Upregulation of angiotensin-(1–7) formation in human podocytes – enzyme activity assay upon fluid flow shear stress

Figures

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  Fig 1. Characterization of ACE2, PRCP, and PREP on protein level in hPC conducted by immunofluorescence. Representative immunofluorescence images of (A) ACE2, (B) PRCP, and (C) PREP, demonstrating protein expression on hPC. (D) Control without primary antibody revealed no specific binding of secondary antibody goat anti-rabbit Alexa Fluor™ Plus 488. Nuclei were stained using DAPI (blue). Scale bar 50 µm.

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  Fig 2. Inhibitor effects on ACE2-, PRCP-, and PREP-mediated Ang II to Ang-(1-7) conversion. (A) Inhibition of Ang-(1-7) formation [ng/mL] by ACE2 (10 ng/mL) was tested with increasing inhibitor concentrations of five different inhibitors. (B) For a complete inhibition of Ang-(1-7) formation by ACE2, 1 ng/mL ACE2 was incubated with increasing ACE2i concentrations [0.01 μM- 100 μM]. (C) Inhibition of Ang-(1-7) formation by PRCP (10 ng/mL) was tested for five different inhibitors [0.01 μM- 100 μM]. (D) For PRCPi, lower inhibitor concentrations [0.01 nM- 100 nM] were used to investigate inhibitory effects on Ang-(1-7) formation by PRCP.

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  Fig 3. Ang-(1-7) formation in hPC lysates and supernatants upon FFSS. (A) Ang-(1-7) formation rate in cell lysates was elevated upon FFSS (blue squares) compared to no flow controls (black circles). Experiments were performed twice on different cell passages and different dates, each consisting of n = 6 technical replicates, respectively. Statistics: ***, p < 0.001, assessed by two-tailed Student´s t-test. (B) Ang-(1-7) formation rate in supernatants was not significantly regulated upon FFSS (blue squares) compared to no flow controls (black circles). Experiments were performed twice on different cell passages and different dates, each consisting of n = 4 technical replicates, respectively. Statistics: assessed by Mann-Whitney test. Each data point represents one technical replicate. Data are plotted as mean ± SD (horizontal lines).

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  Fig 4. Gene expression of ACE2 and PRCP in hPC upon FFSS. (A) ACE2 gene expression increased in hPC upon FFSS (blue squares) compared to no flow control (black circles). (B) PRCP gene expression decreased in hPC upon FFSS (blue squares) compared to no flow control (black circles). qPCR results are presented as relative mRNA expression level normalized to GUSB and referred to unstimulated control group. Each data point represents one technical replicate of four separate experiments. Experiments were performed on different cell passages and different dates, each experiment consisting of n = 5-6 technical replicates, respectively. Data are plotted as mean ± SD (horizontal lines). Statistics: *, p < 0.05, assessed by two-tailed Student´s t-test.

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  Fig 5. Ang-(1-7) formation rate in hPC lysates upon FFSS. (A) Ang-(1-7) formation rate under no flow control condition and (B) after FFSS exposure. Inhibition of ACE2 (blue squares) did not affect the Ang-(1-7) formation rate in hPC cell lysates compared to the total Ang-(1-7) formation rate (black circles). Inhibition of PRCP (green triangles) significantly reduced the Ang-(1-7) formation compared to total Ang-(1-7) formation (black circles) as well as ACE2 inhibition (blue squares). Experiments were performed twice on different cell passages and different dates, each experiment consisting of n = 6 technical replicates, respectively. Each data point represents one technical replicate. Data are plotted as mean ± SD (horizontal lines). Statistics: ****, p < 0.0001, assessed by one-way ANOVA with Tukey`s multiple comparison follow-up test.

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  Fig 6. Ang-(1-7) formation rate from Ang II in hPC supernatants upon FFSS. (A) Under no flow control condition, Ang-(1-7) formation with ACE2 inhibition (blue squares) was significantly lower than total Ang-(1-7) formation (black circles) and formation with PRCP inhibition (green triangles). (B) Upon FFSS exposure, ACE2 inhibition (blue squares) reduced the Ang-(1-7) formation significantly compared to total Ang-(1-7) formation without inhibitors (black circles). Ang-(1-7) formation with PRCP inhibition (green triangles) was no longer significantly different to Ang-(1-7) formation upon ACE2 inhibition. Experiments were performed twice on different cell passages and different dates, each experiment consisting of n = 4 technical replicates, respectively. Statistics: **, p < 0.01; ***, p < 0.001; n.s., not significant, assessed by one-way ANOVA with Tukey`s multiple comparison follow-up test. Each data point represents one technical replicate. Data are plotted as mean ± SD (horizontal lines).