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Biomedical subjects

M A Saleem

Publications and source records attributed to M A Saleem.

At least 19 recordsLinked to original sources
PubMed2006

Direct effects of dexamethasone on human podocytes.

Glucocorticoids are widely used in the treatment of human glomerular diseases, but their mode of action is poorly understood particularly in steroid-sensitive nephrotic syndrome, which is most common in childhood and is characterized by a lack of inflammation in the kidney. The podocyte is a key cell in the glomerulus in health and disease: until recently, human podocytes have been difficult to study in vitro. We have developed a conditionally immortalized human podocyte cell line transfected with a temperature-sensitive simian virus 40 transgene: when the transgene is inactivated in vitro, these cells adopt the phenotype of differentiated podocytes. We have used these cells to evaluate, using immunocytochemistry, reverse transcriptase-polymerase chain reaction, and Western blotting, direct effects of the glucocorticoid dexamethasone at concentrations designed to mimic in vivo therapeutic corticosteroid levels. Dexamethasone upregulated expression of nephrin and tubulin-alpha, and downregulated vascular endothelial growth factor. Effects on cell cycle were complex with downregulation of cyclin kinase inhibitor p21 and augmentation of podocyte survival, without any effect on apoptosis. We report cytokine production by human podocytes, especially interleukin (IL)-6 and -8; IL-6 expression was suppressed by dexamethasone. These potent direct effects on podocytes illustrate a novel mode of action of glucocorticoids and suggest potential new therapeutic strategies for glomerular disease.

Antigens, Polyomavirus Transforming↗
PubMed2006

Podocyte proteoglycan synthesis is involved in the development of nephrotic syndrome.

Proteoglycans (PGs) are important for the glomerular barrier, for cell signaling, and for the anchorage of cells to the glomerular basement membrane. They are, however, complex macromolecules, and their production has not yet been thoroughly investigated in podocytes. In the present study, we studied the biosynthesis of PGs by highly differentiated human podocytes and in rats. The cells were treated with puromycin aminonucleoside (PAN; a nephrosis-inducing agent), steroids (used as primary treatment for nephrotic syndrome), or both. Analysis was made by TaqMan real-time PCR, Western blotting, and by metabolic labeling with (35)S and (3)H. We found that podocytes produce versican, syndecan-1, decorin, and biglycan together with the previously known PG syndecan-4, glypican, and perlecan. PAN treatment downregulated the mRNA and the protein expression of both versican (by 24 +/- 6%, P < 0.01, for mRNA and by 50% for protein) and perlecan (by 14 +/- 5%, P < 0.05, for mRNA and by 50% for protein). The decreased expression was confirmed by studying the glomerular gene expression in rats treated with PAN during a time course study. In addition, puromycin decreased the expression of enzymes involved in the glycosaminoglycan biosynthesis. Steroid treatment decreased perlecan (by 24 +/- 3%, P < 0.01) and syndecan-1 expression (by 30 +/- 4%, P < 0.01) but increased the expression of decorin 2.5-fold. The observed alterations of PG synthesis induced by PAN may lead to decreased glomerular anionic charge and disturbed podocyte morphology, factors that are important for the development of a nephrotic syndrome.

Animals↗
PubMed2006

VEGF-C promotes survival in podocytes.

