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

doi:10.11958/20231036

Abstract

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

CLC Number:

R349.5，R692.4

Figures/Tables 9

References 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^＊＊

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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