Vitamin D receptor-modulated Hsp70/AT1 expression may protect the kidneys of SHRs at the structural and functional levels

Previous hypertension studies have shown that low levels of vitamin D are linked to elevated renin–angiotensin system. The heat shock protein 70 regulates signaling pathways for cellular oxidative stress responses. Hsp70 has been shown to protect against angiotensin II-induced hypertension and exert a cytoprotective effect. Here, we wanted to evaluate whether the vitamin D receptor (VDR) associated with Hsp70/AT₁ expression may be involved in the mechanism by which paricalcitol provides renal protection in spontaneously hypertensive rats (SHRs). One-month-old female SHRs were treated for 4 months with vehicle, paricalcitol, enalapril, or a combination of both paricalcitol and enalapril. The following were determined: blood pressure; biochemical parameters; fibrosis; apoptosis; mitochondrial morphology; and VDR, AT₁ receptor, and Hsp70 expression in the renal cortex. Blood pressure was markedly reduced by enalapril or the combination but not by paricalcitol alone. However, VDR activation, enalapril or combination, prevented fibrosis, the number of TUNEL-positive apoptotic cells, mitochondrial damage, and NADPH oxidase activity in SHRs. Additionally, high AT₁ receptor expression, like low Hsp70 expression (immunohistochemical/immunofluorescence studies), was reversed in the renal cortices of paricalcitol- and/or enalapril-treated animals (SHRs), and these changes were most marked in the combination therapy group. Finally, all of the recovery parameters were consistent with an improvement in VDR expression. Data suggest that Hsp70/AT₁ modulated by VDR is involved in the mechanism by which paricalcitol provides renal protection in SHRs. We propose that low AT₁ expression through VDR induction could be a consequence of the heat shock response Hsp70-mediated cell protection.     

The vitamin D receptor activator paricalcitol prevents fibrosis and diastolic dysfunction in a murine model of pressure overload

BACKGROUND: Activation of the vitamin D-vitamin D receptor (VDR) axis has been shown to reduce blood pressure and left ventricular (LV) hypertrophy. Besides cardiac hypertrophy, cardiac fibrosis is a key element of adverse cardiac remodeling. We hypothesized that activation of the VDR by paricalcitol would prevent fibrosis and LV diastolic dysfunction in an established murine model of cardiac remodeling. METHODS: Mice were subjected to transverse aortic constriction (TAC) to induce cardiac hypertrophy. Mice were treated with paricalcitol, losartan, or a combination of both for a period of four consecutive weeks. RESULTS: The fixed aortic constriction caused similar increase in blood pressure, both in untreated and paricalcitol- or losartan-treated mice. TAC significantly increased LV weight compared to sham operated animals (10.2±0.7 vs. 6.9±0.3mg/mm, p<0.05). Administration of either paricalcitol (10.5±0.7), losartan (10.8±0.4), or a combination of both (9.2±0.6) did not reduce LV weight. Fibrosis was significantly increased in mice undergoing TAC (5.9±1.0 vs. sham 2.4±0.8%, p<0.05). Treatment with losartan and paricalcitol reduced fibrosis (paricalcitol 1.6±0.3% and losartan 2.9±0.6%, both p<0.05 vs. TAC). This reduction in fibrosis in paricalcitol treated mice was associated with improved indices of LV contraction and relaxation, e.g. dPdtmax and dPdtmin and lower LV end diastolic pressure, and relaxation constant Tau. Also, treatment with paricalcitol and losartan reduced mRNA expression of ANP, fibronectin, collagen III and TIMP-1. DISCUSSION: Treatment with the selective VDR activator paricalcitol reduces myocardial fibrosis and preserves diastolic LV function due to pressure overload in a mouse model. This is associated with a reduced percentage of fibrosis and a decreased expression of ANP and several other tissue markers.     

Oxidative stress contributes to sex differences in angiotensin II-mediated hypertension in spontaneously hypertensive rats

NADPH oxidase has been implicated in ANG II-induced oxidative stress and hypertension in males; however, the contribution of oxidative stress to ANG II hypertension in females is unknown. In the present study, we tested the hypothesis that greater antioxidant capacity in female spontaneously hypertensive rats (SHR) blunts ANG II-induced oxidative stress and hypertension relative to males. Whole body and renal cortical oxidative stress levels were assessed in female and male SHR left untreated or following 2 wk of chronic ANG II infusion. Chronic ANG II infusion increased NADPH oxidase enzymatic activity in the renal cortex of both sexes; however, this increase only reached significance in female SHR. In contrast, male SHR demonstrated a greater increase in all measurements of reactive oxygen species production in response to chronic ANG II infusion. ANG II infusion increased plasma superoxide dismutase activity only in female SHR (76 ± 9 vs. 190 ± 7 Units·ml(-1)·mg(-1), P < 0.05); however, cortical antioxidant capacity was unchanged by ANG II in either sex. To assess the functional implication of alterations in NADPH enzymatic activity and oxidative stress levels following ANG II infusion, additional experiments assessed the ability of the in vivo antioxidant apocynin to modulate ANG II hypertension. Apocynin significantly blunted ANG II hypertension in male SHR (174 ± 2 vs. 151 ± 1 mmHg, P < 0.05), with no effect in females (160 ± 11 vs. 163 ± 10 mmHg). These data suggest that ANG II hypertension in male SHR is more dependent on increases in oxidative stress than in female SHR.     

