synthetic ace2 peptide  (Mimetics)

Journal: American Journal of Physiology - Renal Physiology

Article Title: Increased urinary angiotensin converting enzyme 2 and neprilysin in patients with type 2 diabetes

doi: 10.1152/ajprenal.00565.2017

Figure Lengend Snippet: Urinary and plasma ACE2 activity in ND, Dnormo, Dmicro, and Dmacro subjects using a fluorogenic enzyme assay. A: urinary and plasma ACE2 activity was measured in 10–20μl of samples, *P < 0.01 vs ND B: ACE2 activity assay was validated using MLN-4760 (ACE2 inhibitor), ZPP (Prolyl endopeptidase/prolyl carboxypeptidase inhibitor), and thiorphan (NEP inhibitor). Urinary ACE2 activity without addition of inhibitors (control) was set at 100%. *P < 0.0001 vs. control. Each bar represents mean ± SE. ACE2, angiotensin converting enzyme 2; Dmacro, Type 2 diabetic patients with macroalbuminuria; Dmicro, Type-2 diabetic patients with microalbuminuria; Dnormo, Type 2 diabetic patients with normoalbuminuria; ND, nondiabetics; NEP, neprilysin; ZPP, Z-prolyl-prolinal.

Article Snippet: Urine samples equivalent to 10 μg of creatinine or 20 μl of plasma were incubated in the assay buffer (50 mM Tris, 5 mM ZnCl 2 , 150 mM NaCl 2, and 10 μM lisinopril) containing the synthetic ACE2 fluorogenic peptide substrate 7-Mca-APK-(Dnp) (Enzo Life Sciences, Farmingdale, NY) for 0.5–2 h. This fluorogenic peptide emits fluorescence, which was measured at excitation (λ ex ) of 328 nm and emission (λ em ) of 393 nm using a Fusion Packard instrument.

Techniques: Activity Assay, Enzymatic Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: Increased urinary angiotensin converting enzyme 2 and neprilysin in patients with type 2 diabetes

doi: 10.1152/ajprenal.00565.2017

Figure Lengend Snippet: Immunoblot analysis of urinary ACE2, NEP, ADAM17, and albumin in ND, Dnormo, Dmicro, and Dmacro subjects. Immunoblots were depicted using two patients, and the semiquantitative analysis is performed using four to six patients per group. A: urinary ACE2 expression was determined in ND and patients with diabetes with various degrees of albuminuria. Kidney lysate (2 μl) and urine (2 μl) from diabetic mice were used as positive controls. In mice, full length of ACE2 (100 kDa) was observed both in kidney and urine. In addition, fragmented ACE2 bands (75 kDa and 65 kDa) were seen in urine obtained from mice, whereas in humans, a 120-kDa immunoreactive band, as well as fragmented ACE2 immunoreactive bands at 75 kDa, ~70 kDa, and ~50 kDa were seen only in patients with diabetes. Fragmented urinary ACE2 bands (50 kDa) were quantified, and significant increase in urinary ACE2 expression was observed in patients with diabetes compared with ND (*P < 0.0001). B: urinary NEP expression was determined in ND and patients with diabetes using the volume equivalent to 10 μg creatinine. Kidney lysate (2 μl) and urine (2 μl) from diabetic mice were used as positive controls. In mice, full length of NEP (94 kDa) was observed in both kidney and urine. In addition, fragmented NEP immunoreactive bands at 70 kDa and 50 kDa were seen in urine obtained from mice, whereas in humans, full-length as well as fragmented NEP immunoreactive bands were seen at ~110 kDa, ~70 kDa, and ~50 kDa with increased intensity in patients with diabetes. Fragmented NEP immunoreactive bands were not observed in ND patients. Fragmented urinary NEP bands (70 kDa) were quantified, and significant increase in urinary NEP expression was observed in patients with diabetes compared with ND (*P < 0.02). C: urinary ADAM17 expression in ND and patients with diabetes according to volume equivalent to 10 μg creatinine. Kidney lysate (2 μl) and urine (2 μl) from diabetic mice were used as positive controls. The diabetic mouse kidney shows several immunoreactive bands for ADAM17 (~93 kDa, ~65 kDa, and ~55 kDa). A 55-kDa immunoreactive band was observed in urine obtained from diabetic mice. In humans, a 70-kDa immunoreactive band for urinary ADAM17 was only seen in patients with diabetes with increased intensity in patients with diabetes compared with ND subjects (*P < 0.0001). D: urinary albumin was determined in volume equivalent to 10 μg creatinine. A predominant band for albumin at 66 kDa was detected in kidney and urine obtained from diabetic mice and patients with diabetes. Significant increase of urinary albumin expression was observed in patients with diabetes compared with ND (*P < 0.002). ACE2, angiotensin converting enzyme 2; ADAM17, a disintegrin and metalloproteinase 17; Dmacro, patients with type 2 diabetes with macroalbuminuria; Dmicro, patients with type 2 diabetes with microalbuminuria; Dnormo, patients with type 2 diabetes with normoalbuminuria; ND, nondiabetics; NEP, neprilysin.

