Produção Nacional
Revisado por pares
Genetic deletion of the angiotensin-(1–7) receptor Mas leads to glomerular hyperfiltration and microalbuminuria
2009; Elsevier BV; Volume: 75; Issue: 11 Linguagem: Inglês
10.1038/ki.2009.61
ISSN1523-1755
AutoresSérgio Veloso Brant Pinheiro, Anderson J. Ferreira, Gregory T. Kitten, Kátia D. Silveira, Deivid A da Silva, Sérgio Henrique Sousa Santos, Elisandra Gava, Carlos H. Castro, Júnio A Magalhães, Renata K da Mota, Giancarla Aparecida Botelho-Santos, Michael Bäder, Natália Alenina, Robson A.S. Santos, Ana Cristina Simões e Silva,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoAngiotensin-(1–7), an active fragment of both angiotensins I and II, generally opposes the vascular and proliferative actions of angiotensin II. Here we evaluated effects of the angiotensin-(1–7) receptor Mas on renal physiology and morphology using Mas-knockout mice. Compared to the wild-type animals, Mas knockout mice had significant reductions in urine volume and fractional sodium excretion without any significant change in free-water clearance. A significantly higher inulin clearance and microalbuminuria concomitant with a reduced renal blood flow suggest that glomerular hyperfiltration occurs in the knockout mice. Histological analysis found reduced glomerular tuft diameter and increased expression of collagen IV and fibronectin in the both the mesangium and interstitium, along with increased collagen III in the interstitium. These fibrogenic changes and the renal dysfunction of the knockout mice were associated with an upregulation of angiotensin II AT1 receptor and transforming growth factor-β mRNA. Our study suggests that Mas acts as a critical regulator of renal fibrogenesis by controlling effects transduced through angiotensin II AT1 receptors in the kidney. Angiotensin-(1–7), an active fragment of both angiotensins I and II, generally opposes the vascular and proliferative actions of angiotensin II. Here we evaluated effects of the angiotensin-(1–7) receptor Mas on renal physiology and morphology using Mas-knockout mice. Compared to the wild-type animals, Mas knockout mice had significant reductions in urine volume and fractional sodium excretion without any significant change in free-water clearance. A significantly higher inulin clearance and microalbuminuria concomitant with a reduced renal blood flow suggest that glomerular hyperfiltration occurs in the knockout mice. Histological analysis found reduced glomerular tuft diameter and increased expression of collagen IV and fibronectin in the both the mesangium and interstitium, along with increased collagen III in the interstitium. These fibrogenic changes and the renal dysfunction of the knockout mice were associated with an upregulation of angiotensin II AT1 receptor and transforming growth factor-β mRNA. Our study suggests that Mas acts as a critical regulator of renal fibrogenesis by controlling effects transduced through angiotensin II AT1 receptors in the kidney. The renin–angiotensin system (RAS) is classically conceived as a coordinated hormonal cascade in the control of cardiovascular, renal, and adrenal functions, mainly through the actions of Angiotensin (Ang)II.1.Zaman M.A. Oparil S. Calhoun D.A. Drugs targeting the renin angiotensin aldosterone system.Nat Rev Drug Discov. 2002; 1: 621-636Crossref PubMed Scopus (319) Google Scholar Recent advances in cell and molecular biology have led to the recognition of other active fragments of RAS metabolism, such as Ang III, Ang IV, and Ang-(1–7),2.Ferrario C.M. Chappell M.C. Novel angiotensin peptides.Cell Mol Life Sci. 2004; 61: 2720-2727Crossref PubMed Scopus (101) Google Scholar, 3.Simões e Silva A.C. Pinheiro S.V.B. Pereira R.M. et al.The therapeutic potential of Angiotensin-(1–7) as a novel renin angiotensin system mediator.Mini-Rev Med Chem. 2006; 6: 603-609Crossref PubMed Scopus (33) Google Scholar, 4.Santos R.A.S. Ferreira A.J. Simões e Silva A.C. Recent advances in the angiotensin-converting enzyme 2-angiotensin(1–7)-Mas axis.Exp Physiol. 