NADPH oxidase inhibition reduces tubular sodium transport and improves kidney oxygenation in diabetes

Sustained hyperglycemia is associated with increased oxidative stress resulting in decreased intrarenal oxygen tension (Po(2)) due to increased oxygen consumption (Qo(2)). Chronic blockade of the main superoxide radicals producing system, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, normalizes Qo(2) by isolated proximal tubular cells (PTC) and reduces proteinuria in diabetes. The aim was to investigate the effects of acute NADPH oxidase inhibition on tubular Na(+) transport and kidney Po(2) in vivo. Glomerular filtration rate (GFR), renal blood flow (RBF), filtration fraction (FF), Na(+) excretion, fractional Li(+) excretion, and intrarenal Po(2) was measured in control and streptozotocin-diabetic rats during baseline and after acute NADPH oxidase inhibition using apocynin. The effects on tubular transporters were investigated using freshly isolated PTC. GFR was increased in diabetics compared with controls (2.2 ± 0.3 vs. 1.4 ± 0.1 ml·min(-1)·kidney(-1)). RBF was similar in both groups, resulting in increased FF in diabetics. Po(2) was reduced in cortex and medulla in diabetic kidneys compared with controls (34.4 ± 0.7 vs. 42.5 ± 1.2 mmHg and 15.7 ± 1.2 vs. 25.5 ± 2.3 mmHg, respectively). Na(+) excretion was increased in diabetics compared with controls (24.0 ± 4.7 vs. 9.0 ± 2.0 μm·min(-1)·kidney(-1)). In controls, all parameters were unaffected. However, apocynin increased Na(+) excretion (+112%) and decreased fractional lithium reabsorption (-10%) in diabetics, resulting in improved cortical (+14%) and medullary (+28%) Po(2). Qo(2) was higher in PTC isolated from diabetic rats compared with control. Apocynin, dimethylamiloride, and ouabain reduced Qo(2), but the effects of combining apocynin with either dimethylamiloride or ouabain were not additive. In conclusion, NADPH oxidase inhibition reduces tubular Na(+) transport and improves intrarenal Po(2) in diabetes.     

Mitochondrial reactive oxygen species and calcium uptake regulate activation of phagocytic NADPH oxidase

Production of superoxide (O(2)(·-)) by NADPH oxidases contributes to the development of hypertension and atherosclerosis. Factors responsible for activation of NADPH oxidases are not well understood; interestingly, cardiovascular disease is associated with both altered NADPH oxidase activity and age-associated mitochondrial dysfunction. We hypothesized that mitochondrial dysfunction may contribute to activation of NADPH oxidase. The effect of mitochondrial inhibitors on phagocytic NADPH oxidase in human lymphoblasts and whole blood was measured at the basal state and upon PKC-dependent stimulation with PMA using extracellular 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium or mitochondria-targeted 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine spin probes and electron spin resonance (ESR). Intracellular cytosolic calcium [Ca(2+)](i) was measured spectrofluorometrically using fura-2 AM. Incubation of lymphoblasts with the mitochondrial inhibitors rotenone, antimycin A, CCCP, or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter) did not significantly change basal activity of NADPH oxidase. In contrast, preincubation with the mitochondrial inhibitors prior to PMA stimulation of lymphoblasts resulted in two- to three-fold increase of NADPH oxidase activity compared with stimulation with PMA alone. Most notably, the intracellular Ca(2+)-chelating agent BAPTA-AM abolished the effect of mitochondrial inhibitors on NADPH oxidase activity. Cytosolic Ca(2+) measurements with fura-2 AM showed that the mitochondrial inhibitors increased [Ca(2+)](i), while BAPTA-AM abolished the increase in [Ca(2+)](i). Furthermore, depletion of cellular Ca(2+) with thapsigargin attenuated CCCP- and antimycin A-mediated activation of NADPH oxidase in the presence of PMA by 42% and 31%, correspondingly. Our data suggest that mitochondria regulate PKC-dependent activation of phagocytic NADPH oxidase. In summary, increased mitochondrial O(2)(·-) and impaired buffering of cytosolic Ca(2+) by dysfunctional mitochondria result in enhanced NADPH oxidase activity, which may contribute to the development of cardiovascular diseases.     

