Maresins: Specialized Proresolving Lipid Mediators and Their Potential Role in Inflammatory-Related Diseases.

Acute inflammatory responses are host-protective and normally self-limited; these responses can maintain cell homeostasis and promote defense against various infections and damage factors. However, when improperly managed or inappropriately activated, acute inflammation can lead to persistent and uncontrolled chronic inflammation, which is associated with many other chronic diseases including cardiovascular disease and metabolic disease. Recently, studies have shown that resolution of acute inflammation is a biosynthetically active process. Specialized proresolving lipid mediators (SPMs) known as resolvins and protectins are autacoids that resolve inflammation. A new family of anti-inflammatory and proresolving lipid mediators have recently been reported, known as maresins, which are biosynthesized from docosahexaenoic acid (DHA) by macrophages, have a conjugated double-bond system, and display strong anti-inflammatory and proresolving activity. Here, we review the biological actions, pathways, and mechanisms of maresins, which may play pivotal roles in the resolution of inflammation.

The efficacy and safety of SGLT2 inhibitors for adjunctive treatment of type 1 diabetes: a systematic review and meta-analysis.

To assess the efficacy and safety of the SGLT-2 inhibitors as adjunct therapy to insulin in T1DM, clinical trials indexed in PubMed, Cochrane Library, EMbase from inception through April 5, 2016. A meta-analysis was conducted on trials of SGLT-2 inhibitors in patients with T1DM on insulin therapy using RevMan 5.3 software. Of the 371 articles identified, ten met eligibility criteria. Seven clinical trials including four randomized controlled trials and 581 patients were included. Compared with the control group, SGLT-2 inhibitors group had significantly reduced fasting plasma glucose by 0.69 mmol/L [1.32; 0.07], glycosylated hemoglobin A1C by 0.37% [0.54; 0.20], body weight by 2.54 kg [3.48; 1.60] and total daily insulin dose by 6.22 IU [8.04; 4.40]. The total incidence of adverse events (AEs), hypoglycemia, and genital and urinary infections were also similar to placebo, while an increased incidence of diabetic ketoacidosis (DKA) (n = 16) was seen in SGLT-2 inhibitors group. The present study demonstrates that SGLT-2 inhibitors are effective as adjunct therapy to insulin in T1DM, heralding improved glycemic control, reduced body weight and total daily insulin dose without an increase in total AEs, hypoglycemia, or genital and urinary infections. However, the risk of DKA should be carefully monitored in future clinical trials.

Diabetic Kidney Disease and Hypertension.

Achieving blood pressure (BP) goals is an essential goal for both primary and secondary prevention of diabetic kidney disease (DKD). Even though there is universal agreement about the importance of controlling BP, there are many issues about many aspects of hypertension management for DKD patients. These issues include: what is the optimal BP for the prevention or slowing of progression of DKD patients; what is the best method for diagnosing hypertension and monitoring BP, and what are the best medicines to use to treat hypertension in DKD patients. In this review, these issues as well as others will be discussed.

A method for determination of pyridine nucleotides using a single extract.

Intracellular redox levels play an important role in physiology and pathophysiology. The principal intracellular reductant is NADPH, which is required for both the proper activity of the entire antioxidant system and important prooxidant enzymes such as nitric oxide synthase and NADPH oxidase. Thus an easy and accurate measurement of NADPH is very desirable. The method described in this paper is based on the fact that NADH and NADPH (not NAD(+) and NADP(+)) affect absorbance at 340 nm. A single cell extract is separated into three aliquots (A(1), A(2), and A(3)). A(1) is untreated and the absorbance at 340 nm is measured. A(2) is treated with an enzyme that converts all of the NADP(+) to NADPH and then the absorbance at 340 nm is measured. A(3) is treated with an enzyme that converts all of the NADPH to NADP(+) and then the absorbance at 340 nm is measured. A(1) - A(3) is the NADPH content and A(2) - A(1) is the NADP(+) content of the extract. Using this method, we have obtained full recovery of all added nucleotides from cell extracts, thus making the method suitable for the quick determination of NADP(+) and NADPH in living cells. We conclude that this method for the measurement of NADP(+) and NADPH is rapid, simple, accurate, and reliable.

High glucose inhibits glucose-6-phosphate dehydrogenase via cAMP in aortic endothelial cells.

