Catharine I. Whiteside

High Glucose-suppressed Endothelin-1 Ca2+ Signaling via NADPH Oxidase and Diacylglycerol-sensitive Protein Kinase C Isozymes in Mesangial Cells

High glucose (HG) is the underlying factor contributing to long term complications of diabetes mellitus. The molecular mechanisms transforming the glomerular mesangial cell phenotype to cause nephropathy including diacylglycerol-sensitive protein kinase C (PKC) are still being defined. Reactive oxygen species (ROS) have been postulated as a unifying mechanism for HG-induced complications. We hypothesized that in HG an interaction between ROS generation, from NADPH oxidase, and PKC suppresses mesangial Ca2+ signaling in response to endothelin-1 (ET-1). In primary rat mesangial cells, growth-arrested (48 h) in 5.6 mm (NG) or 30 mm (HG) glucose, the total cell peak [Ca2+]i response to ET-1 (50 nm) was 630 ± 102 nm in NG and was reduced to 159 ± 15 nm in HG, measured by confocal imaging. Inhibition of PKC with phorbol ester down-regulation in HG normalized the ET-1-stimulated [Ca2+]i response to 541 ± 74 nm. Conversely, an inhibitory peptide specific for PKC-ζ did not alter Ca2+ signaling in HG. Furthermore, overexpression of conventional PKC-β or novel PKC-δ in NG diminished the [Ca2+]i response to ET-1, reflecting the condition observed in HG. Likewise, catalase or p47phox antisense oligonucleotide normalized the [Ca2+]i response to ET-1 in HG to 521 ± 58 nm and 514 ± 48 nm, respectively. Pretreatment with carbonyl cyanide m-chlorophenylhydrazone or rotenone did not restore Ca2+ signaling in HG. Detection of increased intracellular ROS in HG by dichlorofluorescein was inhibited by catalase, diphenyleneiodonium, or p47phox antisense oligonucleotide. HG increased p47phox mRNA by 1.7 ± 0.1-fold as measured by reverse transcriptase-PCR. In NG, H2O2 increased membrane-enriched PKC-β and -δ, suggesting activation of these isozymes. HG-enhanced immunoreactivity of PKC-δ visualized by confocal imaging was attenuated by diphenyleneiodium chloride. Thus, mesangial cell [Ca2+]i signaling in response to ET-1 in HG is attenuated through an interaction mechanism between NADPH oxidase ROS production and diacylglycerol-sensitive PKC.

Protein kinase A is a negative regulator of renal branching morphogenesis and modulates inhibitory and stimulatory bone morphogenetic proteins.

Protein kinase A (PKA) regulates morphogenetic responses to bone morphogenetic proteins (BMPs) during embryogenesis. However, the mechanisms by which PKA regulates BMP function are unknown. During kidney development, BMP-2 and high doses of BMP-7 inhibit branching morphogenesis, whereas low doses of BMP-7 are stimulatory (Piscione, T. D., Yager, T. D., Gupta, I. R., Grinfeld, B., Pei, Y., Attisano, L., Wrana, J. L., and Rosenblum, N. D. (1997) Am. J. Physiol. 273, F961–F975). We examined the interactions between PKA and these BMPs in embryonic kidney explants and in the mouse inner medullary collecting duct-3 model of collecting duct morphogenesis. H-89, an inhibitor of PKA, stimulated branching morphogenesis and enhanced the stimulatory effect of low doses of BMP-7 on tubule formation. Furthermore, H-89 rescued the inhibition of tubulogenesis by BMP-2 (or high doses of BMP-7) by attenuating BMP-2-induced collecting duct apoptosis. In contrast, 8-bromo-cAMP, an activator of PKA, inhibited tubule formation and attenuated the stimulatory effects of low doses of BMP-7. To determine mechanisms underlying the interdependence of BMP signaling and PKA activity, we examined the effect of PKA on the known signaling events in the BMP-2-dependent Smad1 signaling pathway and the effect of BMP-2 on PKA activity. PKA did not induce endogenous Smad1 phosphorylation, Smad1-Smad4 complex formation, or Smad1 nuclear translocation. In contrast, BMP-2 increased endogenous PKA activity and induced phosphorylation of the PKA effector, cAMP-response element-binding protein, in a PKA-dependent manner. We conclude that BMP-2 induces activation of PKA and that PKA regulates the effects of BMPs on collecting duct morphogenesis without activating the known signaling events in the BMP-2-dependent Smad1 signaling pathway.

