Renal tubular epithelial cells treated with calcium oxalate up-regulate S100A8 and S100A9 expression in M1-polarized macrophages via interleukin 6

Document Type : Original Article


1 Department of Urology, Guizhou Provincial People’s Hospital, Guiyang, Guizhou, 550000, China

2 Zunyi Medical University, Zunyi, Guizhou, 550000, China



Objective(s): Calgranulins S100A8 and S100A9 are common in renal stones and they are up-regulated in both urinary exosomes and kidneys of stone patients. Renal sources and important regulators for S100A8 and S100A9 in nephrolithiasis were explored in this study.
Materials and Methods: We identified S100A8 and S100A9 abundance in various renal cells by searching the Single Cell Type Atlas. Macrophages were polarized from human myeloid leukemia mononuclear cells. Human proximal renal tubular epithelial cells (HK-2) were stimulated with calcium oxalate monohydrate (COM). Coculture experiments involving HK-2 cells and macrophages were conducted. qPCR, Western blotting, ELISA, and immunofluorescence were used for detecting interleukin 6 (IL6), S100A8, and S100A9.
Results: The Single Cell Type Atlas showed that S100A8 and S100A9 in human kidneys primarily originated from macrophages. M1 was the predominant macrophage type expressing S100A8 and S100A9. Direct culture with COM did not affect the expression of these two calgranulins in M1 macrophages but coculture with COM-treated HK-2 cells did. COM could promote HK-2 cells to secrete IL6. IL6 could up-regulate S100A8 and S100A9 expression in macrophages of M1 type. In addition, 0.5 μM SC144 (a kind of IL6 inhibitor) significantly prevented COM-treated HK-2 cells from up-regulating S100A8 and S100A9 expression in macrophages of M1 type.
Conclusion: M1-polarized macrophages were the predominant cell type expressing S100A8 and S100A9 in the kidneys of nephrolithiasis patients. CaOx crystals can promote renal tubular epithelial cells to secrete IL6 to up-regulate S100A8 and S100A9 expression in macrophages of M1 type.


