Biomedical subjects

S P Makker

Publications and source records attributed to S P Makker.

At least 19 recordsLinked to original sources

PubMed2006

Differential capacity of anti-RAP and anti-megalin antibodies to produce progressive passive Heymann nephritis - implications for the pathogenesis of idiopathic human membranous glomerulonephritis.

Passive Heymann nephritis (PHN) induced with heterologous antisera has been described according to various criteria, which may or may not include induction of chronic disease and proteinuria. Characteristics of the glomerular immune deposits determined by the antigenic specificities of the antisera presumably account for differences in disease outcome. In this study, the clinical and immunohistological features in the model produced with monospecific antisera were compared against megalin or receptor associated protein (RAP), two proteins that have been implicated as target antigens in PHN. Rats injected with either anti-megalin or anti-RAP antiserum developed typical glomerular immune deposits of PHN when examined after 7 days. Although the deposits stained for complement, none of the animals had abnormal proteinuria in this time frame. Over a longer time course (7-16 weeks), immune deposits persisted and proteinuria increased to pathological levels in all animals injected with anti-megalin serum. By contrast, immune deposits had cleared from the kidneys of rats injected with anti-RAP antiserum when examined at 7-8 weeks post-injection and the proteinuria levels observed up to 13 weeks remained in the normal range. Additional doses of anti-RAP antiserum given 4 and 17 days after the first injection did not prolong the duration of glomerular immune deposits. These results demonstrate a clear divergence in pathogenic potential of antisera generated against the two renal antigens, which suggest differences in the immune deposits linked to a soluble antigen that is non-covalently bound to the podocyte membrane versus those linked to an integral membrane antigen. These observations could provide clues to the nature of the unknown glomerular autoantigen of idiopathic membranous glomerulonephritis in humans.

Animals↗

PubMed2001

Functional characterization of rat gp600/megalin promoter: combination of proximal Sp1 site and JCV repeat is important in rat gp600/megalin promoter activation.

Gp600/megalin is an endocytic receptor belonging to the low-density lipoprotein receptor family. Up or down regulation of this protein were observed in certain disease states. To understand the mechanisms that control gp600/megalin gene expression, we cloned and functionally characterized a 738-bp fragment of the 5'-flanking region of rat gp600/megalin gene. A transcription start site was mapped to 33 bp downstream of TAGAAA sequence (TATA-like box). Multiple transcription factor binding sites were identified. Serial 5' deletions and transient transfection assays showed that the deletion fragment containing the Sp1 site proximal to the TATA-like box and a JCV repeat retained 80% of the promoter activity. Individual mutations of the proximal Sp1 site and JCV repeat reduced the promoter activity by 60 and 34% respectively. Double mutations of the proximal Sp1 site and JCV repeat produced a dramatic 80% reduction in the promoter activity. However, deletions and mutations or double mutations of other transcription factor binding sites in the promoter region had a minor effect on the promoter activity. These results indicate that the combination of proximal Sp1 site and the JCV repeat are necessary for activation of gp600/megalin expression. Moreover, Sp1 and Sp3 proteins interacted with the proximal and the distal Sp1 sites in the nuclear extracts of gp600/megalin expressing cell lines. TCF site seems to be involved in negative regulation of this promoter but no nuclear protein(s) were found to bind to this site. In addition, Ap2 site responsible for 28% promoter activity is able to bind two dominant unknown nuclear proteins. This functional characterization of the regulation of gp600/megalin gene is likely to advance the knowledge of the regulation of this gene in health and disease.

Animals↗

PubMed2001

Gastrostomy tube leak in a patient receiving peritoneal dialysis.

