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
It is increasingly recognized that there is sexual dimorphism in kidney disease progression; however, this disparity is lost in the presence of diabetes where women progress at a similar rate to men. The renin-angiotensin-aldosterone system (RAAS) is known to regulate diabetes-induced kidney injury, and recent literature would suggest that gender differences exist in RAAS-dependent responses in the kidney. In this regard, these gender differences may be overcome by excessive salt intake. Thereby, we hypothesized that salt would promote proteinuria in transgenic female rats under conditions of excess tissue angiotensin (Ang) II and circulating aldosterone.
Materials and Methods
We utilized young female transgenic (mRen2)27 (Ren2) rats and Sprague-Dawley (SD) littermates and fed a high-salt diet (4%) over 3 weeks.
Compared to SD and Ren2 controls, female Ren2 rats fed a high-salt diet displayed increases in proteinuria, periarterial and interstitial fibrosis as well as ultrastructural evidence of basement membrane thickening, loss of mitochondrial elongation, mitochondrial fragmentation and attenuation of basilar canalicular infoldings. These findings occurred temporally with increases in transforming growth factor-β but not indices of oxidant stress.
Our current data suggest that a diet high in salt promotes progressive kidney injury as measured by proteinuria and fibrosis associated with transforming growth factor-β under conditions of excess tissue Ang II and circulating aldosterone.
Angiotensin II; Transgenic (mRen2)27 rat; Proteinuria; Fibrosis; Reactive oxygen species
Recent data would suggest pre-menopausal insulin resistant women are more prone to diastolic dysfunction than men, yet it is unclear why. We and others have reported that transgenic (mRen2)27 (Ren2) rats overexpressing the murine renin transgene are insulin resistant due to oxidative stress in insulin sensitive tissues. As increased salt intake promotes inflammation and oxidative stress, we hypothesized that excess dietary salt would promote diastolic dysfunction in transgenic females under conditions of excess tissue Ang II and circulating aldosterone levels.
For this purpose we evaluated cardiac function in young female Ren2 rats or age-matched Sprague-Dawley (SD) littermates exposed to a high (4%) salt or normal rat chow intake for three weeks.
Compared to SD littermates, at 10 weeks of age, female Ren2 rats fed normal chow showed elevations in left ventricular (LV) systolic pressures, LV and cardiomyocyte hypertrophy, and displayed reductions in LV initial filling rate accompanied by increases in 3-nitrotyrosine content as a marker of oxidant stress. Following 3 weeks of a salt diet, female Ren2 rats exhibited no further changes in LV systolic pressure, insulin resistance, or markers of hypertrophy but exaggerated increases in type 1 collagen, 3-nitrotryosine content, and diastolic dysfunction. These findings occurred in parallel with ultrastructural findings of pericapillary fibrosis, increased LV remodeling, and mitochondrial biogenesis.
These data suggest that a diet high in salt in hypertensive female Ren2 rats promotes greater oxidative stress, maladaptive LV remodeling, fibrosis, and associated diastolic dysfunction without further changes in LV systolic pressure or hypertrophy.
angiotensin II; TG (mRen2) 27 rat; Diastolic Dysfunction; Cardiac Remodeling; Oxidative Stress; Perivascular Fibrosis
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.
1. The aim of the present study was to test the hypothesis that increasing kidney tissue concentrations of epoxyeicosatrienoic acids (EETs) by preventing their degradation to the biologically inactive dihydroxyeicosatrienoic acids (DHETEs) using blockade of soluble epoxide hydrolase (sEH) would attenuate the progression of chronic kidney disease (CKD).
2. Ren-2 transgenic rats (TGR) after 5/6 renal mass reduction (5/6 NX) served as a model of CKD associated with angiotensin (Ang) II-dependent hypertension. Soluble epoxide hydrolase was inhibited using cis-4-[4-(3-adamantan-1-yl-ureido)cyclohexyloxy]benzoic acid (c-AUCB; 3 mg/L drinking water) for 20 weeks after 5/6 NX. Sham-operated normotensive transgene-negative Hannover Sprague-Dawley (HanSD) rats served as controls.
