Frederick R. DeRubertis

Activation of Protein Kinase C in Glomerular Cells in Diabetes: Mechanisms and Potential Links to the Pathogenesis of Diabetic Glomerulopathy

Protein kinase C (PKC) is activated in rat renal glomerulus within a week of induction of experimental diabetes. Studies in isolated glomeruli and in cultured endothelial and mesangial cells have demonstrated that high ambient concentrations of glucose activate PKC and thus implicate hyperglycemia per se as a mediator of PKC activation in glomerular cells in diabetes. High glucose concentrations activate PKC by increasing cellular levels of diacylglycerol (DAG), the major endogenous modulator of this signalling system. In contrast to physiological extracellular stimuli of PKC that increase cellular DAG levels by receptor-mediated enhancement of membrane inositol phospholipid hydrolysis, in glomerular cells high concentrations of glucose increase DAG by de novo synthesis from glycolytic intermediates. Activation of PKC by glucose or other agonists increases the permeability of endothelial cells to albumin and stimulates matrix protein synthesis in mesangial cells; it thereby may be involved in the pathogenesis of both the functional and structural alterations of the glomerulus in diabetes. Recent studies in isolated glomeruli from diabetic rats have also implicated activation of PKC in suppression of nitric oxide (NO)-mediated increases in glomerular cGMP generation in response to cholinergic stimuli. In mesangial cells, cGMP suppresses PKC-mediated increases in matrix protein synthesis. Thus, impaired NO-mediated cGMP generation in glomeruli of diabetic individuals may amplify matrix protein synthesis in response to hyperglycemia and other stimuli of PKC. These and other observations suggest that activation of the PKC system by hyperglycemia may represent an important pathway by which glucotoxicity is transduced in susceptible cells in diabetes.

Increase in Diacylglycerol Mass in Isolated Glomeruli by Glucose From De Novo Synthesis of Glycerolipids

An increase in glucose concentration in the medium from 5 to 30 mM transiently enhanced diacylglycerol mass and activated protein kinase C in glomeruli isolated from nondiabetic rats as assessed by translocation of enzyme activity from the soluble to participate fraction. Effects of glucose on both diacylglycerol mass and protein kinase C were evident at 5 and 15 min but waned by 30 min. An increase in glucose concentration in the medium also increased the incorporation of [14C]glucose into the glycerol backbone of diacylglycerol, triacylglycerol, and phospholipids. Several observations implied that [14C]glucose was being incorporated into diacylglycerol through the de novo pathway for glycerolipid synthesis rather than being derived from phospholipids. 1) [14C]glucose incorporation into all the lipids was suppressed by 2-deoxyglucose. 2) The incorporation of [14C] glucose into diacylglycerol and triacylglycerol was evident by 1 min and increased linearly for at least 30 min. In contrast, incorporation into phosphatidylcholine occurred with a lag of at least 5 min. 3) Although only 10% of the [14C]glucose incorporated into lipids was present in diacylglycerol versus >50% in phospholipids, the specific activity of [14C]glucose in diacylglycerol was fivefold higher than that in phospholipid when expressed as a function of mass. 4) Glucose had no effect on labeled diacylglycerol or phosphatidic acid production in glomeruli that had been prelabeled with [3H]glycerol. Glucose-induced increases in diacylglycerol may contribute to the activation of glomerular protein kinase C observed in early diabetes.

Role of activation of protein kinase C in the stimulation of colonic epithelial proliferation and reactive oxygen formation by bile acids.

Deoxycholate (DOC), chenodeoxycholate, 12-O-tetradecanoyl phorbol-13-acetate (TPA), or 1-oleoyl-2-acetyl-glycerol (OAG) activated colonic epithelial protein kinase C as reflected by translocation from the soluble to the particulate cell fraction. Activation of protein kinase C was correlated with stimulation of enhanced proliferative activity of colonic mucosa and reactive oxygen production. TPA and OAG, but not DOC, directly activated soluble protein kinase C in vitro. However, DOC rapidly increased labeled inositol phosphate and diacylglycerol accumulation in colonic epithelial cells. Retinoic acid inhibited protein kinase C activity and suppressed DOC-, TPA-, and OAG-induced increases in reactive oxygen production. The results support a role for protein kinase C in the stimulation of colonic epithelial proliferative activity and reactive oxygen production induced by bile acids, TPA and OAG. In contrast to TPA and OAG, which activate protein kinase C directly, bile acids appear to activate protein kinase C indirectly by increasing the diacylglycerol content of colonic epithelium.

