Ca2+诱导HK-2细胞焦亡及黏附性变化对含钙肾结石形成的分子机制研究

doi:10.11958/20231036

天津医药 ›› 2024, Vol. 52 ›› Issue (3): 250-255.doi: 10.11958/20231036

Ca²⁺诱导HK-2细胞焦亡及黏附性变化对含钙肾结石形成的分子机制研究

向近杰(), 吕懋鑫, 王梦悦, 张坤, 李颢^△()

昆明医科大学第一附属医院泌尿外科（邮编650032）

收稿日期:2023-07-12 修回日期:2023-09-02 出版日期:2024-03-15 发布日期:2024-03-13
通讯作者: ^△E-mail：lihao834@sina.com
作者简介:向近杰（1998），男，硕士在读，主要从事肾结石疾病基础方面研究。E-mail：jjx980911@163.com
基金资助:
云南省科技厅科技计划项目(202001AY070001-158)

Molecular mechanisms of Ca²⁺-induced pyroptosis and adhesion changes of HK-2 cells in the formation of calcium-containing kidney stones

XIANG Jinjie(), LYU Maoxin, WANG Mengyue, ZHANG Kun, LI Hao^△()

Department of Urology, the First Affiliated Hospital of Kunming Medical University, Kunming 650032, China

Received:2023-07-12 Revised:2023-09-02 Published:2024-03-15 Online:2024-03-13
Contact: ^△E-mail: lihao834@sina.com

向近杰, 吕懋鑫, 王梦悦, 张坤, 李颢. Ca²⁺诱导HK-2细胞焦亡及黏附性变化对含钙肾结石形成的分子机制研究[J]. 天津医药, 2024, 52(3): 250-255.

XIANG Jinjie, LYU Maoxin, WANG Mengyue, ZHANG Kun, LI Hao. Molecular mechanisms of Ca²⁺-induced pyroptosis and adhesion changes of HK-2 cells in the formation of calcium-containing kidney stones[J]. Tianjin Medical Journal, 2024, 52(3): 250-255.

摘要/Abstract

摘要：

目的探究人肾皮质近曲小管上皮细胞（HK-2）经Ca²⁺作用后的焦亡经典通路激活及黏附性变化对含钙肾结石形成的作用及机制。方法不同质量浓度的CaCl₂（0、0.1、0.5、1.0、2.0、4.0、8.0 g/L）培养HK-2细胞24 h，使用细胞计数试剂盒（CCK-8）及流式细胞凋亡术检测筛选最佳处理浓度。使用透射电镜观察高钙环境下肾小管上皮细胞微结构的变化。在高钙处理后用2，7-二氯荧光素二乙酸酯（DCFH-DA）检测细胞内活性氧（ROS）的产生，并采用实时荧光定量聚合酶链式反应和Western blot法分别检测高钙刺激后HK-2细胞焦亡相关NOD样受体热蛋白结构域相关蛋白3（NLRP3）、胱天蛋白酶1（Caspase-1）、gasderminD（GSDMD）和黏附分子骨桥蛋白（OPN）、CD44的mRNA和蛋白表达水平变化，酶联免疫吸附试验检测白细胞介素（IL）-1β、IL-18和黏附分子单核细胞趋化蛋白1（MCP-1）在高钙刺激后的表达变化。结果 Ca²⁺对HK-2细胞生长具有细胞毒性并且可以促进其凋亡，Ca²⁺浓度越高，对HK-2细胞生长的毒性越大且凋亡率越高。高钙可以促进HK-2细胞发生细胞膜完整性缺失、内容物释放及胞内大量空泡产生等焦亡样形态学改变。与对照组相比，1.0 g/L及2.0 g/L CaCl₂组的ROS表达水平依次升高，焦亡相关基因NLRP3、Caspase-1、GSDMD、IL-1β、IL-18以及黏附相关基因OPN、CD44、MCP-1的mRNA和蛋白表达水平均依次升高（P<0.05）。结论高钙能使HK-2细胞发生氧化应激损伤并产生ROS，从而激活NLRP3炎症小体，进而导致细胞焦亡经典通路的激活和细胞黏附性的增加，最终间接促进肾结石的形成。