3. When applied in TGR subjected to 5/6 NX, c-AUCB treatment improved survival rate, prevented the increase in blood pressure, retarded the progression of cardiac hypertrophy, reduced proteinuria and the degree of glomerular and tubulointerstitial injury and reduced glomerular volume. All these organ-protective actions were associated with normalization of the intrarenal EETs : DHETEs ratio, an index of the availability of biologically active EETs, to levels observed in sham-operated HanSD rats. There were no significant concurrent changes of increased intrarenal AngII content.
4. Together, these results show that 5/6 NX TGR exhibit a profound deficiency of intrarenal availability of active epoxygenase metabolites (EETs), which probably contributes to the progression of CKD in this model of AngII-dependent hypertension, and that restoration of intrarenal availability of EETs using long-term c-AUCB treatment exhibits substantial renoprotective actions.
5/6 nephrectomy; chronic kidney disease; cytochrome P450 enzymes; end-organ damage; epoxyeicosatrienoic acids; hypertension; renin–angiotensin system; soluble epoxide hydrolase
Injury to the renal microvasculature and inflammatory process may be major factors in the progression of renal disease, therefore, protection of the renal endothelial cell and regulation of inflammatory process may be an important therapeutic target of renal disease. Thus, we evaluated the protective effect of cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) in unilateral ureteral obstruction (UUO)-induced renal fibrosis, cyclosporine A (CsA)-induced renal injury, and the diabetic nephropathy model. In the UUO model, morphologic examination indicated less tubular injury and tubulointerstitial fibrosis in mice that received COMP-Ang1 compared to vehicle-treated mice. Interstitial type I collagen, myofibroblast accumulation, renal surface microvasculature and renal blood flow were higher after treatment with COMP-Ang1 compared to vehicle-treated mice. COMP-Ang1 treatment decreased monocyte/macrophage infiltration, tissue levels of transforming growth factor β1, and Smad 2/3 phosphorylation and increased Smad 7 in the obstructed kidney. In CsA-induced renal injury, histologic examination showed significantly decreased CsA-induced tubular damage and tubulointerstitial fibrosis in COMP-Ang1 treated mice. COMP-Ang1 administration also decreased increased macrophage infiltration, adhesion molecule expression, TGF-β1, and Smad 2/3 levels in CsA-treated kidneys, while increasing Smad 7 levels. Laser-Doppler sonographic findings and endothelial factor VIII staining revealed that COMP-Ang1 had a preservative effect on peritubular vasculature. In the diabetic nephropathy model, COMP-Ang1 reduced albuminuria and decreased mesangial expansion, thickening of the glomerular basement membrane and podocyte foot process broadening and effacement. COMP-Ang1 may delay the fibrotic changes in the kidney of diabetic db/db mice through its anti-inflammatory or metabolic effects. In conclusion, COMP-Ang1 may be an endothelium-specific and anti-inflammatory therapeutic modality in fibrotic renal disease.
angiopoietin-1; kidney; endothelial cells
Nephrin, an important structural and signal molecule of podocyte slit-diaphragm (SD), has been suggested to contribute to the angiotensin II (Ang II)-induced podocyte injury. Caveolin-1 has been demonstrated to play a crucial role in signaling transduction. In the present study, we evaluated the role of caveolin-1 in Ang II-induced nephrin phosphorylation in podocytes. Wistar rats-receiving either Ang II (400 ng/kg/min) or normal saline (via subcutaneous osmotic mini-pumps, control) were administered either vehicle or telmisartan (3 mg/kg/min) for 14 or 28 days. Blood pressure, 24-hour urinary albumin and serum biochemical profile were measured at the end of the experimental period. Renal histomorphology was evaluated through light and electron microscopy. In vitro, cultured murine podocytes were exposed to Ang II (10−6 M) pretreated with or without losartan (10−5 M) for variable time periods. Nephrin and caveolin-1 expression and their phosphorylation were analyzed by Western-blotting and immunofluorescence. Caveolar membrane fractions were isolated by sucrose density gradient centrifugation, and then the distribution and interactions between Ang II type 1 receptor (AT1), nephrin, C-terminal Src kinase (Csk) and caveolin-1 were evaluated using Western-blotting and co-immunoprecipitation. Podocyte apoptosis was evaluated by cell nucleus staining with Hoechst-33342.