Article Snippet: Urine samples equivalent to 10 μg of creatinine or 20 μl of plasma were incubated in the assay buffer (50 mM Tris, 5 mM ZnCl 2 , 150 mM NaCl 2, and 10 μM lisinopril) containing the synthetic ACE2 fluorogenic peptide substrate 7-Mca-APK-(Dnp) (Enzo Life Sciences, Farmingdale, NY) for 0.5–2 h. This fluorogenic peptide emits fluorescence, which was measured at excitation (λ ex ) of 328 nm and emission (λ em ) of 393 nm using a Fusion Packard instrument.

Techniques: Western Blot, Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Increased urinary angiotensin converting enzyme 2 and neprilysin in patients with type 2 diabetes

doi: 10.1152/ajprenal.00565.2017

Figure Lengend Snippet: Mass spectrometric based enzyme activity assay for urinary ACE2 and NEP in ND, Dnormo, Dmicro, and Dmacro subjects. A: urinary ACE2 activity was measured in volume equivalent to 25 µg of creatinine. Samples were incubated for 2 h at 37°C in 0.4 M MES buffer pH 6.75 containing 0.1 mg/ml Ang II. B: urinary ACE2 activity inhibition in the presence of specific ACE2 inhibitor, MLN-4760 (0.1 mM) C: urinary NEP activity was measured in volume equivalent to 25 µg of creatinine incubated for 2 h at 37°C in 0.4 M MES buffer pH 6.75 containing 1 mg/ml Ang I. D: urinary NEP activity inhibition in the presence of specific NEP inhibitor thiorphan (0.1 mM). ACE2, angiotensin converting enzyme 2; And I, angiotensin I; Ang II, angiotensin II; Dmacro, patients with type 2 diabetes with macroalbuminuria; Dmicro, patients with type 2 diabetes with microalbuminuria; Dnormo, patients with type 2 diabetes with normoalbuminuria; m/z, mass-to-charge ratio; ND, nondiabetics; NEP, neprilysin.

Article Snippet: Urine samples equivalent to 10 μg of creatinine or 20 μl of plasma were incubated in the assay buffer (50 mM Tris, 5 mM ZnCl 2 , 150 mM NaCl 2, and 10 μM lisinopril) containing the synthetic ACE2 fluorogenic peptide substrate 7-Mca-APK-(Dnp) (Enzo Life Sciences, Farmingdale, NY) for 0.5–2 h. This fluorogenic peptide emits fluorescence, which was measured at excitation (λ ex ) of 328 nm and emission (λ em ) of 393 nm using a Fusion Packard instrument.

Techniques: Enzyme Activity Assay, Activity Assay, Incubation, Inhibition

Journal: American Journal of Physiology - Renal Physiology

Article Title: Increased urinary angiotensin converting enzyme 2 and neprilysin in patients with type 2 diabetes

doi: 10.1152/ajprenal.00565.2017

Figure Lengend Snippet: Linear regression analysis of urinary ACE2 and NEP with renal functional parameters. (A–D): urinary ACE2 activity was significantly and positively correlated with HbA1C (r = 0.42, **P < 0.0003), BUN (r = 0.28, *P < 0.014), and SCr (r = 0.25, *P < 0.03) but negatively correlated with eGFR (r = −0.22, *P < 0.04). (E–H): a linear regression analysis of urinary NEP activity was only significantly and positively correlated with HbA1C (r = 0.33, *P < 0.01) but no correlation with eGFR, BUN, or SCr. ACE2, angiotensin converting enzyme 2; eGFR, estimated glomerular filtration rate (ml/min/1.73 m2); HbA1C, glycated hemoglobin; BUN, blood urea nitrogen (mg/dl); NEP, neprilysin; SCr, serum creatinine (mg/dl).

