Novel Variants of Angiotensin Converting Enzyme-2 of Shorter Molecular Size to Target the Kidney Renin Angiotensin System

Figures

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  Figure 1. Native mouse recombinant (mr)ACE2 (100–110 kD) was spiked into ACEKO urine (Left) or kidney cortex lysate (10 nM mrACE2/~1 mg total protein of the lysate) from one ACE2KO mouse (Right) and incubated at 37 °C for 48 h. Spiked native mrACE2 samples at 0, 2, 4, 8, 24 and 48 h of incubation were subsequently probed in Western blot. Left Panel. Western blot image of ACE2KO urine spiked with native mrACE2 shows gradual weakening of the 100–110 kD native mrACE2 band and the appearance of smaller 75 kD ACE2 immunoreactive band for up to 8 h. At 24 and 48 h, no ACE2 immunoreactive bands and no ACE2 activity are detectable anymore. Right Panel. Western blot image shows the disappearance of the 100–110 kD mrACE2 band and, first the appearance of a smaller 75 kD ACE2 immunoreactive band, and then a ~60–70 kD band in kidney lysates from ACE2KO mice. In the lower panel, ACE2 activity is depicted showing similar enzyme activities of the 75 and ~60 kD bands versus the original 110 kD mrACE2 band throughout the incubation period; a single time-course experiment for kidney and for urine is shown.

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  Figure 2. Native mouse recombinant (mr)ACE2 1-740 AA (100–110kD) was spiked into urine (10 nM mrACE2/~0.5 mL urine) (A) and kidney cortex lysates (10 nM mrACE2/~1 mg total protein of the lysate) from three ACE2KO mice (B) and incubated at 37 °C for 6 h. Spiked mrACE2 samples at 0 h (Start) and at 6 h of incubation were subsequently probed in Western blot using an anti-ACE2 antibody. Western blot image shows the disappearance of the 100–110 kD mrACE2 band and the appearance of a smaller 75 kD ACE2 immunoreactive band at 6 h in urine and kidney lysates from ACE2KO mice. In the lower panel, ACE2 activity is depicted showing the enzyme activity of the 75 kD band and the spiked original 100–110 kD native mrACE2 band; urines and kidney lysates from three independent degradation experiments.

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  Figure 3. The native mrACE2 spiked into urine (A) and kidney (B) samples from ACE2KO mice had a c-terminal 10-His tag. The Western blot membrane shown in Figure 2 was re-probed using an anti-His antibody. Western blot image shows disappearance of the 100–110 kD mrACE2 band and no appearance of a smaller 75 kD ACE2 immunoreactive band in urine and kidney lysates from ACE2KO mice, suggesting the proteolysis of native mrACE2 from its c-terminal end; urines and kidney lysates from three independent degradation experiments.

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  Figure 4. Enzyme activity and molecular size of shorter recombinant ACE2 protein variants. Relative activity of mouse small recombinant ACE2variants and ACE2 protein detection in conditioned media collected 40–60 h after transfection. (A) The cleavage rate of the synthetic fluorogenic substrate Mca-AKP(Dnp) over one hour (shown in relative fluorescence units—RFU) for native rACE2 and small ACE2 variants. Similar to native soluble mrACE2 1-740, mrACE2 variants, 1-619- and 1-605- showed enzymatic activity, respectively, while 1-522 did not exhibit any ACE2 activity. (B) Presence and estimated molecule size were confirmed by Western Blot with antibody against ACE2 ectodomain.

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  Figure 5. Summary of ACE2-activity assessment of highly purified mouse recombinant ACE2 1-619 and ACE2 1-605 compared to native rACE2 1-740 with a synthetic fluorogenic substrate Mca-APK(Dnp) (A) and with the natural substrate, angiotensin II (B). A higher catalytic activity (Kcat/Km) was found by cleavage of Mca-APK(Dnp) for rACE2 1-619 and rACE2 1-605 than for native rACE2 1-740 (Panel A). The catalytic efficiency to convert Angiotensin II to Angiotensin (1–7) (Kcat/Km) was similar for native rACE2 1-740 and for the small rACE2 variants 1-619 and 1-605 (Panel B). For assessment of activity levels, a Michaelis–Menten model was used to derive the parameters of catalytic kinetics such as Km and Kcat. The values were calculated from three to four independent experiments.

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  Figure 6. Pharmacokinetics of small ACE2 variants in vivo after i.v. (A) or i.p. injection (B). Wild-type mice (n = 5–6 per experiment) were injected with 1 µg/g BW of native murine rACE2 1-740 (reference protein) or mouse small rACE2 variants 1-619 and 1-605. Venous blood was collected at defined time points after bolus rACE2 injection and ACE2 activity measured using Mca-APK-Dnp fluorogenic substrate; RFU—relative fluorescence unit.

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  Figure 7. Pre-infusion of 1-619 (A) or 1-605 (B) rACE2 variants (filled circles) cause a faster recovery from acute Ang II-induced hypertension as compared to respective mice pre-infused with PBS as a vehicle (Veh; empty squares). Y-axis is SBP and X-axis indicates time (min) from Ang II bolus (arrow; 0.2 µg/g BW). Two-way ANOVA over time was used for comparisons of the experimental groups; * denotes p < 0.01.

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  Figure 8. Western blot of urines collected before (Baseline) and after i.v. bolus of ACE2 1-619 variant (0–2 h) from five ACE2-deficient mice, showing an ACE2-immunoreactive band at the expected size of ~70 kD, consistent with the molecular size of the short rACE2 1-619 variant after, but not before, the injection. M34-M38 are ID numbers of the individual ACE2 deficient mice (n = 5) from which urines were obtained at baseline and within 2 h after mrACE2 1-619 infusion. Equal volumes (36 μL) of urines with similar creatinine concentrations (~10–20 mg/dL) were loaded per well.

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  Figure 9. Glomerular filtration and tubular uptake of 1-605- (blue, n = 8) and 1-619-mACE2 (green, n = 6) as compared to native 1-740 rACE2 (red, n = 4) in ACE2-deficient mice. Immediately after voiding (baseline urine collection), ACE2-deficient mice were injected i.v. with 1 µg/g BW rACE2. Urine was collected again within the first 2 h after ACE2-injection (“rACE2”). L-Lysine was injected i.p. two hours after rACE2-injection and urine was collected within the next 2 h. (“rACE2/L-Lysine”). Recovery of ACE2-activity in the urine normalized to creatinine excretion is depicted. Repeated measures one-way ANOVA was used for comparisons within the experimental groups, followed by post-hoc analysis; * denotes p < 0.05 or p < 0.01.

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  Figure 10. ACE2-deficient mice were infused i.v. with a bolus of PBS, native mouse rACE2 (1-740) or mouse rACE2 1-619 (1 µg/g BW). After 2 h, mice were perfused with saline and the kidneys removed. (A) A gain in kidney ACE2 activity was only appreciated in 1-619-infused mice. (B) In an ex vivo setting, kidney lysates (2 mg) were incubated with Ang II (10−5M) to assess Ang 1–7 formation over time by 2-way Anova. Higher Ang 1–7 formation was seen in 1-619 mice as compared to the two other groups; ** denotes p < 0.01 against 1-740 and p < 0.001 against PBS.