The mammalian target of rapamycin (mTOR) is a serine kinase that regulates phosphorylation (p) of its target ribosomal S6 kinase (S6K1), whose activation can lead to glomerular and proximal tubular cell (PTC) injury and associated proteinuria. Increased mTOR/S6K1 signaling regulates signaling pathways that target fibrosis through adherens junctions. Recent data indicate aldosterone signaling through the mineralocorticoid receptor (MR) can activate the mTOR pathway. Further, antagonism of the MR has beneficial effects on proteinuria that occur independent of hemodynamics.
Accordingly, hypertensive transgenic TG(mRen2)27 (Ren2) rats, with elevated serum aldosterone and proteinuria, and age-matched Sprague-Dawley rats were treated with either a low dose (1 mg/kg/day) or a conventional dose (30 mg/kg/day) of spironolactone (MR antagonist) or placebo for 3 weeks.
Ren2 rats displayed increases in urine levels of the PTC brush border lysosomal enzyme N-acetyl-β-aminoglycosidase (β-NAG) in conjunction with reductions in PTC megalin, the apical membrane adherens protein T-cadherin and basolateral α-(E)-catenin, and fibrosis. In concert with these abnormalities, Ren2 renal cortical tissue also displayed increased Ser2448 (p)/activation of mTOR and Thr389 (p)-S6K1 and increased 3-nitrotyrosine (3-NT) content, a marker for peroxynitrite. Low-dose spironolactone had no effect on blood pressure but decreased proteinuria and β-NAG comparable to a conventional dose of this MR antagonist. Both doses of spironolactone attenuated ultrastructural maladaptive alterations and led to comparable reductions in (p)-mTOR/(p)-S6K1, 3-NT, fibrosis, and increased expression of α-(E)-catenin, T- and N-cadherin.
Thereby, MR antagonism improves proximal tubule integrity by targeting mTOR/S6K1 signaling and redox status independent of changes in blood pressure.
Cadherin; Megalin; β-NAG; Proteinuria
Enhanced renin-angiotensin-aldosterone system (RAAS) activation contributes to proteinuria and chronic kidney disease by increasing glomerular and tubulointerstitial oxidative stress, promotion of fibrosis. Renin activation is the rate limiting step in angiotensin (Ang II) and aldosterone generation, and recent work suggests direct renin inhibition improves proteinuria comparable to that seen with Ang type 1 receptor (AT1R) blockade. This is important as, even with contemporary use of AT1R blockade, the burden of kidney disease remains high. Thereby, we sought to determine if combination direct renin inhibition with AT1R blockade in vivo, via greater attenuation of kidney oxidative stress, would attenuate glomerular and proximal tubule injury to a greater extent than either intervention alone. We utilized the transgenic Ren2 rat with increased tissue RAS activity and higher serum levels of aldosterone, which manifests hypertension and proteinuria. Ren2 rats were treated with renin inhibition (aliskiren), AT1R blockade (valsartan), the combination (aliskiren+valsartan), or vehicle for 21 days. Compared to Sprague-Dawley controls, Ren2 rats displayed increased systolic pressure (SBP), circulating aldosterone, proteinuria and greater urine levels of the proximal tubule protein excretory marker beta-N-acetylglucosaminidase (β-NAG). These functional and biochemical alterations were accompanied by increases in kidney tissue NADPH oxidase subunit Rac1 and 3-nitrotyrosine (3-NT) content as well as fibronectin and collagen type III. These findings occurred in conjunction with reductions in the podocyte-specific protein podocin as well as the proximal tubule-specific megalin. Further, in transgenic animals there was increased tubulointerstitial fibrosis on light microscopy as well as ultrastructural findings of glomerular podocyte foot-process effacement and reduced tubular apical endosomal/lysosomal activity. Combination therapy led to greater reductions in SBP and serum aldosterone, but did not result in greater improvement in markers of glomerular and tubular injury (ie. β-NAG) compared to either intervention alone. Further, combination therapy did not improve markers of oxidative stress and podocyte and proximal tubule integrity in this transgenic model of RAAS-mediated kidney damage despite greater reductions in serum aldosterone and BP levels.
Aldosterone; Combination; Renin inhibition; AT1R blockade; Podocyte; β-NAG; Oxidative Stress
The impact of nebivolol therapy on the renal proximal tubular cell (PTC) structure and function was investigated in a transgenic (TG) rodent model of hypertension and the cardiometabolic syndrome. The TG Ren2 rat develops nephropathy with proteinuria, increased renal angiotensin II levels and oxidative stress, and PTC remodeling. Nebivolol, a β1-antagonist, has recently been shown to reduce albuminuria, in part, through reductions in renal oxidative stress. Accordingly, we hypothesized that nebivolol therapy would attenuate PTC damage and tubulointerstitial fibrosis.
Young Ren2 (R2-N) and SD (SD-N) rats were treated with nebivolol (10 mg/kg/day) or vehicle (R2-C; SD-C) for 3 weeks. PTC structure and function were tested using transmission electron microscopy and functional measurements.
Nebivolol treatment decreased urinary N-acetyl-β-D-glucosaminidase, tubulointerstitial ultrastructural remodeling and fibrosis, NADPH oxidase activity, 3-nitrotyrosine levels, and increased megalin and lysosomal-associated membrane protein-2 immunostaining in PTCs. Ultrastructural abnormalities that were improved with therapy included altered canalicular structure, reduced endosomes/lysosomes and PTC vacuoles, basement membrane thickening, and mitochondrial remodeling/fragmentation.
