| PART 1: INFLAMMATORY STRESS EXACERBATES LIPID MEDIATED RENAL INJURYObjective:The aim of this part was to observe the influence of in-flammation on lipid metabolism disturbance and the progression of renal injury.Methods:C57BL/6 mice were randomized into normal diet (A1), high fat diet (B1) and high fat diet plus casein injection (C1) (induce inflamma-tion) groups. Animals were sacrificed after 14 weeks, terminal blood sam-ples were taken for cholesterol (TC), triglycerides (TG), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C) and interleukin-6 (IL-6) assays. Red O stain and intracellular cholesterol concentration assay were performed. The morphological changes of kidney were examied by HE, PAS, Masson staining. The mRNA levels and protein expression of transforming growth factor-β(TGF-β1), collagen type I (Collagen I) and type IV (Collagen IV), Fibronectin (FN) were determined by Real-Time Polymerase Chain Reac-tion (RT-PCR) and immunohistochemistry (IHC) assessments. Results: Blood IL-6 levels were higher, while TC and LDL-C were lower in the C1 group compared to B1 group (P<0.05). Lipid accumulation in the kidney was more extensive in C1 group despite lower blood lipid levels by ORO staining and quantitative analysis (P<0.05). PAS, Masson and HE staining demonstrated casein injection induced structural changes characterized by mesangial expansion in the kidney. The GSI was signifi-cantly higher in C2 group than those of other group (P<0.05). These ab-normalities were associated with increased gene and protein expression of Collagen I, Collagen IV, and TGF-β1.Conclusions: Low blood cholesterol levels may be associated with high risk for chronic renal injury under inflammatory stress. Inflammation exacerbates lipid accumulation in kidney by diverting lipid from plasma to kidney.PART 2: THE ROLE OF SREBP2/LDLR/ HMG-COA REDUC-TASE PATHWAY IN INFLAMMATORY STRESS EXACER-BATES LIPID MEDIATED RENAL INJURYObjective: To explore the role of the sterol regulatory element binding protein cleavage protein (SCAP)-sterol regulatory element binding protein 2 (SREBP2)-low density lipoprotein receptor (LDLr) / 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) pathways in the process of inflammation stress exacerbating lipid mediated renal injury.Methods: On the basis of part I, the genes of LDLr, SREBP2, SCAP, HMGCoA reductase involved in cholesterol negative feedback pathways were determined by RT-PCR and western-blotting (WB) as well. Some variables were analysis by Pearson correlation assesment.Results: The mRNA of SCAP, SREBP2, LDLr, and HMGCoA reduc-tase as well as protein levels were markedly enhanced in C1 group com-pared with B1 group (P<0.05). Casein injection increased LDLr, HMGCoA reductase gene and protein expression in the kidney, probably by disrupting the intracellular cholesterol sensor, regulatory function of SREBP cleavage activating protein (SCAP). Futher, the protein level of HMGCoA reductase and LDLr was positively related to CE deposition in kidney (P<0.05).Conclusion: Inflammation possibly exacerbates lipid induced-renal injury through the SCAP-SREBP2-LDLr/HMGCoA reductase pathways by abnormal cholesterol synthesising and uptaking. PART 3: INFLAMMATORY STRESS EXACERBATES LIPID MEDIATED RENAL INJURY IN SR-A/CD36/APOE TRIPLE KNOCK-OUT TRIPLE-KNOCKOUT MICEObjectives: This study was designed to investigate whether inflam-matory stress exacerbates lipid accumulation in lipid-mediated glomerular injury and, thereby exacerbating the progression of renal injury by non–scavenger receptor–mediated pathways in vivo.Methods: Male SR-A/CD36/ApoE triple Knock-out (KO) mice were randomly divided into normal diet (A2), high fat diet (B2) and high fat diet plus inflammation groups (C2). Animals were sacrificed after 14 weeks, terminal blood samples were taken for creatinine (Scr), urea nitrogen (BUN), TC, TG, and LDL-C, HDL-C, amyloid A (SAA) and IL-6 assays. Renal sections were used for histological and IHC assessments. Renal histopa-thology was quantified using a glomerulosclerosis index (GSI) and a tubu-lointerstitial injury index (TII). The lipid accumulation in kidney was evaluated by Oil Red O staining and quantitative analysis. The mRNA and protein expression of TGF-β1, Collagen I, Collagen IV, FN, a-smooth mus-cle actin (a-SMA), SREBP2, SCAP, LDLr were analyzed by RT-PCR and WB analysis.Results: Blood levels of SAA and IL-6 were higher (P<0.05), while TC, LDL-C and HDL-C were lower in the C2 group compared to B2 group (P<0.05). Meanwhile, lipid accumulation in the kidney was more extensive in C2 group despite lower blood lipid levels (P<0.05). Casein injection ex-acerbated renal function failure and proteinuria compared to B2 group (P<0.05). Casein injection induced massive structural changes. The GSI and TII were significantly higher in C2 group than those of B2 group. These abnormalities were associated with increased gene and protein expression of Collagen I, Collagen IV, TGF-β1, a-SMA, and