- Email: [email protected]
A Paracrine Mechanism Involving Renal Tubular Cells, Adipocytes and Macrophages Promotes Kidney Stone Formation in a Simulated Metabolic Syndrome Environment
A Paracrine Mechanism Involving Renal Tubular Cells, Adipocytes and Macrophages Promotes Kidney Stone Formation in a Simulated Metabolic Syndrome Environment Li Zuo,* Keiichi Tozawa,* Atsushi Okada,† Takahiro Yasui, Kazumi Taguchi, Yasuhiko Ito, Yasuhiko Hirose, Yasuhiro Fujii, Kazuhiro Niimi, Shuzo Hamamoto, Ryosuke Ando, Yasunori Itoh, Jiangang Zou and Kenjiro Kohri From the Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences (LZ, AO, TY, KT, YI, YH, YF, KN, SH, RA, YI, KT, KK), Aichi, Japan, and Department of Urology, Changzhou Second Hospital, Nanjing Medical University (LZ, JZ), Nanjing, People’s Republic of China
Abbreviations and Acronyms APN ¼ adiponectin COM ¼ calcium oxalate monohydrate Ct ¼ cycle threshold DMEM ¼ Dulbecco’s modified Eagle’s medium FBS ¼ fetal bovine serum IL-6 ¼ interleukin-6 MCP-1 ¼ monocyte chemoattractant protein-1 MetS ¼ metabolic syndrome OPN ¼ osteopontin PCR ¼ polymerase chain reaction TNF-a ¼ tumor necrosis factor-a Accepted for publication January 7, 2014. Supported by Japan-China Sasakawa Medical Fellowship and Grants-in-Aid for Scientific Research 23249074, 23592374, 23592375, 23791770, 23791774, 23791775, 22591797, 22791481, 22791479, 22791484, 21791517 and 21791520 from the Japanese Ministry of Education, Culture, Sports, Science and Technology. * Equal study contribution. † Correspondence: Department of Nephrourology, Nagoya City University Graduate School of Medical Sciences, Aichi, Japan 4678601 (telephone: þ81-52-853-8266; FAX: þ81-52-8523179; e-mail: [email protected]).
Purpose: We developed an in vitro system composed of renal tubular cells, adipocytes and macrophages to simulate metabolic syndrome conditions. We investigated the molecular communication mechanism of these cells and their involvement in kidney stone formation. Materials and Methods: Mouse renal tubular cells (M-1) were cocultured with adipocytes (3T3-L1) and/or macrophages (RAW264.7). Calcium oxalate monohydrate crystals were exposed to M-1 cells after 48-hour coculture and the number of calcium oxalate monohydrate crystals adherent to the cells was quantified. The expression of cocultured medium and M-1 cell inflammatory factors was analyzed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. Results: The inflammatory markers MCP-1, OPN and TNF-a were markedly up-regulated in cocultured M-1 cells. OPN expression increased in M-1 cells cocultured with RAW264.7 cells while MCP-1 and TNF-a were over expressed in M-1 cells cocultured with 3T3-L1 cells. Coculturing M-1 cells simultaneously with 3T3-L1 and RAW264.7 cells resulted in a significant increase in calcium oxalate monohydrate crystal adherence to M-1 cells. Conclusions: Inflammatory cytokine changes were induced by coculturing renal tubular cells with adipocytes and/or macrophages without direct contact, indicating that crosstalk between adipocytes/macrophages and renal tubular cells was mediated by soluble factors. The susceptibility to urolithiasis of patients with metabolic syndrome might be due to aggravated inflammation of renal tubular cells triggered by a paracrine mechanism involving these 3 cell types. Key Words: kidney, urolithiasis, metabolic syndrome X, inflammation, obesity
UROLITHIASIS is a common urological disorder with a 10% to 12% lifetime risk in the populations of industrialized countries.1 The prevalence of kidney stones has increased worldwide
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in the last few decades. Dietary and lifestyle factors have an increasing role in the risk of stone disease with high protein and fat diets increasing the risk.2 Recent studies indicate
0022-5347/14/1916-1906/0 THE JOURNAL OF UROLOGY® © 2014 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
http://dx.doi.org/10.1016/j.juro.2014.01.013 Vol. 191, 1906-1912, June 2014 Printed in U.S.A.
PARACRINE MECHANISM PROMOTES KIDNEY STONE IN SIMULATED METABOLIC SYNDROME
that nephrolithiasis is linked to other chronic diseases, such as diabetes mellitus,3 obesity and MetS.4 MetS involves various manifestations, including visceral fat obesity, impaired glucose metabolism, atherogenic dyslipidemia and hypertension. There is compelling evidence that obesity is a crucial etiological factor in the development of MetS with obesity also considered a metabolic disease and a chronic, low grade inflammatory disease. The adipose tissue in patients with MetS is highly infiltrated by macrophages, which are responsible for the production of inflammatory cytokines.5 Despite the large amount of epidemiological evidence supporting the association between MetS and kidney stone formation4 the mechanism linking MetS to kidney stone formation is largely unknown. In vivo data from our previous study showed that MetS exacerbated the formation of calcium oxalate kidney stones by enhancing inflammation.6 We hypothesized that cellular interactions increased in adipocytes and macrophages to accelerate stone formation. To further understand how MetS promotes susceptibility to urolithiasis before and during kidney stone formation we simulated MetS by coculturing renal tubular epithelial cells with adipocytes and/or macrophages.
MATERIALS AND METHODS
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cultures were done at 37C in a 5% CO2 humidified atmosphere. Mature 3T3-L1 cells with larger lipid droplets were used after culturing for up to 21 days.