关键词: 细胞焦亡, 高钙尿症, 活性氧, 细胞黏附分子, 肾结石

Abstract:

Objective To investigate the possible role and mechanism of activation of pyroptosis classical pathway and alterations in cell adhesion in calcium-containing kidney stones after the action of high concentration of Ca²⁺ on HK-2 cells. Methods HK-2 cells were cultured in the presence of different concentrations of CaCl₂ (0, 0.1, 0.5, 1.0, 2.0, 4.0 and 8.0 g/L) for 24 hours, and cell counting Kit-8 (CCK-8) and flow cytometry were used to determine the optimal treatment concentration. Subsequently, the ultrastructure of renal tubular epithelial cells under high Ca²⁺ condition was observed by transmission electron microscopy after Ca²⁺ treatment. DCFH-DA staining was used to detect intracellular reactive oxygen species production, and quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were performed to examine the expression of pyroptosis-related proteins NLRP3, Caspase-1, gasdermin D (GSDMD), adhesive molecules osteopontin (OPN) and CD44 at mRNA and protein levels after high concentration Ca²⁺ treatment. The expression levels of pyroptosis-related inflammatory factors interleukin (IL)-1β, IL-18 and adhesive molecule monocyte chemotactic protein-1 (MCP-1) were detected by enzyme-linked immunosorbent assay (ELISA) after high Ca²⁺ stimulation. Results Ca²⁺ showed cytotoxicity for HK-2 cell growth and can promote apoptosis. The higher the Ca²⁺ concentration, the more toxicity and apoptosis rate for HK-2 cell growth. High concentration of Ca²⁺ can promote pyroptosis-like morphological changes in HK-2 cells, including loss of cell membrane integrity, release of contents and numerous intracellular vacuoles. Compared with the control group, the expression levels of ROS were sequentially increased in the 1.0 g/L CaCl₂ group and the 2.0 g/L CaCl₂ group, and the expression levels of pyroptosis-related genes NLRP3, Caspase-1, GSDMD, and the pyroptosis-associated inflammatory factors IL-1β and IL-18, as well as the adhesion molecules OPN, CD44 and MCP-1 were significantly increased (P<0.05). Conclusion High Ca²⁺ treatment can cause oxidative stress damage in HK-2 cells to produce ROS, which activates NLRP3 inflammasome, leads to the activation of the classical pathway of pyroptosis and increase the adhesion of cells, and ultimately leads to the formation of kidney stones.

Key words: pyroptosis, hypercalciuria, reactive oxygen species, cell adhesion molecules, kidney

中图分类号:

R349.5，R692.4

图/表 9

参考文献 22

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组别	存活率	凋亡率
对照组	100.00±2.03	2.15±0.83
0.1 g/L CaCl₂组	96.96±1.99	7.54±2.26
0.5 g/L CaCl₂组	95.63±1.37	12.89±2.35
1.0 g/L CaCl₂组	81.57±3.81^a	17.35±2.28^a
2.0 g/L CaCl₂组	63.14±1.43^a	21.42±1.98^a
4.0 g/L CaCl₂组	46.73±1.15^a	30.82±10.53^a
8.0 g/L CaCl₂组	10.58±0.08^a	39.18±6.74^a
F	820.800^＊＊	19.970^＊

组别	存活率	凋亡率
对照组	100.00±2.03	2.15±0.83
0.1 g/L CaCl₂组	96.96±1.99	7.54±2.26
0.5 g/L CaCl₂组	95.63±1.37	12.89±2.35
1.0 g/L CaCl₂组	81.57±3.81^a	17.35±2.28^a
2.0 g/L CaCl₂组	63.14±1.43^a	21.42±1.98^a
4.0 g/L CaCl₂组	46.73±1.15^a	30.82±10.53^a
8.0 g/L CaCl₂组	10.58±0.08^a	39.18±6.74^a
F	820.800^＊＊	19.970^＊

