RAS blockade decreases blood pressure and proteinuria in transgenic mice overexpressing rat angiotensinogen gene in the kidney
2006; Elsevier BV; Volume: 69; Issue: 6 Linguagem: Inglês
10.1038/sj.ki.5000210
ISSN1523-1755
AutoresSébastien Sachetelli, Qingxue Liu, S.-L. Zhang, F. Liu, Tusty‐Jiuan Hsieh, M.-L. Brezniceanu, Deng‐Fu Guo, János G. Filep, Julie R. Ingelfinger, Curt D. Sigmund, Pavel Hamet, John S.D. Chan,
Tópico(s)Receptor Mechanisms and Signaling
ResumoAngiotensinogen (ANG) is the sole substrate of the renin–angiotensin system (RAS). Clinical studies have shown that RAS activation may lead to hypertension, a major cardiovascular and renal risk factor. To delineate the underlying mechanisms of hypertension-induced nephropathy, we generated transgenic mice that overexpress rat ANG (rANG) in the kidney to establish whether intrarenal RAS activation alone can evoke hypertension and kidney damage and whether RAS blockade can reverse these effects. Transgenic mice overexpressing renal rANG were generated by employing the kidney-specific, androgen-regulated protein promoter linked to rANG cDNA. This promoter targets rANG cDNA to renal proximal tubules and responds to androgen stimulation. Transgenic mice displayed kidney-specific expression of rANG, significantly increased blood pressure (BP) and albuminuria in comparison to non-transgenic littermates. Administration of losartan (an angiotensin II (type 1)-receptor antagonist) or perindopril (an angiotensin-converting enzyme inhibitor) reversed these abnormalities in transgenic animals. Renal injury was evident on examination of the kidneys in transgenic mice, and attenuated by losartan and perindopril treatment. We conclude that the overproduction of ANG alone in the kidney induces an increase in systemic BP, proteinuria, and renal injury. RAS blockers prevent these abnormalities. These data support the role of the intrarenal RAS in the development of hypertension and renal injury. Angiotensinogen (ANG) is the sole substrate of the renin–angiotensin system (RAS). Clinical studies have shown that RAS activation may lead to hypertension, a major cardiovascular and renal risk factor. To delineate the underlying mechanisms of hypertension-induced nephropathy, we generated transgenic mice that overexpress rat ANG (rANG) in the kidney to establish whether intrarenal RAS activation alone can evoke hypertension and kidney damage and whether RAS blockade can reverse these effects. Transgenic mice overexpressing renal rANG were generated by employing the kidney-specific, androgen-regulated protein promoter linked to rANG cDNA. This promoter targets rANG cDNA to renal proximal tubules and responds to androgen stimulation. Transgenic mice displayed kidney-specific expression of rANG, significantly increased blood pressure (BP) and albuminuria in comparison to non-transgenic littermates. Administration of losartan (an angiotensin II (type 1)-receptor antagonist) or perindopril (an angiotensin-converting enzyme inhibitor) reversed these abnormalities in transgenic animals. Renal injury was evident on examination of the kidneys in transgenic mice, and attenuated by losartan and perindopril treatment. We conclude that the overproduction of ANG alone in the kidney induces an increase in systemic BP, proteinuria, and renal injury. RAS blockers prevent these abnormalities. These data support the role of the intrarenal RAS in the development of hypertension and renal injury. Hypertension is a major risk factor for cardiovascular and renal morbidity and mortality. Although numerous studies have implicated the renin–angiotensin system (RAS) in the development of hypertension and nephropathy, the mechanisms by which this occurs remain incompletely delineated. Traditionally, the effects of the RAS were considered to be the result of circulating components of this system. Physiological, biochemical, and molecular studies have provided, however, convincing evidence for other pathways of angiotensin II (Ang II) production. Among these, the intrarenal RAS is of special interest. Renal proximal tubules (RPTs) contain all components of the RAS.1.Darby I.A. Sernia C. In situ hybridization and immunocytochemistry of renal angiotensinogen in neonatal and adult rat kidneys.Cell Tissue Res. 1995; 281: 197-206Crossref PubMed Scopus (96) Google Scholar, 2.Gomez R.A. Lynch K.R. Chevalier R.L. et al.Renin and angiotensinogen gene expression and intrarenal renin distribution during ACE inhibitors.Am J Physiol Renal Fluid Electrolyte Physiol. 1988; 254: F900-F906PubMed Google Scholar, 3.Ingelfinger J.R. Zuo W.M. Fon E.A. et al.In situ hybridization evidence for angiotensinogen messenger RNA in the rat proximal tubule. An hypothesis for the intrarenal renin–angiotensin system.J Clin Invest. 1990; 85: 417-423Crossref PubMed Scopus (305) Google Scholar It has been demonstrated that activation of the Ang II (type 1) receptor (AT1R) in proximal tubules stimulates the apical sodium–hydrogen exchanger4.Saccomani G. Mitchell K.D. Navar L.G. Angiotensin II stimulation of Na+–H+ exchange in proximal tubule cells.Am J Physiol Renal Fluid Electrolyte Physiol. 