Young Mi Seok

Reactive oxygen species/oxidative stress contributes to progression of kidney fibrosis following transient ischemic injury in mice

Recently, kidney fibrosis following transplantation has become recognized as a main contributor of chronic allograft nephropathy. In transplantation, transient ischemia is an inescapable event. Reactive oxygen species (ROS) play a critical role in ischemia and reperfusion (I/R)-induced acute kidney injury, as well as progression of fibrosis in various diseases such as hypertension, diabetes, and ureteral obstruction. However, a role of ROS/oxidative stress in chronic kidney fibrosis following I/R injury remains to be defined. In this study, we investigated the involvement of ROS/oxidative stress in kidney fibrosis following kidney I/R in mice. Mice were subjected to 30 min of bilateral kidney ischemia followed by reperfusion on day 0 and then administered with either manganese (III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP, 5 mg/kg body wt ip), a cell permeable superoxide dismutase (SOD) mimetic, or 0.9% saline (vehicle) beginning at 48 h after I/R for 14 days. I/R significantly increased interstitial extension, collagen deposition, apoptosis of tubular epithelial cells, nitrotyrosine expression, hydrogen peroxide production, and lipid peroxidation and decreased copper-zinc SOD, manganese SOD, and glucose 6-phosphate dehydrogenase activities in the kidneys 16 days after the procedure. MnTMPyP administration minimized these postischemic changes. In addition, MnTMPyP administration significantly attenuated the increases of alpha-smooth muscle actin, PCNA, S100A4, CD68, and heat shock protein 47 expression following I/R. We concluded that kidney fibrosis develops chronically following I/R injury, and this process is associated with the increase of ROS/oxidative stress.

Orchiectomy Attenuates Post-ischemic Oxidative Stress and Ischemia/Reperfusion Injury in Mice A ROLE FOR MANGANESE SUPEROXIDE DISMUTASE

Males are much more susceptible to ischemia/reperfusion (I/R)-induced kidney injury when compared with females. Recently we reported that the presence of testosterone, rather than the absence of estrogen, plays a critical role in gender differences in kidney susceptibility to I/R injury in mice. Although reactive oxygen species and antioxidant defenses have been implicated in I/R injury, their roles remain to be defined. Here we report that the orchiectomized animal had significantly less lipid peroxidation and lower hydrogen peroxide levels in the kidney 4 and 24 h after 30 min of bilateral renal ischemia when compared with intact or dihydrotestosterone-treated orchiectomized males. The post-ischemic kidney expression and activity of manganese superoxide dismutase (MnSOD) in orchiectomized mice was much greater than in intact or dihydrotestosterone-administered orchiectomized mice. Four hours after 30 min of bilateral ischemia, superoxide formation was significantly lower in orchiectomized mice than in intact mice. In Madin-Darby canine kidney cells, a kidney epithelial cell line, 1 mm H2O2 decreased MnSOD activity, an effect that was potentiated by pretreatment with dihydrotestosterone. Orchiectomy prevented the post-ischemic decrease of catalase activity. Treatment of male mice with manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a SOD mimetic, reduced the post-ischemic increase of plasma creatinine, lipid peroxidation, and tissue hydrogen peroxide. These results suggest that orchiectomy accelerates the post-ischemic activation of MnSOD and reduces reactive oxygen species and lipid peroxidation, resulting in reduced kidney susceptibility to I/R injury.

Isoflavone Attenuates Vascular Contraction through Inhibition of the RhoA/Rho-Kinase Signaling Pathway

Isoflavones decrease blood pressure, improve lipid profiles, and restore vascular function. We hypothesized that isoflavone attenuates vascular contraction by inhibiting RhoA/Rho-kinase signaling pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with 11,9 epoxymethano-prostaglandin F2α (U46619), a thromboxane A2 analog, or KCl 30 min after the pretreatment with genistein (4′,5,7-trihydroxyisoflavone), daidzein (4′,7-dihydroxyisoflavone), or vehicle. We determined the phosphorylation level of the myosin light chain (MLC20), myosin phosphatase-targeting subunit 1 (MYPT1), and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light-chain phosphatase of 17 kDa (CPI17) by means of the Western blot. We also measured the amount of GTP RhoA as a marker regarding RhoA activation. The cumulative additions of U46619 or KCl increased vascular tension in a concentration-dependent manner, which were inhibited by pretreatment with genistein or daidzein. Both U46619 (30 nM) and KCl (50 mM) increased MLC20 phosphorylation levels, which were inhibited by genistein and daidzein. Furthermore, both genistein and daidzein decreased the amount of GTP RhoA activated by either U46619 or KCl. U46619 (30 nM) increased phosphorylation of the MYPT1Thr855 and CPI17Thr38, which were also inhibited by genistein or daidzein. However, neither genistein nor daidzein inhibited phorbol 12,13-dibutyrate-induced vascular contraction and CPI17 phosphorylation. In conclusion, isoflavone attenuates vascular contraction, at least in part, through inhibition of the RhoA/Rho-kinase signaling pathway.

