| Objective: To observe whether upregulation of haem oxygenase-1(HO-1)/adiponectin system could reduce urinary albumin levels,regulate imbalance of glomerular vascular endothelial growth factor(VEGF)-nitric oxide(NO)axis,and then improve early damage of obesity rats kidney.Methods: Six-week-old male Wistar rats were randomly divided into the following four groups: a normal control group(NC group)fed with a regular diet;an obesity group(OB group)fed with a high-fat diet to make obesity model;an obesity plus cobalt protoporphyrin(Co PP)group(Co PP group)fed with a high-fat diet and meanwhile induced by injection of Co PP to induce HO-1;and an obesity plus Co PP and stannous protoporphyrin(Sn PP)group(Sn PP group)fed with a high-fat diet and meanwhile induced by injection of Co PP to induce HO-1 and Sn PP to inhibit HO-1.The body weight and visceral fat were measured.Blood and urine samples were collected to measure the blood glucose,serum creatinine,free fatty acid(FFA),triglyceride,high-sensitivity C-reactive protein(hs-CRP),tumour necrosis factor-?(TNF-?),malondialdehyde(MDA),adiponectin and albumin-to-creatinine ratio(ACR).Endothelial function was determined by measuring endothelium-dependent vasodilatation(EDV)of the aorta.Renal tissues were collected for CD34 immunohistochemistry for evaluation of the degree of glomerular endothelial cells proliferation.Glomerular NO levels were measured by the Griess reaction.Glomerular VEGF,e NOS,Akt and AMPK protein were measured by western blot.Results:(1)Comparements of renal cortex HO-1 concentration and serum adiponectin levels: Compared with the NC group,renal cortex HO-1 concentration in OB group rats were significantly reduced(P<0.05).Co PP group rats significantly increased levels of HO-1 concentration compared with the NC and OB groups(P<0.05).In addition,compared with the NC group,serum adiponectin levels were significantly reduced in OB group(12.77 ± 2.77 ?g/m L vs.20.34 ± 2.76 ?g/m L,P<0.05),which increased after induction of HO-1 by Co PP(19.35 ± 2.67 ?g/m L vs.12.77 ± 2.77 ?g/m L,P<0.05),suggesting that Co PP successfully upregulated HO-1/ adiponectin system.(2)Biometric parameters of the four groups: Rats in the OB group exhibited increased body weight and visceral fat compared with rats in the NC group(body weight,702.3 ± 94.4 g vs.501.5 ± 58.8 g;visceral fat,54.90 ± 14.18 g vs.17.7 ± 5.6 g,P<0.01).Co PP group rats had significant decreased body weight and visceral fat than the OB group(body weight,579.7 ± 66.5g vs.702.3 ± 94.4g;visceral fat,24.8 ± 10.3g vs.54.90 ±14.18 g,P<0.01).(3)Comparements of blood parameters: Compared with the NC group,OB group rats exhibited a significant increase in plasma glucose,FFA concentrations and serum concentrations of triglyceride,hs-CRP,TNF-? and MDA(plasma glucose,135.7 ± 12.3 mg/d L vs.110.5 ± 10.4 mg/d L;FFA,1.29 ± 0.36 mmol/L vs.0.35 ± 0.14 mmol/L;triglyceride,1.45 ± 0.36 mmol/L vs.0.55 ± 0.25 mmol/L;hs-CRP,2.28 ± 0.61 mg/L vs.0.72 ±0.18 mg/L;TNF-?,92.5 ± 10.2 pg/m L vs.27.5 ± 3.6 pg/m L;MDA,3.92 ± 0.51?mol/L vs.1.47 ± 0.31 ?mol/L,P<0.01).Induction of HO-1 by Co PP resulted in a significant decrease in the above index in OB group rats when compared with the NC group(plasma glucose,114.7 ± 12.9 mg/d L vs.135.7 ± 12.3 mg/d L;FFA,0.74 ±0.31 mmol/L vs.1.29 ± 0.36 mmol/L;triglyceride,0.83 ± 0.28 mmol/L vs.1.45 ±0.36 mmol/L;hs-CRP,1.04 ± 0.14 mg/L vs.2.28 ± 0.61 mg/L;TNF-?,56.8 ± 8.9pg/m L vs.92.5 ± 10.2 pg/m L;MDA,2.28 ± 0.51 ?mol/L vs.3.92 ± 0.51 ?mol/L,P<0.01).Whereas both FFA,hs-CRP,TNF-? and MDA were higher in Co PP group rats than the NC group(P<0.01).