2008; 93: 519-527Crossref PubMed Scopus (350) Google Scholar the Ang IV insulin-regulated aminopeptidase-binding site,5.Albiston A.L. McDowall S.G. Matsacos D. et al.Evidence that the angiotensin IV (AT(4)) receptor is the enzyme insulin-regulated aminopeptidase.J Biol Chem. 2001; 276: 48623-48626Crossref PubMed Scopus (389) Google Scholar the angiotensin-converting enzyme (ACE)2, a homolog of classic ACE that forms Ang-(1–7) directly from Ang II and indirectly from Ang I,6.Donoghue M. Hsieh F. Baronas E. et al.A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin-(1–9).Circ Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 7.Tipnis S.R. Hooper N.M. Hyde R. et al.A human homolog of Angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase.J Biol Chem. 2000; 275: 33238-33243Crossref PubMed Scopus (1408) Google Scholar and the G-protein-coupled receptor Mas.8.Santos R.A.S. Simões e Silva A.C. Maric C. et al.Angiotensin-(1–7) is an endogenous ligand for the G-protein coupled receptor Mas.Proc Natl Acad Sci USA. 2003; 100: 8258-8263Crossref PubMed Scopus (1229) Google Scholar In general, Ang-(1–7) opposes the vascular and proliferative effects of Ang II and exerts complex renal actions in chronic renal diseases and hypertension.3.Simões e Silva A.C. Pinheiro S.V.B. Pereira R.M. et al.The therapeutic potential of Angiotensin-(1–7) as a novel renin angiotensin system mediator.Mini-Rev Med Chem. 2006; 6: 603-609Crossref PubMed Scopus (33) Google Scholar, 9.Simões e Silva A.C. Diniz J.S. Regueira-Filho A. et al.The Renin Angiotensin System in childhood hypertension: Selective increase of Angiotensin-(1–7) in essential hypertension.J Pediatr. 2004; 145: 93-98Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 10.Simões e Silva A.C. Diniz J.S. Pereira R.M. et al.Circulating Renin Angiotensin System in childhood chronic renal failure: Marked increase of Angiotensin-(1–7) in end-stage renal disease.Pediatr Res. 2006; 60: 734-739Crossref PubMed Scopus (42) Google Scholar Ang-(1–7) is formed from Ang II by prolylendopeptidase, prolyl-carboxypeptidase or ACE2, or directly from Ang I through hydrolysis by prolylendopeptidase and endopeptidase 24.11 and is metabolized by ACE to Ang-(1–5).6.Donoghue M. Hsieh F. Baronas E. et al.A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin-(1–9).Circ Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 7.Tipnis S.R. Hooper N.M. Hyde R. et al.A human homolog of Angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase.J Biol Chem. 2000; 275: 33238-33243Crossref PubMed Scopus (1408) Google Scholar ACE inhibitors elevate Ang-(1–7) concentrations by both increasing Ang I, a substrate for Ang-(1–7) generation, and preventing Ang-(1–7) degradation.2.Ferrario C.M. Chappell M.C. Novel angiotensin peptides.Cell Mol Life Sci. 2004; 61: 2720-2727Crossref PubMed Scopus (101) Google Scholar, 3.Simões e Silva A.C. Pinheiro S.V.B. Pereira R.M. et al.The therapeutic potential of Angiotensin-(1–7) as a novel renin angiotensin system mediator.Mini-Rev Med Chem. 2006; 6: 603-609Crossref PubMed Scopus (33) Google Scholar, 4.Santos R.A.S. Ferreira A.J. Simões e Silva A.C. Recent advances in the angiotensin-converting enzyme 2-angiotensin(1–7)-Mas axis.Exp Physiol. 