1990; 258: F1188-F1195PubMed Google Scholar and, more distally, augments epithelial sodium channel activity in the collecting ducts.5.Komlosi P. Fuson A.L. Fintha A. et al.Angiotensin I conversion to angiotensin II stimulates cortical collecting duct sodium transport.Hypertension. 2003; 42: 195-199Crossref PubMed Scopus (87) Google Scholar, 6.Peti-Peterdi J. Warnock D.G. Bell P.D. Angiotensin II directly stimulates EnaC activity in the cortical collecting duct via AT1 receptors.J Am Soc Nephrol. 2002; 13: 1131-1135Crossref PubMed Scopus (250) Google Scholar Consequently, AT1R activation in the kidney modulates blood pressure (BP) and fluid homeostasis. Furthermore, dysregulation of the intrarenal RAS has been implicated in certain models of hypertension.7.Wang C.T. Navar L.G. Mitchell K.D. Proximal tubular fluid angiotensin II levels in angiotensin II-induced hypertensive rats.J Hypertens. 1996; 28: 290-296Crossref Scopus (25) Google Scholar, 8.Mitchell K.D. Jacinto S.M. Mullins J.J. Proximal tubular fluid, kidney, and plasma levels of angiotensin II in hypertensive ren-2 transgenic rats.Am J Physiol Renal Fluid Electrolyte Physiol. 1997; 273: F246-F253PubMed Google Scholar, 9.Cervenka L. Wang C.T. Mitchell K.D. et al.Proximal tubular angiotensin II levels and renal functional responses to AT1 receptor blockade in nonclipped kidneys of Goldblatt hypertensive rats.Hypertension. 1999; 33: 102-107Crossref PubMed Scopus (98) Google Scholar Overexpression of both human angiotensinogen (hANG) and human renin (hREN) cDNA in RPTs contributes directly to the development of hypertension independently of systemic RAS activation in transgenic mice.10.Ding Y. Davission R.L. Hardy D.O. et al.The kidney androgen-regulated protein promoter confers renal proximal tubule cell-specific and highly androgen-responsive expression on the human angiotensinogen gene in transgenic mice.J Biol Chem. 1997; 272: 28142-28148Crossref PubMed Scopus (112) Google Scholar, 11.Davisson R.L. Ding Y. Stee D.E. et al.Novel mechanism of hypertension revealed by cell specific targeting of human angiotensinogen in transgenic mice.Physiol Genomics. 1999; 1: 3-9Crossref PubMed Scopus (105) Google Scholar, 12.Ding Y. Sigmund C.D. Androgen-dependent regulation of human angiotensinogen expression in KAP-hAGT transgenic mice.Am J Physiol Renal Fluid Electrolyte Physiol. 2001; 280: F54-F60PubMed Google Scholar, 13.Sigmund C.D. Genetic manipulation of the renin–angiotensinogen system: targeted expression of the renin–angiotensin system in kidney.Am J Hypertens. 2001; 14: 33S-37SCrossref PubMed Google Scholar, 14.Lavoie J.L. Bruse-Lake K.D. Sigmund C.D. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in renal proximal tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 2004; 286: F965-F971Crossref PubMed Scopus (96) Google Scholar It appears, however, that losartan (an AT1R antagonist) is not particularly effective in lowering BP in these double transgenic (hANG and hREN) mice.11.Davisson R.L. Ding Y. Stee D.E. et al.Novel mechanism of hypertension revealed by cell specific targeting of human angiotensinogen in transgenic mice.Physiol Genomics. 1999; 1: 3-9Crossref PubMed Scopus (105) Google Scholar, 14.Lavoie J.L. Bruse-Lake K.D. Sigmund C.D. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in renal proximal tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 2004; 286: F965-F971Crossref PubMed Scopus (96) Google Scholar Thus, the question remains whether intrarenal RAS activation alone could induce hypertension and renal injury independently of systemic RAS activation, and whether other RAS blockers could block intrarenal RAS activation. The objective of our present studies was to investigate whether a model characterized by activation of the intrarenal RAS in the absence of activation of the circulating RAS or other tissue RAS could elicit hypertension and renal injury. A second objective was to determine whether RAS blockade could reverse these effects. KAP2-rANG transgenic mice were generated to produce specific and inducible expression of rANG in RPTs. This was accomplished by inserting rANG cDNA into a construct containing the KAP promoter and exons 3–5 of hANG gene, including non-coding DNA at the 3′ terminal (Figure 1a). The non-coding region is necessary to obtain transgene expression since it contains enhancer elements that are present downstream of hANG gene.14.Lavoie J.L. Bruse-Lake K.D. Sigmund C.D. