Experimental Research Of Renal Dysfunction And Mitochondrial Biogenesis In Septic Rats

ObjectiveSepsis is the Systemic Inflammatory Respond Syndrom caused or autoinmmunity injury caused by microorganism or any other immunogenicity. Sepsis remains a serious cause of morbidity and mortality; it may cause severe sepsis, septic shock and MODS. There are 79000 casese of sepsis in Geman annual, 75000 of severe sepsis or septic shock. The pathophysiology of the disease is not clear, it is characterized by inflammation, oxidative damage, hypercoagulation, tissue hypoperfusion, immune suppression and mitochondrial dysfunction. Mitochondrial dysfunction plays an important role in the process of sepsis, but it is unknown in the recovery of sepsis. Normal mitochondrial number, structure, and function are supported by mitochondrial biogenesis, a cellular program that adjust energy production by synthesis of new organelles and organelle components and mediates interorganelle interactions. To elucidate the mechanisms of mitochondrial dysfunction and biogenesis in kidney in the process of sepsis, we assessed the function of the organ and the genes mRNA levels of the transcription factor of biogenesis. All of these researches could provide experiment evidences of diagnosis and treatment of mitochondrial injury in kidney.Method1 .The animal modelFifty male SD rats were randomly divided into five groups according to the detected time. The rats in LPS groups received l0mg/kg lipopolysaccharide by intraperitoneal injection .2. Measurements and Methods2.1 General state of animals 2.1.1 Rectal temperature, heart rate and breathing rate were observed before intraperitoneal injection and at each time point after intraperitoneal injection.2.1.2 LPS was detected by tachypleus amebocyte lysate kinesis quantitative method.2.2 renal functionSerum Cr, Urea levels were detected by automatic biochemistry analyzer.2.3 Renal mitochondrial transmembrane potential and swelling.Mitochondrion of kidney was isolated by differential centrifugation. Swelling and membrane potential of renal mitochondrial in rats were nanalyzed by FCM.2.4 Assessing mitochondrial oxidative stressMitochondrial oxidative stress level (activities of SOD, GSH, NOS MDA, NO) of renal mitochondrion in rats were assessed2.5 mtDNA copy numberTotal cellular DNA was extracted. The mtDNA copy number was obtained by real-time PCR.2.6 Nuclear and mitochondrial mRNA expressionTotal RNA was extracted. Gene transcripts were amplified, and GAPDH mRNA was used to control for variation in efficiency of RNA extraction.3. Statistical analysisThe data was analyzed by SPSS13.0, and the cartograms were drawn using Excel 2003. All results were reported as mean±standard deviation (±s). Differences between groups were determined by ANOVA. If there was statistical significance and homogeneity of variance between groups, Tukey Test was used. Pearson correlation test was used betwent these items.α=0.05.Results1.General state of animalsAll animals in LPS groups had septic symptoms and signs.The content of LPS in plasma of LPS groups was positive.2. Renal function2.1 Serum Cr levelsCompared with control group, serum Cr levels increased significantly in LPS 4h, LPS 24h and LPS 48h (P<0.01, P<0.05, P<0.05). There was no difference in Cr between control group and LPS 72h group.2.2 Serum BUN levelsCompared with control group,serum Urea levels increased significantly in LPS4h, LPS 24h,LPS 48h and LPS 72h groups .(P<0.01, P<0.01, P<0.05, P<0.05).3.Membrane potential and swelling of renal mitochondria in rats.3.1 Renal mitochondrial membrane potential significantly decreased in LPS 4h and LPS 24h groups( P<0.05, P < 0. 05).3.2 Swelling of renal mitochondrion significantly increased in LPS 24h group. (P < 0. 05).4. Renal mitochondrial oxidative stressActivities of TSOD were significantly decreased in LPS 4h and LPS 72h (P < 0. 05, P < 0. 01). Activities of MnSOD were significantly decreased in LPS 48h, LPS72h (P < 0. 01, P < 0. 01). The content of GSH increased significantly in LPS 24h (P < 0. 01) but decreased in LPS 72h (P < 0. 01) compared with the control group. Activities of TNOS increased in all LPS groups (P < 0. 01, P < 0. 01, P < 0. 01, P < 0. 01). But there were no statistical differences in MDA among all the groups (P > 0. 05).5. MtDNA copy numberMtDNA copy number was significantly decreased in LPS 24h compared to the control group (P < 0. 05).6. Nuclear and mitochondrial mRNA expressionTfam mRNA levels began to increase in LPS 4h (P<0.05), but significantly in LPS 24h (P<0.01). Expression of PGC-1α, NRF2 mRNA began to increase significantly in LPS 4h (P<0.01), back to the normal level at 48h.Conclusions1. LPS induced mitochondrial injuries in kidney in the early of septic rats. Mitochondrial injury was relative to the renal function and was one of the causes of renal dysfuncton.2. The damage of mitochondrial DNA copy number caused the decreased of mitochondrial membrane potetial, the recovery of membrane potential was relative to the recovery of mtDNA copy number.3. The mRNA expression of mitochondrial biogenesis regulatory genes began to increase in the early of sepsis and it cuased the recovery of mitochondrial membrane potetial and DNA copy number.4. Oxidative stress caused the mitochondrial injuries, ROS may initiate mitochondrial biogenesis.