分析HK-2腎小管細胞株暴露在草酸、草酸鈣結晶或兩者合併後分子表現的差異：建立理想的草酸鈣結石生物體外模型

劉展榮^1,2、何冠達^1,2,3、黃鶴翔^1,2、蔡曜聲^3,4

1.成大醫院泌尿科;2.成大醫學院泌尿部;3.成大臨床醫學研究所;4.成大醫院臨醫中心

 Molecular analysis of HK-2 cells exposed to oxalate and calcium oxalate crystals: in vitro model of calcium oxalate nephrolithiasis

 Chan-Jung Liu^1,2, Kuan-Ta Ho^1,2,3, Ho-Shiang Huang^1,2, Yau-Sheng Tsai^3,4

1. Department of Urology, National Cheng Kung University Hospital, Tainan, Taiwan

2. Department of Urology, College of Medicine, National Cheng Kung University, Tainan, Taiwan 704302

3. Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan 704302, Taiwan

4. Clinical Medicine Research Center, National Cheng Kung University Hospital, Tainan 704302, Taiwan

Purpose: Several in vitro studies on calcium oxalate (CaOx) nephrolithiasis have been performed under experimental conditions that closely simulate actual stone-forming conditions. HK-2 cell, a proximal tubular epithelial cell line derived from normal human kidney, is most widely used in stable culture. However, there is still no consensus regarding the experimental methods to simulate CaOx nephrolithiasis, including oxalate (Ox) alone, calcium oxalate monohydrate (COM) crystals, CaOx crystals, and the combination of Ox and COM. The aim of the study was to investigate the molecular effects of four in vitro experimental models of CaOx nephrolithiasis on HK-2 cells; thus to determine the optimal pattern of CaOx kidney stones in vitro.

Materials and Methods: HK-2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). When 70–80% confluent, HK-2 cells were harvested under NaOx (0.25, 0.5, 1, 2, 4 mM), COM (10, 20, 40, 80, 160, 320 µg/cm²), CaOx (10, 20, 40, 80 µg/cm²) or combination treatment and used for 24 hrs.

Results: Under low dose of calcium crystals (< 80 µg/cm²), including COM and CaOx, nearly no significantly changes in the expressions of phosphor-NFkB (pNFkB) and NLRP3, which are the two archetypical molecular drivers of the inflammasome. The expressions of  pNFkB significantly increased till the dose of calcium crystals was above 80 µg/cm². Nevertheless, low dose Ox exposure can activate the expression of  pNFkB, and the combination of Ox and COM can generate the most significantly increased expression of pNFkB and NLRP3. The exposure of Ox alone cannot increase the ratio of LC3II/I, that is unable to activate autophagy. However, if calcium crystals are present, autophagy activity is upregulated in a dose-dependent manner. Finally, the expression of full-length osteopontin (OPN), a critical regulator of calcium nephrolithiasis, was increased both by the exposure of Ox alone and calcium crystals. It is worth to mention that the combination of Ox and COM led to OPN cleavage, possibly via activating matrix metalloproteinase-3 (MMP-3), which induces epithelial-mesenchymal transition in kidney.

Conclusions: The presence of calcium crystals upregulated autophagy; whereas Ox activated inflammasome pathway.