrazide and a water-soluble carbodiimide reagent. Optimal specific labeling of rat glomerular structures was obtained with in situ perfusion of the biotinylation reagents, followed by fixation in 4% phosphate-buffered paraformaldehyde, embedment in LR White resin, and post-embedment detection of biotinylated sites using a sequence of anti-biotin antibodies followed by a secondary antibody-colloidal gold conjugate. Attempts to use streptavidin-gold conjugates were not successful. Specificity of labeling was confirmed by enzymatic (neuraminidase) pre-treatment or by modification of carboxyl groups, as evaluated by electron microscopy and by solid-phase assays of solubilized glomerular basement membrane (GBM) components. In two rats with heavy proteinuria induced by doxorubicin (Adriamycin), a marked reduction in sialic acid residues within the GBM and on the epithelial cell surfaces was found, suggesting that reduced charge density attributable to abnormal sialylation may be important in the pathogenesis of nephrotic proteinuria.

Characterization of type IV collagen NC1 monomers and Goodpasture antigen in human renal basement membranes.

Monomeric subunits of the globular domain of type IV collagen from human renal basement membrane were isolated and characterized. The monomers, M24, M26, M28+, and M28 , which have been identified previously in human glomerular basement membrane, were characterized by amino acid analysis, amino-terminal sequencing, and electrophoretic mobility. The results indicate that M24 and M26 are derived from alpha 1(IV) and alpha 2(IV) collagen chains, respectively. Amino-terminal sequencing revealed that M28+, and M28 correspond to the globular domain of novel collagen chains. M28 has been characterized as the principal target antigen in Goodpasture's syndrome and antiglomerular basement membrane (GBM) nephritis, and both M28 species are absent from the GBM in Alport familial nephritis. The cationic charge of M28 appears to be a consequence of a relatively high concentration of basic amino acids when compared with other monomers. Previous studies of bovine GBM have demonstrated chains with amino-terminal sequence homology to M28+ and M28 .

Glomerular platelet-activating factor levels and origin in experimental glomerulonephritis.

The content of platelet activating factor (PAF) in glomeruli isolated from rats with nephrotoxic serum glomerulonephritis (NSGN) was quantified at various stages of the disease and the role of complement, platelets and neutrophils in mediating changes in glomerular PAF levels was evaluated. PAF content was assessed following extraction, isolation and quantification of this alkyl ether lipid using a bioassay based on [3H]-serotonin release from labelled rabbit platelets. Following induction of NSGN using proteinuric doses of rabbit immune serum raised against rat glomerular basement membrane, enhanced glomerular PAF levels were observed at 3 hours, 24 hours and on day 15 following induction of the disease. In complement depleted rats and at three hours following induction of NSGN, glomerular PAF levels were significantly lower than in complement replete controls studied in parallel. At the same time point of the disease, platelet depleted rats with NSGN demonstrated significantly lower glomerular PAF levels than parallel controls, whereas in neutrophil depleted rats glomerular PAF levels were no different than controls. These observations indicate that in infiltrative and complement dependent forms of glomerular immune injury, glomerular PAF levels are increased via a complement mediated mechanism. Infiltrating platelets, but not neutrophils, partially account for the enhanced glomerular PAF levels. The observations are of potential importance in the pathophysiology of glomerulonephritis.

Ovine mesangiocapillary glomerulonephritis type I and crescent formation.

Morphologic examination of four Finnish Landrace mixed-breed lambs, 27 to 35 days of age, affected with mesangiocapillary glomerulonephritis type 1, demonstrated a progressive glomerulonephritis. By 27 days of age, three lambs had crescents in 58 to 93% of glomeruli. These three lambs were also uremic. The accelerated rate of crescent formation was attributed to infiltrating polymorphonuclear leukocytes and monocytes, the result of discontinuities (gaps) in the glomerular basement membrane, and to the loss of the integrity of Bowman's capsule. In the three lambs, platelets were identified adjacent to the endothelium or denuded glomerular basement membrane. Two distinctly different types of crescents were noted, apparently dependent on the integrity of Bowman's capsule. One type resulted from the influx of inflammatory cells and dissociation of parietal epithelial cells from Bowman's capsule. The other type was more extensive and contained collagen and was associated with damage to Bowman's capsule resulting in cellular infiltration from the interstitium and sclerosis. Based on morphologic similarities, ovine mesangiocapillary glomerulonephritis is a suitable model for studying the pathogenesis and treatment of mesangiocapillary glomerulonephritis type 1 in human beings.

Comparison of non-collagenous type IV collagen subunits in human glomerular basement membrane, alveolar basement membrane, and placenta.

This study examines the similarities and differences in the noncollagenous domain (NC1) of type IV collagen from human glomerular basement membrane (hGBM), alveolar basement membrane (hABM), and placenta (hPBM). Following collagenase digestion, NC1 domain was isolated on Bio-Gel A-0.5m or by cation exchange chromatography on S-Sepharose. NC1 from each source was characterized by SDS PAGE, and two dimension NEPHGE/SDS PAGE. Immunoblotting and ELISA inhibition was performed using antibody probes specific for M28 , M28+, M26 and M24 monomer subunits of human NC1. It was observed that all NC1 subunits were present in hGBM and hABM derived material, however M28 and M28+ monomers were absent in hPBM NC1. These findings indicate that while alpha 1(IV) and alpha 2(IV) collagen chains are present in hGBM, hABM and hPBM, alpha 3(IV) and alpha 4(IV) collagen chains are only found in hGBM and hABM but are absent in hPBM. It can now be appreciated that heterogeneity of alpha (IV) chain composition exists in basement membranes from various organs.

Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane.

Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.