2008; 93: 519-527Crossref PubMed Scopus (350) Google Scholar Recent studies suggest that, at least in part, the beneficial effects of ACE inhibitors11.Maia L.G. Ramos M.C. Fernandes L. et al.Angiotensin-(1–7) antagonist A-779 attenuates the potentiation of bradykinin by captopril in rats.J Cardiovasc Pharmacol. 2004; 43: 685-691Crossref PubMed Scopus (39) Google Scholar, 12.Höcht C. Gironacci M.M. Mayer M.A. et al.Involvement of angiotensin-(1–7) in the hypothalamic hypotensive effect of captopril in sinoaortic denervated rats.Regul Pept. 2008; 146: 58-66Crossref PubMed Scopus (26) Google Scholar and AT1 receptor blockade13.Ishiyama Y. Gallagher P.E. Averill D.B. et al.Upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin II receptors.Hypertension. 2004; 43: 970-976Crossref PubMed Scopus (408) Google Scholar may be attributed to Ang-(1–7). These findings are in keeping with the hypothesis that the RAS is capable of self-regulating its activity through the formation of Ang-(1–7).2.Ferrario C.M. Chappell M.C. Novel angiotensin peptides.Cell Mol Life Sci. 2004; 61: 2720-2727Crossref PubMed Scopus (101) Google Scholar, 3.Simões e Silva A.C. Pinheiro S.V.B. Pereira R.M. et al.The therapeutic potential of Angiotensin-(1–7) as a novel renin angiotensin system mediator.Mini-Rev Med Chem. 2006; 6: 603-609Crossref PubMed Scopus (33) Google Scholar, 4.Santos R.A.S. Ferreira A.J. Simões e Silva A.C. Recent advances in the angiotensin-converting enzyme 2-angiotensin(1–7)-Mas axis.Exp Physiol. 2008; 93: 519-527Crossref PubMed Scopus (350) Google Scholar Furthermore, Kostenis et al14.Kostenis E. Milligan G. Christopoulos A. et al.G-protein-coupled receptor Mas is a physiological antagonist of the angiotensin II type 1 receptor.Circulation. 2005; 111: 1806-1813Crossref PubMed Scopus (303) Google Scholar showed that receptor Mas can hetero-oligomerize with AT1 and, by so doing, inhibit the actions of Ang II. Indeed, Mas acts in vitro as an antagonist of the AT1 receptor.15.Canals M. Jenkins L. Kellett E. et al.Up-regulation of the angiotensin II type 1 receptor by the Mas proto-oncogene is due to constitutive activation of Gq/G11 by Mas.J Biol Chem. 2006; 281: 16757-16767Crossref PubMed Scopus (70) Google Scholar Our group showed that deletion of Mas produces an impairment in cardiac function associated with a significant increase in collagen I, III, and fibronectin in the heart.16.Santos R.A.S. Castro C.H. Gava E. et al.Impairment of in vitro and in vivo heart function in angiotensin-(1–7) receptor Mas knockout mice.Hypertension. 2006; 47: 996-1002Crossref PubMed Scopus (177) Google Scholar The aim of this study was to evaluate the role of Mas in kidney structure and function using mice with genetic deletion of this receptor. As the results displayed in Table 1 the 24-h urine flow of Mas−/− animals was significantly lower in comparison with that of Mas+/+. However, no differences in blood glucose levels and in water and food intake were observed. The reduced urinary volume observed in Mas−/− mice was accompanied by a significant increase in urinary osmolality and a decrease in the fractional excretion of sodium (P<0.05, Table 1). Despite the increase in urinary osmolality, plasma osmolality, osmolal, and free-water clearance were unchanged in Mas−/− animals when compared with those in Mas+/+ mice. No changes were detected in the fractional excretion of potassium either. Mean arterial pressure (MAP) in Mas−/− mice was also undistinguished from that in Mas +/+ animals. However, significant reductions of renal blood flow accompanied by increased total renal vascular resistance were detected in knockout mice. Despite the reduced renal blood flow, Mas−/− animals presented a significant elevation in the glomerular filtration rate (GFR), estimated by the creatinine and inulin