Mechanism of dietary salt-mediated increase in intravascular production of TGF-beta1

Abstract

Clinical and preclinical studies have demonstrated an important effect of arterial pathobiology on the progressive loss of renal function that occurs in chronic kidney disease. Chronic kidney disease, in turn, promotes alterations in vascular function. A modulating role for dietary salt has been suggested, with the amount of salt intake regulating endothelial cell production of transforming growth factor-beta1 (TGF-beta1), a fibrogenic growth factor that promotes arteriosclerosis and glomerulosclerosis. The purpose of the present studies was to determine how the interaction between dietary salt intake and vasculature promoted the production of TGF-beta1 in rats. Two different vascular tissues, aortic rings and glomeruli, were chosen for study. Dietary salt induced, in a dose-dependent fashion, activation of proline-rich tyrosine kinase-2 (Pyk2) and further identified c-Src as an important binding partner of Pyk2 in these tissues. Use of pharmacological inhibitors and dominant negative strategies confirmed that dietary salt induced complex formation of Pyk2 and c-Src with downstream activation of p38 and p42/44 mitogen-activated protein kinases and generation of TGF-beta1. The experiments defined the molecular signaling events that promoted the production of TGF-beta1, a key growth factor involved in the vascular response to increased salt intake.

Fig. 1.

Western analyses of the effect of salt intake on the phosphorylation state of proline-rich tyrosine kinase-2 (Pyk2) in lysates from aortic tissue (top) and isolated glomeruli (bottom). Although total levels of Pyk2 did not differ between the 2 groups, compared with rats on the 0.3% NaCl diet, rats on the 8.0% NaCl diet demonstrated increased levels of phospho-Pyk2(Y402), represented as the ratio of density of phospho-Pyk2(Y402) to the density of total Pyk2 in lysates of aortic tissue (0.63 ± 0.03 vs. 0.14 ± 0.02; P < 0.05) and isolated glomeruli (0.708 ± 0.10 vs. 0.06 ± 0.04; P < 0.05). Levels of phospho-Pyk2(Y579/80)/total Pyk2 in both aortic tissue lysates (0.56 ± 0.11 vs. undetectable; top) and glomerular lysates (0.58 ± 0.08 vs. undetectable; bottom) were also dramatically increased in rats on the 8.0% NaCl diet. Immunoblot analysis of phospho-Pyk2(Y881) was performed but was not observed in either group (data not shown). Each column represents data from the same rat (n = 4 rats/group).

Fig. 2.

Increasing dietary salt intake produced a dose-dependent increase in Pyk2 activity. Pyk2 activity assays were performed using lysates of aortic endothelia and isolated glomeruli obtained from groups of rats (n = 4 in each group) that had been fed 0.3, 1.0, 3.0, and 8.0% NaCl diets for 4 days before the study. As dietary salt intake increased, Pyk2 activity in aortic endothelia and glomeruli increased. *P < 0.05 compared with values obtained from rats on the other 3 diets.

Fig. 3.

Effect of tyrphostin A9, an inhibitor of Pyk2, on production of total and active transforming growth factor-β1 (TGF-β1) by aortic rings and isolated glomeruli from rats (n = 6 in each group) on 0.3 and 8.0% NaCl diets. The augmented production of total and active TGF-β1 observed in both tissues with the increase in salt intake was inhibited by 2 μM Pyk2. *P < 0.05 compared with values obtained from rats on the 8.0% NaCl diet. †P < 0.05 compared with untreated tissues from rats on the same diet.

Fig. 4.

Top: a schematic representation of Pyk2 with sites along the molecule that potentially permit binding of SH2 domains of c-Src, phosphatidylinositol 3-kinase (PI3K), and Grb2. Proline-rich sites (P) and a potential alternative-splicing site that removes 42 amino acids also are shown. The Tat fusion proteins Tat-AP, Tat-PBM, and Tat-GBM served as dominant negative constructs that inhibited the respective binding of c-Src, PI3K, and Grb2, respectively, to Pyk2. Middle: a Coomassie-stained gel demonstrating that 4 different preparations of the purified Tat fusion proteins were the expected size. Bottom: the 3 FITC-labeled Tat fusion proteins readily transduced rat endothelial cells in culture. Cells were counterstained with Hoechst to label the nuclei.

Fig. 5.

Administration of 500 nM Tat-AP and Tat-PBM, but not Tat-GBM, inhibited the dietary salt-induced increase in TGF-β1 by aortic rings and isolated glomeruli. Preparations of aortic rings and isolated glomeruli were obtained from rats (n = 4 rats in each group) on 0.3 and 8.0% NaCl diets for 4 days. *P < 0.05 compared with corresponding values obtained from rats on the 0.3% NaCl diet.