Vascular endothelial growth factor (VEGF)-A is an autocrine survival factor for podocytes, which express two VEGF receptors, VEGF-R1 and VEGF-R3. As VEGF-A is not a known ligand for VEGF-R3, the aim of this investigation was to examine whether VEGF-C, a known ligand for VEGF-R3, served a function in podocyte biology and whether this was VEGF-R3 dependent. VEGF-C protein expression was localized to podocytes in contrast to VEGF-D, which was expressed in parietal epithelial cells. Intracellular calcium ([Ca2+]i) experiments demonstrated that VEGF-C induced a 0.74+/-0.09-fold reduction in [Ca2+]i compared with baseline in human conditionally immortalized podocytes (hCIPs; P<0.05, one sample t-test, n=8). Cytotoxicity experiments revealed that in hCIPs VEGF-C reduced cytotoxicity to 81.4+/-1.9% of serum-starved conditions (P<0.001, paired t-test, n=16), similar to VEGF-A (82.8+/-4.5% of serum-starved conditions, P<0.05, paired t-test). MAZ51 (a VEGF-R3 kinase inhibitor) inhibited the VEGF-C-induced reduction in cytotoxicity (106.2+/-2.1% of serum-starved conditions), whereas MAZ51 by itself had no cytotoxic effects on hCIPs. VEGF-C was also shown to induce a 0.5+/-0.13-fold reduction in levels of MAPK phosphorylation compared with VEGF-A and VEGF-A-Mab treatment (P<0.05, ANOVA, n=4), yet had no effect on Akt phosphorylation. Surprisingly, immunoprecipitation studies detected no VEGF-C-induced autophosphorylation of VEGF-R3 in hCIPs but did so in HMVECs. Moreover, SU-5416, a tyrosine kinase inhibitor, blocked the VEGF-C-induced reduction in cytotoxicity (106+/-2.8% of serum-starved conditions) at concentrations specific for VEGF-R1. Together, these results suggest for the first time that VEGF-C acts in an autocrine manner in cultured podocytes to promote survival, although the receptor or receptor complex activated has yet to be elucidated.

Calcium↗
PubMed2006

Conditionally immortalized human glomerular endothelial cells expressing fenestrations in response to VEGF.

Glomerular endothelial cells (GEnC) are specialized cells with important roles in physiological filtration and glomerular disease. Despite their unique features, GEnC have been little studied because of difficulty in maintaining them in cell culture. We have addressed this problem by generation of conditionally immortalized (ci) human GEnC using technology with which we have previously produced ci podocytes. Primary culture GEnC were transduced with temperature-sensitive simian virus 40 large tumour antigen and telomerase using retroviral vectors. Cells were selected, cloned, and then characterized by light and electron microscopy (EM), response to vascular endothelial growth factor (VEGF), and tumour necrosis factor (TNF)alpha, expression of endothelial markers by focused gene array, immunofluorescence and Western blotting, and formation and behaviour of monolayers. CiGEnC proliferated at the permissive temperature (33 degrees C) and became growth arrested at the non-permissive temperature (37 degrees C). CiGEnC retained morphological features of early-passage primary culture GEnC up to at least p41, confirming successful immortalization. EM demonstrated fenestrations, increased in number by VEGF. mRNA analysis confirmed expression of the endothelial markers platelet endothelial cell adhesion molecule 1, intercellular adhesion molecule 2, VEGF receptor 2, and von Willebrand factor, validated by immunofluorescence and Western blotting. CiGEnC also expressed Tie2, and TNFalpha upregulated E-selectin. CiGEnC formed monolayers with barrier properties responsive to cyclic adenosine 3',5' monophosphate (cAMP) and thrombin. CiGEnC retain the markers and behaviour of primary culture GEnC. They express fenestrations which are upregulated in response to VEGF. These cells are a unique resource for further study of GEnC and their roles in glomerular filtration, glomerular disease, and response to glomerular injury.

Biomarkers↗
PubMed2006

A prospective, randomized, multicenter trial of tacrolimus-based therapy with or without basiliximab in pediatric renal transplantation.

In a 6-month, multicenter, randomized, controlled, open-label, parallel-group trial, we investigated the efficacy and safety of adding basiliximab to a standard tacrolimus-based regimen in pediatric renal transplant recipients. Patients < 18 years received tacrolimus/azathioprine/steroids (TAS, n = 93) or tacrolimus/azathioprine/steroids/basiliximab (TAS + B, n = 99). Target tacrolimus levels were 10-20 ng/mL between days 0-21 and 5-15 ng/mL thereafter. Steroid dosing was identical in both groups. Basiliximab was administered at 10 mg (patients < 40 kg) or 20 mg (patients > or = 40 kg) within 4 h of reperfusion; the same dose was repeated on day 4. Biopsy-proven acute rejection rates were 20.4% (TAS) and 19.2% (TAS + B); steroid-resistant acute rejection rates were 3.2% and 3.0%, respectively. Patient survival was 100%; graft survival rates were 95% in both arms. The nature and incidence of adverse events were similar in both arms except toxic nephropathy and abdominal pain, which were significantly higher in the TAS + B arm (14.1% vs. 4.3%; p = 0.03 and 11.1% vs. 2.2%; p = 0.02; respectively). Median serum creatinine concentrations at 6 months were 86 micromol/L in the TAS and 91 micromol/L in the TAS + B arm; glomerular filtration rate was 79.4 and 77.6 (mL/min/1.73 m2), respectively. Adding basiliximab to a tacrolimus-based regimen is safe in pediatric patients, but does not improve clinical efficacy.

