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1.
J Appl Microbiol ; 130(5): 1721-1729, 2021 May.
Article in English | MEDLINE | ID: mdl-32966644

ABSTRACT

AIMS: This study was designed to analyse the prevalence of class 1 and class 2 integron integrase genes among antibiotic-resistant coliform bacteria isolated from paper currency circulating in Pakistan. METHODS AND RESULTS: A total of 500 individual currency notes were collected from different food vending sites at Lahore, Pakistan. Bacterial population were identified by biochemical and PCR techniques. Antimicrobial susceptibility testing was performed by disc diffusion assay. The highest bacterial population on currency was found from street vendors and butcher shops. Escherichia coli was found to be the most prevalent coliform bacteria followed by Klebsiella sp. and Enterobacter sp. PCR amplification of antimicrobial resistance gene showed the presence of ampC, blaTEM , blaNDM-1 , qnrA, tet(A) and tet(B) genes among coliform isolates. A total of 47 integron integrase bearing strains of coliform bacteria were analysed. Sequence analysis showed the presence of dfrA1-aadA1, dfrA1, dfrA5, dfrA7, aadA1, aadA4 cassette arrays in class 1 integron and dfrA1-sat2-aadA1 in class 2 integrase genes. CONCLUSION: Circulating currency was heavily contaminated with antimicrobial-resistant coliform bacteria bearing class 1 and class 2 integron integrase genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a potential threat of severe bacterial infections due to improper hand hygiene and community sanitation when dealing with the currency notes.


Subject(s)
Drug Resistance, Bacterial , Enterobacteriaceae/classification , Enterobacteriaceae/genetics , Integrases/genetics , Integrons/genetics , Paper , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Commerce , DNA, Bacterial , Enterobacteriaceae/drug effects , Hygiene , Microbial Sensitivity Tests , Pakistan , Polymerase Chain Reaction
2.
Nat Commun ; 10(1): 403, 2019 01 24.
Article in English | MEDLINE | ID: mdl-30679422

ABSTRACT

Albuminuria affects millions of people, and is an independent risk factor for kidney failure, cardiovascular morbidity and death. The key cell that prevents albuminuria is the terminally differentiated glomerular podocyte. Here we report the evolutionary importance of the enzyme Glycogen Synthase Kinase 3 (GSK3) for maintaining podocyte function in mice and the equivalent nephrocyte cell in Drosophila. Developmental deletion of both GSK3 isoforms (α and ß) in murine podocytes causes late neonatal death associated with massive albuminuria and renal failure. Similarly, silencing GSK3 in nephrocytes is developmentally lethal for this cell. Mature genetic or pharmacological podocyte/nephrocyte GSK3 inhibition is also detrimental; producing albuminuric kidney disease in mice and nephrocyte depletion in Drosophila. Mechanistically, GSK3 loss causes differentiated podocytes to re-enter the cell cycle and undergo mitotic catastrophe, modulated via the Hippo pathway but independent of Wnt-ß-catenin. This work clearly identifies GSK3 as a critical regulator of podocyte and hence kidney function.


Subject(s)
Albuminuria/metabolism , Glycogen Synthase Kinase 3/metabolism , Kidney Diseases/metabolism , Kidney/physiology , Podocytes/metabolism , Albuminuria/blood , Albuminuria/pathology , Albuminuria/urine , Animals , Cell Cycle , Cell Line , Disease Models, Animal , Drosophila , Gene Deletion , Gene Silencing , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3 beta/drug effects , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Hippo Signaling Pathway , Kaplan-Meier Estimate , Kidney/pathology , Kidney Diseases/blood , Kidney Diseases/pathology , Kidney Diseases/urine , Male , Mice , Podocytes/enzymology , Podocytes/pathology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proteomics , Rats, Wistar , Renal Insufficiency , Verteporfin/pharmacology , beta Catenin/metabolism
3.
Clin Radiol ; 74(2): 166.e9-166.e13, 2019 02.
Article in English | MEDLINE | ID: mdl-30591213

