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Increased crystal–cell interaction in vitro under co-culture of renal tubular cells and adipocytes by in vitro co-culture paracrine systems simulating metabolic syndrome

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Abstract

We established an experimental co-culture system for renal tubular cells and adipocytes to investigate kidney stone formation mechanisms under metabolic syndrome (MetS) conditions and examined the interaction between these cells morphologically and genetically. M-1s and 3T3-L1s were cultured individually (control, CON), with 24-h culture media from each cell type added to the other cell type (replacement, RP) in 2-layer co-culture dishes for 24 h (transwell, TW). M-1s were then exposed to calcium oxalate monohydrate (COM) crystals, and attached 14C-labeled COM crystals were quantified. Expression of kidney stone- and adipocyte-related genes was analyzed. The radioactivity of adherent COM crystals significantly increased in TW and was relatively higher in RP compared to CON. M-1s demonstrated significant upregulation of adiponectin (Adipoq) in RP and secreted phosphoprotein 1 (Spp1) in TW compared to CON before COM crystal exposure, and significant downregulation of Spp1 in TW and upregulation of tumor necrosis factor (Tnf), interleukin 6 (Il-6), and chemokine (C–C motif) ligand 2 (Ccl2) compared to CON after COM crystal exposure. 3T3-L1s showed significant upregulation of Spp1, Adipoq, Tnf-α, and Ccl2 compared to CON. Enzyme-linked immunosorbent assays of co-culture medium revealed significantly increased TNF-α in TW. Our results highlight the potential for paracrine interactions between renal tubular cells and adipocytes and suggest that MetS conditions may lead to kidney stone formation.

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Acknowledgments

We thank Ms. N. Kasuga, Ms. Kawamura, and Ms. Ichikawa for their secretarial assistance. This work was partly supported by Grants-in-Aid for Scientific Research (Nos. 23249074, 23592374, 23592375, 23791770, 23791774, 23791775, 22591797, 22791481, 22791479, 22791484, 21791517, and 21791520) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

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Authors and Affiliations

  1. Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya City, Aichi, 467-8601, Japan

    Jun Ichikawa, Atsushi Okada, Kazumi Taguchi, Yasuhiro Fujii, Li Zuo, Kazuhiro Niimi, Shuzo Hamamoto, Yasue Kubota, Yukihiro Umemoto, Yasunori Itoh, Takahiro Yasui, Noriyasu Kawai, Keiichi Tozawa & Kenjiro Kohri

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  1. Jun Ichikawa
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  2. Atsushi Okada
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  3. Kazumi Taguchi
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  4. Yasuhiro Fujii
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  5. Li Zuo
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Correspondence to Atsushi Okada.

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240_2013_612_MOESM1_ESM.pptx

Supplemental Figure 1. Quantitative PCR of Sod2, Tgfb and Icam1 expression by M-1s Expression values of each gene were determined by quantitative PCR using TaqMan assays. The expression level was calculated by normalization to the internal standard β-actin using the ΔΔCt method. Data are presented as the mean ± SE. *P < 0.05 vs. day 0. †P < 0.05, ††P < 0.01 vs. CON at the same time point. Sod2 expression without COM crystal treatment demonstrated no significant expression changes or difference between the 3 groups. COM exposure did not induce a significant Sod2 expression change or difference. Tgfb expression by COM crystal-untreated M-1s demonstrated no significant change. COM crystal exposure was associated with a significant difference in Tgfb expression by 373-L1s in the RP group. A decline of Icam1 expression was observed in COM crystal-untreated M-1s in the RP group. Icam1 expression of COM-treated M-1s in the TW and CON groups significantly increased after 6 h. COM-treated 3T3-L1s in the TW group demonstrated significantly higher expression of Icam1 at 6 h than CON-group cells. Supplemental Figure 2. Quantitative PCR of Sod2, Tgfb and Icam1 expression of 3T3-L1s Expression values of each gene were determined by quantitative PCR using TaqMan assays. The expression level was calculated by normalization to the internal standard β-actin using the ΔΔCt method. Data are presented as the mean ± SE. P < 0.05 vs. day 0. †P < 0.05, ††P < 0.01 vs. CON at the same time point. COM crystal-exposed 3T3-L1 cells of the TW group demonstrated persistently higher Sod2 expression than CON. Tgfb expression of COM-untreated 3T3-L1s in the TW groups significantly increased but that of the COM-treated cells in the RP and TW groups demonstrated significant decrease than CON group. The Icam1 expression of COM crystal-exposed 3T3-L1s in the TW group significantly increased after 6 h. (PPTX 103 kb)

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Ichikawa, J., Okada, A., Taguchi, K. et al. Increased crystal–cell interaction in vitro under co-culture of renal tubular cells and adipocytes by in vitro co-culture paracrine systems simulating metabolic syndrome. Urolithiasis 42, 17–28 (2014). https://doi.org/10.1007/s00240-013-0612-5

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