Adolescent↗
PubMed2005

Porcine dermal collagen graft in pediatric renal transplantation.

Successful closure of the anterior abdominal wall in infants following renal transplantation of adult organs may present a challenging dilemma to the transplant surgeon. Restricted volume of the recipient abdominal cavity and size discrepancy of donor adult kidney may lead to graft compromise. Pressure on the graft may be exacerbated further in the postoperative period by oedema that may lead to abdominal compartment syndrome. Donor/recipient size disparity remains the major obstacle in infant renal transplantation. We describe the use of a porcine collagen graft to facilitate closure of the abdominal wall following intra-peritoneal transplantation of an adult cadaveric kidney.

Abdominal Wall↗
PubMed2001

Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin.

NPHS2 was recently identified as a gene whose mutations cause autosomal recessive steroid-resistant nephrotic syndrome. Its product, podocin, is a new member of the stomatin family, which consists of hairpin-like integral membrane proteins with intracellular NH(2)- and COOH-termini. Podocin is expressed in glomerular podocytes, but its subcellular distribution and interaction with other proteins are unknown. Here we show, by immunoelectron microscopy, that podocin localizes to the podocyte foot process membrane, at the insertion site of the slit diaphragm. Podocin accumulates in an oligomeric form in lipid rafts of the slit diaphragm. Moreover, GST pull-down experiments reveal that podocin associates via its COOH-terminal domain with CD2AP, a cytoplasmic binding partner of nephrin, and with nephrin itself. That podocin interacts with CD2AP and nephrin in vivo is shown by coimmunoprecipitation of these proteins from glomerular extracts. Furthermore, in vitro studies reveal direct interaction of podocin and CD2AP. Hence, as with the erythrocyte lipid raft protein stomatin, podocin is present in high-order oligomers and may serve a scaffolding function. We postulate that podocin serves in the structural organization of the slit diaphragm and the regulation of its filtration function.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Recurrent focal segmental glomerulosclerosis in grafts treated with plasma exchange and increased immunosuppression.

We report on three children with severe, recurrent focal segmental glomeruloscerosis (FSGS) in their first allografts, treated with methylprednisolone, plasma exchange and cyclophosphamide. This protocol is based on a previous publication showing its successful use in three children. Our patients were 2 girls and 1 boy, aged 14.5, 14.6 and 13.2 years, respectively, at transplant. Concomitant immunosuppression included cyclosporin A and prednisolone. Recurrence occurred in all three patients within 24 h, and specific treatment was commenced within 48 h. All patients developed anuria and were dialysed. The boy stopped dialysis after 4 weeks, and has stable chronic renal failure (CRF) and no proteinuria 3 years later. One girl required dialysis for 4 months, and 3 years later has CRF with non-nephrotic range proteinuria. The other girl remained dialysis-dependent and died from septic complications. We conclude that even anuric patients treated with this protocol may have an improvement in renal function and reduction of proteinuria, which can last for over 3 years. However, treatment may need to be prolonged and carries the substantial risks of heavy immunosuppression.

Adolescent↗
PubMed2000

Treatment of severe complicated Kawasaki disease with oral prednisolone and aspirin.

We report on 7 patients with severe, complicated Kawasaki disease treated with oral prednisolone, after apparently unsuccessful intravenous immunoglobulin treatment. An additional eighth patient was a Jehovah's Witness, who was given steroid and aspirin as first-line treatment. These findings support a beneficial role for steroids in intravenous immunoglobulin-resistant Kawasaki disease.

Anti-Inflammatory Agents↗
PubMed2000

Clinical course of patients with major histocompatibility complex class II deficiency.

The clinical course of 10 children who have been diagnosed with major histocompatibility complex (MHC) class II deficiency (bare lymphocyte syndrome) in the UK over the past eight years is described. They have had a generally poor prognosis, with only two of the 10 still alive despite eight attempts at bone marrow transplantation in six patients. Overwhelming viral infection was the predominant cause of death. Alternative transplant strategies or novel therapies are required for these patients.