Groups, Coculturing and Cell Morphology Observations Four groups were established. The M group (controls) comprised only M-1 cells. Coculture groups included M-1 cells cocultured with mature 3T3-L1 cells (M-T group), M-1 cells cocultured with RAW264.7 cells (M-R group) and M-1 cells cocultured with 3T3-L1 and RAW264.7 cells (M-T-R group). Cocultures were grown in a TranswellÒ system. Initially, 3T3-L1 cells were differentiated in the isolated upper compartments. Medium was replaced with FBS-free medium before coculturing. RAW264.7 cells were then seeded in the upper compartment as specified, and M-1 cells were seeded in the lower compartment (fig. 1). To observe M-1 cells by polarized light microscopy appropriate size glass slides were placed on the bottom of the lower compartments before seeding M-1 cells. Cultured M-1 cells were observed by phase contrast microscopy using a DMI4000 B device (Leica, Wetzlar, Germany) 0, 12, 24 and 48 hours after initiation of the experiment.
COM Crystals Preparation. Sodium oxalate (200 mM, 0.5 ml) and calcium chloride (200 mM, 0.5 ml) were mixed at room temperature to a final concentration of 10 mM. COM crystals were equilibrated for 3 days, washed 3 times with sodium-free and chloride-free distilled water, saturated with calcium oxalate, resuspended to a final concentration of 2.92 mg/ml and adjusted to pH 6.8.
Cell Culture M-1 cells (ATCCÒ), a mouse renal cortex collecting duct cell line, were cultured in DMEM/Ham’s F-12 (Life TechnologiesÔ) supplemented with 5% FBS, 5 mM dexamethasone (Sigma-AldrichÒ) and 72 mg/ml antibiotics (penicillin and streptomycin). Cells were routinely seeded at a density of 5 104/cm2 in culture dishes (Nalge NuncÔ) at 37C in a 5% CO2 humidified atmosphere. Medium was changed every third day and cells were subcultured before forming confluent monolayers. RAW264.7 cells (ATCC), a mouse macrophage cell line, were cultured under conditions similar to those of M-1 cells except they were cultured in DMEM supplemented with 233.6 mg/ml glutamine, 72 mg/ml antibiotics (penicillin and streptomycin) and 10% FBS. Cells were routinely seeded at a density of 2 to 4 105/cm2. We maintained 3T3-L1 cells (ATCC), a mouse adipocyte cell line, in an undifferentiated state using 3T3-L1 Preadipocyte Medium (ZenBio, Research Triangle Park, North Carolina) containing FBS and antibiotics. Medium was replaced every other day. The 3T3-L1 preadipocytes were routinely seeded in culture dishes at a density of 5 104/cm2. Differentiation to mature adipocytes was induced by differentiating confluent 3T3-L1 preadipocytes with differentiation medium (Zen-Bio) containing DMEM, 10% FBS, antibiotics, insulin and the PPARg agonist isobutyl methylxanthine for 3 days. Thereafter cells were maintained in 3T3-L1 Adipocyte Maintenance Medium (Zen-Bio) and medium was changed every 2 days. All
Figure 1. In coculture system 4 groups were established, including M (control)dM-1 cells cultivated independently of other types, M-Tdcocultured M-1 cells and mature 3T3-L1 cells, M-Rdcocultured M-1 and RAW264.7 cells, and M-T-Rd coculture of 3 cell types. Microporous membranes separated upper and lower compartments.
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Exposure and image analysis. COM crystals were exposed to M-1 cells in each group at a density of 20 mg/cm2 after 48 hours of coculture. Six hours after COM exposure M-1 cells and COM crystals were detected using a DMI4000 B polarized light microscope. Images were obtained from 20 random fields at 400 magnification. At this resolution there were about 200 to 300 cells per field. Images were analyzed using Image-ProÒ Plus. The area covered by the crystalline material was determined and expressed as a percent of the whole imaging area.
Quantitative Reverse Transcriptase-PCR Total RNA was extracted from cultured M-1 cells using the RNeasyÒ Mini Kit according to manufacturer instructions. Total RNA concentration and quality were determined using a NanoVueÔ Plus Spectrophotometer. Total RNA (2 mg) was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (InvitrogenÔ) according to manufacturer instructions. Quantitative reverse transcriptase-PCR was performed using TaqManÒ Fast Universal PCR Master Mix (2X) on an ABI PrismÒ 7500 Sequence Detection System. Thermal cycling conditions consisted of incubation at 95C for 20 seconds, followed by 40 cycles at 95C for 3 seconds and at 60C for 30 seconds. We used predesigned primers and probes for mouse secreted phosphoprotein 1 (Spp1, Mm00436767_m1) encoding OPN, chemokine (C-C motif) ligand 2 (Ccl2, Mm00441242_m1) encoding MCP-1, adiponectin (Adipoq, Mm00456425_m1) encoding APN, TNF (Tnf, Mm00443258_m1) encoding TNF-a and IL-6 (Il6, Mm00446190_m1) encoding IL-6 (Applied BiosystemsÒ). Expression levels were calculated with the expression ratio against b-actin (Actb, Mm00607939_s1) as the internal standard using the DDCt method.