These observations support the notion that nebivolol may improve PTC reabsorption of albumin and other glomerular filtered small molecular weight proteins in association with the attenuation of oxidative stress, tubulointerstitial injury and fibrosis in this rat model of metabolic kidney disease.
NADPH oxidase; Proximal tubule cell; Megalin
Endogenous angiotensin (Ang)-(1−7) enhances, while Ang II attenuates, baroreceptor sensitivity (BRS) for reflex control of heart rate (HR) in Sprague-Dawley (SD) rats. In (mRen2)27 renin transgenic rats [(mRen2)], there is overexpression of the mouse Ren2 gene in brain, leading to elevated Ang II and reduced Ang-(1−7) in brain medullary, and associated with hypertension and impaired BRS.
We therefore tested the contribution of endogenous Ang-(1−7) to BRS for control of HR and responses to cardiac vagal chemosensitive afferent fiber activation (CVA) with phenylbiguanide (PBG) in anesthetized SD and (mRen2) 27 rats before and after bilateral nucleus of the solitary tract (nTS) injection of the Ang-(1−7) receptor antagonist (D-Ala7)-Ang-(1−7).
(mRen2) 27 rats exhibited a ∼50% impairment in BRS as compared with SD (P < 0.05). (D-Ala7)-Ang-(1−7) attenuated BRS by ∼50% in SD rats, but was without effect in (mRen2) 27 rats. (D-Ala7)-Ang-(1−7) did not alter the responses to CVA by PBG (iv bolus) in either strain. There were no differences in the depressor effects of Ang-(1−7) injected into the nTS, nor were levels of mRNA different for angiotensin-converting enzyme, angiotensin-converting enzyme 2, neprilysin, or the mas receptor in medullary tissue from SD versus (mRen2)27 rats.
Endogenous Ang-(1−7) does not provide tonic input in the nTS to modulate BRS for control of HR in (mRen2)27 rats, which may contribute to impairment of BRS in these animals.
angiotensins; baroreceptor reflex; control of heart rate; cardiac vagal chemosensitive fiber activation; phenylbiguanide; transgenic rats
Non-alcoholic fatty liver disease (NAFLD) is a common health problem and includes a spectrum of hepatic steatosis, steatohepatitis and fibrosis. The renin–angiotensin system (RAS) plays a vital role in blood pressure regulation and appears to promote hepatic fibrogenesis. We hypothesized that increased RAS activity causes NAFLD due to increased hepatic oxidative stress.
We employed the transgenic TG(mRen2)27(Ren2) hypertensive rat, harboring the mouse renin gene with elevated tissue Angiotensin II (Ang II).
Compared with normotensive Sprague–Dawley (SD) control rats, Ren2 developed significant hepatic steatosis by 9 weeks of age that progressed to marked steatohepatitis and fibrosis by 12 weeks. These changes were associated with increased levels of hepatic reactive oxygen species (ROS) and lipid peroxidation. Accordingly, 9-week-old Ren2 rats were treated for 3 weeks with valsartan, an angiotensin type 1 receptor blocker, or tempol, a superoxide dismutase/catalase mimetic. Hepatic indices for oxidative stress, steatosis, inflammation and fibrosis were markedly attenuated by both valsartan and tempol treatment.
This study suggests that Ang II causes development and progression of NAFLD in the transgenic Ren2 rat model by increasing hepatic ROS. Our findings also support a potential role of RAS in prevention and treatment of NAFLD.
Angiotensin II; Oxidative stress; Non-alcoholic fatty liver disease
Emerging evidence suggests that cardiac angiotensin-converting enzyme 2 (ACE2) may contribute to the regulation of heart function and hypertension-induced cardiac remodeling. We tested the hypothesis that inhibition of ACE2 in the hearts of (mRen2)27 hypertensive rats may accelerate progression of cardiac hypertrophy and fibrosis by preventing conversion of angiotensin II (Ang II) into the antifibrotic peptide, angiotensin-(1–7) (Ang-(1–7)).
Fourteen male (mRen2)27 transgenic hypertensive rats (12 weeks old, 401 ± 7 g) were administered either vehicle (0.9% saline) or the ACE2 inhibitor, MLN-4760 (30 mg/kg/day), subcutaneously via mini-osmotic pumps for 28 days.
Although ACE2 inhibition had no effect on average 24-h blood pressures, left ventricular (LV) Ang II content increased 24% in rats chronically treated with the ACE2 inhibitor (P < 0.05). Chronic ACE2 inhibition had no effect on plasma Ang II or Ang-(1–7) levels. Increased cardiac Ang II levels were associated with significant increases in both LV anterior, posterior, and relative wall thicknesses, as well as interstitial collagen fraction area and cardiomyocyte hypertrophy in the transgenic animals chronically treated with the ACE2 inhibitor. Cardiac remodeling was not accompanied by any further alterations in LV function.
