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Biomedical subjects

M Kashgarian

Publications and source records attributed to M Kashgarian.

At least 37 records · Page 2Linked to original sources
PubMed1998

Antibodies against mesangial cells and their secretory products in chronic renal allograft rejection in the rat.

Antibodies or cell-mediated immunity can cause chronic rejection of vascularized organ grafts, but the nature and specificity of the antigen(s) involved has remained elusive. We have previously demonstrated the presence of antibodies against cryptic glomerular basement membrane antigens and undefined antigens in the mesangial area in rats with chronic renal allograft rejection. Current experiments were designed to study the post-transplant antibody response against cultured mesangial and endothelial cells in rats with chronic rejection using flow cytometry, indirect immunofluorescent staining, immunoelectron microscopy, confocal microscopy, and Western blots. The results were compared with those obtained with alloantisera raised by immunization with cultured mesangial cells. Post-transplant and post-immunization sera contained IgG antibodies against trypsinized mesangial cells detected by flow cytometry. Indirect immunofluorescent studies using mesangial cells grown on coverslips showed autoantibody binding to cytoplasmic granules in cultures early after plating whereas staining of later cultures showed antibody binding in an interrupted, web-like pattern on the outside of the cells. Immunoelectron microscopy showed autoantibody binding to intracellular secretory granules and to cell surface focal adhesion plaques. The latter finding was confirmed in double-labeling experiments with an antiserum against vinculin. Western blots with mesangial cell culture supernatants demonstrated autoantibody reactivity with antigens in the 40-kd and 60- to 70-kd range, and immunoprecipitation identified these molecules as biglycan and decorin. Absorption of the sera with mesangial cell culture supernatant removed most of the antibodies except those that gave a punctate staining with the mesangial cell surface. However, not all immunostaining of mesangial cells could be explained by antibodies against biglycan and decorin. Post-transplant sera, furthermore, contained low-titered antibodies against endothelial cells. We conclude that rats with chronic renal transplant rejection produce a strong autoantibody response against mesangial cell focal adhesion plaques and proteins secreted by these cells in culture. Such antibodies may cause local damage and interfere in the tissue repair process after injury.

Animals↗
PubMed1998

Indinavir nephropathy in an AIDS patient with renal insufficiency and pyuria.

Indinavir has been described to cause crystalluria and nephrolithiasis in a variable number of treated patients. Acute renal failure, often reversible with discontinuation of the medication, induction of a diuresis and correction of urinary obstruction if present, occurs in a smaller percent of patients. One recent report described renal biopsy findings, indinavir crystals within cellular casts in the collecting tubules, in a patient receiving this antiretroviral agent. We report a second case of a patient with mild renal insufficiency and pyuria following indinavir therapy and describe similar renal biopsy findings.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Sorting of P-type ATPases in polarized epithelial cells.

The Na,K-ATPase and the H,K-ATPase are highly homologous members of the P-type family of ion transporting ATPase. Despite their structural similarity, these two pumps are sorted to different destinations in polarized epithelial cells. While the Na,K-ATPase is restricted to the basolateral surfaces of most epithelial cells types, the H,K-ATPase is concentrated at the apical plasmalemma and in a pre-apical vesicular storage compartment in the parietal cells of the stomach. We have generated molecular chimeras composed of complementary portions of these two pumps' alpha-subunits. By expressing these pump constructs in polarized epithelial cells in culture, we have been able to identify sequence domains which participate in the targetting of the holoenzyme. We find that information embedded within the sequence of the fourth transmembrane domain of the H,K-ATPase is sufficient to account for this protein's apical localization. Stimulation of gastric acid secretion results in insertion of the intracellular H,K-ATPase pool into the apical plasma membrane and inactivation of acid secretion is accompanied by the re-internalization of these pumps. We have identified a tyrosine-based signal in the cytoplasmic tail of the H,K-ATPase beta-subunit which appears to be required for this endocytosis. We have mutated the critical tyrosine residue to alanine and expressed the altered protein in transgenic mice. The H,K-ATPase remains continuously at the apical cell surface in parietal cells from these animals, and they constitutively hypersecrete gastric acid. These results demonstrate that the beta-subunit sequence mediates the internalization of the H,K-ATPase and is required for the cessation of gastric acid secretion. Thus, at least two sorting signals are required to ensure the proper targetting and regulation of the gastric H,K pump.

