| Objective To investigate the effects of myo-inositol oxygenase on the expression transforming growth factor-β1 in diabetic nephropathy.Methods(1)Thirty-one Sprague-Dawley rats aged 8 wks were assigned randomly to receive streptozotocin at a dose of 60 mg/kg intraperitoneally(diabetes,n=18)or citrate buffer alone (controls,n=13).One week after injection,only streptozotocin-treated rats with plasma glucose concentrations above 16.7mmol/L were considered diabetic and were subsequently raised for either four weeks (DM group)or twelve weeks(DN group),after which individual rats were placed in metabolic cages for 24h urine collection.Then the rats in three groups were sacrificed for their kidneys and blood samples.The cortex tissues of kidney were stained with hematoxylin and eosin or Masson using 2μm thick sections.Total RNA and protein were also extracted from the cortex tissues of rat kidneys.The expression of MIOX and TGF-β1 mRNA were determined by reverse transcription PCR.The protein levels of MIOX and TGF-β1 were detected by Western blot.(2) The rat renal tubular epithelial cell line NRK-52E was maintained in DMEM containing 5.6mmol/L D-glucose and 10%FBS.When the cells reached near 80%confluency,the concentration of FBS was reduced to 0.5%for 12h to arrest and synchronize cell growth,after which the cells were treated with D-glucose at different concentrations(ranging from 5.6 to 30mmol/L)for 48h.D-mannitol served as control.Gene expression was analyzed at mRNA level by semi-quantitative RT-PCR or at protein level by Western blot.(3)Observe the change of MIOX mRNA and protein in concentration line after the stimulation of TGF-β1 on rat renal tubular epithelial cell.(4)Transient transfection of the normal NRK-52E cells with anti-sense oligodeoxynucleotide(ASODN)was performed to knockdown MIOX using LipofectamineTM2000.NRK-52E cells were cultured and incubated with 4μmol/L SODN or 4μmol/L ASODN in the presence of 10μg/ml LipofectamineTM2000 in DMEM medium for 8h. Cells were then exposed to 30mmol/L D-glucose for 48h,and cell lysate was prepared for RT-PCR and Western blot,respectively.Results(1) Compared with control rats,diabetic rats in DM and DN groups had significiantly elevated serum creatinine,elevated 24h urinary albumin excretion(P＜0.05).Tubulointerstitial fibrosis area was significiantly increased in DN rats as compared with DM rats and controls.The expression of MIOX mRNA and protein were upregulated in DM group and even more significiantly in DN group(P＜0.01).The expression of TGF-β1 mRNA and protein were also increased in DN rats(P＜0.01).(2) In vitro,the expression of MIOX mRNA and protein in NRK-52E cells was induced by high D-glucose in a dose-dependent manner.Paralleled increases were observed with respect to TGF-β1.(3)There was no significant difference of MIOX mRNA and protein between each group after the stimulation of TGF-β1 on rat renal tubular epithelial cell(P＞0.05);(4)Transient transfection of the normal NRK-52E cells with anti-sense oligodeoxynucleotide(ASODN)significantly attenuated the increase of TGF-β1 expression in normal NRK-52E cells induced by high D-glucose(P＜0.05).Conclusion The gene expression of MIOX in diabetic nephropathy was upregulated.It was positively correlated with the expression of TGF-β1.These results suggest that a potential link between the expression of MIOX and TGF-β1 in diabetic nephropathy. |
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