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in renal proximal tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 2004; 286: F965-F971Crossref PubMed Scopus (96) Google Scholar Southern blot analysis revealed the presence of the transgene in heterozygote and homozygote animals (Figure 1b). Figure 2 displays the specific reverse transcription-polymerase chain reaction (RT-PCR) analysis of two (lines #351 and #388) of five transgenic lines. Testosterone strongly evoked transgene expression in the kidney. Transgene expression was not seen in the other tissues (liver, spleen, lung, heart, brain, testis, and ovaries) of transgenic mice as well as in tissues of non-transgenic littermates and transgenic female without testosterone induction. Figure 3 shows that strong ANG protein expression was observed in RPTs of neonatal transgenic mice (F4 generation) in comparison to RPTs of neonatal non-transgenic littermates by employing rabbit anti-rANG polyclonal antibodies. Western blot analysis reveals that recombinant rANG-HA fusion protein was detected in RPT extracts of transgenic mice induced with testosterone by employing anti-HA antibodies (Figure 4a), but no rANG-HA fusion protein was detected in the plasma (Figure 4b). Anti-HA antibodies also interacted well with the recombinant rANG-HA fusion protein that expressed in COS-7 cells. In contrast, immunoreactive ANG was detected in mouse plasma by employing rabbit anti-rANG polyclonal antibodies with an apparent molecular weight slightly lower to recombinant rANG-HA fusion protein that is expressed in COS-7 cells (Figure 4c). Figure 5a shows that BP was significantly elevated (P<0.05) by 20–30 mmHg in transgenic animals at 82 days after testosterone treatment but not in non-transgenic wild-type mice. Testosterone elicited a significant increase (P<0.05) in the kidney to body weight ratio in both transgenic and non-transgenic animals (Figure 5b). Most interestingly, urinary proteins were significantly higher in transgenic mice (P<0.05) but not in non-transgenic controls (Figure 5c). These data indicate that overexpression of intrarenal rANG appears to be capable of inducing high blood pressure (HBP) and proteinuria in transgenic mice. A longitudinal study was performed to examine the development of HBP and kidney injury in non-transgenic and transgenic mice. Figure 6 depicts BP over time in non-transgenic mice and demonstrates that there was no significant change of BP in female (a) and male (b) non-transgenic mice with testosterone induction for a period of 83 days. Figure 7 depicts BP in transgenic mice and illustrates the BP difference from baseline in female and male transgenic mice, respectively, for the #388 line during an 83-day period with or without RAS blockers. Note that the #351 line is not included, since results were essentially the same as to those of the #388 transgenic line. It was apparent that significant HBP (P<0.05) had developed in all transgenic mice after 7 days of induction with testosterone (without treatment with RAS blockers) and continued until 83 days compared to the placebo controls. Treatment with losartan or perindopril (starting at day 14) significantly prevented HBP development in transgenic mice with testosterone induction. Perindopril seemed to be more effective than losartan in decreasing BP in female transgenic mice (Figure 7a) than in male transgenic mice (Figure 7b). There was no significant difference in urinary ketone excretion between transgenic and non-transgenic animals (data not shown). Figure 8 depicts urinary microalbumin excretion after day 75. This figure shows that both female and male transgenic mice (testosterone treated) exhibited microalbuminuria as compared to placebo controls (P<0.05). Treatment with losartan or perindopril significantly reduced albumin levels (P<0.05). Again, perindopril seemed to be more effective than losartan in lowering urinary albumin in female transgenic mice (Figure 8a) than in male transgenic mice (Figure 8b). Hematoxylin–phloxin–saffran (HPS) staining was undertaken to analyze histologic changes in the non-transgenic and transgenic kidneys (Figures 9 and 10). Non-transgenic female littermates had normal kidneys, irrespective of testosterone administration (Figure 9a and b). However, with exogenous testosterone, transgenic female kidneys displayed cellular edema, reabsorption of droplets, and an increase in proximal tubular cell size with narrowing of the tubular lumen (Figure 9d) as compared to the transgenic placebo controls (Figure 9c). Losartan and perindopril appeared to be renoprotective, ameliorating these pathological findings (Figure 9e and f). In contrast, male transgenic mice exhibited no apparent renal pathology with testosterone administration, and their renal histology was similar to that of their non-transgenic littermates (Figure 10).Figure 10Hematoxylin–phloxin–saffran staining in male transgenic mice after 83 days of induction with testosterone (Original magnification × 400). (a) Uninduced male control littermate, (b) testosterone-induced male control littermate, (c) uninduced male transgenic animal, (d) testosterone-induced male transgenic animal, (e) testosterone-induced male transgenic animal+losartan, and (f) testosterone-induced male transgenic animal+perindopril.View Large Image Figure ViewerDownload (PPT) To assess the relative significance of ANG in the proximal tubules, we generated transgenic mice that overexpress rANG gene in the kidney after induction with testosterone. The present studies demonstrate that overexpression of renal rANG alone induces hypertension and albuminuria in transgenic mice and that RAS blockade reverses these abnormalities. The data indicate an important role for the intrarenal RAS in the development of hypertension and renal injury independently of systemic RAS activation. The transgene expression in our model is kidney specific. ANG protein was readily detectable in RPTs of transgenic mice but weakly in RPTs of non-transgenic mice. Our results are consistent with previous investigations in that the KAP2 promoter drives hANG expression in RPTs of transgenic mice.10.Ding Y. Davission R.L. Hardy D.O. et al.The kidney androgen-regulated protein promoter confers renal proximal tubule cell-specific and highly androgen-responsive expression on the human angiotensinogen gene in transgenic mice.J Biol Chem. 1997; 272: 28142-28148Crossref PubMed Scopus (112) Google Scholar, 11.Davisson R.L. Ding Y. Stee D.E. et al.Novel mechanism of hypertension revealed by cell specific targeting of human angiotensinogen in transgenic mice.Physiol Genomics. 