Role for Protein Kinase C in the Mediation of Increased Fibronectin Accumulation by Mesangial Cells Grown in High-Glucose Medium

The fibronectin content of RMC cultures grown for 8–14 days in medium containing 30 mM (540 mg/dl) D-glucose was increased 30–60% relative to that of control cells cultured in 10 mM (180 mg/dl) glucose. Addition of equiosmolar concentrations (20 mM, 360 mg/dl) of L-glucose, 3-O-methylglucose, or mannitol to 10 mM glucose media did not alter fibronectin accumulation compared with values observed in 10 mM glucose alone. The basal phosphorylation of the 80,000-Mr MARCKS protein, which is a substrate for PKC, and the phosphorylation induced by acute (15-min) exposure of cells to 15% FCS or to 0.1 μM (50 ng/ml) PDBu were all increased in cells grown in 30 mM compared with 10 mM glucose. By contrast, phosphorylation of the 80,000-Mr protein in response to a maximal concentration of PDBu (1 μM, 500 ng/ml) was not different in cells grown in 30 mM compared with 10 mM glucose. The acute increases in phosphorylation of the 80,000-Mr protein were blocked by the PKC inhibitor calphostin C. Chronic (7-day) exposure of mesangial cells grown in 10 mM glucose to 0.1 μM of the PKC agonist PDBu also resulted in a sustained 40% increase in 80,000-Mr phosphorylation and a 20–30% increase in fibronectin accumulation. As assessed by [35S]methionine incorporation, mesangial cell fibronectin synthesis was increased by exposure to PDBu, a finding consistent with earlier observations with 30 mM glucose. The increase in 80,000-Mr proteimesangial cells grown in either 0.1 μM PDBu or 30 mM glucose was abolished in cells whose PKC activity had been downregulated by exposure to PMA. Culture of mesangial cells with the thromboxane-endoperoxide analogue U-46619 in 30 mM, but not 10 mM, glucose increased fibronectin accumulation compared withvalues obtained in 30 mM glucose alone. These results demonstrate a sustained activation of PKC in mesangial cells cultured in a high concentration of glucose and support a role for PKC activation in the mediation of enhanced fibronectin accumulation induced by glucose. Because nonmetabolizable glucose analogues and mannitol were ineffective, metabolism of glucose is implicated in expression of these actions. The results also suggest that a high ambient glucose concentration may interact with thromboxane and/or prostaglandin endoperoxide in the activation of PKC and the enhancement of fibronectin accumulation by mesangial cells.

Release of Prostaglandin by Mitogen- and Antigen-Stimulated Leukocytes in Culture

The prostaglandin (PG) content of mitogen- and antigen-stimulated leukocyte cultures was examined by a radioimmunoassay procedure empolying an antiserum reactive with PGB(1) and PGB(2), the alkaline dehydration products of PGE and PGA. At 48 h, mitogen-activated mouse spleen cell cultures showed 2-10-fold increases in the PGE, but not in the PGA, component of immunoreactive PG (iPG) fractionated by silicic acid column chromatography. Increases in iPG were detectable by h 16 in spleen cell cultures incubated with staphylococcal enterotoxin B. Since iPG levels rose only in the culture supernates and not in cells exposed to mitogens for 48 h, increases reflected extracellular release of PG. The validity of the radioimmunoassay determinations of PGE in spleen cell cultures was supported by the results of concomitant assessment of the PGE(2) content of basal and enterotoxin-stimulated cultures by gas chromatography/mass spectrometry. By the latter method, the PGE(2) content was three-fold higher in enterotoxin-activated, compared to basal, cultures at 48 h. Aspirin effectively suppressed increases in both iPG and PGE(2). In spleen cell cultures prepared from mice previously inoculated with an attenuated strain of yellow fever virus in vivo and then incubated with this virus in vitro, iPG levels increased twofold over basal at 48 h. By contrast, iPG content of spleen cell cultures prepared from saline-inoculated mice was not appreciably altered by exposure to the virus in vitro. The enhancement of iPG release from cultured spleen cells by mitogens did not correlate with an ability of these agents to increase cellular cyclic AMP (cAMP) levels. Moreover, epinephrine and cholera toxin markedly increased spleen cell cAMP content but had no demonstrable effect on basal iPG levels, suggesting iPG release from these cells was not mediated by cAMP. Incubation with mitogens also enhanced the iPG content of 72-cultures of human peripheral leukocytes and of human lymphocytes isolated by nylon chromatography. However, the iPG of cultures of human lymphocytes purified by glass bead chromatography and of mouse thymocytes was not appreciably altered when these cells were cultured with mitogens, even though DNA synthesis in both instances was markedly increased. Accordingly, iPG release was not an invariable concomitant of increased DNA synthesis in lymphoid cell cultures. In summary, the results demonstrate that mitogen and antigen stimulation of leukocytes in culture may be accompanied by enhanced release of PGE. The mechanisms mediating this phenomenon and its biologic significance remain to be delineated, but participation of PGE in immunologically induced inflammatory responses seems possible.

