Norepinephrine increases calcium sensitivity of mouse afferent arteriole, thereby enhancing angiotensin II–mediated vasoconstriction

Many agents constrict isolated afferent arterioles only at concentrations higher than their physiological levels. Here we determined if norepinephrine, as released by sympathetic nerve activity, could influence the angiotensin II responsiveness of isolated mouse afferent arterioles. Pretreatment of the arterioles for short periods with norepinephrine significantly increased the ability of 10 picomolar angiotensin II to constrict the vessels, an effect inhibited by the alpha receptor blockers prazosin (α-1) or yohimbine (α-2). Although the intracellular calcium transients induced by angiotensin were not different, phosphorylation of the 20kDa myosin light chain was significantly increased in the presence of norepinephrine. Phosphorylation of the p38 mitogen-activated protein kinase was not changed. Phosphorylation of the myosin phosphatase targeting subunit at Thr696, but not at Thr850, was significantly enhanced by, norepinephrine pretreatment, thus increasing the calcium sensitivity of the arteriolar smooth muscle. Our results show that norepinephrine increases afferent arteriolar sensitivity to angiotensin II by means of alpha receptor activation, causing increased calcium sensitivity through phosphorylation of the myosin phosphatase targeting subunit

Tempol Protects Against Acute Renal Injury by Regulating PI3K/Akt/mTOR and GSK3β Signaling Cascades and Afferent Arteriolar Activity

Background/Aims: Free radical scavenger tempol is a protective antioxidant against ischemic injury. Tubular epithelial apoptosis is one of the main changes in the renal ischemia/reperfusion (I/R) injury. Meanwhile some proteins related with apoptosis and inflammation are also involved in renal I/R injury. We tested the hypothesis that tempol protects against renal I/R injury by activating protein kinase B/mammalian target of rapamycin (PKB, Akt/mTOR) and glycogen synthase kinase 3β (GSK3β) pathways as well as the coordinating apoptosis and inflammation related proteins. Methods: The right renal pedicle of C57Bl/6 mouse was clamped for 30 minutes and the left kidney was removed in the study. The renal injury was assessed with serum parameters by an automatic chemistry analyzer. Renal expressions of Akt/mTOR and GSK3β pathways were measured by western blot in I/R mice treated with saline or tempol (50mg/kg) and compared with sham-operated mice. Results: The levels of blood urea nitrogen (BUN), creatinine and superoxide anion (O2.-) increased, and superoxide dismutase (SOD) and catalase (CAT) decreased significantly after renal I/R injury. However, tempol treatment prevented the changes. Besides, I/R injury reduced renal expression of p-Akt, p-GSK3β, p-mTOR, Bcl2 and increased NF-κB, p-JNK and p53 in kidney, tempol significantly normalized these changes. In addition, renal I/R injury reduced the response of afferent arteriole to Angiotensin II (Ang II), while tempol treatment improved the activity of afferent arteriole. Conclusion: Tempol attenuates renal I/R injury. The protective mechanisms seem to relate with activation of PI3K/Akt/mTOR and GSK3β pathways, inhibition of cellular damage markers and inflammation factors, as well as improvement of afferent arteriolar activity

Effect of PPARα siRNA on ERK1/2 and PI3K-AKT pathways, and the Collagen-IV expression in high glucose treated mesangial cells.

<p>(A) Representative western blot of PPARα. (B) Representative western blot of p-AKT. (C) Representative western blot of p-ERK1/2. (D) Representative western blot of Collagen-IV. Protein levels were determined by western Blot, normalised to GAPDH. Phosphorylated ERK1/2 and AKT were corrected for the loading control, total ERK1/2 and AKT. Values are given as mean ±S.D. from 3 independent experiments in triplicate and p<0.05 is considered statistically significant. ^*P<0.05 vs NG; ^# p<0.05 vs HG. NG, normal glucose; HG, high glucose.</p

PPARα siRNA accelerated high glucose-induced mesangial cell proliferations via ERK1/2 and PI3K-AKT pathways.

<p>PPARα siRNA was transfected into mesangial cells for 5h, followed by stimulating with high glucose for 24h, or high glucose plus Ly294002 (10µmol/L), or high glucose plus U0126 (10µmol/L). Cells were analyzed by flow cytometry after PI staining. Data in the bar graphs represent the average values ± SE of experiments performed in triplicate. ^*P<0.05 vs NG; ^# p<0.05 vs HG. NG, normal glucose; HG, high glucose.</p

Effect of fenofibrate on the phosphorylation of ERK1/2 and PI3K-AKT in rat mesangial cells.

<p>(A) Representative western blot of p-AKT. (B) Representative western blot of p-ERK1/2. The figure shows the average volume density of phosphorylated ERK1/2 and AKT corrected for the loading control, total ERK1/2 and AKT. Values are given as mean±S.D. from 3 separate experiments. ^*P<0.05 vs NG; ^# p<0.05 vs HG. NG, normal glucose (5mM); HG, high glucose (40 mM); FN, fenofibrate; MK, MK886.</p