Experimental colitis is associated with transcriptional inhibition of Na+/Ca2+ exchanger isoform 1 (NCX1) expression by interferon γ in the renal distal convoluted tubules

Abstract

NCX1 is a Na(+)/Ca(2+) exchanger, which is believed to provide a key route for basolateral Ca(2+) efflux in the renal epithelia, thus contributing to renal Ca(2+) reabsorption. Altered mineral homeostasis, including intestinal and renal Ca(2+) transport may represent a significant component of the pathophysiology of the bone mineral density loss associated with Inflammatory Bowel Diseases (IBD). The objective of our research was to investigate the effects of TNBS and DSS colitis and related inflammatory mediators on renal Ncx1 expression. Colitis was associated with decreased renal Ncx1 expression, as examined by real-time RT-PCR, Western blotting, and immunofluorescence. In mIMCD3 cells, IFNγ significantly reduced Ncx1 mRNA and protein expression. Similar effects were observed in cells transiently transfected with a reporter construct bearing the promoter region of the kidney-specific Ncx1 gene. This inhibitory effect of IFNγ is mediated by STAT1 recruitment to the proximal promoter region of Ncx1. Further in vivo study with Stat1(-/-) mice confirmed that STAT1 is indeed required for the IFNγ mediated Ncx1 gene regulation. These results strongly support the hypothesis that impaired renal Ca(2+) handling occurs in experimental colitis. Negative regulation of NCX1- mediated renal Ca(2+) absorption by IFNγ may significantly contribute to the altered Ca(2+) homeostasis in IBD patients and to IBD-associated loss of bone mineral density.

Keywords: colitis; distal convoluted tubule; epithelial transport; gene transcription; inflammatory bowel disease (IBD); interferon; kidney; signal transducers and activators of transcription 1 (STAT1); sodium-calcium exchange.

FIGURE 1.

Real-time RT-PCR analysis of pro-inflammatory cytokine mRNA expression in colonic tissues of TNBS (top panels) and DSS colitis (bottom panels). Cytokine expression was normalized to that of TBP relative to a calibrator (normalized Ct value obtained from healthy control mice) and expressed as 2^−ΔΔCt (*p < 0.05 colitis versus controls; Student's t test).

FIGURE 2.

Reduced expression of renal Ncx1 mRNA and protein in experimental colitis. A, Ncx1 mRNA expression as determined by real-time RT-PCR in the kidneys of controls and TNBS-treated (left panel) or DSS-treated (right panel) mice. Ncx1 expression was normalized to that of TBP relative to a calibrator (normalized Ct value obtained from untreated healthy mice) and expressed as 2^−ΔΔCt (mean ± S.D.; *, p < 0.05 colitis versus controls; Student t test). B, NCX1 Western blot was performed with 20 μg of kidney lysate protein control and TNBS-treated (left panel) or DSS-treated (right panel) mice. Loading was normalized to expression of β-actin. C, immunofluorescent labeling of NCX1 in the kidney of mice with TNBS-induced (top panels) or DSS-induced colitis (bottom panels). NCX1 immunoreactivity is depicted in green; Na/Cl transporter NCCT (encoded by SLC12A3 gene) was used as a marked of DCT and is depicted in pink; nuclei were counterstained with DAPI, blue. Magnification ×400.

FIGURE 3.

A, effects of inflammatory cytokines on gene expression of Ncx1 in mIMCD3 cells. Cells were treated with individual cytokines (100 units/ml of IFNγ, 10 ng/ml of TNFα, and 2 ng/ml of IL1β) for 24 h, and the impact on the Ncx1 mRNA expression was examined by real-time RT-PCR. Ncx1 expression was normalized to that of TATA-box binding protein (TBP) relative to a calibrator (normalized Ct value obtained from untreated cells) and expressed as 2^−ΔΔCt (*, p < 0.05 IFNγ versus control; ANOVA followed by Fisher test; n = 3). B, effects of individual cytokines on the Ncx1 promoter activity in mIMCD3 cells transiently transfected with pGL3–703 Ncx1 promoter construct. 24 h post-transfection, cells were treated for 24 h with indicated cytokines. Luciferase activity (RLU) was normalized to mg protein and expressed relative to the activity of the pGL3–703 construct in PBS-treated cells (mean ± S.E., *, p < 0.05 versus PBS, ANOVA followed by Fisher test; n = 3).

FIGURE 4.