Cytokine Measurement in Culture Medium Enzyme-linked immunosorbent assays were performed to measure levels of soluble OPN (Mouse Osteopontin Assay Kit, IBL, Gunma, Japan), APN (Adiponectin ELISA Kit, Mouse/Rat, Otsuka Pharmaceutical, Tokyo, Japan), TNF-a (TNF-a ELISA Kit), IL-6 (Mouse IL-6 ELISA Kit, Thermo Scientific, Rockford, Illinois) and MCP-1 (Mouse CCL2/JE/MCP-1 Immunoassay, R&D SystemsÒ) produced in the supernatant of each culture dish.
cytokines Tnf, Ccl2 and Spp in M-1 cells (figs. 2 and 3). After 12 hours of coculturing Spp1 expression in M-1 cells was gradually up-regulated in the M-R and M-T-R groups. The peak of Spp1 expression occurred after 48 hours of coculturing in the M-T-R and M-R groups. It was significantly higher than in the control and M-T groups (fig. 2, A). Ccl2 expression in M-1 cells significantly increased after coculturing with adipocytes (M-T and M-T-R groups) for 24 hours. This continued until at least 48 hours of coculturing (fig. 2, B). Tnf expression in M-1 cells increased significantly in the M-T group by 12 hours with a similar tendency in the M-T-R group. After 48 hours of coculturing the level of Tnf expression was markedly increased in adipocyte cocultured M-1 cells (fig. 2, C ). The expression of Il6 and Adipoq did not statistically differ among the groups at any time point (fig. 2, D and E ). In the M-T-R group levels of proinflammatory gene mRNA (Spp1, Ccl2 and Tnf ) were progressively increased during the duration of coculturing (fig. 3). Proinflammatory Factors Induced by Adipocyte/Macrophage Coculturing After 48 hours of coculture the OPN protein level increased significantly in the M-R and M-T-R groups compared with the other 2 groups (fig. 4, A). MCP-1 and TNF-a also showed higher levels in the M-T and M-T-R groups but the IL-6 level was only increased in M-T-R coculture medium (fig. 4, B to D). Adiponectin protein levels did not significantly differ among the groups (fig. 4, E ). Enhancement of COM Crystal Adhesion to Renal Tubular Cells Morphometrically, COM crystals bound to M-1 cells in significantly higher numbers in the M-T-R group than in the other 3 groups (fig. 5).
Statistical Analysis
DISCUSSION
Data are shown as the mean SE. SPSSÒ14.0 for WindowsÒ was used to evaluate data. Statistical analysis was performed using 1-way ANOVA followed by the post hoc t-test with results considered statistically significant at p <0.05.
In patients with MetS adipose tissue is characterized by enhanced infiltration of macrophages, suggesting that inflammation has an important role in this context.7,8 Anatomically, the kidneys are surrounded by a large quantity of perinephric and intrasinus adipose tissue, and interstitial cells with fat drops are observed histologically. Thus, the kidneys could be considered huge fat storing organs and the condition of adipose tissue is expected to affect kidney function. We developed an in vitro coculture system involving renal tubular cells, adipocytes and macrophages to simulate the pathological conditions of MetS. To our knowledge we report for the first time that coculturing with differentiated 3T3-L1 and
RESULTS Cocultured Renal Tubular Cell Morphology and Inflammatory Response Induction M-1 cells showed a normal paving stone morphology in the presence or absence of other cocultured cells. Coculturing with differentiated 3T3-L1 cells and/or RAW264.7 cells in Transwell systems elicited remarkable up-regulation of the proinflammatory
PARACRINE MECHANISM PROMOTES KIDNEY STONE IN SIMULATED METABOLIC SYNDROME
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Figure 2. Induction of inflammatory gene changes by coculture, as assayed by quantitative reverse transcriptase-PCR using TaqMan assays. Open bars indicate M group. Light gray bars indicate M-T group. Dark gray bars indicate M-R group. Black bars indicate M-T-R group. A, Spp1. Single asterisk indicates p <0.05 vs control. B, Ccl2. Double asterisks indicate p <0.01 vs control. C, Tnf. D, Il6. E, Adipoq. h, hours.
RAW264.7 cells resulted in remarkable upregulation of proinflammatory cytokines, including OPN, TNF-a and MCP-1, as well as increased COM crystal adhesion in M-1 cells. OPN expression increased significantly in the M-T-R and M-R coculture groups. There was also much more COM deposition, consistent with in vivo results.9 OPN is a multifunctional protein secreted by different types of cells that has important roles in biomineralization. Approximately 75% of urinary stones are predominantly composed of calcium oxalate and a considerable amount of OPN.10 Importantly, OPN has a crucial role in the morphological conversion of calcium oxalate crystals to stones in mouse kidneys.11 The surface of COM crystals is positively charged and anionic cell surface molecules may lead to crystal retention and renal calculus formation.12,13
OPN contains an aspartic acid rich region called the poly Asp86-93 domain, which serves as the center of the interaction between the OPN molecule and calcium oxalate14 through a strong electrostatic attraction between the carboxyl groups of aspartic acids and Ca2þ ions. In the current study M-1 cells cocultured with adipocytes and/or macrophages had increased OPN levels, which might have enhanced COM crystal adhesion. Furthermore, during the adhesion process COM crystals may have injured the renal tubular cells, allowing more COM crystals to adhere to M-1 cells. The increase in the OPN mRNA level in M-1 cells only occurred when they were cocultured with RAW264.7 cells (M-R and M-T-R groups), suggesting that higher OPN expression was triggered by some soluble factors from RAW264.7 cells. Interestingly, the medium OPN level was higher than in
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Figure 3. In M-T-R group (solid curve) change in Spp1, Ccl2 and Tnf proinflammatory gene mRNA expression depended on coculture duration. Relative expression levels were calculated using 2eDDCt. h, hours. Dashed curve indicates M-T-R group. Single asterisk indicates p <0.05 vs control. Double asterisks indicate p <0.01 vs control. Dagger indicates p <0.05 vs same group at 0 hours.
