慢性代谢性酸中毒显著诱导大鼠系膜细胞的增殖

目的：研究慢性代谢性酸中毒对大鼠肾小球体积、系膜细胞增殖、基质表达的影响及分子机制。 方法： 采用0.28 mol/L NH4Cl喂饲大鼠构建大鼠慢性代谢性酸中毒模型。采用光镜结合图像分析软件观察和分析大鼠肾小球结构。从肾皮质分离大鼠肾小球，肾小球增殖细胞核抗原(PCAN)和p27的表达采用免疫组化和/或Western blotting检测。肾皮质纤维连接蛋白(FN)ｍRNA的表达采用荧光定量RT-PCR检测。分别采用［3H］-TdR掺入法和ELISA方法检测了慢性酸负荷对体外培养的大鼠系膜细胞增殖及FN合成的影响。 结果： 各时点实验组大鼠动脉血pH、［HCO3-］均显著低于对照组(P<0.01)。第3、7、14 d实验组大鼠肾重及肾/体重比值均显著大于对照组，而绝对体重与对照相比无显著差异；平均肾小球面积、肾小球丝球体面积、球内细胞总数以及系膜区细胞数均大于对照组（P<0.05），第14 d大鼠肾小球系膜区面积与肾小球丝球体面积比值高于对照组（P<0.05）；第3、7、14 d Western blotting检测实验组大鼠肾小球PCNA表达增加，p27表达下调，免疫组织化学检测显示肾小球内阳性PCNA细胞主要位于系膜区；第7 d、14 d大鼠肾皮质FN mRNA的表达显著高于对照组（P<0.01）。体外系膜细胞［3H］-TdR掺入量随酸负荷强度而增高，酸负荷24 h和48 h，系膜细胞上清中FN含量显著增高。 结论： 慢性代谢性酸中毒能够诱导大鼠肾小球肥大、系膜细胞增生及细胞外基质增多，其机制与系膜细胞周期调控蛋白P27的表达下调有关。本研究为慢性代谢性酸中毒参与慢性肾小球疾病的硬化提供了体内和体外证据。

Chronic metabolic acidosis markedly induces proliferation of mesangial cells in rats

AIM: To study the effects of chronic metabolic acidosis on glomerulus, mesangial cells and the production of extracellular matrix. METHODS: Chronic metabolic acidosis was induced by addition of 0.28 mol/L NH_4Cl to drinking water for 3, 7, or 14 days in male Wistar rats (n=10). Light microscope combined with computer software (Motic Images Advanced 3.2) was used to determine the effect of chronic acid loading on renal morphologic changes. The expressions of proliferation cell nuclear antigen (PCNA) and p27 in glomeruli were detected by Western blotting or immunohistochemistry. Fibronectin (FN) mRNA was detected by real-time PCR. The proliferation of mesangial cells in vitro was determined by ~3H:-TdR incorporation. The concentration of FN in cultured supernatant was detected by ELISA. RESULTS: On day 1, 3, 7 and 14, the arterial pH and plasma HCO_3~-: in experimental rats were significantly decreased. There was a significantly increased in the kidney weight and the ratio of kidney to body weigh in experimental rats on day 3, 7 and 14. The glomerular area and cell numbers also increased significantly. Immunoblotting demonstrated decreased p27 expression and increased PCNA expression in isolated glomeruli, and the expression of PCNA increased in a time-dependent manner following the time of chronic metabolic acidosis. Immunohistochemistry showed increased positive PCNA expression mainly localized to mesangial cells. The expression of FN mRNA was significantly elevated in experimental rats on day 7 and 14. In vitro, acid loading induced mesangial cell proliferation and synthesis of FN. CONCLUSION: These results suggest that chronic metabolic acidosis induces mesangial cell proliferation, and its mechanism may be associated with the downregulation of cell cycle kinase inhibitor p27.