ABSTRACT
Objective:To investigate the effect of 12(S)-HETE on the p27~(kip1) expression in mesangial cells and glomeruli.Methods:Mesangial cells were exposed to 12(S)-HETE.12(S)-HETE was infused to rats by osmotic mini-pump.Total protein content measurement for cell hypertrophy,RT-PCR for mRNA expression and Western blot for protein expression were performed respectively.Results:12(S)-HETE stimulation induced mesangial cell hypertrophy and p27~(kip1) protein expression,but not p27~(kip1) mRNA expression.Furthermore,p27~(kip1) mRNA and protein expression in the glomeruli were significantly increased by 12(S)-HETE stimulation using osmotic mini-pump.Conclusion:12(S)-HETE plays an important role in the pathogenesis of glomerular cell hypertrophy and senescence through upregulation of p27~(kip1) expression.
ABSTRACT
Objective To investigate the effect of 12-lipoxygenase(12-LO) on the p27Kip1 expression in diabetic glomeruli. Methods Mesangial cells were exposed to 12-LO product 12 (S)-HETE (10-7 mmol/L) with or without p38 MAPK (p38) inhibitor (SB203580, 1 μmol/L) for 24 hours. Rats fed with high fat diet received low dose streptozotoein (ST-Z, 35 mg/kg, IP injection) to develop type 2 diabetes and were divided into 2 groups: low dose STZ, low dose STZ+12-LO inhibitor cinnamyl-3,4-dihydroxy-α-cynanocinnamate (CDC, 8 mg/kg) treatment. Rats fed with regular chow were divided into two groups: controls, CDC treatment. The rats received injection of CDC or vehicle subcutaneously in the hind leg. CDC or vehicle injection was performed three times weekly on alternate days. All the rats were sacrificed after 4 weeks, Wild type and 12-LO knockout C57BL/6 mice were divided into 4 groups: wild type control, 12-LO knockout, STZ-induced wild type type 1 diabetes and STZ-induced 12-LO knockout type 1 diabetes. All the mice were sacrificed after 16 weeks. Urine, blood, kidney cortical tissue and isolated glomeruli by sieving method were collected at the end of study respectively. Western blot and immunohistochemistry for target protein were performed respectively. Results Inhibition of p38 activation could significantly reduce p27Kip1 expression induced by 12 (S)-HETE in mesangial cells (P<0.01). Increased glomerular volume, microalbuminuria, elevated glomeluli p38 activation, p27Kip1 expresssion in type 2 diabetic glomeruli was decreased after CDC treatment (P<0.01). Compared with wild type diabetic mice, glomerular p38 activation, p27Kip1 exprcsssion and extracellular matrix accumulation in the 12-LO knockout diabetic mice were significantly decreased (P <0.01, respectively). Conclusions 12-LO induces p27kipl expression via p38 pathway in diabetic glomeruli.