Article Snippet: Urine samples equivalent to 10 μg of creatinine or 20 μl of plasma were incubated in the assay buffer (50 mM Tris, 5 mM ZnCl 2 , 150 mM NaCl 2, and 10 μM lisinopril) containing the synthetic ACE2 fluorogenic peptide substrate 7-Mca-APK-(Dnp) (Enzo Life Sciences, Farmingdale, NY) for 0.5–2 h. This fluorogenic peptide emits fluorescence, which was measured at excitation (λ ex ) of 328 nm and emission (λ em ) of 393 nm using a Fusion Packard instrument.

Techniques: Functional Assay, Activity Assay, Filtration

Journal: American Journal of Physiology - Renal Physiology

Article Title: Increased urinary angiotensin converting enzyme 2 and neprilysin in patients with type 2 diabetes

doi: 10.1152/ajprenal.00565.2017

Figure Lengend Snippet: The selected predictors for HbA1C and eGFR responses using stepwise regression

Article Snippet: Urine samples equivalent to 10 μg of creatinine or 20 μl of plasma were incubated in the assay buffer (50 mM Tris, 5 mM ZnCl 2 , 150 mM NaCl 2, and 10 μM lisinopril) containing the synthetic ACE2 fluorogenic peptide substrate 7-Mca-APK-(Dnp) (Enzo Life Sciences, Farmingdale, NY) for 0.5–2 h. This fluorogenic peptide emits fluorescence, which was measured at excitation (λ ex ) of 328 nm and emission (λ em ) of 393 nm using a Fusion Packard instrument.

Techniques:

Journal: American Journal of Physiology - Renal Physiology

Article Title: Increased urinary angiotensin converting enzyme 2 and neprilysin in patients with type 2 diabetes

doi: 10.1152/ajprenal.00565.2017

Figure Lengend Snippet: ROC curves to predict HbA1C (>6.5) and eGFR (<60 ml/min/1.73 m2). A: ROC curves for urinary ACE2 (AUC = 0.6117) and urinary NEP (AUC = 0.6211) for the prediction of HbA1C (>6.5%). B: ROC curves for urinary ACE2 (AUC = 0.6117) and plasma NEP (AUC = 0.6211) for the prediction of eGFR (<60 ml/min/1.73 m2). C: the addition of age, urinary ACE2, and urinary NEP to the model for predicting HbA1C (>6.5) in all the patients (AUC = 0.7367). D: inclusion of age, urinary ACE2, and plasma NEP to the model for predicting eGFR (<60ml/min/1.73 m2) in all the patients (AUC = 0.76). ACE2, angiotensin converting enzyme 2; AUC, areas under the ROC; eGFR, estimated glomerular filtration rate; HbA1C, glycated hemoglobin; NEP, neprilysin; ROC, receiver operating characteristic.

Article Snippet: Urine samples equivalent to 10 μg of creatinine or 20 μl of plasma were incubated in the assay buffer (50 mM Tris, 5 mM ZnCl 2 , 150 mM NaCl 2, and 10 μM lisinopril) containing the synthetic ACE2 fluorogenic peptide substrate 7-Mca-APK-(Dnp) (Enzo Life Sciences, Farmingdale, NY) for 0.5–2 h. This fluorogenic peptide emits fluorescence, which was measured at excitation (λ ex ) of 328 nm and emission (λ em ) of 393 nm using a Fusion Packard instrument.

Techniques: Filtration

Journal: Endocrinology

Article Title: The Identification of a Calmodulin-Binding Domain within the Cytoplasmic Tail of Angiotensin-Converting Enzyme-2

doi: 10.1210/en.2008-1274

Figure Lengend Snippet: Sequence comparison of ACE2 to GPVI and L-Selectin. Top panel , Cytoplasmic juxtamembrane sequence of human ACE2 compared with functional CaM-binding juxtamembrane sequences that regulate metalloproteinase-mediated ectodomain shedding of platelet GPVI and leukocyte L-selectin ( , ). Shown are alignments of residues in the cytoplasmic tails of ACE2, GPVI, and L-selectin. Conserved residues are highlighted ( bottom panel ). Synthetic peptides corresponding to residues 762–777 of human ACE2 and peptide variants (mut 1–4) containing mutated amino acid residues.

