Gasdermin E Does Not Limit Apoptotic Cell Disassembly by Promoting Early Onset of Secondary Necrosis in Jurkat T Cells and THP-1 Monocytes.

During the progression of necroptosis and pyroptosis, the plasma membrane will become permeabilized through the activation of mixed lineage kinase domain like pseudokinase (MLKL) or gasdermin D (GSDMD), respectively. Recently, the progression of apoptotic cells into secondary necrotic cells following membrane lysis was shown to be regulated by gasdermin E (GSDME, or DFNA5), a process dependent on caspase 3-mediated cleavage of GSDME. Notably, GSDME was also proposed to negatively regulate the disassembly of apoptotic cells into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs) by promoting earlier onset of membrane permeabilisation. The presence of a process downstream of caspase 3 that would actively drive cell lysis and limit cell disassembly during apoptosis is somewhat surprising as this could favor the release of proinflammatory intracellular contents and hinder efficient clearance of apoptotic materials. In contrast to the latter studies, we present here that GSDME is not involved in regulating secondary necrosis in human T cells and monocytes, and also unlikely in epithelial cells. Furthermore, GSDME is evidently not a negative regulator of apoptotic cell disassembly in our cell models. Thus, the function of GSDME in regulating membrane permeabilization and cell disassembly during apoptosis may be more limited.

Expression of the alpha6 chain of type IV collagen in glomerular basement membranes of healthy adult dogs.

OBJECTIVE: To evaluate expression of the alpha6 chain of type IV collagen in the glomerular basement membranes (GBM) of healthy dogs. SAMPLE POPULATION: Kidney specimens from 12 healthy dogs. For comparison, kidney specimens from 8 human subjects between 25 and 83 years old also were evaluated. PROCEDURE: Sections were immunolabeled with a monospecific antibody that cross-reacts with human and canine alpha6(IV) chains and examined by means of fluorescence microscopy. RESULTS: Immunolabeling of the alpha6(IV) chain was not observed in GBM of 6 dogs < or = 30 months old but was observed in GBM of the remaining 6 dogs, all of which were > or = 45 months old. Expression of the alpha6(IV) chain was not observed in GBM of the human subjects, regardless of the age of the subject. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that the alpha6(IV) chain is expressed in GBM of healthy dogs, but the expression is age-dependent. Composition and structural organization of type IV collagen in the GBM of healthy adult dogs is different from that described for other species.

Alterations of corneal extracellular matrix after multiple refractive procedures: a clinical and immunohistochemical study.

PURPOSE: To describe the clinical course and alterations of the corneal extracellular matrix (ECM) and basement membrane (BM) in a cornea after hexagonal keratotomy, transverse keratotomies, and keratomileusis. METHODS: Frozen sections of this cornea and of 12 normal corneas were studied by immunofluorescence with specific antibodies. The patient history was analyzed to allow a clinical correlation. RESULTS: In the treated cornea, keratotomy scars and subepithelial fibrosis with neovascularization were seen. Around and beneath the epithelial plugs and along the keratotomy scars, deposits of types III, VI, VIII, and XIV collagen; fibrillin-1; fibronectin; and tenascin-C were found, together with short streaks of types IV (alpha 1-alpha 2) and VII collagen, laminin-1 and -5, entactin, and perlecan. alpha 3-alpha 4 Type IV collagen chains were abnormally absent from the BM around the epithelial plugs. At the edges of the keratomileusis flap, subepithelial fibrosis areas were found, with abnormal deposits of eight different collagen types, perlecan, fibronectin, fibrillin-1, and tenascin-C. The major part of the flap interface did not show ECM abnormalities. ECM alterations outside the scarred areas included the appearance of tenascin-C in the stroma and of alpha 1-alpha 2 type IV collagen in the epithelial BM, and the disappearance of fibronectin from Descemet's membrane. CONCLUSION: Five years after surgery, the treated cornea still presented BM abnormalities at sites of keratotomy scars and epithelial plugs. Several ECM components were abnormally expressed outside the scarred areas, consistent with an ongoing fibrosis in the treated cornea.

Alterations in human glomerular epithelial cells interacting with nonenzymatically glycosylated matrix.