ABSTRACT

AIM: To test the effectiveness of a premedication protocol using intravenous bolus of 100 µg fentanyl to reduce pain associated with femoral artery closure device placement for neuro-endovascular procedures. MATERIALS AND METHODS: The severity of pain associated with femoral artery closure device placement was analysed using a numerical rating scale score ranging from 0 (no pain) to 10 (most severe pain) in two cohorts of consecutive adult patients (n=118), those who were (n=64) or were not (n=54) treated with premedication protocol. The primary endpoints were the proportion of patients with excellent (score ≤1) and failed pain control (score ≥8). Stepwise logistic regression analysis was performed to identify the effect of premedication on pain control after adjustment for potential confounders. RESULTS: The median numerical pain rating score at femoral artery closure device placement was significantly lower in patients treated with premedication protocol compared with those who underwent closure without premedication (1 versus 5, p<0.001). There was a significantly higher rate of excellent (56.2% versus 14.8%, p<0.001) and good (68.7% versus 31.2%, p<0.001) pain control at closure device placement among patients treated with premedication protocol. None of the patients treated with premedication protocol reported failed pain control compared with 33.4% of those who underwent closure device placement without premedication. In the multivariate analysis, treatment with the premedication protocol was significantly associated with an increased rate of excellent pain control (odds ratio 2.3; 95% confidence interval 1.9-3.1). CONCLUSION: Premedication with intravenous fentanyl injection prior to femoral artery closure device placement can reduce the intensity of pain associated with closure.


Subject(s)
Endovascular Procedures/methods , Equipment and Supplies , Femoral Artery/surgery , Fentanyl/therapeutic use , Pain/drug therapy , Premedication/methods , Adult , Aged , Aged, 80 and over , Analgesics, Opioid/administration & dosage , Analgesics, Opioid/therapeutic use , Female , Fentanyl/administration & dosage , Humans , Injections, Intravenous , Male , Middle Aged , Pain Measurement/methods , Prospective Studies , Registries , Treatment Outcome
4.
J Biol Regul Homeost Agents ; 32(2): 379-384, 2018.
Article in English | MEDLINE | ID: mdl-29685022

ABSTRACT

The current study was designed to evaluate bioactive compounds under the influence of biotic stress on seedlings of Peganum harmala. Biologically active compounds were determined by using different techniques. The HPLC and GC-MS analyses detected the significant amount of phenolic acids and active biological compound. Total protein content, activity of proteases, α-amylases, catalases and peroxidases were observed to be accelerated under fungal stress. The seedling extracts exhibited prominent antifungal and antimicrobial activity against selected strains. The present study showed that P. harmala is a good candidate to be used in natural therapies and medicine.


Subject(s)
Anti-Bacterial Agents/pharmacology , Peganum/chemistry , Phytochemicals/pharmacology , Plant Extracts/pharmacology
5.
Sci Rep ; 7(1): 2261, 2017 05 23.
Article in English | MEDLINE | ID: mdl-28536464

ABSTRACT

Renal sinus fat (RSF) is a perivascular fat compartment located around renal arteries. In this in vitro and in vivo study we hypothesized that the hepatokine fetuin-A may impair renal function in non alcoholic fatty liver disease (NAFLD) by altering inflammatory signalling in RSF. To study effects of the crosstalk between fetuin-A, RSF and kidney, human renal sinus fat cells (RSFC) were isolated and cocultured with human endothelial cells (EC) or podocytes (PO). RSFC caused downregulation of proinflammatory and upregulation of regenerative factors in cocultured EC and PO, indicating a protective influence of RFSC. However, fetuin-A inverted these benign effects of RSFC from an anti- to a proinflammatory status. RSF was quantified by magnetic resonance imaging and liver fat content by 1H-MR spectroscopy in 449 individuals at risk for type 2 diabetes. Impaired renal function was determined via urinary albumin/creatinine-ratio (uACR). RSF did not correlate with uACR in subjects without NAFLD (n = 212, p = 0.94), but correlated positively in subjects with NAFLD (n = 105, p = 0.0005). Estimated glomerular filtration rate (eGRF) was inversely correlated with RSF, suggesting lower eGFR for subjects with higher RSF (r = 0.24, p < 0.0001). In conclusion, our data suggest that in the presence of NAFLD elevated fetuin-A levels may impair renal function by RSF-induced proinflammatory signalling in glomerular cells.