High glucose activates PKC-ζ and NADPH oxidase through autocrine TGF-β1 signaling in mesangial cells

Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-beta(1) is central to the pathogenesis of diabetic nephropathy. Previously, we reported that mesangial cells respond to high glucose by generating reactive oxygen species (ROS) from NADPH oxidase dependent on protein kinase C (PKC) -zeta activation. We investigated the role of TGF-beta(1) in this action of high glucose on primary rat mesangial cells within 1-48 h. Both high glucose and exogenous TGF-beta(1) stimulated PKC-zeta kinase activity, as measured by an immune complex kinase assay and immunofluorescence confocal cellular imaging. In high glucose, Akt Ser473 phosphorylation appeared within 1 h and Smad2/3 nuclear translocation was prevented with neutralizing TGF-beta(1) antibodies. Neutralizing TGF-beta(1) antibodies, or a TGF-beta receptor kinase inhibitor (LY364947), or a phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC-zeta activation by high glucose. TGF-beta(1) also stimulated cellular membrane translocation of PKC-alpha, -beta(1), -delta, and -epsilon, similar to high glucose. High glucose and TGF-beta(1) enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-beta(1) antibodies, LY364947, or a specific PKC-zeta pseudosubstrate peptide inhibitor. Expression of constitutively active PKC-zeta in normal glucose caused upregulation of p22(phox), a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-beta(1) stimulation of PKC isozymes including PI3 kinase activation of PKC-zeta and consequent generation of ROS by NADPH oxidase.

Heparin Inhibits Mitogen-activated Protein Kinase-dependent and -independent c-fos Induction in Mesangial Cells

Heparin suppresses mitogenic responses in renal mesangial cells, and when quiescent mesangial cells are stimulated with serum, heparin blocks the induction of c-fos seen at 15 min. Because heparin is taken up by cells over a much longer time course, we addressed mechanisms whereby extracellular heparin might suppress c-fos induction at such early times. Quiescent cells were treated with serum, 12-O-tetradecanoylphorbol-13-acetate, or low concentrations of Ca2+ ionophores that produced increases in intracellular Ca2+ concentration ([Ca2+]i) in the physiological range. Each treatment caused an increase in c-fos mRNA, but they did so by different mechanisms. Serum activated mitogen-activated protein kinase (MAPK) and increased [Ca2+]i without affecting protein kinase C. Activation of protein kinase C with phorbol ester activated MAPK without much effect on [Ca2+]i. Ionophores increased [Ca2+]i without affecting basal levels of protein kinase C or MAPK. Heparin (1 μg/ml) suppressed the induction of c-fos initiated by all three treatments. It did not affect the activity of protein kinase C, but inhibited activation of MAPK by either serum or phorbol ester, suggesting a common site of action at or below the probable convergence of the induced signals at Ras/Raf-1 activation. Heparin also inhibited the serum-stimulated entry of extracellular Ca2+ to the same extent as verapamil, consistent with the ability of verapamil to block L-type Ca2+ channels and the known presence of these channels in mesangial cells. However, this effect does not appear to be related to heparins ability to inhibit induction of c-fos. First, verapamil had no effect on induction of c-fos by serum. Second, heparin had no effect on changes in [Ca2+]i achieved by ionophores. We conclude that heparin suppresses induction of c-fos in mesangial cells by blocking at least two different points in signal transduction cascades, one upstream of MAPK and the other independent of MAPK, but dependent on intracellular Ca2+.

O-linked β-N-acetylglucosamine supports p38 MAPK activation by high glucose in glomerular mesangial cells

Hyperglycemia augments flux through the hexosamine biosynthetic pathway and subsequent O-linkage of single β-N-acetyl-d-glucosamine moieties to serine and threonine residues on cytoplasmic and nuclear proteins (O-GlcNAcylation). Perturbations in this posttranslational modification have been proposed to promote glomerular matrix accumulation in diabetic nephropathy, but clear evidence and mechanism are lacking. We tested the hypothesis that O-GlcNAcylation enhances profibrotic signaling in rat mesangial cells. An adenovirus expressing shRNA directed against O-GlcNAc transferase (OGT) markedly reduced basal and high-glucose-stimulated O-GlcNAcylation. Interestingly, O-GlcNAc depletion prevented high-glucose-induced p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase phosphorylation. Downstream of p38, O-GlcNAc controlled the expression of plasminogen activator inhibitor-1, fibronectin, and transforming growth factor-β, important factors in matrix accumulation in diabetic nephropathy. Treating mesangial cells with thiamet-G, a highly selective inhibitor of O-GlcNAc-specific hexosaminidase (O-GlcNAcase), increased O-GlcNAcylation and p38 phosphorylation. The high-glucose-stimulated kinase activity of apoptosis signal-regulating kinase 1 (ASK1), an upstream MAPK kinase kinase for p38 that is negatively regulated by Akt, was inhibited by OGT shRNA. Akt Thr(308) and Ser(473) phosphorylation were enhanced following OGT shRNA expression in high-glucose-exposed mesangial cells, but high-glucose-induced p38 phosphorylation was not attenuated by OGT shRNA in cells pretreated with the phosphatidylinositol 3-kinase inhibitor LY-294002. OGT shRNA also reduced high-glucose-stimulated reactive oxygen species (ROS) formation. In contrast, diminished O-GlcNAcylation caused elevated ERK phosphorylation and PKCδ membrane translocation. Thus, O-GlcNAcylation is coupled to profibrotic p38 MAPK signaling by high glucose in part through Akt and possibly through ROS.