Ang II-receiving rats displayed diminished phosphorylation of nephrin but enhanced glomerular/podocyte injury and proteinuria when compared to control rats. Under control conditions, podocyte displayed expression of caveolin-1 in abundance but only a low level of phospho moiety. Nonetheless, Ang II stimulated caveolin-1 phosphorylation without any change in total protein expression. Nephrin and caveolin-1 were co-localized in caveolae fractions. AT1 receptors and Csk were moved to caveolae fractions and had an interaction with caveolin-1 after the stimulation with Ang II. Transfection of caveolin-1 plasmid (pEGFPC3-cav-1) significantly increased Ang II-induced nephrin dephosphorylation and podocyte apoptosis. Furthermore, knockdown of caveolin-1 expression (using siRNA) inhibited nephrin dephosphorylation and prevented Ang II-induced podocyte apoptosis. These findings indicate that Ang II induces nephrin dephosphorylation and podocyte injury through a caveolin-1-dependent mechanism.
Caveolin-1; Podocyte; Angiotensin II; Nephrin
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
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
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
Strategies that block angiotensin II actions on its angiotensin type 1 receptor or inhibit actions of aldosterone have been shown to reduce myocardial hypertrophy and interstitial fibrosis in states of insulin resistance. Thereby, we sought to determine if combination of direct renin inhibition with angiotensin type 1 receptor blockade in vivo, through greater reductions in systolic blood pressure (SBP) and aldosterone would attenuate left ventricular hypertrophy and interstitial fibrosis to a greater extent than either intervention alone.
We utilized the transgenic Ren2 rat which manifests increased tissue expression of murine renin which, in turn, results in increased renin-angiotensin system activity, aldosterone secretion and insulin resistance. Ren2 rats were treated with aliskiren, valsartan, the combination (aliskiren+valsartan), or vehicle for 21 days.
Compared to Sprague-Dawley controls, Ren2 rats displayed increased systolic blood pressure, elevated serum aldosterone levels, cardiac tissue hypertrophy, interstitial fibrosis and ultrastructural remodeling. These biochemical and functional alterations were accompanied by increases in the NADPH oxidase subunit Nox2 and 3-nitrotyrosine content along with increases in mammalian target of rapamycin and reductions in protein kinase B phosphorylation. Combination therapy contributed to greater reductions in systolic blood pressure and serum aldosterone but did not result in greater improvement in metabolic signaling or markers of oxidative stress, fibrosis or hypertrophy beyond either intervention alone.
Thereby, our data suggest that the greater impact of combination therapy on reductions in aldosterone does not translate into greater reductions in myocardial fibrosis or hypertrophy in this transgenic model of tissue renin overexpression.
Direct Renin Inhibition; Angiotensin II Type 1 receptor; Echocardiography; Ren2 rat
Sex differences exist in the hypertrophic response, cardiac remodeling, and transition to heart failure of hypertensive patients, and while some of these differences are likely influenced by estrogen, the genetic pathways downstream of estrogen that impact on cardioprotection have yet to be fully elucidated. We have previously shown that the cardioprotective effects of adrenomedullin (AM), an emerging clinical biomarker for cardiovascular disease severity, vary with sex in mouse models. AM signaling during cardiovascular stress is strongly modulated by receptor activity-modifying protein 3 (RAMP3) via its interaction with the G protein-coupled receptor calcitonin receptor-like receptor (CLR). Like AM, RAMP3 expression is potently regulated by estrogen, and so we sought to determine the consequences of genetic Ramp3 loss on cardiac adaptation to chronic hypertension, with a particular focus on characterizing potential sex differences. We generated and bred RAMP3−/− mice to RenTgMK mice that consistently display severe angiotensin II-mediated CV disease and compared CV disease progression in RenTgMK to that of RenTgMK:RAMP3−/− offspring. As expected, RAMP3 gene expression was higher in cardiovascular tissues of RenTgMK mice and more strongly up-regulated in female RenTgMK mice relative to wildtype controls. RAMP3 loss did not affect the development of hypertension or the presence and severity of perivascular and interstitial fibrosis in the left ventricle (LV). However, echocardiography revealed that while RenTgMK mice developed concentric cardiac hypertrophy with sustained systolic function, male RenTgMK:RAMP3−/− mice showed evidence of LV chamber dilatation and depressed systolic function, suggestive of cardiac decompensation. Consistent with these measures of heart failure, male RenTgMK:RAMP3−/− mice had increased cardiac apoptosis and elevated activation of Akt. These phenotypes were not present in female RenTgMK:RAMP3−/− mice. Collectively, these data demonstrate a sex-dependant, cardioprotective role of RAMP3 in the setting of chronic hypertension.