Subject(s)
Intra-Abdominal Fat/physiology , Kidney Glomerulus/cytology , Kidney Glomerulus/metabolism , Kidney/anatomy & histology , Kidney/physiology , Renal Artery/anatomy & histology , alpha-2-HS-Glycoprotein/metabolism , Adipocytes/metabolism , Adult , Cells, Cultured , Coculture Techniques , Cytokines/metabolism , Female , Gene Expression , Humans , Immunohistochemistry , Inflammation Mediators/metabolism , Intra-Abdominal Fat/diagnostic imaging , Kidney/diagnostic imaging , Macrophages/metabolism , Male , Middle Aged , RNA, Messenger/genetics , Renal Artery/diagnostic imaging
6.
Cell Death Dis ; 6: e1752, 2015 May 07.
Article in English | MEDLINE | ID: mdl-25950482

ABSTRACT

High serum lipopolysaccharide (LPS) activity in normoalbuminuric patients with type 1 diabetes (T1D) predicts the progression of diabetic nephropathy (DN), but the mechanisms behind this remain unclear. We observed that treatment of cultured human podocytes with sera from normoalbuminuric T1D patients with high LPS activity downregulated 3-phosphoinositide-dependent kinase-1 (PDK1), an activator of the Akt cell survival pathway, and induced apoptosis. Knockdown of PDK1 in cultured human podocytes inhibited antiapoptotic Akt pathway, stimulated proapoptotic p38 MAPK pathway, and increased apoptosis demonstrating an antiapoptotic role for PDK1 in podocytes. Interestingly, PDK1 was downregulated in the glomeruli of diabetic rats and patients with type 2 diabetes before the onset of proteinuria, further suggesting that reduced expression of PDK1 associates with podocyte injury and development of DN. Treatment of podocytes in vitro and mice in vivo with LPS reduced PDK1 expression and induced apoptosis, which were prevented by inhibiting the Toll-like receptor (TLR) signaling pathway with the immunomodulatory agent GIT27. Our data show that LPS downregulates the cell survival factor PDK1 and induces podocyte apoptosis, and that blocking the TLR pathway with GIT27 may provide a non-nephrotoxic means to prevent the progression of DN.


Subject(s)
Podocytes/cytology , Podocytes/metabolism , Toll-Like Receptors/antagonists & inhibitors , Acetates/pharmacology , Animals , Apoptosis/physiology , Diabetes Mellitus, Type 1/blood , Humans , Lipopolysaccharides/blood , Male , Mice , Mice, Inbred BALB C , Oxazoles/pharmacology , Podocytes/drug effects , Protein Serine-Threonine Kinases/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Rats , Signal Transduction , Toll-Like Receptors/metabolism
7.
Apoptosis ; 20(7): 907-20, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25808596

ABSTRACT

Glomerulo-podocytic communication plays an important role in the podocytic injury in IgA nephropathy (IgAN). In this study, we examine the role of podocytic angiotensin II receptor subtype 1 (AT1R) and prorenin receptor (PRR) in podocytic apoptosis in IgAN. Polymeric IgA (pIgA) was isolated from patients with IgAN and healthy controls. Conditioned media were prepared from growth arrested human mesangial cells (HMC) incubated with pIgA from patients with IgAN (IgA-HMC media) or healthy controls (Ctl-HMC media). A human podocyte cell line was used as a model to examine the regulation of the expression of AT1R, PRR, TNF-α and CTGF by IgA-HMC media. Podocytic nephrin expression, annexin V binding and caspase 3 activity were used as the functional readout of podocytic apoptosis. IgA-HMC media had no effect on AngII release by podocytes. IgA-HMC media significantly up-regulated the expression of AT1R and PRR, down-regulated nephrin expression and induced apoptosis in podocytes. Mono-blockade of AT1R, PRR, TNF-α or CTGF partially reduced podocytic apoptosis. IgA-HMC media activated NFκB, notch1 and HEY1 expression by podocytes and dual blockade of AT1R with PRR, or anti-TNF-α with anti-CTGF, effectively rescued the podocytic apoptosis induced by IgA-HMC media. Our data suggests that pIgA-activated HMC up-regulates the expression of AT1R and PRR expression by podocytes and the associated activation of NFκB and notch signalling pathways play an essential role in the podocytic apoptosis induced by glomerulo-podocytic communication in IgAN. Simultaneously targeting the AT1R and PRR could be a potential therapeutic option to reduce the podocytic injury in IgAN.


