Limin Lu

Chronic angiotensin (1-7) injection accelerates STZ-induced diabetic renal injury.

AbstractAim:The renin-angiotensin system (RAS) plays a critical role in blood pressure control and body fluid and electrolyte homeostasis. In the past few years, angio-tensin (Ang) (1–7) has been reported to counteract the effects of Ang II and was even considered as a new therapeutical target in RAS. The present study aimed to investigate the effect of Ang (1–7) administration on a diabetic animal model and the modulation on local RAS.Methods:Streptozotocin (STZ) injection-induced diabetic rats were used in the experiment. The animals were divided into 3 groups: (1) control; (2) STZ-induced diabetes; and (3) STZ-induced diabetes with chronic Ang (1–7) treatment [D+Ang(1–7)]. In the D+Ang(1–7) group, a dose of 25 μg·kg−1·h−1 of Ang (1–7) was continually injected through the jugular vein by embedding mini-osmotic pump for 6 weeks. Plasma glucose, ratio of kidney to body weight, and 24 h urine protein and serum creatinine were monitored by conventional measurement. Plasma and renal Ang II levels were measured by radioimmunoassay. Ang-converting enzyme (ACE), ACE2, Ang II type 1 (AT1) receptor, Ang II type 2 (AT2) receptor, Ang (1–7) Mas receptor, and TGF-β1 mRNA levels were measured by real time PCR; ACE, ACE2, and TGF-β1 protein levels were analyzed by Western blotting.Results:The renal function of diabetic rats was significantly retrogressed when compared with that of control rats. After the treatment by constant Ang (1–7) vein injection for 6 weeks, renal function was found to be even worse than diabetic rats, and both TGF-β1 mRNA and protein levels were elevated in the D+Ang(1–7) group compared with the diabetic rats. The real-time PCR result also showed an increase in ACE mRNA expression and decrease in ACE2 mRNA level in the D+Ang(1–7) group when compared with diabetic rats. The number of AT1 receptors increased in the Ang (1–7)-injected group, while the number of AT2 and Mas receptors decreased.Conclusion:Exogenous Ang (1–7) injection did not ameliorate STZ-induced diabetic rat renal injury; on the contrary, it accelerated the progressive diabetic nephropathies.

Inhibition of renin/prorenin receptor attenuated mesangial cell proliferation and reduced associated fibrotic factor release.

The present study was designed to investigate the physiological roles of renin/prorenin receptor in cultured rat mesangial cells. The presence of renin/prorenin receptor in mesangial cells was determined by immunofluorescence staining, real-time quantitative reverse transcriptase-polymerase chain reaction (real-time PCR) and Western blotting assay. The expression of renin/prorenin receptors was identified in both rat renal slices and cultured mesangial cells. Meanwhile, the cultured mesangial cells were demonstrated synthesizing and secreting (Pro)renin as well. Knockdown of renin/prorenin receptor expression was performed by small interfering RNA (siRNA) transfection. Either knockdown or blockade of renin/prorenin receptor by handle region peptide (HRP) reduced mesangial cell proliferation. In the meantime, release of type IV collagen, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and mRNA level of transforming growth factor-beta1 (TGF-beta1) were decreased. On the other hand, both the production and activity of matrix metalloproteinase-2, a key enzyme to degrade type IV collagen, was increased. The present results indicated that renin/prorenin receptor played a regulatory role in mesangial cells proliferation and extracellular matrix accumulation. Blockade of renin/prorenin receptors may postpone over extracellular accumulation, which occurs in some pathological situations, via suppressed both mesangial cell proliferation and fibrotic factor release.

Angiotensin AT1 receptor activation mediates high glucose-induced epithelial-mesenchymal transition in renal proximal tubular cells.

1. Renal tubular epithelial cells can undergo epithelial to mesenchymal transition (EMT) under hyperglycaemic conditions, which is associated with renal interstitial fibrosis. Activation of the renin–angiotensin system (RAS) is involved in diabetic nephropathy. The present study investigated the positive role of angiotensin AT1 receptors in high glucose‐induced EMT in cultured tubular epithelial cells.

