COX-2-derived prostaglandins as mediators of the deleterious effects of nicotine in chronic kidney disease.

Tobacco smoking has been identified as a risk factor in the progression of chronic kidney disease (CKD). In previous studies, we showed that nicotine induces cyclooxygenase (COX)-2 expression in vivo and in vitro and that the administration of nicotine in vivo worsens the severity of renal injury in a model of subtotal renal ablation. In the present study, we tested the role of COX-2-derived prostaglandins on the deleterious effects of nicotine in CKD. Sham and 5/6 nephrectomy (5/6Nx) rats received tap water or nicotine (100 µg/mL) in the drinking water for 12 wk. Additional groups also systemically received the COX-2 inhibitor NS-398 (1.5 mg·kg-1·day-1 via osmotic minipump). The administration of nicotine worsened renal injury and proteinuria in 5/6Nx rats and increased proteinuria in sham rats. 5/6Nx rats had increased cortical production of the prostaglandins PGE2, PGI2, PGD2, and PGF2α and of thromboxane A2. In these rats, nicotine reduced the production of all prostaglandins examined except thromboxane A2. Treatment with the COX-2 inhibitor NS-398 resulted in complete inhibition of all prostaglandins studied and ameliorated renal injury and proteinuria in 5/6Nx rats on nicotine but not in 5/6 Nx rats on tap water. Nicotine also reduced the expression of megalin in all groups examined, and this was partially prevented by COX-2 inhibition. In the present study, we showed that in CKD, nicotine worsens renal injury at least in part by producing an imbalance in the production of prostaglandins. This imbalance in the production of prostaglandins likely plays a role in the deleterious effects of smoking on the progression of CKD.

Long term renal survival in patients undergoing T-Cell depleted versus conventional hematopoietic stem cell transplants.

Calcineurin inhibitor (CNI)-sparing T-cell depleted (TCD) hematopoietic stem cell transplants (HSCTs) are presumed to be less nephrotoxic than conventional HSCTs. We evaluated incidence and risk factors for kidney failure and chronic kidney disease (CKD) in 231 TCD and 212 conventional HSCT recipients. Kidney failure required a median glomerular filtration rate (GFR) <60 ml/min/1.73 m2 for ⩾100 days anytime after 180-days post-HSCT. Two-year cumulative incidence (CI) of kidney failure was 42% in the conventional versus 31% in the TCD group (P=0.005). TCD, age, acute kidney injury and number of toxic CNI levels all impacted on kidney failure, which was associated with increased all-cause mortality (hazard ratio 2.86 (95% CI: 1.88-4.36), P<0.001). Renal recovery occurred in 28% of kidney failure patients whereas the remaining patients were defined to have CKD. In those with baseline GFR>60 ml/min/1.73 m2, only exposure to nephrotoxic medications was associated with CKD (P=0.033). In the myeloablative-conditioning subgroup only total body irradiation was associated with CKD (P=0.013). Of all patients, five (1.13%) required dialysis. These results confirm an impact of TCD on kidney failure but not CKD for which other risk factors such as radiation or nephrotoxic drug exposure may have a role.

Effects of the reactive oxygen species hydrogen peroxide and hypochlorite on endothelial nitric oxide production.

Reactive oxygen species (ROS) hydrogen peroxide (H(2)O(2)) and hypochlorite (HOCl) participate in the pathogenesis of ischemia/reperfusion injury, inflammation, and atherosclerosis. Both NO and ROS are important modulators of vascular tone and architecture and of adhesive interactions between leukocytes, platelets, and vascular endothelium. We studied the effect of H(2)O(2) and HOCl on receptor-dependent (bradykinin [10(-6) mol/L] and ADP [10(-4) mol/L]) and receptor-independent mechanisms (calcium ionophore A23187 [10(-6) mol/L]) of NO production by porcine aortic endothelial cells (ECs). Changes in the level of EC cGMP (the second messenger of NO) were used as a surrogate of NO production. EC cGMP increased 300% in response to bradykinin and A23187 and 200% in response to ADP. Exposure of ECs to H(2)O(2) (50 micromol/L) for 30 minutes significantly impaired cGMP levels in response to ADP, bradykinin, and the receptor-independent NO agonist A23187. In contrast, preincubation with HOCl (50 micromol/L) impaired cGMP production only in response to ADP and bradykinin but not A23187. These concentrations of H(2)O(2) and HOCl did not result in increased EC lethality as assessed by lactate dehydrogenase release. Neither H(2)O(2) nor HOCl affected EC cGMP production in response to NO donor sodium nitroprusside, which suggests that guanylate cyclase is resistant to these oxidants. We also demonstrated that neither H(2)O(2) nor HOCl affects endothelial NO synthase (eNOS) catalytic activity as measured by conversion of L-arginine to L-citrulline in EC homogenates supplemented with eNOS cofactors. The present studies show that H(2)O(2) impairs NO production in response to both receptor-dependent and receptor-independent agonists and that these effects are due, at least in part, to inactivation of eNOS cofactors, whereas HOCl inhibits NO production by interfering with receptor-operated mechanisms at the level of the cell membrane. Concentrations of H(2)O(2) and HOCl used in the present studies have been shown to be generated in vivo during inflammation and ischemia/reperfusion. Therefore, we infer that these effects of H(2)O(2) and HOCl on EC NO production may contribute to disregulated vascular tone and altered leukocyte-EC interactions that occur in vascular injury as a result of those causes in which ROS generation is involved.