These studies demonstrate that chronic inhibition of ACE2 causes an accumulation of cardiac Ang II, which exacerbates cardiac hypertrophy and fibrosis without having any further impact on blood pressure or cardiac function.
angiotensin-converting enzyme 2; angiotensin II; angiotensin-(1-7); blood pressure; cardiac hypertrophy; hypertension
The immunosuppressive mammalian target of rapamycin (mTOR) inhibitors are widely used in solid organ transplantation, but their effect on kidney disease progression is controversial. mTOR has emerged as one of the main pathways regulating cell growth, proliferation, differentiation, migration, and survival.
The aim of this study was to analyze the effects of delayed inhibition of mTOR pathway with low dose of everolimus on progression of renal disease and TGFβ expression in the 5/6 nephrectomy model in Wistar rats.
This study evaluated the effects of everolimus (0.3 mg/k/day) introduced 15 days after surgical procedure on renal function, proteinuria, renal histology and mechanisms of fibrosis and proliferation.
Everolimus treated group (EveG) showed significantly less proteinuria and albuminuria, less glomerular and tubulointerstitial damage and fibrosis, fibroblast activation cell proliferation, when compared with control group (CG), even though the EveG remained with high blood pressure. Treatment with everolimus also diminished glomerular hypertrophy.
Everolimus effectively inhibited the increase of mTOR developed in 5/6 nephrectomy animals, without changes in AKT mRNA or protein abundance, but with an increase in the pAKT/AKT ratio. Associated with this inhibition, everolimus blunted the increased expression of TGFβ observed in the remnant kidney model.
Delayed mTOR inhibition with low dose of everolimus significantly prevented progressive renal damage and protected the remnant kidney. mTOR and TGFβ mRNA reduction can partially explain this anti fibrotic effect. mTOR can be a new target to attenuate the progression of chronic kidney disease even in those nephropathies of non-immunologic origin.
Chronic activation of the renin-angiotensin system plays a deleterious role in progressive kidney damage, and the renal proximal tubule is known to play an important role in tubulointerstitial fibrosis; however, the underlying molecular mechanism is unclear. Here we report that in the proximal tubule-like LLCPKcl4 cells expressing angiotensin II (Ang II) type 1 receptor, Ang II induced changes in cell morphology and expression of epithelial-to-mesenchymal transition (EMT) markers, which were inhibited by the miotogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK)-activating kinase (MEK) inhibitor PD98059 or the Src kinase inhibitor PP2. Ang II-stimulated phosphorylation of caveolin-1 (Cav) at Y14 and epidermal growth factor receptor (EGFR) at Y845 and induced association of these phosphoproteins in caveolin-enriched lipid rafts, thereby leading to prolonged EGFR-ERK signaling that was inhibited by Nox4 small interfering RNA (siRNA) and Src siRNA. Two different antioxidants not only inhibited phosphorylation of Src at Y416 but also blocked the EGFR-ERK signaling. Moreover, erlotinib (the EGFR tyrosine kinase inhibitor), EGFR siRNA, and Cav siRNA all inhibited both prolonged EGFR-ERK signaling and phenotypic changes induced by Ang II. Thus, this report provides the first evidence that reactive oxygen species (ROS)/Src-dependent activation of persistent Cav-EGFR-ERK signaling mediates renal tubular cell dedifferentiation and identifies a novel molecular mechanism that may be involved in progressive renal injury caused by chronic exposure to Ang II.
Angiotensin-II (Ang-II) contributes to cardiac remodeling and left ventricular dysfunction. In contrast, exercise may have beneficial effects on left ventricular structure and function.
Methods and Results
We investigated the effects of low-intensity exercise training (ET) on in vivo cardiac function in hypertensive TG (mREN-2)27 rats (Ren-2) which develop left ventricular hypertrophy and dysfunction. Ren-2 rats and Sprague Dawley (SD) controls (4–5 weeks) began treadmill exercise every day for 5–6 weeks. Cardiac function was evaluated by echocardiography. Cardiac output and stroke volume were increased by ET in both 8-wk-old SD and Ren-2. Slope of mitral deceleration time, a non-invasive measure of diastolic function, was lower in the Ren-2 rats, but not changed by ET. LV collagen deposition, as assessed by hydroxyproline assay, was not affected by rat strain or ET at 10–11 weeks of age. Left ventricular B-type natriuretic peptide mRNA levels were higher in the Ren-2 rats (100%), but not affected by ET. Both α (~14.5 fold) and β (~2.5 fold) myosin heavy chain mRNA were higher in the LV of Ren-2 rats (p < 0.05), but were not changed by ET.
Low-intensity exercise training in Ren-2 rats, a model of Ang-II-mediated hypertension, maintains cardiac index and stroke volume in the presence of impaired diastolic function at 8 wks of age.
Hypertension; heart; exercise
Hypertension is often associated with increased oxidative stress and systemic insulin resistance. Use of β adrenergic receptor blockers in hypertension is limited due to potential negative influence on insulin sensitivity and glucose homeostasis. We sought to determine the impact of nebivolol, a selective vasodilatory β1adrenergic blocker, on whole-body insulin sensitivity, skeletal muscle oxidative stress, insulin signaling and glucose transport in the transgenic TG(mRen2)27rat (Ren2). This rodent model manifests increased tissue renin angiotensin expression, excess oxidative stress, and whole-body insulin resistance.
Research design and methods
Young (age 6-9 wks) Ren2 and age-matched Sprague-Dawley control rats were treated with nebivolol 10 mg/kg/day or placebo for 21 days. Basal measurements were obtained for glucose and insulin to calculate the Homeostasis Model Assessment (HOMA–IR). Additionally, insulin metabolic signaling, NADPH oxidase activity, reactive oxygen species (ROS), and ultrastructural changes as evaluated by transmission electron microscopy were examined ex vivo in skeletal muscle tissue.