Thromboxane prostanoid receptor activation impairs endothelial nitric oxide-dependent vasorelaxations: The role of Rho kinase

Activation of thromboxane prostanoid (TP) receptors causes potent vasoconstriction, which contributes to increased vascular tone and blood pressure. The present study examined the hypothesis that stimulation of TP receptor impaired endothelial nitric oxide-mediated vasorelaxation via a Rho kinase-dependent mechanism. The common carotid arteries of Sprague-Dawley rats were isolated and suspended in myograph for measurement of changes in isometric tension. The production of nitric oxide in primary cultured aortic endothelial cells was assayed with an imaging technique and phosphorylated levels of endothelial NOS were determined by Western blot analysis. 9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin F(2alpha) (U46619) inhibited isoprenaline-induced relaxations in rings with or without endothelium. Treatment with Rho kinase inhibitors, Y27632 (2 microM) or HA 1077 (10 microM) prevented the effect of U46619 only in rings with endothelium while protein kinase C inhibitors were without effect. Rho kinase inhibitors did not affect isoprenaline-induced relaxations in endothelium-intact rings treated with L-NAME or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ). Isoprenaline stimulated rises in nitric oxide (NO) production in cultured rat endothelial cells. The increased NO production was inhibited by U46619 (100 nM) and this effect was prevented by treatment with Y27632 but unaffected by the absence of extracellular calcium ions. U46619 attenuated isoprenaline-stimulated phosphorylation of eNOS, which was sensitive to inhibition by Y27632 and HA 1077. U46619-mediated effects were abolished by TP receptor antagonist, S18886 and the TP receptor was present in endothelial cells. The present results demonstrate that Rho kinase activation is likely to be the primary mechanism that underlies the U46619-stimulated TP-receptor-mediated inhibition of endothelial NO production and subsequent endothelium-dependent relaxations to isoprenaline.

Counteraction between angiotensin II and angiotensin-(1–7) via activating angiotensin type I and Mas receptor on rat renal mesangial cells

In the updated concept of renin-angiotensin system (RAS), it contains the angiotensin converting enzyme (ACE)-angiotensin (Ang) II-angtiogensin type 1 receptor (AT1) axis and the angiotensin-converting enzyme-related carboxypeptidase (ACE2)-Ang-(1-7)-Mas axis. The former axis has been well demonstrated performing the vasoconstrictive, proliferative and pro-inflammatory functions by activation of AT1 receptors, while the later new identified axis is considered counterbalancing the effects of the former. The present study is aimed at observing the interaction between Ang-(1-7) and Ang II on cultured rat renal mesangial cells (MCs). RT-PCR, Western blot and immunofluorescent staining and confocal microscopy results showed that both AT1 and Mas receptor were co-distributed in rat renal MCs. Ang-(1-7) showed similar effects on Ang II in cultured MCs that stimulated phosphorylated extracellular signal-regulated kinase (ERK)1/2 phosphorylation and transforms growth factor-β1 synthesis, and cell proliferation and extracellular matrix synthesis. Co-treatment of the cell with Ang-(1-7) and Ang II, Ang-(1-7) counteracted AngII-induced effects in a concentration dependent manner, but failed to alter the changes induced by endothelin-1. The stimulating effect of Ang II was mediated by AT1 receptor while all the effects of Ang-(1-7) were blocked by Mas receptor antagonist A-779, but not by AT1 receptor antagonist losartan or AT2 receptor antagonist PD123319. These results suggest that Ang-(1-7) and Ang II specifically interact with each other on rat renal MCs via activation of their specific receptors, Mas and AT1 receptor respectively.

Angiotensin-(1-7) attenuates high glucose-induced proximal tubular epithelial-to-mesenchymal transition via inhibiting ERK1/2 and p38 phosphorylation.

