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Biomedical subjects

Y S Kanwar

Publications and source records attributed to Y S Kanwar.

At least 19 recordsLinked to original sources
PubMed1992

Selective decreased de novo synthesis of glomerular proteoglycans under the influence of reactive oxygen species.

The effect of reactive oxygen species on de novo synthesis of heparan sulfate proteoglycans (HSPGs) of the renal glomerulus was investigated in an organ perfusion system. Isolated kidneys were perfused for 7 hr with a medium containing [35S]sulfate to label sulfated proteoglycans or [35S]methionine to label total glomerular glycoproteins. For the generation of reactive oxygen species, xanthine and xanthine oxidase were included in the perfusion medium, and catalase and superoxide dismutase were used as scavenging agents. Proteoglycans were characterized by Sepharose CL-6B and DEAE-Sephacel chromatographies and SDS/PAGE analysis. The labeled glycoproteins were immunoprecipitated with anti-HSPG, anti-type IV collagen, and anti-laminin, and their specific radioactivities were determined. With exposure to reactive oxygen species, a drastic dose-dependent decrease in de novo synthesis of proteoglycans was seen, and that effect was reversible by catalase treatment. No alterations in the biochemical characteristics of proteoglycans were noted. Immunoprecipitation studies revealed a 16-fold decrease in the synthesis of nascent core peptide of HSPGs, while at comparable concentrations of xanthine and xanthine oxidase, synthesis of type IV collagen and laminin slightly decreased (approximately 15%). Morphologic studies revealed a 14-fold decrease in [35S]sulfate-associated autoradiographic grains overlying the glomerular basement membrane, a critical component of the ultrafiltration apparatus. Relevance of the selective decreased de novo synthesis of HSPGs of the glomerular basement membrane is discussed in terms of increased glomerular permeability to plasma proteins.

Animals↗
PubMed1992

Modulation of glomerular proteoglycans by insulin-like growth factor-1.

Effect of insulin-like growth factor-1 (IGF) on the synthesis of glomerular proteoglycans (PGs) in an ex vivo recirculating organ perfusion system was investigated. Kidneys were perfused with a medium (approximately 80 ml) containing [35S]-sulfate (250 microCi/ml) and IGF (62.5 to 625 ng/ml). After radiolabeling, a small cortical piece was saved for tissue autoradiography, and the remaining kidney and the perfusion medium were utilized for biochemical studies. The glomeruli were isolated; their PGs extracted and characterized. A two- to threefold increase of the total radioactivities in tissue and media fractions was observed with the exposure to IGF. By Sepharose CL-6B chromatography, the tissue PGs eluted as two peaks (A and B) with Kav = 0.24 and 0.48, and the majority of the radioactivity was confined to peak A. This peak contained intact PGs while peak B included glycosaminoglycan (GAG) chains. Elution profiles of the glomerular PGs were similar in the control and IGF groups. However, there was a disproportionate increase of chondroitin/dermatan sulfate in the IGF group. The media fractions also had two peaks, and most of the radioactivity was associated with peak B containing GAG chains. A remarkable accentuation of peak B along with significant increase in the chondroitin/dermatan sulfate were observed in the IGF group. By DEAE-Sephacel chromatography, the PGs/GAGs of IGF group eluted at a relatively lower salt concentration as compared to the control. Autoradiography revealed a relatively high concentration of radioactivity over the mesangium as compared to the other cell types of the glomerulus. [35S]-methionine studies revealed a generalized increase of protein synthesis in the IGF group, but comparatively much less than that of PGs/GAGs. These results indicate that IGF enhances the biosynthesis of PGs/GAGs by various cell types of the renal glomerulus, especially that of the mesangial cell, as reflected by the selective increase of chondroitin/dermatan sulfate.

Animals↗
PubMed1992

Altered synthesis of proteoglycans by cyst-derived cells from autosomal-dominant polycystic kidneys.