Aggressive nutritional support is imperative to ensure an optimal quality of life in the management of children with end stage renal disease (ESRD). Supplemental enteral feeds using a gastrostomy tube (G-tube) are commonly used for nutritional support to overcome the barrier posed by anorexia. Some of the reported complications of G-tube feeds in children simultaneously receiving peritoneal dialysis include G-tube exit site infection with concomitant peritonitis and G-tube obstruction. We are reporting our experience in managing an 8-year-old Caucasian male with ESRD who, while receiving peritoneal dialysis and G-tube feeds, developed medically intractable peritonitis due to separation of the stomach wall from the anterior abdominal wall, resulting in peritoneal contamination with gastric contents. This complication, which has not previously been reported in patients receiving peritoneal dialysis, had devastating consequences, culminating in the death of our patient.

Child↗

PubMed2000

Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin.

gp600/megalin, an endocytic receptor, belongs to the low-density lipoprotein receptor family. It is most abundant in the renal proximal tubular cells, where it is implicated in the reabsorption of a number of molecules filtered through the glomerulus. The cytoplasmic tail (CT) of gp600/megalin contains a number of sequence similarities, which indicate that gp600/megalin might be involved in signal transduction. To find intracellular proteins that would interact with the gp600/megalin CT, a human kidney cDNA library was screened by using the yeast two-hybrid system. The phosphotyrosine interaction domain (PID) of the Disabled protein 2 (Dab2), a mammalian structural analogue of Drosophila Disabled, was found to bind to the gp600/megalin CT in this system. The interaction between these two proteins was confirmed by a binding assay in vitro and by the co-immunoprecipitation of both proteins from renal cell lysates. The gp600/megalin CT contains three PsiXNPXY motifs (in which Psi represents a hydrophobic residue) that are potentially able to interact with PID. Analysis of the CT deletion and point-mutation variants of gp600/megalin by the two-hybrid system revealed that the third PsiXNPXY motif is most probably involved in this interaction. Dab2 is a mitogen-responsive phosphoprotein thought to be an adaptor molecule involved in signal transduction, and a suggested negative regulator of cell growth. Dab2 is the first intracellular ligand identified for gp600/megalin; gp600/megalin is the first known transmembrane receptor that interacts with the cytosolic protein Dab2. We speculate that their interaction might involve gp600/megalin in signal transduction pathways or might mediate the intracellular trafficking of this receptor.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Increased expression of cytoplasmic tail-containing form of gp600/megalin in active Heymann nephritis.

Active Heymann nephritis of rat, an autoimmune glomerular disease, is a model of human membranous glomerulonephropathy. The major target autoantigen of this disease is a 600 kD renal glycoprotein, variously named gp600/gp330/megalin/LRP2. It has been shown previously that there is a marked increase in the gp600/megalin mRNA level in glomeruli of rats with this disease. A rabbit antibody was prepared against a peptide located at the very end of the cytoplasmic tail of rat gp600/megalin. Affinity purified anti-peptide antibody was monospecific and reacted only with gp600/megalin on the immunoblots of total kidney proteins. Immunofluorescence microscopy on frozen kidney sections probed with this antibody revealed the presence of the cytoplasmic tail-containing form of gp600/megalin in both proximal tubules and glomeruli. Expression in glomeruli of normal kidneys was less than in tubules but was clearly detectable. In active Heymann nephritis kidneys, expression of this form was increased most remarkably in glomeruli. These results, showing increased expression of the cytoplasmic tail-containing form in active Heymann nephritis glomeruli, contradict previous reports of its absence. This is the first study to show that there is increased expression of the cytoplasmic tail-containing form of gp600/megalin in the glomeruli of rats with this disease and its role in active Heymann nephritis is discussed.

Animals↗

PubMed2000

Conversion from tacrolimus to neoral for postrenal transplant diabetes.

