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. 2022 Oct 31;8(2):317–329. doi: 10.1016/j.ekir.2022.10.024

Figure 4.

Figure 4

Knockdown of Drosophila actinin in nephrocytes results in decreased ND length and reduced filtration function. (a) Electron micrograph depicting a wild-type nephrocyte. Scale bars indicate 5 μm in A’ and 500 nm in A’’ (b) Protein alignment of the N-terminal actin-binding domain of human ACTN4 and Drosophila actinin. Highlighted are amino acids known to be causative of monogenic nephrotic syndrome. Actinin shares 68.83% overall identity with ACTN4, the actin-binding domain of the 2 proteins shares 78.8% identity. (c,d) Representative micrographs of nephrocytes stained with (c) anti-Pyd and (d) quantification of the ND length. Nephrocytes derived from either control larvae, or larvae with nephrocyte-specific knockdown of actinin (ACTN), by using 2 different UAS-RNAi-lines (actn-RNAi1 and actn-RNAi2). Compared with control nephrocytes, the ND length of actn-depleted nephrocytes is significantly reduced (gray dots show all nephrocytes measured, green dots represent means of n = 3 independent experiments performed in 3 experimental crossings, error bars indicate SD, ∗∗P < 0.01, ∗∗∗P < 0.001, one-way ANOVA with Tukey’s post hoc test). (e,f) Representative micrographs of nephrocytes subjected to (e) FITC-albumin tracer and (f) quantification of fluorescence intensity as a measure of uptake capacity. Control and ACTN knockdown nephrocytes were incubated in 0.2 mg/ml FITC-albumin solution for 30 seconds, and fluorescence intensity was quantified using Fiji. The data are presented as normalized to control levels. Both, actn-RNAi1 and actn-RNAi2 nephrocytes show a significantly reduced capacity of FITC-albumin uptake with respect to control nephrocytes, indicating severe filtration defects (gray dots show all nephrocytes measured, green dots indicate means of n = 3 independent experiments performed in 3 experimental crossings, error bars indicate SD, ∗∗P < 0.01, ∗∗∗∗P < 0.0001, one-way ANOVA with Tukey’s post hoc test). Scale bars indicate 5 μm in (c) and 25 μm in (f). FITC, fluorescein isothiocyanate; ND, nephrocyte diaphragm; Pyd, polychaetoid; RNAi, RNA interference.