Urea treatment (100–300 mM) increased expression of the oxidative stress-responsive transcription factor, Gadd153/CHOP, at the mRNA and protein levels (at ≥4 h) in renal medullary mIMCD3 cells in culture, whereas other solutes did not. Expression of the related protein, CCAAT/enhancer-binding protein (C/EBP-β), was not affected, nor was expression of the sensor of endoplasmic reticulum stress, grp78. Urea modestly increased Gadd153 transcription by reporter gene analysis but failed to influence Gadd153 mRNA stability. Importantly, upregulation of Gadd153 mRNA and protein expression by urea was antioxidant sensitive. Accordingly, urea treatment was associated with oxidative stress, as quantitated by intracellular reduced glutathione content in mIMCD3 cells. In addition, antioxidant treatment partially inhibited the ability of urea to activate transcription of an Egr-1 luciferase reporter gene. Therefore oxidative stress represents a novel solute-signaling pathway in the kidney medulla and, potentially, in other tissues.

Objective-To test the hypothesis that chronic infusion of angiotensin-(1-7) [Ang-(1-7)] may dose-dependently inhibit atherosclerotic lesion formation by targeting vascular smooth muscle cells and a large dose of Ang-(1-7) may stabilize mature plaque by targeting macrophages. Approach and Results-In vivo, the effects of Ang-(1-7) on atherogenesis and plaque stability were observed in ApoE −/− mice fed a high-fat diet and chronic angiotensin II infusion. In vitro, the effects of Ang-(1-7) on vascular smooth muscle cells' proliferation and migration, and macrophage inflammatory cytokines were examined. Ang-(1-7) dosedependently attenuated early atherosclerotic lesions and inhibited vascular smooth muscle cells' proliferation and migration via suppressing extracellular regulated protein kinase/P38 mitogen-activated protein kinase and janus kinase/ signal transducers and activators of transcription activities and enhancing smooth muscle 22α and angiotensin II type 2 receptor expression. Ang-(1-7) treatment resulted in high contents of collagen and vascular smooth muscle cells, and low contents of macrophages and lipids in carotid mature plaques. Ang-(1-7) lowered the expression levels of proinflammatory cytokines and activities of matrix metalloproteinases in mature plaques. Conclusions-Ang-(1-7) treatment inhibits early atherosclerotic lesions and increases plaque stability in ApoE−/− mice, thus providing a novel and promising approach to the treatment of atherosclerosis. Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results Body Weight, Blood Pressure, and Serum Lipid ProfileAt the end of the first part (8 weeks) and the second part (10 weeks) of the in vivo study, both systolic and diastolic blood pressures were substantially increased in comparison with the baseline blood pressure measurements (data not shown). However, no significant differences were found in body weight, blood pressure, and serum lipid levels among vehicle-treated, Ang-(1-7)-treated and Ang-(1-7)+A779-treated groups, indicating that chronic infusion of Ang-(1-7) or A779 had no significant effects on these parameters (Table I in the online-only Data Supplement). In ApoE −/− mice without Ang-II infusion, blood pressure levels were not statistically different among vehicle-treated, Ang-(1-7)-treated, and Ang-(1-7)+A779-treated groups ( Figure 1; Table II in the online-only Data Supplement). Aortic Lesion FormationIn the first part of the in vivo study, the relative en face lesion area of the aorta arches was dose-dependently decreased in the Ang-(1-7)-treated subgroups. However, only the difference between the large dose of Ang-(1-7) subgroup and the vehicle-treated group reached a statistical significance. The antiatherosclerosis effect of Ang-(1-7) was significantly reversed by coadministration of A779 (Figure 2A and 2B). Similarly, the relative cross-sectional area of the aortic lesion also showed a dose-dependent decrease in the Ang-(1-7)-treated subgroups but only the difference between the large do...

Growth factors and other stimuli increase the activity of phosphatidylinositol-3 kinase (PI3K), an SH2 domain-containing lipid kinase. In the murine kidney inner medullary mIMCD3 cell line, urea (200 mM) increased PI3K activity in a time-dependent fashion as measured by immune complex kinase assay. The PI3K effector, Akt, was also activated by urea as measured by anti-phospho-Akt immunoblotting. In addition, the Akt (and PI3K) effector, p70 S6 kinase, was activated by urea treatment in a PI3K-dependent fashion. PI3K inhibition potentiated the proapoptotic effect of hypertonic and urea stress. Urea treatment also induced the tyrosine phosphorylation of Shc and the recruitment to Shc of Grb2. Coexistence of activated Shc and PI3K in a macromolecular complex was suggested by the increase in PI3K activity evident in anti-Shc immunoprecipitates prepared from urea-treated cells. Taken together, these data suggest that PI3K may regulate physiological events in the renal medullary cell response to urea stress and that an upstream tyrosine kinase conferring activation of both PI3K and Shc may govern urea signaling in these cells.

Urea- and NaCl-inducible extracellular signal-regulated kinase (ERK) phosphorylation exhibited dissimilar kinetics. Among cell lines examined, the effect of urea was unique to mIMCD3 inner medullary collecting duct cells and MDCK cells. Urea-inducible ERK activation was ∼10-fold less sensitive to the MEK inhibitor, PD-98059, than was that of NaCl. This difference did not appear to be accounted for by differential activation of MEK isoforms. Interestingly, the inhibitor of p38 activation, SB-203580, abrogated the effect of both urea and NaCl upon both ERK and MEK activation; however, the former was much less sensitive to the inhibitor. Consistent with this observation, NaCl was much more effective than urea at inducing p38 phosphorylation. The effect of hypertonic stress (e.g., sorbitol 100 mM) could be blocked by appropriate medium dilution such that isotonicity was maintained. In marked contrast, the effect of hyperosmotic urea could not be blocked in this fashion, implying the absence of dependence upon cell volume. Together, these data suggest that cells of the renal inner medulla are potentially uniquely responsive to urea and that urea and hypertonic stressors induce ERK activation through distinct mechanisms.