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Follistatin-like 1 (FSTL-1), a secreted glycoprotein, is upregulated in the serum of patients with acute kidney injury. However, it is unknown whether it protects against renal ischemia-reperfusion (I/R) injury. Our present study found that treatment with FSTL-1 (100 mg/kg) intravenous injection alleviated renal injury, as evidenced by reduced serum creatinine (Scr) and blood urea nitrogen (BUN) levels, along with reduced histopathological kidney damage. Moreover, FSTL-1 treatment reduced the number of apoptotic cells and the accumulation of reactive oxygen species (ROS) during I/R injury. The protective effect of FSTL-1 was via AMPK/PPAR-δ pathway, because, after blockade of AMPK/PPAR-δ pathway by individual inhibitor (GSK0660, a PPAR-δ antagonist, or compound C, an AMPK inhibitor), the protective effects of FSTL-1 on oxidative stress and apoptosis were blocked. Taken together, our results reveal that FSTL-1 attenuates renal I/R injury by inhibiting apoptosis in renal tubular epithelial cells, which is meditated by activating AMPK/PPAR-δ pathway.

Contrast-induced nephropathy (CIN), a complication caused by using contrast medium during diagnostic and interventional procedures, occurs frequently and lacks effective treatment. AdipoRon, the agonist of adiponectin receptors, has been shown to benefit many organs including the kidney. This study aimed to investigate the role of AdipoRon in treating CIN. CIN model was established via infusing iopromide (1.8 g/kg) in Sprague–Dawley (SD) rats; NRK52E cells were treated with iopromide (5–50 μM). Renal function, renal histopathology, levels of lactate dehydrogenase (LDH) release, cell vitality, oxidative stress and inflammatory markers were measured to evaluate the protective effects of AdipoRon. The level of pAMPK/AMPK was determined by western blot. AdipoRon (50 mg/kg) significantly reversed serum creatinine, blood urea nitrogen, creatinine clearance and urinary kidney injury molecule-1 levels induced by iopromide in SD rats. Besides, it decreased the renal injury score and apoptosis of renal cells. AdipoRon also reversed the changes of antioxidant markers, pro-oxidant and inflammatory markers induced by iopromide. Moreover, the in vitro studies showed that AdipoRon decreased LDH release and increased cell vitality in NRK52E cells treated with iopromide. Then, we demonstrated that the protection of AdipoRon was accompanied by augmented AMPK phosphorylation. Both in vivo and in vitro studies demonstrated that compound c, an AMPK inhibitor, reversed the AdipoRon-mediated improvement in the CIN model. Our data indicate that AdipoRon protects against the CIN by suppressing oxidative stress and inflammation via activating the AMPK pathway, showing that AdipoRon might be a potential candidate for the prevention and therapy of CIN.

Mitsugumin 53 (MG53) is a tripartite motif family protein that has been reported to attenuate injury via membrane repair in different organs. Contrast-induced acute kidney injury (CI-AKI) is a common complication caused by the administration of iodinated contrast media (CM). While the cytotoxicity induced by CM leading to tubular cell death may be initiated by cell membrane damage, we wondered whether MG53 alleviates CI-AKI. This study was designed to investigate the effect of MG53 on CI-AKI and the underlying mechanism. A rat model of CI-AKI was established, and CI-AKI induced the translocation of MG53 from serum to injury sites on the renal proximal tubular (RPT) epithelia, as illustrated by immunoblot analysis and immunohistochemical staining. Moreover, pretreatment of rats with recombinant human MG53 protein (rhMG53, 2 mg/mL) alleviated iopromide-induced injury in the kidney, which was determined by measuring serum creatinine, blood urea nitrogen and renal histological changes. In vitro studies demonstrated that exposure of RPT cells to iopromide (20, 40, and 80 mg/mL) caused cell membrane injury and cell death, which were attenuated by rhMG53 (10 and 50 μg/mL). Mechanistically, MG53 translocated to the injury site on RPT cells and bound to phosphatidylserine to protect RPT cells from iopromide-induced injury. In conclusion, MG53 protects against CI-AKI through cell membrane repair and reducing cell apoptosis; therefore, rhMG53 might be a potential effective means to treat or prevent CI-AKI.

Irisin, a muscle-origin protein derived from the extracellular domain of the fibronectin domain-containing 5 protein (FNDC5), has been shown to modulate mitochondria welfare through paracrine action. Here, we test the hypothesis that irisin contributes to cardioprotection after myocardial infarction by preserving mitochondrial function in cardiomyocytes. Animal model studies show that intravenous administration of exogenous irisin produces dose-dependent protection against ischemia/reperfusion (I/R)-induced injury to the heart as reflected by the improvement of left ventricular ejection fraction and the reduction in serum level of cTnI (n = 15, P0.05). I/R-induced apoptosis of cardiomyocytes is reduced after irisin treatment. The irisin-mediated protection has, at least in part, an effect on mitochondrial function because administration of irisin increases irisin staining in the mitochondria of the infarct area. Irisin also reduces I/R-induced oxidative stress as determined by mitochondrial membrane potential evaluation and superoxide FLASH event recording (n = 4, P0.05). The interaction between irisin and superoxide dismutase2 (SOD2) plays a key role in the protective process because irisin treatment increases SOD activity (n = 10, P0.05) and restores the mitochondria localization of SOD2 in cardiomyocytes (n = 5, P0.05). These results demonstrate that irisin plays a protective role against I/R injury to the heart. Targeting the action of irisin in mitochondria presents a novel therapeutic intervention for myocardial infarction.

Calorie restriction (CR) extends lifespan and increases resistance to multiple forms of stress, including renal ischemia-reperfusion (I/R) injury. However, whether CR has protective effects on contrast-induced nephropathy (CIN) remains to be determined. In this study, we evaluated the therapeutic effects of CR on CIN and investigated the potential mechanisms. CIN was induced by the intravenous injection of iodinated contrast medium (CM) iopromide (1.8 g/kg) into Sprague Dawley rats with normal food intake or 40% reduced food intake, 4 weeks prior to iopromide administration. We found that CR was protective of CIN, assessed by renal structure and function. CM increased apoptosis, reactive oxygen species (ROS), and inflammation in the renal outer medulla, which were decreased by CR. The silent information regulator 1 (SIRT1) participated in the protective effect of CR on CIN, by upregulating glutathione peroxidase 4 (GPX4), a regulator of ferroptosis, because this protective effect was reversed by EX527, a specific SIRT1 antagonist. Our study showed that CR protected CIN via SIRT1/GPX4 activation. CR may be used to mitigate CIN.

Activation of angiotensin II type 1 receptors increases D 4 dopamine receptor expression in rat renal proximal tubule cells

MG53 is a muscle-specific TRIM-family protein that presides over the cell membrane repair response. Here, we show that MG53 present in blood circulation acts as a myokine to facilitate tissue injury-repair and regeneration. Transgenic mice with sustained elevation of MG53 in the bloodstream (tPA-MG53) have a healthier and longer life-span when compared with littermate wild type mice. The tPA-MG53 mice show normal glucose handling and insulin signaling in skeletal muscle, and sustained elevation of MG53 in the bloodstream does not have a deleterious impact on db/db mice. More importantly, the tPA-MG53 mice display remarkable dermal wound healing capacity, enhanced muscle performance, and improved injury-repair and regeneration. Recombinant human MG53 protein protects against eccentric contraction-induced acute and chronic muscle injury in mice. Our findings highlight the myokine function of MG53 in tissue protection and present MG53 as an attractive biological reagent for regenerative medicine without interference with glucose handling in the body., MG53 is a protein that regulates the cell membrane repair process, and it’s been suggested that it might play a role in diabetes. Here, the authors demonstrate that circulating MG53 functions as a myokine to facilitate tissue injury-repair and regeneration without impacting glucose handling.

