The Gene Expression Of Myo-inositol Oxygenase And Its Potential Role In The Progression Of Tubulointerstitial Fibrosis In Diabetic Nephropathy

Objective To investigate the expression of myo-inositoloxygenase (MIOX) and its potential role in the progression oftubulointerstitial fibrosis in diabetic nephropathy. Methods (1) Thirty-one Sprague-Dawley rats aged eight weeks were assigned randomly toreceive 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 above16.7 mmol/L were considered diabetic and were subsequently raised foreither four weeks (DM group) or twelve weeks (DN group), after whichindividual rats were placed in metabolic cages for 24 h urine collection.Then the rats in four groups were sacrificed for kidney and blood samples.The cortex tissues of kidney were stained with hematoxylin and eosin orMasson using 2-μm-thick sections. Total RNA and protein were alsoextracted from the cortex tissues of rat kidneys. The expression of MIOX,α-SMA, E-cadherin and fibronectin mRNA were determined by reversetranscription PCR or real-time RT-PCR. The protein levels ofα-SMA andE-cadherin were detected by Western blot. (2) The rat renal tubularepithelial 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 12 h toarrest and synchronize cell growth, after which the cells were treated withD-glucose at different concentrations (ranging from 5.6 to 45 mmol/L) for48 h or exposed to 30mmol/L D-glucose for 4, 8, 16, 24 and 48 h, respectively. D-mannitol served as control. Gene expression was analyzedat mRNA level by semi-quantitative RT-PCR or at protein level by Westernblot. (3) Recombinant rat MIOX expression plasmid pcDNA3.1-MIOXthat contains full-length rat MIOX cDNA was cloned. Stable transfectionof normal NRK-52E cells with recombinant plasmid pcDNA3.1-MIOX orempty vector pcDNA3.1 was performed using Lipofectamine^TM 2000. 24 hafter transfection, the cells were re-fed with fresh selective mediumcontaining G418 (geneticin) at a final concentration of 400μg/ml.Neomycin-resistant clones survived in selective medium for 14 d. The cellclones were then individually transferred into six-well plates for expansion.Total RNA and protein were extracted from the cells for evaluation ofMIOX,α-SMA, E-cadherin, fibronectin mRNA expression andα-SMA,E-cadherin protein expression. Transient transfection of the normalNRK-52E cells with anti-sense oligodeoxynucleotide (ASODN) or senseoligodeoxynucleotide (SODN) (served as control) was performed to knockdown MIOX using Lipofectamine^TM 2000. NRK-52E cells were culturedand incubated with 4 mmol/L SODN or 4 mmol/L ASODN in the presenceof 10μg/ml Lipofectamine^TM 2000 in DMEM for 8 h. Cells were thenexposed to 30 mmol/L D-glucose for 48 h, and cell lysate was prepared forRT-PCR and Western blot, respectively. Results (1) Compared withcontrol rats, diabetic rats in DM and DN groups had significiantly elevatedserum creatinine, lower body weight, larger kidneys and elevated 24 hurinary albumin excretion (P＜0.05). Tubulointerstitial fibrosis area wassignificiantly increased in DN rats as compared with DM rats and controls.The expression of MIOX mRNA was upregulated in DM group and evenmore significiantly in DN group, which was confirmed by Real-time PCR(2.40±0.26 versus 1.00±0.07, 3.63±0.38 versus 1.00±0.09, P＜0.01). The expression ofα-SMA and fibronectin were also increased, while theexpression of E-cadherin decreased in DN rats (P＜0.01). (2) In vitro, theexpression of MIOX in NRK-52E cells was induced by high D-glucose ina dose and time-dependent manner, with the highest level at approximately48 h. Paralleled increases were observed with respect toα-SMA andfibronectin, while the level of E-cadherin decreased in NRK-52E cells. (3)NRK-52E cells stably transfected with MIOX gene exhibited higher levelsofα-SMA and fibronectin, and lower levels of E-cadherin relative to emptyvector transfected cells and normal cells (P＜0.05). In contrast, ASODNtransfection significantly attenuated the increase ofα-SMA, fibronectinexpression and the decrease of E-cadherin expression in normal NRK-52Ecells induced by high D-glucose (P＜0.05). Conclusions The expressionof MIOX in diabetic nephropathy was upregulated. It was positivelycorrelated with the expression ofα-SMA and fibronectin, and negativelywith that of E-cadherin, which was implicated in tubulointerstitial fibrosis.These results suggest that MIOX may play a role in the progression oftubulointerstitial fibrosis in diabetic nephropathy.