(4)Urinary albumin excretion and renal function:Compared with the NC group,OB group rats had significant increased ACR(94.10 ±16.27 mg/g vs.17.44 ± 5.32 mg/g,P<0.05).Co PP group rats showed significant decreased ACR compared with the OB group(49.25 ± 14.93 mg/g vs.94.10 ± 16.27mg/g,P<0.05).Serum creatinine concentrations were not different between any of the experimental groups(P > 0.05).(5)Comparement of the vascular endothelial function: Acetylcholine(ACh)-stimulated concentration-dependent vasorelaxation of aortic strips was significantly lower in OB group rats compared with the NC group(P<0.05).Treatment of obese rats with Co PP significantly enhanced ACh-induced vasorelaxation in Co PP group rats compared with the OB group(P < 0.05).And sodium nitroprusside(SNP)-stimulated concentration-dependent vasorelaxation remained unchanged among these four groups(P > 0.05).(6)Results of CD34 histology and immunohistochemistry: Compared with the NC group,OB group rats had marked glomerulomegaly and significantly increased glomerular volume(19.43± 3.20 ?m~3 vs.14.56 ± 3.15 ?m~3,P < 0.05).The Co PP treatment significantly reduced glomerular hypertrophy in Co PP group rats(16.87 ± 2.32 ?m~3 vs.19.43 ±3.20 ?m~3,P < 0.05).Immunohistochemistry showed significant increased CD34 expression in glomeruli of OB group rats compared with the NC group [integrated optical density per stained area(IOD/area): 0.123 ± 0.015 vs.0.094 ± 0.010,P<0.05],suggesting that the degree of glomerular endothelial cells proliferation increased.While Co PP group rats had decreased CD34 expression in glomeruli than the OB group(IOD/area:0.101 ± 0.009 vs.0.123 ± 0.015,P<0.05),suggesting that the degree of glomerular endothelial cells proliferation decreased.Otherwise,severe inflammatory cell infiltration was seen in OB group rats compared with mild inflammatory cell infiltration in the NC group,and inflammatory cell infiltration was moderate in Co PP-treated obese rats.(7)Comparements of the glomerular VEGF and NO levels: Compared with the NC group,glomerular VEGF levels were significantly increased and glomerular NO levels significantly reduced in OB group rats(P<0.05),indicating uncoupling of the glomerular VEGF–NO axis.Compared with OB group rats,treatment of obese rats with Co PP significantly reduced glomerular VEGF levels,enhanced glomerular NO levels and improved the glomerular VEGF–NO axis(P<0.05).(8)Detection of reactive oxygen species in the glomeruli: Compared with the NC group,the production of reactive oxygen species(ROS)in glomeruli was significantly increased in OB group rats(P<0.05).Compared with the OB group,induction of HO-1 with Co PP significantly reduced ROS production in the glomeruli of Co PP group rats(P<0.05).(9)Comparements of renal TNF-? and IL-10 concentrations: Compared with the NC group,renal TNF-? and IL-10 concentrations in OB,Co PP and Sn PP group rats were significantly enhanced(P < 0.05).Compared with OB group rats,treatment with Co PP significantly reduced the renal TNF-? and IL-10 concentrations in the Co PP group(P<0.05).(10)Phosphorylation of glomerular AMPK,Akt and e NOS proteins:Western blot analysis showed significantly reduced phosphorylation of glomerular AMPK,Akt and e NOS proteins in OB group rats compared with the NC group(P<0.05).While Co PP group rats had a significant increase in the above index when compared with the OB group(P<0.05).Conclusions: Upregulation of HO-1/adiponectin system with Co PP,reduces the degree of microalbuminuria and has renal protective effects by improving endothelial dysfunction and regulating the uncoupled glomerular VEGF-NO axis in diet-induced obese rats. |
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