A–F, effects of IFNγ on cellular levels of Ncx1 mRNA and protein. A, Ncx1 mRNA expression as determined by real-time RT-PCR in mIMCD3 cells treated with PBS or 100 units/ml of mIFNγ for 0, 12, 24, 48, and 72 h (mean ± S.D. of three independent experiments). Ncx1 expression was normalized to that of TATA-box binding protein (TBP) relative to a calibrator (normalized Ct value obtained from untreated healthy mice) and expressed as 2^−ΔΔCt; (*, p < 0.05 treated versus control, Student's t test). B, representative Western blot showing NCX1, pTyr⁷⁰¹-STAT1 and GAPDH (as loading control) in mIFNγ-treated mIMCD3 cells. C, Ncx1 mRNA expression of in the kidneys of mIFNγ injected P_TRPV5-EGFP transgenic mice was quantified by real-time RT-PCR as described for panel A. D, representative Western blot analysis of NCX1, pTyr⁷⁰¹-STAT1, GFP, and GAPDH proteins in renal lysates of PBS- and mIFNγ-injected P_TRPV5-EGFP transgenic mice. E, NCX1-mediated Ca²⁺ uptake in GFP⁺ epithelial cells of the renal DCT isolated from mIFNγ-treated P_TRPV5-EGFP transgenic mice. PBS or mIFNγ were injected intraperitoneal daily for 3 days in P_TRPV5-EGFP transgenic mice, which express GFP expression under the control of the TRPV5 promoter (C57Bl/6 background). GFP⁺ cells were flow-sorted using FACAria and used for the NCX1 activity assay as described under “Experimental Procedures.” Data from three independent experiments are presented as Na⁺-dependent Ca²⁺ uptake relative to PBS-injected mice (*, p < 0.05, Student's t test). F, fractional urinary Ca²⁺ excretion (FECa²⁺) in IFNγ-injected mice. Mean ± S.D., *, p < 0.05; Student's t test.

FIGURE 5.

A, identification of the transcription initiation site (+1) of the mouse renal Ncx1. Representative DNA gel of the 5′-RLM-RACE product with mouse kidney RNA. −TAP refers to negative control lacking Tobacco Acid Pyrophosphatase (TAP) used for removing the capping nucleoside. B, luciferase reporter gene activity with mouse kidney specific Ncx1 promoter constructs: pGL3-Basic, pGL3–110, pGL3–269, pGL3–379, and pGL3–703 (promoter length relative to the identified transcription start site) were transiently transfected in to mIMCD3 cells treated with PBS or 100 units/ml mIFNγ for 24 h. Luciferase activity (RLU) was normalized to mg protein and expressed relative to the activity of the promoterless PGL3-basic (pGL3) vector in PBS-treated cells (mean ± S.E., *, p < 0.05 PBS versus IFNγ, Student's t test; n = 3). C, effects of IFNγ on the pGL-703 WT promoter, or construct with mutated core GAS element. Data calculated as above and normalized to the activity of pGL-703 promoter in PBS-treated cells (mean ± S.E., *, p < 0.05 PBS versus IFNγ, Student's t test; n = 3). D, inducible STAT1 recruitment to Ncx1 gene promoter in IFNγ-treated mIMCD3 cells. ChIP assay was performed using a control rabbit IgG or an anti-STAT1 antibody as described under “Experimental Procedures.” Representative DNA gel and the summary of real-time PCR analysis are depicted in left and right panels, respectively. −Ab indicates control IgG, +Ab indicates anti-STAT1 antibody. Bar graph (mean ± S.E.) indicates an over 10-fold difference in STAT1 recruitment (*, p < 0.05 PBS versus IFNγ, Student's t test; n = 3).

FIGURE 6.

STAT1 is required to down-regulate the expression of NCX1 by IFNγ. WT and Stat1^−/− mice (129S6/SvEv background) were injected with PBS or mIFNγ through i.p for 3 days and kidneys were collected for RNA and protein extraction. A, real-time RT-PCR analysis. Ncx1 expression was normalized to that of TBP relative to a calibrator (normalized Ct value obtained from untreated WT or Stat1^−/− mice) and expressed as 2^−ΔΔCt; (*, p < 0.05 treated versus control, Student's t test; n = 5). B, representative Western blot analysis of NCX1, pTyr⁷⁰¹-STAT1, and GAPDH (loading control) in renal lysates of PBS and mIFNγ injected WT and Stat1^−/− mice.