1999; 1: 3-9Crossref PubMed Scopus (105) Google Scholar, 12.Ding Y. Sigmund C.D. Androgen-dependent regulation of human angiotensinogen expression in KAP-hAGT transgenic mice.Am J Physiol Renal Fluid Electrolyte Physiol. 2001; 280: F54-F60PubMed Google Scholar, 13.Sigmund C.D. Genetic manipulation of the renin–angiotensinogen system: targeted expression of the renin–angiotensin system in kidney.Am J Hypertens. 2001; 14: 33S-37SCrossref PubMed Google Scholar, 14.Lavoie J.L. Bruse-Lake K.D. Sigmund C.D. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in renal proximal tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 2004; 286: F965-F971Crossref PubMed Scopus (96) Google Scholar Most importantly, we could not detect rANG-HA fusion protein in the plasma, indicating it is unlikely that rANG-HA fusion protein expressed in RPTs of transgenic mice would leak into the circulation to induce hypertension. We failed to detect HA-staining in RPTs and other organs of transgenic mice by employing mouse anti-HA monoclonal antibodies (data not shown). At present, we have no explanation for this observation. One possible explanation might be that the affinity of our monoclonal antibodies (12CA5) might not be sufficiently high to interact with rANG-HA proteins in transgenic RPTs. In contrast, our monoclonal antibodies interacted with recombinant rANG-HA expressed in mouse transgenic RPTs induced by testosterone and in COS-7 cells by Western blotting. Clearly, more studies are needed to clarify this discrepancy. The basal BP in male transgenic mice is not significantly higher than in non-transgenic males. At the present, the exact reason(s) for these observations are not clear. One likely explanation might be that the renin gene expression is downregulated by intrarenal Ang II (a negative feedback mechanism) in males as shown previously.14.Lavoie J.L. Bruse-Lake K.D. Sigmund C.D. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in renal proximal tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 2004; 286: F965-F971Crossref PubMed Scopus (96) Google Scholar Clearly, more studies are needed to explore these intriguing observations. On the other hand, transgenic mice with exogenous testosterone administration had significantly higher BP than either non-transgenic littermates or transgenic mice that received placebo. In our cross-sectional experiment (82 days), male and female transgenic mice with testosterone induction had on average 20–30 mmHg higher systolic BP than control mice (without testosterone induction) at the end of the study (P<0.05). The kidney to body weight ratio in testosterone-induced transgenic animals was significantly augmented (P<0.05) compared to wild-type mice. Testosterone treatment also resulted in a significant (P<0.05) increase of kidney size in wild-type mice but was not associated with detectable proteinuria. This is not surprising since testosterone is a growth factor and routinely increases renal size. Thus, these results suggest that proteinuria occurs in transgenic mice only in response to the induction of transgene expression. In contrast, both male and female transgenic lines displayed proteinuria (30–36 mg/dl), indicating ongoing kidney injury (transgenic line #351 not shown, but similar to line #388). Proteinuria is important since it is a marker of renal disease.15.Johnson C.A. Levey A.S. Coresh J. et al.Clinical practice guidelines for chronic kidney disease in adults: Part II. Glomerular filtration rate, proteinuria, and other markers.Am Fam Physician. 2004; 70: 1091-1097PubMed Google Scholar Thus, proteinuria following transgene induction suggests that increased intrarenal ANG expression is associated with the development of renal injury. In the longitudinal study, there was no significant difference in BP in male and female non-transgenic mice with or without testosterone induction. The BP of transgenic mice, however, was significantly elevated after 2 weeks of induction with testosterone compared to the placebo controls. The average increase of systolic BP for 83 days was 20–30 mmHg (P<0.05) compared to their non-transgenic littermates or placebo controls. Administration of RAS blockers significantly reduced the BP in these mice, indicating that activation of the intrarenal RAS is important in this process. Perindopril appeared to be more effective than losartan in lowering BP in female but not in male transgenic mice. Since microalbuminuria is an important marker for the early detection of hypertension- or diabetes-induced nephropathy,15.Johnson C.A. Levey A.S. Coresh J. et al.Clinical practice guidelines for chronic kidney disease in adults: Part II. Glomerular filtration rate, proteinuria, and other markers.Am Fam Physician. 