The Ren2 rat demonstrated systemic insulin resistance as examined by HOMA-IR, along with impaired insulin metabolic signaling in skeletal muscle. This was associated with increased oxidative stress and mitochondrial remodeling. Treatment with nebivolol was associated with improvement in insulin resistance and decreased NADPH oxidase activity/levels ROS in skeletal muscle tissue.
Nebivolol treatment for 3 weeks reduces NADPH oxidase activity and improves systemic insulin resistance, in concert with reduced oxidative stress in skeletal muscle in a young rodent model of hypertension, insulin resistance and enhanced tissue RAS expression.
Insulin resistance; oxidative stress; skeletal muscle
Overnutrition characterized by overconsumption of food rich in fat and carbohydrates is a significant contributor to hypertension, type 2 diabetes, and the cardiorenal syndrome. Overnutrition activates the renin-angiotensin-aldosterone system (RAAS) and causes chronic exposure of cardiovascular and renal tissue to increased circulating nutrients, insulin (INS), and angiotensin II (ANG II). Emerging evidence suggests that overnutrition, aldosterone, and ANG II promote INS resistance, a chronic condition that underlies these co-morbidities, through activation of the mammalian target of the rapamycin (mTOR)/S6 kinase 1 (S6K1) signaling pathway in cardiovascular tissue and the kidney. However, a novel ANG II type 2 receptor (AT2R)-mediated cross talk between the RAAS and mTOR pathways ameliorates overnutrition-induced activation of mTOR/S6K1 signaling in cardiovascular tissue of rats, mice, and humans and confers cardioprotection.
Overnutrition; Hypertension; Insulin metabolic signaling; Progressive kidney disease
mTOR, the mammalian target of rapamycin, is a serine-threonine kinase known to regulate cell proliferation and growth. mTOR has also been implicated in neuronal synaptic plasticity as well as in pain transmission in models of chemically induced and neuropathic pain. To date, the role of mTOR as a modulator of inflammatory pain has not been examined. In this study, we investigated the role of mTOR in Sprague Dawley rats using the carrageenan model of inflammatory pain. mRNA of Ras homolog enriched in brain (Rheb), a GTPase that positively regulates mTOR activation, was significantly increased 2 hours following carrageenan injection. Four hours after induction of inflammation phosphorylation (p) of p70S6 kinase (S6K), ribosomal protein S6 (S6) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) was increased, indicating mTOR activation. Inhibition of spinal mTOR with intrathecal (i.t.) injection of rapamycin (0.1–3 μg) led to a dose-dependent decrease in carrageenan-induced thermal hyperalgesia and a reduction of mechanical allodynia. In vitro studies confirmed rapamycin inhibition of the mTOR pathway. Carrageenan-induced activation of the mTOR pathway in rats was localized predominantly to dorsal horn neurons in the superficial lamina. Taken together, these data show that the mTOR pathway is activated in dorsal horn neurons during inflammatory pain, and that inhibition of spinal mTOR attenuates inflammation-induced thermal and tactile hypersensitivity. Hence, our study indicates that spinal mTOR is an important regulator of spinal sensitization and suggests that targeting mTOR may provide a new avenue for pain therapy.
Hypertension and left ventricular (LV) hypertrophy often precede diastolic dysfunction and are risk factors for diastolic heart failure. Although pharmacologic inhibition of the renin angiotensin system (RAS) improves diastolic function and functional capacity in hypertensive patients with LV hypertrophy, the effects of combination therapy with an angiotensin converting enzyme inhibitor (ACEi) and an angiotensin receptor blocker (ARB) are unclear.
We assessed the effects of the combined 10-week administration of lisinopril (10 mg/kg/day, p.o.) and losartan (10 mg/kg/day, p.o.) (LIS/LOS) on diastolic function and LV structure in seven young (5 wks), prehypertensive congenic mRen2.Lewis male rat, a model of tissue renin overexpression and angiotensin II (Ang II)-dependent hypertension compared to vehicle (VEH) treated (n=7), age-matched rats.
Systolic blood pressures were 64% lower with the combination therapy (P<0.001), but there were no differences in heart rate or systolic function between groups. RAS inhibition increased myocardial relaxation, defined by tissue Doppler mitral annular descent (e’), by 2.2 fold (P< 0.001). The preserved lusitropy in the LIS/LOS-treated rats was accompanied by a reduction in phospholamban-to-SERCA2 ratio (P<0.001). Despite lower relative wall thicknesses (VEH: 1.56 ± 0.17 vs. LIS/LOS: 0.78 ± 0.05) and filling pressures, defined by the transmitral Doppler-to-mitral annular descent ratio (E/e’, VEH: 28.7 ± 1.9 vs. LIS/LOS: 17.96 ± 1.5), no differences in cardiac collagen were observed.