adrenomedullin; receptor activity-modifying proteins; cardiovascular disease; sex differences; hypertension; heart failure
Proteinuria is a major marker of the decline of renal function and an important risk factor of coronary heart disease. Elevated proteinuria is associated to the disruption of slit-diaphragm and loss of podocyte foot processes, structural alterations that are considered irreversible. The objective of the present study was to investigate whether proteinuria can be reversed and to identify the structural modifications and the gene/protein regulation associated to this reversal.
We used a novel transgenic strain of mouse (RenTg) that overexpresses renin at a constant high level. At the age of 12-month, RenTg mice showed established lesions typical of chronic renal disease such as peri-vascular and periglomerular inflammation, glomerular ischemia, glomerulosclerosis, mesangial expansion and tubular dilation. Ultrastructural analysis indicated abnormal heterogeneity of basement membrane thickness and disappearance of podocyte foot processes. These structural alterations were accompanied by decreased expressions of proteins specific of podocyte (nephrin, podocin), or tubular epithelial cell (E-cadherin and megalin) integrity. In addition, since TGFβ is considered the major pro-fibrotic agent in renal disease and since exogenous administration of BMP7 is reported to antagonize the TGFβ-induced phenotype changes in kidney, we have screened the expressions of several genes belonging in the TGFβ/BMP superfamily. We found that the endogenous inhibitors of BMPs such as noggin and Usag-1 were several-fold activated inhibiting the action of BMPs and thus reinforcing the deleterious action of TGFβ.Treatment with an AT1 receptor antagonist, at dose that did not decrease arterial pressure, gradually reduced albuminuria. This decrease was accompanied by re-expression of podocin, nephrin, E-cadherin and megalin, and reappearance of podocyte foot processes. In addition, expressions of noggin and Usag-1 were markedly decreased, permitting thus activation of the beneficial action of BMPs.
These findings show that proteinuria and alterations in the expression of proteins involved in the integrity and function of glomerular and renal epithelial phenotype are reversible events when the local action of angiotensin II is blocked, and provide hope that chronic renal disease can be efficiently treated.
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
The cardioprotective effects of estrogen are well recognized, but the mechanisms remain poorly understood. Accumulating evidence suggests that the local cardiac renin-angiotensin system (RAS) is involved in the development and progression of cardiac hypertrophy, remodeling, and heart failure. Estrogen attenuates the effects of an activated circulating RAS; however, its role in regulating the cardiac RAS is unclear. Bilateral oophorectomy (OVX; n = 17) or sham-operation (Sham; n = 13) was performed in 4-week-old, female mRen2.Lewis rats. At 11 weeks of age, the rats were randomized and received either 17 β-estradiol (E2, 36 µg/pellet, 60-day release, n = 8) or vehicle (OVX-V, n = 9) for 4 weeks. The rats were sacrificed, and blood and hearts were used to determine protein and/or gene expression of circulating and tissue RAS components. E2 treatment minimized the rise in circulating angiotensin (Ang) II and aldosterone produced by loss of ovarian estrogens. Chronic E2 also attenuated OVX-associated increases in cardiac Ang II, Ang-(1–7) content, chymase gene expression, and mast cell number. Neither OVX nor OVX+E2 altered cardiac expression or activity of renin, angiotensinogen, angiotensin-converting enzyme (ACE), and Ang II type 1 receptor (AT1R). E2 treatment in OVX rats significantly decreased gene expression of MMP-9, ACE2, and Ang-(1–7) mas receptor, in comparison to sham-operated and OVX littermates. E2 treatment appears to inhibit upsurges in cardiac Ang II expression in the OVX-mRen2 rat, possibly by reducing chymase-dependent Ang II formation. Further studies are warranted to determine whether an E2-mediated reduction in cardiac chymase directly contributes to this response in OVX rats.