FN. Further analysis showed that casein injection increased LDLr, SREBP2, and SCAP gene and protein expression in the kidney.Conclusions: Non scavenger receptor-mediated alternative atherogenic pathways are operational, and SCAP-SREBP2-LDLr pathway may be the dominant mechanism for inflammatory stress exacerbating lipid mediated renal Injury.PART 4: ENO-PROTECTIVE EFFECTS OF ATORVASTATIN INDEPENDENT OF CHOLESTEROL LOWERINGObjective: This part was to investigate whether Atorvastatin de-creased renal lipid accumulation and renal injury independent of the lipid-lowering effect.Methods: Male SR-A/CD36/ApoE triple KO mice were randomly as- signed to receive high fat diet plus Atorvastatinn treatment group (D1) (2mg/kg/d), other groups of A1, A2 as control groups and B2 as model group. Terminal blood samples were taken for cholesterol, SAA, and IL-6 assays. Renal sections were used for histological assessments. The lipid accumulation in kidney was evaluated by Oil Red O staining and quantita-tive analysis. The mRNA expression and protein expression of TGF-β1, Collagen I and Collagen IV, FN, a-SMA were analyzed by RT-PCR and IHC.Results: Blood levels of SAA, IL-6 were lower (P<0.05), while TC, LDL-C and HDL-C were not different in the D1 group, Compared to B2 group. Meanwhile, lipid accumulation in the kidney was less and renal function was better (P<0.05). PAS, Masson and HE staining demonstrated Atorvastatin therapy attenuated massive structural changes. Compared to B2 group, there were decreased gene and protein expression of Collagen I, Collagen IV, TGF-β1, a-SMA, and FN in the Atorvastatin therapy group (P<0.05).Conclusion: The beneficial effect of Atorvastatin might be mediated by the effect of anti-inflammatory action through a reduction TGF-β1 ex-pression independent of the lipid-lowering effect and cellular uptake of SR-A,CD36. PART 5: STATIN RESISTANCE UNDER INFLAMMATION STRESSObjective: Our previous vitro study has demonstrated that inflamma-tion can lead statin resistance in human hepatoma cell line.This part was design to explore if there was statin resisitance in vivo for Eno-protective Effects of Atorvastatin under inflammation stress.Methods: Two doses of atorvastatin were administered in SR-A/CD36/ApoE triple KO mice which were fed with high fat plus in-flammtion, 2mg/kg/d (D2) and 10mg/kg/d (D3), respectively. Terminal blood samples were taken for Scr, BUN, TC, TG, LDL-C, HDL-C, SAA and IL-6 assays. Renal sections were used for histological and IHC assessments. Renal histopathology was quantified using GSI, TII. The lipid accumulation in kidney was evaluated by Oil Red O staining and quantitative analysis. The mRNA of TGF-β1, Collagen I and Collagen IV, FN, a-SMA, were analyzed by RT-PCR.Results: In the inflammatory state, compared to C2 group, D2, D3 group could not effectively reduce the the serum lipid levels (P>0.05).Compared to C2 group, D2 group could not obviously reduce kidney deposition of cholesterol and Scr (P>0.05), and improve the kidney deterioration, while D3 group could reduce kidney tissue cholesterol depo- sition and ameliorate the pathological changes of kidney obviously (P<0.05). Compared to C2 group, the gene expression of Collagen I, Collagen IV was not decreased in D2 group (P>0.05), while D3 group could significantly reduced the TGF-β1, Collagen I and Collagen IV, FN, a-SMA gene expres-sion (P<0.05).Conclusion: Atorvastatin may not as effective on renal protection as it is in the absence of inflammtion. To achieve the same effect, the dose of Atorvastatin should increase accordingly.The potential mechanisms deserve futher exploration.PART 6: MCP-1 GENE POLYMORPHISM AND MCP-1 EX-PRESSION IN CHONGQING HAN CHILDREN WITH TU-BERCULOSISObjective: The aims of this study were to evaluate whether the presence of–2518A/G polymorphism in the distal regulatory region of the MCP-1 was associated with tuberculosis (TB) in Chongqing Han population and to find whether it has a significant impact on the pediatric patient.Method: One hundred children and one hundred adults with TB and 200 healthy controls of comparable age were screened for genotype by PCR-sequence-specific primer (SSP) method. MCP-1 levels in the plasma were detected by ELISA.Result: (1) The -2518GG genotypes was associated with increased TB susceptibility (P<0.05). (2) The odds of developing TB in genotypes GG were higher than those in homozygous AA, and the risk was higher in chil-dren than in adult (P<0.05). (3) Cases of homozygous GG had the highest plasma levels of MCP-1, which increased the likelihood of developing TB. Furthermore, higher levels were observed in children than in adults (P<0.05).Conclusion: These findings suggest that persons bearing the MCP-1 genotype GG produce high concentrations of MCP-1, which increases the risk of active TB infection in Chongqing Han people. These findings are more significant in child patients than in adult patients with TB. |
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