The glomerular epithelial cells and the glomerular basement membrane are important constituents of the permselective barrier in the kidney. These are affected in diabetic nephropathy, one of the long-term complications in diabetic patients. Nonenzymatic glycosylation resulting in the accumulation of advanced glycosylation end products correlates with the development of long-term complications in diabetes. The interaction of cells with extracellular matrix proteins plays a critical role in a variety of biological processes. Recent studies show that cell-matrix interactions mediated by integrins can transduce biochemical signals to the cell interior and regulate cell behavior. In this paper we demonstrate that interactions of human glomerular epithelial cells with a nonenzymatically glycated matrix are altered with defective cell spreading, reduced phosphorylation of focal adhesion kinase and reduced activity of mitogen-activated protein kinase. These data suggest that matrix glycation interferes with normal cell-matrix interactions and intracellular signaling that can potentially result in differential gene expression contributing to the changes seen in diabetic nephropathy.

Altered expression of matrix metalloproteinase-2, TIMP, and TIMP-2 in obstructive nephropathy.

We have previously characterized the evolution of renal cortical interstitial fibrosis in the rabbit model of unilateral ureteral obstruction (UUO). In our earlier report, we examined the extracellular matrix protein composition of the interstitial space. Of note, UUO was associated with the acquisition of prominent interstitial collagen IV immunoreactivity. Interstitial collagens I and III were also increased. In situ hybridization localized increased expression of collagens I and IV to cells of the interstitial space. In the current study, we examine metalloproteinase and metalloproteinase inhibitor expression in the obstructed renal cortex. Matrix metalloproteinase-2 is a metalloproteinase with activity against both collagen IV and denatured collagen I. At day 3 of UUO, both transcripts were significantly increased, although expression of these mRNAs was not different from controls after 7 and 16 days of UUO. Expression of mRNA of tissue inhibitor of the metalloproteinases (TIMP) was significantly increased in the UUO samples at all times, although it was maximal at day 3. Immunohistochemically, increased TIMP reactivity localized to the interstitial space, and TIMP mRNA expression was seen to parallel the interstitial macrophage infiltration that accompanies ureteteral obstruction. In contrast, TIMP-2 mRNA expression appeared to be biphasic, with peaks at both day 3 and day 16 of UUO. At day 7, expression was not different from controls. These data suggest a role for impaired matrix degradation in the development of interstitial fibrosis in the obstructed kidney, particularly at late times when collagen mRNA expression has returned to control values.

Alport syndrome: from bedside to genome to bedside.

Alport syndrome is a genetic disorder of basement membranes manifested clinically by a progressive nephropathy and, in many families, sensorineural hearing loss and ocular lesions. During the 1980s evidence was amassed indicating type IV (basement membrane) collagen as the defective protein in Alport This hypothesis was confirmed in 1990 by the cloning of the X-chromosomal gene COL4A5, which encodes the alpha 5 chain of type IV collagen, and the discovery of mutations in this gene in many Alport kindreds. The results of results of recent studies suggest that the alpha 5(IV) chain forms a distinct collagenous network with the alpha 3 and alpha 4 chains of type IV collagen and that mutations in alpha 5(IV) may prevent the normal incorporation of alpha 3(IV) and alpha 4(IV) into basement membranes. Renal biopsy remains an important modality for making the diagnosis of Alport syndrome, but may eventually be replaced by molecular genetic techniques. Posttransplant anti-glomerular basement membrane nephritis occurs rarely in Alport patients and may be restricted to a subgroup with particular COL4A5 mutations. It is not clear why COL4A5 mutations result in glomerulosclerosis and renal failure, or whether this process may be slowed through dietary or pharmacologic intervention.

Immunochemical studies of the Alport antigen.

The Alport antigen, a component of normal glomerular basement membranes (GBM) which is absent in Alport familial nephritis, is characterized as a 26 kD non-collagenous (NC1) peptide identified by a monoclonal antibody (Mab A7) and an Alport alloantibody. Both antibodies discriminate X-linkage of the Alport defect using indirect immunofluorescence of hemizygous and heterozygous Alport GBM and epidermal basement membrane (EBM). Immunoblotting of SDS-PAGE gels of collagenase-digested Alport renal BM shows absence of monomeric and dimeric components of the Alport antigen, alpha 3(IV) NC1, and alpha 4(IV) NC1. By immunoprecipitation experiments with specific antibodies, the Alport antigen is distinct from the 26 kD NC1 peptide derived from alpha 1(IV). The monoclonal antibody to the Alport antigen and rabbit antiserum to a non-consensus sequence of alpha 5(IV) NC1 react similarly by immunofluorescence with normal kidney and both fail to bind to Alport renal BM. Two dimension Western blots of collagenase-digested BM show that the anti-Alport antigen and the ant-alpha 5(IV) NC1 react similarly with monomeric and dimeric components of BM collagen. These studies are consistent with the likelihood that the Alport antigen and alpha 5(IV) NC1 are the same or are highly homologous molecules. The precise relationship will require characterization of alpha 5(IV) NC1 protein and determination of the nucleotide sequence of the Alport antigen. The associated absence of alpha 3(IV) NC1 and alpha 4(IV) (NC1) from Alport BM is consistent with other observations for a molecular association of these chains in a novel collagen network.