We examined the antihypertensive effects of valsartan, aliskiren or both drugs combined on circulating, cardiac and renal components of the renin-angiotensin system (RAS) in congenic mRen2.Lewis hypertensive rats assigned to: vehicle (n=9), valsartan (via drinking water, 30 mg/kg/day; n=10), aliskiren (s.c. by osmotic mini-pumps, 50 mg/kg/day; n=10), or valsartan (30 mg/kg/day) combined with aliskiren (50 mg/kg/day; n=10). Arterial pressure and heart rate were measured by telemetry before and during two weeks of treatment; trunk blood, heart, urine and kidneys were collected for measures of RAS components. Arterial pressure and left ventricular weight/tibia length ratio were reduced by monotherapy of valsartan, aliskiren and further reduced by the combination therapy. Urinary protein excretion was reduced by valsartan and further reduced by the combination. The increases in plasma Ang II induced by valsartan were reversed by the treatment of aliskiren and partially suppressed by the combination. The decreases in plasma Ang-(1–7) induced by aliskiren recovered in the combination group. Kidney Ang-(1–12) was increased by the combination therapy while the increases in urinary creatinine mediated by valsartan were reversed by addition of aliskiren. The antihypertensive and antiproteinuric actions of the combined therapy were associated with marked worsening of renal parenchymal disease and increased peritubular fibrosis. The data show that despite improvements in the surrogate endpoints of blood pressure, ventricular mass and proteinuria, dual blockade of Ang II receptors and renin activity is accompanied by worsening of renal parenchymal disease reflecting a renal homeostatic stress response due to loss of tubuloglomerular feedback by Ang II.
angiotensin-(1–12); aliskiren; arterial remodeling; direct renin inhibitors; urinary protein; valsartan
Podocyte apoptosis is a critical mechanism for excessive loss of urinary albumin that eventuates in kidney fibrosis. Pharmacological doses of the mammalian target of rapamycin (mTOR) inhibitor rapamycin reduce albuminuria in diabetes. We explored the hypothesis that mTOR mediates podocyte injury in diabetes. High glucose (HG) induces apoptosis of podocytes, inhibits AMP-activated protein kinase (AMPK) activation, inactivates tuberin, and activates mTOR. HG also increases the levels of Nox4 and Nox1 and NADPH oxidase activity. Inhibition of mTOR by low-dose rapamycin decreases HG-induced Nox4 and Nox1, NADPH oxidase activity, and podocyte apoptosis. Inhibition of mTOR had no effect on AMPK or tuberin phosphorylation, indicating that mTOR is downstream of these signaling molecules. In isolated glomeruli of OVE26 mice, there is a similar decrease in the activation of AMPK and tuberin and activation of mTOR with increase in Nox4 and NADPH oxidase activity. Inhibition of mTOR by a small dose of rapamycin reduces podocyte apoptosis and attenuates glomerular injury and albuminuria. Our data provide evidence for a novel function of mTOR in Nox4-derived reactive oxygen species generation and podocyte apoptosis that contributes to urinary albumin excretion in type 1 diabetes. Thus, mTOR and/or NADPH oxidase inhibition may represent a therapeutic modality of diabetic kidney disease.
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
Recent studies have described that the Notch signaling pathway is activated in a wide range of renal diseases. Angiotensin II (AngII) plays a key role in the progression of kidney diseases. AngII contributes to renal fibrosis by upregulation of profibrotic factors, induction of epithelial mesenchymal transition and accumulation of extracellular matrix proteins. In cultured human tubular epithelial cells the Notch activation by transforming growth factor-β1 (TGF-β1) has been involved in epithelial mesenchymal transition. AngII mimics many profibrotic actions of TGF-β1. For these reasons, our aim was to investigate whether AngII could regulate the Notch/Jagged system in the kidney, and its potential role in AngII-induced responses. In cultured human tubular epithelial cells, TGF-β1, but not AngII, increased the Notch pathway-related gene expression, Jagged-1 synthesis, and caused nuclear translocation of the activated Notch. In podocytes and renal fibroblasts, AngII did not modulate the Notch pathway. In tubular epithelial cells, pharmacological Notch inhibition did not modify AngII-induced changes in epithelial mesenchymal markers, profibrotic factors and extracellular matrix proteins. Systemic infusion of AngII into rats for 2 weeks caused tubulointerstitial fibrosis, but did not upregulate renal expression of activated Notch-1 or Jagged-1, as observed in spontaneously hypertensive rats. Moreover, the Notch/Jagged system was not modulated by AngII type I receptor blockade in the model of unilateral ureteral obstruction in mice. These data clearly indicate that AngII does not regulate the Notch/Jagged signaling system in the kidney, in vivo and in vitro. Our findings showing that the Notch pathway is not involved in AngII-induced fibrosis could provide important information to understand the complex role of Notch system in the regulation of renal regeneration vs damage progression.
