Effects of diabetes and hypertension on glomerular transforming growth factor-β receptor expression

Effects of diabetes and hypertension on glomerular transforming growth factor-β receptor expression.BackgroundSeveral studies have suggested that transforming growth factor-β1 (TGF-β1) is an important determinant of diabetic glomerular injury. TGF-β1 forms a heteromeric complex with two cellular receptor subtypes, designated TGF-β RII and TGF-β RI, but the effects of diabetes mellitus on glomerular TGF-β receptor expression have not been completely elucidated. We first compared the effect of experimental type I diabetes mellitus and uninephrectomy on glomerular TGF-β receptor expression in spontaneously hypertensive rats (SHRs), and then sought to determine whether changes in TGF-β receptor expression were strain specific by studying normotensive Wistar-Kyoto (WKY) rats.MethodsFive groups of male SHRs were studied. The first group received streptozotocin (60 mg/kg IV) and was studied after one week. The second group received streptozotocin and was studied after two weeks. The third group received streptozotocin (60 mg/kg IV) but received insulin to maintain euglycemia. The fourth group of age-matched SHRs served as the control group, while a fifth group of SHRs underwent uninephrectomy. Four groups of male WKY rats were also studied. The first group of WKY rats served as the age-matched control group. The second group of WKY rats received streptozotocin, while a third group of WKY rats underwent uninephrectomy. The fourth group underwent uninephrectomy and received streptozotocin. At each time point, glomeruli were isolated for protein extraction, and the protein was subjected to Western blot analysis of TGF-β RII and TGF-β RI expression.ResultsBasal expression of both TGF-β receptors per microgram of glomerular protein was similar in normotensive WKY rats and hypertensive SHRs. Hyperglycemia (blood glucose level, 17.8 ± 2.9 mmol/L) led to an early twofold increase in TGF-β RII protein expression and a fourfold increase in TGF-β RI protein expression in the glomeruli of hypertensive diabetic SHRs compared with euglycemic SHRs (blood glucose level, 5.8 ± 0.8 mmol/L), which was sustained after two weeks. Insulin treatment (blood glucose level, 5.2 ± 0.9 mmol/L) normalized both TGF-β RII and TGF-β RI expression in the glomeruli of SHRs that received streptozotocin. Glomerular capillary hypertension in the uninephrectomized SHRs led to a twofold increase in glomerular TGF-β RII protein expression, but did not reproduce the effect of diabetes mellitus on TGF-β RI expression. In contrast to the findings in SHRs, neither hyperglycemia (blood glucose level, 15.5 ± 2.1 mmol/L), uninephrectomy, nor hyperglycemia (blood glucose level, 16.8 ± 3.0 mmol/L) and uninephrectomy altered TGF-β receptor expression in the glomeruli of normotensive WKY rats.ConclusionThese studies support the hypothesis that hemodynamic factors and metabolic factors influence glomerular TGF-β receptor in vivo in the SHRs