2004; 70: 1091-1097PubMed Google Scholar, 16.Clase C.M. Garg A.X. Kiberd B.A. Classifying kidney problems: can we avoid framing risks as diseases?.BMJ. 2004; 329: 912-915Crossref PubMed Scopus (49) Google Scholar we monitored urinary albumin excretion during the longitudinal study. Animal experiments have shown that early detection of microalbuminuria and subsequent treatment with RAS blockers can slow or even prevent disease progression.16.Clase C.M. Garg A.X. Kiberd B.A. Classifying kidney problems: can we avoid framing risks as diseases?.BMJ. 2004; 329: 912-915Crossref PubMed Scopus (49) Google Scholar, 17.Renaud I.M. Chainey A. Belair M.F. et al.Long-term protection of obese Zucker rat kidneys from fibrosis and renal failure with an angiotensin-converting enzyme inhibitor/diuretic combination.Fundam Clin Pharmacol. 2004; 18: 437-447Crossref PubMed Scopus (18) Google Scholar, 18.Bertram D. Blanc-Brunat N. Sassard J. et al.Differential evolution of blood pressure and renal lesions after RAS blockade in Lyon hypertensive rats.Am J Physiol Regul Integr Comp Physiol. 2002; 283: R1041-R1045Crossref PubMed Scopus (14) Google Scholar Microalbuminuria appeared only after 75 days of induction in our transgenic animals receiving testosterone. Prior to day 75, there was no detectable albuminuria. Microalbuminuria levels reached 0.37 and 0.74 μg albumin/mg of creatinine for females and males at day 83, respectively. Treatment with RAS inhibitors significantly reduced protein levels. Once again, perindopril appeared to be more effective than losartan in lowering albumin in females. In males, RAS blockers were also effective, but perindopril did not confer more advantage than losartan. To the best of our knowledge, this is the first report that RAS blockade could prevent HBP and albuminuria (renal injury) in both male and female transgenic mice with overexpression of rANG gene in RPTs. Taken together, these observations suggest a link between renal ANG gene expression, hypertension, and microalbuminuria. It remains to be seen whether intrarenal RAS activation alone could induce microalbuminuria independently of systemic hypertension. Studies are under way to test this possibility. Interestingly, histological examination revealed that the kidneys of female transgenic mice treated with testosterone displayed cellular edema, reabsorption of droplets, proximal tubular hypertrophy, and glomerular proliferation as compared to wild-type non-transgenic controls treated with testosterone and female transgenic mice treated with placebo. These findings were not observed in male transgenic mice irrespective of testosterone treatment. The exact reason(s) for this sexual dimorphic response is unknown at present, although gender difference or decreased renin expression in the kidneys of male transgenic mice, as observed previously,14.Lavoie J.L. Bruse-Lake K.D. Sigmund C.D. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in renal proximal tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 2004; 286: F965-F971Crossref PubMed Scopus (96) Google Scholar may provide some explanation. Perindopril appeared to be more effective in slowing kidney damage in female mice as compared to losartan. A similar perindopril effect has been reported in humans.19.Mogensen C.E. Viberti G. Halimi S. et al.Preterax in Albuminuria Regression (PREMIER) Study Group. Effect of low-dose perindopril/indapamide on albuminuria in diabetes: preterax in albuminuria regression: PREMIER.Hypertension. 2003; 41: 1063-1071Crossref PubMed Scopus (166) Google Scholar, 20.Cao Z. Cooper M.E. Wu L.L. et al.Blockade of the renin–angiotensin and endothelin systems on progressive renal injury.Hypertension. 2000; 36: 561-568Crossref PubMed Scopus (77) Google Scholar To provide additional evidence that the intrarenal RAS was indeed responsible for the pathology observed, we measured the levels of plasma renin activity as well as plasma testosterone. There was no significant change in plasma renin activity and testosterone levels in the transgenic animals compared to control littermates (data not presented). Although the precise mechanism(s) by which BP increases and causes kidney injury (albuminuria and RPT hypertrophy) in our transgenic mice model remains unclear, several possible hypotheses arise from these observations. One hypothesis is that ANG produced in renal proximal tubular cells (RPTCs) is secreted and converted to Ang II in the proximal tubular lumen or at a more distal point along the nephron, since renin could be filtered into the lumen. As RPTCs also express renin,21.Tang S.-S. Jung F. Diamant D. et al.Temperature-sensitive SV40 immortalized rat proximal tubule cell line has functional renin–angiotensin system.Am J Physiol. 