Normal human renal epithelial cells (NK) and cells from cysts of autosomal-dominant polycystic kidneys (ADPKD) were radiolabeled with [35S]sulfate. A two- to three-fold decrease in the radioactivity incorporated into the proteoglycan (PG) fraction, as ascertained by tissue autoradiography and biochemical techniques, was observed in the ADPKD group. In subconfluent NK cells, PGs eluted as two peaks with different proportions of chondroitin sulfate (CS) and heparan sulfate (HS) in the cellular and media fractions. In the confluent stage, only a single major peak in the media and matrix fractions was seen and had variable proportions of CS and HS. In subconfluent ADPKD monolayers, cellular PGs eluted as two peaks, with the major peak of higher molecular weight compared with NK cells. In confluent stage, there was a single PG peak of a relatively higher molecular weight, with a variable increase in the proportions of CS vs. HS and lower charge-density characteristics. These findings indicate that size and species of PGs vary during subconfluent and confluent stages of culture and elucidate a defect in the biosynthesis of PGs in human ADPKD cells.

Autoradiography↗
PubMed1992

Mannose-induced dysmorphogenesis of metanephric kidney. Role of proteoglycans and adenosine triphosphate.

Because various fetal anomalies are seen in diabetic offspring, we examined the effects of sugars on proteoglycans (PGs): extracellular matrix (ECM) macromolecules modulating morphogenesis. 13-d-old mouse metanephric kidney explants were exposed to mannose for 7 d and labeled with [35S]sulfate, [35S]-methionine, or [3H]thymidine. Mannose exposure caused reduction in kidney size and disorganization of ureteric bud branches with inhibition of glomerulogenesis. Tissue autoradiographic and immunofluorescence studies indicated decreased expression of sulfated PGs in ECMs. Helix pomatia lectin binding to D-GalNAc residues of glomerular epithelial cells was also reduced. Biochemical studies revealed decreased synthesis of sulfated PGs. PGs were of lower molecular weight with reduced charge density and increased chondroitin/heparan sulfate ratio. Immunoprecipitation of [35S]methionine-labeled proteins confirmed the reduction of PG core peptides. Intracellular ATP levels were reduced. The addition of 0.1 mM ATP to culture media restored kidney size, the population of glomeruli, and the synthesis and characteristics of PGs to almost normal, with no detectable effect on the replication of cells as determined by [3H]thymidine incorporation. The effect of ATP could be partially blocked by the P2y-purinoreceptor, i.e., reactive blue-2. Data suggest that mannose causes energy depletion by cellular ATP consumption and thus selectively alters the synthesis of heavily glycosylated proteins with rapid turnover, such as PGs, resulting in renal dysmorphogenesis.

Adenosine Triphosphate↗
PubMed1992

Altered synthesis and intracellular transport of proteoglycans by cyst-derived cells from human polycystic kidneys.

Employing in vitro pulse-chase techniques, we investigated the de novo synthesis and the kinetics of intracellular transport and extracellular matrix incorporation of proteoglycans (PG) by normal human renal epithelial cell and by epithelial cells isolated from cysts of autosomal dominant kidneys (ADPKD). Cell monolayers were pulsed either with (3H)leucine for 15 min and chased for seven intervals between 15 and 270 min or with (35S)sulfate for 150 min and chased for a single interval of 120 min. Total proteins and PG were isolated from cell, media, and matrix fractions and characterized by Sepharose CL-6B and DEAE-Sephacel chromatographies. ADPKD and NK cells synthesized comparable amounts of total proteins; however, the de novo synthesis of PG by ADPKD cells was significantly reduced. ADPKD versus NK cells exhibited a substantial delay in the cellular transport and extracellular release of de novo synthesized PG, indicating an impairment at the level of the Golgi complex and/or secretory vacuoles. PG synthesized by ADPKD versus NK cells had decreased charge density characteristics, probably due to a posttranslational defect in the sulfation of the PG glycosaminoglycan chains. ADPKD versus NK cells synthesized PG of higher molecular weight and had an increased proportion of chondroitin sulfate PG versus heparan sulfate PG. Collectively, these findings suggest a defect in the synthesis and intracellular transport of sulfated PG in human ADPKD cells.

