Study Of The Role Of Oxidative Stress In Preventing Contrast-induced Nephropathy

Background and ObjectiveWith the endovascular contrast media being widely utilized, imaging diagnosticand intervention have made great progress. Meanwhile the incidence ofcontrast-induced nephropathy (CIN) is on the rise. The pathogenesis and treatmentmeasures of CIN are not clear. We observe the effects of nephrotoxicity of iohexol onrenal function and renal tissue nitric oxide (NO), nitric oxide synthetase (NOS) andglutathione (GSH) in male aging rats with diabetes, and investgae the roles of the NO,NOS and GSH in the pathogenesis of contrast-induced nephropathy. We createdchimeras by swapping the ferredoxin-NADP+reductase (FNR)-like module betweennNOS and eNOS to evaluate the role of the FNR module in controlling NOS catalyticactivities. In clinical practice, the creatinine-based GFR and creatinine may not beable to accurately response the change of renal function for elderly patients.weinvestigate the concentration of interleukin-18(IL-18) and Cystatin C (CysC) afteriohexol-enhanced computed tomography (CT) procedures to find more sensitive biological parameters for predicting kidney injury. Antioxidants may be effective inpreventing CIN in risk patients for the role of oxidative stress in pathogenesis of CIN.GSH is known as complex antioxidant network center. GSH can either directlyscavenge free radicals or act as a substrate for glutathione peroxidase and glutathioneS-transferase during the detoxification of hydrogen peroxide, lipid hydroperoxides.We investigate the efficacy of reduced glutathione injection in the prevention of CIN.Because there are a large number of GSH in the blood, we investigate the interactionsof GSH and hemoglobin by spectroscopic methods to understand the antioxidantmechanism of GSH.The content of this thesis includes three chapters.Part ⅠMaterials and MethodsTo investigate the effects of nephrotoxicity of iohexol in male aging rats withdiabetes．40male SD rats were included in the study.10rats were divided intonormal control group (SD group，n=10)，the other30rats were induced byintra-peritoneal administration of one dosage of streptotocin．At the end of the eighthweeks，the diabetic rats were divided into two groups at random：diabetic controlgroup (DM group，n=13), diabetic rats injected with iohexol (10ml/kg)(LOCMgroup，n=13)．After having finished the injection for48hours，the levels of serumcreatinine (Scr),blood urea nitrogen (BUN) and blood glucose were examined underanesthetization；NO, NOS and GSH in renal cortex were measured．We createdchimeras by swapping the ferredoxin-NADP+reductase (FNR)-like module betweennNOS and eNOS to evaluate the role of the FNR module in controlling NOS catalyticactivities.ResultsThe levels of Scr and BUN increased in DM group compared with SD group(P<0.05). The levels of NO and GSH decreased in DM group compared with SDgroup (P<0.01and P<0.05, respectively). The level of Scr significantly increased inLOCM group compared with the SD group (P <0.01)．The level of NO and NOSsignificantly decreased in LOCM group compared with DM group (P<0.05)． NOand NOS maybe play an important role in the nephrotoxicty of iohexo in diabetic rats．The eNOS chimera (eNOSnFNR) had a cytochrome c reductase activity that wasover10-fold higher than that of native eNOS in the absence of calmodulin (CaM),and did not increase upon CaM binding.Its rate of NO synthesis was similar to nativeeNOS. In contrast, the nNOS chimera (nNOSeFNR) had a cytochrome c reductaseactivity that was~10%of native nNOS in the absence of CaM, and its NO synthesisactivity that was~12%of the native nNOS, respectively. We demonstrated that theFNR module is critical in controlling electrons flow through the reductase domainand out of the FMN module. We propose that the FNR module plays a role inadjusting the electron transfer capacities of the FMN module, likely by determiningthe domain-domain interaction.Part ⅡMaterials and Methods30elderly diabetic patients without renal impairment who scheduled forcontrast-enhanced CT were enrolled from January2011to October2011in ourhospital. All patients were given low-osmolar nonionic contrast media. Theconcentration of interleukin-18was measured with ELISA.ResultsThere was no significant difference between the level of serum IL-18adoptedbefore contrast-enhanced CT and those adopted at the24and48hours afterexamination (P>0.05). The levels of urine IL-18and serum cystatin C adopted at the24and48hours after contrast-enhanced CT significantly rose (P <0.05) than thatadopted before examination. The levels of serum creatinine rose at the48hours aftercontrast-enhanced CT (P <0.05) than that adopted before examination.Part ⅢMaterials and Methods96elderly patients (age≥60years) with DM and apparently normal renalfunction scheduled for iohexol-enhanced computed tomography (CT) procedureswere randomized to have either reduced glutathione injection (1800mg in100ml ofphysiological saline intravenously before procedures, then1800mg/d for two daysafter the procedures) plus1ml/kg BW/h physiological saline intravenously (GSHgroup, n=48) or only physiological saline intravenously (control group, n=48). Serum creatinine (SCr), blood urea nitrogen (BUN), Cystatin C and urinary interleukin-18were measured prior to and48hours after procedures. CIN was defined as0.5mg/dLelevation of creatinine from baseline and/or an increment of25%over baselinecreatinine. The interaction of GSH with hemoglobin (Hb) was studied byspectroscopic methods. It was confirmed that the quenching of GSH to the intrinsicfluorescence of trypsin is a static quenching procedure.ResultsThere were no significant differences between the GSH group and control groupin baseline demographics and baseline renal function. The incidence of CIN in GSHgroup was slightly lower compared with control group (2.08%vs.8.33%, P=0.364).The level of SCr, BUN, CysC and urinary IL-18increased and estimated glomerularfiltration rate (eGFR) decreased after the procedures in both groups. Significantdifferences between-group in SCr (P=0.001), eGFR (P=0.002), CysC (P <0.001),BUN (P=0.001) and urinary IL-18(P <0.001) were presented after the procedures.The thermodynamics parameter (ΔH, ΔS, ΔG) implied that major force type in thebinding reaction. The distance r was determined to be less than7nm with F sterenergy theory. By using the method of synchronous fluorescence, UV/Vis absorptionand three-dimensional fluorescence spectra, the conformational change of Hb causedby GSH was analyzed.Conclusions1. Iohexol can cause nephrotoxicity in aging diabetic rats．2. FNR module is critical in controlling electrons flow through the reductase domainand out of the FMN module.3. The urine IL-18and CysC are sensitive biological parameters for predictingkidney injury. So it can be clinically applied to monitor CIN.4. The reduced glutathione injection plus physiological saline intravenously maybebeneficial to the prevention of CIN in risk people by modulating oxidative andantioxidant systems.5. Interaction of GSH with hemoglobin (Hb) was studied by spectroscopic methods. It wasconfirmed that the quenching of GSH to the intrinsic fluorescence of trypsin is astatic quenching procedure.