Subject(s)
Apoptosis , Mesangial Cells/cytology , Podocytes/metabolism , Receptors, Angiotensin/metabolism , Receptors, Cell Surface/metabolism , Asian People , Female , Humans , Immunoglobulin A/metabolism , Male , Mesangial Cells/metabolism , Receptors, Notch/metabolism , Renin-Angiotensin System , Signal Transduction , Prorenin Receptor
8.
Exp Cell Res ; 326(2): 251-8, 2014 Aug 15.
Article in English | MEDLINE | ID: mdl-24815572

ABSTRACT

Evidence is emerging that podocytes are able to endocytose proteins such as albumin using kinetics consistent with a receptor-mediated process. To date the role of the fatty acid moiety on albumin uptake kinetics has not been delineated and the receptor responsible for uptake is yet to be identified. Albumin uptake studies were carried out on cultured human podocytes exposed to FITC-labelled human serum albumin either carrying fatty acids (HSA+FA) or depleted of them (HSA-FA). Receptor-mediated endocytosis of FITC-HSA+FA over 60 min was 5 times greater than that of FITC-HSA-FA. 24h exposure of podocytes to albumin up-regulated nephrin expression and induced the activation of caspase-3. These effects were more pronounced in response to HSA-FA. Individually, anti-CD36 antibodies had no effect upon endocytosis of FITC-HSA. However, a cocktail of 2 antibodies reduced uptake by nearly 50%. Albumin endocytosis was enhanced in the presence of the CD36 specific inhibitor sulfo-N-succinimidyl oleate (SSO) while knock-down of CD36 using CD36siRNA had no effect on uptake. These data suggest that receptor-mediated endocytosis of albumin by podocytes is regulated by the fatty acid moiety, although, some of the detrimental effects are induced independently of it. CD36 does not play a direct role in the uptake of albumin.


Subject(s)
CD36 Antigens/metabolism , Fatty Acids/metabolism , Fluorescein-5-isothiocyanate/analogs & derivatives , Podocytes/metabolism , Serum Albumin/metabolism , CD36 Antigens/antagonists & inhibitors , CD36 Antigens/genetics , Caspase 3/metabolism , Cell Line , Endocytosis , Fatty Acids/chemistry , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/metabolism , Gene Knockdown Techniques , Humans , Membrane Proteins/metabolism , Oleic Acids/pharmacology , Protein Binding , Serum Albumin/chemistry , Succinimides/pharmacology
9.
Am J Physiol Renal Physiol ; 305(2): F182-8, 2013 Jul 15.
Article in English | MEDLINE | ID: mdl-23698113

ABSTRACT

Podocytes are critically important for maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Recently, it has become clear that to achieve this, they need to be insulin sensitive and produce an optimal amount of VEGF-A. In other tissues, insulin has been shown to regulate VEGF-A release, but this has not been previously examined in the podocyte. Using in vitro and in vivo approaches, in the present study, we now show that insulin regulates VEGF-A in the podocyte in both mice and humans via the insulin receptor (IR). Insulin directly increased VEGF-A mRNA levels and protein production in conditionally immortalized wild-type human and murine podocytes. Furthermore, when podocytes were rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the IR) or in vivo (using transgenic podocyte-specific IR knockout mice), podocyte VEGF-A production was impaired. Importantly, in vivo, this occurs before the development of any podocyte damage due to podocyte insulin resistance. Modulation of VEGF-A by insulin in the podocyte may be another important factor in the development of glomerular disease associated with conditions in which insulin signaling to the podocyte is deranged.