1995; 268: F435-F446PubMed Google Scholar it is conceivable that RPTC-renin could cleave ANG to produce angiotensin I and then Ang II by angiotensin-converting enzyme. Subsequently, Ang II could affect BP via stimulation of the sodium/hydrogen exchanger in RPTs,4.Saccomani G. Mitchell K.D. Navar L.G. Angiotensin II stimulation of Na+–H+ exchange in proximal tubule cells.Am J Physiol Renal Fluid Electrolyte Physiol. 1990; 258: F1188-F1195PubMed Google Scholar, 7.Wang C.T. Navar L.G. Mitchell K.D. Proximal tubular fluid angiotensin II levels in angiotensin II-induced hypertensive rats.J Hypertens. 1996; 28: 290-296Crossref Scopus (25) Google Scholar or by the activation of epithelial sodium channels in collecting duct cells,5.Komlosi P. Fuson A.L. Fintha A. et al.Angiotensin I conversion to angiotensin II stimulates cortical collecting duct sodium transport.Hypertension. 2003; 42: 195-199Crossref PubMed Scopus (87) Google Scholar, 6.Peti-Peterdi J. Warnock D.G. Bell P.D. Angiotensin II directly stimulates EnaC activity in the cortical collecting duct via AT1 receptors.J Am Soc Nephrol. 2002; 13: 1131-1135Crossref PubMed Scopus (250) Google Scholar thereby promoting sodium reabsorption. Furthermore, Ang II could increase transforming growth factor-β1 gene expression via AT1R in RPTCs, leading to renal injury (cellular hypertrophy). We have observed this effect in vitro where Ang II stimulation of immortalized rat proximal tubular cells induces cell hypertrophy.22.Zhang S.-L. To C. Chen X. et al.Effect of renin–angiotensin system blockade on the expression of the angiotensinogen gene and induction of hypertrophy in rat kidney proximal tubular cells.Exp Nephrol. 2001; 9: 109-117Crossref PubMed Scopus (35) Google Scholar, 23.Zhang S.-L. To C. Chen X. et al.Essential role(s) in intrarenal renin–angiotensin system on transforming growth factor-beta 1 expression and induction of hypertrophy in rat kidney proximal tubular cells in high glucose.J Am Soc Nephrol. 2002; 13: 302-312Crossref PubMed Google Scholar Indeed, further work is needed to confirm this possibility. In conclusion, our data strongly suggest that activation of the intrarenal RAS alone evokes a significant increase of BP and leads to renal injury. The administration of RAS blockers prevents these abnormalities. Losartan (a non-peptide Ang II (AT1)-receptor blocker) and perindopril (an inhibitor of angiotensin-converting enzyme) were obtained from Dr Ronald D Smith (Dupont Merck, Wilmington, DE, USA) and Dr Serge Carrière (Servier Amérique, Laval, QC, Canada), respectively. COS-7 cells (Green monkey kidney cells) were obtained from the American Type Tissue Culture Collection (Rockville, MD, USA). The pKAP2 plasmid that contains the kidney androgen-regulated promoter (KAP), which is responsive to testosterone stimulation, is constructed by us (CD Sigmund's laboratory) and has been described elsewhere.10.Ding Y. Davission R.L. Hardy D.O. et al.The kidney androgen-regulated protein promoter confers renal proximal tubule cell-specific and highly androgen-responsive expression on the human angiotensinogen gene in transgenic mice.J Biol Chem. 1997; 272: 28142-28148Crossref PubMed Scopus (112) Google Scholar, 14.Lavoie J.L. Bruse-Lake K.D. Sigmund C.D. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in renal proximal tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 2004; 286: F965-F971Crossref PubMed Scopus (96) Google Scholar Placebo pellets or pellets containing 5 mg testosterone with a 91-day release schedule (A-192) were purchased from Innovative Research of America (Sarasota, FL, USA). Mouse anti-HA (a sequence encoding amino-acid residues 98–106 (YPYDVPDYA) of human influenza virus hemagglutinin) monoclonal antibodies (12CA5) were obtained from La Roche Biochemicals (Dorval, QC, Canada). Rabbit anti-rANG antibodies were generated in our laboratory (JSD Chan).24.Wang L. Lei C. Zhang S.-L. et al.Synergistic effect of dexamethasone and isoproterenol on the expression of angiotensinogen in immortalized rat proximal tubular cells.Kidney Int. 1998; 53: 287-295Abstract Full Text PDF PubMed Scopus (38) Google Scholar These antibodies are specific for intact rat and mouse ANG (i.e. 55–62 kDa ANG) and do not crossreact with pituitary hormone preparations or other rat or mouse plasma proteins.24.Wang L. Lei C. Zhang S.-L. et al.Synergistic effect of dexamethasone and isoproterenol on the expression of angiotensinogen in immortalized rat proximal tubular cells.Kidney Int. 1998; 53: 287-295Abstract Full Text PDF PubMed Scopus (38) Google Scholar The plasmid vector pcDNA 3.1 and oligonucleotides were procured from InVitrogen, Inc. (Burlington, ON, Canada). Restriction and modifying enzymes were purchased from either InVitrogen, Inc., La Roche Biochemicals, or Amersham-Pharmacia Biotech, Inc. (Baie d'Urfé, QC, Canada). A cDNA encoding full-length rANG fused with HA-tag at the carboxyl terminal and NotI restriction enzyme site attached at both 5′- and 3′-termini was inserted into the pcDNA 3.1 plasmid at the NotI site. The orientation of the insert was confirmed by DNA sequencing (performed at the DNA Sequencing Facility of the CHUM). The T7 promoter primer (InVitrogen, Inc.) was used in DNA sequencing. The plasmid pcDNA 