Cldn14基因敲除對大鼠草酸鈣結(jié)石誘導(dǎo)形成的影響及其機制

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

LUO Peiyue^1，2，ZHENG Liying¹，CHEN Tao^1，2，ZOU Jun^1，2，LI Wei^1，2，CHEN Qi^1，2，CHENG Le^1，2，GAN Lifeng^1，2，ZHANG Fangtao^1，2，QIAN Biao^1，2

（1.Department of Urology，The First Affiliated Hospital of Gannan Medical University，Ganzhou 341000; 2.Key Laboratory of Urology and Andrology of Ganzhou，Ganzhou 341000，China）

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，Pizzolattos 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.

KEY WORDS：urinary calium metabolism；molecular biology；Cldn14 gene; kidney stones; tight junction protein; ethylene glycol

摘要：目的 探究緊密連接蛋白14（Cldn14）基因敲除對大鼠腎臟代謝及結(jié)石誘導(dǎo)形成的影響及其機制，為尿鈣代謝及結(jié)石領(lǐng)域研究提供參考。方法 將野生型大鼠與同周齡Cldn14基因敲除的純合子大鼠分別隨機分為野生型對照（WC）組、野生型乙二醇（WE）組、基因敲除對照（KC）組及基因敲除乙二醇（KE）組，每組10只。其中WE、KE組采用乙二醇+氯化銨方式誘導(dǎo)腎結(jié)石形成，WC和KC 組采用生理鹽水灌胃。標(biāo)準(zhǔn)維持飼料喂養(yǎng)4周后收集大鼠尿液，并進行靜脈血檢測，同時收集大鼠腎臟行HE、鈣鹽染色及透射電鏡觀察并提取腎組織蛋白。采用蛋白質(zhì)印跡法檢測緊密連接蛋白16（Claudin16）及緊密連接蛋白19（Claudin19）的表達水平。結(jié)果 WE組與KE組腎小管腔內(nèi)均可見晶體沉積，且KE組晶體多于WE組；相比于KC組，KE組在透射電鏡下的腎小管上皮細胞中可見大量胞質(zhì)內(nèi)黑色結(jié)晶性包涵體，而WE組包涵體數(shù)量顯著多于KE組。相比于WC和WE組，KC和KE組大鼠的血鈣及血鎂水平顯著降低而尿鈣水平則明顯升高。此外，WE組相對于WC組、KE組相對于KC組尿鈣水平進一步上升。KE組大鼠腎臟組織Claudin16水平顯著降低，然而四組大鼠腎臟組織Claudin19水平差異均無統(tǒng)計學(xué)意義（P＞0.05）。結(jié)論 單純敲除Cldn14基因并不能有效減少大鼠尿鈣排泄及降低結(jié)石形成的風(fēng)險，這可能與Claudin16水平降低有關(guān)。

關(guān)鍵詞：尿鈣代謝；分子生物學(xué)；Cldn14基因；腎結(jié)石；緊密連接蛋白；乙二醇

中圖分類號：R691.4

文獻標(biāo)志碼：ADOI：10.3969/j.issn.1009-8291.2025.02.015

鈣性結(jié)石是臨床最常見的腎結(jié)石類型^［1］。盡管病因復(fù)雜，但已有充分的證據(jù)表明，高鈣尿癥是引起腎結(jié)石的主要危險因素^［2］。……