Suppression of the development of diabetic nephropathy by expression of non-smooth muscle calponin

碱性钙钙蛋白（calponin-h1），平滑肌分化特异性基因，可能改善肾小球对损伤的反应。相反，没有证据表明calponion-h2异构体对肾脏疾病的影响。中性钙钙蛋白（calponin -h2, CN2）是一种非平滑肌亚型，可能在细胞骨架组织中发挥生理作用。CN2还能有效抑制平滑肌细胞的迁移。我们假设CN2参与了糖尿病间质损伤的过程。我们利用CRE-loxP位点特异性重组系统建立了CN2转基因（CN2- tg）小鼠，其中CN2仅在硫酸镉（0.5mg/kg BW/day）处理时才被诱导表达。用链脲佐菌素（STZ， 100mg/kg BW × 2次/3天）诱导CN2-Tg小鼠和野生型小鼠（WT）发生糖尿病。在第一次STZ注射前5天开始腹腔注射硫酸镉。分别于疾病诱发后第8天、30天及第0天行肾切除术（n=6例，各≥n例）。对所有小鼠进行增殖细胞（ki-67）、巨噬细胞（F4/80）和间质细胞（a-平滑肌肌动蛋白，aSMA）表型变化的免疫染色，评估间质炎症，并采用计算机分析系统对所有数据进行评估。在第8天，CN2-Tg组间质细胞增殖（ki-67^+细胞/高倍视野，hpf）明显受到抑制（CN2-Tg组3.7±0.5 (m±SE)， WT组5.7±0.4,p<0.05），但在第30天无明显抑制（1.1±0.4 vs 1.2±0.2）。CN2-Tg在第8天（3.7±0.1 vs 6.9±1.3,p<0.02）和第30天（2.2±0.7 vs 5.4±0.5,p<0.01）诱导间质巨噬细胞募集减少（F4/80^+细胞/hpf）。两组患者aSMA表达及血压（收缩压和舒张压）无显著差异。因此，CN2过表达抑制了糖尿病早期间质细胞增殖和巨噬细胞浸润。总之，CN2先前的抗炎特性可能是糖尿病间质性肾损伤随后发展的关键。少

Basic calponin (calponin-h1), smooth muscle differentiation-specific gene, may improve the glomerular response to injury. To the contrary, there was no evidence about calponion-h2 isoform, on the effect of kidney disease. Neutral calponin (Calponin-h2, CN2) is a non-smooth muscle isoform, and a may play a physiological role in cytoskeletal organization. CN2 also powerfully suppresses smooth muscle cell migration. We hypothesized that CN2 is involved in the course of diabetic interstitial injury. We established CN2 transgenic (CN2-Tg) mouse with the CRE-loxP site-specific recombination system, in that CN2 expression is induced only during the treatment of cadmium sulfate (0.5mg/kg BW/day). Diabetes was induced by streptozocin (STZ, 100mg/kg BW x 2 times/3days) on both of CN2-Tg mouse and wild type ones (WT). Cadmium sulfate intraperitoneal treatment was started 5 days before the first STZ injection. Nephrectomy was done on 8 and 30 days after disease induction as well as on day 0 (n=6 i … More n each). Immunostaining for proliferating cells (ki-67), macrophages (F4/80) and phenotypic change of interstitial cells (a-smooth muscle actin, aSMA) for evaluation of interstitial inflammation on all mice and all data were evaluated by computer-analysis system.The interstitial cell proliferation (ki-67^+ cells/high power field, hpf) was significantly suppressed in CN2-Tg at day 8 (3.7±0.5 (m±SE) in CN2-Tg, vs 5.7±0.4 in WT, p<0.05), but not at day 30 (1.1±0.4 vs 1.2±0.2). Reduction of macrophages recruitment in the interstitium (F4/80^+ cells/hpf) was induced in CN2-Tg at day 8 (3.7±0.1 vs 6.9±1.3, p<0.02), and day 30 (2.2±0.7 vs 5.4±0.5, p<0.01). There was no significant difference in both of aSMA expression and blood pressure (both systolic and diastolic) between two groups. Thus, CN2 overexpression suppressed both of interstitial cell proliferation and macrophages infiltration from early phase in diabetes mellitus.In conclusion, precedent anti-inflammatory property of CN2 may be critical for subsequebt development of interstitial renal injuriy in diabetes mellitus. Less