Renal entactin (nidogen): isolation, characterization and tissue distribution.

Entactin/nidogen (E/N) was isolated from bovine renal tubular basement membrane. Apparent molecular weight, amino acid composition, and molecular configuration by electron microscopy rotary shadowing were similar to that of nidogen from EHS mouse tumor. The identity of bovine E/N was confirmed using a thrombin derived peptide, the sequence of which corresponded to a region within mouse and human E/N. Monoclonal and polyclonal anti-E/N antibodies were used to determine the distribution of E/N in human kidney by immunofluorescent and immunoelectron microscopy. E/N was present in all renal basement membranes and was distributed through the full width of the glomerular basement membrane (GBM) with accentuation along its epithelial aspects. E/N distribution was similar to that of novel collagen chain alpha 3(IV) NC domain in the GBM. In the mesangium, E/N was distributed mainly in the peripheral mesangial region that is bounded by the GBM, while classical collagen chain alpha 1(IV) NC as present diffusely throughout the mesangium. In the developing nephron, E/N was present in basement membranes of the ureteric bud, primitive vesicle and S-form. In all instances, E/N co-localized with laminin B2 chain. Prominent E/N detection within the mesangium was observed in diseases where mesangial expansion was present. This process was also seen in early diabetic nephropathy, but disappeared with disease progression. However, all thickened diabetic renal basement membranes showed an increase in E/N which was also present in Kimmelstiel-Wilson lesions. E/N was observed in the GBM "spikes" of membranous glomerulonephritis and in epithelial crescents associated with various disorders. The association between E/N, laminin and type IV collagen chains observed in the normal kidney were maintained in disorders with altered E/N distribution. We could not detect any changes in the distribution of E/N in other acquired and hereditary kidney diseases. These observations reflect the involvement of E/N in the structure and disease alteration of renal basement membranes and mesangial matrix.

Lymphohaemopoietic antigens of cultured human glomerular epithelial cells.

Glomerular visceral epithelial cells (GVEC) from normal human glomeruli were grown in tissue culture. Cell surface markers were studied by immunofluorescence microscopy using antibodies against lymphohaemopoietic differentiation antigens which are known to be present early (BA-1, OKB2, BA-2) and late (J5, anti CR1) in renal ontogenesis. Like foetal human glomerular epithelium, the cultured cells reacted with BA-1 and OKB2 (identifying an antigen expressed on B cells and polymorphonuclear leucocytes), and BA-2 (leukaemia-associated antigen), but were consistently negative for CR1 (C3b receptor); J5 which identifies the common acute lymphoblastic leukaemia antigen (CALLA) stained variably. Reactivity with antimyosin or anti factor VIII were absent. The cells produced an extracellular matrix containing laminin, type IV collagen, and fibronectin. This study supports the notion that GVEC undergo dedifferentiation as shown by the acquisition of lymphohaemopoietic differentiation antigens present early in renal ontogeny. In addition, the production of extracellular matrix constituents in vitro may be useful for the investigation of human glomerular basement membranes.

Cellular antigens in nephroblastoma: identification with monoclonal antibodies which recognize hemopoietic cells.

The correlation between expression of differentiation antigens and morphogenesis was examined in 11 human nephroblastomas using monoclonal antibodies which recognize both hemopoietic and renal cells. Analysis of frozen tissue sections by indirect immunofluorescence revealed that blastema cells and some tubules were recognized by BA-1 and OKB2, which identify unstimulated B cells and granulocytes. Stroma, some tubules, and focal blastema were recognized by BA-2, which identifies a 24-kDa antigen on leukemic cells and platelets. Mature epithelium in glomerular bodies was identified by C3bR and J5, which recognize CR1 and the common acute lymphoblastic leukemia antigen, respectively. Tissue sections from a clear cell sarcoma and a mesoblastic nephroma were notably reactive only with BA-2. These observations demonstrate that the relationship between antigen expression and morphology in nephroblastomas is similar to that observed in fetal kidney and suggest that expression of these cell surface antigens may have a role in morphogenesis.