Biological Transport, Active↗
PubMed1992

Interleukin-1-induced alterations in glomerular proteoglycans: biochemical and tissue autoradiographic aspects.

The effect of interleukin-1 alpha (IL-1) on the synthesis of glomerular basement membrane heparan sulfate-proteoglycan (HS-PG) was investigated. An ex vivo recirculating organ perfusion system was used. Kidneys were perfused with a medium containing (35S) sulfate and IL-1 (0.625 to 6.25 ng/mL). After radiolabeling was performed, a small cortical piece was saved for tissue autoradiography; the remaining kidney and perfusion medium were used for biochemical studies. Renal cortices were dissected out, and glomeruli were isolated; the PG were extracted and characterized. With exposure to IL-1 (5 ng/mL), an approximately twofold increase of the radioactivity in the glomerular fraction was noted. The increase was unaffected by indomethacin treatment. By Sepharose CL-6B chromatography, a single peak of radioactivity with Kav = 0.25 (macromolecular form of PG) was observed in the control group. The IL-treated group had two peaks of radioactivity with Kav = 0.25 and 0.45: the first peak contained PG, and the second peak consisted of free glycosaminoglycan (GAG) chains. Elution profiles of hydrolyzed tissue GAG chains were similar. No change in the ratio of chondroitin to heparan sulfate was observed. By DEAE-Sephacel chromatography, the glomerular PG/GAG of the IL-treated group eluted at a relatively lower salt concentration, suggesting a change in the charge density characteristics. No differences in the elution profiles of media PG/GAG were observed. (35S)methionine labeling of proteins showed no significant increase of the total radioincorporation in the IL-treated group. Immunoprecipitation studies revealed an approximately 88% increase in the de novo synthesis of PG. Tissue autoradiography revealed an approximately twofold increase of (35S) sulfate radioactivity over the glomerular mesangium, epithelium, and basement membranes. These results indicate that IL-1 enhances the synthesis of the macromolecular form of PG and the generation of free chains. Conceivably, such alterations may lead to defective macromolecular interactions among various components of the glomerular basement membrane and compromise the integrity of the ultrafiltration unit of the glomerulus.

Animals↗
PubMed1992

Sequential tubular cell and basement membrane changes in polycystic kidney disease.

Tubular basement membrane (BM) changes (dysmorphogenesis), cell proliferation, and fluid accumulation related to the altered location of Na,K-ATPase are purported essential key events in the development and progression of renal cysts. These changes were assessed daily in Phenol II (2-amino-4-hydroxyphenyl-5-phenyl thiazole)-treated rats, which rapidly develop marked and progressive cystic change of all collecting tubules (CT). At Day 1, 12% of CT were cystic and their BM were thickened severalfold. At Day 4, 30% of CT were cystic and their BM remained thickened. BM of cystic tubules showed decreased staining for heparan sulfate proteoglycan and increased staining for fibronectin. Proliferation, as determined by (3H)thymidine, incorporation, was not significant until Day 2 and involved cystic and noncystic tubular cells as well as interstitial cells. As cystic changes progressed, cell proliferation decreased. By immunohistochemistry, the altered location of Na,K-ATPase in epithelial cells lining cysts was primarily detected after Day 2 and consisted of focal loss from basal and/or lateral cell membranes and localization in the cell cytoplasm. Only rarely was Na,K-ATPase localized to the apical cell membrane. After the removal of Phenol II, cystic tubular cells, BM, and Na,K-ATPase returned to normal. Thus, in this model of polycystic kidney disease, initial cyst formation occurred in tandem with BM structural change whereas cell proliferation and altered location of Na,K-ATPase occurred after the appearance of cysts.

Animals↗
PubMed1992

Nephritogenicity of anti-proteoglycan antibodies in experimental murine lupus nephritis.