Importance of changes in gastric emptying for postprandial plasma glucose fluxes in healthy humans

Angiotensin II (Ang II) stimulation of the Ang type 1 receptor (AT(1)R) facilitates myocardial remodeling through NADPH oxidase-mediated generation of oxidative stress. Components of the renin-angiotensin system constitute an autocrine/paracrine unit in the myocardium, including renin, which is the rate-limiting step in the generation of Ang II. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo renin inhibition and/or AT(1)R blockade in a rodent model of chronically elevated tissue Ang II levels, the transgenic (mRen2)27 rat (Ren2). The Ren2 overexpresses the mouse renin transgene with resultant hypertension, insulin resistance, and cardiovascular damage. Young (6- to 7-wk-old) heterozygous (+/-) male Ren2 and age-matched Sprague-Dawley rats were treated with the renin inhibitor aliskiren, which has high preferential affinity for human and mouse renin, an AT(1)R blocker, irbesartan, or placebo for 3 wk. Myocardial NADPH oxidase activity and immunostaining for NADPH oxidase subunits and 3-nitrotyrosine were evaluated and remodeling changes assessed by light and transmission electron microscopy. Blood pressure, myocardial NADPH oxidase activity and subunit immunostaining, 3-nitrotyrosine, perivascular fibrosis, mitochondrial content, and markers of activity were significantly increased in Ren2 compared with SD littermates. Both renin inhibition and blockade of the AT(1)R significantly attenuated cardiac functional and structural alterations, although irbesartan treatment resulted in greater reductions of both blood pressure and markers of oxidative stress. Collectively, these data suggest that both reduce changes driven, in part, by Ang II-mediated increases in NADPH oxidase and, in part, increases in blood pressure.     

Effect of paricalcitol and calcitriol on aortic wall remodeling in uninephrectomized ApoE knockout mice

Despite an only minor reduction in the glomerular filtration rate, uninephrectomy (UNX) markedly accelerates the rate of growth of atherosclerotic plaques in ApoE-/- mice. It has been suggested that vitamin D receptor (VDR) activation exerts an antiproliferative effect on vascular smooth muscle cells, but the side effects may limit its use. To assess a potentially different spectrum of actions, we compared the effects of paricalcitol and calcitriol on remodeling and calcification of the aortic wall in sham-operated and UNX ApoE-/- mice on a diet with normal cholesterol content. Sham-operated and UNX mice were randomly allotted to treatment with solvent, calcitriol (0.03 μg/kg) or paricalcitol (0.1 μg/kg) 5 times/wk intraperitoneally for 10 wk. Semithin (0.6 μm) sections of the aorta were analyzed by 1) morphometry, 2) immunohistochemistry, and 3) Western blotting of key proteins involved in vascular calcification and growth. Compared with sham-operated animals (5.6 ± 0.24), the wall-to-lumen ratio (x100) of the aorta was significantly higher in solvent- and calcitriol-treated UNX animals (6.64 ± 0.27 and 7.17 ± 0.81, respectively, P < 0.05), but not in paricalcitol-treated UNX (6.1 5 ± 0.32). Similar differences were seen with respect to maximal plaque height. Expression of transforming growth factor (TGF)-β1 in aortic intima/plaque was also significantly higher in UNX solvent and UNX calcitriol compared with sham-operated and UNX paricalcitol animals. Treatment with both paricalcitol and calcitriol caused significant elevation of VDR expression in the aorta. While at the dose employed paricalcitol significantly reduced TGF-β expression in plaques, calcitriol in contrast caused significant vascular calcification and elevated expression of related proteins (BMP2, RANKL, and Runx2).     

Paricalcitol Reduces Peritoneal Fibrosis in Mice through the Activation of Regulatory T Cells and Reduction in IL-17 Production

Fibrosis is a significant health problem associated with a chronic inflammatory reaction. The precise mechanisms involved in the fibrotic process are still poorly understood. However, given that inflammation is a major causative factor, immunomodulation is a possible therapeutic approach to reduce fibrosis. The vitamin D receptor (VDR) that is present in all hematopoietic cells has been associated with immunomodulation. We investigated whether the intraperitoneal administration of paricalcitol, a specific activator of the VDR, modulates peritoneal dialysis fluid (PDF)-induced peritoneal fibrosis. We characterized the inflammatory process in the peritoneal cavity of mice treated or not treated with paricalcitol and analyzed the ensuing fibrosis. The treatment reduced peritoneal IL-17 levels, which strongly correlated with a significantly lower peritoneal fibrotic response. In vitro studies demonstrate that both CD4⁺ and CD8⁺ regulatory T cells appear to impact the regulation of IL-17. Paricalcitol treatment resulted in a significantly increased frequency of CD8⁺ T cells showing a regulatory phenotype. The frequency of CD4⁺ Tregs tends to be increased, but it did not achieve statistical significance. However, paricalcitol treatment increased the number of CD4⁺ and CD8⁺ Treg cells in vivo. In conclusion, the activation of immunological regulatory mechanisms by VDR signaling could prevent or reduce fibrosis, as shown in peritoneal fibrosis induced by PDF exposure in mice.     