Glomerular Mesangial Cell Altered Contractility in High Glucose Is Ca2+ Independent

In diabetes, loss of renal arteriolar smooth-muscle cell contractility leads to intraglomerular hypertension. In glomeruli isolated from streptozotocin (STZ)-induced diabetic rats, the mesangial cells (smooth muscle-like) display loss of contractile responsiveness to angiotensin II. This study examines the mechanistic relationship between altered mesangial cell contractility and vasopressor hormone-stimulated Ca2+ signaling in high glucose. Glomeruli were isolated from normal or STZ-induced diabetic rats to observe ex vivo mesangial cell contractile function. Also, rat mesangial cells were cultured (10–20 passages) in normal (5.6 mmol/1) or high (10–25.6 mmol/1) glucose for 1–5 days. Reduction of glomerular volume and decreased planar surface area of cultured mesangial cells in response to vasoconstrictor stimulation over 60 min were measured by videomicroscopy and personal computer—based morphometry. Contraction of glomeruli isolated from STZ-administered rats in response to endothelin (ET)-1 (0.1 μmol/1) or the Ca2+ ionophore A23187 (5 μmol/l) was impaired significantly compared with that in normal glucose. In the presence of arginine vasopressin (AVP) (1.0 μmol/1) or ET-1 (0.1 μmol/1), mesangial cells demonstrated a dose-dependent loss of contractile response to increasing glucose concentrations (5.6–25.6 mmol/1) within 24 h of high-glucose exposure, which was sustained for 5 days. Mesangial cells in high glucose were consistently smaller in size compared with those in normal glucose. Mesangial cells were preloaded with myo-[2-3H]inositol and intracellular [3H] inositol phosphate release in response to AVP (1.0 μmol/1) was analyzed by Dowex chromatography. Comparing cells in normal (5.6 mmol/1) versus high (25.6 mmol/1) glucose, we observed no significant difference in stimulated inositol phosphate levels from 10 to 60 s. The Ca2+-signaling response of cultured mesangial cells preloaded with Fura 2 or Indo 1 was measured by spectrofluorometry. After culture in 25.6 mmol/1 glucose, 0.1 μmol/l AVP or 0.1 μmol/1 ET-1 stimulated a cytosolic Ca2+ signal, with first and second phases, identical to that exhibited by mesangial cells in normal glucose. Fluorescence imaging of cultured mesangial cell cytoskeletal filamentous actin (F-actin) stained with rhodamine-phalloidin demonstrated reduced F-actin staining in the basal state and loss of the normal F-actin disassembly response to ET-1 in high glucose. Glomerular mesangial cells display glucose-induced altered contraction and F-actin disassembly to vasoactive stimuli, which occur in the presence of normal Ca2+ signaling.

Accurate measurement of impaired glomerular filtration using single-dose oral cimetidine☆☆☆

To improve the validity of a timed creatinine clearance as a measure of glomerular filtration rate (GFR), we investigated whether a single 800-mg dose of oral cimetidine was sufficient to inhibit tubular secretion of creatinine (TScr). Forty-five 3-hour timed creatinine clearances (Clcr) with single 800-mg dose oral cimetidine (TCC) in 17 renal transplant recipients with marked renal function impairment (creatinine 2.0 to 7.1 mg/dL) were compared with simultaneous [125I]-iothalamate GFR (Cliothal). For comparison, 13 timed Clcr without cimetidine (TC), and 36 24-hour Clcr were performed. The TCC was the most accurate: the ratio (mean +/- SD) of TCC:Cliothal was 1.12 +/- 0.02, compared with 1.33 +/- 0.08 for Clcr:Cliothal and 1.53 +/- 1.02 for TC:Cliothal. The difference between Cliothal and TCC was small over the range of GFRs tested (mean +/- 2 SD), 0.9 +/- 2.5 mL/min/1.73 m2. The intraclass correlation (R) for within-subject reproducibility of the TCC in five subjects was 0.8 (95 percent CI; 0.5, 0.9), and in 11 subjects who had at least three GFR determinations over 24 weeks, the TCC was as responsive to change in GFR as Cliothal. There was an inverse relationship between fractional excretion of cimetidine and GFR (r2 = -0.70), suggesting increased tubular secretion of cimetidine with decreasing GFR. In conclusion, a single 800-mg oral dose of cimetidine was effective in inhibiting TScr such that the TCC was an accurate, reproducible, and responsive test of GFR.