Diabetes mellitus (DM) is a well-recognized complication of immunosuppressive therapy in the post-transplant (Tx) period. The DM encountered in the setting of tacrolimus therapy has been managed in the past by tacrolimus dose reduction and a rapid corticosteroid taper; frequently insulin therapy is also required to maintain normoglycemia. However, the dose reduction of immunosuppressive agents has often resulted in acute allograft rejection. We are reporting our experience in managing three pediatric renal Tx recipients who developed DM in the post-Tx period while on triple immunosuppressive therapy including tacrolimus and corticosteroids. All of our patients were managed by substitution of tacrolimus with conventional doses of neoral while maintaining their usual corticosteroid dose. All three patients had resolution of hyperglycemia and none experienced acute rejection. Tacrolimus was then successfully reinitiated 4.6 months later; at last follow-up, all of our patients have good allograft function and have maintained a normal blood sugar. In conclusion, we feel that conversion of patients from tacrolimus to neoral should be attempted as a safer alternative to tacrolimus dose reduction, for managing post-Tx DM in tacrolimus treated patients.

Adrenal Cortex Hormones↗

PubMed1996

Membranous nephropathy, interstitial nephritis, and Fanconi syndrome-- glomerular antigen.

We characterized the glomerular antigen of membranous nephropathy (MN) in a child with the triad of MN, proximal renal tubular basement membrane autoantibody (TBMAb)-associated interstitial nephritis (ITN), and Fanconi syndrome. Granular staining was demonstrated for human gp600 in the vicinity of immune deposits of MN along glomerular capillary loops, using a monospecific polyclonal antibody to human gp600 by indirect immunofluorescence. However, no staining was observed in the MN deposits for receptor-associated protein. Membranous nephropathy preceded the development of TBMAbs, ITN, and Fanconi syndrome by 1 year, showing that the MN lesion does not result from the initial immunological injury to the tubulointerstitium, as postulated earlier. We confirmed the reactivity of TBMAbs with the recently described rabbit 58-kilodalton (kDa) tubular basement membrane antigen (TBMAg). However, this is the first report to show reactivity of these antibodies with the human 58-kDa protein. Also, we found that TBMAg is comprised of a single protein band of 58 kDa, unlike the previously described combination of two protein band (58 kDa and 175 kDa). In this patient, following prednisone treatment, the TBMAbs became undetectable, and the nephrotic syndrome and Fanconi syndrome resolved, thus suggesting a causal role of TBMAbs in the pathogenesis of Fanconi syndrome. We postulate that in this rare disorder, renal lesions result from an autoimmune response to the 58-kDa TBMAg and possibly to gp600, and that the predisposition to autoimmunity is genetically linked to the HLA B7 serotype.

Anti-Inflammatory Agents↗

PubMed1995

Different molecular forms of rat kidney gp330, the dominant autoantigen of active Heymann nephritis.

The primary structure, consisting of 1650 amino acid residues, of the C-terminal end of the dominant autoantigen of active Heymann Nephritis, gp330, from rat kidney was obtained by cloning and sequencing of cDNA clones. Comparison of this sequence with the previously published sequences of fragments of the C-terminal end of gp330 [Raychowdhury, Niles, McCluskey and Smith (1989) Science 244, 1163-1165] revealed certain differences in their primary structures. These differences included several single amino acid substitutions, replacement of a stretch of 15 amino acid residues by a different stretch of six amino acid residues, and different lengths of cytoplasmic domain (188 versus 213 amino acid residues). These findings of two different primary structures of gp330 provide direct evidence for the existence of two molecular forms of gp330.

Amino Acid Sequence↗

PubMed1995

Role of receptor-associated 39/45 kD protein in active Heymann nephritis.