组别	NLRP3		GSDMD	IL-1β	IL-18	OPN	CD44	MCP-1
对照组	1.01±0.16	1.00±0.08	1.00±0.03	1.00±0.06	1.00±0.11	1.00±0.04	1.01±0.13	1.00±0.06
1.0 g/L CaCl₂组	5.42±0.30^a	3.13±0.40^a	1.76±0.25^a	2.14±0.10^a	4.10±0.45^a	74.43±8.66^a	19.42±2.94^a	10.23±1.54^a
2.0 g/L CaCl₂组	8.24±0.48^ab	4.87±0.57^ab	2.77±0.31^ab	4.63±0.16^ab	5.84±0.73^ab	93.43±5.10^ab	29.24±3.98^ab	15.45±1.83^ab
F	345.700^＊＊	68.850^＊＊	43.350^＊	831.300^＊＊	72.350^＊＊	212.900^＊＊	75.530^＊＊	84.250^＊＊

组别	NLRP3		GSDMD	IL-1β	IL-18	OPN	CD44	MCP-1
对照组	1.01±0.16	1.00±0.08	1.00±0.03	1.00±0.06	1.00±0.11	1.00±0.04	1.01±0.13	1.00±0.06
1.0 g/L CaCl₂组	5.42±0.30^a	3.13±0.40^a	1.76±0.25^a	2.14±0.10^a	4.10±0.45^a	74.43±8.66^a	19.42±2.94^a	10.23±1.54^a
2.0 g/L CaCl₂组	8.24±0.48^ab	4.87±0.57^ab	2.77±0.31^ab	4.63±0.16^ab	5.84±0.73^ab	93.43±5.10^ab	29.24±3.98^ab	15.45±1.83^ab
F	345.700^＊＊	68.850^＊＊	43.350^＊	831.300^＊＊	72.350^＊＊	212.900^＊＊	75.530^＊＊	84.250^＊＊

组别	NLRP3	Caspase-1	GSDMD	OPN	CD44
对照组	0.37±0.05	0.41±0.04	0.51±0.06	0.49±0.04	0.58±0.05
1.0 g/L CaCl₂组	0.79±0.06^a	0.74±0.05^a	0.80±0.05^a	0.96±0.07^a	0.96±0.07^a
2.0 g/L CaCl₂组	1.08±0.06^ab	1.03±0.07^ab	1.04±0.06^ab	1.18±0.04^ab	1.15±0.05^ab
F	123.500^＊＊	92.860^＊＊	62.880^＊＊	136.100^＊＊	80.870^＊＊

Ca²⁺诱导HK-2细胞焦亡及黏附性变化对含钙肾结石形成的分子机制研究

Molecular mechanisms of Ca²⁺-induced pyroptosis and adhesion changes of HK-2 cells in the formation of calcium-containing kidney stones

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基因名称	引物序列（5'→3'）	产物大小/bp
NLRP3	上游：CAAGAATCCACAGTGTAA	101
	下游：TCTGATTAGTGCTGAGTA	101
Caspase-1	上游：GAACAAGGAAGAGATGGAGAA 下游：TCGGAATAACGGAGTCAATC	86
GSDMD	上游：CTACTGCCTGGTGGTTAG 下游：TTGATAGACAGGTTGACACA	78
IL-1β	上游：AATGACCTGAGCACCTTCT 下游：GCACATAAGCCTCGTTATCC	82
IL-18	上游：GACCAAGTTCTCTTCATT 下游：ATAGTTACAGCCATACCT	143
OPN	上游：AATGATGAGAGCAATGAG 下游：GTCTACAACCAGCATATC	114
CD44	上游：AACGCAGCAGAGTAATTC 下游：GATGTCAGAGTAGAAGTTGTT	90
MCP-1	上游：CCAGTCACCTGCTGTTAT 下游：GCTTCTTTGGGACACTTG	98
GAPDH	上游：TTGCCCTCAACGACCACTTT 下游：TGGTCCAGGGGTCTTACTCC	120

组别	IL-1β	IL-18	MCP-1
对照组	16.13±2.11	46.42±3.93	203.50±20.85
1.0 g/L CaCl₂组	49.32±3.91^a	99.00±5.66^a	423.00±27.30^a
2.0 g/L CaCl₂组	67.95±3.99^ab	121.50±5.63^ab	579.60±44.19^ab
F	231.700^＊＊	224.800^＊＊	102.500^＊＊

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