AIMS The kidney is an important target for both Angiotensin II and angiotensin-(1-7) [Ang-(1-7)] in the renin-angiotensin system. However, the renal function of Ang-(1-7) remains unclear. This study is aimed at investigating the effect of Ang-(1-7) on high glucose-induced epithelial to mesenchymal transition (EMT) in cultured renal epithelial cells. MAIN METHODS Cultured renal epithelial (NRK-52E) cell line was used in the experiment. Fluorescence immunocytochemistry was performed to observe α-smooth muscle actin (α-SMA). Real-time PCR and Western blot were used to determine mRNA and protein levels. Enzyme-linked immunosorbent assay was used to measure the concentration of transforming growth factor-β1 (TGF-β1) in the culture media. KEY FINDINGS High glucose-induced decreased in both angiotensin-converting enzyme-related carboxypeptidase (ACE2) and Mas mRNA levels. Meanwhile, high glucose induced increases in α-SMA and vimentin, decreases in E-cadherin, elevations in TGF-β1 and fibronectin secretions. Ang-(1-7) partially reversed high glucose-induced changes in α-SMA, vimentin, E-cadherin, TGF-β1 and fibronectin. High glucose stimulated ERK, p38 and JNK phosphorylation and Ang-(1-7) reversed the changes in ERK and p38 but not JNK phosphorylation. SIGNIFICANCE Inhibition and insufficiency in ACE2-Ang-(1-7)-Mas axis under high glucose condition participate EMT. Supplementation of Ang-(1-7) attenuates high glucose-induced EMT. ERK and p38 intracellular signaling pathways, not JNK, mediate the effect of Ang-(1-7) on EMT.

Suppression of XBP1S mediates high glucose-induced oxidative stress and extracellular matrix synthesis in renal mesangial cell and kidney of diabetic rats.

Recent evidences suggest that endoplasmic reticulum (ER) stress was involved in multi pathological conditions, including diabetic nephropathy (DN). X-box binding protein 1(XBP1), as a key mediator of ER stress, has been proved having the capability of preventing oxidative stress. In this study, we investigated the effects of spliced XBP1 (XBP1S), the dominant active form of XBP1, on high glucose (HG)-induced reactive oxygen species (ROS) production and extracellular matrix (ECM) synthesis in cultured renal mesangial cells (MCs) and renal cortex of STZ-induced diabetic rats. Real time PCR and Western blot were used to evaluate the mRNA and protein levels respectively. Transfection of recombinant adenovirus vector carrying XBP1S gene (Ad-XBP1S) was used to upregulate XBP1S expression. XBP1S siRNA was used to knockdown XBP1S expression. ROS level was detected by dihydroethidium (DHE) fluorescent probe assay. The results showed that HG treatment significantly reduced XBP1S protein and mRNA level in the cultured MCs while no obvious change was observed in unspliced XBP1 (XBP1U). In the mean time, the ROS production, collagen IV and fibronectin expressions were increased. Diphenylene-chloride iodonium (DPI), a NADPH oxidase inhibtor, prevented HG-induced increases in ROS as well as collagen IV and fibronectin expressions. Transfection of Ad-XBP1S reversed HG-induced ROS production and ECM expressions. Knockdown intrinsic XBP1S expression induced increases in ROS production and ECM expressions. Supplementation of supreoxide reversed the inhibitory effect of Ad-XBP1S transfection on ECM synthesis. P47phox was increased in HG-treated MCs. Ad-XBP1S transfection reversed HG-induced p47phox increase while XBP1S knockdown upregulated p47phox expression. In the renal cortex of diabetic rats, the expression of XBP1S was reduced while p47phox, collagen IV and fibronectin expression were elevated. These results suggested that XBP1S pathway of ER stress was involved in HG-induced oxidative stress and ECM synthesis. A downstream target of XBP1S in regulating ROS formation might be NADPH oxidase.

T-Cell Mineralocorticoid Receptor Controls Blood Pressure by Regulating Interferon-GammaNovelty and Significance

Rationale: Hypertension remains to be a global public health burden and demands novel intervention strategies such as targeting T cells and T-cell–derived cytokines. Mineralocorticoid receptor (MR) antagonists have been clinically used to treat hypertension. However, the function of T-cell MR in blood pressure (BP) regulation has not been elucidated. Objective: We aim to determine the role of T-cell MR in BP regulation and to explore the mechanism. Methods and Results: Using T-cell MR knockout mouse in combination with angiotensin II–induced hypertensive mouse model, we demonstrated that MR deficiency in T cells strikingly decreased both systolic and diastolic BP and attenuated renal and vascular damage. Flow cytometric analysis showed that T-cell MR knockout mitigated angiotensin II–induced accumulation of interferon-gamma (IFN-&ggr;)–producing T cells, particularly CD8+ population, in both kidneys and aortas. Similarly, eplerenone attenuated angiotensin II–induced elevation of BP and accumulation of IFN-&ggr;–producing T cells in wild-type mice. In cultured CD8+ T cells, T-cell MR knockout suppressed IFN-&ggr; expression whereas T-cell MR overexpression and aldosterone both enhanced IFN-&ggr; expression. At the molecular level, MR interacted with NFAT1 (nuclear factor of activated T-cells 1) and activator protein-1 in T cells. Finally, T-cell MR overexpressing mice manifested more elevated BP compared with control mice after angiotensin II infusion and such difference was abolished by IFN-&ggr;–neutralizing antibodies. Conclusions: MR may interact with NFAT1 and activator protein-1 to control IFN-&ggr; in T cells and to regulate target organ damage and ultimately BP. Targeting MR in T cells specifically may be an effective novel approach for hypertension treatment.