Paraoxonase 2 decreases renal reactive oxygen species production, lowers blood pressure, and mediates dopamine D2 receptor-induced inhibition of NADPH oxidase

The dopamine D(2) receptor (D(2)R) regulates renal reactive oxygen species (ROS) production, and impaired D(2)R function results in ROS-dependent hypertension. Paraoxonase 2 (PON2), which belongs to the paraoxonase gene family, is expressed in various tissues, acting to protect against cellular oxidative stress. We hypothesized that PON2 may be involved in preventing excessive renal ROS production and thus may contribute to maintenance of normal blood pressure. Moreover, D(2)R may decrease ROS production, in part, through regulation of PON2. D(2)R colocalized with PON2 in the brush border of mouse renal proximal tubules. Renal PON2 protein was decreased (-33±6%) in D(2)(-/-) relative to D(2)(+/+) mice. Renal subcapsular infusion of PON2 siRNA decreased PON2 protein expression (-55%), increased renal oxidative stress (2.2-fold), associated with increased renal NADPH oxidase expression (Nox1, 1.9-fold; Nox2, 2.9-fold; and Nox4, 1.6-fold) and activity (1.9-fold), and elevated arterial blood pressure (systolic, 134±5 vs 93±6mmHg; diastolic, 97±4 vs 65±7mmHg; mean 113±4 vs 75±7mmHg). To determine the relevance of the PON2 and D(2)R interaction in humans, we studied human renal proximal tubule cells. Both D(2)R and PON2 were found in nonlipid and lipid rafts and physically interacted with each other. Treatment of these cells with the D(2)R/D(3)R agonist quinpirole (1μM, 24h) decreased ROS production (-35±6%), associated with decreased NADPH oxidase activity (-32±3%) and expression of Nox2 (-41±7%) and Nox4 (-47±8%) protein, and increased expression of PON2 mRNA (2.1-fold) and protein (1.6-fold) at 24h. Silencing PON2 (siRNA, 10nM, 48h) not only partially prevented the quinpirole-induced decrease in ROS production by 36%, but also increased basal ROS production (1.3-fold), which was associated with an increase in NADPH oxidase activity (1.4-fold) and expression of Nox2 (2.1-fold) and Nox4 (1.8-fold) protein. Inhibition of NADPH oxidase with diphenylene iodonium (10μM/30 min) inhibited the increase in ROS production caused by PON2 silencing. Our results suggest that renal PON2 is involved in the inhibition of renal NADPH oxidase activity and ROS production and contributes to the maintenance of normal blood pressure. PON2 is positively regulated by D(2)R and may, in part, mediate the inhibitory effect of renal D(2)R on NADPH oxidase activity and ROS production.     

Activating Nrf-2 Signaling Depresses Unilateral Ureteral Obstruction-Evoked Mitochondrial Stress-Related Autophagy,Apoptosis and Pyroptosis in Kidney

Exacerbated oxidative stress and inflammation may induce three types of programmed cell death, autophagy, apoptosis and pyroptosis in unilateral ureteral obstruction (UUO) kidney. Sulforaphane activating NF-E2-related nuclear factor erythroid-2 (Nrf-2) signaling may ameliorate UUO-induced renal damage. UUO was induced in the left kidney of female Wistar rats. The level of renal blood flow, cortical and medullary oxygen tension and reactive oxygen species (ROS) was evaluated. Fibrosis, ED-1 (macrophage/monocyte) infiltration, oxidative stress, autophagy, apoptosis and pyroptosis were evaluated by immunohistochemistry and Western blot in UUO kidneys. Effects of sulforaphane, an Nrf-2 activator, on Nrf-2- and mitochondrial stress-related proteins and renal injury were examined. UUO decreased renal blood flow and oxygen tension and increased renal ROS, 3-nitrotyrosine stain, ED-1 infiltration and fibrosis. Enhanced renal tubular Beclin-1 expression started at 4 h UUO and further enhanced at 3d UUO, whereas increased Atg-5-Atg12 and LC3-II expression were found at 3d UUO. Increased renal Bax/Bcl-2 ratio, caspase 3 and PARP fragments, apoptosis formation associated with increased caspase 1 and IL-1β expression for pyroptosis formation were started from 3d UUO. UUO reduced nuclear Nrf-2 translocation, increased cytosolic and inhibitory Nrf-2 expression, increased cytosolic Bax translocation to mitochondrial and enhanced mitochondrial Cytochrome c release into cytosol of the UUO kidneys. Sulforaphane significantly increased nuclear Nrf-2 translocation and decreased mitochondrial Bax translocation and Cytochrome c release into cytosol resulting in decreased renal injury. In conclusion, sulforaphane via activating Nrf-2 signaling preserved mitochondrial function and suppressed UUO-induced renal oxidative stress, inflammation, fibrosis, autophagy, apoptosis and pyroptosis.     