Irisin, a myokine, is cleaved from the extracellular portion of fibronectin domain-containing 5 protein in skeletal muscle and myocardium and secreted into circulation as a hormone during exercise. Irisin has been found to exert protective effects against lung and heart injuries. However, whether irisin influences myocardial infarction (MI) remains unclear. In this study we investigated the therapeutic effects of irisin in an acute MI model and its underlying mechanisms. Adult C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery and treated with irisin for 2 weeks after MI. Cardiac function was assessed using echocardiography. We found that irisin administration significantly alleviated MI-induced cardiac dysfunction and ventricular dilation at 4 weeks post-MI. Irisin significantly reduced infarct size and fibrosis in post-MI hearts. Irisin administration significantly increased angiogenesis in the infarct border zone and decreased cardiomyocyte apoptosis, but did not influence cardiomyocyte proliferation. In human umbilical vein endothelial cells (HUVEC), irisin significantly increased the phosphorylation of ERK, and promoted the migration of HUVEC detected in wound-healing and transwell chamber migration assay. The effects of irisin were blocked by the ERK inhibitor U0126. In conclusion, irisin improves cardiac function and reduces infarct size in post-MI mouse heart. The therapeutic effect is associated with its pro-angiogenic function through activating ERK signaling pathway.

Ischemic injury to neurons represents the underlying cause of stroke to the brain. Our previous studies identified MG53 as an essential component of the cell membrane repair machinery. Here we show that the recombinant human (rh)MG53 protein facilitates repair of ischemia-reperfusion (IR) injury to the brain. MG53 rapidly moves to acute injury sites on neuronal cells to form a membrane repair patch. IR-induced brain injury increases permeability of the blood-brain-barrier, providing access of MG53 from blood circulation to target the injured brain tissues. Exogenous rhMG53 protein can protect cultured neurons against hypoxia/reoxygenation-induced damages. Transgenic mice with increased levels of MG53 in the bloodstream are resistant to IR-induced brain injury. Intravenous administration of rhMG53, either prior to or after ischemia, can effectively alleviate brain injuries in rats. rhMG53-mediated neuroprotection involves suppression of apoptotic neuronal cell death, as well as activation of the pro-survival RISK signaling pathway. Our data indicate a physiological function for MG53 in the brain and suggest that targeting membrane repair or RISK signaling may be an effective means to treat ischemic brain injury.

Aims The proliferation of VSMCs is the pathologic basis for intimal hyperplasia after angioplasty in diabetic patients. Translocator protein (TSPO), located in the outer mitochondrial membrane, has been found to regulate redox intermediate components in cell dysfunction. We hypothesized that TSPO may regulate VSMC proliferation and migration, and be involved in the intimal hyperplasia after angioplasty in diabetes. Materials and methods Cell proliferation was measured by cell counting and MTT assays. Cell migration was measured by Transwell® and scratch-wound assays. TSPO expression in arteries of rats and high glucose-treated A10 cells were detected by immunoblotting and immunofluorescence staining. Neointimal formation of carotid artery was induced by balloon injury in type 2 diabetic rat. Key findings TSPO expression was increased in the arterial samples from diabetic rats and A10 cells treated with high glucose. Down-regulation of TSPO expression by siRNA decreased the high-glucose-induced VSMC proliferation and migration in A10 cells. This phenomenon could be simulated by using TSPO ligands, PK 11195 and Ro5-4864. cGMP/PKG signals were involved in the TSPO ligand action, since in the presence of cGMP or PKG inhibitor ODQ or KT5823 respectively, the effect of PK 11195 on VSMC proliferation was blocked. Furthermore, PK 11195 significantly inhibited neointimal formation by the inhibition of VSMC proliferation. Significance This study suggests that TSPO inhibition suppresses the proliferation and migration of VSMCs induced by hyperglycemia, consequently, preventing atherosclerosis and restenosis after angioplasty in diabetic conditions. TSPO may be a potential therapeutic target to reduce arterial remodeling induced by angioplasty in diabetes.

The dopaminergic and renin–angiotensin systems interact to regulate blood pressure. Disruption of the D 4 dopamine receptor gene in mice produces hypertension that is associated with increased renal angiotensin type 1 (AT 1 ) receptor expression. We hypothesize that the D 4 receptor can inhibit AT 1 receptor expression and function in renal proximal tubule cells from Wistar–Kyoto (WKY) rats, but the D 4 receptor regulation of AT 1 receptor is aberrant in renal proximal tubule cells from spontaneously hypertensive rats (SHRs). The D 4 receptor agonist, PD168077, decreased AT 1 receptor protein expression in a time- and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT 1 receptor protein expression. The inhibitory effect of D 4 receptor on AT 1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D 4 receptor–mediated signaling pathway. Angiotensin II increased Na + -K + ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na + -K + ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D 4 receptor on angiotensin II–mediated stimulation of Na + -K + ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT 1 receptor on Na + -K + ATPase activity in SHR cells. This was confirmed in vivo; pretreatment with PD128077 for 1 week augmented the antihypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D 4 and AT 1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension.

The abnormal migration of vascular smooth muscle cells (VSMCs) has been implicated to contribute to lesion formation in the adult vasculature. The renin-angiotensin-aldosterone system (RAAS) is intensively involved in the pathogenesis of a variety of cardiovascular diseases. There are increasing pieces of evidence for interactions between RAAS and dopamine receptors. We hypothesize that the D3 receptor has an inhibitory effect on angiotensin II (Ang II)/aldosterone-induced VSMC migration.In this study, embryonic thoracic aortic smooth muscle cells were cultured. VSMC migration was determined by the Boyden chamber and wound healing assays.VSMC migration was increased by Ang II (10(-10)-10(-7) mol/L) in a concentration-dependent manner, but not by aldosterone (10(-10)-10(-7) mol/L), and a synergistic effect of Ang II (10(-10) mol/L)/aldosterone (10(-10)mol/L) was also observed in VSMC migration. The migratory effects of Ang II alone/with aldosterone were attenuated by the activation of D3 receptors (10(-10)-10(-7) mol/L), although a D3 receptor agonist, PD128907, by itself, had no effect on VSMC migration. The inhibitory effect of the D3 receptor on Ang II/ aldosterone-mediated VSMC migration was blocked by the blocker of PKA (14-22 amide, 10(-7) mol/L), indicating that PKA was involved in the signaling pathway.These results indicate that activation of vascular D3 receptors inhibits Ang II/aldosterone-induced VSMC migration through the PKA signal pathway, which may be important in the regulation of vascular remodeling.

Limb remote ischemic preconditioning (RIPC) is an effective means of protection against ischemia/reperfusion (IR)-induced injury to multiple organs. Many studies are focused on identifying endocrine mechanisms that underlie the cross-talk between muscle and RIPC-mediated organ protection. We report that RIPC releases irisin, a myokine derived from the extracellular portion of fibronectin domain-containing 5 protein (FNDC5) in skeletal muscle, to protect against injury to the lung. Human patients with neonatal respiratory distress syndrome show reduced concentrations of irisin in the serum and increased irisin concentrations in the bronchoalveolar lavage fluid, suggesting transfer of irisin from circulation to the lung under physiologic stress. In mice, application of brief periods of ischemia preconditioning stimulates release of irisin into circulation and transfer of irisin to the lung subjected to IR injury. Irisin, via lipid raft-mediated endocytosis, enters alveolar cells and targets mitochondria. Interaction between irisin and mitochondrial uncoupling protein 2 (UCP2) allows for prevention of IR-induced oxidative stress and preservation of mitochondrial function. Animal model studies show that intravenous administration of exogenous irisin protects against IR-induced injury to the lung via improvement of mitochondrial function, whereas in UCP2-deficient mice or in the presence of a UCP2 inhibitor, the protective effect of irisin is compromised. These results demonstrate that irisin is a myokine that facilitates RIPC-mediated lung protection. Targeting the action of irisin in mitochondria presents a potential therapeutic intervention for pulmonary IR injury.