1. Thongprayoon C, Krambeck AE, Rule AD. Determining the true burden of kidney stone disease. Nat Rev Nephrol 2020;16:736-746.
2. Romero V, Akpinar H, Assimos DG. Kidney stones: A global picture of prevalence, incidence, and associated risk factors. Rev Urol 2010;12:e86-96.
3. Khan SR, Canales BK, Dominguez-Gutierrez PR. Randall’s plaque and calcium oxalate stone formation: Role for immunity and inflammation. Nat Rev Nephrol 2021; 17: 417-433.
4. Khan SR, Pearle MS, Robertson WG, Gambaro G, Canales BK, Doizi S, et al. Kidney stones. Nat Rev Dis Primers 2016, 2: 16008.
5. Antonelli JA, Maalouf NM, Pearle MS, Lotan Y. Use of the national health and nutrition examination survey to calculate the impact of obesity and diabetes on cost and prevalence of urolithiasis in 2030. Eur Urol 2014; 66: 724-729.
6. Yang Y, Hong S, Li C, Zhang J, Hu H, Chen X, et al. Proteomic analysis reveals some common proteins in the kidney stone matrix. PeerJ 2021; 9: e11872. 
7. Wang Q, Sun Y, Yang Y, Li C, Zhang J, Wang S. Quantitative proteomic analysis of urinary exosomes in kidney stone patients. Transl Androl Urol 2020; 9: 1572-1584. 
8. Chaiyarit S, Thongboonkerd V. Changes in mitochondrial proteome of renal tubular cells induced by calcium oxalate monohydrate crystal adhesion and internalization are related to mitochondrial dysfunction. J Proteome Res 2012; 11: 3269-3280. 
9. Tsuji H, Wang W, Sunil J, Shimizu N, Yoshimura K, Uemura H, et al. Involvement of renin-angiotensin-aldosterone system in calcium oxalate crystal induced activation of NADPH oxidase and renal cell injury. World J Urol 2016; 34: 89-95.
10. Khan A, Khan SR, Gilani AH. Studies on the in vitro and in vivo antiurolithic activity of Holarrhena antidysenterica. Urol Res 2012; 40: 671-681. 
11. Khan A, Wang W, Khan SR. Calcium oxalate nephrolithiasis and expression of matrix GLA protein in the kidneys. World J Urol 2014; 32: 123-130.
12. Wang XW, Seed B. A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res 2003; 31: e154.
13. Karlsson M, Zhang C, Méar L, Zhong W, Digre A, Katona B, et al. A single-cell type transcriptomics map of human tissues. Sci Adv 2021; 7: eabh2169.
14. Wang Z, Zhang JW, Zhang Y, Zhang SP, Hu QY, Liang H. Analyses of long non-coding RNA and mRNA profiling using RNA sequencing in calcium oxalate monohydrate-stimulated renal tubular epithelial cells. Urolithiasis 2019; 47: 225–234. 
15. Wang S, Song R, Wang Z, Jing Z, Wang S, Ma J. S1000A8/A9 in inflammation. Front Immunol 2018; 9: 1298. 
16. Ding Z, Du F, Averitt V RG, Jakobsson G, Rönnow CF, Rahman M, et al. Targeting S100A9 reduces neutrophil recruitment, inflammation and lung damage in abdominal sepsis. Int J Mol Sci 2021; 22: 12923. 
17. He GY, Zhao CH, Wu DG, Cheng H, Sun LA, Zhang DL, et al. S100A8 promotes inflammation via Toll-like receptor 4 after experimental traumatic brain injury. Front Neurosci 2021; 14: 616559. 
18. Pruenster M, Vogl T, Roth J, Sperandio M. S100A8/A9: From basic science to clinical application. Pharmacol Ther 2016; 167: 120-131.
19. New SE, Goettsch C, Aikawa M, Marchini JF, Shibasaki M, Yabusaki K, et al. Macrophage-derived matrix vesicles: An alternative novel mechanism for microcalcification in atherosclerotic plaques. Circ Res 2013; 113: 72-77. 
20. Taguchi K, Okada A, Unno R, Hamamoto S, Yasui T. Macrophage function in calcium oxalate kidney stone formation: A systematic review of literature. Front Immunol 2021; 12: 673690.
21. Dominguez-Gutierrez PR, Kwenda EP, Khan SR, Canales BK. Immunotherapy for stone disease. Curr Opin Urol 2020; 30: 183-189.
22. Oishi Y, Manabe I. Macrophages in inflammation, repair and regeneration. Int Immunol 2018; 30: 511-528.
23. Taguchi K, Okada A, Hamamoto S, Unno R, Moritoki Y, Ando R, et al. M1/M2-macrophage phenotypes regulate renal calcium oxalate crystal development. Sci Rep 2016; 6: 35167. 
24. Zhu J, Wang Q, Li C, Lu Y, Hu H, Qin B, et al. Inhibiting inflammation and modulating oxidative stress in oxalate-induced nephrolithiasis with the Nrf2 activator dimethyl fumarate. Free Radic Biol Med 2019; 134: 9-22. 
25. Qian X, Wu W, Hu H, Yu X, Wang S, Zhu J, et al. The role of reactive oxygen species derived from different NADPH oxidase isoforms and mitochondria in oxalate-induced oxidative stress and cell injury. Urolithiasis 2022; 50: 149-158.  
26. Wu Y, Xun Y, Zhang J, Hu H, Qin B, Wang T, et al. Resveratrol attenuates oxalate-induced renal oxidative injury and calcium oxalate crystal deposition by regulating TFEB-induced autophagy pathway. Front Cell Dev Biol 2021; 9: 638759.
27. Deng YL, Liu YL, Tao ZW, Wang X. The role of cell-crystal reaction mediated inflammation in the formation of intrarenal calcium oxalate crystals. Zhonghua Wai Ke Za Zhi 2018; 56: 733-736. 
28. Liu X, Yuan P, Sun X, Chen Z. Hydroxycitric acid inhibits renal calcium oxalate deposition by reducing oxidative stress and inflammation. Curr Mol Med 2019; 20: 527-535.
29. Eggers K, Sikora K, Lorenz M, Taubert T, Moobed M, Baumann G, et al. RAGE-dependent regulation of calcium-binding proteins S100A8 and S100A9 in human THP-1. Exp Clin Endocrinol Diabetes 2011; 119: 353-357.