P300-dependent STAT3 acetylation is necessary for angiotensin II-induced pro-fibrotic responses in renal tubular epithelial cells.

Aim:To explore the signal transducer and activator of transcription 3 (STAT3) signaling pathway, especially STAT3 acetylation, in angiotensin II (Ang II)-induced pro-fibrotic responses in renal tubular epithelial cells.Methods:Rat renal tubular epithelial cell line (NRK-52E) was used. STAT3 acetylation and phosphorylation, as well as the expression of fibronectin, collagen IV and transforming growth factor-β1 (TGF-β1) were examined using Western blotting. The level and localization of STAT3 phosphorylation on Tyr705 were detected with fluorescence immunocytochemistry. The cells were transfected with a plasmid vector carrying p300 gene or siRNA targeting p300 to regulate p300 expression.Results:Overexpression of p300 significantly increased STAT3 acetylation on Lys685, STAT3 phosphorylation on Tyr705, and the expression of TGF-β1, collagen IV and fibronectin in the cells. Treatment of the cells with Ang II (1 μmol/L) significantly increased STAT3 phosphorylation on Tyr705 through JAK2 activation, and dose-dependently increased the expression of fibronectin, collagen IV and TGF-β1. Pretreatment with curcumin, an inhibitor of JAK2 and p300, blocked Ang II-induced effects. Knockdown of p300 significantly decreased STAT3 acetylation on Lys685, and abolished Ang II-stimulated STAT3 phosphorylation on Tyr705, whereas pretreatment of the cells with C646, a selective inhibitor of p300, inhibited Ang II-induced STAT3 nuclear translocation and the expression of TGF-β1, collagen IV and fibronectin. Pretreatment of the cells with AG490, a JAK2 inhibitor, markedly inhibited Ang II-induced STAT3 phosphorylation on Tyr705 and fibronectin expression.Conclusion:p300-dependent STAT3 acetylation is necessary for Ang II-induced STAT3 phosphorylation and the consequent pro-fibrotic responses in renal tubular epithelial cells in vitro.

Effect of adrenotensin on cell proliferation is mediated by angiotensin II in cultured rat mesangial cells.

AbstractAim:Both adrenomedullin (ADM) and adrenotensin (ADT) are derived from the same propeptide precursor, and both act as circulating hormones and local paracrine mediators with multiple biological activities. Compared with ADM, little is known about how ADT achieves its functions. In the present study, we investigated the effect of ADT on cell proliferation and transforming growth factor-β (TGF-β) secretion in cultured renal mesangial cells (MCs) and determined whether angiotensin II (Ang II) was involved in mediating this process.Methods:Cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation assay, Ang II levels were assayed using an enzyme immunoassay, and real time PCR was used to measure Ang II type 1 (AT1) receptor, Ang II type 2 (AT2) receptor, angiotensinogen (AGT), renin, angiotensin converting enzyme (ACE) and TGF-β1 mRNA levels. TGF-β1 and collagen type IV protein levels in cell media were measured using enzyme-linked immunoassays.Results:ADT treatment induced cell proliferation in a concentration-dependent manner; it also increased the levels of TGF-β1 mRNA and protein as well as collagen type IV excretion by cultured MCs. ADT treatment increased renin and AGT mRNAs as well as Ang II protein, but did not affect the ACE mRNA level. ADT up-regulated angiotensin AT1 receptor mRNA, but not that of the AT2 receptor. The angiotensin AT1 receptor antagonist losartan blocked the effects of ADT-induced cell proliferation, TGF-β1 and collagen type IV synthesis and secretion.Conclusion:ADT has a stimulating role in cell proliferation in cultured MCs. Increases in the levels of Ang II and the AT1 receptor after ADT treatment mediate the stimulating effects of ADT on cell proliferation and extracellular matrix synthesis and secretion.