| Background:Type 1 diabetes is resulted from autoimmune attack of the insulin-producing pancreaticβ cell, while type 2 diabetes occurs when pancreatic β cells fail in face of insulin resistance(IR). It is well known that diabetic nephropathy(DN) is the leading cause of the end-stage renal disease in developed countries. Albuminuria is a hallmark of early DN. Recently,numerous studies have focused on type 2 DN(T2DN), which is characterized clinically by the development of IR and microalbuminuria(MAU) in the early period. IR, an independent risk factor of MAU, is emerged as the direct cause of hyperglycemia in type 2 diabetes. IR may exist with MAU before the onset of diabetes and lead to pathophysiological changes in the kidney. Unlike the insulin-dependent therapy for type 1 diabetes, treatment of type 2diabetes is focused on interference of IR to correct hyperglycemia and hyperinsulinemia.Lipoxygenases(LO) are a group of non-heme iron enzymes that insert molecular oxygen into 1, 4-cis, cis-pentadiene containing polyunsaturated fatty acids. They are classified as 5-, 8-, 12-, and 15-LO according to the carbon atom of arachidonic acid at which the oxygen is inserted. 12-LO activation contributed to the formation of oxidized lipids such as 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE]. A number of studies on renal biological activities demonstrated for suggested roles of 12-LO along with12(S)-HETE in renal disorders. Interestingly, inhibition of 12-LO could ameliorate MAU in type 2 diabetic animal models evidently, but the inhibition could not reduce MAU under type1 diabetic condition. Moreover, IR also had a strong, independent relationship with MAU in patients with type 2 diabetes and 12-LO contributed to IR induced by high fat diet. However,the underlying mechanism that whether 12-LO inhibition could reduce MAU by ameliorating IR is still unclear.Previous observation showed that dysregulation of renin-angiotensin(Ang) system(RAS) in the metabolic syndrome favors type 2 diabetes. AngⅡ type 1 receptor(AT1R), as one of the key mediator of Ang Ⅱ physiological action, is also considered to be related to pathogenesis of IR. Furthermore, previous studies suggest a functional interplay between AT1R and 12-LO in DN conditions. It is well known that RAS inhibitor Ang-converting enzyme inhibitor(ACEI) or AngⅡ receptor blocker(ARB) can increase insulin sensitivity and diminish MAU in type 2 diabetes.Therefore, we hypothesized that 12-LO inhibition could potentially ameliorate MAU via down-regulation of glomerular AT1 R through preventing IR in type 2 DN.Methods:1. Rat mesangial cells(RMCs) were treated with insulin(10-6 mol/L) for 24 h, and RMCs were harvested for AT1 R detection.2. RMCs were treated with 12(S)-HETE(10-7 mol/L) or p38 mitogen-activated protein kinase(MAPK) inhibitor SB202190(10-6 mol/L) for 24 h, and RMCs were harvested for AT1 R associated protein(ATRAP) detection.3. Rats were randomly assigned to receive either vehicle(ethanolamine) or 12(S)-HETE(infusion rate 1 mg/kg/d) through osmotic mini-pumps. 7 days later, glomeruli were isolated for AngⅡ, AT1 R and ATRAP detection.4. Wild type(WT) and 12-LO knockout(LOKO) C57BL/6 mice were randomly assigned to receive either vehicle(ethanolamine) or Ang Ⅱ(infusion rate 1.1 mg/kg/d)through osmotic mini-pumps. 14 days later, mice were sacrificed and glomeruli were isolated for AT1 R detection.5. Type 1 diabetes was induced by single injection of streptozocin(STZ)intraperitoneally(200 mg/kg). WT and LOKO C57BL/6 mice were divided into 4 groups:WT, LOKO, WT+STZ and LOKO+STZ. Fasting blood glucose levels of mice were monitored. 6 weeks later, mice were sacrificed. 24 h urine was collected.6. Type 1 diabetes was induced by high doze STZ injection(65 mg/kg) intraperitoneally.Once type 1 diabetes onset, rats were randomly divided into 2 groups: type 1 diabetes(T1DN), type 1 diabetes + cinnamyl-3,4-dihydroxy-α-cynanocinnamate(CDC, 12-LO inhibitor) treatment(8 mg/kg/d, 3 times/week, by subcutaneous injection in the hind leg).Rats fed regular chow were used as control. Fasting blood glucose levels of rats were monitored. 6 weeks later, rats were sacrificed. Blood and 24 h urine were collected.Glomeruli were isolated for AT1 R detection.7. Type 2 diabetes was induced by high fat diet combined with low doze STZ(35 mg/kg)injection. After the onset of diabetes, rats were randomly divided into 2 groups: type 2diabetes(T2DN), type 2 diabetes + CDC treatment(8 mg/kg/d, 3 times/week, by subcutaneous injection in the hind leg). Rats fed regular chow were used as control. Fasting blood glucose levels of rats and fasting insulin levels were monitored. 