Compromised junctional integrity phenocopies age-dependent renal dysfunction in Drosophila Snakeskin mutants

ABSTRACT Transporting epithelia provide a protective barrier against pathogenic insults while allowing the controlled exchange of ions, solutes and water with the external environment. In invertebrates, these functions depend on formation and maintenance of ‘tight’ septate junctions (SJs). However, the mechanism by which SJs affect transport competence and tissue homeostasis, and how these are modulated by ageing, remain incompletely understood. Here, we demonstrate that the Drosophila renal (Malpighian) tubules undergo an age-dependent decline in secretory capacity, which correlates with mislocalisation of SJ proteins and progressive degeneration in cellular morphology and tissue homeostasis. Acute loss of the SJ protein Snakeskin in adult tubules induced progressive changes in cellular and tissue architecture, including altered expression and localisation of junctional proteins with concomitant loss of cell polarity and barrier integrity, demonstrating that compromised junctional integrity is sufficient to replicate these ageing-related phenotypes. Taken together, our work demonstrates a crucial link between epithelial barrier integrity, tubule transport competence, renal homeostasis and organismal viability, as well as providing novel insights into the mechanisms underpinning ageing and renal disease.

(A) 5 day adult SC SskRNAi (KK) MT.SCs exhibiting absence of mature stellar morphology, appearing vacuolar and extruded from the plane of the MT.Junctional complexes, as realised by anti-discs large (white), appear disorganised or missing with accretions associated with SCs.(B) 5 day adult SC SskRNAi (GD)   MT.SCs, clustered within the MT, range from those displaying some stellar-like morphology to those exhibiting complete absence of mature stellar morphology.The more compromised SCs again appear vacuolar and extruded from the plane of the MT.Junctional complexes, as realised by anti-discs large (white) appear disorganised or missing with accretions associated with the SCs.(C) Demonstration of significant difference for water content between experimental (29ºC) SC SskRNAi (KK) and SC SskRNAi (GD) females versus

Fig. S2 .
Fig. S2.SC-specific depletion of Ssk using alternative independent RNAi lines in adult Malpighian tubules iterating observed compromised cellular and junctional morphology.

Fig. S3 .
Fig. S3.ClC-aGAL4 knockdown of Ssk in adult Malpighian tubules.(A) ClC-aGAL4 driving membrane-bound (mGFP) in 5 day adult MT, with expression in evenly spaced SCs exhibiting stereotypical stellar morphology, and smoothly organised junctions throughout realized by anti-discs large (Dlg).SCs are stained with anti-ClC-a demonstrating specific localisation to the SC basolateral membrane.(B) ClC-aGAL4 driving Ssk RNAi (Furuse) and mGFP in 5 day adult MT.SCs exhibiting absence of mature stellar morphology (arrowheads).Junctional complexes, as realised by Dlg staining, appear disorganised or missing and the tubule itself is distended.ClC-a staining is largely absent, what staining remains is disorganised with absence of localisation to the basolateral membrane indicative of loss of apicobasal polarity in the mutants cells.(C) Demonstration of significant difference between ClC-aGAL4>Ssk RNAi (Furuse) experimental (29ºC) flies versus (18ºC) controls for water content, indicative of accumulation of haemolymph.Box-plot whiskers, min to max.*P<0.05,Student's T-Test, N's in parentheses.Dapi, blue; mGFP, green; ClC-a, magenta; Dlg, white.Scale bars = 50 µm.

Fig. S4 .
Fig. S4.Cell-specific depletion of Ssk in adult Malpighian tubules results in loss of cellular, junctional and cytoarchitectural organisation.(A) UroGAL4 driving membrane-bound green fluorescent protein (mGFP) in 5 day adult MT. mGFP in PCs exhibiting apical bias of expression (arrows), with smoothly organised junctions throughout realized by anti-discs large (Dlg).(B) 5 day experimental adult PC SskRNAi MT.PCs exhibit disorganised, or missing, junctional complexes and apparent loss of apical bias in mGFP expression.Complete absence of staining indicating apoptotic cells indicated (arrowheads).(C) 5 day experimental adult PC SskRNAi MT exhibiting absence of cellular architecture, realised by Phalloidin (Phall) staining as well as junctional expression, mGFP and Dapi staining (white box).mGFP, green; Phall, magenta; Dlg, white; Dapi, blue.Scale bars = 50 µm.

