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Abstract
Calcineurin (CaN) is activated in diabetes and plays a role in glomerular hypertrophy and extracellular matrix (ECM) accumulation. Here, kidneys from diabetic model mice were investigated for the expression of the regulator of CaN 1 (RCAN1) isoform 4 (RCAN1.4) which had been shown to be transcriptionally upregulated by CaN activation. We found the increased immunoreactivity for RCAN1 in the glomerular cells of db/db mice and streptozotocin-induced diabetic mice. In concordance, the expression of RCAN1 protein and RCAN1.4 mRNA were elevated in the whole kidney sample from db/db mice. Interleukin-1β (IL-1β), tumor necrosis factor-α, and glycated albumin (AGE-BSA) were identified as inducers of RCAN1.4 in mesangial cells. Pretreatment of cyclosporine A blocked the increases of RCAN1.4 stimulated by IL-1β or AGE-BSA, suggesting that activation of CaN is required for the RCAN1.4 induction. Stable transfection of RCAN1.4 in Mes-13 mesangial cells upregulated several factors relevant to ECM production and degradation. These results suggested that RCAN1.4 might act as a link between CaN activation and ECM turnover in diabetic nephropathy.
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Fig. 1.
RCAN1.4 expression in the kidneys of diabetic mouse models. (A) Representative photomicrographs of RCAN1 immunohistochemistry in the glomerulus of kidney sections that were obtained from diabetic (db/db) and non-diabetic (db/m) mice at the age of 20 weeks, and from diabetic (STZ+) and non-diabetic (STZ-) C57BL mice at 16 weeks after treatment (400×). (B) Western blot analysis shows increased levels of RCAN1 protein in the whole kidney homogenates of diabetic (db/db) mice compared to non-diabetic (db/m) mice. Alpha-tubulin (α-tubulin) served as a loading control. (C) Specific induction of RCAN1.4 mRNA. The relative amount of mRNAs was estimated by real-time PCR, normalized by GAPDH, and the values for db/db mice (open bars) are relative to the expression observed in db/m mice (filled bars; arbitrarily designated a value of 1). Data are means±SE of five experiments. ∗p<0.01, db/m vs. db/db.
Fig. 2.
RCAN1.4 mRNA induction by diverse stimuli in mouse primary mesangial cells. (A) Mesangial cells were isolated from glomeruli by differential sieving, and then the cells of passages 7 to 10 in quiescent state were stimulated with the agents indicated below for 24 h or cultured for 48 h in high glucose condition (33 mM) or in osmotically balanced control medium (5.9 mM glucose plus 27 mM mannitol). Effect of the each stimulation was tested for RCAN1.4 mRNA induction, as described in the Methods. Concentrations were: TGF-β1 (4 ng/ml), ANG II (2μM), VEGF (10 ng/ml), IL-1β (20 ng/ml), TNF-α (20 ng/ml), MCP-1 (100 ng/ml), oxidative stress (300 μM of H2O2), BSA (50 μg/ml), and AGE-BSA (50 μg/ml). ∗p<0.01 compared to no-treatment. (B) Effect of CsA on IL-1β- or AGE-BSA-mediated RCAN1.4 induction was tested. Quiescent mesangial cells were stimulated with IL-1β (20 ng/ml) or AGE-BSA (50 μg/ml) in presence of CsA (1 μM; open bars) or vehicle (filled bars). After 24 h, mRNAs were isolated to determine RCAN1.4 mRNA induction. All the values are means±SE of five experiments/group. Values are expressed relative to the expression observed in untreated controls (arbitrarily assigned a value of 1). ∗p<0.01 vs. the corresponding control (vehicle) group.
Fig. 3.
Expression patterns of ECM-related factors in Mes-13 cells stably transfected with RCAN1.4. Mes-13 cells were stably transfected with an expression vector containing RCAN1.4 construct (RCAN1.4; open bars) or vector alone (GFP; filled bars). From the actively dividing cells in complete medium, mRNAs were prepared and then subjected to analysis for the ECM-related gene expression with specific primers (Table 1). All values are means±SE of at least 5 experiments/group and the values of RCAN1.4-transfected cells are expressed relative to the expression observed in GFP-transfected cells (arbitrarily designated a value of 1). ∗p<0.01, RCAN1.4 vs. GFP group. Col, collagen.
Table 1.
Primer sequences used for real-time RT PCR
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