Doobay MF, Talman LS, Obr TD, Tian X, Davisson RL, Lazartigues E. ... Ye M, Wysocki J, William J, Soler MJ, Cokic I, Batlle D. Glomerular localization.
ACE/ACE2 IN DIABETIC MICE: IMPLICATIONS FOR NEPHROPATHY María José Soler*, Jan Wysocki, Minghao Ye, Eva Rodriguez and Daniel Batlle. Division of Nephrology & Hypertension, Department of Medicine. The Feinberg School of Medicine, Northwestern University. Chicago, USA. * Nephrology Department Hospital del Mar Universitat Autònoma de Barcelona. Barcelona, Spain.
Introduction Diabetic nephropathy (DN) is a microvascular complication of type 1 and type 2 diabetes, which is associated with end-stage renal disease (ESRD) and premature death from cardiovascular disease. The development of clinical nephropathy is insidious, and macroalbuminuria [urinary albumin excretion rate > 300 mg/24 hours], of the condition, is preceded by a phase of microalbuminuria (UAE 30–300 mg/24 hours), which usually lasts 5 to 10 years. As albuminuria worsens and blood pressure increases, there is a relentless decline in GFR and progression to ESRD. DN is now the most common single cause of end-stage renal disease (ESRD) in the US and Europe. Up to 40% of patients with type 2 diabetes will eventually develop diabetic nephropathy, and the number of patients progressing to ESRD has increased over the past 50 years. Although there is no cure for diabetic nephropathy, the rate of decline in renal function, and therefore progression to ESRD, can be slowed if it is detected and treated at an early stage. The renin-angiotensin system (RAS) plays an important role in the pathophysiology of many progressive renal diseases including diabetic nephropathy, and blockade of the RAS system with either angiotensin converting enzyme (ACE) or Angiotensin (ang) II receptor blockade attenuates progression of glomerular disease including DN . The recent discovery of Angiotensin converting enzyme 2, an active homologue of ACE, has expanded the knowledge of the RAS system. Whereas ACE promotes ang II formation from ang I, ACE2 promotes ang II degradation to the vasodilator peptide ang 1-7 (Figure 1). Moreover, ACE2 also catalyzes ang I to the inactive peptide ang 1-9 (Figure 1) .
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Figure 1: Renin-angiotensin system pathways for formation and degradation of angiotensin II.
ACE/ACE2 in the kidney Initially, ACE2 was thought to be restricted to the kidney, heart and testes. Subsequently, ACE2 was found in other organs such as lungs, central nervous system and placenta. Using a dual fluorometric method for the concurrent determination of ACE and ACE2 activity, our laboratory found that the activity of both enzymes was higher in kidney than in heart tissue samples . This finding suggests that the high ACE2 expression in the kidney tissue may play a crucial role in the regulation of the intrarenal RAS. We also found that in the kidney, ACE and ACE2 are co-localized in the brush border of proximal tubules, but within the glomerulus both enzymes are localized in distinct structures. Glomerular ACE2 is mainly present in podocytes and, to a lesser extent, in glomerular mesangial cells, whereas glomerular ACE, by contrast, is only in endothelial cells. In kidneys from healthy control subjects, Lely et al. have found ACE2 expression in tubular and glomerular epithelium and in vascular muscular smooth muscle cells and the endothelium of interlobular arteries. In agreement with our findings in mice they did not find ACE2 expression in glomerular endothelium.
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ACE/ACE2 in diabetic mice We examined the ACE2 and ACE glomerular expression in kidney samples from genetically obese-diabetic mouse (db/db). For this study, we used young female db/db mice to study an early phase of diabetes (3 to 4 wk of onset) without renal pathology by light microscopy. By immunohistochemistry, glomeruli from 8-wk-old db/db mice, showed increased ACE staining as compared with db/m. ACE2, by contrast, was decreased in db/db as compared with db/m. As mentioned before, glomerular ACE is mainly located in the endothelial cells, it seems that the increased ACE staining observed in the glomeruli from db/db mice points to an increase at the level of glomerular endothelial cells. On the other hand, the decreased ACE2 glomerular expression in db/db mice maybe reflects a decrease in protein expression at the level of the podocyte and possibly mesangial cells (or both). Taken these results together, we propose that in the diabetic mice there is an imbalance in the RAS with an excess of glomerular angiotensin II accumulation from increased ACE and decreased ACE2 in the glomeruli. The increased intraglomerular angiotensin II peptide in diabetic nephropathy results in an increase in albumin excretion rate, plasma creatinine, glomerular basement membrane width and a reduction in glomerular filtration rate. We also studied the effect of MLN-4760, a specific ACE2 inhibitor, on diabetic nephropathy. For this purpose we used two experimental models of diabetes: db/db mice (type 2 diabetes) and streptozotocin (STZ)-diabetic mice (type 1 diabetes). After chronic ACE2 inhibition, there was a significant increase in albumin excretion in the db/db mice as compared with vehicle treated db/db mice (figure 2). At the end of the study, 16 weeks of treatment, the albumin excretion was approximately three-fold higher in ML-4760treated db/db mice as compared with vehicle-treated db/db mice (figure 2). Moreover, glomerular staining for fibronectin, an extracellular matrix protein, was increased in db/db treated with MLN-4760. In addition, the specific AT1 blocker, telmisartan, prevented the increase in urinary albumin excretion that was associated with MLN-4760.