clearances. This finding was made along with an elevated urinary albumin excretion in Mas−/− mice (P<0.05, Table 1).Table 1General measurements and renal function parameters in Mas+/+ and Mas−/− miceParametersMas+/+Mas−/−Body weight (g)25.7±0.525.7±0.6Blood glucose (mg per 100 ml)116.8±3.9111.8±3.2Daily food intake (g)5.18±0.255.69±0.27Daily water intake (ml)6.37±0.345.90±0.32Daily urine volume (ml)1.65±0.191.07±0.14*P<0.05. Data were analyzed by unpaired Student's t-test.Mean arterial pressure (mm Hg)80.22±1.7878.75±2.23RBF (ml/min per g)19.16±4.0910.76±1.39*P<0.05. Data were analyzed by unpaired Student's t-test.RVR (mm Hg ml/min per g)5.39±1.208.25±1.07*P<0.05. Data were analyzed by unpaired Student's t-test.Creatinine clearance (μl/min per g)1.80±0.393.20±0.50*P<0.05. Data were analyzed by unpaired Student's t-test.Inulin clearance (μl/min per g)2.54±0.494.61±0.74*P<0.05. Data were analyzed by unpaired Student's t-test.Microalbuminuria (mg per 24 h)0.03±0.010.20±0.07*P<0.05. Data were analyzed by unpaired Student's t-test.Urine osmolality (mOsm/kg H2O)3245±1964030±330*P<0.05. Data were analyzed by unpaired Student's t-test.Serum osmolality (mOsm/kg H2O)275±7273±5Osmolal clearance (μl/min)10.2±0.89.1±0.8Free water clearance (μl/min)-9.2±0.7-8.4±0.7Serum sodium (mmol/l)149±2147±2Sodium FE (%)1.05±0.140.38±0.05*P<0.05. Data were analyzed by unpaired Student's t-test.Serum potassium (mmol/l)4.8±0.45.1±0.4Potassium FE (%)15.5±3.515.3±5.6FE, fractional excretion; RBF, renal blood flow; RVR, renal vascular resistance. Values are mean±s.e.m.* P<0.05. Data were analyzed by unpaired Student's t-test. Open table in a new tab FE, fractional excretion; RBF, renal blood flow; RVR, renal vascular resistance. Values are mean±s.e.m. The wet weights of kidneys from Mas+/+ and Mas−/− animals normalized for body weight (BW) were similar (Table 2). However, microscopic changes in the kidney structure of Mas−/− mice were observed, including a significant reduction in the diameters of the glomerular tuft and Bowman's capsule in comparison with those in Mas+/+ animals (Figure 1 and Table 2). Bowman's spaces and the diameter of the cortical tubule were unaltered in Mas−/− animals when compared with those in Mas+/+ mice.Table 2Morphological parameters in Mas+/+ and Mas−/− miceParametersMas+/+Mas−/−Kidney index (mg/g of body weight)6.0 (5.6–6.4)5.9 (5.5–6.3)Cortical tubule diameter (μm)32.5 (30.0–37.5)32.5 (30–37.5)Bowman's capsule diameter (μm)62.5 (57.5–70.0)57.5 (52.5–62.5)*P<0.05. Data were analyzed by the Mann–Whitney test.Glomerulus tuft diameter (μm)57.5 (52.5–62.5)50.0 (47.5–55.0)*P<0.05. Data were analyzed by the Mann–Whitney test.Bowman's space diameter (μm)6.0 (5.7–6.3)6.1 (5.8–6.3)Values are medians (25 and 75% percentile)* P<0.05. Data were analyzed by the Mann–Whitney test. Open table in a new tab Values are medians (25 and 75% percentile) Renal tissue analysis by confocal microscopy revealed that the fluorescence of many extracellular matrix proteins was significantly higher in the cortex and medulla of Mas−/− kidneys when compared with those in Mas+/+(Figures 2 and 3). Mas−/− kidneys exhibited a significant increase in collagen III in the cortex (9.08±1.63 versus 1.83±0.77 arbitrary units in Mas+/+ mice, P<0.05, Figure 2) as well as in the medulla (3.60±0.79 versus 0.26±0.05 arbitrary units in Mas+/+ mice, P<0.05, Figure 3). Similar results were obtained with collagen IV and fibronectin. Kidneys from Mas−/− animals presented higher levels of both extracellular matrix proteins than did Mas+/+ kidneys in the renal cortex (collagen IV: 8.49±0.61 versus 6.49±0.43 arbitrary units and fibronectin: 11.22±2.40 versus 4.07±0.47 arbitrary units, P<0.05 for both