Recent studies have shown that hyperglycemia is a principal cause of cellular damage in patients with diabetes mellitus. A major consequence of hyperglycemia is increased oxidative stress. Glucose-6-phosphate dehydrogenase (G6PD) plays an essential role in the regulation of oxidative stress by primarily regulating NADPH, the main intracellular reductant. In this paper we show that increased glucose (10-25 mm) caused inhibition of G6PD resulting in decreased NADPH levels in bovine aortic endothelial cells (BAEC). Inhibition was seen within 15 min. High glucose-induced inhibition of G6PD predisposed cells to cell death. High glucose via increased activity of adenylate cyclase also stimulated an increase in cAMP levels in BAEC. Agents that increased cAMP caused a decrease in G6PD activity. Inhibition of cAMP-dependent protein kinase A ameliorated the high glucose-induced inhibition of G6PD. Finally, high glucose stimulated phosphorylation of G6PD. These results suggest that, in BAEC, high glucose stimulated increased cAMP, which led to increased protein kinase A activity, phosphorylation of G6PD, and inhibition of G6PD activity. We conclude that these changes in G6PD activity play an important role in high glucose-induced cell damage/death.

Importance of glucose-6-phosphate dehydrogenase activity in cell death.

The intracellular redox potential plays an important role in cell survival. The principal intracellular reductant NADPH is mainly produced by the pentose phosphate pathway by glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme, and by 6-phosphogluconate dehydrogenase. Considering the importance of NADPH, we hypothesized that G6PDH plays a critical role in cell death. Our results show that 1) G6PDH inhibitors potentiated H2O2-induced cell death; 2) overexpression of G6PDH increased resistance to H2O2-induced cell death; 3) serum deprivation, a stimulator of cell death, was associated with decreased G6PDH activity and resulted in elevated reactive oxygen species (ROS); 4) additions of substrates for G6PDH to serum-deprived cells almost completely abrogated the serum deprivation-induced rise in ROS; 5) consequences of G6PDH inhibition included a significant increase in apoptosis, loss of protein thiols, and degradation of G6PDH; and 6) G6PDH inhibition caused changes in mitogen-activated protein kinase phosphorylation that were similar to the changes seen with H2O2. We conclude that G6PDH plays a critical role in cell death by affecting the redox potential.

Importance of glucose-6-phosphate dehydrogenase activity for cell growth.

The intracellular redox potential, which is determined by the level of oxidants and reductants, has been shown to play an important role in the regulation of cell growth. The principal intracellular reductant is NADPH, which is mainly produced by the pentose phosphate pathway through the actions of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, and by 6-phosphogluconate dehydrogenase. Previous research has suggested that an increase in G6PD activity is important for cell growth. In this article, we suggest that G6PD activity plays a critical role in cell growth by providing NADPH for redox regulation. The results show the following: 1) inhibition of G6PD activity abrogated growth factor stimulation of [3H]thymidine incorporation in all cell lines tested; 2) overexpression of G6PD stimulated cell growth, as measured by an increase in [3H]thymidine incorporations as compared with cells transfected with vector alone; 3) inhibition of G6PD caused cells to be more susceptible to the growth inhibitory effects of H2O2; 4) inhibition of G6PD led to a 30-40% decrease in the NADPH/NADP ratio; and 5) inhibition of G6PD inhibited cell anchorage and significantly decreased the growth-related stimulation of tyrosine phosphorylation.

beta Amyloid does not activate the antioxidant pentose phosphate pathway within the B12 neural cell line.

The aim of this study was to determine whether neural cells exposed to beta amyloid (A beta) activate the pentose phosphate pathway (PPP), a critical oxidative stress defense mechanism. A beta stimulated H2O2 production in neural (B12) and non-neural (HepG2) cells and stimulated PPP activity, the source of the main intracellular reductant NADPH, in HepG2 cells (67% increase). Catalase blocked the A beta-induced increase in PPP, demonstrating that H2O2 mediated the increase in PPP activity. B12 cells showed no increase in PPP following A beta exposure. Fifty-five per cent of HepG2 cells but only 11.1% of B12 cells remained viable after A beta exposure. Lack of PPP activation may contribute to A beta cytotoxicity in neural calls and may lead to differences in survival between neural and non-neural cells.

Signal transduction proteins that associate with the platelet-derived growth factor (PDGF) receptor mediate the PDGF-induced release of glucose-6-phosphate dehydrogenase from permeabilized cells.