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Figure 2: Monitoring urinary albumin excretion (UAE) in db/db and in STZ mice receiving vehicle or a specific ACE2 inhibitor, MLN-4760. Left panel: diabetic db/db mice receiving MLN-4760 (db/db + MLN-4760) had significantly increased UAR after 12 wk of treatment as compared to vehicle treated db/db mice (db/db + vehicle). The increase in albuminuria remained until the mice were sacrificed. Right panel: diabetic STZ mice receiving MLN-4760 (STZ + MLN-4760) had significantly increased UAE after 4 wk of treatment as compared to vehicle treated STZ mice (STZ).
In concordance with the study mentioned above in db/db mice, chronic ACE2 inhibition, in another model of diabetes produced by STZ-mice increased albumin expression as compared
to
vehicle-treated
STZ-mice
(Figure
2).
Furthermore,
MLN-4760
administration worsened kidney histological lesions such as matrix mesangial expansion (Figure 3) and tubular hypertrophy in STZ-diabetic mice. It should be noted that ACE2 inhibition was associated with enhanced expression of ACE in both glomeruli and vasculature, suggesting that augmentation of this enzyme may play a role in the observed glomerular lesions following ACE2 inhibition. A combination of high ACE and low ACE2 is apt to increase Ang II formation, while decreasing Ang II degradation. Interestingly, renal medulla and papilla were clearly atrophic in STZ-mice treated with MLN-4760. It should be mentioned that in an ACE knockout, marked medullary and papillary atrophy are prominent findings.
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Figure 3: Ultrastructural analysis of glomeruli from non-diabetic control mice (left panel), STZ-diabetic mice treated with vehicle (middle panel) and STZ-diabetic mice treated with MLN-4760 (right panel): STZ-diabetic mice treated with vehicle shows mild mesangial expansion (M, middle panel), whereas STZdiabetic mice treated with MLN-4760 shows severe mesangial expansion (M, right panel). (magnification X 9500). M:mesangium
It has also been shown that the loss of ACE2 gene in male mice leads to the agedependent development of glomerular mesangial expansion with increased deposition of fibrillar collagens I/III and the extracellular matrix protein fibronectin. Wong et al. studied the effect of deletion of the angiotensin-converting enzyme 2 gene on diabetic kidney injury. For this purpose, ACE2-/- mice were crossed with Akita mice (Ins2WT/C96Y), a model of type 1 diabetes mellitus, and their respective wild type. At three months, ACE2-/y Ins2WT/C96Y mice showed increased urinary albumin excretion rate, in association with increased glomerular volume, increased mesangial matrix expansion, increased fibronectin, and α-SMA expression, and increased glomerular basement membrane thickening as compared with the ACE2+/yIns2WT/C96Y mice. Moreover, angiotensin II type 1 receptor blockade administration in the ACE2-/y Ins2WT/C96Y mice prevented the increase in albumin associated with ACE2 gene deletion. In conclusion, both the deletion of the ACE2 gene and chronic pharmacologic ACE2 inhibition are associated with accelerated kidney injury in diabetic mice. This suggests that ACE2 plays a protective role in diabetic kidney, and its lack leads to increased albumin excretion and mesangial matrix expansion. The fact that the administration of angiotensin II receptor blocker prevents the increase in albumin excretion, suggests that the deleterious effect of ACE2 gene deletion or pharmacological inhibition is mediated by angiotensin II via stimulation of the angiotensin II type 1 receptor. Based on their findings we have surmised that the ACE2 amplification either by gene therapy or with the
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