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

[1]	FRANK D, VINCE J E. Pyroptosis versus necroptosis: similarities,differences,and crosstalk[J]. Cell Death Differ, 2019, 26(1):99-114. doi:10.1038/s41418-018-0212-6.
[2]	JACKSON D N, THEISS A L. Gut bacteria signaling to mitochondria in intestinal inflammation and cancer[J]. Gut Microbes, 2020, 11(3):285-304. doi:10.1080/19490976.2019.1592421.
[3]	HUTTON H L, OOI J D, HOLDSWORTH S R, et al. The NLRP3 inflammasome in kidney disease and autoimmunity[J]. Nephrology (Carlton), 2016, 21(9):736-744. doi:10.1111/nep.12785.
[4]	LI X, ZENG L, CAO C, et al. Long noncoding RNA MALAT1 regulates renal tubular epithelial pyroptosis by modulated miR-23c targeting of ELAVL1 in diabetic nephropathy[J]. Exp Cell Res, 2017, 350(2):327-335. doi:10.1016/j.yexcr.2016.12.006.
[5]	XU G, YUE F, HUANG H, et al. Defects in MAP1S-mediated autophagy turnover of fibronectin cause renal fibrosis[J]. Aging(Albany NY), 2016, 8(5):977-985. doi:10.18632/aging.100957.
[6]	HOU J, ZHAO R, XIA W, et al. PD-L1-mediated gasdermin C expression switches apoptosis to pyroptosis in cancer cells and facilitates tumour necrosis[J]. Nat Cell Biol, 2020, 22(10):1264-1275. doi:10.1038/s41556-020-0575-z.
[7]	THONGPRAYOON C, KRAMBECK A E, RULE A D. Determining the true burden of kidney stone disease[J]. Nat Rev Nephrol, 2020, 16(12):736-746. doi:10.1038/s41581-020-0320-7.
[8]	THONGBOONKERD V. Proteomics of crystal-cell interactions:a model for kidney stone research[J]. Cells, 2019, 8(9):1076. doi:10.3390/cells8091076.
[9]	COE F L, WORCESTER E M, EVAN A P. Idiopathic hypercalciuria and formation of calcium renal stones[J]. Nat Rev Nephrol, 2016, 12(9):519-533. doi:10.1038/nrneph.2016.101.
[10]	DAUDON M, HENNEQUIN C, BOUJELBEN G, et al. Serial crystalluria determination and the risk of recurrence in calcium stone formers[J]. Kidney Int, 2005, 67(5):1934-1943. doi:10.1111/j.1523-1755.2005.00292.x.
[11]	ANDERS H J, SUAREZ-ALVAREZ B, GRIGORESCU M, et al. The macrophage phenotype and inflammasome component NLRP3 contributes to nephrocalcinosis-related chronic kidney disease independent from IL-1-mediated tissue injury[J]. Kidney Int, 2018, 93(3):656-669. doi:10.1016/j.kint.2017.09.022.
[12]	KHAN S R, CANALES B K, DOMINGUEZ-GUTIERREZ P R. Randall's plaque and calcium oxalate stone formation:role for immunity and inflammation[J]. Nat Rev Nephrol, 2021, 17(6):417-433. doi:10.1038/s41581-020-00392-1.
[13]	RIZVI S H M, PARVEEN A, AHMAD I, et al. Aluminum activates PERK-EIF2α signaling and inflammatory proteins in human neuroblastoma SH-SY5Y cells[J]. Biol Trace Elem Res, 2016, 172(1):108-119. doi:10.1007/s12011-015-0553-7.
[14]	KHASKHALI M H, BYER K J, KHAN S R. The effect of calcium on calcium oxalate monohydrate crystal-induced renal epithelial injury[J]. Urol Res, 2009, 37(1):1-6. doi:10.1007/s00240-008-0160-6.
[15]	JOSHI S, WANG W, PECK A B, et al. Activation of the NLRP3 inflammasome in association with calcium oxalate crystal induced reactive oxygen species in kidneys[J]. J Urol, 2015, 193(5):1684-1691. doi:10.1016/j.juro.2014.11.093.
[16]	LIN Q, LI S, JIANG N, et al. PINK1-parkin pathway of mitophagy protects against contrast-induced acute kidney injury via decreasing mitochondrial ROS and NLRP3 inflammasome activation[J]. Redox Biol, 2019, 26:101254. doi:10.1016/j.redox.2019.101254.
[17]	LIU Q, LIU Y, GUAN X, et al. Effect of M2 macrophages on injury and apoptosis of renal tubular epithelial cells induced by calcium oxalate crystals[J]. Kidney Blood Press Res, 2019, 44(4):777-791. doi:10.1159/000501558.
[18]	QI S, WANG Q, XIE B, et al. P38 MAPK signaling pathway mediates COM crystal-induced crystal adhesion change in rat renal tubular epithelial cells[J]. Urolithiasis, 2020, 48(1):9-18. doi:10.1007/s00240-019-01143-z.
[19]	KHAN S R. Reactive oxygen species,inflammation and calcium oxalate nephrolithiasis[J]. Transl Androl Urol, 2014, 3(3):256-276. doi:10.3978/j.issn.2223-4683.2014.06.04.
[20]	QIN B, WANG Q, LU Y, et al. Losartan ameliorates calcium oxalate-induced elevation of stone-related proteins in renal tubular cells by inhibiting NADPH oxidase and oxidative stress[J]. Oxid Med Cell Longev, 2018, 2018:1271864. doi:10.1155/2018/1271864.
[21]	ASSELMAN M, VERHULST A, DE BROE M E, et al. Calcium oxalate crystal adherence to hyaluronan-,osteopontin-,and CD44-expressing injured/regenerating tubular epithelial cells in rat kidneys[J]. J Am Soc Nephrol, 2003, 14(12):3155-3166. doi:10.1097/01.asn.0000099380.18995.f7.
[22]	WANG Z, LI M X, XU C Z, et al. Comprehensive study of altered proteomic landscape in proximal renal tubular epithelial cells in response to calcium oxalate monohydrate crystals[J]. BMC Urol, 2020, 20(1):136. doi:10.1186/s12894-020-00709-z.

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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