Inhibition of Endocytosis of Clathrin-Mediated Angiotensin II Receptor Type 1 in Podocytes Augments Glomerular Injury

Abstract

BackgroundInhibition of the renin-angiotensin system remains a cornerstone in reducing proteinuria and progression of kidney failure, effects believed to be the result of reduction in BP and glomerular hyperfiltration. However, studies have yielded conflicting results on whether podocyte-specific angiotensin II (AngII) signaling directly induces podocyte injury. Previous research has found that after AngII stimulation, β-arrestin–bound angiotensin II receptor type 1 (AT1R) is internalized in a clathrin- and dynamin-dependent manner, and that Dynamin1 and Dynamin2 double-knockout mice exhibit impaired clathrin-mediated endocytosis.MethodsWe used podocyte-specific Dyn double-knockout mice to examine AngII-stimulated AT1R internalization and signaling in primary podocytes and controls. We also examined the in vivo effect of AngII in these double-knockout mice through renin-angiotensin system blockers and through deletion of Agtr1a (which encodes the predominant AT1R isoform expressed in kidney, AT1aR). We tested calcium influx, Rac1 activation, and lamellipodial extension in control and primary podocytes of Dnm double-knockout mice treated with AngII.ResultsWe confirmed augmented AngII-stimulated AT1R signaling in primary Dnm double-knockout podocytes resulting from arrest of clathrin-coated pit turnover. Genetic ablation of podocyte Agtr1a in Dnm double-knockout mice demonstrated improved albuminuria and kidney function compared with the double-knockout mice. Isolation of podocytes from Dnm double-knockout mice revealed abnormal membrane dynamics, with increased Rac1 activation and lamellipodial extension, which was attenuated in Dnm double-knockout podocytes lacking AT1aR.ConclusionsOur results indicate that inhibiting aberrant podocyte-associated AT1aR signaling pathways has a protective effect in maintaining the integrity of the glomerular filtration barrier.

Inhibiting calpain 1 and 2 in cyclin G associated kinase–knockout mice mitigates podocyte injury

“…Previous studies, including ours, have suggested the importance of the clathrin-mediated endocytic pathway in maintaining the integrity of the glomerular filtration barrier (1,(32)(33)(34)(35). In order to examine the clinical relevance of endocytosis in human disease, we probed a human database, Nephroseq, and discovered that decreased glomerular expression of clathrin-mediated endocytic pathways correlated with the reduction in GFR.…”

“…Administration of calpain-1/-2 inhibitors, or targeted deletion of the calpain-1/-2 heterodimer's common regulatory domain, Capns1, reduced both podocyte injury and damage to the glomerular filtration barrier. Endocytosis can be controlled and triggered by calcium channels at the cell membrane and intracellular calcium concentrations (38,39), suggesting that impairment in endocytosis induced by the loss of Gak, similar to the loss of Dnm1/2 inhibiting angiotensin II receptor type 1 internalization, results in the dysregulation of cytoplasmic calcium homeostasis (32) and following calpain activation, explaining the observed increase. However, it is unclear in our current study whether loss of podocyte-associated Gak directly leads to calpain activation because podocyte injury in itself (37).…”

Angiotensin II promotes podocyte injury by activating Arf6-Erk1/2-Nox4 signaling pathway

“…In our study, inhibition of Arf6 activity by both secinH3 and Arf6 knockdown dramatically prevented Ang IIinduced caspase 3 activation as well as Nox4 and ROS production, suggesting that elevation of active Arf6-GTP by Ang II is required for Nox4 upregulation and ROS production. Just recently, it has been reported that in dynamin1/2-deficient primary podocytes, Ang II induces abnormal membrane dynamics with increased Rac1 activation and lamellipodial extension, which was attenuated in deficiency of AT1R [32]. This finding suggests that the internalization of AT1R is blunted and Ang II signal is prolonged in podocytes with dynamin1/2 deficiency.…”

Identification of Podocyte Cargo Proteins by Proteomic Analysis of Clathrin-Coated Vesicles

“…Under physiological circumstances, podocytes heavily rely on CME for maintenance of the slit-diaphragm for internalizing nephrin (5,8) as well as receptors such as the type Ia angiotensin II receptor (9). However, to our knowledge, CCV proteomics to identify CCV-associated proteins have not been elucidated in podocytes.…”