3.1/rANG-HA was then transiently transfected into COS-7 cells with Lipofectamine according to the instruction manual provided by the supplier (InVitrogen, Inc.). The cells were harvested 24 h after transfection and extracted with lysis buffer containing 1% Triton-100 (Laboratoires MAT, Montreal, QC, Canada), 0.1% sodium deocyl sulfate (SDS), 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.5, 1 mM phenylmethylsulphonyl fluoride, and cocktail inhibitor (1 tablet/10 ml lysis buffer, La Roche Biochemicals, Dorval, QC, Canada). The extracts were centrifuged at 13 000 g and 4°C. The supernatant was taken for quantification of protein concentration and Western blot analysis. Cellular proteins (30–50 μg) or 1 μl of mouse plasma were loaded on SDS-10% polyacrylamide gel. After transfer by wet blotting onto a polyvinylidine difluoride membrane (Hybond-P, Amersham-Pharmacia Biotech, Inc.), the membrane was blocked in 5% non-fat milk powder and 0.05% Tween 20 in phosphate-buffered saline. It was first blotted with mouse anti-HA monoclonal antibodies and developed with chemiluminescent developing reagent (Roche Biochemicals, Inc.). The membrane was then re-blotted with rabbit anti-rANG polyclonal antibodies and developed with anti-rabbit horseraddish peroxidase conjugates (HRP) and the avidin–HRP conjugates according to the protocol of the supplier (Bio-Rad Laboratories, Richmond, CA, USA). A full-length rANG cDNA fused with HA-tag at the carboxyl terminal and NotI restriction enzyme site attached at both 5′- and 3′-termini (as described above) was inserted into pKAP2 plasmid at the NotI site. The orientation of the insert was confirmed by DNA sequencing. The primer 5′ CCA GCC AAC TGT GGA AAA A 3′ was used in DNA sequencing. The final 17 kb KAP2-rANG transgene was excised by digestion with NdeI and SpeI, purified by agarose gel electrophoresis, and recovered by gel extraction. The isolated KAP2-rANG transgene was then microinjected into one-cell fertilized mouse embryos obtained from superovulated C57Bl6 × C3H mice, using a standard procedure (performed at the Clinical Research Institute of Montreal, Montreal, QC, Canada). Founder mice were identified by Southern blot analysis of mouse tail genomic DNA digested with BamHI. The positive transgenic founders were then crossed with wild-type C57Bl6 mice (Charles River, St-Constant, QC, Canada) to obtain an F1 generation. Breeding was continued until homozygous F4 mice were generated. The mice used in these experiments were 15–20 weeks of age at the time of data collection. Non-transgenic and sex-matched littermates served as controls. All animals received standard mouse chow and water ad libitum. Their care in these experiments met the standards set forth by the Canadian Council on Animal Care, and the procedures utilized were approved by the Institutional Animal Care Committee of the CHUM. Various tissues were harvested and snap-frozen on dry ice. Total RNA was isolated by TRIzol (InVitrogen) after tissue homogenization. Before proceeding with RT, RNA was subjected to DNase-1 treatment (InVitrogen) to remove contaminating genomic DNA. RNA was subjected to RT-PCR analysis as described previously.25.Hsieh T.-J. Zhang S.-L. Filep J.G. et al.High glucose stimulates angiotensinogen gene expression via reactive oxygen species (ROS) generation in rat kidney proximal tubular cells.Endocrinology. 2002; 143: 2975-2985Crossref PubMed Scopus (147) Google Scholar, 26.Hsieh T.-J. Fustier P. Zhang S.-L. et al.High glucose stimulates angiotensinogen gene expression and cellular hypertrophy via activation of the hexosamine biosynthesis pathway in rat kidney proximal tubular cells.Endocrinology. 2003; 144: 4338-4349Crossref PubMed Scopus (86) Google Scholar, 27.Hsieh T.-J. Fustier P. Wei C.-C. et al.Reactive oxygen species blockade and action of insulin on expression of angiotensinogen gene in proximal tubular cells.J Endocrinol. 2004; 183: 535-550Crossref PubMed Scopus (43) Google Scholar The rANG sense primer (5′-CCT CGC TCT CTG GAC TTA TC-3′) corresponding to the nucleotide sequences N+676 to N+695 of rANG cDNA,28.Ohkubo H. Kageyama R. Ujihara M. et al.Cloning and sequence analysis of cDNA for rat angiotensinogen.Proc Natl Acad Sci USA. 1983; 80: 2196-3000Crossref PubMed Scopus (210) Google Scholar HA anti-sense primer (5′-GGC GTA GTC AGG CAC GTC GT-3′), β-actin forward primer and reverse primer corresponding to the nucleotide sequences N+155 to N+179 of exon 3 (5′-ATG CCA TCC TGC GTC TGG ACC TGG C-3′) and N+115 to N+139 of exon 5 (5′-AGC ATT TGC GGT GCA CGA TGG AGG G-3′) of the rat β-actin gene,29.Nudel U. Zakut R. Shani M. et al.The nucleotide sequence of the rat cytoplasmic β-actin gene.Nucleic Acids Res. 1983; 11: 1759-1771Crossref PubMed Scopus (1018) Google Scholar respectively, were used for PCR. Kidneys from neonatal mice were removed immediately after they were killed. Formalin-fixed, paraffin-embedded kidney sections of 5 μm were deparaffinized in xylene and rehydrated. Immunohistochemical examination was performed by the standard avidin–biotin–peroxidase complex method (ABC Staining System) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA). Endogenous