We previously demonstrated that there are acute and delayed phases of renal protection against renal ischemia and reperfusion (IR) injury with renal ischemic preconditioning (IPC). This study assessed whether hepatic IPC could also reduce distant renal IR injury through the blood stream-mediated supply of reactive oxygen species (ROS). Male C57BL/6 mice were randomly divided into four groups: group I, sham operated including right nephrectomy; group II (IR), left renal ischemia for 30 min and reperfusion injury; group III (IPC-IR), hepatic ischemia for 10 min followed by 10 min of reperfusion before left renal IR injury; group IV (MPG - IPC + IR), pretreated with 100 mg/kg N-(2-mercaptopropionyl)-glycine (MPG) 15 min before hepatic IPC and left renal IR injury. Renal function, histopathologic findings, proinflammatory cytokines, and cytoprotective proteins were evaluated 15 min or 24 hr after reperfusion. Hepatic IPC attenuated the expression of proinflammatory cytokines, tumor necrosis factor alpha, intercellular adhesion molecule 1, and induced inducible nitric-oxide synthase, and the phosphorylation of Akt in the murine kidney. Renal function was better preserved in mice with hepatic IPC (group III) than groups II or IV. Hepatic IPC protects against distant renal IR injury through the blood stream-delivery of hepatic IPC-induced ROS, by inducing cytoprotective proteins, and by inhibiting inflammatory reactions.
Animals ; Intercellular Adhesion Molecule-1/genetics/metabolism ; *Ischemic Preconditioning ; Kidney/drug effects/metabolism/pathology/physiopathology ; Liver/blood supply/drug effects/physiopathology ; Male ; Mice ; Mice, Inbred C57BL ; Nitric Oxide Synthase Type II/metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism ; Reactive Oxygen Species/metabolism ; Reperfusion Injury/*metabolism/pathology/prevention & control ; Tiopronin/pharmacology ; Tumor Necrosis Factor-alpha/genetics/metabolism

OBJECTIVETo explore the effects of the exogenous and endogenous reactive nitrogen metabolites (RNM) as NK cell inhibitors on NK cell-mediated killing of K562 cells and the influence of Tiopronin (TIP), glutamylcysteinylglycine (GSH) and histamine dihydrochloride (DHT) as RNM scavengers on reversing the suppressing effect of RNM.

METHODSThe exogenous ONOO(-) was administered in the NK+K562 culture system, then the RNM scavengers were added in the NK+K562+ONOO(-) culture system, respectively. The concentrations of RNM, TNF-beta and IFN-gamma, K562 cell inhibition rate (KIR) and the percentage of living NK cells were examined. IL-2+PHA were used as monocyte (MO) activators in the culture system of MO+NK+K562. Then TIP, GSH and DHT were administered and the parameters of NK cell activity were analyzed.

RESULTSAfter exogenous ONOO(-) was administered in NK+K562 culture system, the percentage of living NK cells was decreased from (93.17 +/- 2.57)% to (71.87 +/- 1.02)% (P < 0.01) and KIR was decreased from (67.47 +/- 2.64)% to (43.44 +/- 2.87)% (P < 0.01). When TIP, GSH and DHT were administered into the systems, the percentage of living NK cells was increased to (91.13 +/- 3.67)% (P < 0.05), (88.03 +/- 1.46)% (P < 0.05), (73.60 +/- 2.76)% (P > 0.05), respectively; KIR was increased to (61.58 +/- 1.89)% (P < 0.05), (60.68 +/- 2.07)% (P < 0.05) and (45.26 +/- 3.31)% (P > 0.05), respectively. When IL-2/PHA were administered in the NK+K562+MO culture system, RNM products was increased from (82.10 +/- 6.60) micromom/L to (193.65 +/- 5.95) micromom/L(P < 0.01);KIR was decreased from (90.64 +/- 3.06)% to (61.29 +/- 2.22)% (P < 0.01). When the TIP, GSH and DHT were administered in the systems, RNM products were decreased to (91.32 +/- 6.81) micromom/L (P < 0.05), (84.66 +/- 5.99) micromom/L (P < 0.05) and (188.92 +/- 5.00) micromom/L (P > 0.05), respectively; KIR was increased to (84.31 +/- 4.56)%(P < 0.05), (81.65 +/- 3.09)% (P < 0.05) and (72.20 +/- 4.10)% (P < 0.05), respectively.

CONCLUSIONNK Cell-mediated killing of K562 cells can be suppressed by exogenous and endogenous RNM administration. Both of TIP and GSH can protect NK cells by scavenging RNM and enhance the antineoplasmic activity of NK cells.