the M-R group. This indicates a role for adipocytes in increasing excreted OPN and crystal binding of M-1 cells since adipocytes can up-regulate OPN expression in mononuclear/macrophage cells.15 Increased MCP-1 and TNF-a levels were detected in each coculture group containing adipocytes (M-T and M-T-R groups). An in vivo study also showed that MCP-1 was over expressed in the kidney of a stone forming MetS rat model.9 MCP-1 is a mediator of inflammatory processes, extracellular matrix production and the deposition of various urinary crystals in the kidneys.16e18 Kidney cells produced MCP-1 in response to various proinflammatory stimuli18,19 and its increased expression was identified in kidney diseases involving significant inflammation.20 In our previous study renal macrophage migration and crystal phagocytosis were associated with the expression of inflammation related genes, including MCP-1, during calcium oxalate kidney stone formation.21 Adipose tissue is the principal source of TNF-a in obesity.22 However, macrophages are another primary source of adipose derived TNF-a and
increased TNF-a in obesity is due to increased macrophage infiltration into adipose tissue.23 TNF-a is also a cell marker of oxidative stress after lithotripsy.24 In the current study MCP-1 and TNF-a were increased in groups containing 3T3-L1 cells (M-T and M-T-R groups), suggesting that adipocytes stimulated the inflammatory responses in renal tubular cells. Up-regulation of MCP-1 and TNF-a expression was associated with increased COM crystal adhesion in the M-T-R group, suggesting an essential role of inflammation at the early stages of kidney stone formation in a MetS environment. Interestingly, although the IL-6 mRNA level did not change in the groups, the medium level of IL-6 in the M-T-R group was significantly higher than in the other 3 groups, suggesting that the higher IL-6 level resulted from interaction between RAW264.7 and 3T3-L1 cells.25 Increased IL-6 levels were also reported in the urine of patients with urolithiasis26 and in human renal epithelial cells exposed to oxalate,27 demonstrating that IL-6 might have a critical role in the initiation/progression of urolithiasis.28
PARACRINE MECHANISM PROMOTES KIDNEY STONE IN SIMULATED METABOLIC SYNDROME
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Figure 4. Soluble cytokines in coculture medium were detected using enzyme-linked immunosorbent assays. Graphs represent results after 48-hour coculture. A, OPN increased significantly in M-1/RAW264.7 and M-1/RAW264.7/3T3-L1 coculture groups. Single asterisk indicates p <0.05 vs control. Double asterisks indicate p <0.01 vs control. B and C, MCP-1 and TNF-a were higher in M-1/3T3-L1 and M-1/RAW264.7/3T3-L1 coculture groups. D, IL-6 was only increased in M-1/RAW264.7/3T3-L1 coculture medium. E, APN changed significantly among groups.
The changes in inflammatory cytokine levels induced by coculturing renal tubular cells with adipocytes and/or macrophages without direct
contact indicate that the crosstalk between adipocytes/macrophages and renal tubular cells is mediated by soluble factors released by these cells.
Figure 5. COM crystals bound to M-1 cells in significantly higher numbers in M-1/RAW264.7/3T3-L1 coculture group than in other 3 groups. A, Polarized light microscopy reveals M-1 cells containing COM crystals. Reduced from 400. B, area occupied by crystalline material was measured and expressed as percent of image area. Asterisk indicates p <0.05.
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This reflects the paracrine interaction between renal tubular cells with adipose tissue adipocytes and macrophages in a MetS environment. However, our study does not prove certain points, including 1) the origin of these soluble factors, 2) the mechanism of producing these factors under coculture and 3) the signal mechanism needed to change the characteristics of each cell. Further studies are required to resolve these points.
CONCLUSIONS Inflammatory cytokines were increased in a simulated MetS environment, resulting in increased COM crystal adhesion to renal tubular epithelial cells. In MetS macrophages and adipocytes elicited chronic, low grade inflammation in renal tubular epithelial cells via a paracrine mechanism. Inflammation related injury may then initiate a cascade of events leading to further crystallization, crystal retention and stone development.
REFERENCES 1. Moe OW: Kidney stones: pathophysiology and medical management. Lancet 2006; 367: 333. 2. Curhan GC: Epidemiology of stone disease. Urol Clin North Am 2007; 34: 287. 3. Taylor EN, Stampfer MJ and Curhan GC: Diabetes mellitus and the risk of nephrolithiasis. Kidney Int 2005; 68: 1230. 4. Taylor EN, Stampfer MJ and Curhan GC: Obesity, weight gain, and the risk of kidney stones. JAMA 2005; 293: 455. 5. Surmi BK and Hasty AH: Macrophage infiltration into adipose tissue: initiation, propagation and remodeling. Future Lipidol 2008; 3: 545. 6. Fujii Y, Okada A, Yasui T et al: Effect of adiponectin on kidney crystal formation in metabolic syndrome model mice via inhibition of inflammation and apoptosis. PLoS One 2013; 8: e61343. 7. Cancello R and Clement K: Is obesity an inflammatory illness? Role of low-grade inflammation and macrophage infiltration in human white adipose tissue. BJOG 2006; 113: 1141. 8. Heilbronn LK and Campbell LV: Adipose tissue macrophages, low grade inflammation and insulin resistance in human obesity. Curr Pharm Des 2008; 14: 1225. 9. Okamoto M, Kohjimoto Y, Iba A et al: Calcium oxalate crystal deposition in metabolic syndrome model rat kidneys. Int J Urol 2010; 17: 996.
12. Farell G, Huang E, Kim SY et al: Modulation of proliferating renal epithelial cell affinity for calcium oxalate monohydrate crystals. J Am Soc Nephrol 2004; 15: 3052.
20. Rovin BH, Rumancik M, Tan L et al: Glomerular expression of monocyte chemoattractant protein-1 in experimental and human glomerulonephritis. Lab Invest 1994; 71: 536.
13. Lieske JC, Leonard R and Swift H: Adhesion of calcium oxalate monohydrate crystals to anionic sites on the surface of renal epithelial cells. Am J Physiol 1996; 270: F192.