BACKGROUND: Cross-reactivity between anti-DNA antibodies and heparan sulfate (HS)/heparan sulfate-proteoglycan (HS-PG) of glomerular basement membrane has been previously reported. Conceivably, this determines the final outcome of glomerular injury in lupus nephritis. EXPERIMENTAL DESIGN: We investigated the status of glomerular injury in NZB/NZW F1 mice after the administration of rabbit anti-HS-PG antibody (experiment group). The controls received normal rabbit IgG only. RESULTS: All experimental animals became proteinuric 2 weeks after the administration of anti-HS-PG. The animals of the older age group (16 weeks) had significant hematuria as well. Their glomeruli exhibited hypercellularity with a heavy influx of polymorphonuclear leukocytes and monocytes into their capillaries, and some of them exhibited crescentic changes. Electron-dense deposits were present in subepithelial, subendothelial, and mesangial regions of the glomeruli. The control group had normocellular glomeruli with a few mesangial deposits. Mouse IgG and C3 displayed a granular pattern of immunofluorescence in the experimental group. Anti-rabbit IgG titers in the serum were higher in the control group, which lower in the renal glomerular eluates. No significant differences were observed in the concentrations of anti-dsDNA and -ssDNA either in the sera or in the eluates. There was also no difference between the control and experimental group in terms of antibody synthesis by the splenic lymphocytes and their proliferation subsequent to antigenic challenge. CONCLUSIONS: Data suggest that administration of anti-HS-PG accentuates the glomerular injury during the natural course of lupus nephritis in (NZB/NZW F1 mice; seemingly these two antibodies (anti-HS-PG and -DNA) do not competitively inhibit the binding of the other to the same anionic sites of glomerular basement membrane enriched with heparan sulfate in vivo.

Animals↗
PubMed1992

Influence of genetics on the nephritogenic potential of proteoglycans.

Nephritogenic potential of antibodies directed against one of the glomerular basement membrane (GBM) components, i.e., heparan sulfate-proteoglycan (HS-PG), was investigated in different strain of rats, i.e., Brown Norway, Lewis, Long Evans, and Sprague-Dawley. The rats were given two intravenous injections of anti-HS-PG antibody on days 1 and 3, and killed 2 to 8 weeks later. Before killing, blood and urine were collected for determination of anti-rabbit IgG levels and excretion of proteins, respectively. In addition, the right kidney was perfused with 125I-anti-rat IgG to quantitate the amount of immune-complexes present within the GBM. The tissues were processed for morphologic, autoradiographic, and immunofluorescent studies. The anti-HS-PG antibody was seen uniformly bound to GBM equally in all strains of rats. However, the protein-uric response was as follows: Brown Norway much much greater than Lewis much greater than Long Evans greater than Sprague Dawley. Also, the glomerular cells, monocytes in the glomerular capillaries, immunoreactivity of rat IgG and C3 frequency of subepithelial immune deposits, serum levels of anti-rabbit IgG, and the amount of 125I-anti-rat IgG bound to the GBM were proportionately increased among different strains of rats. The data suggest that the sustained presence of anti-HS-PG antibodies in the subepithelial aspect of the GBM with differential humoral response in the production of the antibody by the host most likely attributed to the variable glomerular damage in different strains of rats. Thus, it seems that the genetic makeup of a given strain of rat heavily influences the nephritogenic potential of an antibody and consequentially the outcome of the immune complex-mediated glomerular injury.

Animals↗
PubMed1991

Enzymatic isolation of chondrocytes from immature rabbit articular cartilage and maintenance of phenotypic expression in culture.