Ursodeoxycholate modulates bile flow and bile salt pool independently from the cystic fibrosis transmembrane regulator (Cftr) in mice

Cystic fibrosis liver disease (CFLD) is treated with ursodeoxycholate (UDCA). Our aim was to evaluate, in cystic fibrosis transmembrane regulator knockout (Cftr(-/-)) mice and wild-type controls, whether the supposed therapeutic action of UDCA is mediated via choleretic activity or effects on bile salt metabolism. Cftr(-/-) mice and controls, under general anesthesia, were intravenously infused with tauroursodeoxycholate (TUDCA) in increasing dosage or were fed either standard or UDCA-enriched chow (0.5% wt/wt) for 3 wk. Bile flow and bile composition were characterized. In chow-fed mice, we analyzed bile salt synthesis and pool size of cholate (CA). In both Cftr(-/-) and controls intravenous TUDCA stimulated bile flow by ～250% and dietary UDCA by ～500%, compared with untreated animals (P < 0.05). In non-UDCA-treated Cftr(-/-) mice, the proportion of CA in bile was higher compared with that in controls (61 ± 4 vs. 46 ± 4%; P < 0.05), accompanied by an increased CA synthesis [16 ± 1 vs. 10 ± 2 μmol·h(-1)·100 g body wt (BW)(-1); P < 0.05] and CA pool size (28 ± 3 vs. 19 ± 1 μmol/100 g BW; P < 0.05). In both Cftr(-/-) and controls, UDCA treatment drastically reduced the proportion of CA in bile below 5% and diminished CA synthesis (2.3 ± 0.3 vs. 2.2 ± 0.4 μmol·day(-1)·100 g BW(-1); nonsignificant) and CA pool size (3.6 ± 0.6 vs. 1.5 ± 0.3 μmol/100 g BW; P < 0.05). Acute TUDCA infusion and chronic UDCA treatment both stimulate bile flow in cystic fibrosis conditions independently from Cftr function. Chronic UDCA treatment reduces the hydrophobicity of the bile salt pool in Cftr(-/-) mice. These results support a potential beneficial effect of UDCA on bile flow and bile salt metabolism in cystic fibrosis conditions.     

Enhanced angiotensin II-mediated central sympathoexcitation in streptozotocin-induced diabetes: role of superoxide anion

Studies have shown that the superoxide mechanism is involved in angiotensin II (ANG II) signaling in the central nervous system. We hypothesized that ANG II activates sympathetic outflow by stimulation of superoxide anion in the paraventricular nucleus (PVN) of streptozotocin (STZ)-induced diabetic rats. In α-chloralose- and urethane-anesthetized rats, microinjection of ANG II into the PVN (50, 100, and 200 pmol) produced dose-dependent increases in renal sympathetic nerve activity (RSNA), arterial pressure (AP), and heart rate (HR) in control and STZ-induced diabetic rats. There was a potentiation of the increase in RSNA (35.0 ± 5.0 vs. 23.0 ± 4.3%, P < 0.05), AP, and HR due to ANG II type I (AT(1)) receptor activation in diabetic rats compared with control rats. Blocking endogenous AT(1) receptors within the PVN with AT(1) receptor antagonist losartan produced significantly greater decreases in RSNA, AP, and HR in diabetic rats compared with control rats. Concomitantly, there were significant increases in mRNA and protein expression of AT(1) receptor with increased superoxide levels and expression of NAD(P)H oxidase subunits p22(phox), p47(phox), and p67(phox) in the PVN of rats with diabetes. Pretreatment with losartan (10 mg·kg(-1)·day(-1) in drinking water for 3 wk) significantly reduced protein expression of NAD(P)H oxidase subunits (p22(phox) and p47(phox)) in the PVN of diabetic rats. Pretreatment with adenoviral vector-mediated overexpression of human cytoplasmic superoxide dismutase (AdCuZnSOD) within the PVN attenuated the increased central responses to ANG II in diabetes (RSNA: 20.4 ± 0.7 vs. 27.7 ± 2.1%, n = 6, P < 0.05). These data support the concept that superoxide anion contributes to an enhanced ANG II-mediated signaling in the PVN involved with the exaggerated sympathoexcitation in diabetes.     