Bone Marrow Transplantation↗
PubMed1998

Effect of Tri-O-cresyl phosphate (TOCP) on proteolytic enzyme activities in mouse liver in vivo.

To develop a clearer understanding of the biochemical mechanisms of toxicity in higher animals, we determined the effect of four sublethal dosages (0.25 to 1.0 g/Kg) of organophosphate (OP), and of tri-ortho-cresyl phosphate (TOCP) on the activities of a representative range of cytoplasmic and lysosomal proteolytic enzymes in mouse liver in vivo. Administration of the lowest sublethal dosage of TOCP (0.25g/1Kg, 24 hours) resulted in significant inhibition (10 to 30% of control activity) of the cytoplasmic proteases tripeptidyl aminopeptidase and proline endopeptidase only. An increasing dosage of TOCP (1g/Kg for 7 days) resulted in significant inhibition (10 to 50% of control activity) of the cytoplasmic proteases alanyl aminopeptidase, leucyl aminopeptidase, tripeptidyl aminopeptidase, and proline endopeptidase, as well as the lysosomal proteases dipeptidyl aminopeptidase I and cathepsins B, D, and L. The activities of the cytoplasmic proteases arginyl aminopeptidase and dipeptidyl aminopeptidase IV, and of lysosomal proteases dipeptidyl aminopeptidase II and cathepsin H were not significantly altered following TOCP administration at this higher dosage. All of the above proteases are involved in the general process of intracellular protein turnover (common to all tissues), and may also have more specialized functions such as the processing of physiologically active hormonal peptides, and the processing of antigenic proteins in the immune response. Data obtained in the present study suggest that inhibition of the above enzymes by OP such as TOCP may represent a previously unrecognized toxicity hazard induced by the latter compound in higher animals.

Aminopeptidases↗
PubMed1997

Effect of pirimiphos-methyl on proteolytic enzyme activities in rat heart, kidney, brain and liver tissues in vivo.

To elucidate whether pesticide toxicity in higher animals involves pesticide-induced dysfunction of the intracellular protein catabolic process, we have determined the effect in vivo of the organophosphate insecticide pirimiphos-methyl on the activities of representative protein catabolising cytoplasmic and lysosomal proteases (responsible for the various stages of the protein degradation cascade and essential for normal cell functioning) in heart, kidney, brain and liver target tissues in the rat. In liver tissue (the major site of pesticide metabolism), the activities of all of the cytoplasmic proteases investigated (alanyl-, arginyl-, leucyl aminopeptidases, dipeptidyl aminopeptidase IV, tripeptidyl aminopeptidase, proline endopeptidase) were significantly inhibited (by 20-40% of control activity) following administration of 10 mg pirimiphos-methyl/kg bodyweight, whereas of the lysosomal proteases investigated, only the activities of dipeptidyl aminopeptidase I and cathepsin D were significantly reduced (by 15-20% of control activity). In contrast, there was no insecticide-induced inhibition of protease activities in heart, kidney or brain tissues; some lysosomal enzymes (dipeptidyl aminopeptidase I, cathepsins L and D) showed significantly increased activities in these tissues (the reason for which remains to be determined). We conclude that the effect of pirimiphos-methyl on proteolytic enzyme activities differs in different target tissues, and that pirimiphos-methyl induced inhibition of proteases in liver tissue may represent a previously unrecognised toxicity hazard in higher animals.

Animals↗
PubMed1995

Hypercalciuria and ultrasound abnormalities in children with cystinosis.

We noted microscopic haematuria in children with cystinosis. To investigate this we studied urinary calcium excretion and undertook renal ultrasound scans. Most patients had elevated urinary calcium excretion and all had abnormal appearances on ultrasound scan, ranging from increased cortical echogenicity only to those with increased cortical and medullary echogenicity. The ultrasound scan appearance was graded and correlated with laboratory parameters. It remains unclear as to the aetiology of the ultrasound findings and whether they are a consequence of treatment or a hitherto unrecognised feature of the disease.