Histone Deacetylase Inhibition Attenuates Cardiac Hypertrophy and Fibrosis through Acetylation of Mineralocorticoid Receptor in Spontaneously Hypertensive Rats

Inhibition of histone deacetylases (HDACs) by valproic acid (VPA) attenuates inflammatory, hypertrophic, and fibrotic responses in the hearts of spontaneously hypertensive rats (SHRs); however, the molecular mechanism is still unclear. We hypothesized that HDAC inhibition (HDACi) attenuates cardiac hypertrophy and fibrosis through acetylation of mineralocorticoid receptor (MR) in SHRs. Seven-week-old SHRs and Wistar-Kyoto rats were treated with an HDAC class I inhibitor (0.71% w/v in drinking water; VPA) for 11 weeks. Sections of heart were visualized after trichrome stain as well as H&E stain. Histone modifications, such as acetylation (H3Ac [acetylated histone 3]) and fourth lysine trimethylation (H3K4me3) of histone 3, and recruitment of MR and RNA polymerase II (Pol II) into promoters of target genes were measured by quantitative real-time polymerase chain reaction after chromatin immunoprecipitation assay. MR acetylation was determined by Western blot with anti–acetyl-lysine antibody after immunoprecipitation with anti-MR antibody. Treatment with VPA attenuated cardiac hypertrophy and fibrosis. Although treatment with VPA increased H3Ac and H3K4me3 on promoter regions of MR target genes, expression of MR target genes as well as recruitment of MR and Pol II on promoters of target genes were decreased. Although HDACi did not affect MR expression, it increased MR acetylation. These results indicate that HDACi attenuates cardiac hypertrophy and fibrosis through acetylation of MR in spontaneously hypertensive rats.

Promoter hypomethylation upregulates Na+-K+-2Cl- cotransporter 1 in spontaneously hypertensive rats.

The Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) is one of several transporters that have been implicated for development of hypertension since NKCC1 activity is elevated in hypertensive aorta and vascular contractions are inhibited by bumetanide, an inhibitor of NKCC1. We hypothesized that promoter hypomethylation upregulates the NKCC1 in spontaneously hypertensive rats (SHR). Thoracic aortae and mesenteric arteries were excised, cut into rings, mounted in organ baths and subjected to vascular contraction. The expression levels of nkcc1 mRNA and protein in aortae and heart tissues were measured by real-time PCR and Western blot, respectively. The methylation status of nkcc1 promoter region was analyzed by combined bisulfite restriction assay (COBRA) and bisulfite sequencing. Phenylephrine-induced vascular contraction in a dose-dependent manner, which was inhibited by bumetanide. The inhibition of dose-response curves by bumetanide was much greater in SHR than in Wistar Kyoto (WKY) normotensive rats. The expression levels of nkcc1 mRNA and of NKCC1 protein in aortae and heart tissues were higher in SHR than in WKY. Nkcc1 gene promoter was hypomethylated in aortae and heart than those of WKY. These results suggest that promoter hypomethylation upregulates the NKCC1 expression in aortae and heart of SHR.

Enhanced Ca2+-Dependent Activation of Phosphoinositide 3-Kinase Class IIα Isoform-Rho Axis in Blood Vessels of Spontaneously Hypertensive Rats

Rho-mediated inhibition of myosin light chain (MLC) phosphatase (MLCP), together with Ca2+-dependent MLC kinase activation, constitutes the major signaling mechanisms for vascular smooth muscle contraction. We recently unveiled the involvement of Ca2+-induced, phosphoinositide 3-kinase (PI3K) class II&agr; isoform (PI3K-C2&agr;)–dependent Rho activation and resultant Rho kinase-dependent MLCP suppression in membrane depolarization- and receptor agonist-induced contraction. It is unknown whether Ca2+- and PI3K-C2&agr;–dependent regulation of MLCP is altered in vascular smooth muscle of hypertensive animals and is involved in hypertension. Therefore, we studied the role of the Ca2+-PI3K-C2&agr;-Rho-MLCP pathway in spontaneously hypertensive rats (SHRs). PI3K-C2&agr; was readily detected in various vascular beds of Wistar-Kyoto rats and activated by high KCl. High KCl also stimulated vascular Rho activity and phosphorylation of the MLCP regulatory subunit MYPT1 at Thr853 in a PI3K inhibitor wortmannin-sensitive manner. In mesenteric and other vessels of SHRs at the hypertensive but not the prehypertensive stage, the activity of PI3K-C2&agr; but not class I PI3K p110&agr; was elevated with concomitant rises of Rho activity and Thr853-phosphorylation of MYPT1, as compared with normotensive controls. Infusion of the Ca2+ channel antagonist nicardipine reduced blood pressure with suppression of vascular activity of PI3K-C2&agr;-Rho and phosphorylation of MYPT1 in hypertensive SHRs. Infusion of wortmannin lowered blood pressure with inhibition of PI3K-C2&agr;-Rho activities and MYPT1 phosphorylation in hypertensive SHRs. These observations suggest that an increased activity of the Ca2+-PI3K-C2&agr;-Rho signaling pathway with resultant augmented MLCP suppression contributes to hypertension in SHRs. The Ca2+- and PI3K-C2&agr;–dependent Rho stimulation in vascular smooth muscle may be a novel, promising target for treating hypertension.

