Angiotensin II increases glomerular permeability by β-arrestin mediated nephrin endocytosis

Figures

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  Figure 1. Ang II increases the glomerular permeability and enhances the interaction of β-arrestin to nephrin. (a) Ang II increases the glomerular permeability significantly in FVB mice (n = 5 per data point, p < 0.004 tested by Kruskal-Wallis test). The glomerular permeability was measured by FITC-Ficoll 70 appearance in the urine before (white columns, 0 min), 60 minutes after the begin of Ang II stimulation (black columns, 60 min) and after additional 60 minutes of Ang II stimulation or 60 minutes of Ang II discontinuation washout (grey columns, 120 min or +60 min). The measured FITC-Ficoll 70 concentrations in the urine were referenced to the urine creatinine concentration. Pretreatment with candesartan blocks the Ang II mediated increased glomerular permeability (control n = 9, 0 min vs 60 min p = 0.35, 60 min vs 120 min p = 0.87; Ang II n = 9, 0 min vs 60 min p = 0.0036, 60 min vs 120 min p = 0.18, 0 min vs 120 min p = 0.16; Ang II + washout n = 5, 0 min vs 60 min p = 0.038, 60 min vs 120 min p = 0.25, 0 min vs 120 min p = 0.58; Ang II + ARB n = 3, 0 min vs 60 min p = 0.99). (b) Systolic blood pressure was controlled by tail cuff measurement. No significant systolic blood pressure…

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  Figure 2. Ang II promotes nephrin endocytosis. (a) Ang II induces the endocytosis of nephrin in HEK293T cells significantly in a time dependent fashion. The biotinylated nephrin fraction decreases the longer the Ang II exposure lasts (Kruskal-Wallis test: *n = 5, p = 0.04; **n = 5, p = 0.009). The AT1-receptor plasmid was cotransfected in all conditions to the AT1-receptor deficient HEK293T cells. (b) Nephrin endocytosis in murine podocytes is significantly enhanced by Ang II (*n = 5, p = 0.03). Biotinylated nephrin is significantly decreased in mouse podocytes when they are stimulated with Ang II. (c) Nephrin endocytosis in mice without and with Ang II stimulation and additional candesartan treatment. Biotinylated nephrin is significantly decreased in Ang II treated animals compared to control mice and restored to nearly control levels by additional candesartan treatment. (*Kruskal-Wallis test: n = 5, p = 0.02).

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  Figure 3. Ang II enhances the interaction of nephrin with β-arrestin2 through AT1 receptor activation. (a) HEK293T cells expressing nephrin, β-arrestin2 and the AT1-receptor were stimulated with Ang II. Ang II stimulation enhances the binding of β-arrestin2 to nephrin in a time dependent fashion. The maximum enhancement of 3-fold is seen after 60 min of Ang II stimulation (**Kruskal-Wallis test n = 12, p = 0.026). (b) HEK293T cells lacking the AT1 receptor do not show enhanced binding of nephrin to β-arrestin2 under stimulation with Ang II (**n = 9, p = 0.0019). The Ang II mediated enhanced binding of β-arrestin2 to nephrin is blocked by the AT1 receptor antagonist (c) Candesartan [100 nM] (*n = 3, p = 0.03, ns = 0.314). Comparable amounts of nephrin fusion protein expression and its control were controlled by western blot (experiments were conducted in HEK293T cells). The expression of the transfected nephrin fusion proteins and its control is shown in the supplemental Fig. 1Sb–d. The Ang II mediated activation of ERK is shown in the supplemental Fig. 3Sa.

