Soluble ACE2 Is Filtered into the Urine.

Background: ACE2 is a key enzyme in the renin-angiotensin system (RAS) capable of balancing the RAS by metabolizing angiotensin II (AngII). First described in cardiac tissue, abundance of ACE2 is highest in the kidney, and it is also expressed in several extrarenal tissues. Previously, we reported an association between enhanced susceptibility to hypertension and elevated renal AngII levels in global ACE2-knockout mice. Methods: To examine the effect of ACE2 expressed in the kidney, relative to extrarenal expression, on the development of hypertension, we used a kidney crosstransplantation strategy with ACE2-KO and WT mice. In this model, both native kidneys are removed and renal function is provided entirely by the transplanted kidney, such that four experimental groups with restricted ACE2 expression are generated: WT→WT (WT), KO→WT (KidneyKO), WT→KO (SystemicKO), and KO→KO (TotalKO). Additionally, we used nanoscale mass spectrometry-based proteomics to identify ACE2 fragments in early glomerular filtrate of mice. Results: Although significant differences in BP were not detected, a major finding of our study is that shed or soluble ACE2 (sACE2) was present in urine of KidneyKO mice that lack renal ACE2 expression. Detection of sACE2 in the urine of KidneyKO mice during AngII-mediated hypertension suggests that sACE2 originating from extrarenal tissues can reach the kidney and be excreted in urine. To confirm glomerular filtration of ACE2, we used micropuncture and nanoscale proteomics to detect peptides derived from ACE2 in the Bowman's space. Conclusions: Our findings suggest that both systemic and renal tissues may contribute to sACE2 in urine, identifying the kidney as a major site for ACE2 actions. Moreover, filtration of sACE2 into the lumen of the nephron may contribute to the pathophysiology of kidney diseases characterized by disruption of the glomerular filtration barrier.

Interleukin-9 mediates chronic kidney disease-dependent vein graft disease: a role for mast cells.

AIMS: Chronic kidney disease (CKD) is a powerful independent risk factor for cardiovascular events, including vein graft failure. Because CKD impairs the clearance of small proteins, we tested the hypothesis that CKD exacerbates vein graft disease by elevating serum levels of critical cytokines that promote vein graft neointimal hyperplasia. METHODS AND RESULTS: We modelled CKD in C57BL/6 mice with 5/6ths nephrectomy, which reduced glomerular filtration rate by 60%, and we modelled vein grafting with inferior-vena-cava-to-carotid interposition grafting. CKD increased vein graft neointimal hyperplasia four-fold, decreased vein graft re-endothelialization two-fold, and increased serum levels of interleukin-9 (IL-9) five-fold. By quantitative immunofluorescence and histochemical staining, vein grafts from CKD mice demonstrated a ∼two-fold higher prevalence of mast cells, and a six-fold higher prevalence of activated mast cells. Concordantly, vein grafts from CKD mice showed higher levels of TNF and NFκB activation, as judged by phosphorylation of NFκB p65 on Ser536 and by expression of VCAM-1. Arteriovenous fistula veins from humans with CKD also showed up-regulation of mast cells and IL-9. Treating CKD mice with IL-9-neutralizing IgG reduced vein graft neointimal area four-fold, increased vein graft re-endothelialization ∼two-fold, and reduced vein graft total and activated mast cell levels two- and four-fold, respectively. Treating CKD mice with the mast cell stabilizer cromolyn reduced neointimal hyperplasia and increased re-endothelialization in vein grafts. In vitro, IL-9 promoted endothelial cell apoptosis but had no effect on smooth muscle cell proliferation. CONCLUSION: CKD aggravates vein graft disease through mechanisms involving IL-9 and mast cell activation.

ACE2 deficiency increases NADPH-mediated oxidative stress in the kidney.

Abstract Angiotensin-converting enzyme 2 (ACE2) is highly expressed in the kidney and hydrolyzes angiotensin II (Ang II) to Ang(1-7). Since Ang II is a strong activator of oxidative stress, we reasoned that ACE2 could be involved in the regulation of renal oxidative stress by governing the levels of Ang II. We, therefore, assessed levels of oxidative stress in kidney cortex of ACE2 knockout and wild-type littermate mice under baseline conditions. We found multiple markers of increased oxidative stress in ACE2KO mice. NADPH oxidase activity was increased in kidney cortex from ACE2KO mice as compared to WT (227 ± 24% vs.100 ± 19%, P < 0.001). However, kidney catalase and superoxide dismutase activities were not different between groups. Exogenous Ang II was degraded less efficiently by kidneys from ACE2KO mice than WT mice, and administration of an AT1R blocker (losartan 30 mg/kg/day) resulted in normalization of NADPH oxidase activity in the ACE2KO. These findings suggest that an AT1R-dependent mechanism contributes to increased ROS observed in the ACE2KO. This study demonstrates that genetic deficiency of ACE2 activity in mice fosters oxidative stress in the kidney in the absence of overt hypertension and is associated with reduced kidney capacity to hydrolyze Ang II. ACE2KO mice serve as a novel in vivo model to examine the role of overactivity of NADPH oxidase in kidney function.

Diabetic kidney disease in FVB/NJ Akita mice: temporal pattern of kidney injury and urinary nephrin excretion.

Akita mice are a genetic model of type 1 diabetes. In the present studies, we investigated the phenotype of Akita mice on the FVB/NJ background and examined urinary nephrin excretion as a marker of kidney injury. Male Akita mice were compared with non-diabetic controls for functional and structural characteristics of renal and cardiac disease. Podocyte number and apoptosis as well as urinary nephrin excretion were determined in both groups. Male FVB/NJ Akita mice developed sustained hyperglycemia and albuminuria by 4 and 8 weeks of age, respectively. These abnormalities were accompanied by a significant increase in systolic blood pressure in 10-week old Akita mice, which was associated with functional, structural and molecular characteristics of cardiac hypertrophy. By 20 weeks of age, Akita mice developed a 10-fold increase in albuminuria, renal and glomerular hypertrophy and a decrease in the number of podocytes. Mild-to-moderate glomerular mesangial expansion was observed in Akita mice at 30 weeks of age. In 4-week old Akita mice, the onset of hyperglycemia was accompanied by increased podocyte apoptosis and enhanced excretion of nephrin in urine before the development of albuminuria. Urinary nephrin excretion was also significantly increased in albuminuric Akita mice at 16 and 20 weeks of age and correlated with the albumin excretion rate. These data suggest that: 1. FVB/NJ Akita mice have phenotypic characteristics that may be useful for studying the mechanisms of kidney and cardiac injury in diabetes, and 2. Enhanced urinary nephrin excretion is associated with kidney injury in FVB/NJ Akita mice and is detectable early in the disease process.