Angiotensin-converting enzyme 2 amplification limited to the circulation does not protect mice from development of diabetic nephropathy
2016; Elsevier BV; Volume: 91; Issue: 6 Linguagem: Inglês
10.1016/j.kint.2016.09.032
ISSN1523-1755
AutoresJan Wysocki, Minghao Ye, Ahmed Khattab, Agnes B. Fogo, Aline Martin, Valentin David, Yashpal S. Kanwar, Mark J. Osborn, Daniel Batlle,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoBlockers of the renin-angiotensin system are effective in the treatment of experimental and clinical diabetic nephropathy. An approach different from blocking the formation or action of angiotensin II (1-8) that could also be effective involves fostering its degradation. Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that cleaves angiotensin II (1-8) to form angiotensin (1-7). Therefore, we examined the renal effects of murine recombinant ACE2 in mice with streptozotocin-induced diabetic nephropathy as well as that of amplification of circulating ACE2 using minicircle DNA delivery prior to induction of experimental diabetes. This delivery resulted in a long-term sustained and profound increase in serum ACE2 activity and enhanced ability to metabolize an acute angiotensin II (1-8) load. In mice with streptozotocin-induced diabetes pretreated with minicircle ACE2, ACE2 protein in plasma increased markedly and this was associated with a more than 100-fold increase in serum ACE2 activity. However, minicircle ACE2 did not result in changes in urinary ACE2 activity as compared to untreated diabetic mice. In both diabetic groups, glomerular filtration rate increased significantly and to the same extent as compared to non-diabetic controls. Albuminuria, glomerular mesangial expansion, glomerular cellularity, and glomerular size were all increased to a similar extent in minicircle ACE2-treated and untreated diabetic mice, as compared to non-diabetic controls. Recombinant mouse ACE2 given for 4 weeks by intraperitoneal daily injections in mice with streptozotocin-induced diabetic nephropathy also failed to improve albuminuria or kidney pathology. Thus, a profound augmentation of ACE2 confined to the circulation failed to ameliorate the glomerular lesions and hyperfiltration characteristic of early diabetic nephropathy. These findings emphasize the importance of targeting the kidney rather than the circulatory renin angiotensin system to combat diabetic nephropathy. Blockers of the renin-angiotensin system are effective in the treatment of experimental and clinical diabetic nephropathy. An approach different from blocking the formation or action of angiotensin II (1-8) that could also be effective involves fostering its degradation. Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that cleaves angiotensin II (1-8) to form angiotensin (1-7). Therefore, we examined the renal effects of murine recombinant ACE2 in mice with streptozotocin-induced diabetic nephropathy as well as that of amplification of circulating ACE2 using minicircle DNA delivery prior to induction of experimental diabetes. This delivery resulted in a long-term sustained and profound increase in serum ACE2 activity and enhanced ability to metabolize an acute angiotensin II (1-8) load. In mice with streptozotocin-induced diabetes pretreated with minicircle ACE2, ACE2 protein in plasma increased markedly and this was associated with a more than 100-fold increase in serum ACE2 activity. However, minicircle ACE2 did not result in changes in urinary ACE2 activity as compared to untreated diabetic mice. In both diabetic groups, glomerular filtration rate increased significantly and to the same extent as compared to non-diabetic controls. Albuminuria, glomerular mesangial expansion, glomerular cellularity, and glomerular size were all increased to a similar extent in minicircle ACE2-treated and untreated diabetic mice, as compared to non-diabetic controls. Recombinant mouse ACE2 given for 4 weeks by intraperitoneal daily injections in mice with streptozotocin-induced diabetic nephropathy also failed to improve albuminuria or kidney pathology. Thus, a profound augmentation of ACE2 confined to the circulation failed to ameliorate the glomerular lesions and hyperfiltration characteristic of early diabetic nephropathy. These findings emphasize the importance of targeting the kidney rather than the circulatory renin angiotensin system to combat diabetic nephropathy. Clinical interventions targeting the renin-angiotensin system (RAS) in diabetic kidney disease center on the use of RAS blockers.1Raij L. The pathophysiologic basis for blocking the renin-angiotensin system in hypertensive patients with renal disease.Am J Hypertens. 