We conclude that the lusitropic benefit of early dual RAS blockade may be due to improved vascular hemodynamics and/or cardiac calcium handling rather than effects on extracellular matrix reduction.
diastolic function; fibrosis; hypertension; myocardial relaxation; renin-angiotensin system (RAS)
The pregnant female human angiotensinogen (hAGN) transgenic rat (TGR) mated with the male hrenin (hREN) TGR is a model of preeclampsia with increased blood pressure, proteinuria, and placenta alterations of edema and necrosis. The reverse mating (RM) of female hREN × male hAGN does not show preeclamptic features. Since the placenta is well recognized to be a key contributor to the preeclamptic syndrome, our hypothesis is that local angiotensin peptide concentrations found in the placenta (Pla) and its associated mesometrial triangle (MT) of the preeclamptic TGR differ from the RM. We characterized the Ang peptide content and the mRNA expression of hREN and hAGN of the MT and the Pla. Three groups of pregnant rats from the following matings [Sprague Dawley (SD) × SD, RM, and female hAGN × male hREN] were studied on day 21 of gestation. In the hAGNxhREN TGR Ang II is significantly increased in the Pla and MT vs SD (24.2 ± 3.9 vs 8.6 ± 1.5 pg/mg protein; 27.8 ± 5.5 vs 5.6 ± 1.3 pg/mg protein, p<0.05), whereas in the RM Ang II is increased in the Pla (19.1 ± 1.7 vs 5.6 ± 1.3 pg/mg protein, p<0.05) but unchanged in the MT (4.2 ± 0.2 vs 8.6 ± 1.5 pg/mg protein). The marked contrast in the expression of Ang II in the MT of the preeclamptic model vs the RM suggests that local Ang II generated from the maternal parts of the uteroplacental unit may play a critical role in preeclampsia.
preeclampsia; renin-angiotensin system; angiotensinogen; placenta; fetal; maternal
Human and animal studies support the idea that there are sex differences in the development of diabetic renal disease. Our lab and others have determined that in addition to Ang II (through the AT1R), growth hormone (GH) contributes to renal damage in models of renal failure; however, the impact of sex and GH on the mechanisms initiating diabetic renal disease is not known. This study examined the effect of sex and GH on parameters of renal damage in early, uncontrolled streptozotocin (STZ)-induced diabetes.
Adult male and female Sprague–Dawley rats were injected with vehicle (control), STZ, or STZ + GH and euthanized after 8 weeks.
Mild but significant glomerulosclerosis (GS) and tubulointerstitial fibrosis (TIF) was observed in both kidneys from male and female diabetic rats, with GH significantly increasing GS and TIF by 30% and 25% in male rats, but not in female rats. STZ increased TGF-β expression in both kidneys from male and female rats; however, while GH had no further effect on TGF-β protein in diabetic females, GH increased TGF-β protein in the male rat’s kidneys by an additional 30%. This sex-specific increase in renal injury following GH treatment was marked by increased MCP-1 and CD-68+ cell density. STZ also reduced renal MMP-2 and MMP-9 protein expression in both kidneys from male and female rats, but additional decreases were only observed in GH-treated diabetic male rats. The sex differences were independent of AT1R activity.
These studies indicate that GH affects renal injury in diabetes in a sex-specific manner and is associated with an increase in pro-inflammatory mediators.
Sex Differences; Gender; Glomerulosclerosis; Renal Injury; TGF-β; Inflammation
Cardiac insulin resistance is a metabolic and functional disorder that is often associated with obesity and/or the cardiorenal metabolic syndrome (CRS), and this disorder may be accentuated by chronic alcohol consumption. In conditions of over-nutrition, increased insulin (INS) and angiotensin II (Ang II) activate mammalian target for rapamycin (mTOR)/p70 S6 kinase (S6K1) signaling, whereas chronic alcohol consumption inhibits mTOR/S6K1 activation in cardiac tissue. Although excessive activation of mTOR/S6K1 induces cardiac INS resistance via serine phosphorylation of INS receptor substrates (IRS-1/2), it also renders cardioprotection via increased Ang II receptor 2 (AT2R) upregulation and adaptive hypertrophy. In the INS-resistant and hyperinsulinemic Zucker obese (ZO) rat, a rodent model for CRS, activation of mTOR/S6K1signaling in cardiac tissue is regulated by protective feed-back mechanisms involving mTOR↔AT2R signaling loop and profile changes of microRNA that target S6K1. Such regulation may play a role in attenuating progressive heart failure. Conversely, alcohol-mediated inhibition of mTOR/S6K1, down-regulation of INS receptor and growth-inhibitory mir-200 family, and upregulation of mir-212 that promotes fetal gene program may exacerbate CRS-related cardiomyopathy.
Renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system activation are crucial in the pathogenesis of hypertension, cardiovascular and renal disease. NADPH oxidase-mediated increases in reactive oxygen species (ROS) are an important mediator for RAAS-induced cardiovascular and renal injury. Increased levels of ROS can diminish the bioactivity of nitric oxide (NO), a critical modulator of RAAS effects on the kidney. Thereby, we hypothesized that in vivo nebivolol therapy in a rodent model of activated RAAS would attenuate glomerular damage and proteinuria through its actions to reduce NADPH oxidase activity/ROS and increase bioavailable NO.
We utilized the transgenic Ren2 rat which displays heightened tissue RAAS, hypertension, and proteinuria. Ren2 rats (6–9 weeks of age) and age-matched Sprague-Dawley littermates were treated with nebivolol 10 mg/kg/day (osmotic mini-pump) for 21 days.
