不同草酸/钙摩尔比条件下草酸钙晶体的生长及对HK-2细胞的毒性

doi:10.7503/cjcu20190713

摘要/Abstract

摘要：

研究了不同草酸/钙(Ox/Ca)摩尔比对CaOx晶体在损伤前后的人肾近曲小管上皮细胞(HK-2)表面的生长差异及形成的晶体对细胞的毒性差异. 实验结果表明, CaOx过饱和溶液对正常细胞和损伤细胞均会产生进一步的损伤, 导致细胞活力、溶酶体的完整性和线粒体膜电位降低, 而细胞内活性氧(ROS)、细胞骨架的紊乱程度、磷酯酰丝氨酸(PS)外翻比例和骨桥蛋白(OPN)表达量均增加; 且随着过饱和溶液中Ox/Ca摩尔比的增加而损伤加重. 正常细胞主要诱导二水草酸钙(COD)晶体形成, 且COD的含量与Ox/Ca摩尔比成正相关. 损伤细胞表面主要生成一水草酸钙(COM), 且晶体的数量和聚集程度与Ox/Ca摩尔比成正相关. 相比于正常细胞, 损伤细胞诱导的晶体棱角更加尖锐, 其对细胞的损伤大于棱角圆钝的晶体. 实验结果还表明, 降低CaOx的过饱和度、减小Ox/Ca摩尔比和修复受损伤的肾上皮细胞均有利于抑制CaOx结石形成.

关键词: 草酸钙, Ox/Ca摩尔比, 细胞毒性, 肾结石, 过饱和度

Abstract:

We investigated the growth differences of CaOx crystals with different oxalic acid/calcium(Ox/Ca) molar ratios on the surface of human renal proximal tubule epithelial cells(HK-2) before and after injury and the toxicity of the crystals formed on the cells. CaOx supersaturated solution caused further damage to both the normal and damaged cells, resulting in the decrease of cell viability, lysosomal integrity and mitochondrial membrane potential, while the increase of intracellular reactive oxygen species(ROS), cytoskeleton disorder, phosphatidylserine(PS) eversion ratio and osteopontin(OPN) expression. With the increase of Ox/Ca molar ratio in saturated solution, the cell damage increased. Normal cells mainly induce the formation of calcium oxalate dihydrate(COD) crystals, and the percentage of COD is positively correlated with the Ox/Ca ratio. Damaged cells mainly induced calcium oxalate monohydrate(COM), and the number and aggregation of crystals are positively correlated with Ox/Ca molar ratio. Compared with the control group cells, the crystal edges induced by the damaged group cells are sharper, and the damage of sharp crystals to the cells is greater than that of round and blunt crystals. The results of this study suggest that reducing the supersaturation of CaOx, reducing the Ox/Ca molar ratio and repairing the damaged renal epithelial cells are all beneficial to inhibit CaOx stone formation.

Key words: Calcium oxalate, Ox/Ca molar ratio, Cytotoxicity, Kidney stones, Supersaturation degree

中图分类号:

O614.23
R69

TrendMD:

刘虹,邹国均,孙新园,欧阳健明. 不同草酸/钙摩尔比条件下草酸钙晶体的生长及对HK-2细胞的毒性. 高等学校化学学报, 2020, 41(6): 1231.

LIU Hong,ZOU Guojun,SUN Xinyuan,OUYANG Jianming. Differences of Growth and Cytotoxicity of Calcium Oxalate Crystals Formed on HK-2 Cells Under Different Oxalic Acid/Calcium Ratios ^†. Chem. J. Chinese Universities, 2020, 41(6): 1231.

图/表 12

Table 1 Composition and labeling of CaOx supersaturated solution with different Ox/Ca molar ratios

Group	c(Ca²⁺)/(mmol·L^-1)	c(Ox^2-)/(mmol·L^-1)	Ox/Ca molar ratio	Labeling	RS^* of CaOx
Ⅰ	1.00	0.25	0.25	1.00×0.25	9.27
Ⅱ	0.50	0.50	1.00	0.50×0.50	9.27
Ⅲ	0.25	1.00	4.00	0.25×1.00	9.27

Fig.1 Cell viability of HK-2 cells before and after injury with CaOx supersaturated solutions with various Ox/Ca molar ratios Action time: 12 h; ** P<0.01, * P<0.05 vs. normal blank group.

Fig.2 Intracellular ROS level of HK-2 cells before and after injury with CaOx supersaturated solutions with various Ox/Ca molar ratios (A) Fluorescence microscopy images of ROS; (B) quantitative fluorescence intensity.Action time: 12 h; ** p<0.01, * p<0.05 vs. normal blank group.

