重组人血管非炎症分子1促进梗阻性肾病肾小管细胞分泌TGF-β加重肾纤维化的实验研究

VNN1 promoted the secretion of transforming growth factor beta of renal tubular cell and aggravated renal fibrosis in obstructive renal injury

目的本研究拟明确重组人血管非炎症分子1(recombinant human vascular non-inflammatory molecule 1,VNN1)在梗阻性肾病后肾间质纤维化过程中的作用及相关机制。方法选取Balb/c野生型、VNN1基因敲除雄鼠,分别设置假手术组、手术组。术后当天、14 d留取梗阻侧肾盂尿液、肾组织标本,检测尿液转化生长因子β(transforming growth factor-β,TGF-β)水平,免疫组化明确肾组织VNN1表达水平。肾组织完善过碘酸雪夫染色(Periodic Acid-Schiff stain,PAS)明确肾脏损伤严重程度,马松染色(Masson)、免疫组化检测肾组织α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达水平,明确肾组织纤维化严重程度。提取野生型和VNN1基因敲除小鼠原代肾小管上皮细胞,传代至第2代,予以血管紧张素刺激,模拟体内梗阻性肾病模型,收取细胞和上清,检测VNN1蛋白水平和上清TGF-β表达水平。结果梗阻性肾病后第14天,手术组PAS染色提示肾组织损伤明显,免疫组化提示肾组织VNN1表达水平明显升高。进一步完善肾组织Masson染色,免疫组化检测α-SMA水平,发现野生型小鼠间质纤维化水平较VNN1基因敲除鼠明显加重,α-SMA水平明显升高。进一步检测梗阻侧尿液TGF-β水平发现,VNN1基因敲除鼠梗阻侧尿液TGF-β水平明显降低。我们进一步通过细胞实验证实,予以血管紧张素刺激后,野生型小鼠肾小管细胞VNN1蛋白表达水平明显升高,野生型小鼠肾小管细胞上清中TGF-β水平明显高于VNN1基因敲除鼠。结论VNN1可促进梗阻性肾病肾小管上皮细胞TGF-β分泌,加重肾纤维化。

Objective To elucidate the role and related mechanisms of VNN1 in the process of renal interstitial fibrosis after obstructive renal injury.Methods Balb/c wild-type and VNN1 gene knockout male mice were selected for sham operation and operation groups respectively.Urine and kidney tissue specimens from obstructed renal pelvis were collected at Day 0 and 14 post-operation and the urinary level of transforming growth factor beta(TGF-β)was detected.The expression level of VNN1 in renal tissue was measured by immunohistochemistry.Renal tissue is improved by periodic acid-Schiff(PAS)stain to determine the severity of renalinjury.Masson stain and immunohistochemistry were utilized for detecting the expression ofα-smooth muscle actin(α-SMA)in renal tissue to determine the severity of renal tissue fibrosis.Primary renal tubular epithelial cells were harvested from wild-type and VNN1 knockout mice,propagated to the second generation and stimulated with angiotensin forsimulate anobstructive kidney injury model in vivo.Both cells and supernatant were collected for detecting the level of VNN1 protein and supernatant TGF-βlevel.Results At Day 14 after obstructive renal injury,PAS stain in operation group hinted at overt renal tissue damageand immunohistochemistry indicated a significant up-regulation of VNN1 in renal tissue.Masson stain of renaltissue was further improved and the level ofα-SMA was detected by immunohistochemistry.The level of interstitial fibrosis worsened morein wild-type mice than that of VNN1 knockout counterparts while the level ofα-SMA spiked greatly.Further testing the urinary level of TGF-βon the obstructive side indicated that the urinary level of TGF-βon the obstructive side of VNN1 gene knockout mice declined obviously.It was further confirmed through cell experiments that after a stimulation of angiotensin,there was a marked up-regulation of VNN1 protein in wild-type murine renal tubular cells and the supernatant level of TGF-βwas significantly higher in wild-type murine renal tubular cells than that of VNN1 gene knockout counterparts.Conclusion VNN1 can promote the secretion of TGF-βand aggravate fibrosis in renal tubular epithelial cells after obstructive injury.