Animals↗
PubMed1997

A tyrosine-based signal targets H/K-ATPase to a regulated compartment and is required for the cessation of gastric acid secretion.

Gastric acid secretion is mediated by the H/K-ATPase of parietal cells. Activation of acid secretion involves insertion of H/K-ATPase into the parietal cell plasmalemma, while its cessation is associated with reinternalization of the H/K-ATPase into an intracellular storage compartment. The cytoplasmic tail of the H/K-ATPase beta subunit includes a four residue sequence homologous to tyrosine-based endocytosis signals. We generated transgenic mice expressing H/K-ATPase beta subunit in which this motif's tyrosine residue is mutated to alanine. Gastric glands from animals expressing mutant beta subunit constitutively secrete acid and continuously express H/K-ATPase at their cell surfaces. Thus, the beta subunit's tyrosine-based signal is required for the internalization of H/K-ATPase and for the termination of acid secretion. As a consequence of chronic hyperacidity, the mice develop gastric ulcers and a hypertrophic gastropathy resembling Menetrier's disease.

Animals↗
PubMed1997

Regulation of colonic H-K-ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion.

Active K absorption in the rat distal colon is energized by an apical membrane H-K-ATPase, whereas K absorption in the distal collecting duct is generally believed to be modulated by a related renal H-K-ATPase. Experiments were performed to establish the mechanism(s) by which dietary Na depletion (with resulting elevated aldosterone levels) and K depletion stimulate K absorption. A colonic H-K-ATPase-specific cDNA probe and a polyclonal antibody were utilized to measure mRNA (Northern blot analyses) and protein (Western blot and immunofluorescence studies) abundance in the distal and proximal colon and renal collecting ducts and cortex of dietary Na- and K-depleted rats. Dietary Na depletion, but not K depletion, upregulated H-K-ATPase-specific mRNA and protein expression in the distal and proximal colon; Na depletion also stimulated H-K-ATPase activity in the distal colon. In contrast to the distal colon, H-K-ATPase-specific protein level in the outer medulla was enhanced by dietary K depletion, but not by Na depletion. This study establishes that 1) dietary Na depletion stimulates colonic H-K-ATPase activity most likely by a transcriptional process and 2) the regulation of colonic H-K-ATPase expression by dietary Na depletion and dietary K depletion is not identical in the large intestine and differs in the kidney from the colon, suggesting the presence of two (or more) H-K-ATPase isoforms in the rat colon.

Animals↗
PubMed1997

Evidence that tumor necrosis factor triggers apoptosis in human endothelial cells by interleukin-1-converting enzyme-like protease-dependent and -independent pathways.

Cultured human endothelial cells (EC) resist tumor necrosis factor (TNF)-mediated apoptosis. However, the combination of TNF and the protein synthesis inhibitor cycloheximide (CHX) induces apoptosis in up to 50% of EC within 24 hours. TNF + CHX killing is effectively blocked by transfected CrmA protein or treatment with Z-VAD.fmk peptide-both inhibitors of interleukin-1-converting enzyme-like proteases-but not by transfected antiapoptotic proteins Bcl-2, Bcl-XL, or A1. C6-ceramide (cer) can also sensitize EC to TNF-induced apoptosis. TNF + cer killing, which can affect more than 50% of EC, is not effectively inhibited by CrmA or Z-VAD frank, but can be readily blocked by Bcl-2, Bcl-XL, or A1. Both TNF + CHX and TNF+ cer killing are induced by a TNF mutein that only interacts with the type 1 TNF receptor, and both responses can be inhibited by the antiapoptotic protein A20. These data suggest that TNF activates two biochemically separable pathways of EC injury.

Apoptosis↗
PubMed1996

Identification of a small cytoplasmic ankyrin (AnkG119) in the kidney and muscle that binds beta I sigma spectrin and associates with the Golgi apparatus.