Fig.3 Lysosomal integrity observation of HK-2 cells before and after injury with CaOx supersaturated solution with various Ox/Ca molar ratios (A) Fluorescence microscopy images; (B) quantitative fluorescence intensity.Action time: 12 h; ** P<0.01, * P<0.05 vs. normal blank group.

Fig.4 OPN expression of HK-2 cells before and after injury with CaOx supersaturated solutions with various Ox/Ca molar ratios (A) Fluorescence microscopy images; (B) quantitative results of OPN expression. Nucleus: blue;OPN: green. Action time: 12 h; ** P<0.01, * P<0.05 vs. normal blank group.

Fig.5 Confocal observation images of cytoskeleton in HK-2 cells before and after injury with CaOx supersaturated solutions with various Ox/Ca molar ratios Actin: green; nuclei: blue; action time: 12 h.

Fig.6 Mitochondrial membrane potential detection(Δψm) of HK-2 cells before and after injury with CaOx supersaturated solutions with various Ox/Ca molar ratios (A1—A8) Flow cytometric data of mitochondrial membrane potential(Δψm), green R2 value represents decreasing membrane potential; (A1)—(A4) normal cell; (A5)—(A8): damage cell; (A1), (A5) blank; n(Ox)/n(Ca): (A2), (A6) 0.25; (A3), (A7) 1; (A4), (A8) 4. (B) Quantitative histogram of Δψm. Action time: 12 h; ** P<0.01, * P<0.05 vs. normal blank group.

Fig.7 Flow cytometry analysis of PS eversion in HK-2 cells before and after injury with CaOx supersaturated solutions with various Ox/Ca molar ratios (A1—A8) Histogram of the percentage of PS eversion; abscissa: the fluorescence intensity; ordinate: the number of cells; (A1)—(A4) normal cell; (A5)—(A8): damage cell; (A1), (A5) blank; n(Ox)/n(Ca): (A2), (A6) 0.25; (A3), (A7) 1; (A4), (A8) 4. (B) Quantitative histogram. Action time: 12 h; ** P<0.01, * P<0.05 vs. normal blank group.

Fig.8 Amount of CaOx crystals on HK-2 cells injury with CaOx supersaturated solutions with various Ox/Ca molar ratios Action time: 12 h; * p<0.05.

Fig.9 SEM images of CaOx crystals on HK-2 cells before and after injury with CaOx supersaturated solutions with various Ox/Ca molar ratios (A1)—(A3) Normal cells; (A4)—(A6) damage cell; n(Ox)/n(Ca): (A1), (A4) 0.25;(A2), (A5) 1; (A3), (A7) 4. Action time: 12 h.

Fig.10 XRD patterns of CaOx crystals on HK-2 cells before and after injury with CaOx supersaturated solutions with various Ox/Ca molar ratios * COD; # COM. (A1)—(A3) normal cell; (A4)—(A6) damage cell; n(Ox)/n(Ca): (A1), (A4) 0.25; (A2), (A5) 1; (A3), (A6) 4. Action time: 12 h.

Fig.11 Mechanism diagram of cytotoxic difference and CaOx crystal difference induced by HK-2 cells before and after injury with CaOx supersaturated solutions with various Ox/Ca molar ratios