comparisons, Figure 2) and medulla (collagen IV: 18.98±1.11 versus 10.70±0.84 arbitrary units and fibronectin: 21.56±2.71 versus 11.04±2.24 arbitrary units, P<0.05 for both comparisons, Figure 3). However, no differences in the expression of collagen I in the cortex and medulla were observed in the kidneys from Mas−/− and Mas+/+ animals (Figures 2 and 3). In the mesangium, Mas−/− kidneys exhibited a selective increase in collagen IV (13.25±0.99 versus 10.07±0.60 arbitrary units in Mas+/+ mice, P<0.05) and fibronectin (29.43±4.17 versus 14.22±2.66 arbitrary units in Mas+/+ mice, P<0.05). In contrast, collagen types I and III exhibited a similar pattern in the mesangium in Mas−/− and Mas+/+ kidneys (Figure 4). As shown in Figure 5, results obtained with immunoblotting confirmed the increased levels of fibronectin and collagen types III and IV in Mas−/− animals.Figure 3Immunofluorescence of extracellular matrix (ECM) proteins in the medulla of kidneys from Mas+/+ (left column) and Mas−/− (right column) mice. (a) Fluorescence (Cy3-labeled anti-rabbit IgG) reveals the immunolabeling of ECM proteins. Expression of type III collagen (Col III), type IV collagen (Col IV), and fibronectin (Fn) were increased in the medulla of Mas−/− compared with that of Mas+/+ mice, whereas the expression of type I collagen (Col I) was unaltered. (b) Quantification of ECM proteins in the medulla of Mas+/+ and Mas−/− mice. Data are shown as mean±s.e.m. *P<0.05; **P<0.01. A.U. indicates arbitrary unit.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Immunofluorescence of extracellular matrix (ECM) proteins in the mesangium of kidneys from Mas+/+ (left column) and Mas−/− (right column) mice. (a) Fluorescence (Cy3-labeled anti-rabbit IgG) reveals the immunolabeling of ECM proteins. Expression of type IV collagen (Col IV) and fibronectin (Fn) was increased in the mesangium of Mas−/− compared with Mas+/+ mice, whereas the expression of type I collagen (Col I) and type III collagen (Col III) was unaltered. (b) Quantification of ECM proteins in the mesangium of Mas+/+ and Mas−/− mice. Data are shown as the mean±s.e.m. *P<0.05; *P<0.01. A.U. indicates arbitrary unit.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Immunoblotting of extracellular matrix (ECM) proteins in kidneys of Mas+/+ and Mas−/− animals. (a) Immunoblotting shows no difference of Collagen I expression in Mas+/+ and Mas−/− mice kidneys. Significant increases in (b) Collagen III, (c) Collagen IV, and (d) fibronectin expression were detected by comparing immunoblots of Mas−/− mouse kidneys with those of Mas+/+ controls. Each band represents one mouse kidney from either Mas+/+ or Mas−/− mice. Data are shown as the mean±s.e.m. *P<0.05. A.U. indicates arbitrary unit.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the distribution of Mas in the kidneys, confocal immunofluorescence analysis of renal tissue from Mas+/+ mice was performed. Mas immunolabeling was detected in different segments of the nephron, such as the juxtaglomerular apparatus, proximal cortical tubules, and collecting ducts (Figure 6). As observed in Figure 7, real-time PCR revealed a threefold increase in RNA expression of AT1 receptor and a twofold elevation of total transforming growth factor-β (TGF-β) RNA in kidneys from Mas−/− mice compared with those in Mas +/+ mice (2.65±0.29 versus 0.85±0.19 relative expression for AT1 RNA and 1.75±0.23 versus 0.85±0.17 relative expression for TGF-β RNA, P<0.05 for both comparisons). Genetic deletion of the Ang-(1–7) receptor Mas produces an extremely rich phenotype that includes cardiac dysfunction,16.Santos R.A.S. Castro C.H. Gava E. et al.Impairment of in vitro and in vivo heart function in angiotensin-(1–7) receptor Mas knockout mice.Hypertension. 