Ankyrins are a family of large, membrane-associated proteins that mediate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. A repetitive 33-residue motif characteristic of domain I of ankyrin has also been identified in proteins involved with cell cycle control and development. We have cloned and characterized a novel ankyrin isoform, AnkG119 (GenBank accession No. U43965), from the human kidney which lacks part of this repetitive domain and associates in MDCK cells with beta I sigma spectrin and the Golgi apparatus, but not the plasma membrane. Sequence comparison reveals this ankyrin to be an alternative transcript of AnkG, a much larger ankyrin recently cloned from brain. AnkG119 has a predicted size of 119,201 D, and contains a 47-kD domain I consisting of 13 ankyrin repeat units, a 67-kD domain II with a highly conserved spectrin-binding motif, and a truncated 5-kD putative regulatory domain. An AnkG119 cDNA probe hybridized to a 6.0-kb message in human and rat kidney, placenta, and skeletal muscle. An antibody raised to AnkG119 recognized an apparent 116-kD peptide in rat kidney cortical tissue and MDCK cell lysates, and did not react with larger isoforms of ankyrin at 190 and 210 kD in these tissues, nor in bovine brain, nor with ankyrin from human erythrocytes. AnkG119 remains extractable in 0.5% Triton X-100, and assumes a punctuate cytoplasmic distribution in mature MDCK cells, in contrast to the Triton-stable plasma membrane localization of all previously described renal ankyrins. AnkG119 immunocreativity in subconfluent MDCK cells distributes with the Golgi complex in a pattern coincident with beta -COP and beta I sigma spectrin immunoreactivity. A fusion peptide containing residues 669-860 of AnkG119 interacts with beta I sigma 1 spectrin in vitro with a Kd = 4.2 +/- 4.0 ( +/- 2 SD) nM, and avidly binds the beta spectrin in MDCK cell lysates. Collectively, these data identify AnkG119 as a novel small ankyrin that binds and colocalizes with beta I sigma spectrin in the ER and Golgi apparatus, and possible on a subset of endosomes during the early stages of polarity development. We hypothesize that AnkG119 and beta I spectrin form a vesicular Golgi-associated membrane skeleton, promote the organization of protein microdomains within the Golgi and trans-Golgi networks, and contribute to polarized vesicle transport.

Amino Acid Sequence↗
PubMed1996

Na-K-ATPase alpha-isoform expression in heart and vascular endothelia: cellular and developmental regulation.

The Na pump (Na-K-ATPase) is important for regulation of membrane potential and transport in smooth muscle and heart. The alpha (catalytic)-subunit of this pump has three isoforms: alpha 1 is ubiquitous, but alpha 2 and alpha 3 are mainly localized to excitable tissue. Physiological differences between isoforms are not completely understood, but alpha 3 pumps appear to have a lower affinity for intracellular Na and a higher ouabain affinity than alpha 1 pumps. The alpha 2-and alpha 3-isoform mRNAs are expressed at high levels in the normal adult rat cardiac conduction system. Although alpha 1 and alpha 3 are both globally expressed in neonatal rat myocardia, there is a switch in the myocardial isoform pattern from alpha 3 to alpha 2 after birth. There are also important species differences in cardiac isoform patterns. Furthermore, changes in Na-K-ATPase isoforms in heart and vascular tissue have been reported in association with hypertension, but little is known about isoform expression in normal endothelia. We therefore studied the cellular distribution of Na pump protein isoforms in neonatal and adult myocardia and endothelia. Immunohistochemical analysis of rat tissues showed that the alpha 1-isoform was expressed throughout atrial and ventricular myocardium, with alpha 1 the only isoform detectable in the adult t tubule system. Although alpha 2 was also present in ventricular myocytes, the signal was markedly stronger in conduction tissue and papillary muscle. In hearts from neonatal rats, the alpha 3-isoform predominated in the cardiac conduction system, whereas alpha 2 was not detectable in any structure except vascular endothelium. In tissues and in cell lines representing a variety of species and vessel sizes, endothelia of large vessels expressed primarily alpha 1, whereas alpha 2 could be detected in endothelia of small vessels in rat heart. No evidence of alpha 3 expression in endothelium was found. Thus the complex spatial and developmental regulation of Na pump isoform expression in cardiovascular tissues may provide additional correlates to distinct physiological roles of these transporters.

