JAK<sub>2</sub>/STAT<sub>3</sub>信号通路在局灶节段性肾小球硬化足细胞损伤中的作用及机制

doi:10.3877/cma.j.issn.1674-0785.2021.09.008

目的

建立阿霉素诱导的局灶节段性肾小球硬化（FSGS）小鼠模型，探讨JAK₂/STAT₃信号通路在FSGS足细胞损伤中的作用及机制。

方法

选用8周龄雄性C57BL/6小鼠建立阿霉素诱导的FSGS小鼠模型，随机分为FSGS组和对照组，每组各20只。FSGS组予以25 mg/kg单次尾静脉注射阿霉素，对照组予同等剂量的PBS溶液。观察24 h尿蛋白定量、尿素氮、血肌酐等一般指标。HE染色观察肾组织病理；透射电镜观察肾小球基底膜厚度、足细胞形态；双重免疫荧光染色及激光共聚焦观察足细胞nephrin和podocin表达；TUNEL法检测足细胞凋亡。采用实时定量聚合酶链反应（PCR）法检测2组肾组织Janus激酶2（JAK₂）、信号转导和转录活化因子3（STAT₃）mRNA水平；Western印迹法检测肾组织JAK₂、STAT₃蛋白水平；酶联免疫吸附法检测肾组织白细胞介素-6（IL-6）、单核细胞趋化蛋白-1（MCP-1）、α-平滑肌肌动蛋白（α-SMA）、转化生长因子-β₁（TGF-β₁）水平。

结果

（1）与对照组相比，FSGS组24 h尿蛋白定量、血肌酐、尿素氮均升高，差异均有统计学意义（P均＜0.01）。（2）与对照组相比，光镜下，FSGS组小鼠系膜区基质增生，肾小管上皮细胞凋亡、坏死，节段中度加重，毛细血管结构受压狭窄，足细胞增生明显；电镜下FSGS组肾小球基底膜增厚，足细胞融合；激光共聚焦观察显示，FSGS组足细胞nephrin和podocin表达减少；光镜下FSGS组足细胞凋亡增多。（3）与对照组相比，FSGS组肾组织JAK₂、STAT₃ mRNA及蛋白水平增加（P均＜0.01），肾组织IL-6、MCP-1、α-SMA、TGF-β₁水平升高（P均＜0.01），差异有统计学意义。

结论

阿霉素建立FSGS小鼠模型稳定可靠，JAK₂/STAT₃信号通路可通过促进下游因子活化、氧化应激、细胞凋亡等途径参与FSGS足细胞损伤的发生发展。

关键词: 局灶节段性肾小球硬化, 足细胞, Janus激酶2/信号转导和转录活化因子3信号通路, 阿霉素

Objective

To establish a mouse model of adriamycin-induced focal segmental glomerulosclerosis and explore the mechanism of action of the JAK₂/STAT₃ signaling pathway in FSGS podocyte injury.

Methods

Eight-week-old male C57BL/6 mice were randomly divided into either FSGS group (n=20) or control group (n=20) randomly. The FSGS group was given a single tail vein injection of 25 mg/kg adriamycin, and the control group was given the same dose of PBS solution. Indexes including 24-h urinary protein, blood urea nitrogen (BUN), and serum creatinine (Scr) were measured. Renal histopathology was observed by HE staining. The glomerular basement membrane thickness and the morphology of podocytes were observed by transmission electron microscopy. Dual immunofluorescence staining for nephrin and podocin in podocytes were observed by laser confocal microscopy. Podocyte apoptosis was detected by TUNEL. The expression of Janus kinase 2 (JAK₂) and signal transducer and activator of transcription 3 (STAT₃) mRNA was measured by real-time PCR. The expression of JAK₂ and STAT₃ proteins was measured by Western blot. The contents of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), α-smooth muscle actin (α-SMA), and transforming growth factor-β₁ (TGF-β₁) were detected by enzyme linked immunosorbent assay (ELISA).

Results

Compared with the control group, 24-h urinary protein, BUN, and Scr were significantly increased in the FSGS group (P＜0.01 for all). In the FSGS group, there was stromal proliferation in mesangial area, apoptosis and necrosis of renal tubular epithelial cells, moderately aggravated segments, compressed and narrowed capillary structure, and obvious proliferation of podocytes; electron microscopy showed that glomerular basement membrane was thickened and podocytes were fused; the expression of nephrin and podocin in podocytes was decreased, and the apoptosis of podocytes was increased. Compared with the control group, the expression of JAK₂/STAT₃ mRNA and proteins, as well as the contents of IL-6, MCP-1, α-SMA, and TGF-β1, was significantly elevated in the FSGS group (P＜0.01 for all).

Conclusion

Adriamycin-induced FSGS mouse model is stable and reliable. The JAK₂/STAT₃ signaling pathway activation participates in the development of podocyte injury in FSGS by promoting downstream factor activation, oxidative stress, apoptosis and other pathways.

