Angiotensin II- and glucose-stimulated extracellular matrix production: mediation by the insulin-like growth factor (IGF) axis in a murine mesangial cell line

Abstract

In diabetic nephropathy, glomerular mesangial cells exhibit aberrant anabolic activity that includes excessive production of extracellular matrix (ECM) proteins, leading to crowding of filtration surface areas and possible renal failure. In the present study, a murine mesangial cell line (MES-13 cells) was studied to determine the roles of the renin-angiotensin system (RAS) and the insulin-like growth factor (IGF) axis in the anabolic response to elevated glucose levels. Culture of MES-13 cells in medium containing supra-physiological glucose concentrations (>5.5 mmol/l) resulted in increased production of ECM proteins including laminin, fibronectin, and heparan sulfate proteoglycan with concurrent increases in IGF-binding protein (IGFBP)-2 production. These responses were blocked by the angiotensin receptor antagonists saralasin and losartan, while exogenous angiotensin II (Ang II) treatment directly stimulated increases in ECM and IGFBP-2. In all experiments, IGFBP-2 levels were correlated with anabolic activity implicating IGFBP-2 as a possible mediator in cellular responses to high glucose and Ang II. Such mediation appears to involve IGFBP-2 modulation of IGF-I signaling, since all responses to high glucose or Ang II were blocked by immuno-neutralization of IGF-I. These data suggest alterations in the IGF axis as key mechanisms underlying nephropathic responses of mesangial cells to Ang II and high glucose.

Fig. 1

Effect of increased ambient glucose concentration on production of IGFBP-2 and ECM components in cultured MES-13 cells. Data shown are from cells cultured for 72 h in medium containing 5.5 or 25 mmol/l glucose. IGFBP-2 (a), laminin (b), fibronectin (c), and heparan sulfate (d) were each measured using Western immunoblot analysis as described in Methods. Data illustrated are representative of experiments repeated four separate times (n = 4). MES-13 cells cultured in 25 mmol/l glucose concentrations exhibited >3-fold increases in each measured component as compared with corresponding levels in cells cultured in 5.5 mmol/l glucose

Fig. 2

Effect of increasing Ang II concentrations on IGFBP-2 production in MES-13 cells. In cells cultured in 5.5 mmol/l glucose medium, addition of Ang II at concentrations between 10⁻⁸ and 10⁻⁵ M elicits a dose-related increase in IGFBP-2. Recombinant bovine IGFBP-2 (rBP-2; 1 μg; far left lane) serves as a positive control for the Western immunoblot procedure; data shown represent 72 h culture experiments

Fig. 3

Effect of increasing Ang II concentrations on fibronectin production in MES-13 cells as measured by ELISA. In cells cultured in 5.5 mmol/l glucose medium, addition of Ang II at concentrations between 10⁻⁸ and 10⁻⁵ M resulted in a dose-related increase in fibronectin levels in conditioned medium (P<0.01). Data shown were generated from four separate 72 h culture experiments. ^a,b,c Superscripts denote significantly different mean fibronectin concentrations (P < 0.05)

Fig. 4

Effect of angiotensin receptor antagonists on Ang II-induced IGFBP-2 secretion. Addition of 10⁻⁶ M Ang II increased IGFBP-2 to the level observed in high glucose (25 mmol/l)-treated cells, whereas this effect was blocked in the presence of saralasin (to 13.8% of Ang II-stimulated IGFBP-2 at 10⁻⁵ M saralasin dose; upper panel) or losartan (to undetectable levels at the two highest losartan doses; lower panel). Data shown represent 72 h culture experiments, which were repeated three times with the same result

Fig. 5

Effect of the AT1 angiotensin receptor antagonist, losartan, on Ang II-induced ECM production. Fibronectin production stimulated by 10⁻⁶ M Ang II (upper panel) is reduced by addition of losartan at concentrations ≥10⁻⁷ M (to <30% of Ang II-stimulated levels at 10⁻⁶ and 10⁻⁵ M losartan, P < 0.05). Ang II-stimulated laminin production is also reduced by losartan to undetectable levels at 10⁻⁶ M (lower panel). Data shown represent 72 h experiments, which were repeated three times with the same result. Similar effects of the general antagonist, saralasin, are observed under the same experimental conditions (data not shown)

Fig. 6

Effect of the AT1 receptor antagonist, losartan, on fibronectin (upper panel) and laminin (lower panel) production in MES-13 cells cultured in high glucose (25 mmol/l) medium. Losartan reduced levels of both ECM proteins in a dose-dependent manner in MES-13 cells cultured in 25 mmol/l glucose medium (by >50% at 10⁻⁶ M for both fibronectin and laminin). Data shown represent 72 h experiments, which were repeated three times with the same result. Similar effects of the general antagonist, saralasin, are observed under the same experimental conditions (data not shown).

Fig. 7

Effect of angiotensin receptor antagonists on high glucose-induced IGFBP-2 production. MES-13 cells cultured in the presence of 25 mmol/l glucose (“C” lanes) exhibit a 4.5 ± 0.7-fold increase in IGFBP-2 (P < 0.05 vs. cells in 5.5 mmol/l glucose medium, n = 4). However, the addition of 10⁻⁶ M saralasin (“+Sar” lane) or losartan (“+Los” lane) blocks this effect, resulting in undetectable levels of IGFBP-2. Data shown represent 72 h experiments, which were repeated three times with the same result

Fig. 8

Effect of IGF-I immuno-neutralization on high glucose-induced fibronectin and IGFBP-2 production. MES-13 cells were cultured under basal (5.5 mmol/l) or high (25 mmol/l) glucose conditions without (“C” = controls) or with IGF-I antiserum added at 1:10,000 or 1:5,000 titers. Fibronectin levels were measured using ELISA, as described in Methods; bars represent mean ± SE values (n = 6/mean), with different ^a,b,c superscripts indicating significantly different values (P < 0.05). IGFBP-2, measured by Western immunoblot procedure, is shown in the lower panels. Immuno-neutralization of IGF-I inhibits the high glucose-induced increases in fibronectin and IGFBP-2

Fig. 9

Effect of IGF-I immuno-neutralization on Ang II-induced fibronectin and IGFBP-2 production. MES-13 cells were cultured under basal glucose conditions (5.5 mmol/l; “C” indicates non-hormone-treated control) and treated with 10⁻⁷ or 10⁻⁶ M Ang II in the absence [“(−)” lanes] or presence [“(+)” lanes] of IGF-I antiserum at a titer of 1:5,000. Fibronectin levels (upper panel) were measured using ELISA, as described in Methods; bars represent mean ± SD values (n = 6/mean), with ^a,b superscripts indicating significantly different values (P < 0.05). IGFBP-2, measured by Western immunoblot procedure, is shown in the lower panels. Immuno-neutralization of IGF-I inhibits Ang II-induced increases in fibronectin and decreases IGFBP-2 to non-detectable levels