Aging↗
PubMed1996

The alpha3 isoform protein of the Na+, K(+)-ATPase is associated with the sites of cardiac and neuromuscular impulse transmission.

The alpha (catalytic) subunit of the Na+ pump (Na+, K(+)-ATPase) has three isoforms; alpha1 is ubiquitous, skeletal muscle expresses predominantly alpha2, and alpha3 has been localized to specific types of neurons and, possibly, to axonal processes. The alpha3 isoform mRNA is also expressed in the rat cardiac conduction system. Thus, we studied rat heart and quadriceps muscles by immunohistochemistry using isoform-specific antibodies to the Na+ pump alpha subunit and labeled alpha-bungarotoxin as a probe for the neuromuscular junction (NMJ). We found that alpha3 pump protein is localized to three sites important for impulse transmission: the junctional complex between cardiac myocytes, the heart conduction system, and the NMJ. Specifically, all levels of the conduction system expressed alpha3 immunoreactive protein, as assessed by two isoform-specific antibodies and histological conduction system markers. Specific expression at the junctional complex was confirmed by immuno-EM. Double-labeling and denervation analysis indicated that alpha3-positive areas in skeletal muscle were presynaptic and adjacent to postsynaptic bungarotoxin-positive regions, which had the classic morphology of NMJs. Thus, specific Na+,K(+)-ATPase pump isoforms may be adapted to maintenance of membrane potential and/or intracellular ion concentrations required for impulse transmission in both heart and presynaptic motor terminals contacting skeletal muscle.

Animals↗
PubMed1996

The role of the kidney biopsy in the treatment of lupus nephritis.

The renal biopsy plays an important role in the clinical evaluation of patients with lupus erythematosus. From numerous studies of renal biopsies in lupus nephritis, it has become clear that although the renal lesions are quite varied, the pattern of renal involvement correlates with the clinical outcome. The broad spectrum of lesions seen in lupus nephritis has been attributed to individual differences in the immune response in different patients or in the same patient during the course of disease. Classification of lupus nephritis authorized by the World Health Organization has provided a standardized approach to the findings on renal biopsy. This classification combines all of the morphologic modalities of biopsy interpretation including light, immuno-fluorescence, and electron microscopy as well as providing a semiquantitative assessment of activity and chronicity. The clinical correlations utilizing this histologic classifications have demonstrated that the specific nature of the renal histopathology can predict both the acute and long-term outcome of the renal disease in patients with lupus erythematosus and can be useful in determining the management of individual patients.

Animals↗
PubMed1995

A unique subset of rat and human intestinal villus cells express the cystic fibrosis transmembrane conductance regulator.

BACKGROUND/AIMS: In the intestine, the cystic fibrosis transmembrane conductance regulator (CFTR) has been localized to the apical pole of crypt epithelial cells. Recent data indicate that some villus cells may also express CFTR, although the identity of these cells has not been established. The aim of the current study was to characterize the distribution, morphology, and surface marker expression of CFTR-expressing villus cells. METHODS: Immunofluorescence and immunoelectron microscopy was performed using anti-CFTR and enzyme marker antibodies. RESULTS: In the rat and human proximal small intestine, a subpopulation of scattered villus and superficial crypt epithelial cells label brightly with anti-CFTR antibodies. The fluorescent signal is detected throughout the cells with its greatest concentration apically. At the ultrastructural level, labeling involves the brush border and a prominent subapical vesicular compartment. The cells resemble adjacent villus enterocytes in their abundance of mitochondria and expression of basolateral Na(+)-K(+)-adenosine triphosphatase yet differ in their absence of brush-border sucrase and lactase expression. CONCLUSIONS: A previously uncharacterized subpopulation of villus cells with high levels of intracellular CFTR expression exists in the proximal small intestine. Morphological and cytochemical studies suggest that this subset of villus cells has a unique transport function.

Animals↗
PubMed1995

Heat-insoluble cryoglobulin in a patient with essential type I cryoglobulinemia and massive cryoglobulin-occlusive glomerulonephritis.