参考文献 35

[1]	Daudon M., Jungers P., Bazin D., Bazin D., Williams J. C. Jr., Urolithiasis, 2018, 46(5), 459—470
[2]	Khan A., Int. Urol. Nephrol., 2018, 50(5), 799—806
[3]	Yuen J. W. M., Gohel M. D. I., Poon N., Shum D. K., Tam P. C., Au D. W., Clin. Chim. Acta, 2010, 411, 1018—1026
[4]	Yao X. Q., Deng S. P., Ouyang J. M. , Chem. J. Chinese Universities 2011, 32(2), 236—240
	( 姚秀琼, 邓穗平, 欧阳健明 . 高等学校化学学报, 2011, 32(2) 236—240)
[5]	Rimer J. D., Kolbach-Mandel A. M., Ward M. D., Wesson J. A., Urolithiasis, 2017, 45(1), 57—74
[6]	Zuo J., Khan A., Glenton P. A., Khan S. R., Nephrol. Dial. Transplant., 2011, 26(6), 1785—1796
[7]	Gan Q. Z., Sun X. Y., Yao X. Q., Ouyang J. M. , Chem. J. Chinese Universities 2016, 37(6), 1050—1058
	( 甘琼枝, 孙新园, 姚秀琼, 欧阳健明 . 高等学校化学学报, 2016, 37(6) 1050—1058)
[8]	Zhang S., Yang R. E., Ouyang J. M. , Acta Chimica Sinica 2011, 3, 284—290
	( 张生, 杨如娥, 欧阳健明化学学报, 2011, 3, 284—290)
[9]	Chai W., Liebman M., Kynast-Gales S., Massey L., Am. J. Kidney Dis., 2004, 44(6), 1060—1069
[10]	Guerra A., Allegri F., Meschi T., Adorni G., Prati B., Nouvenne A., Novarini A., Maggiore U., Fiaccadori E., Borghi L., Clin. Chem. Lab. Med. , 2005, 43(6), 585—589
[11]	Bretherton T., Rodgers A., Cryst. Growth., 1998, 192, 448—455
[12]	Jung T., Kim W. S., Choi C. K., Mater. Sci. Eng. C, 2004, 24, 31—33
[13]	Seeger H., Kaelin A., Ferraro P. M., Weber D., Jaeger P., Ambuehl P., Robertson W., Unwin R., Wagner C. R., Mohebbi N., BMC Nephrol., 2017, 18(1), 349
[14]	Mitchell T., Kumar P., Reddy T., Wood K. D., Knight J., Assimos D. J., Holmes R. P., Am. J. Physiol. Renal Physiol., 2019, 316(3), F409—F413
[15]	Borghi L., Guerra A., Meschi T., Briganti A., Schianchi T., Allegri F., Novarini A., Kidney Int., 1999, 55(3), 1041—1050
[16]	Carvalho M., Vieira M. A., Int. Braz. J. Urol., 2004, 30(3), 205—209
[17]	Schepers M. S. J., Van Ballegooijen E. S., Bangma C. H., Verkoelen C. F., Kidney Int., 2005, 68(4), 1660—1669
[18]	Grohe B., Taller A., Vincent P. L., Tieu L. D., Rogers K. A., Heiss A., Sørensen E. S., Mittler S., Goldberg H. A., Hunter G. K., Langmuir, 2009, 25(19), 11635—11646
[19]	King M., Mcclure W. F., Andrews L. C., International Center for Diffraction Data: Newtown Square, PA, 1992
[20]	Ouyang J. M., Yao X. Q., Tan J., Wang F. X., J. Biol. Inorg. Chem., 2011, 16(3), 405—416
[21]	Yao X. Q., Ouyang J. M., Peng H., Zhu W. Y., Chen H. Q., Carbohydr. Polym., 2012, 90(1), 392—398
[22]	Yuen J. W. M., Gohel M. D. I., Poon N. W., Shum D. K. Y., Tam P. C., Au D. W. T., Clin. Chim. Acta, 2010, 411, 1018—1026
[23]	Lieske J. C., Toback F. G., Deganello S., Kidney Int., 1998, 54(3), 796—803 doi: 10.1046/j.1523-1755.1998.00058.x URL
[24]	Ouyang J. M., Yao X. Q., Su Z. X., Cui F. Z ., Science in China Series B: Chemistry, 2003, 1, 14—20
	( 欧阳健明, 姚秀琼, 苏泽轩, 崔福斋中国科学B辑, 2003, 1, 14—20)
[25]	Lieske J. C., Toback F. G., Deganello S., Calcif. Tissue Int., 1996, 58, 195—200
[26]	Scheid C. R., Cao L. C., Honeyman T., Jonassen J. A., Front Biosci., 2004, 9(797), 797—808
[27]	Deng S. P., Ouyang J. M., Cryst. Growth Des., 2008, 9(1), 82—87
[28]	Zhang S., Su Z. X., Yao X. Q., Peng H., Deng S. P., Ouyang J. M., Mater. Sci. Eng. C, 2012, 32(4), 840—847
[29]	Yao X. Q., Peng H., Tan J., Ouyang J. M., Chinese J. Inorg. Chem., 2012, 28(3), 459—464
[30]	Rodgers A., J. Am. Soc. Nephrology: JASN, 1999, 10, S351—S354
[31]	Robertson W. G., Peacock M., Nephron, 1980, 26(3), 105—110
[32]	Ebisuno S., Umehara M., Kohjimoto Y., Ohkawa T., BJU Int, l999, 84(1), 118—122
[33]	Zorov D. B., Juhaszova M., Sollott S. J., Biochim. Biophys. Acta, 2006, 1757, 509—517
[34]	Sun X. Y., Yao X. Q., Yu K., Ouyang J. M. , Chinese J. Inorg. Chem. 2016, 32(5), 818—826
	( 孙新园, 姚秀琼, 余凯, 欧阳健明 . 无机化学学报, 2016, 32(5) 818—826)
[35]	Sun X. Y., Xu M., Ouyang J. M., ACS Omega, 2017, 2(9), 6039—6052