Permeabilized rat kidney cells rapidly released glucose 6-phosphate dehydrogenase (G6PD) following stimulation with peptide growth factors (Stanton, R.C., Seifter, J.L., Boxer, D.C., Zimmerman, E., and Cantley, L. C. (1991) J. Biol. Chem. 266, 12442-12448). To evaluate the signal transduction pathways mediating release of G6PD, two cell lines transfected with wild type or mutant platelet-derived growth factor (PDGF) receptors (PDGFR) were studied using two permeabilization protocols. G6PD release was evaluated by enzyme activity and Western blot analysis. PDGF caused a significant increase in G6PD release in 1 min in cells transfected with wild type PDGFR. PDGF did not stimulate G6PD release in cells transfected with tyrosine kinase-deficient PDGFR. PDGF did not stimulate G6PD release in cells transfected with partially autophosphorylation-deficient PDGFR in which four known signaling proteins do not associate with the PDGFR. The PDGF-stimulated release of G6PD was partially restored in PDGFR mutants in which either phosphatidylinositol-3-kinase or phospholipase C gamma 1 could associate with the PDGFR. Lastly, there was no basal or PDGF-stimulated phosphorylation of G6PD. We conclude that release of G6PD: 1) requires intrinsic PDGFR tyrosine kinase activity; 2) requires PDGFR autophosphorylation; 3) is mediated by signaling proteins that associate with the PDGFR; 4) is not mediated by direct phosphorylation of G6PD.

Rapid release of bound glucose-6-phosphate dehydrogenase by growth factors. Correlation with increased enzymatic activity.

Epidermal growth factor (EGF), a mitogen for renal proximal tubule cells, activated the hexose monophosphate (HMP) shunt in renal proximal tubule cells (Stanton, R. C., and Seifter, J. L. (1988) Am. J. Physiol. 254, C267-C271). We therefore evaluated the effect of EGF on the HMP shunt enzymes glucose 6-phosphate dehydrogenase (G6PD, the rate-limiting enzyme) and 6-phosphogluconate dehydrogenase. Rat renal cortical cells (RCC) were incubated with either EGF or platelet-derived growth factor (PDGF) and then assayed for G6PD and 6-phosphogluconate dehydrogenase activities. EGF and PDGF increased G6PD activity by 25 and 27% respectively. Although phorbol myristate acetate (PMA), ionomycin, PMA + ionomycin, and 8-bromo-cyclic AMP had no significant effect on the activity, a 5-min preincubation with PMA potentiated the activation of G6PD by PDGF. Growth factor activation of G6PD was also seen in a fibroblast and epithelial cell line. None of the agents affected 6-phosphogluconate dehydrogenase activity in the RCC or in the cell lines. Further exploration into a possible mechanism for G6PD activation revealed that growth factors caused release of G6PD from a structural element within the cell. Streptolysin O permeabilization of RCC did not cause significant release of G6PD. However, within 1 min of addition of EGF or PDGF to permeabilized cells, G6PD was released into the cell supernatant. The nonhydrolyzable analog of GTP, guanosine 5'-O-(thiotriphosphate), caused a similar release of G6PD. Preincubation with pertussis toxin or guanyl-5'-yl thiophosphate inhibited the PDGF but not the EGF effect. Although the data do not establish a definitive proof linking G6PD release and G6PD activation, these results suggest that they are related. Thus, growth factor stimulation of the HMP shunt likely occurs by a novel mechanism associated with release of bound G6PD.

Expression of Na(+)-H+ exchange and ATP-dependent proton extrusion in growing rat IMCD cells.

As the last step of urinary acidification, the inner medullary collecting duct (IMCD) is thought to secrete protons into the tubular lumens by means of a H(+)-translocating adenosinetriphosphatase (H(+)-ATPase). However, recent studies have also shown the existence of Na(+)-H+ exchange activity in IMCD cells. Although the physiological function of the antiporter in IMCD cells is unknown, activation of Na(+)-H+ exchange in other cell-culture systems has been suggested to be closely associated with the process of cell growth. Thus presence of Na(+)-H+ exchange may relate to the growth phase of these cells. To examine intracellular pH (pHi) regulation in growing IMCD cells, we studied proton transport by Na(+)-dependent and Na(+)-independent mechanisms by microfluorimetry using the pHi-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF/AM). Actively growing cells, defined by [3H]thymidine incorporations, demonstrated an amiloride-sensitive Na(+)-dependent pHi recovery after an acid load. No pHi recovery was evident in the absence of Na+, indicating the importance of Na(+)-H+ exchange for pHi recovery. However, when evaluated in quiescent cells, Na(+)-dependent pHi recovery appeared to be diminished. Instead, a Na(+)-independent pHi recovery which was inhibitable by ATP depletion and by 1 mM N-ethylmaleimide was present, suggesting function of a H(+)-ATPase. These findings indicate that Na(+)-dependent proton extrusion activity (Na(+)-H+ exchange) but not Na(+)-independent proton extrusion activity is expressed during the rapid growth phase of IMCD cells, whereas the more quiescent cells express Na(+)-independent ATP-dependent proton extrusion activity and a possibly less active Na(+)-H+ exchanger.