Key words: Focal segmental glomerulosclerosis, Podocytes, JAK₂/STAT₃ signaling pathway, Adriamycin

表1 聚合酶链反应引物序列

名称	引物
JAK₂	上游	5'-GCTACCAGATGGAAACTGTGCG-3'
JAK₂	下游	5'-GCCTCTGTAATGTTGGTGAGATC-3'
STAT₃	上游	5'-AGGAGTCTAACAACGGCAGCCT-3'
STAT₃	下游	5'-GTGGTACACCTCAGTCTCGAAG-3'
GAPDH	上游	5'-TGCGACTTCAACAGCAACTCC-3'
GAPDH	下游	5'-TGCTGTAGCCGTATTCATTGTC-3'

表1 聚合酶链反应引物序列

名称	引物
JAK₂	上游	5'-GCTACCAGATGGAAACTGTGCG-3'
JAK₂	下游	5'-GCCTCTGTAATGTTGGTGAGATC-3'
STAT₃	上游	5'-AGGAGTCTAACAACGGCAGCCT-3'
STAT₃	下游	5'-GTGGTACACCTCAGTCTCGAAG-3'
GAPDH	上游	5'-TGCGACTTCAACAGCAACTCC-3'
GAPDH	下游	5'-TGCTGTAGCCGTATTCATTGTC-3'

表2 2组小鼠生化指标比较（

x ˉ

±s）

组别	只数	尿蛋白定量（mg/24 h）	尿素（mmol/L）	血肌酐（μmol/L）
对照组	20	64.552±13.370	6.435±1.244	19.845±3.091
FSGS组	20	224.261±29.539	41.138±5.497	70.513±12.683
t值		-22.203	-27.537	-17.358
P值		＜0.001	＜0.001	＜0.001

表2 2组小鼠生化指标比较（

x ˉ

±s）

组别	只数	尿蛋白定量（mg/24 h）	尿素（mmol/L）	血肌酐（μmol/L）
对照组	20	64.552±13.370	6.435±1.244	19.845±3.091
FSGS组	20	224.261±29.539	41.138±5.497	70.513±12.683
t值		-22.203	-27.537	-17.358
P值		＜0.001	＜0.001	＜0.001

图1 2组小鼠肾组织HE染色。对照组小鼠肾小球、肾小管结构清晰完整；FSGS组小鼠系膜区基质增生，肾小管上皮细胞凋亡、坏死，节段中度加重，毛细血管结构受压狭窄，足细胞增生明显

图2 2组小鼠肾组织电镜表现。对照组基底膜清晰可见，足突清晰完整，未见融合；FSGS组肾小球基底膜增厚，足细胞融合

图3 2组小鼠肾组织足细胞nephrin和podocin表达情况（免疫荧光，×400）。nephrin阳性表达为绿色荧光，podocin阳性表达为红色荧光，DAPI染细胞核为蓝色荧光；FSGS组足细胞nephrin和podocin表达量较对照组减少

图4 2组小鼠足细胞凋亡情况（TUNEL染色，×400）。TUNEL阳性细胞呈棕褐色染色；FSGS组足细胞凋亡较对照组增多

图5 2组小鼠肾组织JAK₂、STAT₃ mRNA及蛋白表达情况注：与对照组比较，^aP＜0.01；JAK₂为Janus激酶2；STAT₃为信号转导和转录活化因子3；GAPDH为磷酸甘油醛脱氢酶

表3 2组小鼠肾组织JAK₂/STAT₃信号通路下游因子表达水平比较（

x ˉ

±s）

组别	只数	IL-6（ng/L）	MCP-1（ng/L）	α-SMA（ng/L）	TGF-β₁（μg/L）
对照组	20	23.698±4.142	33.089±4.089	34.974±5.759	29.847±4.047
FSGS组	20	35.554±4.079	47.751±7.831	52.407±7.951	38.606±5.396
t值		-9.121	-7.422	-7.941	-5.808
P值		＜0.001	＜0.001	＜0.001	＜0.001

表3 2组小鼠肾组织JAK₂/STAT₃信号通路下游因子表达水平比较（

x ˉ

±s）

组别	只数	IL-6（ng/L）	MCP-1（ng/L）	α-SMA（ng/L）	TGF-β₁（μg/L）
对照组	20	23.698±4.142	33.089±4.089	34.974±5.759	29.847±4.047
FSGS组	20	35.554±4.079	47.751±7.831	52.407±7.951	38.606±5.396
t值		-9.121	-7.422	-7.941	-5.808
P值		＜0.001	＜0.001	＜0.001	＜0.001

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Mechanism of action of JAK₂/STAT₃ signaling pathway in podocyte injury in focal segmental glomerulosclerosis

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Mechanism of action of JAK2/STAT3 signaling pathway in podocyte injury in focal segmental glomerulosclerosis

Mechanism of action of JAK₂/STAT₃ signaling pathway in podocyte injury in focal segmental glomerulosclerosis