FIGURE SUMMARY
Title

A high throughput zebrafish chemical screen reveals ALK5 and non-canonical androgen signalling as modulators of the pkd2-/- phenotype

Authors
Metzner, A., Griffiths, J.D., Streets, A.J., Markham, E., Philippou, T., Van Eeden, F.J.M., Ong, A.C.M.
Source
Full text @ Sci. Rep.

A high through-put zebrafish screen of pkd2−/− phenotype modulators. (a) Workflow of compound screen on pkd2 tail curvature phenotype. (b) pkd2−/− embryo at 3 dpf with schematic indication of curvature measurement. 180° - straight tail, 0° - tail crosses body axis. (c) Curvature analysis of pkd2 mutants exposed from 27 hpf to DMSO or 200 nM TSA. Example images of curvature next to y-axis. Significance via Mann-Whitney test; ****p ≤ 0.0001. (d) Combined data on hit compounds of the Spectrum library after initial compound screen, validation and cherry-picked compound exposures. Enhancers of pkd2 curvature in red, repressors in green. Representative tail curvature images on the left of results. Chemical classes as indicated. Mean of DMSO baseline indicated with black line. Significances via Kruskal-Wallis test; ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 and non-significant (ns): p > 0.05. (a) created using BioRender.com.

Androgens modulate tail curvature in pkd2−/− embryos independently of AR. (a) Dose-response effects of androstandione on tail curvature in pkd2−/− zebrafish embryos. (b) Effects on tail curvature in pkd2−/− zebrafish embryos with co-exposure of androstandione and the anti-androgen flutamide. (c) The effects of androstandione on tail curvature in pkd2−/−;ar−/− embryos. (d) Effects of co-exposure of androstandione (10 µM) and the LTCC inhibitor, nifedipine (10 µM) or agonist, BayK8644 (30 µM), on tail curvature of pkd2−/− zebrafish embryos. All results represented by mean +/− SEM. Significance via one-way ANOVA with Dunnett’s multiple comparisons. P values represented by * (****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 and non-significant (ns): p > 0.05.). (e) Chemical structures of androgens and their potency on pkd2−/− tail curvature with androstandione being the most potent and norethynodrel the least potent. Circles are positioned at C3 and C17, with the complexity of side groups at these locations correlating with potency of the compound.

PKIS zebrafish screen revealed four PKIS repressors of the pkd2−/− phenotype. (a) Hit compounds identified from the PKIS library after initial compound screening and validation. Enhancers of pkd2 curvature in red, repressors in green. Mean of DMSO baseline indicated with black line. Effect of re-ordered PKIS repressor compounds on pkd2 curvature (b) and glomerular area (c). Statistical significances by one-way ANOVA with Dunnett’s multiple comparison, results represented by mean +/− SEM. P values represented by * (****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 and non-significant (ns): p > 0.05.). (d) Chemical structure of the three validated PKIS tail curvature repressor compounds.

Chemical compounds and PKIS inhibitors identified from zebrafish screen in two cystic cell culture models, MDCKII and Ox161c1. Cyst area of MDCKII (a) and Ox161c1 (b) cells after 17 and 14 days of compound exposure respectively to compounds identified in the zebrafish screen. Chemical classes as indicated, concentrations determined via prior dose response assays to exclude toxicity. Cyst area of MDCKII (c) and Ox161c1 (d) cells after 17 and 20 days of compound exposure respectively to compounds identified in the zebrafish screen compared to DMSO control exposed cells. PKIS inhibitors as indicated at 10 µM concentrations and 5 µM forskolin. Results represented by median +/− IQR. P values represented by * (****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 and non-significant (ns): p > 0.05.). Median of DMSO baseline indicated with black line and significances via Kruskal-Wallis test with Dunn’s multiple comparison.

Identification of ALK5 as a pkd2−/− phenotype modulator in zebrafish. (a) Heat maps of known kinases inhibited by PKIS repressor compounds with the top 25 inhibited kinase targets in decreasing order. Numbers represent percentage of inhibition; dark yellow ≥65%, yellow ≥40%, light grey 35–40%, dark grey 30–35%, grey/blue 20–30%, light blue 15–20% and dark blue >15%. (b) Effects of potent KDR and (c) MAP4K4 inhibitors on pkd2−/− zebrafish embryo tail curvature. All compounds tested at 10 µM. Results represented by mean ± SEM. No significance via one-way ANOVA and Dunnett’s multiple comparison. (d) Effects of different concentrations of the ALK5 inhibitor SD208 on pkd2−/− curvature. Results represented by mean ± SEM. Significance via one-way ANOVA and Dunnett’s multiple comparison, p values represented by * (****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 and non-significant (ns): p > 0.05).

ALK5 as a modulator of cystic growth in cell culture and expression of ALK4/5 in human ADPKD kidneys. Effects of SD208 (1 and 0.1 µM) after 10 days exposure on cyst area (a), proliferation (b) and apoptosis (c) in MDCKII cells compared to DMSO and forskolin controls. Results represented by median +/− IQR. Significance via Kruskal-Wallis test with Dunn’s multiple comparison, p values represented by * (****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 and non-significant (ns): p > 0.05.). (di, ii, iii) Representative images of SD208 effects on cyst area, proliferation and apoptosis respectively compared to DMSO or forskolin. Podocalyxin/gp135 was used to define lumen formation with nuclear DAPI marker (di), Ki67 and cleaved caspase 3 antibodies used to measure proliferation and apoptosis respectively with phalloidin apical markers (dii and diii). Microarray expression profile of ALK5 (e) and ALK4 (f) in human kidney tissues derived from 5 polycystic kidney samples and 3 non-polycystic kidney samples. Polycystic sample tissues are separated into minimally cystic, small cysts (<1 ml), medium cysts (10–20 ml) and large cysts (>50 ml). Results represented by mean +/− SEM. Significance via one-way ANOVA with Dunnett’s multiple comparison, p values represented by * (****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 and non-significant (ns): p > 0.05).

Model for role of ALK5 and non-canonical androgen signaling in ADPKD. (a) TGFβ binds to ALK5 (TGFβ-receptor 1) or ALK4 (not shown) at the plasma membrane to activate the transcription factors SMAD2/3. SMAD2/3 in turn activate other TGFβ regulated genes to promote cyst expansion and disease progression by effects on inflammation, extracellular matrix accumulation, proliferation and apoptosis. (b) Androgens bind to androgen receptors (AR) in the cytoplasm resulting in translocation of the complex to the nucleus. The AR-androgen complex modulates gene transcription of androgen related genes in the canonical signaling pathway. Androgens can also bind to and inhibit non-genomic receptors such as L-type calcium channels, ZIP9, GPRC6A or OXER1 in a non-canonical, AR-independent pathway. Androgen activation of this non-canonical pathway promotes cyst expansion and upward tail curvature. Figure created with BioRender.com.

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Acknowledgments
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