2005; 18: 95S-99SCrossref PubMed Scopus (34) Google Scholar, 2Gurley S.B. Coffman T.M. 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William J. et al.Glomerular localization and expression of angiotensin-converting enzyme 2 and angiotensin-converting enzyme: implications for albuminuria in diabetes.J Am Soc Nephrol. 2006; 17: 3067-3075Crossref PubMed Scopus (379) Google Scholar administration of an ACE2 inhibitor caused worsening of albuminuria. In agreement with the studies in which a pharmacologic ACE2 inhibitor was given to diabetic mice, the deletion of the Ace2 gene was reported to accentuate,19Tikellis C. Bialkowski K. Pete J. et al.ACE2 deficiency modifies renoprotection afforded by ACE inhibition in experimental diabetes.Diabetes. 2008; 57: 1018-1025Crossref PubMed Scopus (150) Google Scholar and the transgenic glomerular ACE2 overexpression ameliorated, diabetes-related kidney lesions.26Nadarajah R. Milagres R. Dilauro M. et al.Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice.Kidney Int. 2012; 82: 292-303Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Moreover, a beneficial effect of human recombinant (r)ACE2 given by i.p. injections was reported to ameliorate albuminuria and diabetic kidney lesions in the Akita model of diabetic kidney disease.36Oudit G.Y. Liu G.C. Zhong J. et al.Human recombinant ACE2 reduces the progression of diabetic nephropathy.Diabetes. 2010; 59: 529-538Crossref PubMed Scopus (244) Google Scholar This finding was surprising because human rACE2, when given to mice for more than 2 weeks, results in the formation of neutralizing antibodies and the attendant loss of ACE2 activity.8Wysocki J. Ye M. Rodriguez E. et al.Targeting the degradation of angiotensin II with recombinant angiotensin-converting enzyme 2: prevention of angiotensin II-dependent hypertension.Hypertension. 2010; 55: 90-98Crossref PubMed Scopus (236) Google Scholar, 37Ye M. Wysocki J. Gonzalez-Pacheco F.R. et al.Murine recombinant angiotensin-converting enzyme 2: effect on angiotensin II-dependent hypertension and distinctive angiotensin-converting enzyme 2 inhibitor characteristics on rodent and human angiotensin-converting enzyme 2.Hypertension. 2012; 60: 730-740Crossref PubMed Scopus (74) Google Scholar We therefore developed murine recombinant ACE237Ye M. Wysocki J. Gonzalez-Pacheco F.R. et al.Murine recombinant angiotensin-converting enzyme 2: effect on angiotensin II-dependent hypertension and distinctive angiotensin-converting enzyme 2 inhibitor characteristics on rodent and human angiotensin-converting enzyme 2.Hypertension. 2012; 60: 730-740Crossref PubMed Scopus (74) Google Scholar and reasoned that ACE2 amplification using this rodent form of rACE2 would circumvent the problem of immunogenicity arising from chronic administration of xenogeneic human rACE2 to mice. Accordingly, in the present study we administered soluble mouse rACE2 protein by daily i.p. injections in mice that had been given STZ 4 weeks earlier to produce early DN. To increase and sustain high levels of ACE2 activity for a much longer period, murine rACE2 was administered by minicircle (Mc) ace2 DNA delivery. The Mc system uses a phiC31 integrase recombination event to remove the bacterial backbone elements of the plasmid that are required for plasmid amplification and replication in bacteria but subsequently contribute to gene silencing.38Chen Z.Y. He C.Y. Ehrhardt A. et al.Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo.Mol Ther. 2003; 8: 495-500Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 39Osborn M.J. McElmurry R.T. Lees C.J. et al.Minicircle DNA-based gene therapy coupled with immune modulation permits long-term expression of alpha-L-iduronidase in mice with mucopolysaccharidosis type I.Mol Ther. 2011; 19: 450-460Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar By removing these sequences and isolating the circular expression cassette, long-term gene expression can be achieved from the Mc that is maintained as an extrachromosomal episome.39Osborn M.J. McElmurry R.T. Lees C.J. et al.Minicircle DNA-based gene therapy coupled with immune modulation permits long-term expression of alpha-L-iduronidase in mice with mucopolysaccharidosis type I.Mol Ther. 2011; 19: 450-460Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar This approach also permitted examination of a potential preventative effect of preexisting high levels of circulating ACE2 on STZ-induced DN. Finally, the impact of markedly increasing ACE2 in the circulation on urinary ACE2 was examined not only in STZ-treated mice with mild albuminuria, but also in a model of CKD caused by a Col4A3 gene deficiency that results in advanced alterations in the glomerular basement membrane and robust proteinuria.40Cosgrove D. Meehan D.T. Grunkemeyer J.A. et al.Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev. 1996; 10: 2981-2992Crossref PubMed Scopus (296) Google Scholar We previously showed that when human rACE2 is administered to normal mice there is a loss of ACE2 activity by 2 weeks that is attributable to the formation of neutralizing antibodies.8Wysocki J. Ye M. Rodriguez E. et al.Targeting the degradation of angiotensin II with recombinant angiotensin-converting enzyme 2: prevention of angiotensin II-dependent hypertension.Hypertension. 2010; 55: 90-98Crossref PubMed Scopus (236) Google Scholar Therefore, in this study we used mouse rACE2, which results in a sustainable increase in serum ACE2 activity for at least 4 weeks.37Ye M. Wysocki J. Gonzalez-Pacheco F.R. et al.Murine recombinant angiotensin-converting enzyme 2: effect on angiotensin II-dependent hypertension and distinctive angiotensin-converting enzyme 2 inhibitor characteristics on rodent and human angiotensin-converting enzyme 2.Hypertension. 2012; 60: 730-740Crossref PubMed Scopus (74) Google Scholar Studies using murine recombinant (mr)ACE2 administration for 4 weeks by i.p. injections to STZ mice showed that mrACE2 does not have a protective effect on kidney pathology or urinary albumin excretion as compared with STZ-treated mice receiving phosphate-buffered saline (Supplementary Results, Supplementary Table S1, and Supplementary Figure S1). ACE2 Mc was administered as a single injection38Chen Z.Y. He C.Y. Ehrhardt A. et al.Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo.Mol Ther. 2003; 8: 495-500Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 39Osborn M.J. McElmurry R.T. Lees C.J. et al.Minicircle DNA-based gene therapy coupled with immune modulation permits long-term expression of alpha-L-iduronidase in mice with mucopolysaccharidosis type I.Mol Ther. 2011; 19: 450-460Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar to mice in the FVB/N background. Two doses (10 and 30 ug) were used to determine whether Mc injection results in dose-dependent increases in serum ACE2 activity. At 3 to 9 days after Mc administration, serum ACE2 activity in female FVB/N mice that received 10 ug McACE2 (n = 9) increased markedly compared with controls (n = 14) (138 ± 48 vs. 0.5 ± 0.1 relative fluorescence unit (RFU)/ul/h; P < 0.01). In FVB/N mice that received 30 ug McACE2 (n = 8), serum ACE2 activity increased even further (480 ± 153 RFU/ul/h; P < 0.001). The marked increase in serum ACE2 activity achieved initially (3–9 days) was sustained over 10 weeks of observation in McACE2-treated (n = 7) compared with untreated mice (n = 9) (108 ± 46 vs. 1.0 ± 0.2 RFU/ul/h, respectively; P < 0.05). In urine from McACE2-treated mice, however, ACE2 activity was not significantly different than in urine from mice that did not receive McACE2 (3.5 ± 0.9 vs. 4.4 ± 0.6 RFU/ug creatinine per hour, respectively). At the end of this experiment (10–12 weeks after sham or McACE2 injection), mice were given an i.p. bolus of Ang II (0.2 ug/body weight) or vehicle (phosphate-buffered saline) to examine their ability to handle an acute Ang II load. Five minutes after the Ang II bolus, ACE2 activity in serum and Ang II levels in plasma were evaluated from cardiac blood obtained at death. Plasma Ang II levels in samples obtained 5 minutes after Ang II bolus were significantly lower in McACE2 mice compared with sham mice also infused with Ang II (915 ± 154 vs. 1420 ± 131 fmol/ml, respectively; P < 0.05) (Figure 1, middle panel). Serum ACE2 activity was markedly higher in McACE2 mice (n = 6) compared with sham mice (n = 8) (63.0 ± 32.6 vs. 0.96 ± 0.18 RFU/ul/h, respectively) (Figure 1, left panel). Kidneys harvested after Ang II infusion, however, did not show any detectable increase in kidney ACE2 activity compared with kidneys from vehicle-treated mice (26.9 ± 5.3 vs. 29.4 ± 4.6 RFU/ug/h) (Figure 1, right panel). In additional experiments urinary Ang II (1-8) was evaluated after 1 week of Ang II administration. McACE2-pretreated (n = 8) and sham-pretreated control mice (n = 7) were administered Ang II (40 pmol/min) for 7 days using subcutaneous osmotic minipumps. No significant differences in urinary Ang II levels were found between mice pretreated with McACE2 or not (1781 ± 248 vs. 1794 ± 166 fmol Ang II per mg creatinine, respectively; P = 0.967). The levels of urinary Ang II in animals infused with this peptide were markedly higher than those in mice that did not receive Ang II infusion (n = 9) (217 ± 61 fmol Ang II per mg creatinine; P < 0.001). Altogether, these findings in wild-type mice showed that McACE2 delivery results in a marked increase in serum ACE2 activity, which facilitates decreasing plasma Ang II levels when this peptide is infused acutely. By contrast, urinary Ang II levels after 1 week of Ang II infusion are not affected significantly by prior McACE2 administration despite a large and sustained increase in serum ACE2 levels. The scheme for ACE2 amplification via minicircle DNA delivery and later induction of diabetes by STZ is shown in Figure 2. Six to 9 weeks after minicircle ace2 DNA injection or sham injection, STZ or vehicle was given. At 40 weeks of age, 20 weeks after diabetes induction and 26 to 29 weeks after ace2 DNA mini-circle or sham injection, the levels of blood glucose were increased similarly in both groups with STZ-induced diabetes (Table 1). The levels of serum ACE2 activity were increased profoundly in the diabetic group treated with ACE2 minicircles (n = 14) compared with untreated diabetic (n = 15) and nondiabetic controls (n = 9). Systolic blood pressure was not significantly different between STZ-McACE2 mice and STZ mice not treated with McACE2 (109 ± 5 and 106 ± 6 mm Hg, respectively). Body weight was similar in the 3 groups. Kidneys to body weight ratios were significantly higher in sham STZ and STZ/McACE2 compared with a nondiabetic control group but not different from each other. The heart to body weight ratio was not significantly different between the 3 groups. No significant difference in the albumin-to-creatinine ratio between the 2 diabetic groups was observed over the entire 20-week period of observation (Figure 3).Table 1General parameters in mice 20 weeks after diabetes induction with STZ and in vehicle-treated mice that served as a nondiabetic control groupParameterControlsSTZSTZ/McACE2Blood glucose, mg/dl177 ± 16aSignificant differences between controls and both STZ and STZ/McACE2 (at least at P < 0.05).457 ± 36449 ± 32Body weight, g24.3 ± 0.425.9 ± 0.626.1 ± 0.6Serum ACE2 activity, RFU/ul/h1.4 ± 0.3aSignificant differences between controls and both STZ and STZ/McACE2 (at least at P < 0.05).2.4 ± 0.3497 ± 135bSignificant difference versus controls and STZ mice not pretreated with McACE2 (P < 0.05). Otherwise there were no significant differences between untreated and McACE2-treated STZ mice.L + R kidney weight, g0.291 ± 0.0087aSignificant differences between controls and both STZ and STZ/McACE2 (at least at P < 0.05).0.437 ± 0.01380.415 ± 0.0204Kidney/body weight ratio, mg/g12.2 ± 0.4aSignificant differences between controls and both STZ and STZ/McACE2 (at least at P < 0.05).17.2 ± 0.516.3 ± 1.0Heart weight, g0.093 ± 0.0040.097 ± 0.0040.094 ± 0.003Heart/body weight, mg/g3.89 ± 0.183.79 ± 0.113.65 ± 0.11SBP, mm Hg125 ± 8106 ± 6109 ± 5L, left; McACE2, minicircle angiotensin-converting enzyme 2; R, right; RFU, relative fluorescence unit; SBP, systolic blood pressure; STZ, streptozotocin.Six to 9 weeks before induction of diabetes with STZ, animals were either injected with ace2 DNA minicircle (McACE2) or given a sham injection.a Significant differences between controls and both STZ and STZ/McACE2 (at least at P < 0.05).b Significant difference versus controls and STZ mice not pretreated with McACE2 (P < 0.05). Otherwise there were no significant differences between untreated and McACE2-treated STZ mice. Open table in a new tab Figure 3Urinary albumin-to-creatinine ratio (ACR) in streptozotocin (STZ) mice that were pretreated with angiotensin-converting enzyme 2 (ace2) DNA minicircle (gray bars, n = 14) or sham injected (black bars, n = 15–16). Vehicle-injected mice (white bars, that served as nondiabetic control group; n = 9) had ACR values significantly lower than both STZ-treated groups. There were no significant differences in ACR between sham STZ mice and minicircle angiotensin-converting enzyme (McACE2)-pretreated STZ mice at any of the time points measured. *Significantly different at least at a P < .05 level versus vehicle-injected nondiabetic controls.View Large Image Figure ViewerDownload Hi-res image Download (PPT) L, left; McACE2, minicircle angiotensin-converting enzyme 2; R, right; RFU, relative fluorescence unit; SBP, systolic blood pressure; STZ, streptozotocin. Six to 9 weeks before induction of diabetes with STZ, animals were either injected with ace2 DNA minicircle (McACE2) or given a sham injection. The glomerular filtration rate was increased markedly in both diabetic groups compared with controls, but it was not significantly different from eac
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