Background— Long noncoding RNAs (lncRNAs) have recently been implicated in many biological processes and diseases. Atherosclerosis is a major risk factor for cardiovascular disease. However, the functional role of lncRNAs in atherosclerosis is largely unknown. Methods and Results— We identified lincRNA-p21 as a key regulator of cell proliferation and apoptosis during atherosclerosis. The expression of lincRNA-p21 was dramatically downregulated in atherosclerotic plaques of ApoE −/− mice, an animal model for atherosclerosis. Through loss- and gain-of-function approaches, we showed that lincRNA-p21 represses cell proliferation and induces apoptosis in vascular smooth muscle cells and mouse mononuclear macrophage cells in vitro. Moreover, we found that inhibition of lincRNA-p21 results in neointimal hyperplasia in vivo in a carotid artery injury model. Genome-wide analysis revealed that lincRNA-p21 inhibition dysregulated many p53 targets. Furthermore, lincRNA-p21, a transcriptional target of p53, feeds back to enhance p53 transcriptional activity, at least in part, via binding to mouse double minute 2 (MDM2), an E3 ubiquitin-protein ligase. The association of lincRNA-p21 and MDM2 releases MDM2 repression of p53, enabling p53 to interact with p300 and to bind to the promoters/enhancers of its target genes. Finally, we show that lincRNA-p21 expression is decreased in patients with coronary artery disease. Conclusions— Our studies identify lincRNA-p21 as a novel regulator of cell proliferation and apoptosis and suggest that this lncRNA could serve as a therapeutic target to treat atherosclerosis and related cardiovascular disorders.

The dopaminergic and sympathetic systems interact to regulate blood pressure. Our previous studies showed regulation of α1-adrenergic receptor function by D1-like dopamine receptors in vascular smooth muscle cells. Because renalase could regulate circulating epinephrine levels and dopamine production in renal proximal tubules (RPTs), we tested the hypothesis that D1-like receptors regulate renalase expression in kidney. The effect of D1-like receptor stimulation on renalase expression and function was measured in immortalized RPT cells from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). We found that the D1-like receptor agonist fenoldopam (10−7–10−5 mol/l) increased renalase protein expression and function in WKY RPT cells but decreased them in SHR cells. Fenoldopam also increased renalase mRNA levels in WKY but not in SHR cells. In contrast, fenoldopam increased the degradation of renalase protein in SHR cells but not in WKY cells. The regulation of renalase by the D1-like receptor was mainly via the D5 receptor because silencing of the D5 but not D1 receptor by antisense oligonucleotides blocked the stimulatory effect of the D1-like receptor on renalase expression in WKY cells. Moreover, inhibition of PKC, by the PKC inhibitor 19–31, blocked the stimulatory effect of fenoldopam on renalase expression while stimulation of PKC, by a PKC agonist (PMA), increased renalase expression, indicating that PKC is involved in the process. Our studies suggest that the D5 receptor positively regulates renalase expression in WKY but not SHR RPT cells; aberrant regulation of renalase by the D5 receptor may be involved in the pathogenesis of hypertension.

Extracellular vesicles (EVs) carry signals within or at their limiting membranes, providing a mechanism by which cells can exchange more complex information than what was previously thought. In addition to mRNAs and microRNAs, there are DNA fragments in EVs. Solexa sequencing indicated the presence of at least 16434 genomic DNA (gDNA) fragments in the EVs from human plasma. Immunofluorescence study showed direct evidence that acridine orange-stained EV DNAs could be transferred into the cells and localize to and inside the nuclear membrane. However, whether the transferred EV DNAs are functional or not is not clear. We found that EV gDNAs could be homologously or heterologously transferred from donor cells to recipient cells, and increase gDNA-coding mRNA, protein expression, and function (e.g. AT1 receptor). An endogenous promoter of the AT1 receptor, NF-κB, could be recruited to the transferred DNAs in the nucleus, and increase the transcription of AT1 receptor in the recipient cells. Moreover, the transferred EV gDNAs have pathophysiological significance. BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia, could be transferred from K562 EVs to HEK293 cells or neutrophils. Our present study shows that the gDNAs transferred from EVs to cells have physiological significance, not only to increase the gDNA-coding mRNA and protein levels, but also to influence function in recipient cells.

Aims Vascular inflammation is a key factor in the pathogenesis of diabetes-related vascular complications. Our previous study showed that (−)-epigallocatechin-3-gallate (EGCG) inhibits high glucose-induced vascular smooth muscle cell proliferation, thus it may have beneficial effects in diabetes and its complications. However, the effect of EGCG on inflammation in diabetes is not known. In the present study, we investigated whether EGCG suppresses the vascular inflammation induced by high glucose in human umbilical vein endothelial cells (HUVECs). Main methods The inhibitory effect of EGCG on high glucose-induced up-regulation of the expression of vascular cell adhesion molecule 1 (VCAM-1) was measured using enzyme-linked immunosorbent, RT-PCR, immunoblotting and cell adhesion assays. The effect of EGCG on high glucose-induced nuclear factor-kappa B (NF-κB) activation was investigated by immunoblotting, immunofluorescence and electrophoretic mobility shift assays. Key findings High glucose increased VCAM-1 expression and enhanced the adhesion of monocytes to HUVECs. Pretreatment with EGCG in a concentration-dependent manner (1.0–50 μM) significantly attenuated these effects. High glucose (25 mM)-mediated vascular inflammation was blocked by PKC pseudosubstrate (PKC inhibitor 19–31) or the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Stimulation with high glucose increased the NF-κB translocation from the cytoplasm to the nucleus, and increased IκB-α phosphorylation, decreased its expression, and in the presence of EGCG, the effect of high glucose on NF-κB and IκB-α were blocked. Significance EGCG suppresses high glucose-induced vascular inflammatory process via the inhibition of PKC and NF-κB activation in HUVECs, suggesting that EGCG may be a potential candidate for the treatment and prevention of diabetic vascular complications.

Both the dopaminergic and renin-angiotensin systems play important roles in the regulation of blood pressure. Our previous study showed that the stimulation of dopaminergic D

Background Ion transport in the renal proximal tubule ( RPT ), which is increased in essential hypertension, is regulated by numerous hormones and humoral factors, including insulin and dopamine. Activation of dopamine receptor inhibits sodium reabsorption, whereas activation of insulin receptor increases sodium reabsorption in RPT s, and hyperinsulinemic animals and patients have defective renal dopaminergic system. We presume that there is an inhibition of D 4 receptor on insulin receptor expression and effect, and the regulation is lost in spontaneously hypertensive rats ( SHR s). Methods and Results Insulin receptor expression was determined by immunoblotting, and Na + ‐K + ‐ ATP ase activity was detected in both Wistar‐Kyoto ( WKY ) and SHR RPT cells. Stimulation of D 4 receptor with PD 168077 decreased expression of insulin receptors, which was blocked in the presence of the calcium‐channel blocker, nicardipine (10 −6 mol/L per 24 hours), in cell culture medium without calcium or in the presence of inositol 1,4,5‐trisphosphate ( IP 3) receptor blocker (2‐aminoethyl diphenylborinate [2‐ ADB] ; 10 −6 mol/L per 24 hours), indicating that extracellular calcium entry and calcium release from the endoplasmic reticulum were involved in the signal pathway. Stimulation of the insulin receptor stimulated Na + ‐K + ‐ ATP ase activity, whereas pretreatment with PD 168077 for 24 hours decreased the inhibitory effects of insulin receptor on Na + ‐K + ‐ ATP ase activity in WKY cells. However, in SHR cells, inhibition of D 4 receptor on insulin receptor expression and effect were lost. Conclusions Activation of D 4 receptor inhibits insulin receptor expression in RPT cells from WKY rats. The aberrant inhibition of D 4 receptor on insulin receptor expression and effect might be involved in the pathogenesis of essential hypertension.