FLAVONE INHIBITS VASCULAR CONTRACTION BY DECREASING PHOSPHORYLATION OF THE MYOSIN PHOSPHATASE TARGET SUBUNIT

1 Flavonoids modulate vascular tone through an endothelium‐dependent or ‐independent mechanism. Although a few mechanisms for endothelium‐independent relaxation have been suggested, such as interference with protein kinase C or cAMP or cGMP phosphodiesterase, the inhibition of Ca2+ release from intracellular stores or Ca2+ influx from extracellular fluids, the mode of action of flavonoids remains elusive. 2 We hypothesized that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the myosin phosphatase target subunit (MYPT1). 3 Rat aortic rings were denuded of endothelium, mounted in organ baths and contracted with U46619, a thromboxane A2 analogue. 4 Flavone dose‐dependently inhibited the U46619‐induced contractile response and myosin light chain (MLC20) phosphorylation. At 10−7 mol/L, U46619 induced vascular contraction with the concomitant phosphorylation of MYPT1 at Thr855, but not at Thr697. Incubation with flavone (100 or 300 µmol/L) for 30 min attenuated the phosphorylation of MYPT1Thr855, but not MYPT1Thr697. 5 It is concluded that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the MYPT1. These results suggest that flavone causes endothelium‐independent relaxation through, at least in part, the inhibition of p160 Rho‐associated coiled‐coil‐containing protein kinase (ROCK) signalling.

Wen‐pi‐tang‐Hab‐Wu‐ling‐san attenuates kidney fibrosis induced by ischemia/reperfusion in mice

Renal fibrosis is highly implicated as a cause of chronic renal failure, for which suitable therapeutics have not yet been developed. Recently, it was reported that Wen‐pi‐tang‐Hab‐Wu‐ling‐san (WHW) extract attenuates epithelial cells undergoing mesenchymal transition in cultured Madin‐Darby canine kidney cells. This study investigated whether WHW extract prevents renal fibrosis induced by ischemia/reperfusion (I/R) in mice. Ischemia/reperfusion resulted in kidney fibrosis at 14 days after the procedure. When WHW was administered orally to mice beginning from 2 days after the onset of ischemia until killing, the fibrosis was significantly reduced. WHW administration significantly prevented a post‐ischemic decrease of copper‐zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) activities, leading to decreased lipid peroxidation and hydrogen peroxide production. In addition, WHW administration attenuated the phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and c‐Jun N‐terminal kinase 1/2 (JNK1/2) and attenuated the activation of nuclear factor‐kappa B (NF‐κB) in the kidneys subjected to ischemia. In conclusion, WHW extract attenuated the renal fibrosis and the attenuation was associated with a reduction of oxidative stress and an inhibition of ERK1/2, JNK1/2, p38 and NF‐κB activation. WHW extract may be an attractive agent to attenuate the progression of fibrosis. Copyright

GPER-1 agonist G1 induces vasorelaxation through activation of epidermal growth factor receptor-dependent signalling pathway.

The G protein‐coupled oestrogen receptor‐1 (GPER‐1) agonist G1 induces endothelium‐dependent relaxation. Activation of the epidermal growth factor (EGF) receptor leads to transduction of signals from the plasma membrane for the release of nitric oxide. We tested the hypothesis that G1 induces endothelium‐dependent vasorelaxation through activation of the EGF receptor.

A ROLE FOR RHO-KINASE IN Ca2+-INDEPENDENT CONTRACTIONS INDUCED BY PHORBOL-12,13-DIBUTYRATE

1 Phorbol‐12,13‐dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca2+‐depleted solutions. In the present study, we tested the hypothesis that Rho‐kinase plays a role in Ca2+‐independent contractions induced by PDBu in vascular smooth muscles. 2 In Ca2+‐free solution, 0.1 and 1 µmol/L PDBu induced contraction and myosin light chain (MLC20) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs’ solution. Hydroxyfasudil (H1152; 1 µmol/L), an inhibitor of Rho‐kinase, but not ML7 (10 µmol/L), an inhibitor of myosin light chain kinase, inhibited Ca2+‐independent contractions induced by PDBu. 3 In Ca2+‐free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI‐17 (PKC‐potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI‐17 almost completely abolished by 1 µmol/L Ro31‐8220, an inhibitor of PKC. 4 In Ca2+‐free solution, PDBu increased the amount of GTP‐RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31‐8220, but not by the Rho kinase inhibitor H1152. 5 In conclusion, RhoA/Rho‐kinase plays an important role in Ca2+‐independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca2+‐independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP‐RhoA and subsequent phosphorylation of MYPT1 and CPI‐17.