| Background:As one of the most common microvascular complications of diabetes mellitus, diabetic nephropathy(DN) is the most common cause of end-stage renal disease(ESRD) worldwide. Early pathological changes of diabetic nephropathy is manifested mainly glomerular hypertrophy, and increased extracellular matrix(ECM). Studies indicate that glomerular hypertrophy and expansion of the mesangial ECM in DN are mediated by multiple mechanisms including the activation of angiotensinⅡ(AngⅡ). 12- lipoxygenase(12-LO) is a polyunsaturated fatty acid oxygenase. Studies have indicated that 12-LO increased in glomerular cell exposed to high glucose and in diabetic glomeruli, and had similar effect with AngⅡ. Therefore, 12-LO and its products 12(S)-HETE were regarded the critical factor in the development of DN by enhancing oxidative stress and inflammation. Studies have indicated that AngⅡ could upregulate 12-LO activity, and ARB could downregulate 12-LO activity in the glomeruli of DM rats and attenuated kidney injury.12-LO promots DN by ECM increasing induced by p38 MAPK activation. However, it remains unclear how AngⅡ-12-LO pathway interacts and affects glomerular hyperphrophy in DN.The glomerular hypertrophy is related to cyclin-dependent kinase inhibitors(CKIs), which associated with cell cycle regulation,espicially the CIP/KIP family. It was reported that p21WAF1/CIP1(p21) and p27KIP1(p27) expression increased accompanied by hypertrophy of renal cell and glomerular in DN. To explore whether or not 12-LO can cause glomerular hypertrophy in DN, it is crucial to investigate effect of 12-LO and its products 12(S)-HETE on CKIs in CIP/KIP family including p21, p27 and p57KIP2(p57), which result in cell cycle arrest and cell hypertrophy. In this study, we evaluated whether there is an interplay between 12-LO and Ang II on CIP/KIP family CKIs associated with glomerular hypertrophy in DN.Methods:(1) Rats were treated with either vehicle(ethanolamine) or AngⅡ( 400 ng?Kg-1?min-1) through osmotic mini-pumps for 14 days. Glomerular p21, p27, p57 and 12(S)-HETE were detected by ELISA or Western blot.(2) Rats were treated with either vehicle(ethanolamine) or 12(S)-HETE(1 mg?Kg-1?d-1) through osmotic mini-pumps for 7 days. Glomerular p21, p27, p57 and AngⅡ were detected by ELISA or Western blot. Glomeruli were isolated with a sieving method and classified into large glomeruli and small glomeruli for AT1 detection.(3) Mesangial cells were treated with high glucose(30mmol/L) for 24 h and 48 h, and cells were harvested for p21, p27 and p57 detection. Protein expressions of p21, p27 and p57 in db/db mice and db/m mice were detected.(4) Rats fed regular chow were used as control. High fat diet combined with low doze streptozocin(STZ) injection were used to induce type 2 diabetes. Diabetic rats were randomly divided into 2 groups: diabetic nephropathy(DN), ARB Losartan treatment(5 mg? Kg-1?d-1, by gavage) for 6 weeks. 24 h urine albumin and kidney weight to body weight were detected. Renal slices were stained with periodic acid schiff(PAS). Glomerular p21, p27, p57 and 12(S)-HETE were detected.(5) Rats fed regular chow were used as control. High fat diet combined with low doze STZ injection were used to induce type 2 diabetes. Diabetic rats were randomly divided into 2 groups: diabetic nephropathy(DN), 12-LO inhibitor CDC treatment(8 mg?Kg-1?d-1, 3 times/week, subcutaneously in the hind leg) for 8 weeks. 24 h urine albumin and kidney weight to body weight were detected. Renal slices were stained with PAS. Glomerularp 21, p27, p57 and AngⅡwere detected. Glomeruli were isolated with a sieving method and AT1 protein expression of large glomeruli(LG) and small glomeruli(SG) in control group, DN group and CDC group were detected. AT1 m RNA in DN group were assayed using competitive quantitative reverse transcription polymerase chain reaction(RT-PCR). Glomerular AngⅡ,12(S)-HETE, p21, p27, p57 in DN LG and SG were detected.(6) AngⅡ(1.1mg·Kg-1·d-1) were infused in wild type(WT) and 12-LO gene knock-out mice(LOKO) through osmotic mini-pumps for 7 days.Glomerular p21 and p27 were detected by Western blot.(7) Glomeruli isolated with a sieving method were treated with 12(S)–HETE(10-6mol/L) for 6 h. Glomerular AT1 were detected by Western blot.