6 weeks later, rats were sacrificed. Blood, 24 h urine and glomeruli were collected. Renal slices were stained with periodic acid schiff(PAS). AT1 R and Ang Ⅱ in glomeruli, adenosine5’-monophosphate-activated protein kinase(AMPK) and p-AMPK in skeletal muscle were detected.Glomeruli were isolated with a sieving method, the levels of Ang Ⅱ and urinary albumin were tested by enzyme-linked immunosorbent assay(ELISA). Fasting insulin levels were determined by radioimmunoassay. Protein and m RNA expression were detected by reverse transcription-polymerase chain reaction(RT-PCR) and western blot analysis,respectively. Renal slices were stained with PAS.Results:1. PAS staining showed remarkable enlargement of the glomeruli with mesangial expansion in type 2 diabetic rats compared to control. However, these changes were attenuated in rats receiving CDC. The glomerular volume of diabetic rats was significantly higher than control(P<0.01) and this change was also ameliorated by CDC treatment(P<0.05).2. The fasting blood glucose levels in LOKO+STZ significantly decreased compared to WT+STZ(P<0.01) at 2nd and 3rd week after onset of diabetes. A decrease in blood glucose levels was also observed at 2nd week and 3rd week after STZ injection in CDC-treated rats compared to that in type 1 and type 2 diabetic rat models, but did not reach statistical significance.3. The level of MAU significantly increased in WT+STZ mice and LOKO+STZ mice compared with corresponding controls(P<0.01). 12-LO knockout could not prevent STZ-induced MAU elevation. The level of MAU significantly increased in type 1 and type 2diabetic rat compared with corresponding controls(P<0.01), CDC treatment cannot reduce type 1 diabetic-induced MAU but can ameliorate MAU in type 2 diabetic rat(P<0.05).4. Reduction of insulin sensitivity index as well as increases of fasting insulin levels were observed in type 2 diabetic rats compared with control at 2nd, 4th and 6th week after onset of diabetes(P<0.01). However, these abnormalities were significantly ameliorated by12-LO inhibition at 6th week(P<0.05). Reduction of p-AMPK in skeletal muscle of type 2diabetic rats was observed compared to control(P<0.01), CDC abolished p-AMPK decreases in type 2 diabetic rats(P<0.05).5. Subcutaneous injection of 12(S)-HETE with osmotic minipumps increased Ang Ⅱlevels in rat glomeruli compared to control(P<0.01). Increase of glomerular Ang Ⅱ was observed in type 2 diabetic rats compared to control(P<0.01), CDC evidently abolished the abnormality(P<0.05).6. AT1 R m RNA expressions decreased in glomeruli of LOKO mice compared to WT.Subcutaneous injection of Ang Ⅱ with osmotic minipumps decreased AT1 R m RNA expression in glomeruli of both WT and LOKO.7. Subcutaneous injection of 12(S)-HETE with osmotic minipumps increased AT1 R expressions in rat glomeruli compared to control(P<0.01), but decreased glomerular ATRAP expressions compared to control. 12(S)-HETE stimulation alone decreased the ATRAP protein expression in RMCs significantly compared to control(P<0.01). In the presence of SB202190, 12(S)-HETE induced ATRAP reduction was clearly attenuated(P<0.05).8. Insulin significantly increased AT1 R protein expression in RMCs compared with control(P<0.01).9. A decrease in AT1 R expression was observed in glomeruli of type 1 diabetic rat compared with control, but the decrease did not reach statistical significance, 12-LO inhibition did not alter AT1 R protein expression. Increase of glomerular AT1 R as well as reduction of ATRAP were observed in type 2 diabetic rats compared to control(P<0.01),CDC evidently abolished these abnormalities(P<0.05).Conclusions:1. 12-LO inhibition did not alter fasting blood glucose in type 1 and type 2 diabetic rats.While 12-LO inhibition attenuated MAU increases in type 2 diabetic rats, but not in type 1diabetic rats. Considering IR is the major distinction between type 1 and type 2 diabetes, we concluded that 12-LO inhibition prevent MAU by ameliorating IR.2. 12-LO inhibition did not influence AT1 R expression in glomeruli of type 1 diabetic rats, but significantly abolished AT1 R increases in type 2 diabetic rat glomeruli. Further, IR could upregulate the AT1 R expression in glomerular cells, suggesting that 12-LO inhibition modulates AT1 R expression by ameliorating IR.3. 12-LO exerted a pivotal role in ATRAP expression through p38 MAPK pathway,resulting in internalization of AT1 R.4. Attenuating IR by inhibiting 12-LO activation to downregulate glomerular AT1R levels prevented the development of MAU in type 2 diabetes. |
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