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  Figure 4. The Gαi signaling is essential in conveying the Ang II mediated, enhanced β-arrestin2 binding to the nephrin-c-terminus and nephrin endocytosis. (a) The AT1 receptor D125AR126L mutant abolishes G-protein signaling and disables the Ang II induced enhanced β-arrestin2 binding to the nephrin-c-terminus (*n = 4, p = 0.014). (b) The inhibition of Gαi by pertussis toxin (PTX) does not prevent the Ang II mediated enhanced β-arrestin2 binding to the nephrin-c-terminus (**n = 5, p = 0.0079). (c) The inhibition of the PI3kinase with Ly294002 does not influence the Ang II enhanced interaction of the nephrin-c-terminus-β-arrestin2 excluding the Gβγ pathway (*n = 4, p = 0.0143, p = 0.15 for Ly294002). (d) Gαq mediates the AT1 receptor activation resulting in enhanced β-arrestin2 binding to the nephrin-c-terminus. The expression of GNAQ siRNA blocks the Ang II mediated enhancement of the β-arrestin2 binding to the nephrin-c-terminus. (e) The PLC inhibitor U73122 reduces the Ang II mediated nephrin endocytosis in HEK293T cells. (f) The PLC inhibitor U73122 abolishes the Ang II mediated enhanced β-arrestin2 binding to the nephrin-c-terminus in HEK293T (*n = 3, p = 0.0318). The use of the PLC…

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  Figure 5. Nephrin Y1217 is critical for the Ang II mediated enhancement of the β-arrestin2 binding to nephrin. Several distinct nephrin residues have influence on the nephrin β-arrestin2 binding and the regulatory sensitivity to Ang II stimulation. The nephrin motif T-GERD-T at nephrin T1120/T1125 forms the β-arrestin2 binding site. (a) The nephrin T1120A mutant does not impair the binding of β -arrestin2 to nephrin and is still susceptible to Ang II. (b) The nephrin double mutant T1120A/T1125A impairs binding of β-arrestin2 completely. (c) The truncation mapping of the nephrin c-terminus unravels the nephrin aminoacid residues 1215–1221 to harbor the region that mediates the Ang II dependent enhanced β-arrestin2 binding to nephrin. The c-terminal nephrin truncations nephrin 1087–1221 show the Ang II mediated enhanced β-arrestin2 binding to nephrin whereas the nephrin truncations nephrin 1087–1208 and nephrin 1087–1215 lack the Ang II mediated enhanced β-arrestin2 binding to nephrin. P-values β-arrestin binding to nephrin for the comparison of Ang II stimulation versus without Ang II stimulation: Nephrin 1087–1241 n = 8, p = 0.0006; Nephrin 1087–1208 n = 4, p = 0.27; Nephrin 1087–1215 n =…

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  Figure 6. Ang II decreases nck2 binding to nephrin. (a) The tyrosine kinase inhibitor PP2 enhances the β-arrestin2 binding to nephrin under Ang II stimulation. PP2 alone without Ang II stimulation does not influence the β-arrestin2 binding to nephrin (**DMSO: n = 5, p = 0.004; PP2: n = 5, p = 0.0079). The use of the tyrosine kinase inhibitor PP2 causes a reduced protein expression compared to conditions without PP2. (b) Ang II stimulation attenuates nephrin nck2 binding (**n = 5, p = 0.0079). (c) Nephrin Y1217WT demonstrates under Ang II stimulation a significantly decreased binding to nck2 Y1217WT (n = 6, p < 0.008). The nephrin mutants Y1217A and Y1217D failed to demonstrate an effect to the nck2 binding to nephrin under Ang II stimulation. But the nephrin Y1217A mutant showed a significantly decreased binding to nck2 whereas the Y1217D mutant presented no different binding to nck2 than the nephrin. (d) Densitometry of nck2 binding to nephrin and its mutants without Ang II stimulation (n = 6, **p = 0.0023, ns p = 0.17). All experiments were performed in HEK293T cells. Comparable amounts of nephrin fusion protein expression and its control were controlled by western blot. The expression…

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  Figure 7. This scheme of function depicts how Ang II promotes glomerular permeability by nephrin endocytosis. Ang II activates the AT1-receptor (AT1-R). The activated AT1-receptor transfers the signal through Gαq/11 and PLC. In turn nephrin gets phosphorylated at T1120/T1125 forming a β-arrestin2 binding site16. The binding of β-arrestin2 leads to increased nephrin endocytosis. This could be the mode of action how the glomerular permeability is increased beyond hemodynamic effects. In contrast, inhibition of the AT1-receptor by sartans promotes less β-arrestin2 nephrin interaction and less nephrin endocytosis leading to decreased glomerular permeability.