Article Snippet: Complexes formed between CaM and a 16-residue synthetic peptide corresponding to residues 762–777 within the cytoplasmic domain of ACE2 (ACE2T2 = CFTGIRDRKKKNKARSG-amide; Auspep) were analyzed by gel shift assay as described by Erickson-Viitanen and Degrado ( ) and previously used to identify CaM-binding peptides ( , – ).

Techniques: Sequencing, Comparison, Functional Assay, Binding Assay

Journal: Endocrinology

Article Title: The Identification of a Calmodulin-Binding Domain within the Cytoplasmic Tail of Angiotensin-Converting Enzyme-2

doi: 10.1210/en.2008-1274

Figure Lengend Snippet: Binding of ACE2 from cell lysates by GST-CaM. Cell lysates (∼2 mg protein) from ACE2-transfected CHOP cells were incubated with glutathione Sepharose beads and GST (20 μg), or GST-CaM (20 μg) bait for 16 h. After incubation, samples were separated by SDS-PAGE and immunoblotted for ACE2 (A) and GST (B) antibodies. Cell lysates (transfected with ACE2) and soluble ACE2 were used as controls for Western blotting.

Article Snippet: Complexes formed between CaM and a 16-residue synthetic peptide corresponding to residues 762–777 within the cytoplasmic domain of ACE2 (ACE2T2 = CFTGIRDRKKKNKARSG-amide; Auspep) were analyzed by gel shift assay as described by Erickson-Viitanen and Degrado ( ) and previously used to identify CaM-binding peptides ( , – ).

Techniques: Binding Assay, Transfection, Incubation, SDS Page, Western Blot

Journal: Endocrinology

Article Title: The Identification of a Calmodulin-Binding Domain within the Cytoplasmic Tail of Angiotensin-Converting Enzyme-2

doi: 10.1210/en.2008-1274

Figure Lengend Snippet: Shedding of ACE2 is increased by CaM inhibitors. A, Huh-7 cells that endogenously express ACE2 were incubated in OptiMEM containing 25 μmol/liter TFP or in an equal volume of carrier (Me 2 SO) for 16 h. The medium was subsequently harvested and concentrated 50-fold, whereas the cells were pelleted and detergent solubilized, as described in Materials and Methods . Media (25 μg protein) (A) and cell lysates (25 μg protein) (B) were assayed for their ability to cleave an ACE2 fluorogenic substrate. The individual mean control ACE2 activity with TFP treatment in media and cell lysate were calculated. The data are normalized against the controls from at least four independent experiments (n ≥ 4, Student’s t test; unpaired, two tailed). Asterisk denotes significant difference ( P < 0.05) compared against control. C, One hundred micrograms total protein from the media and cell lysates were separated by SDS-PAGE and Western blotted with monoclonal ACE2 antibody. Soluble ACE2 (SACE2) was used as positive control for immunoblotting. D, Huh-7 cells were incubated in OptiMEM containing combinations of 25 μmol/liter CMZ, BIM (2 μmol/liter), or PMA (1 μmol/liter) and an equal volume of carrier (Me 2 SO) for 1 h. The medium was prepared and analyzed as described above. The mean control ACE2 activity after CMZ chloride treatment in the media was calculated. The data are normalized against the controls from at least four independent experiments (n ≥ 4, one-way ANOVA). Asterisk denotes significant difference ( P < 0.05) compared against control.

Article Snippet: Complexes formed between CaM and a 16-residue synthetic peptide corresponding to residues 762–777 within the cytoplasmic domain of ACE2 (ACE2T2 = CFTGIRDRKKKNKARSG-amide; Auspep) were analyzed by gel shift assay as described by Erickson-Viitanen and Degrado ( ) and previously used to identify CaM-binding peptides ( , – ).

Techniques: Incubation, Control, Activity Assay, Two Tailed Test, SDS Page, Western Blot, Positive Control