Subject(s)
Insulin/metabolism , Podocytes/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Humans , Insulin Resistance , Mice , Mice, Knockout , RNA, Messenger/metabolism
10.
J Pathol ; 230(1): 95-106, 2013 May.
Article in English | MEDLINE | ID: mdl-23299523

ABSTRACT

Podocytes are crucial for preventing the passage of albumin into the urine and, when lost, are associated with the development of albuminuria, renal failure and cardiovascular disease. Podocytes have limited capacity to regenerate, therefore pro-survival mechanisms are critically important. Insulin-like growth factor-II (IGF-II) is a potent survival and growth factor; however, its major function is thought to be in prenatal development, when circulating levels are high. IGF-II has only previously been reported to continue to be expressed in discrete regions of the brain into adulthood in rodents, with systemic levels being undetectable. Using conditionally immortalized human and ex vivo adult mouse cells of the glomerulus, we demonstrated the podocyte to be the major glomerular source and target of IGF-II; it signals to this cell via the IGF-I receptor via the PI3 kinase and MAPK pathways. Functionally, a reduction in IGF signalling causes podocyte cell death in vitro and glomerular disease in vivo in an aged IGF-II transgenic mouse that produces approximately 60% of IGF-II due to a lack of the P2 promoter of this gene. Collectively, this work reveals the fundamental importance of IGF-II in the mature podocyte for glomerular health across mammalian species.


Subject(s)
Insulin-Like Growth Factor II/metabolism , Podocytes/cytology , Podocytes/metabolism , Signal Transduction/physiology , Aging/physiology , Animals , Cell Line, Transformed , Cell Survival/physiology , Humans , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor II/genetics , Kidney Glomerulus/cytology , Kidney Glomerulus/physiology , Mesangial Cells/cytology , Mesangial Cells/metabolism , Mice , Mice, Transgenic , RNA, Small Interfering/genetics
11.
Am J Physiol Renal Physiol ; 302(12): F1630-9, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22419694

ABSTRACT

Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291-299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.


Subject(s)
Glomerulonephritis/metabolism , Inflammation/metabolism , Kidney/metabolism , MicroRNAs/metabolism , Vascular Cell Adhesion Molecule-1/metabolism , Animals , Cell Line , Cells, Cultured , Disease Models, Animal , E-Selectin/genetics , E-Selectin/metabolism , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Glomerulonephritis/genetics , Humans , Inflammation/genetics , Kidney Glomerulus/metabolism , Mice , MicroRNAs/genetics , Tumor Necrosis Factor-alpha/pharmacology , Vascular Cell Adhesion Molecule-1/genetics
13.
Am J Physiol Renal Physiol ; 299(3): F664-73, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20630938

ABSTRACT

Human immunodeficiency virus (HIV)-1 has been demonstrated to contribute to the pathogenesis of HIV-associated nephropathy. In renal biopsy studies, podocytes have been reported to be infected by HIV-1. However, the mechanism involved in HIV-1 internalization into podocytes is not clear. In the present study, we evaluated the occurrence of HIV-1 internalization into conditionally immortalized human podocytes and the mechanism involved. Human podocytes rapidly internalized R5 and X4 HIV-1 primary strains via an endocytosis-dependent pathway, without establishing a productive infection. The HIV-1 internalization was dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) receptor mediated. The role of DC-SIGN was confirmed by using specific blocking antibodies and transfection with small interfering (si) RNA/DC-SIGN. Since podocyte HIV-1 trafficking was not altered by pH-modulating agents, it appeared that HIV-1 routing occurred through nonacid vesicular compartments. Interestingly, transfection of podocytes with neither siRNA/caveolin-1 nor siRNA/clathrin heavy chain inhibited podocyte viral accumulation. Thus it appears that clathrin-coated vesicles and caveosomes may not be contributing to HIV-1-associated membrane traffic.