21. Okada A, Yasui T, Fujii Y et al: Renal macrophage migration and crystal phagocytosis via inflammatory-related gene expression during kidney stone formation and elimination in mice: Detection by association analysis of stonerelated gene expression and microstructural observation. J Bone Miner Res 2010; 25: 2701.
14. Chien YC, Masica DL, Gray JJ et al: Modulation of calcium oxalate dihydrate growth by selective crystal-face binding of phosphorylated osteopontin and polyaspartate peptide showing occlusion by sectoral (compositional) zoning. J Biol Chem 2009; 284: 23491. 15. Samuvel DJ, Sundararaj KP, Li Y et al: Adipocytemononuclear cell interaction, Toll-like receptor 4 activation, and high glucose synergistically up-regulate osteopontin expression via an interleukin 6-mediated mechanism. J Biol Chem 2010; 285: 3916. 16. Khan SR: Role of renal epithelial cells in the initiation of calcium oxalate stones. Nephron Exp Nephrol 2004; 98: e55. 17. Umekawa T, Chegini N and Khan SR: Increased expression of monocyte chemoattractant protein-1 (MCP-1) by renal epithelial cells in culture on exposure to calcium oxalate, phosphate and uric acid crystals. Nephrol Dial Transplant 2003; 18: 664.
10. Kohri K, Nomura S, Kitamura Y et al: Structure and expression of the mRNA encoding urinary stone protein (osteopontin). J Biol Chem 1993; 268: 15180.
18. Umekawa T, Chegini N and Khan SR: Oxalate ions and calcium oxalate crystals stimulate MCP-1 expression by renal epithelial cells. Kidney Int 2002; 61: 105.
11. Okada A, Nomura S, Saeki Y et al: Morphological conversion of calcium oxalate crystals into stones is regulated by osteopontin in mouse kidney. J Bone Miner Res 2008; 23: 1629.
19. Rovin BH, Yoshiumura T and Tan L: Cytokineinduced production of monocyte chemoattractant protein-1 by cultured human mesangial cells. J Immunol 1992; 148: 2148.
22. Halle M, Berg A, Northoff H et al: Importance of TNF-alpha and leptin in obesity and insulin resistance: a hypothesis on the impact of physical exercise. Exerc Immunol Rev 1998; 4: 77. 23. Weisberg SP, McCann D, Desai M et al: Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003; 112: 1796. 24. Clark DL, Connors BA, Evan AP et al: Localization of renal oxidative stress and inflammatory response after lithotripsy. BJU Int 2009; 103: 1562. 25. Suganami T, Nishida J and Ogawa Y: A paracrine loop between adipocytes and macrophages aggravates inflammatory changes: role of free fatty acids and tumor necrosis factor alpha. Arterioscler Thromb Vasc Biol 2005; 25: 2062. 26. Rhee E, Santiago L, Park E et al: Urinary IL-6 is elevated in patients with urolithiasis. J Urol 1998; 160: 2284. 27. Huang MY, Chaturvedi LS, Koul S et al: Oxalate stimulates IL-6 production in HK-2 cells, a line of human renal proximal tubular epithelial cells. Kidney Int 2005; 68: 497. 28. Hirano T: Interleukin-6. In: The Cytokine Handbook. Edited by AW Thomson. San Diego: Academic Press 1994; pp 145e168.
Abbreviations and Acronyms APN ¼ adiponectin COM ¼ calcium oxalate monohydrate Ct ¼ cycle threshold DMEM ¼ Dulbecco’s modified Eagle’s medium FBS ¼ fetal bovine serum IL-6 ¼ interleukin-6 MCP-1 ¼ monocyte chemoattractant protein-1 MetS ¼ metabolic syndrome OPN ¼ osteopontin PCR ¼ polymerase chain reaction TNF-a ¼ tumor necrosis factor-a Accepted for publication January 7, 2014. Supported by Japan-China Sasakawa Medical Fellowship and Grants-in-Aid for Scientific Research 23249074, 23592374, 23592375, 23791770, 23791774, 23791775, 22591797, 22791481, 22791479, 22791484, 21791517 and 21791520 from the Japanese Ministry of Education, Culture, Sports, Science and Technology. * Equal study contribution. † Correspondence: Department of Nephrourology, Nagoya City University Graduate School of Medical Sciences, Aichi, Japan 4678601 (telephone: þ81-52-853-8266; FAX: þ81-52-8523179; e-mail: [email protected]).
Purpose: We developed an in vitro system composed of renal tubular cells, adipocytes and macrophages to simulate metabolic syndrome conditions. We investigated the molecular communication mechanism of these cells and their involvement in kidney stone formation. Materials and Methods: Mouse renal tubular cells (M-1) were cocultured with adipocytes (3T3-L1) and/or macrophages (RAW264.7). Calcium oxalate monohydrate crystals were exposed to M-1 cells after 48-hour coculture and the number of calcium oxalate monohydrate crystals adherent to the cells was quantified. The expression of cocultured medium and M-1 cell inflammatory factors was analyzed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. Results: The inflammatory markers MCP-1, OPN and TNF-a were markedly up-regulated in cocultured M-1 cells. OPN expression increased in M-1 cells cocultured with RAW264.7 cells while MCP-1 and TNF-a were over expressed in M-1 cells cocultured with 3T3-L1 cells. Coculturing M-1 cells simultaneously with 3T3-L1 and RAW264.7 cells resulted in a significant increase in calcium oxalate monohydrate crystal adherence to M-1 cells. Conclusions: Inflammatory cytokine changes were induced by coculturing renal tubular cells with adipocytes and/or macrophages without direct contact, indicating that crosstalk between adipocytes/macrophages and renal tubular cells was mediated by soluble factors. The susceptibility to urolithiasis of patients with metabolic syndrome might be due to aggravated inflammation of renal tubular cells triggered by a paracrine mechanism involving these 3 cell types. Key Words: kidney, urolithiasis, metabolic syndrome X, inflammation, obesity
UROLITHIASIS is a common urological disorder with a 10% to 12% lifetime risk in the populations of industrialized countries.1 The prevalence of kidney stones has increased worldwide
1906
j
www.jurology.com
in the last few decades. Dietary and lifestyle factors have an increasing role in the risk of stone disease with high protein and fat diets increasing the risk.2 Recent studies indicate
0022-5347/14/1916-1906/0 THE JOURNAL OF UROLOGY® © 2014 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
http://dx.doi.org/10.1016/j.juro.2014.01.013 Vol. 191, 1906-1912, June 2014 Printed in U.S.A.