2006; 47: 996-1002Crossref PubMed Scopus (177) Google Scholar decreased baroreflex function, endothelial dysfunction,17.Xu P. Costa-Goncalves A.C. Todiras M. et al.Endothelial dysfunction and elevated blood pressure in Mas gene-deleted mice.Hypertension. 2008; 51: 574-580Crossref PubMed Scopus (151) Google Scholar reduced reproductive function,18.Costa Gonçalves A.C. Leite R. Fraga-Silva R.A. et al.Evidence that the vasodilator angiotensin-(1–7)-Mas axis plays an important role in erectile function.Am J Physiol Heart Circ Physiol. 2007; 293: H2588-H2596Crossref PubMed Scopus (44) Google Scholar increased thrombogenesis,19.Fraga-Silva R.A. Pinheiro S.V. Gonçalves A.C. et al.The antithrombotic effect of angiotensin-(1–7) involves Mas-mediated NO release from platelets.Mol Med. 2008; 14: 28-35Crossref PubMed Scopus (99) Google Scholar and, depending on the genetic background, increased blood pressure and marked changes in lipid and glycidic metabolism, leading to a metabolic syndrome-like state.17.Xu P. Costa-Goncalves A.C. Todiras M. et al.Endothelial dysfunction and elevated blood pressure in Mas gene-deleted mice.Hypertension. 2008; 51: 574-580Crossref PubMed Scopus (151) Google Scholar, 20.Santos S.H. Fernandes L.R. Mario E.G. et al.Mas deficiency in FVB/N mice produces marked changes in lipid and glycemic metabolism.Diabetes. 2008; 57: 340-347Crossref PubMed Scopus (190) Google Scholar Phenotypic differences have been reported according to genetic background. This is particularly true for blood pressure and plasma glucose, which are increased in FVB/N Mas−/−17.Xu P. Costa-Goncalves A.C. Todiras M. et al.Endothelial dysfunction and elevated blood pressure in Mas gene-deleted mice.Hypertension. 2008; 51: 574-580Crossref PubMed Scopus (151) Google Scholar, 20.Santos S.H. Fernandes L.R. Mario E.G. et al.Mas deficiency in FVB/N mice produces marked changes in lipid and glycemic metabolism.Diabetes. 2008; 57: 340-347Crossref PubMed Scopus (190) Google Scholar and are normal in C57BL/6 mice, as shown in this study. These differences could be related to the fact that C57BL/6 presents only one renin gene compared with two genes of other mice strains, including FVB/N.21.Hansen P.B. Yang T. Huang Y. et al.Plasma renin in mice with one or two renin genes.Acta Physiol Scand. 2004; 181: 431-437Crossref PubMed Scopus (22) Google Scholar However, despite the potentially lower sensitivity to the genetic deletion of Mas in C57BL/6 mice, we showed that Mas is critical for the regulation of renal homeostasis even in young animals. Our data suggest that the genetic deletion of Mas probably leads to glomerular hypertension, and also produces structural and molecular changes, which stimulates renal fibrosis. Furthermore, the observed changes were not related to arterial hypertension or to hyperglycemia. The ability of the kidney to generate high concentrations of Ang II and Ang-(1–7) allows it to regulate intrarenal levels of these angiotensins in accordance with homeostatic needs for the regulation of renal hemodynamics.22.Navar L.G. Nishiyama A. Why are angiotensin concentrations so high in the kidney?.Curr Opin Nephrol Hypertens. 