(8) Mesangial cells were treated with 12(S)–HETE(10-6mol/L) and/or transcriptional inhibitor(Actinomycin D) for 6 h, and cells were harvested for AT1 m RNA detection.Results:(1) Subcutaneous injection of 12(S)-HETE increased AngⅡ levels(P<0.01), p21 and p27 protein expression in glomeruli significantly(P<0.01), but had no effect on protein expression of p57. Compared with control, 12(S) –HETE increased AT1 protein expression significantly in glomeruli.Compared with small glomeruli, 12(S) –HETE increased AT1 protein expression significantly in large glomeruli.(2) Subcutaneous injection of AngⅡ increased 12(S)-HETE levels(P<0.01) and p27 protein expression in glomeruli significantly(P<0.01), but had no effect on protein expression of p21 and p57.(3) High glucose increased p21, p27 protein expression in mesangial cells significantly compared with the relative control, but had no effect on p57. Protein expression of p21, p27 in glomeruli of db/db mice were significantly increased compared with db/m mice(P<0.01). There was no difference in p57 protein expression between db/db mice and db/m mice.(4) Blood glucose, kidney weight/body weight ratio, 24 h urinary albumin levels and glomerular 12(S)-HETE levels were increased significantly in DN group compared with the relative control. Glomerular p21, p27 protein expression also increased in DN group compared with the relative control(P<0.01). DM had no effect on p57. PAS stainings showed glomerular hypertrophy and ECM accumulation in DN group. Losartan had no effect on blood glucose levels nor protein expression of p21 and p57, but decreased kidney weight/body weight ratio, 24 h urinary albumin and 12(S)-HETE levels compared with DN group significantly. Losartan treatment also ameliorated p27 protein expression and kidney injury induced by type 2 diabetes.(5) Blood glucose, kidney weight/body weight ratio, 24 h urinary albumin levels and AngⅡ levels were increased significantly in DN group compared with the relative control. Glomerular p21, p27 protein expression also increased in DN group compared with the relative control(P<0.01). DM had no effect on p57. PAS stainings showed glomerular hypertrophy and ECM accumulation in DN group. CDC had no effect on blood glucose levels and glomerular p57 protein expressions, but decreased kidney weight/body weight ratio, 24 h urinary albumin and glomerular AngⅡlevels in type 2 diabetic rats significantly. CDC treatment also ameliorated p21 and p27 protein expression and kidney injury induced by type 2 diabetes. Compared with control. AT1 protein expression was increased in type 2 diabetic glomeruli, especially in large glomeruli. CDC treatment ameliorated AT1 over-expression induced by type 2 diabetes, especially in DN large glomeruli. AT1 m RNA expression increased in DN large glomeruli. Compared with DN small glomeruli, 12(S)–HETE, AngⅡ, p21 and p27 increased significantly in DN large glomeruli.(6) Protein expression of p21, p27 were decreased significantly in LOKO group compared with WT. Subcutaneous injection of AngⅡ increased p27 protein expression in WT+AngⅡ group, but were attenuated in LOKO mice. There were no difference in protein expression of p21 between WT+ AngⅡgroup and LOKO+ AngⅡ group.(7) 12(S)–HETE increased AT1 m RNA expression in mesangial cells without transcriptional inhibitor(Actinomycin D).12(S)–HETE had no effect on AT1 m RNA expression with Actinomycin D in the culture media.Conclusions:(1) The glomerular hypertrophy in DN is related to cyclin-dependent kinase inhibitor(CKIs), which associated with cell cycle regulation,espicially the CIP/KIP family. Increased expressions of p21 and p27 are accompanied with hypertrophy of renal cell and glomeruli in DN.(2) There are crosstalk/ interaction between 12(S)-HETE and Ang Ⅱ in diabetic glomeruli,and 12(S)-HETE enhance effcet of AngⅡby increasing AT1 expression.(3) The glomerular hypertrophy in DN is closed related to the levels of 12(S)-HETE and AngⅡ. AngⅡ can induce p27 expression, but has no effect on p21. 12(S)-HETE can induce p21 and p27 expression. 12-LO-12(S)-HETE pathway, Ang II –AT1 pathway, and their interaction closely related to the glomerular hypertrophy and p21, p27 expression in diabetic glomeruli.(4) The inhibitions of 12-LO and RAS pathways can prevent glomerular from hypertrophy, ameliorate the progression of DN. |
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