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Dietary salt intake activates MAP kinases in the rat kidney¹

Nephrology Research and Training Center, Comprehensive Cancer Center, and Cell Adhesion and Matrix Research Center; Division of Nephrology, Department of Medicine, and Department of Physiology and Biophysics, University of Alabama at Birmingham; and Department of Veterans Affairs Medical Center, Birmingham, Alabama, USA

²Correspondence: Division of Nephrology/Department of Medicine, 642 Lyons-Harrison Research Bldg., 1530 Third Ave., South, University of Alabama at Birmingham, Birmingham, AL 35294-0007, USA. E-mail: psanders{at}uab.edu

SPECIFIC AIM

Previous studies from this laboratory have demonstrated that dietary salt regulates the renal expression of transforming growth factor ß1 (TGF-ß1), which in turn modulates expression of the endothelial isoform of nitric oxide synthase (NOS3). These effector molecules contribute to the renal response to dietary salt by modulating renal blood flow and solute excretion. The present study demonstrated the signal transduction events by which dietary salt regulates renal expression of TGF-ß1 and NOS3.

PRINCIPAL FINDINGS

1. Activities of p38 MAPK and p42/44 MAPK were increased in kidneys of rats on 3.0% and 8.0% NaCl diets
Without changing blood pressure, an increase in dietary salt intake produced a dose-dependent increase in activities of p38 MAPK and p42/44 MAPK in lysates from kidney cortex and outer and inner medulla (Fig. 1 ). As dietary salt was increased to 3.0% and higher, activities of p38 MAPK and p42/44 MAPK in cortex and the outer and inner medulla increased (P<0.05) vs. findings obtained from rats on 0.3% and 1.0% NaCl diets for the same duration. Phosphorylation of two nuclear targets (ATF-2 and Elk-1) of these MAPKs was increased in lysates from cortex and outer and inner medulla of rats on the 8.0% NaCl diet.

View larger version (45K): [in this window] [in a new window]   Figure 1. A series of in vitro kinase activity assays were performed by immunoprecipitating the MAPK of interest from tissue lysates containing 200 µg total protein, using either an anti-phospho-p42/44 MAPK (T202/Y204) or anti-phospho-p38 MAPK (T180/Y182) monoclonal antibodies. Each lane represents a single animal (n=4 in each group). Lysates of kidney cortex (top panels) and outer (middle panels) and inner (bottom panels) medulla from rats on the 3.0% and 8.0% NaCl diets contained increased (P<0.05) activities of p38 MAPK and p42/44 MAPK with lysates from animals on 0.3% and 1.0% NaCl diets.

2. Increased glomerular expression of TGF-ß1 and NOS3 by dietary salt was dependent on activation of both p38 and p42/44 MAPK pathways
Pharmacological inhibitors of the p38 MAPK and p42/44 MAPK pathways were used to determine the contribution of these pathways in regulation of TGF-ß1 and NOS3 by dietary salt. Glomeruli from rats on 8.0% NaCl diet produced greater (P<0.05) amounts of total and active TGF-ß1 and nitrite plus nitrate (NO_x) than glomeruli from rats on the 0.3% NaCl diet (Fig. 2 ). Addition of either PD-098059, a specific cell-permeable inhibitor of activation of MAPK kinase-1 (MEK1), or SB-203580, a cell-permeable inhibitor of p38 MAPK-, abolished production of TGF-ß1 and NO_x. The decrease in production of NO_x was related to decreased expression of NOS3, determined by immunoblot analysis. NOS3 expression correlated (r²=0.773; P<0.001) directly with NO_x production. In these studies, a direct correlation (r²=0.922; P<0.001) between production of total and active TGF-ß1 was observed. A direct correlation (r²=0.744; P<0.001) between production of active TGF-ß1 and NO_x was also found.

View larger version (35K): [in this window] [in a new window]   Figure 2. Glomerular incubation studies (n=6 rats in each group) were performed in standard fashion. Glomeruli from rats on the 8.0% NaCl diet produced more (P<0.05) total and active TGF-ß1 and NOx than glomeruli from rats on the 0.3% NaCl diet. Administration of PD-098059 (50 µM) or SB-203580 (10 µM) reduced levels of these compounds to levels that were close to background.

3. Dietary salt increased MAPK activities through a TEA-sensitive mechanism
Five minutes before harvesting the kidney, rats on a 8.0% NaCl diet were injected intravenously with a 1 mL bolus of tetraethylammonium chloride (TEA); findings were from rats on the same diet and received vehicle alone. Administration of TEA decreased (P<0.05) activities of both p38 MAPK and p42/44 MAPK obtained from the lysates of kidney cortex and medulla with findings from animals that received vehicle alone. These data were consistent with our previous in vitro experiments, which showed that TEA inhibited the augmented glomerular expression of TGF-ß1 and NOS3 induced by an increase in dietary salt.

CONCLUSIONS AND SIGNIFICANCE

These findings represented the first demonstration that dietary salt intake modulates MAPK signaling events in the kidney in the absence of changes in blood pressure. These studies further provided the potential mechanism of activation of the MAPK pathways and the "downstream" targets that included TGF-ß1 and NOS3. The data fit the known paradigm of TGF-ß1 gene expression, which involves cooperation of transcription factors (including ATF-2) with Smads to form a heteromeric nuclear complex for efficient and specific gene transcription. The data were also consistent with shear forces mediating this process (Fig. 3 ). An increase in renal blood flow activates a TEA-sensitive process that increases activities of p38 MAPK and p42/44 MAPK. Both pathways are required to promote expression of TGF-ß1, which in turn increases nitric oxide (NO) production through NOS3. The increase in NO promotes vasodilation, which completes a negative feedback loop by decreasing shear forces.

View larger version (17K): [in this window] [in a new window]   Figure 3. Proposed mechanism whereby dietary salt modulates production of TGF-ß1 and NO. As dietary salt increases, renal blood flow increases. An increase in flow increases shear stress, which opens an inwardly rectifying potassium channel (Kir). Through a mechanism that is not completely defined, p38 MAPK and p42/44 MAPK are both activated. Both work in concert to increase the production of TGF-ß1, which stimulates NO production by increasing glomerular expression of NOS3. The increase in NO promotes vasodilation and decrease in shear forces, creating a feedback loop. The process can be inhibited by TEA, SB-203580, or PD-098059.

These studies are relevant to renal physiology and pathophysiology, since long-term exposure of rats to a diet high in salt appears to promote renal arteriolonephrosclerosis. This laboratory hypothesizes that the closely coordinated expression of TGF-ß1 and NOS3 is required to promote optimum renal functioning in response to changes in dietary salt intake. Disruption of this coordination, for example, by abnormal production of free radicals that scavenge NO may result in unopposed and augmented expression of TGF-ß1. The outcome might be hypertension, glomerulosclerosis, arteriosclerosis, and impaired renal vasodilation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0982fje; to cite this article, use FASEB J. (August 19, 2002) 10.1096/fj.02-0982fje

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