Role of reactive oxygen in bile salt stimulation of colonic epithelial proliferation.

Our previous studies had suggested a link between bile salt stimulation of colonic epithelial proliferation and the release and oxygenation of arachidonate via the lipoxygenase pathway. In the present study, we examined the role of reactive oxygen versus end products of arachidonate metabolism via the cyclooxygenase and lipoxygenase pathways in bile salt stimulation of rat colonic epithelial proliferation. Intracolonic instillation of 5 mM deoxycholate increased mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA. Responses to deoxycholate were abolished by the superoxide dismutase mimetic CuII (3,5 diisopropylsalicylic acid)2 (CuDIPS), and by phenidone or esculetin, which inhibit both lipoxygenase and cyclooxygenase activities. By contrast, indomethacin potentiated the response. Phenidone and esculetin suppressed deoxycholate-induced increases in prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 5, 12, and 15-hydroxyeicosatetraenoic acid (HETE), whereas CuDIPS had no effect. Indomethacin suppressed only PGE2. Deoxycholate (0.5-5 mM) increased superoxide dismutase sensitive chemiluminescence 2-10-fold and stimulated superoxide production as measured by cytochrome c reduction in colonic mucosal scrapings or crypt epithelium. Bile salt-induced increases in chemiluminescence were abolished by CuDIPS, phenidone, and esculetin, but not by indomethacin. Intracolonic generation of reactive oxygen by xanthine-xanthine oxidase increased colonic mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA approximately twofold. These effects were abolished by superoxide dismutase. The findings support a key role for reactive oxygen, rather than more distal products of either the lipoxygenase or cyclooxygenase pathways, in the stimulation of colonic mucosal proliferation by bile salts.

Impaired water excretion in myxedema

Abstract The response to acute water ingestion (20 ml/kg body weight) was determined in sixteen patients with myxedema and eighteen control subjects. Comparison of the results in the two groups indicated an abnormality in water excretion in the patients with myxedema. This impaired water diuresis in the patients with myxedema, including those with significant hyponatremia, appeared to be related to decreased volume delivery to the distal diluting segment of the nephron rather than to persistent secretion of antidiuretic hormone. The reevaluation of eleven patients with myxedema after thyroid replacement therapy revealed significant improvement in the response to water ingestion.

Nitric Oxide Inhibition of Transforming Growth Factor-β and Collagen Synthesis in Mesangial Cells

Culture of mesangial cells (MCs) in 5.6 vs. 30.0 mmol/1 glucose for 3 weeks induced a sustained increase in protein kinase C (PKC) activity, transforming growth factor (TGF)-β1 mRNA, bioactive TGF-β, and collagen synthesis. Nitric oxide (NO), generated exogenously by the NO donor S-nitroso-N-acetyl, D,L-penicillamine (SNAP) or endogenously after the exposure of MC to interleukin-1β (IL-1β), suppressed bioactive TGF-β in MCs cultured in 5.6 or 30.0 mmol/1 glucose and suppressed or abolished increases in TGF-β1 mRNA and collagen synthesis induced by high concentrations of glucose or phorbol 12,13-dibutyrate without altering values obtained with normal glucose concentrations. SNAP had a transient suppressive effect on PKC activity, which may explain at least in part some of the actions of SNAP. The selective inhibitor of PKC, bisindolylmaleimide (GFX), mimicked NO action. The ability of SNAP and IL-lβ to suppress TGF-β and collagen synthesis was not mediated by cGMP, since the cGMP analog, 8-Br-PET-cGMP, did not mimic NO action and an antagonist of cGMP-dependent protein kinase, Rp-8-pCPT-cGMPs, did not prevent the inhibitory actions of SNAP. N-ω-L-arginine methyl ester (NMMA) increased TGF-β in glomerular capillary endothelial cells (GCECs) and stimulated collagen synthesis by MC in a co-culture with GCECs. Captopril inhibited TGF-β and collagen synthesis and increased cGMP in co-cultures of GCECs and MCs. These effects of captopril were abolished by NMMA, implying mediation by NO. Thus, endogenous NO produced by GCECs may modulate TGF-β production by both GCECs and MCs and act to suppress matrix protein synthesis by MCs.