Subject(s)
Cell Adhesion Molecules/physiology , HIV-1/physiology , Lectins, C-Type/physiology , Podocytes/virology , Receptors, Cell Surface/physiology , Virus Internalization , Caveolin 1/genetics , Cells, Cultured , Clathrin/genetics , Endocytosis/physiology , Humans , Hydrogen-Ion Concentration , Podocytes/cytology , RNA, Small Interfering/genetics , Transfection
14.
Indian J Pharm Sci ; 72(6): 710-8, 2010 Nov.
Article in English | MEDLINE | ID: mdl-21969742

ABSTRACT

The ability of ß-cyclodextrin, hydroxypropyl-ß-cyclodextrin, polyvinyl pyrrolidone and urea to influence the percutaneous absorption of meloxicam through isolated rat skin was evaluated. Carrier complex were prepared by kneading method in 1:1 and 1:2 in molar ratios for ß-cyclodextrin and hydroxypropyl-ß-cyclodextrin and in 1:1, 1:3 and 1:5 in weight ratios for polyvinyl pyrrolidone and urea. The complexes were characterized by IR, DSC and evaluated for solubility, dissolution and skin permeability. The solubility, dissolution and permeability of meloxicam were enhanced by using the carriers. The influence of cyclodextrins, polyvinyl pyrrolidone and urea on in vitro permeation of meloxicam through rat skin was investigated by incorporation of prepared carrier complex in 1% carbopol gel. The prepared gel was evaluated for drug content, pH and viscosity and in vitro permeation. All the percutaneous parameters like flux (Jss), amount permeated (Q(6)), diffusivity (D), permeability coefficient (K(p)), partition coefficient (K) and release rate constant (k) were calculated statistically. In vitro permeation study showed the trend that the penetration flux and enhancement factor increases with increasing concentration of ß-cyclodextrin and hydroxypropyl-ß-cyclodextrin and then decrease dramatically in case of hydroxypropyl-ß-cyclodextrin gel formulation with the increase to 1:2 ratio. Similar changes in pattern of permeation were also observed with polyvinyl pyrrolidone and urea carrier complex. These findings concluded that the carriers cyclodextrins, polyvinyl pyrrolidone and urea could be used as transdermal permeation enhancer in topical preparation of meloxicam.

15.
Diabetologia ; 52(9): 1944-52, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19533082

ABSTRACT

AIMS/HYPOTHESIS: Peroxisome proliferator-activated receptor (PPAR) gamma agonists are used increasingly in the treatment of type 2 diabetes. In the context of renal disease, PPARgamma agonists reduce microalbuminuria in diabetic nephropathy; however, the mechanisms underlying this effect are unknown. Glomerular podocytes are newly characterised insulin-sensitive cells and there is good evidence that they are targeted in diabetic nephropathy. In this study we investigated the functional and molecular effects of the PPARgamma agonist rosiglitazone on human podocytes. METHODS: Conditionally immortalised human podocytes were cultured with rosiglitazone and functional effects were measured with glucose-uptake assays. The effect of rosiglitazone on glucose uptake was also measured in 3T3-L1 adipocytes, nephrin-deficient podocytes, human glomerular endothelial cells, proximal tubular cells and podocytes treated with the NEFA palmitate. The role of the glucose transporter GLUT1 was investigated with immunofluorescence and small interfering RNA knockdown and the plasma membrane expression of GLUT1 was determined with bis-mannose photolabelling. RESULTS: Rosiglitazone significantly increased glucose uptake in wild-type podocytes and this was associated with translocation of GLUT1 to the plasma membrane. This effect was blocked with GLUT1 small interfering RNA. Nephrin-deficient podocytes, glomerular endothelial cells and proximal tubular cells did not increase glucose uptake in response to either insulin or rosiglitazone. Furthermore, rosiglitazone significantly increased basal and insulin-stimulated glucose uptake when podocytes were treated with the NEFA palmitate. CONCLUSIONS/INTERPRETATION: In conclusion, rosiglitazone has a direct and protective effect on glucose uptake in wild-type human podocytes. This represents a novel mechanism by which PPARgamma agonists may improve podocyte function in diabetic nephropathy.