The renin-angiotensin (Ang) system controls blood pressure, in part, by regulating renal tubular sodium transport. In the kidney, activation of the angiotensin II type 1 (AT(1)) receptor increases renal sodium reabsorption, whereas the angiotensin II type 2 (AT(2)) receptor produces the opposite effect. We hypothesized that the AT(2) receptor regulates AT(1) receptor expression and function in the kidney.In immortalized renal proximal tubule (RPT) cells from Wistar-Kyoto rats, CGP42112, an AT(2) receptor agonist, decreased AT(1) receptor mRNA and protein expression (P 0.05), as assessed by reverse transcriptase-polymerase chain reaction and immunoblotting. The inhibitory effect of the AT(2) receptor on AT(1) receptor expression was blocked by the AT(2) receptor antagonist, PD123319 (10 (-6)mol/l), the nitric oxide synthase inhibitor N(w)-nitro-L-arginine methyl ester (10(-4) mol/l), or the nitric oxide-dependent soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10(-5) mol/l), indicating that both nitric oxide and cyclic guanosine monophosphate (cGMP) were involved in the signaling pathway. Furthermore, CGP42112 decreased Sp1 serine phosphorylation and reduced the binding of Sp1 to AT(1) receptor DNA. Stimulation with Ang II (10(-11) mol/l per 30 min) enhanced Na(+)-K(+)-ATPase activity in RPT cells, which was prevented by pretreatment with CGP42112 (10(-7) mol/l per 24 h) (P 0.05). The above-mentioned results were confirmed in RPT cells from AT(2) receptor knockout mice; AT(1) receptor expression and Ang II-stimulated Na-K-ATPase activity were greater in these cells than in RPT cells from wild-type mice (P 0.05). AT(1)/AT(2) receptors co-localized and co-immunoprecipitated in RPT cells; short-term CGP42112 (10 mol/l per 30 min) treatment increased AT(1)/AT(2) receptor co-immunoprecipitation (P 0.05).These results indicate that the renal AT(2) receptor, via nitric oxide/cGMP/Sp1 pathway, regulates AT(1 )receptor expression and function, which may be important in the regulation of sodium excretion and blood pressure.

Abnormal vascular smooth muscle cell (VSMC) proliferation, which is central to the development of vascular diseases, including hypertension, is regulated by numerous hormones and humoral factors.1–3 Epidemiological evidence supports a link between insulin resistance and hypertension.2,3 Insulin and its receptor stimulate VSMC proliferation and insulin levels are increased in patients with essential hypertension. The high levels of insulin may play an important role in the risk factors and pathogenesis of hypertension by increasing conduit vessel stiffness and peripheral vascular resistance by stimulating VSMC proliferation.2,3 Dopamine is an endogenous catecholamine that regulates or modulates many cellular and physiological processes, including behavior, hormone synthesis and release, blood pressure, and transmembrane ion transport.4 Dopamine receptors are classified into the D1- and D2-like subtypes based on their structure and pharmacology. D1-like receptors, comprised of D1 and D5 receptors, stimulate adenylyl cyclase activity, whereas D2-like receptors, comprised of D2, D3, and D4 receptors, inhibit adenylyl cyclase activity and regulate/modulate the activity of several ion channels.4 Our previous studies found that stimulation of the D3 receptor causes vasodilation, which is mediated by activation of small- and/or large-conductance calcium-activated potassium channels.5 Stimulation of α1-adrenergic receptor increases VSMC proliferation; in the presence of the D3 receptor agonist, PD128907, the α1-adrenergic receptor-mediated proliferative effect is inhibited.1 There is increasing evidence for an interaction between insulin and dopamine receptors. Hyperinsulinemic animals and patients have a defective renal dopaminergic system; activation of the D2-like dopamine receptor decreases insulin levels in obese women6 and activation of D1-like receptors improves peripheral insulin sensitivity and renal function in rats with streptozotocin-induced type 2 diabetes.7 The D3 receptor, as with the insulin receptor, is expressed in VSMCs.5 We hypothesize that the D3 receptor, as with D1-like receptors, may have an inhibitory effect on insulin receptor expression and function, which is involved in the regulation of blood pressure. Therefore, this study was designed to investigate the role of the D3 receptor on insulin receptor expression and function, and to determine the underlying mechanisms by which the D3 receptor regulates the insulin receptor in A10 cell, a VSMC line from embryonic thoracic aorta of normotensive Berlin-Druckrey IX rats. In addition, insulin receptor expression was quantified in aorta of D3 receptor deficient mice.

Background: Activation of renal D3 receptor induces natriuresis and diuresis in Wistar-Kyoto (WKY) rats; in the presence of ETB receptor antagonist, the natriuretic effect of D3 receptor in WKY rats is reduced. We hypothesize that ETB receptor activation may regulate D3 receptor expression in renal proximal tubule (RPT) cells from WKY rats, which is impaired in RPT cells from spontaneously hypertensive rats (SHRs). Methods: D3 receptor expression was determined by immunoblotting; the D3/ETB receptor linkage was checked by coimmunoprecipitation; Na+-K+-ATPase activity was determined as the rate of inorganic phosphate released in the presence or absence of ouabain. Results: In RPT cells from WKY rats, the ETB receptor agonist BQ3020 increased D3 receptor protein. In contrast, in RPT cells from SHRs, BQ3020 did not increase D3 receptor. There was coimmunoprecipitation between D3 and ETB receptors in RPT cells from WKY and SHRs. Activation of ETB receptor increased D3/ETB coimmunoprecipitation in RPT cells from WKY rats, but not from SHRs. The basal levels of D3/ETB receptor coimmunoprecipitation were greater in RPT cells from WKY rats than in those from SHRs. Stimulation of D3 receptor inhibited Na+-K+-ATPase activity, which was augmented by the pretreatment with the ETB receptor agonist BQ3020 in WKY RPT cells, but not in SHR RPT cells. Conclusion: ETB receptors regulate and physically interact with D3 receptors differently in WKY rats and SHRs. The impaired natriuretic effect in SHRs may be, in part, related to impaired ETB and D3 receptor interactions.