PARACRINE MECHANISM PROMOTES KIDNEY STONE IN SIMULATED METABOLIC SYNDROME
that nephrolithiasis is linked to other chronic diseases, such as diabetes mellitus,3 obesity and MetS.4 MetS involves various manifestations, including visceral fat obesity, impaired glucose metabolism, atherogenic dyslipidemia and hypertension. There is compelling evidence that obesity is a crucial etiological factor in the development of MetS with obesity also considered a metabolic disease and a chronic, low grade inflammatory disease. The adipose tissue in patients with MetS is highly infiltrated by macrophages, which are responsible for the production of inflammatory cytokines.5 Despite the large amount of epidemiological evidence supporting the association between MetS and kidney stone formation4 the mechanism linking MetS to kidney stone formation is largely unknown. In vivo data from our previous study showed that MetS exacerbated the formation of calcium oxalate kidney stones by enhancing inflammation.6 We hypothesized that cellular interactions increased in adipocytes and macrophages to accelerate stone formation. To further understand how MetS promotes susceptibility to urolithiasis before and during kidney stone formation we simulated MetS by coculturing renal tubular epithelial cells with adipocytes and/or macrophages.
MATERIALS AND METHODS
1907
cultures were done at 37C in a 5% CO2 humidified atmosphere. Mature 3T3-L1 cells with larger lipid droplets were used after culturing for up to 21 days.
Groups, Coculturing and Cell Morphology Observations Four groups were established. The M group (controls) comprised only M-1 cells. Coculture groups included M-1 cells cocultured with mature 3T3-L1 cells (M-T group), M-1 cells cocultured with RAW264.7 cells (M-R group) and M-1 cells cocultured with 3T3-L1 and RAW264.7 cells (M-T-R group). Cocultures were grown in a TranswellÒ system. Initially, 3T3-L1 cells were differentiated in the isolated upper compartments. Medium was replaced with FBS-free medium before coculturing. RAW264.7 cells were then seeded in the upper compartment as specified, and M-1 cells were seeded in the lower compartment (fig. 1). To observe M-1 cells by polarized light microscopy appropriate size glass slides were placed on the bottom of the lower compartments before seeding M-1 cells. Cultured M-1 cells were observed by phase contrast microscopy using a DMI4000 B device (Leica, Wetzlar, Germany) 0, 12, 24 and 48 hours after initiation of the experiment.
COM Crystals Preparation. Sodium oxalate (200 mM, 0.5 ml) and calcium chloride (200 mM, 0.5 ml) were mixed at room temperature to a final concentration of 10 mM. COM crystals were equilibrated for 3 days, washed 3 times with sodium-free and chloride-free distilled water, saturated with calcium oxalate, resuspended to a final concentration of 2.92 mg/ml and adjusted to pH 6.8.
Cell Culture M-1 cells (ATCCÒ), a mouse renal cortex collecting duct cell line, were cultured in DMEM/Ham’s F-12 (Life TechnologiesÔ) supplemented with 5% FBS, 5 mM dexamethasone (Sigma-AldrichÒ) and 72 mg/ml antibiotics (penicillin and streptomycin). Cells were routinely seeded at a density of 5 104/cm2 in culture dishes (Nalge NuncÔ) at 37C in a 5% CO2 humidified atmosphere. Medium was changed every third day and cells were subcultured before forming confluent monolayers. RAW264.7 cells (ATCC), a mouse macrophage cell line, were cultured under conditions similar to those of M-1 cells except they were cultured in DMEM supplemented with 233.6 mg/ml glutamine, 72 mg/ml antibiotics (penicillin and streptomycin) and 10% FBS. Cells were routinely seeded at a density of 2 to 4 105/cm2. We maintained 3T3-L1 cells (ATCC), a mouse adipocyte cell line, in an undifferentiated state using 3T3-L1 Preadipocyte Medium (ZenBio, Research Triangle Park, North Carolina) containing FBS and antibiotics. Medium was replaced every other day. The 3T3-L1 preadipocytes were routinely seeded in culture dishes at a density of 5 104/cm2. Differentiation to mature adipocytes was induced by differentiating confluent 3T3-L1 preadipocytes with differentiation medium (Zen-Bio) containing DMEM, 10% FBS, antibiotics, insulin and the PPARg agonist isobutyl methylxanthine for 3 days. Thereafter cells were maintained in 3T3-L1 Adipocyte Maintenance Medium (Zen-Bio) and medium was changed every 2 days. All
Figure 1. In coculture system 4 groups were established, including M (control)dM-1 cells cultivated independently of other types, M-Tdcocultured M-1 cells and mature 3T3-L1 cells, M-Rdcocultured M-1 and RAW264.7 cells, and M-T-Rd coculture of 3 cell types. Microporous membranes separated upper and lower compartments.