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
Currently available rodent models exhibit characteristics of early diabetic nephropathy (DN) such as hyperfiltration, mesangial expansion, and albuminuria yet features of late DN (hypertension, GFR decline, tubulointerstitial fibrosis) are absent or require a significant time investment for full phenotype development. Accordingly, the aim of the present study was to develop a mouse model of advanced DN with hypertension superimposed (HD mice). Mice transgenic for human renin cDNA under the control of the transthyretin promoter (TTRhRen) were employed as a model of angiotensin-dependent hypertension. Diabetes was induced in TTRhRen mice through low dose streptozotocin (HD-STZ mice) or by intercrossing with OVE26 diabetic mice (HD-OVE mice). Both HD-STZ and HD-OVE mice displayed more pronounced increases in urinary albumin levels as compared with their diabetic littermates. Additionally, HD mice displayed renal hypertrophy, advanced glomerular scarring and evidence of tubulointerstitial fibrosis. Both HD-OVE and HD-STZ mice showed evidence of GFR decline as FITC-inulin clearance was decreased compared to hyperfiltering STZ and OVE mice. Taken together our results suggest that HD mice represent a robust model of type I DN that recapitulates key features of human disease which may be significant in studying the pathogenesis of DN and in the assessment of putative therapeutics.
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
Background. In glomerulonephritis (GN), an overload of free fatty acids (FFA) bound to albumin in urinary protein may induce oxidative stress in the proximal tubules. Human liver-type fatty acid-binding protein (hL-FABP) expressed in human proximal tubules, but not rodents, participates in intracellular FFA metabolism and exerts anti-oxidative effects on the progression of tubulointerstitial damage. We examined whether tubular enhancement of this anti-oxidative action modulates the progression of glomerular damage in immune-mediated GN in hL-FABP chromosomal gene transgenic (Tg) mice.
Methods. Anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN) was induced in Tg and wild-type mice (WT). Proteinuria, histopathology, polymorphonuclear (PMN) influx, expression of tubulointerstitial markers for oxidative stress 4-hydroxy-2-Nonenal (HNE) and fibrosis (α-smooth muscle actin), proximal tubular damage (Kim-1), Peroxisome Proliferator-Activated Receptor γ (PPAR γ) and inflammatory cytokines [Monocyte Chemotactic Protein-1, tumor necrosis factor-alpha (TNF-α) and Transforming growth factor beta (TGF-β)] were analyzed. The mice were also treated with an angiotensin type II receptor blocker (ARB).
Results. The urinary protein level in Tg mice decreased significantly during the acute phase (∼Day 5). Tg mice survived for a significantly longer time than WT mice, with an attenuation of tubulointerstitial damage score and expression of each tubulointerstitial damage marker observed at Day 7. Expression of inflammatory cytokines on Day 7 was higher in WT mice than Tg mice and correlated strongly with PPARγ expression in WT mice, but not in Tg mice. Interestingly, Tg mice showed insufficient PMN influx at 3 and 6 h, with simultaneous elevation of urinary L-FABP and reduction in HNE expression. The two strains of mice showed different types of glomerular damage, with mild mesangial proliferation in Tg mice and severe endothelial swelling with vascular thrombosis in WT mice. The glomerular damage in Tg mice was improved by administration of an ARB.
Conclusions. The present experimental model suggests that tubular enhancement of L-FABP may protect mice with anti-GBM GN from progression of both tubulointerstitial and glomerular injury.
L-FABP; oxidative stress; tubulointerstitial damage