Essential hypertension is one of the most common risk factors in developed and developing countries, affecting ~25% of the middle-aged adult population. The long-term regulation of blood pressure rests on renal and nonrenal mechanisms.1 Ion transport in the renal proximal tubule (RPT) and thick ascending limb of Henle, which is increased in essential hypertension, is regulated by numerous hormones and humoral factors, including insulin and dopamine. Activation of dopamine receptor inhibits sodium reabsorption, whereas activation of insulin receptor increases sodium reabsorption in RPTs.2–5 Insulin is an important regulator of renal salt and water excretion, and hyperinsulinemia has been implicated to play a role in hypertension.3–5 Diabetes mellitus is associated with Na+ and water retention and extracellular fluid volume expansion.4 There is evidence that insulin directly stimulates fluid absorption in rabbit proximal convoluted tubules.5 This stimulatory effect on the proximal tubule is associated with increased Na+/H+ exchanger and Na+–K+ ATPase activities. Dopamine is an endogenous catecholamine that regulates/modulates many cellular activities, including blood pressure and transmembrane ion transport. Dopamine receptors are classified into the D1- and D2-like subtypes based on their structure and pharmacology. D1-like receptors, consisted of D1 and D5 receptors, stimulate adenylyl cyclase activity, whereas D2-like receptors, consisted of D2, D3, and D4 receptors, inhibit adenylyl cyclase activity and regulate/modulate the activity of several ion channels.2 Increasing pieces of evidence show an interaction between insulin and dopamine receptors.3,6,7 Hyperinsulinemic animals and patients with noninsulin-dependent diabetes have defective renal dopaminergic system.6 In obese Zucker rats, a model of type 2 diabetes, renal D1 receptor is downregulated and dopamine fails to produce both diuresis and natriuresis. Treatment with the insulin sensitizer rosiglitazone, a peroxisome proliferator–activated receptor-γ agonist, decreases plasma insulin levels and restores renal D1-receptor function. Chronic exposure of cells to insulin causes both a reduction in D1-receptor abundance, and uncoupling of D1 receptor from G protein/effector complex;3 there is also a diminution of the inhibitory effect of dopamine on Na+–K+ ATPase activity in hyperinsulinemic rats.7 As a major receptor subtype, the D5 receptor, like the Dl receptor, is expressed in RPTs.2,8 Disruption of the D5 receptor gene produces hypertension in mice, and a high salt diet increases their blood pressure further.9,10 Activation of the D5 receptor stimulates cyclic adenosine monophosphate (cAMP) production.9,11 Therefore, it is possible that the natriuretic effect of dopamine is partially mediated by the D5 receptor. Hence, we hypothesized that insulin may increase D5 receptor expression and function, which may be aberrant in hypertensive states. To test the hypothesis mentioned earlier, we evaluated insulin and D5 receptor interaction in immortalized RPT cells from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs), and the physiological significance of any interaction in D5 receptor cDNA-transfected HEK293 (HEK293-D5) cells. These RPT cells behave similarly to freshly obtained RPT cells, at least with regard to dopamine receptors and responses to G protein stimulation.12,13

Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53(-/-) mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing.

Renal ischemia-reperfusion (I/R) injury causes renal tubular necrosis, apoptosis, and inflammation leading to acute renal dysfunction. Recent studies have revealed that deletion of Gα12 mitigates the renal damage due to I/R injury. Our previous study showed that activation of dopamine D3 receptor (D3R) increased its linkage with Gα12, and hampered Gα12-mediated stimulation of renal sodium transport. In the present study, we used an in-vivo rat model and an in vitro study of the renal epithelial cell line (NRK52E) to investigate whether or not an increased linkage between D3R and Gα12 contributes to the protective effect of D3R on renal I/R injury.For in vivo studies, I/R injury was induced in a rat renal unilateral clamping model. For in vitro studies, hypoxia/reoxygenation and cold storage/rewarming injuries were performed in NRK52E cells. PD128907, a D3R agonist, or vehicle, was administered 15 minutes before clamping (or hypoxia) in both the in vivo or in vitro studies.In the rat renal unilateral clamping model, pretreatment with PD128907 (0.2 mg/kg, intravenous) protected against renal I/R injury and increased survival rate during a long-term follow-up after 7 days. A decrease in the generation of reactive oxygen species, apoptosis, and inflammation may be involved in the D3R-mediated protection because pretreatment with PD128907 increased renal glutathione and superoxide dismutase levels and decreased malondialdehyde levels in the I/R group. The increase in cytokines (TNF-α, IL-1β, and IL-10) and myeloperoxidase in I/R injured kidney was also prevented with a simultaneous decrease in the apoptosis of the epithelial cells and expression of apoptosis biomarkers in kidney harvested 1 day after I/R injury. The increase in the coimmunoprecipitation between D3R and Gα12 with D3R stimulation paralleled the observed renal protection from I/R injury. Moreover, in vitro studies showed that transient overexpression of Gα12 in the NRK52E cells attenuated the protective effect of PD128907 on hypoxia/reoxygenation injury. The protective effect of PD128907 might be of significance to renal transplantation because cold storage/rewarming induced injury increased lactate dehydrogenase release and decreased cell viability in NRK52E cells. Conversely, in the presence of PD128907, the increased lactate dehydrogenase release and decreased cell viability were reversed.These results suggest that activation of D3R, by decreasing Gα12-induced renal damage, may exert a protective effect from I/R injury.

We set out to investigate whether and how SRY (sex-determining region, Y) DNAs in plasma EVs (extracellular vesicles) is involved in the pathogenesis of atherosclerosis. PCR and gene sequencing found the SRY gene fragment in plasma EVs from male, but not female, patients; EVs from male patients with CAD (coronary artery disease) had a higher SRY GCN (gene copy number) than healthy subjects. Additional studies found that leucocytes, the major source of plasma EVs, had higher SRY GCN and mRNA and protein expression in male CAD patients than controls. After incubation with EVs from SRY -transfected HEK (human embryonic kidney)-293 cells, monocytes (THP-1) and HUVECs (human umbilical vein endothelial cells), which do not endogenously express SRY protein, were found to express newly synthesized SRY protein. This resulted in an increase in the adherence factors CD11-a in THP-1 cells and ICAM-1 (intercellular adhesion molecule 1) in HUVECs. EMSA showed that SRY protein increased the promoter activity of CD11-a in THP-1 cells and ICAM-1 in HUVECs. There was an increase in THP-1 cells adherent to HUVECs after incubation with SRY-EVs. SRY DNAs transferred from EVs have pathophysiological significance in vivo ; injection of SRY EVs into ApoE−/− (apolipoprotein-knockout) mice accelerated atherosclerosis. The SRY gene in plasma EVs transferred to vascular endothelial cells may play an important role in the pathogenesis of atherosclerosis; this mechanism provides a new approach to the understanding of inheritable CAD in men. Abbreviations: ApoE−/−, apolipoprotein E-knockout; AT1R, angiotensin type 1 receptor; BCECF, 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein; BCECF/AM, BCECF acetoxymethyl ester; CAD, coronary artery disease; CML, chronic myeloid leukaemia; CMV, cytomegalovirus; EV, extracellular vesicle; GCN, gene copy number; HEK, human embryonic kidney; HUVEC, human umbilical vein endothelial cell; ICAM-1, intercellular adhesion molecule 1; RT, reverse transcription; SRY, sex-determining region, Y

Renal denervation is a novel method for hypertension treatment. In this study, we aimed to investigate the safety and efficacy of radiofrequency ablation (RFA) at ablation temperatures of 45 °C or 50 °C and its possible mechanisms.A hypertensive canine model was established by abdominal aortic constriction in 20 healthy hybrid dogs. These dogs were then randomly assigned to the treatment and the control groups, with dogs in the treatment group further randomly assigned to receive RFA at ablation temperatures of 45 °C or 50 °C. In the treatment group, RFA was performed at 1 month after modeling; renal angiography was performed at 2 months after ablation. The arterial vessels of the dogs were examined histologically with hematoxylin and eosin staining. Changes in blood pressure in the foreleg and whole-body norepinephrine spillover rate were also assessed.No arterial stenosis, dissection, thrombosis or other abnormalities were detected in the treated vessels by renal angiography, yet histology results showed minimal to mild renal arterial injury. Renal denervation resulted in a marked decrease in the whole-body norepinephrine spillover rate (p0.05) in addition to significantly reducing blood pressure (p0.05), with no significant differences detected between the 45 °C and 50 °C subgroups for both (p0.05).Renal denervation can be performed without acute major adverse events, using catheter-based RFA with temperature control. The procedure was feasible in reducing blood pressure by at least partially inhibiting sympathetic drive and systemic sympathetic outflow.

