Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells.
Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin (Ang) II to Ang-(1-7), and protects against diabetic renal injury. Soluble ACE2 fragments are shed from the proximal tubule, and appear at high levels in the urine with diabetes. High glucose-induced shedding of ACE2 from proximal tubular cells is mediated by the enzyme "a disintegrin and metalloproteinase-17″ (ADAM17). Here, we investigated the mechanism for constitutive shedding of ACE2. Mouse proximal tubular cells were cultured and ACE2 shedding into the media was assessed by enzyme activity assay and immunoblot analysis. Cells were incubated with pharmacologic inhibitors, or transfected with silencing (si) RNA. Incubation of proximal tubular cells with increasing concentrations of D-glucose stimulated ACE2 shedding, which peaked at 16 mM, while L-glucose (osmotic control) had no effect on shedding. In cells maintained in 7.8 mM D-glucose, ACE2 shedding was significantly inhibited by the pan-protein kinase C (PKC) competitive inhibitor sotrastaurin, but not by an inhibitor of ADAM17. Incubation of cells with the PKC-α and -β1-specific inhibitor Go6976, the PKC β1 and β2-specific inhibitor ruboxistaurin, inhibitors of matrix metalloproteinases-2,-8, and -9, or an inhibitor of ADAM10 (GI250423X) had no effect on basal ACE2 shedding. By contrast, the PKC-δ inhibitor rottlerin significantly inhibited both constitutive and high glucose-induced ACE2 shedding. Transfection of cells with siRNA directed against PKC-δ reduced ACE2 shedding by 20%, while knockdown of PKC-ε was without effect. These results indicate that constitutive shedding of ACE2 from proximal tubular cells is mediated by PKC-δ, which is also linked to high glucose-induced shedding. Targeting PKC-δ may preserve membrane-bound ACE2 in proximal tubule in disease states and diminish Ang II-stimulated adverse signaling.
Division of Nephrology, Department of Medicine, Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa Ottawa, ON, Canada.
Fengxia
F
Xiao
Division of Nephrology, Department of Medicine, Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa Ottawa, ON, Canada.
Joseph
J
Zimpelmann
Division of Nephrology, Department of Medicine, Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa Ottawa, ON, Canada.
Dylan
D
Burger
Division of Nephrology, Department of Medicine, Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa Ottawa, ON, Canada.
Christopher
C
Kennedy
Division of Nephrology, Department of Medicine, Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa Ottawa, ON, Canada.
Richard L
RL
Hébert
Division of Nephrology, Department of Medicine, Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa Ottawa, ON, Canada.
Kevin D
KD
Burns
ACE2; ADAM17; PKC; diabetes; kidney; renin-angiotensin system; sheddase