We report a case of type I essential cryoglobulinemia with massive cryoglobulin-occlusive glomerulonephritis, in which the clinical course and the physical characteristics of the cryoglobulin were unusual. Nine years before appearance of cryoglobulin, this 54-year-old man noted edema and purpura of the lower extremities. Renal biopsy performed 2 years later showed large amounts of amorphous, weakly eosinophilic, weakly periodic acid-Schiff (PAS)-positive materials occluding the glomerular capillaries. Immunostaining showed the material to be weakly immunoglobulin (Ig) G positive, and electron microscopy showed homogeneous, electron-dense deposits. Nephrotic syndrome and azotemia did not respond to steroid treatment, and dialysis was begun 5 years after the biopsy. A small amount of cryoglobulin was first detected 2 years later, 9 years after the onset of disease. The cryoglobulin had a white gelatinous appearance, was resistant to resuspension, and did not redissolve when rewarmed to 37 degrees C. Immunoelectrophoresis of the cryoglobulin, which partially dissolved at 54 degrees C, showed it to be composed of monoclonal IgG-kappa and a small amount of albumin. We consider that the unusual physical characteristics of the cryoglobulin in this case precipitated a massive cryoglobulin-occlusive glomerulonephritis, which progressed to end-stage renal failure in the absence of significant cryoglobulinemia during the initial onset of disease.

Cryoglobulinemia↗
PubMed1995

PDGF and TGF-beta mediate collagen production by mesangial cells exposed to advanced glycosylation end products.

Deposition of type III collagen protein is increased when cultured rat mesangial cells are cultured in media containing high glucose concentrations. Possible mechanisms for this effect include the production of growth factors, such as PDGF and TGF-beta, and the formation of abnormal glucose-protein adducts called advanced glycosylation end products (AGEs). In our studies, neutralizing antibodies to PDGF and TGF-beta prevented increased type III collagen deposition by mesangial cells exposed either to high glucose media or to low glucose media containing AGEs. Daily addition of PDGF or TGF-beta stimulated type III collagen production. However, while co-incubation with the TGF-beta Ab prevented PDGF-stimulated type III collagen production, the PDGF Ab did not prevent TGF-beta-stimulated type III collagen production. Daily addition of PDGF or TGF-beta stimulated, while AGEs inhibited, mesangial cell proliferation after 96 hours. We propose that high extracellular glucose and AGEs stimulate type III collagen production by pathways that involve the intermediate formation of PDGF and TGF-beta by mesangial cells. PDGF may increase type III collagen production by stimulating the intermediate production of TGF-beta. Exposure to high glucose, AGEs, or TGF-beta also leads to impaired mesangial cell proliferation. The autocrine effects of TGF-beta and PDGF play important roles in the effects of high extracellular glucose and AGEs on cultured mesangial cells.

Analysis of Variance↗
PubMed1995

Periodic high extracellular glucose enhances production of collagens III and IV by mesangial cells.

We examined the effects of periodic changes in extracellular glucose concentration on matrix production and proliferation using three groups of cultured rat mesangial cells (MCs): 1) MCs in medium with continuous 5 mM glucose (CL), 2) MCs in medium alternating daily between 5 and 25 mM glucose (PH), and 3) MCs in medium with continuous 25 mM glucose (CH). MCs cultured in PH for 10 days produced 329 and 110% more type III collagen protein than MCs cultured in CL and CH, respectively. MCs cultured in PH induced 31 and 14% more type IV collagen than MCs cultured in CL and CH, respectively. Extracellular glucose concentration had no effect on the amount of type I collagen produced. MCs cultured in PH or CH for 5 days also expressed increased levels of type I, III, and IV collagen mRNA compared with MCs cultured in CL. MCs cultured in PH for 8-10 days also produced significantly more DNA than MCs in CL or CH. These data suggest that the temporal pattern of exposure to high extracellular glucose plays a role in regulating matrix formation and cellular proliferation by MCs. Furthermore, periodic elevations of extracellular glucose had a greater stimulatory effect on collagen production than a sustained elevation. These results suggest that decreasing the variability of blood glucose concentration may decrease the adverse effect of elevated glucose levels on MC matrix production and the progression of diabetic glomerulopathy.

Analysis of Variance↗