局灶节段性肾小球硬化症大鼠尿足细胞动态检测及其意义

Dynamic examination and significance of urinary podocytes in experimental focal segmental glomerulosclerosis rats

目的建立局灶节段性肾小球硬化症(FSGS)大鼠模型,观察尿中脱落的肾小球足细胞的变化,并探讨其意义。方法FSGS组(n=7):采用左颈静脉插管一次性缓慢注射嘌呤霉素核苷酸(PAN,9mg/100g体重的方法建立)FSGS大鼠模型。正常对照组(n=5):注射等量生理盐水,实验周期20周。动态检测24h尿蛋白定量;间接免疫荧光方法动态检测尿沉渣足细胞特异性标志蛋白podocalyxin(PCX)、Wilm'stumor鄄1(WT鄄1);20周末肾组织光镜下观察肾小球病变;电镜观察肾小球足细胞的改变;免疫荧光染色观察肾小球内PCX和成熟足细胞特异性标志蛋白synaptopodin(PP44)的表达。结果FSGS组大鼠24h尿蛋白定量在第天较注射前明显升高,第312天达到高峰[(672.74±98.72)比(19.31±3.15)mg/24h,P<0.01),此后开始下降,于第6周接近注射前水平,持续至11周[(35.46±14.88)比(19.31±3.15)mg/24h,P=0.0238];从12周开始再度缓慢升高,持续高水平至20周,为注射前近7倍[(140.61±68.90)比(19.31±3.15)mg/24h,P<0.01]。FSGS组大鼠从12周开始,尿足细胞出现阳性(>1～5个/HP),持续至20周。光镜显示,30%～50%肾小球出现不同程度的局灶节段性硬化;电镜证实足突部分融合并可见足突与基底膜剥离。足细胞特异标记蛋白PCX、PP44在肾小球节段性硬化部位呈现节段性缺失。对照?

Objective To establish a rat model of focal segmental glomerulosclerosis (FSGS) and observe the occurrence of urinary podocyte and its relevant significance for FSGS. Methods FSGS model rats (n=7) were injected slowly with a single dose of puromycin aminonucleoside(PAN 9 mg/100 g body weight) via left jugular vein, while control rats(n=5) received an equal volume of saline injection in the same procedure. Rats were sacrificed at week 20. 24 h proteinuria and urinary podocytes were mensurated dynamically. Podocyte-specific marker protein-podocalyxin(PCX) and Wilm′s tumor-1(WT-1)were detected by indirection immunofluorescence. Blood was collected via ventral artery and the levels of serum creatinine(Scr) were examined. Kidney tissues stained by HE?PAS?PASM were observed with light microscopy. The change of glomerular podocytes was observed by electron microscopy. The expression and localization of PCX and synaptopodin (PP44) were assessed by indirection immunofluorescence, and the lackage of podocytes in glomeruli was judged by the absence of fluorescence. Results The protein level of 24 h urine increased dramatically at day 3 after PAN injection, reached the peak level at day 12 [(672.74±98.72) mg/24 h vs the basal level (19.31±3.15) mg/24 h,P < 0.01=. Then the level declined progressively and closed to the basal level at the 6th week and kept low level until the 11th week[(35.46±14.88) vs (19.31±3.15)mg/24 h,P=0.0238]. At the beging of the 12th week, the protein level of 24 h urine slowly rose again, kept the high level until the 20th week [(140.61±68.90) vs control guoup (19.31±3.15) mg/24 h,P < 0.01]. Urinary podocytes were negative within 11 weeks, but became positive( >1～5个/HP)from the beginning of 12th week to 20th week in FSGS-group. In control group rats, levels of 24 h proteinuria were not changed obviously[(23.05±1.40) vs (19.31±3.15),P=0.8414], as well as urinary podocytes were negative during the 20 weeks. There was no significant difference for the levels of Scr between FSGS group and control group [(60.86±3.93) vs (54.57±6.24)μmol/L,P=0.1037]. PAN rats developed focal segmental glomerular sclerosis. Electron microscopy demonstrated the foot processes effaced diffusively, some of them detached from GBM. Focal absence of the expression of PCX and PP44 was seen in the gromeruli from PAN group rats, and the locations of absence were consistent with the lesions of focal sclerosis in glomeruli. Conclusion Injury and loss of podocytes may lead to the initiation and progression of FSGS. Urinary podocyte can be one of the markers to predict the development of FSGS.