Antibody specificity of human glomerular basement membrane type IV collagen NC1 subunits. Species variation in subunit composition.

NC1 subunits were purified from gel filtration pools of acid-extracted, collagenase-digested human glomerular basement membranes (hGBM). This methodology, which enriches 28-kDa monomers (M28) in the total digest, allowed purification of these monomers and 24-kDa (M24) and 26-kDa (M26) monomers free from dimers. Reactivity of these subunits with Goodpasture autoantibodies using immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional nonequilibrium pH gradient electrophoresis gels showed strong reactivity with the purified M28 subunits. Inhibition enzyme-linked immunosorbent assay, used to quantitate the reactivity of the purified NC1 subunits, indicated that M28 had a greater than 10-fold increase in ability to inhibit binding to NC1 than NC1 itself. Comparison of hGBM NC1 components were made with those obtained from collagenase digests of salt and acid-extracted bovine and sheep GBM and Englebreth-Holm-Swarm tumor similarly purified by gel filtration and reverse-phase high performance liquid chromatography. Two-dimensional gel analysis of these NC1 isolates revealed absence of the very cationic M28 monomers. Reactivity with antibodies eluted from diseased kidneys of sheep immunized with hGBM (Steblay nephritis) was compared with Goodpasture autoantibody reactivity by immunoblotting two-dimensional gels of hGBM NC1. We conclude that a very cationic M28 monomer (M28 ) found only in hGBM is the probable target in Goodpasture syndrome, that the epitope is present on most NC1 components from extracted and unextracted hGBM, and is exposed by urea denaturation which is enhanced by acid treatment. A weakly cationic M28 monomer (M28+) is present in GBM from other species and is the probable target in Steblay nephritis. Differential recognition of the two M28 components by these antibodies points to different genetic origins or possibly distinct post-translational modifications for these components. This is supported by their presence or absence in different species and tissues, as well as biochemical differences from the M24/26 monomers which presumably are derived from alpha 1(IV) and alpha 2(IV) collagen chains.

Comparison of non-collagenous type IV collagen components in the human glomerulus and EHS tumor.

A method for the isolation of the NC1 domain of type IV collagen has been developed using the EHS sarcoma, a basement membrane-producing mouse tumor. This NC1 domain has been compared to the NC1 of human glomerular basement membrane (hGBM) to assess its usefulness in the biochemical characterization of the Goodpasture antigen which is associated with NC1. Both NC1 isolates appeared to migrate by gel filtration as hexamers (Mr 160,000) and in SDS-polyacrylamide gel electrophoresis as dimers and monomers (Mr 54,000 and 26,000), and were shown to share biochemical identity by amino acid analysis. The hGBM NC1 showed greater complexity in the monomer region, and when compared by two-dimensional gel electrophoresis was found to contain more components in both regions than EHS NC1. Anti-GBM autoantibodies from patients with Goodpasture's syndrome reacted with the EHS NC1 by immunoblotting of two-dimensional gels. The EHS NC1 isolated by reverse phase HPLC partially inhibited the reactivity of the anti-GBM autoantibodies against hGBM NC1 by inhibition ELISA assay. Reverse phase HPLC elution of EHS and hGBM NC1 showed differences in subunit composition and interaction; complete dissociation of the EHS monomers and dimers in 0.1% trifluoroacetic acid was observed, whereas hGBM monomers and dimers eluted together. Rotary shadowing of hGBM NC1 domains revealed size heterogeneity of globular domains, compared with greater uniformity of EHS NC1 hexamers. We conclude that EHS NC1 contains an epitope(s) that is reactive with human autoantibodies to hGBM NC1. However, the immune response in Goodpasture's syndrome may involve antibodies directed against epitopes which are present in greater density and on a more complex array of peptides in the hGBM NC1 than in EHS NC1.

Recurrent diabetes mellitus in the pancreas iso- and allograft. A light and electron microscopic and immunohistochemical analysis of four cases.