Activation of mesangial cell MAPK in responseto homocysteine

Activation of mesangial cell MAPK in response to homocysteine.BackgroundAlteration in mesangial cell function is central to the progression of glomerular disease in numerous models of chronic renal failure (CRF). Animal models of chronic glomerular disease are characterized by mesangial cell proliferation and elaboration of extracellular matrix protein (ECM), resulting in glomerulosclerosis. Elevated plasma levels of homocysteine (Hcy) are seen in both animal models and humans with CRF, and have been proposed to contribute to the high prevalence of vascular disease in this group. Some of the pathogenetic effects of Hcy are thought to be mediated via the induction of endoplasmic reticulum stress. Thus, Hcy effects on mesangial cells could contribute to the progression of CRF. Previous work has shown Hcy- mediated induction of Erk mitogen-activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). Erk induces increases in activator protein-1 (AP-1) transcription factor activity which may augment mesangial cell proliferation and ECM protein production. Consequently, we studied the effect of Hcy on mesangial cell Erk signaling.MethodsMesangial cells were exposed to Hcy after 24 hours of serum starvation and Erk activity assessed. Nuclear translocation of phospho-Erk was visualized by confocal microscopy. AP-1 nuclear protein binding was measured in response to Hcy by mobility shift assay. Hcy-induced mesangial cell calcium flux was measured in Fura-2 loaded cells. Mesangial cell DNA synthesis in response to Hcy was assessed by [3H]-thymidine incorporation and proliferation by Western blotting for proliferating cell nuclear antigen (PCNA). Expression of endoplasmic reticulum stress response genes were determined by Northern and Western analysis.ResultsHcy led to an increase in Erk activity that was maximal at 50 μmol/L and 20 minutes of treatment. Subsequent experiments used this concentration and time point. Erk activity in response to Hcy was insensitive to n-acetylcysteine and catalase, indicating oxidative stress did not play a role. However, Hcy50 μmol/L induced a brief increase in intracellular mesangial cell calcium within 5 minutes, and the calcium ionophores A23187 and ionomycin increased Erk activity while chelation of intracellular calcium with BAPTA-AM abrogated the Erk response to Hcy. Confocal microscopy of activated Erk nuclear translocation mirrored these results as did mesangial cell nuclear protein binding to AP-1 consensus sequences. Hcy- induced increases in thymidine incorporation and PCNA expression at 24 hours were Erk dependent. The expression of endoplasmic reticulum stress response genes was significantly elevated by Hcy in an Erk-dependent manner.ConclusionHcy increases Erk activity in mesangial cells via a calcium-dependent mechanism, resulting in increased AP-1 nuclear protein binding, cell DNA synthesis and proliferation and induction of endoplasmic reticulum stress. These observations suggest potential mechanisms by which Hcy may contribute to progressive glomerular injury

Improving the public house in Britain, 1920-40: Sir Sydney Nevile and 'social work'

The ‘improved public house’ movement in the inter-war years was a central part of the shift towards retailing by the brewing industry. An important part of the reform movement was the alliance between certain brewers, notably Whitbread, and ‘social workers’, particularly those associated with the University settlement movement in London. Using the papers of Sydney Nevile, the importance of a particular social milieu is outlined, calling into question attempts to align the movement to improve public houses with transatlantic Progressivism. Rather, this alliance drew upon longstanding English traditions of public service and religious affiliation amongst a fraction of the gentry

Multi-faceted intervention to improve management of antibiotics for children presenting to primary care with acute cough and respiratory tract infection (CHICO): efficient cluster randomised controlled trial

Objective: To assess whether an easy-to-use multifaceted intervention for children presenting to primary care with respiratory tract infections would reduce antibiotic dispensing, without increasing hospital admissions for respiratory tract infection. Design: Two arm randomised controlled trial clustered by general practice, using routine outcome data, with qualitative and economic evaluations. Setting: English primary care practices using the EMIS electronic medical record system. Participants: Children aged 0-9 years presenting with respiratory tract infection at 294 general practices, before and during the covid-19 pandemic. Intervention: Elicitation of parental concerns during consultation; a clinician focused prognostic algorithm to identify children at very low, normal, or elevated 30 day risk of hospital admission accompanied by antibiotic prescribing guidance; and a leaflet for carers including safety netting advice. Main outcome measures: Rate of dispensed amoxicillin and macrolide antibiotics (superiority comparison) and hospital admissions for respiratory tract infection (non-inferiority comparison) for children aged 0-9 years over 12 months (same age practice list size as denominator). Results: Of 310 practices needed, 294 (95%) were randomised (144 intervention and 150 controls) representing 5% of all registered 0-9 year olds in England. Of these, 12 (4%) subsequently withdrew (six owing to the pandemic). Median intervention use per practice was 70 (by a median of 9 clinicians). No evidence was found that antibiotic dispensing differed between intervention practices (155 (95% confidence interval 138 to 174) items/year/1000 children) and control practices (157 (140 to 176) items/year/1000 children) (rate ratio 1.011, 95% confidence interval 0.992 to 1.029; P=0.25). Pre-specified subgroup analyses suggested reduced dispensing in intervention practices with fewer prescribing nurses, in single site (compared with multisite) practices, and in practices located in areas of lower socioeconomic deprivation, which may warrant future investigation. Pre-specified sensitivity analysis suggested reduced dispensing among older children in the intervention arm (P=0.03). A post hoc sensitivity analysis suggested less dispensing in intervention practices before the pandemic (rate ratio 0.967, 0.946 to 0.989; P=0.003). The rate of hospital admission for respiratory tract infections in the intervention practices (13 (95% confidence interval 10 to 18) admissions/1000 children) was non-inferior compared with control practices (15 (12 to 20) admissions/1000 children) (rate ratio 0.952, 0.905 to 1.003). Conclusions: This multifaceted antibiotic stewardship intervention for children with respiratory tract infections did not reduce overall antibiotic dispensing or increase respiratory tract infection related hospital admissions. Evidence suggested that in some subgroups and situations (for example, under non-pandemic conditions) the intervention slightly reduced prescribing rates but not in a clinically relevant way. Trial registration: ISRCTN11405239ISRCTN registry ISRCTN1140523