Active Care Management Supported by Home Telemonitoring in Veterans with Type 2 Diabetes: (The DiaTel Randomized Controlled Trial)

OBJECTIVE We compared the short-term efficacy of home telemonitoring coupled with active medication management by a nurse practitioner with a monthly care coordination telephone call on glycemic control in veterans with type 2 diabetes and entry A1C ≥7.5%. RESEARCH DESIGN AND METHODS Veterans who received primary care at the VA Pittsburgh Healthcare System from June 2004 to December 2005, who were taking oral hypoglycemic agents and/or insulin for ≥1 year, and who had A1C ≥7.5% at enrollment were randomly assigned to either active care management with home telemonitoring (ACM+HT group, n = 73) or a monthly care coordination telephone call (CC group, n = 77). Both groups received monthly calls for diabetes education and self-management review. ACM+HT group participants transmitted blood glucose, blood pressure, and weight to a nurse practitioner using the Viterion 100 TeleHealth Monitor; the nurse practitioner adjusted medications for glucose, blood pressure, and lipid control based on established American Diabetes Association targets. Measures were obtained at baseline, 3-month, and 6-month visits. RESULTS Baseline characteristics were similar in both groups, with mean A1C of 9.4% (CC group) and 9.6% (ACM+HT group). Compared with the CC group, the ACM+HT group demonstrated significantly larger decreases in A1C at 3 months (1.7 vs. 0.7%) and 6 months (1.7 vs. 0.8%; P < 0.001 for each), with most improvement occurring by 3 months. CONCLUSIONS Compared with the CC group, the ACM+HT group demonstrated significantly greater reductions in A1C by 3 and 6 months. However, both interventions improved glycemic control in primary care patients with previously inadequate control.

Antioxidant Inhibition of Protein Kinase C-Signaled Increases in Transforming Growth Factor-Beta in Mesangial Cells

Protein kinase C (PKC)-signaled increases in transforming growth factor beta (TGF beta) have been implicated in the stimulation of matrix protein synthesis induced by high concentrations of glucose, thromboxane, angiotension II (AII), and other stimuli in cultured glomerular mesangial cells. In the present study, the effects of several antioxidants on mesangial cell responses to high glucose, thromboxane, and AII were examined. alpha-Tocopherol blocked increases in PKC, TGF beta bioactivity, collagen, and/or fibronectin synthesis induced in mesangial cells by high glucose, the thromboxane analog U46619, and AII. By contrast, alpha-tocopherol did not alter increases in matrix protein synthesis in mesangial cells in response to exogenous TGF beta, a cytokine that does not activate PKC in mesangial cells and whose actions to stimulate matrix protein synthesis in these cells are not blocked by PKC inhibition or downregulation. Taurine and N-acetylcystein similarly inhibited activation of PKC and increases in TGF beta in response to high glucose, U46619, and AII. alpha-Tocopherol but not taurine or N-acetylcysteine partially blocked increases in PKC activity in mesangial cells in response to the diacylglycerol (DAG) analog, phorbol dibutyrate (PDBu). Thus, alpha-tocopherol may have direct effects on interaction of the PKC system of mesangial cells with DAG that are not shared by N-acetylcysteine or taurine. Increases in TGF beta have been implicated in the pathogenesis of glomerulosclerosis in diabetes and other nephropathies. The capacity of antioxidants to block increases in TGF beta in mesangial cells in response to high glucose, thromboxane, and All suggests their potential therapeutic utility to attenuate glomerulosclerosis.