Structuring communication relationships for interprofessional teamwork (SCRIPT): A Canadian initiative aimed at improving patient-centred care

There is a growing movement in health care that advocates the use of interprofessional education to help deliver collaborative patient-centred care (Oandasan et al., 2004). For example, the Romanow Commission (2002) and the First Ministers’ Accord (Health Canada 2003) both stress the need for collaborative practice to help ensure that the quality of health care delivered to Canadians can be enhanced. Both reports identified that introducing interprofessional education within the health professional education system was the key to achieving this aim. Evidence of the effectiveness of interprofessional education suggests that it can generate a number of positive outcomes for professionals and for patients (Barr et al., 2005). However, at present, this evidence base is generally weak and fragmentary in nature (Zwarenstein & Reeves, 2006). To help generate a more informed understanding of interprofessional education and its potential impact on collaborative relationships and the delivery of patient care, the Canadian federal government has recently funded eleven projects across the country. This paper describes one of these projects based at the University of Toronto. The project involves the development of an intervention designed to improve interprofessional collaboration across three separate clinical settings: general internal medicine (GIM); primary care; and rehabilitation and complex continuing care. Each of these settings was selected as they represent key trajectories along which patients travel while receiving care in the Toronto Academic Health Science Network (TAHSN), the network of partnerships between the University of Toronto and its fully affiliated health services’ institutes. Journal of Interprofessional Care, January 2007; 21(1): 111 – 114

Rosiglitazone Prevents High Glucose-Induced Vascular Endothelial Growth Factor and Collagen IV Expression in Cultured Mesangial Cells

Peroxisome proliferator-activated receptor (PPARγ), a ligand-dependent transcription factor, negatively modulates high glucose effects. We postulated that rosiglitazone (RSG), an activator of PPARγ prevents the upregulation of vascular endothelial growth factor (VEGF) and collagen IV by mesangial cells exposed to high glucose. Primary cultured rat mesangial cells were growth-arrested in 5.6 mM (NG) or 25 mM D-glucose (HG) for up to 48 hours. In HG, PPARγ mRNA and protein were reduced within 3 h, and enhanced ROS generation, expression of p22phox, VEGF and collagen IV, and PKC-ζ membrane association were prevented by RSG. In NG, inhibition of PPARγ caused ROS generation and VEGF expression that were unchanged by RSG. Reduced AMP-activated protein kinase (AMPK) phosphorylation in HG was unchanged with RSG, and VEGF expression was unaffected by AMPK inhibition. Hence, PPARγ is a negative modulator of HG-induced signaling that acts through PKC-ζ but not AMPK and regulates VEGF and collagen IV expression by mesangial cells.

High glucose-induced mesangial cell altered contractility: role of the polyol pathway

Summary Glomerular mesangial cells cultured in high glucose conditions display impaired contractile responsiveness. It was postulated that glucose metabolism through the polyol pathway leads to altered mesangial cell contractility involving protein kinase C. Rat mesangial cells were growth-arrested for 24 h with 0.5 % fetal bovine serum in either normal (5.6 mmol/l) or high (30 mmol/l) glucose concentrations or high glucose plus the aldose reductase inhibitor, ARI-509 (100 μmol/l). The reduction of cell planar surface area (contraction) in response to endothelin-1 (0.1 μmol/l), or to phorbol 12-myristate 13-acetate (50 pmol/l), was studied by videomicroscopy. In response to endothelin-1, mesangial cells in normal glucose contracted to 52 ± 3 % of initial planar area. In high glucose, the significantly (p < 0.05) smaller cell size and no contractile responsiveness to endothelin-1 were normalized with ARI-509. Membrane-associated diacylglycerol, measured by a kinase specific 32P-phosphorylation assay, in high glucose was unchanged after 3 h, but significantly increased (p < 0.05) after 24 h which was normalized with ARI-509. Protein kinase C activity, measured by in situ 32P-phosphorylation of the epidermal growth factor receptor substrate was: increased by 32 % at 3 h of high glucose, unchanged by ARI-509; and decreased significantly (p < 0.05) at 24 h compared to cells in normal glucose, normalized by ARI-509. Total cellular protein kinase C-alpha, -delta and -epsilon, analysed by immunoblotting, were unchanged in high glucose at 24 h. Only protein kinase C-epsilon content was reduced by ARI-509 in both normal and high glucose. Therefore, high glucose-induced loss of mesangial cell contractility, diacylglycerol accumulation and altered protein kinase C activity are mediated through activation of the polyol-pathway, although no specific relationship between elevated diacylglycerol and protein kinase C activity was observed. In high glucose, altered protein kinase C function, or another mechanism related to the polyol pathway, contribute to loss of mesangial cell contractile responsiveness. [Diabetologia (1998) 41: 507–515]