Calcium Metabolism Disorders↗
PubMed1993

Renal parenchymal malakoplakia--a case report and review of the literature.

Malakoplakia is an inflammatory condition associated with persisting bacterial antigen in macrophages and characterized histologically by the Michaelis-Gutmann body, containing bacterial fragments. We review the pathogenesis of malakoplakia and report a novel form of treatment successfully used in an 8-week-old infant with bilateral renal malakoplakia. The patient presented with an acute Escherichia coli urinary tract infection and enlarged kidneys. Antibiotic regimes were ineffective, but once the diagnosis was made treatment was changed to an immunosuppressive regime of prednisolone and azathioprine, to which she responded promptly. Renal malakoplakia should be considered in any patient with a urinary tract infection unresponsive to antibiotics and enlarged kidneys. Although a large proportion of patients with malakoplakia have an underlying systemic disorder, which may account for their abnormal macrophage function, the rest demonstrate either an isolated macrophage defect or no detectable anomaly at all. It is in this latter group we suggest that an immunomodulating regime can be curative.

Azathioprine↗
PubMed1992

Studies on mitogen-induced lymphocyte transformation and the effect of pregnancy serum on mitogen-induced normal lymphocyte culture.

It is recognised that there is a non-reactive status in the maternal immune system during normal pregnancy as it fails to recognise the fetus which is an allograft. The present study was aimed to understand the nature of cell-mediated immune response and the presence of immunosuppressive factors during human pregnancy. The functional reactivity of lymphocytes was found to be reduced during pregnancy, particularly in the third trimester, as phytohaemagglutinin- and concanavalin-A-induced lymphocyte transformation was affected. It was also observed that in primigravidae and pregnant women with a history of repeated abortions the lymphocyte reactivity was not reduced during the first and second trimesters, but there was a marked reduction in the third trimester. However, in the case of multigravidae there was no change in lymphocyte transformation between the trimesters. When pregnancy serum from various trimesters was tested for its suppressive activity, it was found to suppress the mitogen-induced proliferation of lymphocytes from normal donors. Sera from primigravidae and patients with a history of abortion showed the highest suppressive activity in the third trimester, whereas sera from multigravidae showed high suppression in all the trimesters.

Concanavalin A↗
PubMed1992

Studies on the immunosuppressive role of steroid hormones during pregnancy.

It has been recognised that steroids can exert a profound influence over immunological reactivity. The present study analyzes the role of steroid hormones--estrogen, progesterone and cortisol--and their involvement in immunoregulation during pregnancy. As is known, the endogenous levels of all the three hormones increase during pregnancy. When the steroid levels in pregnancy serum were correlated with the lymphocyte response to mitogen, no correlation was observed. The suppressive effect of pregnancy serum was found to have no correlation with its steroid content. In general, steroids did not seem to affect the maternal immune system as evidenced by the present study.

Estradiol↗
PubMed1988

Effects of six months' feeding of cypermethrin on the blood and liver of albino rats.

A cypermethrin-mixed diet was fed uninterrupted to male albino rats for six months to evaluate toxicity in nontarget organisms. The rats consumed cypermethrin at a dose of 420 mg active ingredient (AI) per kilogram body weight per day. At the end of the stipulated period, the blood and liver were analyzed for insecticidal toxicity. The hemoglobin content and white blood cell (WBC) count remained unaltered, while the red blood cell (RBC) count and packed-cell volume (PCV) decreased significantly. The blood serum lactate dehydrogenase (LDH), isocitrate dehydrogenase (ICDH), and amylase activities were elevated 61%, 30%, and 46%, respectively, after six months of insecticide feeding, suggesting liver and possibly pancreas malfunction. The glutamate oxaloacetate transaminase (GOT) and creatine phosphokinase (CPK) activities, on the other hand, decreased 37% and 40%, respectively. The blood serum protein and free amino acids (FAA) content increased 12% and 31%, respectively, while cholesterol content decreased 49%. Consequent to cypermethrin administration the hepatic GOT, LDH, and ICDH activities increased 250%, 20%, and 30%, respectively. The soluble proteins, FAA, and glucose contents exhibited significant increases of 28%, 61%, and 71%, respectively. Histological changes were marked by hypertrophied hepatic cells and nuclei.

Amino Acids↗