Nitric oxide inhibits glomerular TGF-beta signaling via SMOC-1

Abstract

Cytokines and nitric oxide (NO) stimulate rat mesangial cells to synthesize and secrete inflammatory mediators. To understand better the signaling pathways that contribute to this response, we exposed rat mesangial cells to the prototypic inflammatory cytokine IL-1beta and analyzed the changes in the pattern of gene expression. IL-1beta downregulated the gene encoding the matricellular glycoprotein secreted modular calcium-binding protein 1 (SMOC-1) in mesangial cells. Inflammatory cytokines attenuated SMOC-1 mRNA and protein expression through endogenous production of NO, which activated the soluble guanylyl cyclase. Silencing SMOC-1 expression with small interfering RNA decreased the formation of TGF-beta, reduced SMAD binding to DNA, and decreased mRNA expression of genes regulated by TGF-beta. In a rat model of anti-Thy-1 glomerulonephritis, glomerular SMOC-1 mRNA and protein decreased and inducible NO synthase expression increased simultaneously. Treatment of nephritic rats with the inducible NO synthase-specific inhibitor l-N(6)-(1-iminoethyl)-lysine prevented SMOC-1 downregulation. In summary, these data suggest that NO attenuates SMOC-1 expression in acute glomerular inflammation, thereby limiting TGF-beta-mediated profibrotic signaling.

Figure 1.

Effects of IL-1β, 1400W, and TNF-α on SMOC-1 mRNA and protein expression. Quiescent mesangial cells were treated as indicated for 24 h with IL-1β (1 nM) in the presence (+) or absence (−) of the iNOS inhibitor 1400W (2 mM). (A) Representative Northern blot of three independent experiments for SMOC-1 mRNA. (B) The data obtained from the analysis of SMOC-1 protein levels in conditioned media by direct ELISA for SMOC-1. (C) Nitrite levels as analyzed in conditioned media. (D) Quiescent mesangial cells were treated with the indicated concentrations of TNF-α in absence or presence of IL-1β as indicated for 24 h, and conditioned media were subjected to direct ELISA for SMOC-1. Data are means ± SD (n = 3). ***P < 0.001, **P < 0.01 versus vehicle-treated controls; ^§§§P < 0.001, ^§P < 0.05 versus IL-1β (1 nM).

Figure 2.

Effects of IL-1β and DETA-NO on SMOC-1 mRNA and protein expression. Quiescent mesangial cells were treated with IL-1β and the NO donating reagent DETA-NO as indicated for 8, 16, or 24 h. Total RNA (20 μg each) was subjected to Northern blotting with probes specific for SMOC-1 mRNA and 18S rRNA. (A) Densitometric evaluation of SMOC-1/18S ratios. (B) Conditioned media were subjected to direct ELISA using an antibody specific for SMOC-1. Data are expressed as percentage of SMOC-1 protein level in untreated cells (control). (C) Time course of nitrite formation in mesangial cells. Data are means ± SD (n = 3 to 6). ***P < 0.001, **P < 0.01, *P < 0.05 versus vehicle-treated controls; ^§§§P < 0.001, ^§P < 0.05 versus IL-1β (1 nM).

Figure 3.

Effects of the sGC inhibitor ODQ and the sGC activator YC-1 on cytokine-mediated SMOC-1 mRNA and protein expression. Quiescent mesangial cells were treated with IL-1β, ODQ, or combinations of IL-1β plus ODQ as indicated for 24 h. (A and B) Thereafter, mRNA was isolated for Northern blotting against a SMOC-1 and 18S rRNA probe (A) and conditioned media were subjected to direct ELISA for SMOC-1 (B). Quiescent mesangial cells were treated with the indicated concentrations of YC-1 for 8 h. Total RNA (20 μg each) was subjected to Northern blotting with probes specific for SMOC-1 mRNA and 18S rRNA. (C) Representative Northern blot experiment with the densitometric evaluation of SMOC-1/18S ratios of seven experiments. Conditioned media were analyzed for SMOC-1 protein expression by ELISA. (D) SMOC-1 protein levels of six experiments. Data are means ± SD (n = 3). ***P < 0.001, **P < 0.01, *P < 0.05 versus vehicle-treated controls; ^§§P < 0.01 versus IL-1β alone; ^§P < 0.05 versus 10 μM YC-1.

Figure 4.