Angiotensin II-NADPH oxidase-derived superoxide mediates diabetes-attenuated cell excitability of aortic baroreceptor neurons

Overactivation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is involved in diabetes-depressed excitability of aortic baroreceptor neurons in nodose ganglia. This involvement links to the autonomic dysfunction associated with high morbidity and mortality in diabetic patients. The present study examined the effects of an angiotensin II type I receptor (AT(1)R) antagonist (losartan), a NADPH oxidase inhibitor (apocynin), and a superoxide dismutase mimetic (tempol) on the enhanced HCN currents and attenuated cell excitability in diabetic nodose neurons. In sham and streptozotocin-induced type 1 diabetic rats, HCN currents and cell excitability of aortic baroreceptor neurons were recorded by the whole cell patch-clamp technique. The angiotensin II level in nodose ganglia from diabetic rats was higher than that from sham rats (101.6 ± 4.8 vs. 38.9 ± 4.2 pg/mg protein, P < 0.05). Single-cell RT-PCR, Western blot, immunofluorescence staining, and chemiluminescence data showed that mRNA and protein expression of AT(1)R, protein expression of NADPH oxidase components, and superoxide production in nodose neurons were increased in diabetic rats compared with those from sham rats. HCN current density was higher and cell excitability was lower in aortic baroreceptor neurons from diabetic rats than that from sham rats. Losartan (1 μM), apocynin (100 μM), and tempol (1 mM) normalized the enhanced HCN current density and increased the cell excitability in the aortic baroreceptor neurons of diabetic rats. These findings suggest that endogenous angiotensin II-NADPH oxidase-superoxide signaling contributes to the enhanced HCN currents and the depressed cell excitation in the aortic baroreceptor neurons of diabetic rats.     

Angiotensin II-mediated oxidative stress promotes myocardial tissue remodeling in the transgenic (mRen2) 27 Ren2 rat

Angiotensin II (ANG II) contributes to cardiac remodeling, hypertrophy, and left ventricular dysfunction. ANG II stimulation of the ANG type 1 receptor (AT(1)R) generates reactive oxygen species via NADPH oxidase, which facilitates this hypertrophy and remodeling. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo AT(1)R blockade (AT(1)B) (valsartan) or superoxide dismutase/catalase mimetic (tempol) treatment in a rodent model of chronically elevated tissue levels of ANG II, the transgenic (mRen2) 27 rat (Ren2). Ren2 rats overexpress the mouse renin transgene with resultant hypertension, insulin resistance, proteinuria, and cardiovascular damage. Young (6-7 wk old) male Ren2 and age-matched Sprague-Dawley rats were treated with valsartan (30 mg/kg), tempol (1 mmol/l), or placebo for 3 wk. Heart tissue NADPH oxidase (NOX) activity and immunohistochemical analysis of subunits NOX2, Rac1, and p22(phox), heart tissue malondialdehyde, and insulin-stimulated protein kinase B (Akt) activation were measured. Structural changes were assessed with cine MRI, transmission electron microscopy, and light microscopy. Increases in septal wall thickness and altered systolic function (cine MRI) were associated with perivascular fibrosis and increased mitochondria in Ren2 on light and transmission electron microscopy (P < 0.05). AT(1)B, but not tempol, reduced blood pressure (P < 0.05); significant improvements were seen with both AT(1)B and tempol on NOX activity, subunit expression, malondialdehyde, and insulin-mediated activation/phosphorylation of Akt (each P < 0.05). Collectively, these data suggest cardiac oxidative stress-induced structural and functional changes are driven, in part, by AT(1)R-mediated increases in NADPH oxidase activity.     

Estimation of the anaerobic threshold from heart rate variability in an incremental swimming test

ABSTRACT: Di Michele, R, Gatta, G, Di Leo, A, Cortesi, M, Andina, F, Tam, E, Da Boit, M, and Merni, F. Estimation of the anaerobic threshold from heart rate variability in an incremental swimming test. J Strength Cond Res 26(11): 3059-3066, 2012-This study aimed to evaluate, in swimming, the agreement between the anaerobic threshold (AT) as determined from the analysis of blood lactate concentration ([La]) and from a new method based on the heart rate (HR) variability (HRV). Fourteen high-level swimmers completed an incremental 7 × 200-m front crawl test, during which the HRV was measured continuously and [La] was collected after each step. To individuate the AT, the trends of the high-frequency HRV spectral power (HFPOW) and of the fraction of HFPOW relative to the respiratory sinus arrhythmia (HFPOW-RSA) were analyzed. In all the subjects, an abrupt increase of both HFPOW and HFPOW-RSA was observed and associated with the AT. The AT parameters determined, respectively, from [La] and HFPOW-RSA were similar (p > 0.05) and highly correlated (HR: 182.0 ± 8.1 vs. 181.1 ± 8.2 b·min, r = 0.93, 95% limits of agreement [LoA]: -6.7 to 4.9 b·min; velocity: 1.47 ± 0.11 vs. 1.47 ± 0.11 m·s, r = 0.98, 95% LoA: -0.05 to 0.05 m·s). Instead, the AT HR and velocity obtained from HFPOW (179.2 ± 8.4 b·min; 1.45 ± 0.11 m·s) were correlated to the corresponding parameters determined from [La] (HR: r = 0.84; velocity: r = 0.94) but underestimated them slightly (95% LoA: -11.9 to 6.3 b·min and -0.11 to 0.05 m·s). These results demonstrate that the AT can be assessed from the HRV in swimming, providing an important testing tool for coaches. Furthermore, using the actual respiratory spectral component, rather than the total HF spectral power, allows us to obtain a more accurate estimate of AT parameters.     