2004; 13: 107-115Crossref PubMed Scopus (93) Google Scholar, 23.Brewster U.C. Perazella M.A. The renin-angiotensin-aldosterone system and the kidney: effects on kidney disease.Am J Med. 2004; 116: 263-272Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar In this context, Mas-deficient mice presented significant changes in renal hemodynamic and in glomerular filtration. When compared with wild-type mice, Mas−/− animals had lower renal blood flow accompanied by elevated renal vascular resistance. Both alterations occurred along with a 1.8-fold increase in GFR according to creatinine as well as inulin clearance measurements, and a 6.7-fold increase in urinary albumin excretion. As blood pressure was similar in Mas−/− and in wild-type animals, the increased GFR in conjunction with reduced renal blood flow was probably because of elevated vascular tonus in the efferent arterioles. Ang II markedly raises efferent glomerular arteriolar resistance but does not change afferent arteriolar resistance unless the renal perfusion pressure rises.24.Arendshorst W.J. Brannstrom K. Ruan X. Actions of angiotensin II on the renal microvasculature.J Am Soc Nephrol. 1999: S149-S161Google Scholar The consequence of the disproportionate increase in efferent (over afferent) resistance is a marked increase in intraglomerular pressure. Thus, the Ang II-induced decrease in renal plasma flow is offset by the increase in mean transcapillary ultrafiltration pressure and this maintains or even increases the GFR.22.Navar L.G. Nishiyama A. Why are angiotensin concentrations so high in the kidney?.Curr Opin Nephrol Hypertens. 2004; 13: 107-115Crossref PubMed Scopus (93) Google Scholar, 23.Brewster U.C. Perazella M.A. The renin-angiotensin-aldosterone system and the kidney: effects on kidney disease.Am J Med. 2004; 116: 263-272Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 24.Arendshorst W.J. Brannstrom K. Ruan X. Actions of angiotensin II on the renal microvasculature.J Am Soc Nephrol. 1999: S149-S161Google Scholar, 25.Hall J.E. Brands M.W. Henegar J.R. Angiotensin II and long-term arterial pressure regulation: the overriding dominance of the kidney.J Am Soc Nephrol. 1999: S258-S265Google Scholar Ang-(1–7) directly and indirectly vasodilates afferent arterioles and increases renal blood flow.26.Ren Y. Garvin J.L. Carretero A.O. Vasodilator action of angiotensin-(1–7) on isolated rabbit afferent arterioles.Hypertension. 2002; 39: 799-802Crossref PubMed Scopus (165) Google Scholar, 27.Sampaio W.O. Nascimento A.A. Santos R.A.S. Systemic and regional hemodynamics effects of angiotensin-(1–7) in rats.Am J Physiol. 2003; 284: H1985-H1994PubMed Google Scholar, 28.Gironacci M.M. Adler-Graschinsky E. Pena C. et al.Effects of angiotensin II and angiotensin-(1–7) on the release of [3H]norepinephrine from rat atria.Hypertension. 1994; 24: 457-460Crossref PubMed Scopus (74) Google Scholar Although the relative role of each angiotensin on glomerular hemodynamics is still unknown, our results indicated that Ang-(1–7) may act as a physiological regulator of intraglomerular pressure by opposing the glomerular hypertensive effects of Ang II. Therefore, without the physiological antagonism of Ang-(1–7), intrarenal and/or plasma Ang II, acting on higher expressed AT1 receptors, might increase efferent arteriolar resistance and glomerular filtration pressure, thus contributing to glomerular capillary hypertension. Mas-knockout animals also exhibited lower urinary volume accompanied by sodium retention without BW gain when compared with controls. As young Mas−/− mice (9–10 weeks of age) were used, it would be possible that the persistence of these alterations could lead to edema and BW elevations in older animals. Moreover, no changes in serum osmolality, osmolal, or free-water clearance were observed, indicating that fluid retention reflects a direct effect on sodium handling without significant changes in water transport.29.Frokiaer J. Nielsen S. Knepper M.A. Molecular physiology of renal aquaporins and sodium transporters: Exciting approaches to understand regulation of renal water handling.J Am Soc Nephrol. 