Cldn14基因敲除对大鼠草酸钙结石诱导形成的影响及其机制

Effects of Cldn14 gene knockout on the formation of calcium oxalate stones in rats and its mechanism

摘要

目的 探究紧密连接蛋白14（Cldn14）基因敲除对大鼠肾脏代谢及结石诱导形成的影响及其机制，为尿钙代谢及结石领域研究提供参考。方法 将野生型大鼠与同周龄Cldn14基因敲除的纯合子大鼠分别随机分为野生型对照（WC）组、野生型乙二醇（WE）组、基因敲除对照（KC）组及基因敲除乙二醇（KE）组，每组10只。其中WE、KE组采用乙二醇+氯化铵方式诱导肾结石形成，WC和KC组采用生理盐水灌胃。标准维持饲料喂养4周后收集大鼠尿液，并进行静脉血检测，同时收集大鼠肾脏行HE、钙盐染色及透射电镜观察并提取肾组织蛋白。采用蛋白质印迹法检测紧密连接蛋白16（Claudin16）及紧密连接蛋白19（Claudin19）的表达水平。结果 WE组与KE组肾小管腔内均可见晶体沉积，且KE组晶体多于WE组；相比于KC组，KE组在透射电镜下的肾小管上皮细胞中可见大量胞质内黑色结晶性包涵体，而WE组包涵体数量显著多于KE组。相比于WC和WE组，KC和KE组大鼠的血钙及血镁水平显著降低而尿钙水平则明显升高。此外，WE组相对于WC组、KE组相对于KC组尿钙水平进一步上升。KE组大鼠肾脏组织Claudin16水平显著降低，然而四组大鼠肾脏组织Claudin19水平差异均无统计学意义（P＞0.05）。结论 单纯敲除Cldn14基因并不能有效减少大鼠尿钙排泄及降低结石形成的风险，这可能与Claudin16水平降低有关。

Abstract

Objective To explore the effects of Cldn14 gene knockout on renal metabolism and stone formation in rats, so as to provide reference for research in the field of urinary calium metabolism and stone formation. Methods Cldn14 gene knockout homozygous rats and wild-type rats of the same age were randomly divided into 4 groups: wild-type control (WC) group, wild-type ethylene glycol (WE) group, gene knockout control (KC) group and gene knockout ethylene glycol (KE) group, with 10 rats in each group. The WE and KE groups were induced with ethylene glycol + ammonium chloride to form kidney stones, while the WC and KC groups received normal saline gavage. After 4 weeks of standard maintenance feeding, the urine samples were collected to detect the venous blood. The kidneys were collected for HE, Pizzolatto's staining and transmission electron microscopy. The protein in renal tissues was extracted to detect the expressions of Claudin16 and Claudin19. Results Crystal deposition was observed in the renal tubular lumen of the WE and the KE groups, and more crystals were detected in the KE group. The WE group had a large number of intracytoplasmic black crystalline inclusions observed in renal tubular epithelial cells under transmission electron microscope, followed by the KE and KC groups. Compared with WC and WE groups, KC and KE groups had significantly decreased serum calcium and magnesium levels but significantly increased urinary calcium level. In addition, the urinary calcium level was higher in the WE group than in the WC group and higher in the KE group than in the KC group. The KE group had lower level of Claudin16, but there was no significant difference in the level of Claudin19 among the 4 groups (P>0.05). Conclusion Knockout of Cldn14 gene alone cannot effectively reduce urinary calcium excretion or reduce the risk of stone formation in rats, which may be related to the decrease of Claudin16 level.

关键词

尿钙代谢 / 分子生物学 / Cldn14基因 / 肾结石 / 紧密连接蛋白 / 乙二醇

Key words

urinary calium metabolism / molecular biology / Cldn14 gene / kidney stones / tight junction protein / ethylene glycol

参考文献

[1] KNOLL T, SCHUBERT AB, FAHLENKAMP D, et al. Urolithiasis through the ages: data on more than 200 000 urinary stone analyses[J]. J Urol, 2011, 185(4): 1304-1311.

[2] LEVY FL, ADAMS-HUET B, PAK CY. Ambulatory evaluation of nephrolithiasis: an update of a 1980 protocol[J]. Am J Med, 1995, 98(1): 50-59.

[3] EDWARDS A, BONNY O. A model of calcium transport and regulation in the proximal tubule[J]. Am J Physiol Renal Physiol, 2018, 315(4): F942-F953.

[4] THORLEIFSSON G, HOLM H, EDVARDSSON V, et al. Sequence variants in the CLDN14 gene associate with kidney stones and bone mineral density[J]. Nat Genet, 2009, 41(8): 926-930.

[5] ARCIDIACONO T, SIMONINI M, LANZANI C, et al. Claudin-14 gene polymorphisms and urine calcium excretion[J]. Clin J Am Soc Nephrol, 2018, 13(10): 1542-1549.

[6] 申茂磊, 王勤章, 钱成, 等. CaSR和Claudin-14在纳米细菌肾结石形成中表达的动态研究[J]. 医学研究生学报, 2018, 31(5): 466-469.

[7] 申茂磊, 王勤章, 钱成, 等. Claudin-14在纳米细菌肾结石形成中表达的动态研究[J]. 现代泌尿外科杂志, 2019, 24(1): 64-67.

[8] 杨恒, 钱彪, 王勤章, 等. Claudin-14在两种肾结石模型肾组织中表达的对比研究[J]. 医学信息, 2022, 35(9): 87-89.

[9] 王钰, 王梦婧, 陈靖. Claudin家族在肾脏疾病中的研究进展[J]. 肾脏病与透析肾移植杂志, 2023, 32(6): 575-579.

[10] HOU J, RAJAGOPAL M, YU ASL. Claudins and the kidney[J]. J Am Soc Nephrol, 2013, 75: 479-501.

[11] ELKOUBY-NAOR L, ABASSI Z, LAGZIEL A, et al. Double gene deletion reveals lack of cooperation between Claudin11 and Claudin14 tight junction proteins[J]. Cell Tissue Res, 2008, 333(3): 427-438.

[12] GONG Y, RENIGUNTA V, HIMMERKUS N, et al. Claudin-14 regulates renal Ca²⁺ transport in response to CaSR signalling via a novel microRNA pathway [J]. EMBO J, 2012, 31(8): 1999-2012.

[13] GUHA M, BANKURA B, GHOSH S, et al. Polymorphisms in CaSR and CLDN14 genes associated with increased risk of kidney stone disease in patients from the eastern part of India[J]. PLoS One, 2015, 10(6): e130790.

[14] 孙稳, 丁国富, 王勤章. 肾草酸钙结石大鼠肾组织中Claudin-14的表达变化及意义[J]. 石河子大学学报(自然科学版), 2014, 32(3): 340-344.

[15] 王敬珅. 选择性调控CaSR-Claudin-14在大鼠草酸钙肾结石形成中的作用的初步研究[D]. 石河子大学, 2021.

[16] LEE JJ, ALZAMIL J, REHMAN S, et al. Activation of the calcium sensing receptor increases claudin-14 expression via a PLC-p38-Sp1 pathway[J]. FASEB J, 2021, 35(11): e21982.

[17] OH IH, JO CH, KIM S, et al. Thick ascending limb claudins are altered to increase calciuria and magnesiuria in metabolic acidosis[J]. Am J Physiol Renal Physiol, 2021, 320(3): F418-F428.

基金资助

国家自然科学基金项目(82060135)