Multiple pyarthrosis in human immunodeficiency virus-infected hemophiliacs

Classically, a swollen, painful joint in a patient with hemophilia has been considered to be due to a hemarthrosis until otherwise proven, and treated immediately with appropriate coagulation factor replacement. Two cases of human immunodeficiency virus (hiv)-infected hemophiliacs presenting with an initial apparent hemarthrosis, complicated subsequently by numerous pyarthroses and sepsis are described. In light of the prevalence of hiv infection in the adult hemophiliac population with arthropathy, a reappraisal of the clinical caveat of immediate infusion without joint aspiration is required

Formation and maintenance of DU145 spheres occurs independently of exogenous mitogens, with EGF treatment enhancing sphere formation.

<p>A) EGF enhances sphere formation independent of the cell seeding density. DU145 spheres were generated and maintained in serum-free media containing 0.4% BSA and 0.2× B27 (SF), and supplemented with or without EGF (10 or 20 ng/ml; 10EGF or 20EGF, respectively) for three passages to establish EGF-free (-EGF) and EGF-stimulated sphere cultures prior to experimentation. Sphere cells were seeded at a density of 5×10² and 1×10³ cells/well (1×10³ and 2×10³ cells/ml, respectively) in 24-well plates (three replicates per treatment). Six experiments were conducted. Spheres maintained in EGF-supplemented SF media display enhanced sphere numbers compared to -EGF sphere cultures. Spheres maintained in SF media containing >10 ng/ml EGF do not display additional increases in sphere-forming capacity. B) Treatment of -EGF spheres with increasing concentrations of EGF (+10EGF or +20EGF, respectively) increased sphere formation, and C) enhanced activation of EGFR signaling (EGFR phosphorylation at Tyr1068; EGFR-P). Sphere numbers are displayed as mean ± S.E.M. of four independent experiments (*<i>p</i><0.05 in comparison to the respective -EGF media condition; two-tailed independent Student’s <i>t</i>-test).</p

DU145 sphere formation is significantly reduced by ERK1 and ERK2 knockdown.

<p>A) Western blot analysis of endogenous ERK1 and ERK2 (ERK1/2) protein levels following ERK1 or ERK2 knockdown in DU145 monolayer cells. B) ERK1 or ERK2 knockdown results in a significant reduction in primary sphere formation compared to shRNA control (shLacZ) cells. Cells were seeded at a density of 2.5×10³ cells/well (0.5 ml/well; three replicates per treatment). The number of primary (1°) spheres formed in the absence (-EGF) or presence of 10 ng/ml EGF (10EGF) is displayed as mean ± S.E.M. of three independent experiments (the <i>p</i> values for the indicated comparisons were obtained by two-tailed independent Student’s <i>t</i>-test).</p

EGFR knockdown reduces the sphere-forming capacity and self-renewal activity of DU145 PCSCs.

