| Lipoxygenases (LO) are a family of nonheme iron-containing enzymes that insert molecular oxygen into polyunsaturated fatty acids such as arachidonic and linoleic acids. They are classified as 5-,8-,12-, and 15-LO on the basis of the carbon atom of arachidonic acid at which oxygen is inserted.12-LO activation can lead to the formation of oxidized lipids such as 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE]. Human and rabbit 15-LO as well as the leukocyte-type 12-LO have high homology and are classified as 12/15-LO because they can form both 12(S)-HETE and 15(S)-HETE from arachidonic acid. Recently, there has been heightened interest in 12/15-LO pathway because of key data implicating it in the pathogenesis of atherosclerosis, retinosis, hypertension and diabetes. It has been documented leukocyte-type 12-LO mainly expressed in the glomeruli studies also demonstrated it is highly expressed in the mesangial cells, podocyte treated by high glucose or glomeruli from diabetic mouse.Diabetic nephropathy (DN) is one of the major complications of diabetes, which could cause end stage renal dysfunction. Currently end-stage renal disease in the proportion of DN has reached 30%to 40%in the western developed countries, including type 2 diabetes caused by more than 80%of cases. The number of its incidence in our country is increasing at an alarming rate. DN has become an serious social health problem. Therefore, medical research focus on the pathogenesis of DN has become a hot research area.It's well known Ang II binds to its type 1 receptor (AT1R), leading to AT1R activation, and then activate a series of signal transduction pathway, causing proteinuria, glomerular sclerosis, and renal failure through the pressure-and non-pressure-dependent pathway in the DN patients. Enhanced angiotensinogen (AGT) expression and increased generation of Ang II are induced by RAS activity in the diabetic glomeruli. Mostly, the effects of Ang II is not only related to it's content, but also related to the amount of it's receptor. Large number of trials has confirmed the expression of ATIR are regulated by various factors. Many studies demonstrated that mesangial cells (MC) extracted from 12-LO knockout mice are not sensitive to Ang II stimulation. Takai observed that 12-HETE pretreatment can enhance Ang II effect in vessels. These studies indicated the possibility of ATIR upregulation by 12-LO.The main purpose of this study was to explore the role of 12-LO in AT1R expression in glomerular cells stimulated by high glucose and type 2 diabetic glomeruli, and provide new therapeutic method to ameliorate the progression of DN. Glomerular cells, rat models that received constant speed infusion of 12(S)-HETE using osmotic mini-pump, as well as high-fat diet with low-dose STZ-induced type 2 diabetes models were used in this study. Our aim is to investigate:â‘ Effect of high-glucose and 12 (S)-HETE stimulation on the expression of ATIR;â‘¡Direct influence of constant infusion 12 (S)-HETE using osmotic mini-pump on the glomerular AT1R expression;â‘¢The 12-LO expression in the type 2 diabetic glomeruli;â‘£The AT1R expression in the type 2 diabetic glomeruli, and the influence of 12-LO inhibitor-CDC treatment on ATIR expression in type 2 diabetic rat glomeruli.Our findings in this study as follows:1. High glucose induced a significant increment of AT1R expression in glomerular mesangial cells and podocytes compared with the control group (p<0.01).2. High glucose increased 12 (S)-HETE excretion in glomerular mesangial cells and podocytes, and CDC treatment significantly reduced this change (p<0.01). 3.12 (S)-HETE directly induced ATIR upregulation in cultured mesangial cells and podocytes.4. Quantitative competitive RT-PCR and Western blot confirmed that the constant infusion 12 (S)-HETE for 7 days induced ATIR protein and mRNA upregulation in rat glomeruli.5. Glomerular volume and proteinuria, and glomeruli expression of p27kipl were significantly increased in high-fat diet combined with low-dose STZ-induced type 2 diabetic rats, which are coincided with major characteristic changes of diabetic renal hypertrophy and proteinuria.6.12-LO expression in glomeruli was significantly increased in type 2 diabetic rat at 2 and 4 weeks.7.12 (S)-HETE levels in glomeruli were significantly increased in type 2 diabetic rats at 2 and 4 weeks, and these changes were significantly reduced by CDC treatment, which indicated effective dosage of CDC were used in the present study.8. ATIR expresson in type 2 diabetes glomeruli were significantly increased relative to control (p<0.01), while CDC treatment significantly reduced this increment (p<0.01).9. p38MAPK pathway was activated in type 2 diabetic glomeruli, which is parallel with changes of 12-LO. CDC can reduce p38MAPK activation in type 2 diabetic glomeruli.Our results demonstrated for the first time that:1. High glucose treatment significantly induce ATIR protein expression in MCs and podocytes at least in part by 12-LO pathway;2. Constant speed infusion of 12 (S)-HETE to the experiment rat can increase ATIR protein and mRNA expression in glomeruli. We demonstrate for the first time that 112 (S)-HETE directly increase ATIR expression in vivo.3. CDC treatment can directly reduce renal hypertrophy index and proteinuria in type 2 diabetes rat. Similarly, hypertrophy associated markers such as p27kipl protein which increased in type 2 diabetic kidney is also decreased by CDC treatment. These results demonstrated that 12-LO participate in the development of glomerular hypertrophy in DN.4.12-LO, AT1R protein expression and p38MAPK activation are increased significantly in the renal cortex of type 2 diabetic rat. CDC treatment can significantly attenuate AT1R protein expression and p38MAPK activity, suggest that increased expression of glomerular AT1R is mediated at least in part by 12-LO-p38MAPK pathway.In summary, with the use of the type 2 diabetic rat model which similar to human type 2 diabetes mellitus,12-LO metabolite 12 (S)-HETE and 12-LO inhibitor-CDC, we demonstrated that AT1R is increased in the type 2 diabetic glomeuli at least in part by the 12-LO-p38MAPK pathway. Our study indicate that increased AT1R induced by 12-LO can enhance AngII effect that related to the progression of DN. |
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