Oral NaCl produces a greater natriuresis and diuresis than the intravenous infusion of the same amount of NaCl. Gastrin is the major gastrointestinal hormone taken up by renal proximal tubule (RPT) cells. We hypothesized that renal gastrin and dopamine receptors interact to synergistically increase sodium excretion, an impaired interaction of which may be involved in the pathogenesis of hypertension. In Wistar-Kyoto (WKY) rats, infusion of gastrin induced natriuresis and diuresis, which was abrogated in the presence of a gastrin (CCK B R; CI-988) or D 1 -like receptor antagonist (SCH23390). Similarly, the natriuretic and diuretic effects of fenoldopam, a D 1 -like receptor agonist, were blocked by SCH23390, as well as by CI-988. However, the natriuretic effects of gastrin and fenoldopam were not observed in spontaneously hypertensive rats (SHRs). The gastrin/D 1 -like receptor interaction was also confirmed in RPT cells. In RPT cells from WKY but not SHRs, stimulation of either D 1 -like or gastrin receptor inhibited Na + -K + -ATPase activity, an effect that was blocked in the presence of SCH23390 or CI-988. In RPT cells from WKY and SHRs, CCK B R and D 1 receptor (D 1 R) co-immunoprecipitated, which was increased after stimulation of either D 1 R or CCK B R in RPT cells from WKY rats; stimulation of one receptor increased RPT cell membrane expression of the other receptor, effects that were not observed in SHRs. These data suggest that there is a synergism between CCK B R and D 1 -like receptors to increase sodium excretion. An aberrant interaction between the renal CCK B R and D 1 -like receptors (e.g., D 1 R) may play a role in the pathogenesis of hypertension.

Summary: A dramatic increase in catecholamine (CA) concentration is believed to be a primary trigger of the neurally mediated syncope (NMS) in elderly subjects. The hypercontractile state of the heart might be alleviated by angiotensin-converting enzyme (ACE) inhibitor through depression of CA release from the sympathetic nerve ending. Thus ACE inhibitor might have positive effect on the prevention of NMS. In this study, 24 elderly subjects who had reproducible NMS induced with head-up tilt test (HUT) were randomized and double-blind divided into placebo and ACE-inhibitor groups. The plasma CA concentration [norepinephrine (NE) and epinephrine (E)] were measured during HUT, and the effects of enalapril on NMS were observed in the two groups. Before administration of enalapril, plasma CA concentrations were significantly increased during HUT compared with those in the supine position; In contrast, administration of the enalapril (10 mg/day) for >1 year inhibited the concentration of plasma CA increase and prevented syncope in all 12 patients (p < 0.05); however, placebo had no effect on plasma CA concentrations and syncope disappeared in only two of 12 patients after administration of placebo. From this study, we conclude that enalapril can prevent NMS in patients, presumably because of its part in the inhibition of CA release from sympathetic nerve endings.

G-protein–coupled receptor kinase 4 (GRK4) gene variants, via impairment of renal dopamine receptor and enhancement of renin–angiotensin system functions, cause sodium retention and increase blood pressure. Whether GRK4 and the angiotensin type 1 receptor (AT 1 R) interact in the aorta is not known. We report that GRK4 is expressed in vascular smooth muscle cells of the aorta. Heterologous expression of the GRK4γ variant 142V in A10 cells increased AT 1 R protein expression and AT 1 R-mediated increase in intracellular calcium concentration. The increase in AT 1 R expression was related to an increase in AT 1 R mRNA expression via the NF-κB pathway. As compared with control, cells expressing GRK4γ 142V had greater NF-κB activity with more NF-κB bound to the AT 1 R promoter. The increased AT 1 R expression in cells expressing GRK4γ 142V was also associated with decreased AT 1 R degradation, which may be ascribed to lower AT 1 R phosphorylation. There was a direct interaction between GRK4γ and AT 1 R that was decreased by GRK4γ 142V. The regulation of AT 1 R expression by GRK4γ 142V in A10 cells was confirmed in GRK4γ 142V transgenic mice; AT 1 R expression was higher in the aorta of GRK4γ 142V transgenic mice than control GRK4γ wild-type mice. Angiotensin II–mediated vasoconstriction of the aorta was also higher in GRK4γ 142V than in wild-type transgenic mice. This study provides a mechanism by which GRK4, via regulation of arterial AT 1 R expression and function, participates in the pathogenesis of conduit vessel abnormalities in hypertension.

Oral NaCl produces a greater natriuresis and diuresis than the intravenous infusion of the same amount of NaCl. Gastrin is the major gastrointestinal hormone taken up by renal proximal tubule (RPT) cells. We hypothesized that renal gastrin and dopamine receptors interact to synergistically increase sodium excretion, an impaired interaction of which may be involved in the pathogenesis of hypertension. In Wistar-Kyoto rats, infusion of gastrin induced natriuresis and diuresis, which was abrogated in the presence of a gastrin (cholecystokinin B receptor [CCK B R]; CI-988) or a D 1 -like receptor antagonist (SCH23390). Similarly, the natriuretic and diuretic effects of fenoldopam, a D 1 -like receptor agonist, were blocked by SCH23390, as well as by CI-988. However, the natriuretic effects of gastrin and fenoldopam were not observed in spontaneously hypertensive rats. The gastrin/D 1 -like receptor interaction was also confirmed in RPT cells. In RPT cells from Wistar-Kyoto but not spontaneously hypertensive rats, stimulation of either D 1 -like receptor or gastrin receptor inhibited Na + -K + -ATPase activity, an effect that was blocked in the presence of SCH23390 or CI-988. In RPT cells from Wistar-Kyoto and spontaneously hypertensive rats, CCK B R and D 1 receptor coimmunoprecipitated, which was increased after stimulation of either D 1 receptor or CCK B R in RPT cells from Wistar-Kyoto rats; stimulation of one receptor increased the RPT cell membrane expression of the other receptor, effects that were not observed in spontaneously hypertensive rats. These data suggest that there is a synergism between CCK B R and D 1 -like receptors to increase sodium excretion. An aberrant interaction between the renal CCK B R and D 1 -like receptors (eg, D 1 receptor) may play a role in the pathogenesis of hypertension.

Proliferation of vascular smooth muscle cells (VSMCs) participates in the pathogenesis and development of cardiovascular diseases, including essential hypertension and atherosclerosis. Our previous study found that stimulation of D1-like dopamine receptors inhibited insulin-induced proliferation of VSMCs. Insulin-like growth factor-1 (IGF-1) and insulin share similar structure and biological effect. However, whether or not there is any effect of D1-like receptors on IGF-1-induced proliferation of VSMCs is not known. Therefore, we investigated the inhibitory effect of D1-like dopamine receptors on the IGF-1-induced VSMCs proliferation in this study.VSMC proliferation was determined by [(3)H]-thymidine incorporation, the uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and cell number. Phosphorylated/non-phosphorylated IGF-1 receptor, Akt, mTOR and p70S6K expressions were determined by immunoblotting. The oligodeoxynucleotides were transfected to A10 cells to identify the effect of D1 and D5 receptors, respectively.IGF-1 increased the proliferation of VSMCs, while in the presence of fenoldopam, IGF-1-mediated stimulatory effect was reduced. Use of either antisense for D1 or D5 receptor partially inhibited the fenoldopam-induced antiproliferation effect of VSMCs. Use of both D1 and D5 receptor antisenses completely blocked the inhibitory effect of fenoldopam. In the presence of PI3k and mTOR inhibitors, the IGF-1-mediated proliferation of VSMCs was blocked. Moreover, IGF-1 increased the phosphorylation of PI3k and mTOR. The inhibitory effect of fenoldopam on VSMC proliferation might be due to the inhibition of IGF-1 receptor expression and IGF-1 phosphorylation, because in the presence of fenoldopam, the stimulatory effect of IGF-1 on phosphorylation of IGF-1 receptor, PI3k and mTOR is reduced, the IGF-1 receptor expression was reduced in A10 cells.Activation of the D1-like receptors suppressed the proliferative effect of IGF-1 in A10 cells via the inhibition of the IGF-1R/Akt/mTOR/p70S6K pathway and downregulated the expression of IGF-1 receptor.