The studies included here identify factors affecting cartilage digestion by crude bacterial collagenase (cCGN) and describe a cartilage digestion medium that maximizes both tissue digestion rate and viable cell yield. The basal digestion medium contained 100 mM NaCl, 3 mM K2HPO4, 1 mM CaCl2, 1 mM MgSO4, 10 mM NaHCO3, 60 mM sorbitol, 5 mg/ml of dextrose, 1 mg/ml of albumin, and 2 mg/ml of cCGN in 25 mM HEPES at pH 7.2. Approximately 45% of articular cartilage tissue was digested in this basal medium in 6 h at 37 degrees C, yielding 6.8 x 10(6) viable cells per g tissue digested. The addition of 30 microM tosyllysylchloromethane (TLCM) increased the fraction of tissue digested in 6 h to 68% (p less than 0.05) and doubled viable cell yields to 13.6 x 10(6) per g tissue digested (p less than 0.05). Withholding Mg, decreasing NaCl to 70 mM, and adding 30 mM KCl increased fractional tissue digestion to 81% (p less than 0.01) and doubled viable cell yield yet again (to 29.9 x 10(6) viable cells per g tissue digested). Supplementation with TLCM increased the rate of cartilage digestion and the yield of viable cells regardless of cCGN source or lot. Additional trypsin (0.25%) inhibited tissue digestion and decreased cell yield; this effect was reversible with the addition of TLCM. The cartilage digestion medium developed in these studies (low Mg with added K and TLCM) was very effective in digesting articular, scapular, rib, and growth plate cartilage, as well as in yielding a large number of viable chondrocytes. These cells grew well in culture and maintained their chondrocytic characteristics, secreting predominantly type II collagen and large macromolecular forms of chondroitin sulfate-rich proteoglycans.

Animals↗
PubMed1991

Effect of puromycin on metanephric differentiation: morphological, autoradiographic and biochemical studies.

Effect of aminonucleoside of puromycin (PAN) on metanephric development and proteoglycans (PGs) was investigated. Murine metanephric tissues, obtained on the thirteenth day of gestation, were exposed to PAN in a culture medium for one to seven days and processed for morphological, histochemical and immunofluorescent studies. For tissue autoradiographic and biochemical studies, kidneys were labelled with a precursor product of PGs, that is, [35S]-sulfate. A generalized decrease in the glomerular population along with swelling and deformation in the ureteric bud branches was observed. These changes were accompanied with a diminution in the total incorporated radioactivity and a reduction in the autoradiographic grains, especially over the tips of ureteric bud branches. Sepharose CL-4B chromatography revealed a major high molecular weight PG (Mr greater than 2.5 x 10(6], and a relative increase in the chondroitinase-ABC sensitive PGs. The media PGs were of relatively smaller size. Immunoprecipitation experiments with [35S]-methionine-labeled tissues and immunofluorescent studies revealed a significant decrease of PGs in metanephric tissues, while type IV collagen and laminin were relatively unaffected. Significant glomerular changes included failure in differentiation of the visceral epithelial foot processes, formation of villi and in maturation of glomerular basement membrane. The latter was seen as fragments of extracellular matrices interspersed among undifferentiated podocytes and had reduced staining with ruthenium red--a dye marker for the PGs. This deficiency of PGs was confirmed by electron microscopic autoradiography, where a reduction in the number of silver grains was observed. The fact that the PAN-induced cellular and extracellular alterations were associated with perturbances in biosynthesis of PGs, suggests that the morphogenetic regulators, that is, PGs play a vital role in various differentiation processes involved during metanephric development.

Animals↗
PubMed1990

Occurrence of intercapillary nodular glomerulosclerosis in the absence of glucose intolerance.

The case of a 58-year-old man with nephrotic syndrome and characteristic pathologic renal lesions of KW disease is presented. No evidence of diabetes was found by oral or intravenous glucose tolerance tests. The possibilities of light-chain disease, membranoproliferative form of glomerulonephritis, and amyloidosis were excluded by histochemistry and immunofluorescent microscopy.

Blood Glucose↗
PubMed1990

Dexamethasone increases heparan sulfate proteoglycan core protein content of glomerular epithelial cells.