peroxidase was inhibited in 1% hydrogen peroxide–methanol for 10 min at room temperature and followed by trypsin treatment for 8 min in a moist chamber at 37°C. After serum blocking, the sections were incubated with primary anti-rANG polyclonal antibody (rabbit #16) diluted 1:1000 for 1 h at room temperature; then, biotinylated secondary antibody was added, followed by the addition of preformed ABC reagent supplied by kit. The rANG protein was visualized by color development with 3, 3′-diaminobenzidine tetrahydrochloride. All sections were counterstained with hematoxylin, dehydrated, and covered with glass slips. Animals were killed under CO2. Their kidneys were removed, rinsed in ice-cold DMEM, decapsulated, and the cortices were separated from the medulla. Proximal tubules were isolated by Percoll gradient30.Vinay P. Gougoux A. Lemieux G. Isolation of a pure suspension of rat proximal tubules.Am J Physiol. 1981; 241: F403-F411PubMed Google Scholar with slight modifications as described previously.31.Zhang S.-L. Chen X. Hsieh T.-J. et al.Hyperglycemia induces insulin resistance on angiotensinogen gene expression in diabetic rat kidney proximal tubular cells.J Endocrinol. 2002; 172: 333-344Crossref PubMed Scopus (35) Google Scholar Homozygous transgenic mice (males and females) and non-transgenic littermates were anesthetized with isofurane and implanted surgically with placebo pellets or pellets containing 5 mg testosterone that was gradually released over 91 days. Before implantation, bupivacaine HCl (0.25%, Abbott Laboratories, Montreal, QC, Canada) was applied topically at the incision site. The pellet was implanted subcutaneously in the back and tunneled to the nape of the neck with a 10-gauge trocar. The incision was closed with a stainless steel staple, and the mice were given 2 days to recover. After a baseline period and then starting 7 days after the implantation of testosterone pellets to induce renal rANG expression, BP was monitored with a BP-2000 tail-cuff pressure machine (Visitech Systems, Apex, NC, USA) once to twice a week for a period of 21–83 days, depending on the experimental protocol. However, before measuring BP, the mice were trained for at least 30 min per day for 5 days prior to the first BP measurements. Thus, after assessing baseline BP for 1 week, non-transgenic littermates and transgenic mice were divided into four equal groups: (1) placebo (with placebo pellet) (n=4), (2) testosterone (n=4), (3) testosterone with losartan treatment (30 mg/kg/day in drinking water, n=4), and (4) testosterone with perindopril (5 mg/kg/day in drinking water, n=4). At the end of treatment, body weights were recorded, and the mice were anesthetized with isofurane, then killed by cervical dislocation. Trunk blood was collected in chilled tubes containing EDTA for plasma renin measurement or in tubes containing clotting agents for serum separation to quantify testosterone levels (Biochemistry Laboratories, CHUM-Hôtel-Dieu, Montreal, QC, Canada). Mice were housed in metabolic cages to obtain 24-h urine samples for assessing proteinuria. Animals had free access to water and food during this period. The next day, urine volume was recorded. Proteinuria was estimated by using Chemstrip 9 (La Roche Biochemicals) and/or albuminuria was assessed with an enzyme-linkedimmunosorbent assay kit according to the manufacturer's protocol (Cedarlane Laboratoires Ltd, Hornby, ON, Canada). Several tissues, such as the liver, brain, heart, lungs, spleen, testis, and ovaries, were collected for RNA extraction and analyzed by RT-PCR for rANG mRNA expression. The kidneys were weighed carefully. One kidney was used for immunohistochemistry and the other for RNA extraction. Kidneys were always taken from the same side of the animals for different procedures. Tissues were collected in Tissue-Tek cassettes (VWR Canlab, Montreal, QC, Canada), dipped immediately in ice-cold phosphate-buffered saline–4% paraformaldehyde, and fixed for 24 h at 4°C. The cassettes were processed by the Pathology Department at the CHUM. Briefly, the tissues were dehydrated by successive ethanol baths (from 50 to 100%), then rehydrated with a mix of toluene and water (from 50 to 100%). The cassettes are dipped in hot paraffin solution and embedded for 16 h. The tissue blocks were cut with a microtome to obtain the desired tissue specimens. Statistical significance between experimental groups was analyzed initially by Student's t-test or by one-way analysis of variance and the Bonferroni test as appropriate. Data are expressed as means±s.e. Values of P<0.05 were considered to be statistically significant. This work was supported in part by grants from the Kidney Foundation of Canada, the Canadian Diabetes Association (#1061), the Canadian Institutes of Health Research (MOP-13420 and MOP 62920 to JSDC, MOP-12573 to JGF, and MT-14726 to D-FG), and the National Institutes of Health (NIH) of USA (HL-48455 to JRI). We thank Dr Julie Lavoie for her valuable advice in writing this paper. The editorial assistance of Mr Ovid M Da Silva, Editor, Research Support Office, Research Centre, CHUM, is acknowledged.
Referência(s)