Cells, Cultured ; Coculture Techniques ; Glutathione ; pharmacology ; Histamine ; pharmacology ; Humans ; Interferon-gamma ; metabolism ; Interleukin-2 ; immunology ; pharmacology ; K562 Cells ; Killer Cells, Natural ; cytology ; immunology ; metabolism ; Lymphotoxin-alpha ; metabolism ; Monocytes ; cytology ; Peroxynitrous Acid ; pharmacology ; Reactive Nitrogen Species ; antagonists & inhibitors ; metabolism ; Tiopronin ; pharmacology

To investigate the effect of a new reactive oxygen metabolites (ROM) scavenger as immune adjuvant in NK cell-mediated killing effect on K562 cell, IL-2 and PHA were used to activate monocyte to produce ROM, and different concentrations of tiopronin as ROM scavenger was used in the cultivated systems with different ratio of monocytes plus NK cells and K562 cells, while histamine dihydrochloride (DHT) with different concentrations was used as positive control. The reuslts indicated that after IL-2 and PHA were supplemented in the cultivated systems mixing with NK cells and K562 cells as the E/T ratio was 10/1, the ROM production increased from 33.17 +/- 25.02 U/ml to 223.59 +/- 59.41 U/ml (P < 0.05) while K562 cell inhibition rate (KIR) increased from 65.56% to 85.89% (P < 0.05). When the monocytes as the E/MO ratios of 10/2, 10/5 and 10/10 were supplemented respectively, ROM production increased correspondingly (ROM production was 389.79 +/- 43.83 U/ml, 456.74 +/- 42.77 U/ml, 601.42 +/- 21.92 U/ml, respectively), and KIR was on the other round (KIR was 82.36%, 81.36%, 48.09% respectively). Tiopronin, DHT were used in the K562 + NK + MO + IL-2/PHA cultivated systems as the E/MO ratio was 10/2, the ROM production also decreased from 389.79 +/- 43.83 U/ml to -1.20 +/- 60.70 U/ml, 50.21 +/- 22.4 U/ml (P < 0.05), respectively, however KIR increased from 82.53% to 96.09% and 94.64% either (P < 0.05). Higher concentrations of tiopronin and DHT were used, ROM production decreased accordingly. There showed a reverse correlation between ROM production and KIR (r = -0.518). When E/MO ratio was 10/5 or 10/10, tiopronin at any testing concentration and DHT at the higher testing concentration could reduce the ROM production (P < 0.05), but did not improve KIR significantly (P > 0.05). Tiopronin was as good as DHT in ameliorating KIR (P > 0.05) and better than DHT in scavenging ROM (P < 0.05). It is concluded that (1) Monocytes are the major resources of ROM, and the ROM derived from monocytes can disable NK cells in killing neoplasm cells (K562 cells); (2) A new ROM scavenger, tiopronin, can scavenge ROM effectively, and reverse the ROM induced inhibition of NK cell-mediated killing of K562 cell in a certain extent. And tiopronin is better than DHT in scavenging ROM, and as good as DHT in up-regulating KIR. The new ROM scavenger tiopronin with less side effect may take the place of DHT as adjuvant during the adoptive immuno-therapy in leukemia.
Coculture Techniques ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; drug effects ; immunology ; Free Radical Scavengers ; pharmacology ; Humans ; K562 Cells ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; Reactive Oxygen Species ; antagonists & inhibitors ; metabolism ; Tiopronin ; pharmacology

OBJECTIVETo observe the harmful effects of oxygen free radicals and the protective roles of Tiopronin in severely scalded rats after delayed fluid resuscitation.

METHODSRats inflicted with 30% III degree scald on the back were employed as the model. They were divided into delayed resuscitation (D) and Tiopronin treatment (T) groups. The changes in superoxide dismutase (SOD) and malonyldialdehyde (MDA) in plasma and subeschar fluid were determined at 24 to 48 postburn hours (PBHs) by means of electron spin resonance (ESR) technique and other routine methods. And the pathomorphological changes in the heart, liver, kidneys and small intestine, and changes in the blood biochemical indices were simultaneously determined. Normal rats were taken as control group (N).

RESULTSThe plasma SOD level was was lower than that in N group, while the MDA content in plasma and subeschar fluid in D group was much higher than that in N group. Changes in all the blood biochemical and internal organ pathomorphology were more obvious in the D group. on the other hand, the rat plasma SOD level in T group increased obviously (P < 0.01) while the MDA contents was decreased in T group (P < 0.05) when compared with those rats of D group. In addition, the internal organ pathomorphology and blood biochemical indices were improved evidently in T group.

CONCLUSIONOxidative stress injury was evoked in severely scalded rats after delayed fluid resuscitation, and it could be protected to some extent by Tiopronin.

Animals ; Burns ; drug therapy ; metabolism ; Female ; Male ; Malondialdehyde ; blood ; Oxidative Stress ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; blood ; Tiopronin ; pharmacology ; therapeutic use