UNLABELLED: Active Heymann nephritis (HN) of rat, an autoimmune glomerular disease, is an experimental model of human membranous glomerulonephropathy (MGN), a common human glomerular disease. The putative autoantigen of HN is believed to be a large glycoprotein (gp330) present in kidneys of rat and human. Gp330 has sequence homology to low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor and co-purifies with another 45 kD protein called receptor-associated protein (RAP) which binds to both gp330 and LRP/alpha 2-macroglobulin receptor. Since RAP co-purifies with gp330 and is thus present in the immunizing material used to induce active HN, the present study was undertaken to determine if gp330 or RAP individually could produce active HN and whether RAP had a role in the pathogenesis of active HN. Rats immunized with the traditional crude antigen (Fx1A) containing both gp330 and RAP developed typical HN. Although these rats developed low titers of autoantibody to RAP in their sera, they had no deposition of antibody to RAP in their kidneys. Rats immunized with gp330 also developed typical HN but had no autoantibody to RAP in their sera or kidneys. Rats immunized with RAP developed high titers of autoantibody to RAP in their sera but had no antibody in their kidneys and did not develop HN. Three rats were injected intravenously with polyclonal antibody to RAP and assessed seven days later for the development of passive HN. All three developed mild passive HN characterized by granular staining of polyclonal antibody along the capillary loops. CONCLUSIONS: (1) This is the first report to show that gp330 alone without the accompanying RAP can induce active HN; (2) RAP by itself does not induce active HN; (3) Autoantibodies to RAP do not appear to be involved in the pathogenesis of active HN induced with the traditional crude antigen, Fx1A; and (4) We confirm that polyclonal antibody to RAP can induce passive HN.

Animals↗

PubMed1995

Transcription and translation of gp600 and receptor-associated protein (RAP) in active Heymann nephritis.

UNLABELLED: Active Heymann nephritis (HN) of rat, an autoimmune glomerular disease, is a model for human membranous glomerulonephropathy. The autoantigen of HN is a glycoprotein of approximately 600 kd that is present in both rat and human kidneys. Another kidney protein of 39-45 kd called receptor-associated protein (RAP) is associated with gp600. In normal kidney very little gp600 and RAP can be detected in glomeruli. This study was undertaken to determine whether the synthesis of gp600 and RAP would increase after development of active HN. Kidneys from normal (n = 5) and active (n = 11) HN rats were studied for expression of gp600 and RAP and their mRNAs by immunofluorescence and in situ hybridization. In all HN kidneys in contrast to normal kidneys both the transcription and translation of gp600 were markedly increased in glomeruli and proximal tubules. Transcription and translation of RAP were also increased but less so than gp600. The site of increased transcription of gp600 and RAP in glomeruli was clearly localized to the visceral glomerular epithelial cells. CONCLUSIONS: This is the first study to show increased transcription of gp600 and RAP in active HN and the first study to identify the visceral glomerular epithelial cell as the cell for the increased transcription.

Animals↗

PubMed1994

A novel autoantibody to plasminogen and its characterization in Heymann nephritis.

Heymann nephritis (HN) of rat is an experimental model of human membranous glomerulonephropathy (MGN). It is generally accepted that the glomerular lesion of HN results from the binding of antibodies (autoantibodies in active HN and heterologous antibodies in passive HN) to gp330, a receptor of the low-density lipoprotein receptor superfamily located on the glomerular epithelial cell. We have previously shown that plasminogen (plg) is a ligand for gp330. This report shows that in addition to antibodies to receptor, gp330, novel autoantibodies (Aab) to ligand, plg, also develop in the course of HN and accumulate selectively and in parallel with gp330 antibodies in the glomeruli of both active and passive HN. Aab to plg are present in serum and in immunoglobulin G (IgG) eluted from glomeruli of diseased animals with the concentration severalfold greater in glomerular IgG than in an equivalent amount of serum IgG. Characterization of the Aab to plg shows that they are directed against the conformational epitopes of plg with some of those epitopes being located in the lysine-binding domain of plg. Aab to plg develop without exogenous immunization with plg. In active HN Aab to ligand, plg, develop following immunization with its autologous receptor, gp330, and in passive HN they develop following administration of polyclonal antibody to the receptor. Although the mechanism of their induction is not known, their selective accumulation in glomeruli suggests a role in the development of the MGN lesions in both active and passive HN.

Animals↗

PubMed1993

Analysis of glomeruli-eluted Gp330 autoantibodies and of Gp330 antigen of Heymann nephritis.

