| 【Objective】To observe the expression of DMP-1,E11and Sclerostin involved invasucular calcification in remnant kidney rats with hyperphosphatemia,Try to discuss the mechanism of vascular calcification of chronic renalfailure.Another objective is to observe the early effect of sodiumthiosulfate (STS) on the progression of vascular calcification.【Methods】Sprague Dawley rats (n=35) underwent5/6nephrectomy (n=21) or shamoperation (n=14) were fed with diet containing high phosphorus (HP)[dietformular: phosphate(P)1.2%, calcium (Ca)1.6%] or normal phosphorus (NP)[diet formular: P (0.9%), Ca (1.2%)] for16weeks after surgery. They weredivided into5groups as follows:(1) sham operation rats receiving NPdiet (SNP, n=7),(2) sham operation rats receiving HP diet (SHP, n=7),(3) remnant kidney rats receiving NP diet (NNP, n=7),(4) remnant kidneyrats receiving HP diet (NHP, n=7),(5) remnant kidney rats receiving HPdiet with STS (THP, n=7). Rats in the THP group were intraperitoneallyinjected with STS0.4g/mL·kg three times a week for16weeks after surgery.At the end of the16th week, tail artery blood pressure of rats was tested,serum creatinine(SCr), blood urea nitrogen(BUN), urine protein, serumcalcium(Ca2+), serum phosphorus(P3+) and intact parathyroid hormone(iPTH)were examined. Throacic aorta of each rat was then removed. Vascularcalcification was confirmed by Von kossa staining and Alizarin redstaining.E11expression in aorta was determined by immunohistochemistryand Western blot. DMP1, E11and Sclerostin mRNA in aorta were determined by RT-PCR.DMP1mRNA in aorta was determined by Real-time PCR.2. The explants derived from thoracic aorta were used for primary cultureof vascular smooth muscle cells and identification of cells was carriedon by morphological identification and direct immunohistochemicalstaining of α-SMA, Passage3to8of VSMC were used for experiments. VSMCwere divided in three groups, normal control group(Pi0.9mmol/L)，normal phosphorus group(Pi1.3mmol/L), high phosphorus group (Pi2.6mmol/L), calcium deposition was visualized by Alizarin stain method atday6. DMP-1,E11and Sclerostin mRNA levels were determined by RT-PCR andReal-Time PCR. E11expression was semi-quantified by Western Blot at day6. All of the experiments were repeated for3times at least.【Results】1. After16weeks high phosphorus diet, SCr, BUN, P3+, Ca2+, uric protein,iPTH and blood pressure were significantly higher in NHP rats thanthose in SNP rats(P<0.05). With the treatment of STS, THP rats showeda marked decrease in SCr, BUN, UA(P<0.05) by comparison to NHP groupand lower P3+, iPTH and blood pressure levels(P<0.05) than NHP rats.After the16th week, von kossa staining showed that significantvascular calcification was found in NHP group while NNP group and SHPgroup occasionally had vascular calcification and SNP group had nosignificant vascular calcification.The expression of DMP1, E11and Sclerostin mRNA in arota increasedsignificantly in NHP rats compared with SNP group by RT-PCR (P<0.05).After16-week-treatment, the expression of DMP1, E11and SclerostinmRNA decreased significantly in THP group compared with those in NHPgroup(P<0.05). The expression of DMP1mRNA in arota increasedsignificantly in NHP rats compared with SNP group by Real-Time PCR(P<0.05). After16-week-treatment, the expression of DMP1mRNAdecreased significantly in THP group compared with those in NHP group(P<0.05)The expression of E11inarota increased significantly in NHP ratscompared with SNP group by immunohistochemistry(P<0.05).After16-week-treatment, the expression of E11decreased significantly inTHP group compared with those in NHP group(P<0.05). The expression ofE11in arota increased significantly in NHP rats compared with SNP groupby Western blot (P<0.05). After16-week-treatment, the expression ofE11decreased significantly in THP group compared with those in NHPgroup(P<0.05).2. Compared with normal control group and the normal phosphorus group,the calcium deposition was significantly increased in high phosphorusgroup (P<0.05);DMP-1,E11and Sclerostin mRNA expression wassignificantly increased in high phosphorus group (P<0.05); Theexpression of E11in high phosphorus group was significantlyincreased compared with normal control group the normal phosphorusgroup by Western Blot(P<0.05).【Conclusion】1. The model of vascular calcification was successfully established on16-weeks high phosphorus diet after5/6nephrectomy in rats，andphosphorus induces VSMC calcification in vitro; The increase ofexpression of DMP1, E11and Sclerostin in aorta and VSMC may showvascular calcification involves a transition to an osteocyte-likephenotype.2. Sodium thiosulfate may delay the progression of vascular calcificationby down-regulation of DMP1, E11and Sclerostin. |
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