Kidney Disease Center, the First Affiliated Hospital, College of Medicine, Zhejiang University, Qingchun Road 79, Hangzhou, 310003, China.
Key Laboratory of Nephropathy, Hangzhou, Zhejiang Province, China.
BMC Nephrol. 2020 Sep 9;21(1):392. doi: 10.1186/s12882-020-02050-x.
This study aimed to understand the mechanistic role of N-methyl-D-aspartate receptor (NMDAR) in acute fibrogenesis using models of in vivo ureter obstruction and in vitro TGF-β administration.
Acute renal fibrosis (RF) was induced in mice by unilateral ureteral obstruction (UUO). Histological changes were observed using Masson's trichrome staining. The expression levels of NR1, which is the functional subunit of NMDAR, and fibrotic and epithelial-to-mesenchymal transition markers were measured by immunohistochemical and Western blot analysis. HK-2 cells were incubated with TGF-β, and NMDAR antagonist MK-801 and Ca/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 were administered for pathway determination. Chronic RF was introduced by sublethal ischemia-reperfusion injury in mice, and NMDAR inhibitor dextromethorphan hydrobromide (DXM) was administered orally.
The expression of NR1 was upregulated in obstructed kidneys, while NR1 knockdown significantly reduced both interstitial volume expansion and the changes in the expression of α-smooth muscle actin, S100A4, fibronectin, COL1A1, Snail, and E-cadherin in acute RF. TGF-β1 treatment increased the elongation phenotype of HK-2 cells and the expression of membrane-located NR1 and phosphorylated CaMKII and extracellular signal-regulated kinase (ERK). MK801 and KN93 reduced CaMKII and ERK phosphorylation levels, while MK801, but not KN93, reduced the membrane NR1 signal. The levels of phosphorylated CaMKII and ERK also increased in kidneys with obstruction but were decreased by NR1 knockdown. The 4-week administration of DXM preserved renal cortex volume in kidneys with moderate ischemic-reperfusion injury.
NMDAR participates in both acute and chronic renal fibrogenesis potentially via CaMKII-induced ERK activation.
本研究旨在通过体内输尿管梗阻模型和体外 TGF-β给药模型，了解 N-甲基-D-天冬氨酸受体（NMDAR）在急性纤维发生中的作用机制。
通过单侧输尿管梗阻（UUO）在小鼠中诱导急性肾纤维化（RF）。通过 Masson 三色染色观察组织学变化。通过免疫组织化学和 Western blot 分析测量 NMDAR 的功能亚基 NR1 以及纤维化和上皮间质转化标志物的表达水平。将 HK-2 细胞与 TGF-β孵育，并给予 NMDAR 拮抗剂 MK-801 和 Ca/钙调蛋白依赖性蛋白激酶 II（CaMKII）拮抗剂 KN-93 以确定途径。通过亚致死性缺血再灌注损伤在小鼠中引入慢性 RF，并给予 NMDAR 抑制剂右美沙芬氢溴酸盐（DXM）口服。
NR1 在梗阻肾脏中表达上调，而 NR1 敲低显着减少了急性 RF 中的间质体积扩张和α-平滑肌肌动蛋白、S100A4、纤维连接蛋白、COL1A1、Snail 和 E-钙粘蛋白表达的变化。TGF-β1 处理增加了 HK-2 细胞的伸长表型以及膜定位的 NR1 和磷酸化 CaMKII 和细胞外信号调节激酶（ERK）的表达。MK801 和 KN93 降低了 CaMKII 和 ERK 磷酸化水平，而 MK801 但不是 KN93 降低了膜 NR1 信号。在梗阻的肾脏中，磷酸化 CaMKII 和 ERK 的水平也增加，但 NR1 敲低后减少。4 周 DXM 给药可在中等缺血再灌注损伤的肾脏中保留肾皮质体积。
NMDAR 通过 CaMKII 诱导的 ERK 激活参与急性和慢性肾纤维化。