Heparan sulfate proteoglycan (HSPG) has been identified as an important determinant of glomerular permselectivity. We have previously reported that glomerular epithelial cells in culture synthesize HSPG, suggesting that in vivo these cells contribute to the HSPG present in the glomerular basement membrane. In this study we examined the effects of dexamethasone on the metabolism of HSPG core protein in cultured glomerular epithelial cells. Dexamethasone caused a dose-dependent and time-dependent increase in the HSPG core protein content of the cells. This effect was not seen with an equimolar concentration of aldosterone, indicating it was selective for dexamethasone. Dexamethasone caused a significant inhibition in 3H-leucine incorporation into de novo synthesized proteins at concentrations that caused maximum increment in the HSPG core protein content. These findings support the interpretation that HSPG core protein is a selective target for dexamethasone. Actinomycin-D completely abrogated the dexamethasone effect on HSPG core protein content, implying that enhanced transcription may be the major mechanism underlying the dexamethasone-induced increment in HSPG core protein content. Our findings suggest that glucocorticoids have important effects on the metabolism of the core protein moiety of heparan sulfate proteoglycan. Furthermore, these data imply that the glucocorticoid-induced amelioration of proteinuria could involve metabolic effects on the local determinants of glomerular permselectivity (e.g., HSPG) in addition to their well-known systemic anti-inflammatory effects.

Aldosterone↗
PubMed1988

cDNA-derived primary structure of the glycoprotein component of canine microsomal signal peptidase complex.

Canine microsomal signal peptidase activity has been shown previously to co-migrate as an apparent complex of six polypeptides with molecular masses of 25, 23, 22, 21, 18, and 12 kDa. The 22- and 23-kDa species are differentially glycosylated forms of the same protein, designated SPC 22/23. The amino acid sequence of SPC 22/23 was deduced from cDNA clones. The protein is synthesized without a cleavable amino-terminal signal sequence and contains a single site for N-linked glycosylation. SPC 22/23 appears to be anchored to the rough endoplasmic reticulum membrane by a single hydrophobic segment near its amino terminus, with the remainder of the protein positioned on the lumenal side of the membrane. The amino acid sequence of SPC 22/23 shares homology with tryptic peptides derived from the hen oviduct signal peptidase glycoprotein, one of two possible proteins required for signal peptide processing in the avian system (Baker, R.K., and Lively, M.O. (1987) Biochemistry 26, 8561-8567). Therefore, the complete amino acid sequence of SPC 22/23 presented in this report corresponds to one of two possible proteins required for signal peptide processing in higher eukaryotic cells.

Amino Acid Sequence↗
PubMed1988

Role of proteoglycans in renal development.

The role of proteoglycans (PGs) in morphogenesis was investigated. Fetal kidneys were obtained from 13-day-old mouse embryos and maintained for 7 days in culture. The biosynthesis of PGs was perturbed by addition of p-nitrophenyl-beta-D-xylopyranoside in the culture medium. The kidneys were processed for morphological and biochemical studies. The morphological studies included staining of tissues with anti-basement membrane antibodies and ruthenium red. [35S]sulfate was used as the precursor product for biosynthetic and autoradiographic studies. The kidneys treated with xyloside had loose mesenchyme, inhibition of ureteric bud branching, diminution in the population of developing nephron elements, decreased immunofluorescence with anti-proteoglycan antibodies and staining with ruthenium red, and a reduced [35S]sulfate incorporation into poorly organized extracellular matrices. The biochemical studies included characterization of PGs/glycosaminoglycans (GAGs) by Sepharose CL-4B, -6B, and DEAE-Sephacel chromatographies and cellulose acetate electrophoresis. Under the influence of xyloside, the total radioactivities decreased 2 to 4-fold in tissues and increased 18 to 42-fold in media fractions. A reduction in the size of macromolecular form of PGs, i.e., from MW approximately 2.5 X 10(6) to approximately 2.5 X 10(4), was noted. The PGs/GAGs synthesized were mainly made up of heparan sulfate and small amounts of chondroitin sulfate. They eluted at a lower salt concentration as compared to the controls. A similar diminution in the size of media PGs, i.e., from MW approximately 1.8 X 10(5) to approximately 2.8 X 10(4), was observed. Additional studies with [3H]xyloside indicated that the chains initiated on xyloside residues were similar in size and composition to GAG-chains. These findings indicate that a perturbance in the biosynthesis of PGs/GAGs leads to abnormalities in renal organogenesis.

Animals↗