Regulator of G protein signaling 2 (RGS2) deficiency accelerates the progression of kidney fibrosis

The regulator of G protein signaling 2 (RGS2) is a potent negative regulator of Gq protein signals including the angiotensin II (AngII)/AngII receptor signal, which plays a critical role in the progression of fibrosis. However, the role of RGS2 on the progression of kidney fibrosis has not been assessed. Here, we investigated the role of RGS2 in kidney fibrosis induced by unilateral ureteral obstruction (UUO) in mice. UUO resulted in increased expression of RGS2 mRNA and protein in the kidney along with increases of AngII and its type 1 receptor (AT1R) signaling and fibrosis. Furthermore, UUO increased the levels of F4/80, Ly6G, myeloperoxidase, and CXCR4 in the kidneys. RGS2 deficiency significantly enhanced these changes in the kidney. RGS2 deletion in the bone marrow-derived cells by transplanting the bone marrow of RGS2 knock-out mice into wild type mice enhanced UUO-induced kidney fibrosis. Overexpression of RGS2 in HEK293 cells, a human embryonic kidney cell line, and RAW264.7 cells, a monocyte/macrophage line, inhibited the AngII-induced activation of ERK and increase of CXCR4 expression. These findings provide the first evidence that RGS2 negatively regulates the progression of kidney fibrosis following UUO, likely by suppressing fibrogenic and inflammatory responses through the inhibition of AngII/AT1R signaling.     

Increased renin excretion is associated with augmented urinary angiotensin II levels in chronic angiotensin II-infused hypertensive rats

Renin expression in principal cells of collecting ducts (CD) is upregulated in angiotensin II (ANG II)-dependent hypertensive rats; however, it remains unclear whether increased CD-derived renin undergoes tubular secretion. Accordingly, urinary levels of renin (uRen), angiotensinogen (uAGT), and ANG II (uANG II) were measured in chronic ANG II-infused Sprague-Dawley rats (80 ng/min for 14 days, n = 10) and sham-operated rats (n = 10). Systolic blood pressure increased in the ANG II rats by day 5 and continued to increase throughout the study (day 13; ANG II: 175 ± 10 vs. sham: 116 ± 2 mmHg; P < 0.05). ANG II infusion increased renal cortical and medullary ANG II levels (cortical ANG II: 606 ± 72 vs. 247 ± 43 fmol/g; P < 0.05; medullary ANG II: 2,066 ± 116 vs. 646 ± 36 fmol/g; P < 0.05). Although plasma renin activity (PRA) was suppressed in the ANG II-infused rats (0.3 ± 0.2 vs. 5.5 ± 1.8 ng ANG I·ml(-1)·h(-1); P < 0.05), renin content in renal medulla was increased (12,605 ± 1,343 vs. 7,956 ± 765 ng ANG I·h(-1)·mg(-1); P < 0.05). Excretion of uAGT and uANG II increased in the ANG II rats [uAGT: 1,107 ± 106 vs. 60 ± 26 ng/day; P < 0.0001; uANG II: 3,813 ± 431 vs. 2,080 ± 361 fmol/day; P < 0.05]. By day 13, despite suppression of PRA, urinary prorenin content increased in ANG II rats [15.7 ± 3 vs. 2.6 ± 1 × 10(-3) enzyme units excreted (EUE)/day, P < 0.01] as was the excretion rate of renin (8.6 ± 2 × 10(-6) EUE/day) compared with sham (2.8 ± 1 × 10(-6) EUE/day; P < 0.05). Urinary renin and prorenin protein levels examined by Western blot were augmented ～10-fold in the ANG II-infused rats. Concomitant AT(1) receptor blockade with candesartan prevented the increase. Thus, in ANG II-dependent hypertensive rats with marked PRA suppression, increased urinary levels of renin and prorenin reflect their augmented secretion by CD cells into the luminal fluid. The greater availability of renin and AGT in the urine reflects the capability for intratubular ANG II formation which stimulates sodium reabsorption in distal nephron segments.     

Apocynin attenuates renal fibrosis via inhibition of NOXs-ROS-ERK-myofibroblast accumulation in UUO rats

Background: Oxidative stress has been identified as an important pathogenesis mechanism in the development of renal interstitial fibrosis in unilateral ureteral obstruction (UUO). Previous studies have demonstrated increased expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOXs) in response to UUO. We aimed to investigate whether NOXs activation was involved in the development of renal fibrosis in UUO by contribution to oxidative stress and the potential mechanism in the present study.