Dopamine receptor, via D(1)-like and D(2)-like receptors, increases sodium excretion in kidney. We have reported positive interactions between D(3) and D(1) receptors in renal proximal tubule (RPT) cells. These reports, however do not preclude that there may be also interaction between D(3) and D(5) receptors, because of the lack of selective D(1) and D(5) receptor agonists or antagonists. We hypothesize that D(3) receptors can regulate D(5) receptors, and that D(3) receptor regulation of D(5) receptors in RPTs is impaired in spontaneously hypertensive rats (SHRs). It showed that a D(3) receptor agonist, PD128907, by the activation of protein kinase C activity, increased the expression of D(5) receptors in a concentration- and time-dependent manner in RPT cells from Wistar-Kyoto (WKY) rats. The stimulatory effect of the D(3) receptor on D(5) receptor expression was impaired in RPT cells from SHRs. The effect of D(3) receptor on D(5) receptor is functionally relevant; stimulation of D(5) receptor decreases Na(+)-K(+) adenosine triphosphatase (ATPase) activity in WKY cells. Pretreatment with D(3) receptor agonist for 24 h enhances the D(5) receptor expression and D(5) receptor-mediated inhibitory effect on Na(+)-K(+) ATPase activity in WKY cells, but decreases them in SHR cells. The effect of D(3) receptor on D(5) receptor expression and function was also confirmed in the D(5) receptor-transfected HEK293 cells. It indicates that activation of D(3) receptor increases D(5) receptor expression and function. Altered regulation of D(3) receptor on D(5) receptors may have a role in the pathogenesis of hypertension.

Proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development and progression of diabetes-related vascular complications. (-)-Epigallocatechin gallate (EGCG), the major catechin derived from green tea, is able to exert antidiabetes effects in animal models. However, it is not known whether or not EGCG inhibits VSMC proliferation induced by high glucose. This study tested the hypothesis that EGCG might have an inhibitory effect on VSMC proliferation induced by high glucose. VSMC proliferation was determined by [(3)H]-thymidine incorporation and uptake of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT). Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was determined by immunoblotting, and ERK 1/2 activity was detected by measuring the ability to phosphorylate its substrate Elk-1. Glucose increased VSMC proliferation in a concentration-dependent manner, which was reduced in the presence of EGCG. VSMC proliferation mediated by high glucose (30 mM) was involved in protein kinase C (PKC) and ERK1/2 signalings, because its effect was blocked by PKC inhibitor (PKC inhibitor 19-31) and ERK1/2 inhibitor (PD98059). Pretreatment of VSMCs with EGCG significantly inhibited the stimulatory effect of high glucose on PKC and ERK1/2 activation, followed by attenuation of its downstream transcription factor Elk-1 phosphorylation. Taken together, these results suggest that EGCG could suppress VSMC proliferation induced by high glucose by inhibition of PKC and ERK1/2 signalings in VSMCs, which indicates that EGCG might be a possible medicine to reduce vascular complications in diabetes.

The D(3) dopamine receptor is the major D(2)-like receptor that regulates sodium transport in the renal proximal tubule (RPT) and helps maintain blood pressure in the normal range. In Wistar-Kyoto (WKY) rats chronically fed high-salt diet, the intrarenal arterial infusion of a D(3) receptor agonist, PD128907, increased absolute and fractional sodium excretion. We have reported that Gα(12) and Gα(13), which participate in the signal transduction of the D(5) receptor, are expressed in RPTs. As the D(3) receptor is also expressed in RPTs, we hypothesized that it may also interact with Gα(12)/Gα(13) in RPTs from WKY rats. There were co-localization and co-immunoprecipitation of D(3) receptor and Gα(12)/Gα(13) in renal brush border membranes (BBMs) and RPT cells. The intrarenal infusion of PD128907 (1 μg kg(-1) min(-1)) that increased sodium excretion also increased the co-immunoprecipitations of D(3)/Gα(12) and D(3)/Gα(13) in renal BBMs; their co-immunoprecipitation was confirmed in RPT cells. As Gα(12) and Gα(13) increase sodium pump and transporter activity (for example, Na(+)-K(+)-ATPase, NHE3), an increased association of D(3) receptors with Gα(12)/Gα(13) receptors after D(3) receptor activation may be a mechanism to prevent Gα(12)/Gα(13)-mediated stimulation of sodium transport (and thus enhance natriuresis). We conclude that a D(3) receptor interaction with Gα(12)/Gα(13) that increases sodium excretion may have a role in the regulation of blood pressure.

Endothelin, originally characterized in vascular endothelial cells, is now known to be secreted by renal tubules where it can regulate sodium transport in an autocrine and/or paracrine manner.1–4 Three endothelin isoforms (ET-1, ET-2, and ET-3) interact with two receptors, ETA and ETB. ETA receptors may contribute to the pathogenesis of hypertension by stimulating vasopressor centers in the brain, and increasing the secretion of aldosterone and the release of catecholamines, renal sodium transport, growth factors, reactive oxygen species, and vascular smooth muscle contractility.2–5 However, endothelins can also relax vascular smooth muscles by releasing endothelium-derived vasodilators.3 In addition, ETB receptors can lower blood pressure by decreasing ET-1 levels, and promoting renal loss of sodium and water.1,4 Indeed, in uninephrectomized rats given deoxycorticosterone acetate and a high-salt diet, spontaneously hypertensive rats (SHRs) and humans with essential hypertension, ETB receptor activation may be a counter regulatory mechanism to the increase in blood pressure.1,4,6,7 Naturally occurring or induced deletion of the ETB receptor gene and chronic pharmacological blockade of ETB receptors in rats result in salt-sensitive hypertension.1 The actions of dopamine on cardiovascular centers in the brain, renal hemodynamics, intestinal and renal epithelial transport, production of reactive oxygen species, and secretion of hormonal/humoral agents, such as aldosterone, catecholamines, endothelin, prolactin, pro-opiomelanocortin, renin, and vasopressin, place dopamine in a central homeostatic position for regulating extracellular fluid volume and blood pressure.8,9 There are two D1-like receptors, D1 and D5, and three D2-like receptors, D2, D3, and D4, expressed in mammalian kidneys. Renal dopamine receptors,8,9 like ETB receptors,7,10–14 inhibit sodium transport. Specifically, dopamine, via D1 and D3 receptors, decreases renal sodium transport in various segments of the nephron, including the renal proximal tubule (RPT).8,9 Disruption of either the D1 or D3 dopamine receptor gene in mice produces hypertension.9,15 The hypertension in the D3 receptor null (D3–/–) mouse is associated with a decreased ability to excrete a sodium load.15 In a previous study, we found that the natriuretic consequence of D3 receptor stimulation is attenuated by an ETB receptor antagonist in Wistar-Kyoto (WKY) rats.16 Because D3 receptors in the kidney are predominantly expressed in RPTs where ETB receptors are also expressed,17–24 we hypothesize that the ETB receptor is regulated by the D3 receptor. Renal D3 receptor function is impaired in SHRs;16,18,19 therefore, we also hypothesize that D3 receptor regulation of the ETB receptor may also be aberrant in SHRs. In order to test the above hypotheses, we studied D3 and ETB receptor interaction in immortalized RPT cells. These RPT cells behave similarly to nonimmortalized RPT cells, at least with regard to dopamine receptors and responses to G protein stimulation.25–27

The sympathetic nervous system plays an important role in the regulation of blood pressure. There is increasing evidence for positive and negative interactions between dopamine and adrenergic receptors; the activation of the α-adrenergic receptor induces vasoconstriction, whereas the activation of dopamine receptor induces vasorelaxation. We hypothesize that the D1-like receptor and/or D3receptor also inhibit α1-adrenergic receptor-mediated proliferation in vascular smooth muscle cells (VSMCs). In this study, VSMC proliferation was determined by measuring [3H]thymidine incorporation, cell number, and uptake of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT). Norepinephrine increased VSMC number and MTT uptake, as well as [3H]thymidine incorporation via the α1-adrenergic receptor in aortic VSMCs from Sprague-Dawley rats. The proliferative effects of norepinephrine were attenuated by the activation of D1-like receptors or D3receptors, although a D1-like receptor agonist, fenoldopam, and a D3receptor agonist, PD-128907, by themselves, at low concentrations, had no effect on VSMC proliferation. Simultaneous stimulation of both D1-like and D3receptors had an additive inhibitory effect. The inhibitory effect of D3receptor was via protein kinase A, whereas the D1-like receptor effect was via protein kinase C-ζ. The interaction between α1-adrenergic and dopamine receptors, especially D1-like and D3receptors in VSMCs, could be involved in the pathogenesis of hypertension.