UNLABELLED: Quantity, charge, IgG isotype, Ag reactivity of glomerular gp330 autoantibodies (gp330Ab), and the charge of the putative Ag, gp330, were studied in active Heymann nephritis. Gp330 was anionic with an isoelectric point of 4.6. Despite variation in C3 glomerular immunofluorescence staining, C5b-9 staining was seen in all rats. Positive correlation was seen between glomerular gp330Ab and abnormal 24-h proteinuria (r = 0.637, p = 0.008), which appeared to require a certain threshold level of gp330Ab. Positive correlation was also seen between serum and glomerular gp330Ab at time of death (r = 0.55, p < 0.05). No differences were seen in charge, IgG isotypes of glomerular gp330Ab, or reactivity to gp330 by Western analysis in rats with or without abnormal proteinuria. Comparison of gp330Ab IgG isotypes in immune sera and glomeruli showed no difference in IgG1 and IgG2a, but IgG2b was significantly lower in glomeruli (p < 0.0001). CONCLUSIONS: 1) this is the first documentation that gp330 is highly anionic; 2) regardless of the degree of proteinuria, all immunized rats deposit in their glomeruli a qualitatively similar type of gp330Ab (charge, IgG isotype, complement activation, gp330 reactivity); 3) despite the highly anionic nature of gp330, anionic gp330Ab accumulate in glomeruli; and 4) complement activation appears to occur in every rat with gp330Ab in glomerulus and development of proteinuria is dependent on a certain threshold level of gp330Ab in the glomerulus which, in turn, correlates with the serum level of gp330Ab.

Animals↗

PubMed1993

Analysis of a 45-kDa protein that binds to the Heymann nephritis autoantigen GP330.

Previously, we have shown that gp330, the putative autoantigen of Heymann nephritis which is located on the glomerular and tubular epithelial cells, is a receptor for plasminogen. During our analysis of the function of gp330, we also observed by means of Western analysis the binding of gp330 to a 45-kDa protein. This 45-kDa protein was obtained from extracts of rat kidney and purified to apparent homogeneity. Enzyme-linked immunosorbent assay analysis indicate that gp330 can bind to the 45-kDa protein. In fact, both the 45-kDa protein and plasminogen can bind to gp330 simultaneously, indicating that gp330 has two separate sites for each respective protein. No inhibition of binding of either ligand to gp330 is observed when both ligands were simultaneously incubated with immobilized gp330. A rabbit polyclonal antibody raised against this 45-kDa protein was used to screen a rat kidney lambda gt11 expression library. Sequence analysis of the isolated clones revealed that this protein is similar to the human alpha 2-macroglobulin receptor-related protein and murine heparin-binding protein-44. It is also identical to a previously reported, supposedly pathogenic, domain of gp330. However, our results show that the cDNAs encode for a 45-kDa protein, which is not a part of gp330 but a separate protein which binds to gp330.

Amino Acid Sequence↗

PubMed1993

Fatal Pneumocystis pneumonia in a child treated for focal segmental glomerulosclerosis.

A 4-year-old female with nephrotic syndrome secondary to focal segmental glomerulosclerosis (FSGS) was treated with pulse methylprednisolone and chlorambucil therapy. She subsequently developed fatal Pneumocystis carinii pneumonia (PCP). This is the first report of a pediatric patient whose treatment for FSGS resulted in PCP, raising the issue of PCP prophylaxis for these patients.

Child, Preschool↗

PubMed1993

Mediators of immune glomerular injury.

Mediators of immune glomerular injury may be divided into primary and secondary. Primary mediators include antibody and T cells and secondary include complement, infiltrating inflammatory cells, such as neutrophils, monocytes/macrophages and platelets, coagulation system, resident glomerular cells including mesangial, endothelial and epithelial cells, reactive oxygen metabolites, eicosanoids, proteolytic enzymes and a host of cytokines. Following initiation of immune glomerular injury with primary mediators, which in most cases is antibody, a complex set of interactions involving some or all of the secondary mediators occurs in the glomerulus, ultimately leading to the clinical manifestations of glomerular injury. The precise sequence and the mechanisms of these interactions are not fully defined but are under intense study. The identity of the putative antigens and why and how an autoimmune response develops are also not fully known.