Four patients with type 1 diabetes mellitus received segmental pancreatic grafts. The donors were HLA-identical twins in three patients and an HLA-identical sibling in one. Each patient had normal glucose metabolism in the posttransplantation period but impaired graft function developed after 6 to 12 weeks. Complete loss of function developed in three patients. The fourth patient received immunosuppressive therapy but continues to require a low dose of insulin 15 months following transplantation. Pancreatic graft biopsy at the time of declining graft function in three patients revealed a mononuclear cell infiltrate centered upon islets consisting of variable numbers of T11 (pan T), OKT8 (suppressor-killer), OKT9 (transferrin receptor), OKT10 (activated), and HLA-DR-reactive mononuclear cells, as well as 63D3 and OKM1 reactive monocytes. Biopsies obtained following loss of graft function revealed resolution of the inflammatory process and selective destruction of all islet beta-cells in two patients, whereas graft biopsy in one patient demonstrated a mononuclear cell infiltrate in islets containing demonstrable beta-cells but no infiltrate in islets without beta-cells. Following immunosuppressive therapy the fourth patient showed resolution of the insulitis and destruction of beta-cells in 70% of the islets. The variable numbers of beta-cells observed in the remaining islets likely account for the relatively low amount of exogenous insulin required by this patient. There was no immunohistologic evidence of humoral mediated immune reaction in any of the biopsies. It is postulated that selective beta-cell destruction was a consequence of cell-mediated immunity leading to recurrent diabetes mellitus.

Enzyme immunoassay of anti-glomerular basement membrane antibodies.

An immunoassay has been developed to detect anti-glomerular basement membrane (GBM) antibodies in human sera. Various plating conditions, types of microtiter plates, and the use of biotinylated or peroxidase-labeled secondary antibodies were examined. The described assay is reliable, fast, and convenient. Sera with positive reactivity in anti-GBM nephritis and Goodpasture's syndrome are readily discriminated from sera obtained from normal individuals or patients with a variety of other diseases.

Urinary albumin excretion in renal transplant donors.

Twenty-four hour urinary albumin excretion was measured using sensitive methods in 129 renal transplant donors 1 to 22 years (mean 7.3 years) after kidney donation. The 24 hour urinary albumin excretion values for the donor group was 6 +/- 9.9 mg (mean +/- standard deviation) (range of 0.1 to 65.2 mg) and was 7.7 +/- 4.5 mg for the control group. Five donors (3.9 percent) had 24 hour urinary albumin excretion levels of 25.5 to 65.2 mg 6 to 13 years after donation, values that were greater than a single value from any member of the control group. Elevated diastolic blood pressure (90 mm Hg or greater) was present in these five donors, and in three, labile or established hypertension was present at the time of donation. It is likely that more careful screening of potential donors with labile or fixed hypertension would further reduce the incidence of microalbuminuria in the renal transplant donor. We conclude that urinary albumin excretion values are within the normal range in most renal transplant donors studied several years after renal donation.

Idiopathic immune complex glomerulonephritis in dogs with multisystem involvement.

Renal specimens obtained by biopsy and/or at necropsy from 4 dogs with nephrotic syndrome were studied using light, immunofluorescence, and electron microscopies. The glomerulonephritis observed in these dogs was considered an idiopathic immune complex glomerulonephritis associated with multisystem involvement because causes of glomerulonephritis in these dogs could not be established. Immunoglobulin A was observed in granular deposits in the mesangial and subendothelial regions of the glomeruli. The relationship of the clinical and pathologic features of this disease in dogs to various renal syndromes in human beings are described.

No evidence for a specific role of IgM in mesangial proliferation of idiopathic nephrotic syndrome.

To define the relationship of mesangial IgM to various morphologic categories of idiopathic nephrotic syndrome (INS), an analysis of 100 patients was carried out in which five morphologic subgroups were evaluated: group 1, minimal glomerular change (38 patients); group 2, minimal change with focal global sclerosis (18 patients); group 3, focal segmental glomerulosclerosis ( FSG ) (23 patients); group 4, mesangial proliferation (12 patients); group 5, focal segmental glomerulosclerosis with mesangial proliferation (9 patients). Immunohistochemical studies failed to demonstrate any differences between these five groups. The intensities of immunofluorescence and the percentage of tissue samples demonstrating IgM and/or C3 in the glomerular mesangium and subendothelial regions were similar. In addition, the presence or intensity of mesangial IgM did not predict the patients' current status or responsiveness to steroid therapy. Morphologic transitions were observed in patients who had more than one biopsy: one of five in group 2 and two of eight in group 3 developed mesangial proliferation; and nine of ten patients with mesangial proliferation in the first biopsy continued to show this finding in the second. In general, a complete response to steroid therapy and a favorable outcome is less likely in patients with this morphologic abnormality. In nine of the 27 repeat biopsies, there was lack of agreement between the first and second tissue samples with respect to the presence or absence of mesangial IgM. Although mesangial proliferation is a consistent feature of the morphology of certain patients with INS, these studies do not support a unique association with mesangial IgM.