Ren2 rats exhibited increases in systolic blood pressure, proteinuria, kidney cortical tissue total NADPH oxidase activity and subunits (Rac1, p67phox, and p47phox), ROS and 3-nitrotyrosine, as well as reductions in podocyte protein markers; each of these parameters improved with nebivolol treatment along with increases in renal endothelial NO synthase expression.
Our data suggest that nebivolol improves proteinuria through reductions in renal RAAS-mediated increases in NADPH oxidase/ROS and increases in bioavailable NO.
Nebivolol; Proteinuria; NADPH oxidase; Reactive oxygen species
Recent studies have shown that the renal cytochrome P-450 metabolites of arachidonic acid: the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE), and the vasodilator epoxyeicosatrienoic acids (EETs) play an important role in the pathophysiology of angiotensin II (ANG II)-dependent forms of hypertension and the associated target organ damage. The present studies were performed in Ren-2 renin transgenic rats (TGR) to evaluate the effects of chronic selective inhibition of 20-HETE formation or elevation of the level of EETs, alone or in combination, on the course of hypertension and hypertension-associated end-organ damage. Both young (30 days of age) prehypertensive TGR and adult (190 days of age) TGR with established hypertension were examined. Normotensive Hannover Sprague-Dawley (HanSD) rats served as controls. The rats were treated with N-methylsulfonyl-12,12-dibromododec-11-enamide to inhibit 20-HETE formation and/or with N-cyclohexyl-N-dodecyl urea to inhibit soluble epoxide hydrolase and prevent degradation of EETs. Inhibition in TGR rats of 20-HETE formation combined with enhanced bioavailability of EETs attenuated the development of hypertension, cardiac hypertrophy, proteinuria, glomerular hypertrophy and sclerosis as well as renal tubulointerstitial injury. This was also associated with an attenuation of the responsiveness of the systemic and renal vascular beds to ANG II without modifying their responses to norepinephrine. Our data suggest that altered production and/or action of 20-HETE and EETs plays a permissive role in the development of hypertension and hypertension-associated end-organ damage in this model of ANG II-dependent hypertension. This information provides a basis for a search of new therapeutic approaches to the treatment of hypertension.
cytochrome P-450 metabolites; renin-angiotensin system; hypertension; end-organ damage; soluble epoxide hydrolase
ACE (angiotensin-converting enzyme) 2 is expressed in the heart and kidney and metabolizes Ang (angiotensin) II to Ang-(1–7) a peptide that acts via the Ang-(1–7) or mas receptor. The aim of the present study was to assess the effect of Ang-(1–7) on blood pressure and cardiac remodelling in a rat model of renal mass ablation. Male SD (Sprague–Dawley) rats underwent STNx (subtotal nephrectomy) and were treated for 10 days with vehicle, the ACE inhibitor ramipril (oral 1 mg·kg−1 of body weight·day−1) or Ang-(1–7) (subcutaneous 24 μg·kg−1 of body weight·h−1) (all n = 15 per group). A control group (n = 10) of sham-operated rats were also studied. STNx rats were hypertensive (P<0.01) with renal impairment (P<0.001), cardiac hypertrophy (P<0.001) and fibrosis (P<0.05), and increased cardiac ACE (P<0.001) and ACE2 activity (P<0.05). Ramipril reduced blood pressure (P<0.01), improved cardiac hypertrophy (P<0.001) and inhibited cardiac ACE (P<0.001). By contrast, Ang-(1–7) infusion in STNx was associated with further increases in blood pressure (P<0.05), cardiac hypertrophy (P<0.05) and fibrosis (P<0.01). Ang-(1–7) infusion also increased cardiac ACE activity (P<0.001) and reduced cardiac ACE2 activity (P<0.05) compared with STNx-vehicle rats. Our results add to the increasing evidence that Ang-(1–7) may have deleterious cardiovascular effects in kidney failure and highlight the need for further in vivo studies of the ACE2/Ang-(1–7)/mas receptor axis in kidney disease.
angiotensin peptide; cardiac fibrosis; kidney failure; renal mass reduction; renin–angiotensin system; ACE, angiotensin-converting enzyme; Ang, angiotensin; AT1R etc., angiotensin type 1 receptor etc.; BNP, brain natriuretic peptide; EMT, epithelial-to-mesenchymal transition; LV, left ventricle; LVEDP, left ventricular end-diastolic pressure; KO, knockout; PRA, plasma renin activity; QFS, quenched fluorescent substrate; qRT, quantitative real-time; RAS, renin–angiotensin system; SD, Sprague–Dawley; STNx, subtotal nephrectomy; TGF-β1, transforming growth factor-β1; 2K1C, two-kidney/one-clip
Cyp1a1-Ren2 transgenic rats [strain name: TGR(Cyp1a1Ren2)], administered indole-3-carbinol (I3C) develop angiotensin (ANG) II-dependent hypertension due to hepatic expression of the Ren2 renin gene. Although AT1 receptor blockade prevents the development of hypertension and normalizes the elevated arterial blood pressure of Cyp1-Ren2 rats, little information is available regarding the blood pressure and renal functional responses to direct inhibition of renin in this high circulating renin model of ANG II-dependent hypertension. The present study was performed to determine the effects of acute direct renin inhibition with aliskiren on blood pressure and renal hemodynamics in Cyp1a1-Ren2 rats with ANG II-dependent malignant hypertension.