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Exposure and image analysis. COM crystals were exposed to M-1 cells in each group at a density of 20 mg/cm2 after 48 hours of coculture. Six hours after COM exposure M-1 cells and COM crystals were detected using a DMI4000 B polarized light microscope. Images were obtained from 20 random fields at 400 magnification. At this resolution there were about 200 to 300 cells per field. Images were analyzed using Image-ProÒ Plus. The area covered by the crystalline material was determined and expressed as a percent of the whole imaging area.
Quantitative Reverse Transcriptase-PCR Total RNA was extracted from cultured M-1 cells using the RNeasyÒ Mini Kit according to manufacturer instructions. Total RNA concentration and quality were determined using a NanoVueÔ Plus Spectrophotometer. Total RNA (2 mg) was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (InvitrogenÔ) according to manufacturer instructions. Quantitative reverse transcriptase-PCR was performed using TaqManÒ Fast Universal PCR Master Mix (2X) on an ABI PrismÒ 7500 Sequence Detection System. Thermal cycling conditions consisted of incubation at 95C for 20 seconds, followed by 40 cycles at 95C for 3 seconds and at 60C for 30 seconds. We used predesigned primers and probes for mouse secreted phosphoprotein 1 (Spp1, Mm00436767_m1) encoding OPN, chemokine (C-C motif) ligand 2 (Ccl2, Mm00441242_m1) encoding MCP-1, adiponectin (Adipoq, Mm00456425_m1) encoding APN, TNF (Tnf, Mm00443258_m1) encoding TNF-a and IL-6 (Il6, Mm00446190_m1) encoding IL-6 (Applied BiosystemsÒ). Expression levels were calculated with the expression ratio against b-actin (Actb, Mm00607939_s1) as the internal standard using the DDCt method.
Cytokine Measurement in Culture Medium Enzyme-linked immunosorbent assays were performed to measure levels of soluble OPN (Mouse Osteopontin Assay Kit, IBL, Gunma, Japan), APN (Adiponectin ELISA Kit, Mouse/Rat, Otsuka Pharmaceutical, Tokyo, Japan), TNF-a (TNF-a ELISA Kit), IL-6 (Mouse IL-6 ELISA Kit, Thermo Scientific, Rockford, Illinois) and MCP-1 (Mouse CCL2/JE/MCP-1 Immunoassay, R&D SystemsÒ) produced in the supernatant of each culture dish.
cytokines Tnf, Ccl2 and Spp in M-1 cells (figs. 2 and 3). After 12 hours of coculturing Spp1 expression in M-1 cells was gradually up-regulated in the M-R and M-T-R groups. The peak of Spp1 expression occurred after 48 hours of coculturing in the M-T-R and M-R groups. It was significantly higher than in the control and M-T groups (fig. 2, A). Ccl2 expression in M-1 cells significantly increased after coculturing with adipocytes (M-T and M-T-R groups) for 24 hours. This continued until at least 48 hours of coculturing (fig. 2, B). Tnf expression in M-1 cells increased significantly in the M-T group by 12 hours with a similar tendency in the M-T-R group. After 48 hours of coculturing the level of Tnf expression was markedly increased in adipocyte cocultured M-1 cells (fig. 2, C ). The expression of Il6 and Adipoq did not statistically differ among the groups at any time point (fig. 2, D and E ). In the M-T-R group levels of proinflammatory gene mRNA (Spp1, Ccl2 and Tnf ) were progressively increased during the duration of coculturing (fig. 3). Proinflammatory Factors Induced by Adipocyte/Macrophage Coculturing After 48 hours of coculture the OPN protein level increased significantly in the M-R and M-T-R groups compared with the other 2 groups (fig. 4, A). MCP-1 and TNF-a also showed higher levels in the M-T and M-T-R groups but the IL-6 level was only increased in M-T-R coculture medium (fig. 4, B to D). Adiponectin protein levels did not significantly differ among the groups (fig. 4, E ). Enhancement of COM Crystal Adhesion to Renal Tubular Cells Morphometrically, COM crystals bound to M-1 cells in significantly higher numbers in the M-T-R group than in the other 3 groups (fig. 5).
Statistical Analysis
DISCUSSION
Data are shown as the mean SE. SPSSÒ14.0 for WindowsÒ was used to evaluate data. Statistical analysis was performed using 1-way ANOVA followed by the post hoc t-test with results considered statistically significant at p <0.05.
In patients with MetS adipose tissue is characterized by enhanced infiltration of macrophages, suggesting that inflammation has an important role in this context.7,8 Anatomically, the kidneys are surrounded by a large quantity of perinephric and intrasinus adipose tissue, and interstitial cells with fat drops are observed histologically. Thus, the kidneys could be considered huge fat storing organs and the condition of adipose tissue is expected to affect kidney function. We developed an in vitro coculture system involving renal tubular cells, adipocytes and macrophages to simulate the pathological conditions of MetS. To our knowledge we report for the first time that coculturing with differentiated 3T3-L1 and
RESULTS Cocultured Renal Tubular Cell Morphology and Inflammatory Response Induction M-1 cells showed a normal paving stone morphology in the presence or absence of other cocultured cells. Coculturing with differentiated 3T3-L1 cells and/or RAW264.7 cells in Transwell systems elicited remarkable up-regulation of the proinflammatory
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Figure 2. Induction of inflammatory gene changes by coculture, as assayed by quantitative reverse transcriptase-PCR using TaqMan assays. Open bars indicate M group. Light gray bars indicate M-T group. Dark gray bars indicate M-R group. Black bars indicate M-T-R group. A, Spp1. Single asterisk indicates p <0.05 vs control. B, Ccl2. Double asterisks indicate p <0.01 vs control. C, Tnf. D, Il6. E, Adipoq. h, hours.
