蛋白激酶C-alpha对小鼠尿浓缩功能的调节机制初探

目的通过观察蛋白激酶C(PKC)-alpha基因敲除鼠血清加压素(AVP)水平及髓内水通道蛋白2(AQP-2)分布、表达和转运状态的变化,初步探讨PKC-alpha在小鼠尿浓缩功能中的调节机制。方法使用代谢箱收集PKC-alpha基因敲除鼠及SV129野生鼠24 h尿液并取血,采用渗透计检测尿渗透浓度。ELISA法检测正常饮食下24 h尿尿素排泄量。放射性免疫法(RIA)检测血清AVP水平。免疫荧光及半定量免疫印迹技术检测小鼠内髓AQP-2的分布和表达情况。分别使用V2受体拮抗剂SR141263及不同浓度去氨基精加压素(DdAVP)腹腔内注射,检测3 h内尿渗透浓度、尿量以观察两组小鼠AQP-2转运状态。结果正常饮食下PKC- alpha基因敲除鼠24 h尿尿素排泄量[(3．25±0．18)mmol／24 h比(3．83±0．42)mmol／24 h,P= 0．24]以及血清AVP水平[(4．64±0．43)pmol／L比[(5．03±0．44)pmol／L,P=0．55]与野生鼠相比,差异均无统计学意义。两组小鼠内髓AQP-2的分布及表达相似(P=0．48)。不同浓度DdAVP腹腔注射后两组小鼠尿量变化曲线完全一致。SR141263腹腔注射后PKC-alpha基因敲除鼠及野生鼠尿渗透浓度改变之间差异也无统计学意义[(0．20±0．02)mmol／L比(0．20±0．04) mmol／L,P=0．97]。结论PKC-alpha参与调节的小鼠尿浓缩功能与血清AVP水平、髓内AQP-2状态无关。

Study on the mechanism of protein kinase C alpha in mediating urine concentration ability

Objective To study the plasma level of vasopressin (AVP or ADH) and the localization, expression as well as trafficking status of aquaporin-2 on protein kinase C (PKC)-alpha knockout mice and to elucidate the potential mechanism of PKC-alpha in mediating urine concentration ability. Methods PKC alpha knockout and wild type (SV129)mice(n=6, each group) were placed in metabolic cages to assess 24 hours urine output, urinary osmolality by freezing-point depression, urinary urea excretion by ELISA, plasma AVP level by radioimmunoassay (RIA). The localization and expression of aquaporin-2(AQP-2) in inner medulla were determined by immunohistochemical and semi-quantitative Western blotting techniques. After i.p. injected with vasopressin V2-receptor antagonist SR121463 and different concentrations of DdAVP separately, the urinary osmolality and urinary output of 3 hours were checked to observe trafficking status of AQP-2. Results As compared to wild type mice, urinary urea excretion and plasma AVP level [(5.03±0.44) pmol/L vs (4.64±0.43) pmol/L,P = 0.55] were not different between tow groups under basal condition consisting with high urinary volume [(2.85±0.35) ml vs (1.80±0.18) ml, P = 0.02] and lower urinary osmolality in PKC-alpha knockout mice [(2.41±0.04) mmol/L vs (3.13±0.11) mmol/L, P = 0.00]. Immunohistochemical and semi-quantitative Western blotting studies revealed that the localization and expression of AQP-2 (P = 0.48) in inner medulla were not different between these two groups. The urinary output presented no difference under DdAVP experiment as well as changes of urinary osmolality [(0.20±0.02) ml vs. (0.20±0.04) ml,P = 0.97] under V2 receptor experiment. Conclusions PKC alpha mediates mouse urine concentration ability independent of inner medulla AQP-2 statues and plasma AVP level.