Objective:Oral NaCl intake produces stronger natriuresis and diuresis than venous infusion of the same amount NaCl, indicating the potential existence of renal-gastric axis. Gastrin, from gastrointestinal tract, is dominant one due to its natriuretic effects and taken-up by the renal proximal tubule

The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D 3 dopamine receptor gene in mice produces renin-dependent hypertension. In rats, D 2 -like receptors reduce angiotensin II binding sites in renal proximal tubules (RPTs). Because the major D 2 -like receptor in RPTs is the D 3 receptor, we examined whether D 3 receptors regulate angiotensin II type 1 (AT 1 ) receptors in rat RPT cells. The effect of D 3 receptors on AT 1 receptors was studied in vitro and in vivo. The D 3 receptor agonist PD128907 decreased AT 1 receptor protein and mRNA in WKY RPT cells and increased it in SHR cells. PD128907 increased D 3 receptors in WKY cells but had no effect in SHR cells. D 3 /AT 1 receptors colocalized in RPT cells; D 3 receptor stimulation decreased the percent amount of D 3 receptors that coimmunoprecipitated with AT 1 receptors to a greater extent in WKY than in SHR cells. However, D 3 receptor stimulation did not change the percent amount of AT 1 receptors that coimmunoprecipitated with D 3 receptors in WKY cells and markedly decreased the coimmunoprecipitation in SHR cells. The D 3 receptor also regulated the AT 1 receptor in vivo because AT 1 receptor expression was increased in kidneys of D 3 receptor–null mice compared with wild type littermates. D 3 receptors may regulate AT 1 receptor function by direct interaction with and regulation of AT 1 receptor expression. One mechanism of hypertension may be related to increased renal expression of AT 1 receptors due decreased D 3 receptor regulation.

Objective Oral NaCl intake produces stronger natriuresis and diuresis than venous infusion of same amount, indicating the existence of renal-gastric axis. Gastrin, from gastrointestinal tract, is dominant one due to its natriuretic effects and taken-up by the renal proximal tubule (RPT) cells. We hypothesise that gastrin interact with dopamine receptors in kidney, resulting in synergistically increased sodium excretion. The impaired interaction might be involved in hypertension. Method Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR) and RPT cells were stimulated or blocked through D 1 -like dopamine and gastrin receptors to observe Na + -K + -ATPase activity and natriuresis. Result Gastrin infusing WKY rats via renal artery induced natriuresis and diuresis, which was blocked in the presence of CI988, a gastrin receptor blocker. Similarly, effect hereinbefore of fenoldopam, a D 1 -like receptor agonist, was blocked by D 1 -like receptor antagonist, SCH23390, indicating gastrin and fenoldopam play natriuretic and diuretic effect through individual receptors. Lower dosages of gastrin or fenoldopam failed to induce natriuresis and diuresis alone, while putting together induced the effects. The above-mentioned effects were lost in SHRs. Natriuresis and diuresis was partially blocked by SCH23390 or CI988, indicating the interaction between gastrin and D1-like receptor. Stimulation of either receptor increased the expression of the other and inhibited Na + -K + -ATPase activity, while the inhibitory effect of Na + -K + -ATPase activity was partially blocked through its corresponding receptors due to respective existence of SCH23390 and CI988. Conclusion It indicated the synergistic effect between gastrin and D1-like receptor would increase the sodium excretion in WKY rats; the impaired interaction might be involved in the pathogenesis of hypertension.

Objective The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D 4 dopamine receptor gene in mice produces hypertension that is associated with increased renal AT 1 receptor expression. We hypothesise that the D 4 receptor can inhibit AT 1 receptor expression and function in renal proximal tubules (RPTs) cells from Wistar-Kyoto (WKY) rats but the D 4 receptor regulation of AT 1 receptor is aberrant in RPT cells from spontaneously hypertensive rats (SHRs). Methods Immortalised RPT cells from WKY and SHRs were treated by The D 4 receptor agonist, PD168077 to determine the AT 1 receptor protein expression and Na + -K + ATPase activity, and treated by calcium channel blocker, nicardipine to determine the signalling molecule. Rats were treated in vivo by D 4 receptor agonist and losartan to obverse the anti-hypertensive and natriuretic effect. Results The D 4 receptor agonist, PD168077, decreased AT 1 receptor protein expression in a time (0–30 hrs)- and concentration (10 –9 –10 –5 M)-dependent manner in WKY RPT cells. By contrast, in SHR cells, PD168077 (10 –6 M) increased AT 1 receptor protein expression. The inhibitory effect of D 4 receptor on AT 1 receptor expression in WKY RPT cells was blocked by a calcium channel blocker, nicardipine (10 –6 M), or calcium-free medium, indicating that calcium is involved as a signalling molecule in the D 4 receptor-mediated signalling pathway. Angiotensin II (10 –11 M) increased Na + -K + ATPase activity in WKY cells. Pretreatment with PD168077 (10 –6 M/24hrs) decreased the stimulatory effect of angiotensin II on Na + -K + ATPase activity in WKY cells. However, in SHR cells, the inhibitory effect of D 4 receptor on angiotensin II-mediated stimulation of Na + -K + ATPase activity was aberrant; pretreatment with D 4 receptor agonist augmented the stimulatory effect of AT 1 receptor on Na + -K + ATPase activity in SHR cells. This was confirmed in in vivo studies; pre-treatment with PD128077 for one week augmented the anti-hypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. Conclusions We suggest that an aberrant interaction between D 4 and AT 1 receptors may play a role in the abnormal regulation of sodium excretion and blood pressure in hypertension.

Our previous study showed that besides mRNAs and microRNAs, there are DNA fragments within extracellular vesicles (EVs). The BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia (CML), could be transferred from K562 EVs to neutrophils and decrease their phagocytic activity in vitro. Our present study provides evidence that BCR/ABL DNAs transferred from EVs have pathophysiological significance in vivo. Two months after injection of K562 EVs into the tail vein of Sprague-Dawley (SD) rats, they showed some characteristics of CML, e.g., feeble, febrile, and thin, with splenomegaly and neutrophilia but with reduced neutrophil phagocytic activity. These findings were also observed in immunodeficient NOD/SCID mice treated with K562 EVs; BCR/ABL mRNA and protein were found in their neutrophils. The administration of actinomycin D, an inhibitor of de novo mRNA synthesis, prevented the abnormalities caused by K562 EVs in NOD/SCID mice related to CML, including neutrophilia and bone marrow hyperplasia. As a specific inhibitor of tyrosine kinases, imatinib blocked the activity of tyrosine kinases and the expression of phospho-Crkl, induced by the de novo BCR/ABL protein caused by K562 EVs bearing BCR/ABL DNA. Our current study shows the pathophysiological significance of transferred tumor gene from EVs in vivo, which may represent an important mechanism for tumorigenesis, tumor progression, and metastasis.