Fig. 6.

MAPK activity assays were performed in standard fashion by immunoprecipitation of the MAPK of interest and in vitro incubation with substrate (Elk-1 for p42/44 MAPK and ATF-2 for p38 MAPK). Lysates were obtained from aortic tissue and isolated glomeruli from rats (n = 4 rats in each group) on 0.3 and 8.0% NaCl diets for 4 days and injected intravenously with 2.5 μM Tat-AP, Tat-PBM, or Tat-GBM. Top: a representative experiment using 2 rats in each group. Bottom: graphs summarize the data from the 8 rats used in these studies. Both Tat-AP and Tat-PBM inhibited (P < 0.05) the activities of p38 and p42/44 MAPK. Although the effect was less for Tat-AP, it was sufficient to inhibit the production of TGF-β1. *P < 0.05 compared with the other 5 groups.

Fig. 7.

Intravenous administration of 2.5 μM Tat-AP and Tat-PBM, but not Tat-GBM, the day before tissue harvest inhibited the dietary salt-induced increase in TGF-β1 by aortic rings and isolated glomeruli (n = 4 rats in each group). *P < 0.05 compared with corresponding values obtained from rats on the 0.3% NaCl diet.

Fig. 8.

Effect of a single intravenous injection of the Tat fusion proteins (2.5 μM) on subsequent urinary excretion of total TGF-β and active TGF-β. Urine was obtained the day after the intravenous injection. Although the excretion rates did not fall to levels observed in rats on the 0.3% NaCl diet, the increases in urinary total and active TGF-β that occurred in response to increased salt intake were inhibited by intravenous injection of Tat-AP and Tat-PBM (n = 4 rats in each group). *P < 0.05 compared with the other 5 groups. †P < 0.05 compared with the groups of rats that received the 3% NaCl diet and the Tat fusion proteins.

Fig. 9.

Coimmunoprecipitation assays examining the interactions between Pyk2 and c-Src were performed by immunoprecipitation of Pyk2 or c-Src from aortic lysates harvested from rats on 0.3 or 8.0% NaCl diets for 4 days. A: immunoblot (IB) analysis of the immunoprecipitate (IP) generated by using an antibody to Pyk2 showed interaction of phospho-c-Src(Y416) with Pyk2, particularly in rats on the 8.0% NaCl diet. A nonspecific IgG was used in the immunoprecipitation step in the 2nd and 4th lanes. B: IB analysis of the IP generated by using an antibody to c-Src showed interaction with phospho-Pyk2(Y402). A nonspecific IgG was used in the immunoprecipitation step in the 2nd and 4th lanes. C: pull-down assays were performed using the anti-Pyk2 antibody and lysates of aortic tissue from rats on either the high- or low-salt diet treated on the day before harvest with Tat-AP, Tat-PBM, or Tat-GBM. As anticipated, interaction of phospho-c-Src(Y416) with Pyk2 was inhibited by Tat-AP, but interaction was also reduced by Tat-PBM. D: pull-down assays were performed using the anti-c-Src antibody and lysates of aortic tissue from rats on either the high- or low-salt diet treated on the day before harvest with Tat-AP, Tat-PBM, or Tat-GBM. The findings were similar to those shown in C and showed that interaction of phospho-Pyk2(Y402) with c-Src was inhibited by Tat-AP, but interaction was also reduced by Tat-PBM. Arrowheads indicate c-Src in A and C and Pyk2 in B and D.

Fig. 10.

Activity of c-Src was determined by IB analysis of phospho-c-Src(Y416) in lysates of aortic endothelial cells and isolated glomeruli. Compared with lysates from rats on the 0.3% NaCl diet, phospho-c-Src(Y416) levels relative to total c-Src levels were greater (0.62 ± 0.07 vs. 0.16 ± 0.02; P < 0.05) in endothelial lysates and greater (0.856 ± 0.046 vs. 0.410 ± 0.055; P < 0.05) in glomerular lysates from rats on the 8.0% NaCl diet. Each column represents data from a single rat (n = 6 rats in each group).

Fig. 11.

Effect of pharmacological inhibition of c-Src on total (top) and active (bottom) TGF-β1 was determined using PP2, administered in the medium following harvest of tissues from rats on either 0.3 or 8.0% NaCl for 4 days (n = 12 rats in each group). Addition of PP2 inhibited production of both total and active TGF-β1. *P < 0.05 compared with untreated tissues from rats on the same diet. †P < 0.05 compared with untreated tissues from rats on the 8.0% NaCl diet.