Methods: Apocynin, a NOXs inhibitor, was initiated immediately by gavage after UUO was performed on Wistar rats and continued until 7 days after UUO. Changes of markers of oxidative stress, renal macrophage infiltration and fibrosis, TGF-β1 expression, NOXs expression and activity, and ERK activation were evaluated.

Results: Apocynin significantly attenuated the activity of NOXs, accompanied with decreased expression of NOX2, NOX4, and oxidative stress markers in the obstructed kidneys of UUO. Additionally, collagen deposition and renal fibrosis induced by UUO were attenuated by apocynin treatment. Furthermore, apocynin treatment significantly attenuated the phosphorylation of ERK, accumulation of myofibroblast and infiltration of macrophage in obstructed kidneys. No significant effect of apocynin on UUO-induced increased TGF-β1 expression could be observed. And there was no significant change of anti-oxidants enzyme activities in the obstructed kidneys of apocynin-treated rats.

Conclusions: These results suggested that apocynin might exert beneficial effects on renal fibrosis by inhibition of NOXs activation and subsequent reduction of oxidative stress, ERK activation, and myofibroblast accumulation in UUO rats. Targeting NOXs may serve as a therapeutic strategy for the treatment of renal fibrosis.     

羟苯磺酸钙通过抑制Ⅰ型胶原表达减轻肾间质纤维化

目的:研究羟苯磺酸钙对小鼠肾间质纤维化、Ⅰ型胶原表达的影响。方法:将C57小鼠随机分为假手术组(Sham组,n=4)、肾间质纤维化模型组(UUO组,n=5)及羟苯磺酸钙治疗组(CDT组,n=4);采用单侧输尿管梗阻制备肾间质纤维化模型,CDT组给予羟苯磺酸钙灌胃、Sham组和UUO组给予双蒸水灌胃;采用HE染色、Masson染色、免疫组化、实时定量PCR以及蛋白免疫印迹观察单侧输尿管梗阻术后14 d小鼠术侧肾脏的肾间质纤维化程度和Ⅰ型胶原表达情况。结果:与Sham组比较,UUO组小鼠术后14 d术侧肾脏肾发生显著肾间质纤维化,Ⅰ型胶原表达显著增强(Ⅰ型胶原基因相对表达量:Sham组:1.00000,UUO组:114.92289,P0.0001)。与UUO组比较,CDT组小鼠术后14 d术侧肾间质纤维化程度显著减轻,Ⅰ型胶原表达显著减弱(Ⅰ型胶原基因相对表达量:UUO组:114.92289,CDT组:45.33516,P0.005)。结论:羟苯磺酸钙通过抑制小鼠肾间质Ⅰ型胶原表达从而减轻单侧输尿管结扎小鼠肾间质纤维化。     

Effect of combining an ACE inhibitor and a VDR activator on glomerulosclerosis, proteinuria, and renal oxidative stress in uremic rats

Angiotensin-converting enzyme (ACE) inhibitors ameliorate the progression of renal disease. In combination with vitamin D receptor activators, they provide additional benefits. In the present study, uremic (U) rats were treated as follows: U+vehicle (UC), U+enalapril (UE; 25 mg/l in drinking water), U+paricalcitol (UP; 0.8 μg/kg ip, 3 × wk), or U+enalapril+paricalcitol (UEP). Despite hypertension in UP rats, proteinuria decreased by 32% vs. UC rats. Enalapril alone, or in combination with paricalcitol, further decreased proteinuria (≈70%). Glomerulosclerosis and interstitial infiltration increased in UC rats. Paricalcitol and enalapril inhibited this. The increase in cardiac atrial natriuretic peptide (ANP) seen in UC rats was significantly decreased by paricalcitol. Enalapril produced a more dramatic reduction in ANP. Renal oxidative stress plays a critical role in inflammation and progression of sclerosis. The marked increase in p22(phox), a subunit of NADPH oxidase, and decrease in endothelial nitric oxide synthase were inhibited in all treated groups. Cotreatment with both compounds inhibited the uremia-induced increase in proinflammatory inducible nitric oxide synthase (iNOS) and glutathione peroxidase activity better than either compound alone. Glutathione reductase was also increased in UE and UP rats vs. UC. Kidney 4-hydroxynonenal was significantly increased in the UC group compared with the normal group. Combined treatment with both compounds significantly blunted this increase, P < 0.05, while either compound alone had no effect. Additionally, the expression of Mn-SOD was increased and CuZn-SOD decreased by uremia. This was ameliorated in all treatment groups. Cotreatment with enalapril and paricalcitol had an additive effect in increasing CuZn-SOD expression. In conclusion, like enalapril, paricalcitol alone can improve proteinuria, glomerulosclerosis, and interstitial infiltration and reduce renal oxidative stress. The effects of paricalcitol may be amplified when an ACE inhibitor is added since cotreatment with both compounds seems to have an additive effect on ameliorating uremia-induced changes in iNOS and CuZn-SOD expression, peroxidase activity, and renal histomorphometry.