Subject(s)
Glucose Transporter Type 1/metabolism , Glucose/metabolism , Hypoglycemic Agents/pharmacology , Kidney Glomerulus/metabolism , Podocytes/metabolism , Thiazolidinediones/pharmacology , Biological Transport/drug effects , Cell Culture Techniques , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , DNA Primers , Glucose Transporter Type 1/drug effects , Glucose Transporter Type 1/genetics , Humans , Kidney Glomerulus/drug effects , Kinetics , Podocytes/drug effects , RNA/genetics , Rosiglitazone , Transfection
16.
Singapore Med J ; 50(12): e410-1, 2009 Dec.
Article in English | MEDLINE | ID: mdl-20087542

ABSTRACT

We report a rare case of spontaneous "idiopathic" rupture of the urinary bladder in a 38-year-old previously-fit and -well woman, who presented with symptoms of severe lower abdominal pain. Computed tomography of the abdomen and pelvis indicated the preliminary findings, but the final diagnosis was confirmed only at laparoscopy. The bladder was repaired. The postoperative cystogram showed excellent healing of the bladder, with no further intra-abdominal urinary leakage.


Subject(s)
Urinary Bladder Diseases/diagnostic imaging , Urinary Bladder Diseases/surgery , Urologic Surgical Procedures/methods , Abdominal Pain/etiology , Adult , Alcoholic Intoxication/complications , Female , Humans , Laparoscopy/methods , Rupture, Spontaneous/diagnostic imaging , Rupture, Spontaneous/surgery , Tomography, X-Ray Computed , Urinary Bladder Diseases/etiology
17.
Indian J Pharm Sci ; 70(5): 640-4, 2008 Sep.
Article in English | MEDLINE | ID: mdl-21394263

ABSTRACT

Rectal suppositories of tramadol hydrochloride were prepared using different bases and polymers like PEG, cocoa butter, agar and the effect of different additives on in vitro release of tramadol hydrochloride was studied. The agar-based suppositories were non-disintegrating/non-dissolving, whereas PEGs were disintegrating/dissolving and cocoa butter were melting suppositories. All the prepared suppositories were evaluated for various physical parameters like weight variation, drug content and hardness. The PEG and cocoa butter suppositories were evaluated for macromelting range, disintegration and liquefaction time. In vitro release study was performed by USP type I apparatus. The prepared suppositories were within the permissible range of all physical parameters. In vitro drug release was in the order of PEG>Agar>cocoa butter. Addition of PVP, HPMC in agar suppositories retards the release. The mechanism of drug release was diffusion controlled and follows first order kinetics. The results suggested that blends of PEG of low molecular weight (1000) with high molecular weight (4000 and 6000) in different percentage and agar in 10% w/w as base used to formulate rapid release suppositories. The sustained release suppositories can be prepared by addition of PVP, HPMC in agar-based suppositories and by use of cocoa butter as base.

18.
Kidney Int ; 70(6): 1038-45, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16837924

ABSTRACT

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.


Subject(s)
Antigens, Polyomavirus Transforming , Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Podocytes/drug effects , Simian virus 40/immunology , Cell Line, Transformed , Cell Survival/drug effects , Cell Transformation, Viral , Child, Preschool , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Humans , Immunohistochemistry , Interleukin-6/metabolism , Membrane Proteins/metabolism , Tubulin/metabolism , Vascular Endothelial Growth Factor A/metabolism
19.
Am J Transplant ; 6(7): 1666-72, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16827869

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/pharmacology , Kidney Transplantation , Recombinant Fusion Proteins/pharmacology , Tacrolimus/pharmacology , Adolescent , Antibodies, Monoclonal/adverse effects , Basiliximab , Biopsy , Child , Child, Preschool , Female , Follow-Up Studies , Graft Rejection , Graft Survival/drug effects , Humans , Male , Recombinant Fusion Proteins/adverse effects , Tacrolimus/adverse effects , Tacrolimus/blood
20.
Am J Physiol Renal Physiol ; 291(4): F722-30, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16622173

ABSTRACT

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.


Subject(s)
Nephrotic Syndrome/physiopathology , Podocytes/physiology , Proteoglycans/biosynthesis , Animals , Cell Line , DNA Primers , DNA Probes , Dexamethasone/pharmacology , Female , Glycosaminoglycans/genetics , Podocytes/drug effects , Proteoglycans/genetics , Puromycin Aminonucleoside/pharmacology , Rats , Rats, Sprague-Dawley
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