Epidermal growth factor rapidly activates the hexose monophosphate shunt in kidney cells.

Epidermal growth factor (EGF) is a potent mitogen that rapidly activates plasma membrane Na+-H+ exchangers, thereby causing intracellular alkalinization. The rise in intracellular pH (pHi) may be an important signal for cell growth. However, recent studies have dissociated Na+-H+ exchange activity and/or alkalinization from cellular proliferation. We have studied the role of EGF in the growth of rat renal proximal tubule (PT) cells in primary culture and monitored the early effects of EGF on pHi in these cells using microfluorimetry and the pHi probe, 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). EGF increased DNA synthesis in growing PT cells and produced transient alkalinization (2-3 min) due to activation of Na+-H+ exchange. In contrast, in the absence of extracellular Na+, EGF administration caused pHi to decrease. This acidification was prevented by 2-deoxy-D-glucose and 6-aminonicotinamide, inhibitors of glucose utilization and the hexose monophosphate shunt (HMP), respectively. EGF was also found to stimulate HMP shunt activity in PT cells using an isotopic method for distinguishing between glucose utilization through the HMP shunt vs. glycolysis. Because EGF caused both cytoplasmic acidifying (HMP activation) and alkalinizing (Na+-H+ exchange activation) processes, we propose that the primary role for the activation of Na+-H+ exchange during growth may be to extrude acid from the cell in order to maintain pHi at levels permissive for cell growth.

Use of monoclonal antibodies to culture rat proximal tubule cells.

Current renal cell culture techniques are limited by either a low yield of cells or by heterogeneity of cell types. We have used monoclonal antibodies to microvillus membrane proteins to isolate and culture a pure population of proximal tubule cells. The cells were characterized as proximal tubule cells by phase microscopy, enzyme histochemistry for alkaline phosphatase, butyrate esterase, and gamma-glutamyltransferase, electron microscopy, and specific reactivity with a variety of monoclonal antibodies for proximal tubule cells. Growth over 2-7 days yielded cell numbers up to 1,000-fold greater than obtained by single tubule microdissection. Dome formation was observed, suggesting intact fluid transport. In addition, Na+-H+ exchange and Na+-dependent D-hexose transport, known transport processes of the proximal tubule, were demonstrated by microfluorimetry of single cells and methyl-alpha-D-glucopyranoside uptake, respectively. Our results indicate that large numbers of homogeneous, cultured rat proximal tubule cells that maintain characteristics of in vivo proximal tubule cells can be obtained using a monoclonal antibody technique of isolation.

Role of the kidney in congestive heart failure.

The changes in renal function observed in congestive heart failure include altered pressures and flows and increased reabsorption of sodium and water leading to expanded extracellular fluid volume. These renal effects are mediated by a variety of volume and pressure sensors that stimulate various effectors which act on the kidney. The role of these sensors and effectors, the relationship between left ventricular function (LVF) and urinary sodium excretion (UNaV) and the role of angiotensin II in mediating the renal hemodynamic changes are reviewed. Rats with experimentally induced myocardial infarction (MI) were studied 3 weeks after infarction. Although UNaV decreased as LVF worsened, the decrease in UNaV was evident even in rats with MI and minimal LVF impairment. Infusion of teprotide (an inhibitor of angiotensin I converting enzyme) returned the hemodynamic parameters to or toward values seen in rats without MI, thereby documenting an important role for angiotensin II in congestive heart failure.

cAMP response of vascular smooth muscle cells to bovine parathyroid hormone.

Parathyroid hormone (PTH) is a vasodilator of vascular smooth muscle tissue. It has been shown to produce this vasodilation in normotensive and hypertensive laboratory rats. The effect is log dose dependent, maximal at 1 min and persists for 3-5 min. The cellular mechanisms involved in PTH-mediated vasodilation are unknown. In this study, we sought to determine the cellular changes of cAMP after administration of bovine (b)PTH (1-34). cAMP content of vascular smooth muscle cells was measured at 30 s, 1, 3, and 5 min after incubation with synthetic bPTH (1-34). Tissue cAMP content was decreased by 55% at 1 min (4.1 +/- 0.5 pmol/mg protein at time 0 vs. 1.9 +/- 0.2 pmol/mg protein at 1 min, P less than 0.001). After 5 min, cAMP levels returned to base-line values and increased over the next 5-10 min to levels above base line (P less than 0.01). In conclusion, our data suggest that the initial response of vascular smooth muscle cells to short-term incubation with bPTH (1-34) is an acute decrease in cAMP content.