<p>A) DU145 monolayer cells were infected with EGFR shRNA lentiviral constructs and selected with puromycin in order to generate stable cell lines. Western blot analysis of various EGFR knockdown (shEGFR) cells was carried out to evaluate the EGFR knockdown efficiency relative to the non-specific shRNA control (shCTRL) cell line. B) Stable EGFR knockdown cells form fewer primary (1°) spheres compared to shCTRL cells. Cells were seeded at a density of 2.5×10³ cells/well (0.5 ml/well; three replicates per treatment). Sphere numbers are displayed as mean ± S.E.M. of three independent experiments (*<i>p</i><0.05 in comparison to the respective -EGF media condition for each cell line; two-tailed independent Student’s <i>t</i>-test). C) Efficient EGFR knockdown in established DU145 spheres can also be achieved. Cells from EGF-free (-EGF) spheres were infected with shCTRL and shEGFR2 lentivirus. Loss of EGFR protein expression significantly reduces the D) number of DU145 spheres grown under EGF-free media conditions. EGFR knockdown (shEGFR2) and shCTRL sphere cells were seeded at a density of 1×10³ cells/well (three replicates per cell line). The number of spheres that formed was counted 12 days post-seeding. Sphere number is displayed as mean ± S.E.M. of three independent experiments (**<i>p</i><0.01; two-tailed independent Student’s <i>t</i>-test). E) EGFR knockdown in DU145 sphere cells significantly reduced their in vitro anchorage-independent growth. The total number of colonies in 5 random fields (at 25X magnification) was counted and represented as mean ± S.E.M. of three independent experiments (**<i>p</i><0.01; two-tailed independent Student’s <i>t</i>-test). Representative phase contrast images of colonies are shown at 50X magnification (inset). Scale bar is equal to 100 µm.</p

MEK1-dependent signal blockage inhibits DU145 sphere formation.

<p>A) Inhibition of ERK signaling following treatment of DU145 sphere cells with increasing concentrations (µM) of U0126 (MEK inhibitor) reduces their capacity to form subsequent spheres. U0126 or DMSO (mock) treatment was administered at the time of seeding. Sphere cells were seeded at a density of 1×10³ cells/well (0.5 ml/well; three replicates per treatment). The number of spheres (mean ± S.E.M.) that formed after 12 days of culture was counted (**<i>p</i><0.01 compared to DMSO control; two-tailed independent Student’s <i>t</i>-test). B) Dominant-negative MEK1(K97M) (pLHCX) cells display reduced ERK1/2 activation compared to empty vector (EV; pLHCX) control cells. DU145 EV and MEK1(K97M) cells were serum-starved for 12 h, and subsequently stimulated with serum-supplemented (10% FBS) or serum-free (SF) media for 60 min. C) MEK1(K97M) cells display reduced primary (1^o) sphere-forming capacity compared to EV cells when seeded in serum-free media lacking EGF (-EGF) or containing 10 ng/ml EGF (10EGF). Cells were seeded at a density of 2.5×10³ cells/well (three replicates per treatment). Sphere numbers are displayed as mean ± S.E.M. of three independent experiments (the <i>p</i> values for the indicated comparisons were obtained by two-tailed independent Student’s <i>t</i>-test).</p

EGFR signaling blockage reduces the self-renewal activity of DU145 PCSCs.

<p>A) Western blot analysis of whole cell lysates following 24 hour treatment of DU145 spheres with DMSO (D) or increasing concentrations of AG1478 (0.1–20 µM concentration range), in the presence or absence of exogenous EGF (10 ng/ml; 10EGF). Sphere formation of EGF-free sphere cells was inhibited following treatment with EGFR inhibitors B) AG1478 or C) PD168393 at various doses in the presence (10EGF) or absence (-EGF) of exogenous EGF (10 ng/ml). Sphere cells were seeded at a density of 2×10³ cells/well (0.5 ml/well; three replicates per treatment). EGFR inhibitor (AG1478 or PD168393) or DMSO (mock) treatment was administered at the time of seeding. Spheres that formed were counted 12 days post-seeding. Sphere numbers are displayed as mean ± S.E.M. (the <i>p</i> values for the indicated comparisons were obtained by two-tailed independent Student’s <i>t</i>-test).</p