Mean arterial pressure (MAP) and renal hemodynamics were measured in pentobarbital-anesthetized male Cyp1a1-Ren2 rats during control conditions and following administration of the renin inhibitor, aliskiren (10 mg/kg, iv).
Rats induced with I3C had higher MAP (194±7 vs. 141±2 mmHg, P<0.001), lower renal plasma flow (RPF; 2.47±0.23 vs. 4.17±0.35 ml/min.g, P<0.001), and lower glomerular filtration rate (GFR; 1.01±0.07 vs. 1.34±0.06 ml/min.g, P=0.01) than noninduced Cyp1a1-Ren2 rats (n=5). Aliskiren administration decreased MAP (194±7 to 136±2 mmHg, P<0.001) and increased RPF (2.47±0.23 vs. 4.31±0.20 ml/min.g, P<0.001) in hypertensive but not in normotensive rats, without altering GFR.
Acute renin inhibition with aliskiren normalizes MAP and RPF in Cyp1a1-Ren2 rats with malignant hypertension. The normalization of MAP and RPF following acute renin inhibition indicates that renin generated by expression of the Ren2 gene is responsible for the maintenance of malignant hypertension and the associated reduction in renal hemodynamic function in Cyp1a1-Ren2 rats.
kidney; renin-angiotensin system; malignant hypertension; renin inhibitor; renal hemodynamics
Angiotensin-converting enzyme-2 (ACE-2) is a homolog of ACE that preferentially forms angiotensin-(ANG)-1-7 from angiotensin II (ANG II). We investigated the cardioprotective effects of telmisartan, a well-known angiotensin receptor blockers (ARBs) against experimental autoimmune myocarditis (EAM). EAM was induced in Lewis rats by immunization with porcine cardiac myosin. The rats were divided into two groups and treated with telmisartan (10 mg/kg/day) or vehicle for 21 days. Myocardial functional parameters were significantly improved by treatment with telmisartan compared with vehicle-treated rats. Telmisartan lowered myocardial protein expressions of NADPH oxidase subunits 3-nitrotyrosine, p47phox, p67 phox, Nox-4 and superoxide production significantly than vehicle-treated rats. In contrast myocardial protein levels of ACE-2, ANG 1-7 mas receptor were upregulated in the telmisartan treated group compared with those of vehicle-treated rats. The myocardial protein expression levels of tumor necrosis factor receptor (TNFR)-associated factor (TRAF)-2, C/EBP homologous protein (CHOP) and glucose-regulated protein (GRP) 78 were decreased in the telmisartan treated rats compared with those of vehicle-treated rats. In addition, telmisartan treatment significantly decreased the protein expression levels of phospho-p38 mitogen-activated protein kinase (MAPK), phospho-JNK, phospho-ERK and phospho (MAPK) activated protein kinase-2 than with those of vehicle-treated rats. Moreover, telmisartan significantly decreased the production of proinflammatory cytokines, myocardial apoptotic markers and caspase-3 positive cells compared with those of vehicle-treated rats. Therefore, we suggest that telmisartan was beneficial protection against heart failure in rats, at least in part by suppressing inflammation, oxidative stress, ER stress as well as signaling pathways through the modulation of ACE2/ANG1-7/Mas receptor axis.
Experimental autoimmune myocarditis; endoplasmic reticulum stress; inflammation; telmisartan; oxidative stress; signaling pathways
A chronic increase in physical activity and (or) endurance training can improve insulin sensitivity in insulin-resistant skeletal muscle. Cellular mechanisms responsible for the development of insulin resistance are unclear, though one proposed mechanism is that nutrient overload chronically increases available energy, over-activating the mammalian target of rapamycin (mTOR) and ribosomal S6 kinase 1 (S6K1) signaling pathway leading to increased phosphorylation of serine residues on insulin receptor substrate-1 (IRS-1). The objective of this study was to determine if increased physical activity would inhibit mTOR/S6K1 signaling and reduce IRS-1 serine phosphorylation in rat skeletal muscle. Soleus muscle was collected from fed male Sprague-Dawley sedentary rats (Inactive) and rats with free access to running wheels for 9 weeks (Active). Immunoblotting methods were used to measure phosphorylation status of mTOR, S6K1, IRS-1, and PKB/Akt (protein kinase B/AKT), and total abundance of proteins associated with the mTOR pathway. Muscle citrate synthase activity and plasma insulin and glucose concentrations were measured. Phosphorylation of mTOR (Ser2448), S6K1 (Thr389), and IRS-1 (Ser636-639) was reduced in Active rats (p < 0.05). Total protein abundance of mTOR, S6K1, IRS-1, 4E-BP1, eEF2, PKB/Akt and AMPKα, and phosphorylation of PKB/Akt were unaffected (p > 0.05). Total SKAR protein, a downstream target of S6K1, and citrate synthase activity increased in Active rats (p < 0.05), though plasma insulin and glucose levels were unchanged (p > 0.05). Reduced mTOR/S6K1 signaling during chronic increases in physical activity may play an important regulatory role in the serine phosphorylation of IRS-1, which should be examined as a potential mechanism for attenuation of insulin resistance associated with increased IRS-1 serine phosphorylation.