2005; 16: 2827-2829Crossref PubMed Scopus (6) Google Scholar These alterations are in agreement with the tubular effects of Ang II.25.Hall J.E. Brands M.W. Henegar J.R. Angiotensin II and long-term arterial pressure regulation: the overriding dominance of the kidney.J Am Soc Nephrol. 1999: S258-S265Google Scholar Through efferent arteriolar vasoconstriction, Ang II causes changes in peritubular capillary dynamics that could increase renal tubular fluid reabsorption.24.Arendshorst W.J. Brannstrom K. Ruan X. Actions of angiotensin II on the renal microvasculature.J Am Soc Nephrol. 1999: S149-S161Google Scholar, 25.Hall J.E. Brands M.W. Henegar J.R. Angiotensin II and long-term arterial pressure regulation: the overriding dominance of the kidney.J Am Soc Nephrol. 1999: S258-S265Google Scholar In addition, Ang II significantly stimulates proximal and distal sodium reabsorption through the activation of AT1 receptors.22.Navar L.G. Nishiyama A. Why are angiotensin concentrations so high in the kidney?.Curr Opin Nephrol Hypertens. 2004; 13: 107-115Crossref PubMed Scopus (93) Google Scholar, 23.Brewster U.C. Perazella M.A. The renin-angiotensin-aldosterone system and the kidney: effects on kidney disease.Am J Med. 2004; 116: 263-272Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar Although further experiments with AT1 blockade would be helpful, our findings suggest that ACE–Ang II–AT1 axis could influence the direction of renal tubular changes detected in Mas−/− mice. The major morphological alterations found in kidneys from young Mas−/− animals were a reduction in glomerular tuft diameter and an increased expression of collagen III, IV, and fibronectin in the kidney interstitium as well as of collagen IV and fibronectin in the mesangium. Despite the presence of matrix proteins deposition in kidneys of Mas−/− mice, we did not observe the classical findings of glomerular hypertension, such as glomerular hyperthrophy. However, as this study reflected only a time point after Mas deletion (9- to 10-week-old mice), we were not able to show dynamic changes in glomerular size. Glomerular hypertrophy may not be evident in young animals. Similarly, the kidneys of young (3-month-old) mice with the deletion of ACE2 gene showed no gross abnormalities having normal architecture of the cortex and medulla, comparable with those of age-matched wild-type mice. However, electron microscopy of these mice evidenced mesangial injury with small foci of collagen deposition suggestive of an early disease process.30.Oudit G.Y. Herzenberg A.M. Kassiri Z. et al.Loss of angiotensin-converting enzyme-2 leads to the late development of angiotensin II-dependent glomerulosclerosis.Am J Pathol. 2006; 168: 1808-1820Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar We also cannot rule out the possibility that the genetic deletion of Mas could interfere with fetal glomerular development by producing smaller glomerulus than that of wild-type animals. Consequently, even if glomerular hypertrophy has occurred in Mas−/− mice, it might not be detected. Further studies are obviously necessary to clarify the time course of histological glomerular changes in Mas−/− animals. In addition, changes in glomerular permeability, not evaluated in this study, may be also involved in the glomerular dysfunction present in Mas−/− mice. Our earlier studies have indicated a role for the Ang-(1–7)–Mas interaction in the regulation of matrix proteins deposition in the heart and liver.16.Santos R.A.S. Castro C.H. Gava E. et al.Impairment of in vitro and in vivo heart function in angiotensin-(1–7) receptor Mas knockout mice.Hypertension. 2006; 47: 996-1002Crossref PubMed Scopus (177) Google Sch
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