Animals↗

PubMed1991

Identification of the rat Heymann nephritis autoantigen (GP330) as a receptor site for plasminogen.

Previous results have demonstrated the binding of a 76- and 80-kDa serum protein to the Heymann nephritis autoantigen, gp330. This 76-kDa serum protein was purified by column chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A rabbit polyclonal antibody for the serum protein was produced and used to screen a rat liver cDNA expression library. Sequence analysis of an isolated clone identified the serum protein as plasminogen. Plasminogen was isolated from rat serum by standard techniques, and the binding of plasminogen to gp330 was confirmed by Western analysis. Enzyme-linked immunosorbent assay results demonstrated a time-dependent, saturable, and specifically inhibitable binding of plasminogen to gp330. There was no significant difference in the binding of the two carbohydrate forms of plasminogen to gp330. Plasminogen binding to gp330 could be completely inhibited by the addition of exogenous gp330. This binding could also be partially inhibited by benzamidine but only slightly by the lysine analogue, epsilon-aminocaproic acid. However, even a combination of these two inhibitors could not completely block the binding of plasminogen to gp330 indicating that gp330 may be binding to plasminogen through some other unknown interactions. These results demonstrate that gp330 is a receptor site for plasminogen.

Amino Acid Sequence↗

PubMed1991

Luminal surfaces of fetal rat alveolar type II and Clara lung cells react with antibody to Heymann nephritis antigen.

We have used affinity purified antibody, which reacts with the renal antigen gp600, to examine the immunoreactivity of pulmonary cells in the quick frozen lung tissue of fetal rats. On immunoelectron microscopy, we found that the reaction was specifically localized to the luminal surfaces of Clara cell and, as previously reported, to alveolar type II cell (ATII) membranes. No immunoreaction was seen on or in type I cells, endothelial cells or any other lung or blood cell. These studies suggest that the anti-gp600 immunoselection can be used as a marker to identify and quantify Clara and ATII cells in rat lung tissue.

Animals↗

PubMed1990

Renal antigens in mercuric chloride induced, anti-GBM autoantibody glomerular disease.

Two antibody probes were used to characterize the putative renal antigens of HgCl2-induced antiglomerular basement membrane renal disease in Brown Norway (BN) rat. The first probe was the linear immunofluorescence imparting, in vivo bound, nephritogenic antiglomerular-basement-membrane autoantibody (anti-GBM-Ab). The second probe was a rat monoclonal antibody to the B subunit of laminin that was obtained from fusion of spleen cells of HgCl2 injected BN rat. By enzyme-linked immunosorbent assay (ELISA) the anti-GBM-Ab reacted with laminin, type IV collagen, collagenase-resistant noncollagenous portion of glomerular basement membrane (GBM), saline soluble proteins of kidney cortex homogenate and fibronectin. Western blot analysis of laminin indicated that the reactive epitopes detected by both probes were on the B chain subunit but not the A subunit. In nonreduced collagenase-digested GBM the epitopes were present on 27 kD and 42 to 48 kD polypeptides. A similar pattern was seen on collagenase-digested human GBM. On rat and human GBM the patterns obtained with rat autoantibody and autoantibody from a patient with Goodpasture syndrome were similar, suggesting that some of the in vivo bound anti-GBM autoantibodies in HgCl2-induced disease in rat are directed against epitopes which are similar to the Goodpasture antigen of human. Reactive epitopes were also detected on saline soluble proteins of kidney cortex homogenate with the predominant antigen being a 31 kD polypeptide. In the saline soluble proteins the reactive polypeptides including the major 31 kD polypeptide did not originate from laminin, type IV collagen, or the collagenase-resistant noncollagenous part of GBM. The precise structural origin of soluble proteins was not defined.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