mTOR; S6K1; IRS-1; physical activity; skeletal muscle; insulin resistance
Tubulointerstitial disease, a prominent phenomenon in diabetic nephropathy, correlates with decline in renal function. The underlying pathogenic link between chronic hyperglycemia and the development of tubulointerstitial injury has not been fully elucidated, but myofibroblast formation represents a key step in the development of tubulointerstitial fibrosis. RAGE, the receptor for advanced glycation end products (AGEs), induces the expression of TGF-β and other cytokines that are proposed to mediate the transdifferentiation of epithelial cells to form myofibroblasts. Here we report specific binding of 125I-AGE-BSA to cell membranes prepared from a rat proximal tubule cell line and show that the binding site was RAGE. AGE exposure induced dose-dependent epithelial-myofibroblast transdifferentiation determined by morphological changes, de novo alpha smooth-muscle actin expression, and loss of epithelial E-cadherin staining. These effects could be blocked with neutralizing Ab’s to RAGE or to TGF-β. Transdifferentiation was also apparent in the proximal tubules of diabetic rats and in a renal biopsy from a patient with type 1 diabetes. The AGE cross-link breaker, phenyl-4,5-dimethylthiazolium bromide (ALT 711) reduced transdifferentiation in diabetic rats in association with reduced tubular AGE and TGF-β expression. This study provides a novel mechanism to explain the development of tubulointerstitial disease in diabetic nephropathy and provides a new treatment target.
Angiotensin II (ANG II)-infused rats exhibit increases in distal nephron renin expressed in principal cells of connecting tubules and collecting ducts. This study was performed to determine whether the augmentation of distal nephron renin involves ANG II type 1 (AT1) receptor activation. Male Sprague-Dawley rats (200 -220 g) were divided into three groups: 1) sham operated (n = 8); 2) ANG II infused (80 ng/min, 13 days, n = 8); and 3) ANG II infused plus AT1 receptor blocker (ARB), olmesartan (5 mg/days, n = 8). ANG II infusion increased systolic blood pressure (BP; 178 ± 4 vs. 122 ± 1 mmHg; P < 0.001) and suppressed plasma renin activity (PRA; 0.08 ± 0.1 vs. 5.3 ± 0.8 ng ANG I · ml-1·h-1). ARB treatment prevented the increase in BP (113 ± 6 mmHg) and led to increases in PRA (15.8 ± 1.5 ng ANG I·ml-1h-1). Renin protein levels measured in the kidney medulla, to avoid contribution from juxtaglomerular appartus cells, were higher in ANG II-infused rats [1.64 ± 0.3 vs. 1.00 ± 0.1 densitometric units (DU) compared with sham-operated rats; P < 0.05], and ARB treatment prevented this increase (1.01 ± 0.1). Similarly, renin immunoreactivity increased in medullary collecting ducts of ANG II-infused compared with sham-operated rats (2.5 ± 0.3 vs. 1.0 ± 0.2 DU; P < 0.001), which was also prevented by ARB (1.01 ± 0.06). Renin qRTPCR in ANG II-infused rats showed higher mRNA levels in the kidney medulla compared with sham-operated rats (5.5 ± 2.3 vs. 0.04 ± 0.02 ratio to GAPDH mRNA levels; P < 0.001); however, renin transcript levels were normalized in the ARB-treated rats. These data demonstrate that the augmentation of distal nephron renin in ANG II-infused hypertensive rats is AT1 receptor mediated. The augmented distal tubular renin may contribute to increased intratubular ANG II levels and distal nephron sodium reabsorption in ANG II-dependent hypertension.
distal nephron; immunohistochemistry; Western blotting; qRT-PCR
Angiotensin receptor (type 1) blockers (ARBs) can reduce both hypertension and insulin resistance induced by local and systemic activation of the renin-angiotensin-aldosterone system. The effectiveness of azilsartan medoxomil (AZIL-M), a novel imidazole-based ARB, to facilitate metabolic improvements in conditions of angiotensin II (Ang II)-associated insulin resistance is currently unknown. The aim of this study was to determine the impact of chronic AZIL-M treatment on glucose transport activity and key insulin signaling elements in red skeletal muscle of Ang II-treated rats. Male Sprague-Dawley rats were treated for 8 weeks with or without Ang II (200 ng/kg/min) combined with either vehicle or AZIL-M (1 mg/kg/day). Ang II induced significant (p < 0.05) increases in blood pressure, which were completely prevented by AZIL-M. Furthermore, Ang II reduced insulin-mediated glucose transport activity in incubated soleus muscle, and AZIL-M co-treatment increased this parameter. Moreover, AZIL-M treatment of Ang II-infused animals increased the absolute phosphorylation of insulin signaling molecules, including Akt [both Ser473 (81%) and Thr308 (23%)] and AS160 Thr642 (42%), in red gastrocnemius muscle frozen in situ. Absolute AMPKα (Thr172) phosphorylation increased (98%) by AZIL-M treatment, and relative Thr389 phosphorylation of p70 S6K1, a negative regulator of insulin signaling, decreased (51%) with AZIL-M treatment. These results indicate that ARB AZIL-M improves the in vitro insulin action on glucose transport in red soleus muscle and the functionality of the Akt/AS160 axis in red gastrocnemius muscle in situ in Ang II-induced insulin-resistant rats, with the latter modification possibly associated with enhanced AMPKα and suppressed p70 S6K1 activation.
Angiotensin II; Glucose transport; Red skeletal muscle; Akt; AS160; AMPK; p70 S6K1