Cigarette smoke-induced endothelium dysfunction: role of superoxide anion.

OBJECTIVES: Cigarette smoking is strongly associated with coronary artery disease and atherosclerosis. While smoking has been shown to impair endothelium-dependent vasorelaxation, the mechanisms involved are not completely understood. We investigated the role of superoxide anion and vasoconstricting prostanoids in cigarette smoke induced endothelial dysfunction. METHODS: Endothelial function was assessed in rat aortic rings exposed to cigarette smoke-treated Krebs buffer, by measuring agonist stimulated endothelium-dependent vasorelaxation. Treatment with superoxide dismutase (SOD) as well as ifetroban, thromboxane A2/prostaglandin endoperoxide H2 (TxA2/PGH2) receptor blocker and indomethacin (cyclooxygenase inhibitor) was used to investigate the role of superoxide anion and vasoconstricting eicosanoids on cigarette smoke-induced endothelial dysfunction. The effect of cigarette smoke on endothelial nitric oxide synthase (eNOS) catalytic activity was measured by conversion of L-arginine to L-citrulline in rat aortas and rat endothelial cell homogenates supplemented with eNOS cofactors. RESULTS: Relaxations to receptor-dependent agonists, acetylcholine and adenosine diphosphate (ADP), as well as to a receptor-independent agonist, A23187 (Ca2+ ionophore) were significantly impaired by cigarette smoke. Cigarette smoke did not impair relaxations to sodium nitroprusside, indicating preserved guanylate cyclase activity. Further, cigarette smoke did not affect eNOS catalytic activity in homogenates from either endothelial cells or aortas previously exposed to cigarette-smoketreated Krebs buffer. Treatment with SOD or ifetroban and in a lesser degree by indomethacin prevented cigarette-smoke-induced endothelial dysfunction. CONCLUSIONS: Taken together, our results suggest that cigarette smoking causes an increase in vascular superoxide production which results in decreased nitric oxide (NO) bioactivity and concomitantly increases production of cyclooxygenase dependent and independent vasoconstricting eicosanoids.

Angiotensin II induces superoxide anion production by mesangial cells.

BACKGROUND: The recognized role of angiotensin II (Ang II) in the pathogenesis of the progression of renal disease cannot be solely attributed to Ang II's hemodynamic effects. Indeed, growth stimulating signals driven by Ang II promote mesangial cell (MC) hypertrophy and extracellular matrix production, prominent features of progressive glomerular injury. Superoxide anion (O2-) avidly interacts with nitric oxide, an endogenous vasodilator that inhibits growth factor stimulated MC growth and matrix production. In addition, O2- acting as an intracellular signal is linked to growth related responses such as activation of mitogen activated protein (MAP) kinases. The studies reported herein were designed to investigate: (a) whether Ang II induces MC O2-production and (b) if increased O2- production elicits growth responses in MC. METHODS: MC were exposed to Ang II for 24 or 48 hours. In some experiments, in addition to Ang II, MC were exposed to: diphenylenieodonium (DPI), an inhibitor of the flavin containing NADH/NADPH oxidase; losartan (LOS), an Ang II type 1 (AT1) receptor blocker; PD 98059, a MAP kinases inhibitor; the protein kinase C inhibitors Calphostin C or H-7; and the tyrosine kinase inhibitors, herbymycin A or genistein. RESULTS: Ang II (10(-5) M to 10(-8) M) dose dependently increased MC O2- production up to 125% above control (ED 50 5 x 10(-7) M). LOS as well as DPI, and the PKC inhibitors blocked Ang II stimulated MC O2- production. Ang II dose dependently increased MC 3H-leucine incorporation, and MC protein content, two markers of MC hypertrophy, as well as 3H-thymidine incorporation, a marker of MC hyperplasia. PD98059, a specific inhibitor of MAP kinases prevented Ang II induced MC hypertrophy. Moreover, LOS, DPI, and the PKC inhibitors each independently inhibited MC 3H-leucine incorporation, thereby establishing the specificity of Ang II induced O2- in driving MC hypertrophy. CONCLUSIONS: The current studies demonstrate a previously unrecognized link between Ang II and MC O2- production that may participate in the pathophysiology of progressive renal disease by concomitantly affecting the hemodynamics of the glomerular microcirculation as well as growth related responses of MC to injury.