RAW264.7 cells resulted in remarkable upregulation of proinflammatory cytokines, including OPN, TNF-a and MCP-1, as well as increased COM crystal adhesion in M-1 cells. OPN expression increased significantly in the M-T-R and M-R coculture groups. There was also much more COM deposition, consistent with in vivo results.9 OPN is a multifunctional protein secreted by different types of cells that has important roles in biomineralization. Approximately 75% of urinary stones are predominantly composed of calcium oxalate and a considerable amount of OPN.10 Importantly, OPN has a crucial role in the morphological conversion of calcium oxalate crystals to stones in mouse kidneys.11 The surface of COM crystals is positively charged and anionic cell surface molecules may lead to crystal retention and renal calculus formation.12,13
OPN contains an aspartic acid rich region called the poly Asp86-93 domain, which serves as the center of the interaction between the OPN molecule and calcium oxalate14 through a strong electrostatic attraction between the carboxyl groups of aspartic acids and Ca2þ ions. In the current study M-1 cells cocultured with adipocytes and/or macrophages had increased OPN levels, which might have enhanced COM crystal adhesion. Furthermore, during the adhesion process COM crystals may have injured the renal tubular cells, allowing more COM crystals to adhere to M-1 cells. The increase in the OPN mRNA level in M-1 cells only occurred when they were cocultured with RAW264.7 cells (M-R and M-T-R groups), suggesting that higher OPN expression was triggered by some soluble factors from RAW264.7 cells. Interestingly, the medium OPN level was higher than in
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Figure 3. In M-T-R group (solid curve) change in Spp1, Ccl2 and Tnf proinflammatory gene mRNA expression depended on coculture duration. Relative expression levels were calculated using 2eDDCt. h, hours. Dashed curve indicates M-T-R group. Single asterisk indicates p <0.05 vs control. Double asterisks indicate p <0.01 vs control. Dagger indicates p <0.05 vs same group at 0 hours.
the M-R group. This indicates a role for adipocytes in increasing excreted OPN and crystal binding of M-1 cells since adipocytes can up-regulate OPN expression in mononuclear/macrophage cells.15 Increased MCP-1 and TNF-a levels were detected in each coculture group containing adipocytes (M-T and M-T-R groups). An in vivo study also showed that MCP-1 was over expressed in the kidney of a stone forming MetS rat model.9 MCP-1 is a mediator of inflammatory processes, extracellular matrix production and the deposition of various urinary crystals in the kidneys.16e18 Kidney cells produced MCP-1 in response to various proinflammatory stimuli18,19 and its increased expression was identified in kidney diseases involving significant inflammation.20 In our previous study renal macrophage migration and crystal phagocytosis were associated with the expression of inflammation related genes, including MCP-1, during calcium oxalate kidney stone formation.21 Adipose tissue is the principal source of TNF-a in obesity.22 However, macrophages are another primary source of adipose derived TNF-a and
increased TNF-a in obesity is due to increased macrophage infiltration into adipose tissue.23 TNF-a is also a cell marker of oxidative stress after lithotripsy.24 In the current study MCP-1 and TNF-a were increased in groups containing 3T3-L1 cells (M-T and M-T-R groups), suggesting that adipocytes stimulated the inflammatory responses in renal tubular cells. Up-regulation of MCP-1 and TNF-a expression was associated with increased COM crystal adhesion in the M-T-R group, suggesting an essential role of inflammation at the early stages of kidney stone formation in a MetS environment. Interestingly, although the IL-6 mRNA level did not change in the groups, the medium level of IL-6 in the M-T-R group was significantly higher than in the other 3 groups, suggesting that the higher IL-6 level resulted from interaction between RAW264.7 and 3T3-L1 cells.25 Increased IL-6 levels were also reported in the urine of patients with urolithiasis26 and in human renal epithelial cells exposed to oxalate,27 demonstrating that IL-6 might have a critical role in the initiation/progression of urolithiasis.28
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Figure 4. Soluble cytokines in coculture medium were detected using enzyme-linked immunosorbent assays. Graphs represent results after 48-hour coculture. A, OPN increased significantly in M-1/RAW264.7 and M-1/RAW264.7/3T3-L1 coculture groups. Single asterisk indicates p <0.05 vs control. Double asterisks indicate p <0.01 vs control. B and C, MCP-1 and TNF-a were higher in M-1/3T3-L1 and M-1/RAW264.7/3T3-L1 coculture groups. D, IL-6 was only increased in M-1/RAW264.7/3T3-L1 coculture medium. E, APN changed significantly among groups.
The changes in inflammatory cytokine levels induced by coculturing renal tubular cells with adipocytes and/or macrophages without direct
contact indicate that the crosstalk between adipocytes/macrophages and renal tubular cells is mediated by soluble factors released by these cells.
Figure 5. COM crystals bound to M-1 cells in significantly higher numbers in M-1/RAW264.7/3T3-L1 coculture group than in other 3 groups. A, Polarized light microscopy reveals M-1 cells containing COM crystals. Reduced from 400. B, area occupied by crystalline material was measured and expressed as percent of image area. Asterisk indicates p <0.05.
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This reflects the paracrine interaction between renal tubular cells with adipose tissue adipocytes and macrophages in a MetS environment. However, our study does not prove certain points, including 1) the origin of these soluble factors, 2) the mechanism of producing these factors under coculture and 3) the signal mechanism needed to change the characteristics of each cell. Further studies are required to resolve these points.
CONCLUSIONS Inflammatory cytokines were increased in a simulated MetS environment, resulting in increased COM crystal adhesion to renal tubular epithelial cells. In MetS macrophages and adipocytes elicited chronic, low grade inflammation in renal tubular epithelial cells via a paracrine mechanism. Inflammation related injury may then initiate a cascade of events leading to further crystallization, crystal retention and stone development.
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