Silencing of SMOC-1 decreases mRNA expression of TGF-associated genes. Mesangial cells were left untreated (control) or transfected with vehicle, 10 nM of specific siRNA targeting SMOC-1, or control siRNA. After transfection, serum-starved mesangial cells were kept in serum-free medium for 24 h. Total RNA (20 μg each) was subjected to Northern blotting with probes specific for CTGF mRNA, SMOC-1 mRNA, TGF-β1 mRNA, PAI-1 mRNA, biglycan mRNA, and 18S rRNA. (A) Densitometric evaluation of CTGF/18S ratios is displayed as means ± SD (n = 5). (B) Representative blots for TGF-β1, PAI-1, and biglycan. ***P < 0.001, **P < 0.01, *P < 0.05 versus untransfected controls; ^$$P < 0.01 versus nonspecific control siRNA.

Figure 5.

Silencing of SMOC-1 decreases active and total TGF-β1 protein in conditioned media. Mesangial cells were transfected with specific siRNA targeting SMOC-1 (10 nM) or negative control siRNA. After transfection, serum-starved mesangial cells were kept in serum-free medium for 24 h. Conditioned media were analyzed for active and—after acidic activation of TGF-β—total TGF-β using a commercially available TGF-β1 ELISA kit. Data are means ± SD (n = 4). **P < 0.01 versus nonspecific control siRNA.

Figure 6.

Silencing of SMOC-1 decreases binding of Smad proteins on a consensus SBE. (A) Quiescent mesangial cells were transfected with or without control siRNA or siRNA directed against SMOC-1. Twenty-four hours after transfection, media were removed and cells were treated with or without TGF-β1 (10 ng/ml) for 2 h. Nuclear extracts were prepared, and formation of SMAD-SBE complexes was analyzed by EMSA. (B) Supershift analysis with nuclear extracts of TGF-β1–treated mesangial cells. The indicated antibodies were added 60 min before the addition of the labeled oligonucleotides. (C) Competition analysis with unlabeled SMAD-SBE oligonucleotide. Thirty minutes before the addition of the labeled oligonucleotides, nuclear extracts of TGF-β1–treated mesangial cells were preincubated with a 100-fold excess of the unlabeled competitor oligonucleotide. The data shown are representative of three similar experiments.

Figure 7.

In situ hybridization for SMOC-1 in normal and Thy-1 nephritic kidneys (16 h). (a, A through C) In the normal kidney, SMOC-1 is expressed in mesangial cells (A and B, black arrow), podocytes (A and B, white arrowhead), and in distal tubular epithelial cells (A and C, black arrowhead). (A, insert) Sense riboprobe. (E) In Thy-1 GN, the expression of SMOC-1 was slightly reduced 16 h after the induction of nephritis. (F versus E) Pretreatment with the specific iNOS inhibitor L-NIL prevented the decline in SMOC-1 expression in glomeruli from Thy-1 nephritic rats. (D) Expression of SMOC-1 in the normal kidney. (G) Sense riboprobe. There was no difference in controls receiving L-NIL or vehicle (data not shown). Bars indicate magnification. (b) Time course of SMOC-1 expression in glomeruli from control and anti–Thy-1 nephritic kidneys by in situ hybridization. Reduction of SMOC-1 expression was observed at days 1 (B) and 3 (C) after anti–Thy-1–induced GN compared with control kidneys (A). (D and E) Levels of SMOC-1 expression 7 (D) and 14 (E) days after induction of disease were comparable with control kidneys.

Figure 8.

Analysis of SMOC-1 mRNA and protein expression in the course of anti–Thy-1 GN. (A) Total mRNA from glomeruli obtained at days 1, 3, and 7 after induction of anti–Thy-1 GN was subjected to RT-PCR using primers SMOC-1 and SMOC-2 for 30 cycles (40 s at 94°C, 2 min at 53°C, and 2 min at 72°C). Transcripts were separated on a 1% agarose gel. The intensity of the bands was analyzed densitometrically and corrected for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Data are means ± SD, P < 0.05 versus vehicle-treated controls (n = 4). (B) Glomerular proteins from control animals and anti–Thy-1 nephritic rats (16 h) treated with or without L-NIL were subjected to direct ELISA for SMOC-1. Data are means ± SD (n = 3). **P < 0.01 versus vehicle-treated control animals.