Glomerular and vascular tissues do not down-regulate nitric oxide synthesis during protracted endotoxemia.

Down-regulation of cytokines is implicated as an important component of the phenomenon of tolerance to bacterial products in humans and animals. Since many effects of endotoxin and cytokines are mediated by nitric oxide, this study was designed to evaluate in vivo the L-arginine:NO pathway in endotoxin tolerance. Sublethal injections of E. coli lipopolysaccharide (LPS, 1 mg/kg body wt, i.p.) were given to rats daily for five days. Blood levels of NO2-/NO3-, stable metabolites of nitric oxide (NO), significantly increased on day 1 (baseline, 89.64 +/- 40, day 1, 260.32 +/- 36 nmol/ml; P < 0.05) but returned to baseline levels on day 5 (77.60 +/- 5 nmol/ml). However, urinary NO2-/NO3- remained significantly elevated several-fold throughout the study period (baseline, 121.25 +/- 11.4, day 1, 899.35 +/- 15.8, day 5, 250.23 +/- 21.4 nmol/hr/100 g body wt). Glomeruli and aortae obtained from these rats showed increased NO production that was maintained at similar levels even at day 5 (glomeruli: control, 0.01 +/- 0.0, day 1, 22.4 +/- 0.3, and day 5, 22.0 +/- 2.5, P < 0.05 vs. control; aortae; control, 0.01 +/- 0.0, day 1, 4.3 +/- 2.2, and day 5, 5.4 +/- 1.0 nmol/hr/mg protein, P < 0.05 vs. control, respectively); this further increased significantly in response to in vitro LPS challenge. However, peritoneal macrophages, liver and spleen showed an initial increase in NO production that decreased significantly by the fifth day of LPS and could not be further stimulated by in vitro LPS challenge. Thus, in vivo NO synthesis is down-regulated during protracted LPS. Our results show that the process is relatively specific to the liver, spleen and macrophages, and is qualitatively and quantitatively different in vascular tissues such as aortae and glomeruli.

Hydrogen peroxide downregulates IL-1-driven mesangial iNOS activity: implications for glomerulonephritis.

In glomerulonephritides, autacoids such as nitric oxide (NO), reactive oxygen species, and prostanoids are produced in increased amounts in response to cytokines such as interleukin-1 (IL-1). These autacoids influence the expression of glomerular injury by their direct as well as interactive actions. We studied the effect of hydrogen peroxide (H2O2) on NO production in rat mesangial cells. We demonstrate that transient exposure of mesangial cells to H2O2 prior to sustained exposure to IL-1 decreased extracellular accumulation of NO2/NO3 and cellular guanosine 3,'5'-cyclic monophosphate (cGMP) content. H2O2 markedly impaired inducible nitric oxide synthase (iNOS) activity induced by IL-1 directly measured by the conversion of L-[14C]arginine to L-[14C]citrulline. Such impairment in iNOS activity was accompanied by a parallel reduction in iNOS protein abundance but not by a reduced expression of iNOS mRNA. The inhibitory effect of H2O2 on NOS activity was further supported by peroxide-induced impairment in IL-1-driven, NO-dependent synthesis of prostaglandin E2. Our studies thus provide the first direct evidence of a posttranscriptional inhibitory effect of H2O2 on iNOS activity. Additionally, our studies uncover the existence of a previously unrecognized effect of H2O2 on the production of NO that may exert influence on the severity of glomerular injury during glomerular inflammation.