Fig
Fig. S5.cell-specific depletion of Ssk in adult Malpighian tubules results in loss of apicobasal polarity marker.(A) Subset of z-stack of c724GAL4 driving membrane-bound green fluorescent protein (mGFP) in 5 day adult MT.A1, merged image of confocal stack subset, exhibiting the basal staining for Na+/K+ alpha subunit (Na/K ATPase; arrow).A2, Na/K ATPase alone, highlighting expression at basal cell boundaries.A3, 2.5D rendering of A1 to illustrate Na/K ATPase expression biased to the basal cell boundary of SC (arrow).(B) Subset of z-stack of 5 day adult SC SskRNAi MT.B1, merged image of confocal stack subset, detailing basal edge of SC exhibiting absence of staining for Na/K ATPase at the basal cell boundary (arrow).Second SC with absent Na/K ATPase at the basal cell boundary (arrowhead).B2, Na/K ATPase alone highlighting presence or absence of expression at basal cell boundaries (arrow and arrowhead).B3, 2.5D rendering B1 to illustrate absence of Na/K ATPase expression specific to mutant SC as compared with surrounding principal cells (arrow).2.5D rendering -intensity values in a 2D image are converted into a height map, with highest intensity values represented by the greatest extension in the Z direction.Note-both confocal stacks were collected using identical settings.mGFP, green; Na/K ATPase, red; Dapi, blue.Scale bars = 20 µm.

Fig. S6 .
Fig. S6.SC-specific depletion of Ssk in adult Malpighian tubules results in loss of the SC-specific chloride channel-a with increased sensitivity to starvation but not salt stress conditions.(A) c724GAL4 driving membrane-bound green fluorescent protein (mGFP) in 5 day adult MT, demonstrating co-expression with SC-specific anti-Chloride Channel-a (ClC-a).(B) 5 day adult SC SskRNAi MT.SCs exhibit absence of SC-specific ClC-a expression.(C) 5 day adult PC SskRNAi MT with with unaffected expression of ClC-a in SCs.Note SC stellar morphology is also unaffected.mGFP, green; ClC-a, magenta; Dlg, white; Dapi, blue.Scale bars = 50 µm.Both male and female 5 -7 day old adult SC SskRNAi flies demonstrate a significant reduction in viability during (D) (non-dessicating) starvation conditions compared to controls.This sensitivity is absent when these flies are challenged with (E) salt stress, with no significant difference in viability compared with controls.Mantel-Cox (Log rank) test.N's in parentheses.*P<0.05.**P<0.0001.

Fig
Fig. S7.Cell-specific depletion of Ssk results in proliferation of renal nephritic stem cells (RNSCs) and hyperplasia of trachea supplying adult Malpighian tubules.(A) Quantitative RT-PCR analysis demonstrating ~35% knock down in Ssk mRNA expression levels in MTs between 5 day old adult SC SskRNAi flies raised at the restrictive temperature (29ºC) compared with those raised at the permissive temperature (18ºC); 0.802 x 10 -4 ng ± 0.038 vs 1.237 x 10 -4 ng ± 0.043 mRNA respectively.*P<0.05,Student's T-Test ±SEM, N's in parentheses.(B) UroGAL4 driving membrane-bound green fluorescent protein (mGFP) in the initial segment day of 5 D adult MT, demonstrating control levels of Ssk expression associated with junctions and, at lower levels, trachea.(C) Hyperplasia of trachea supplying MTs in adult PC SskRNAi flies as realised by anti-Ssk.As with SC SskRNAi flies, hyperplasia levels increase from initial to main segments.Nb-both confocal stacks were collected using identical settings.mGFP, green; Ssk, magenta; Dlg, white.Scale bars = 50 µm.(D) Tiled bright field image of 15 D SC SskRNAi (Furuse) adult MT raised at the permissive temperature (18ºC) demonstrating normal distribution of trachea supplying the MT.(E) Tiled bright field image of 15 D c724GAL4>mGFP adult MT raised at the restrictive temperature (29ºC) demonstrating normal distribution of trachea supplying the MT.Bright field images of 15 D SC SskRNAi (KK) (F) and SC SskRNAi (Furuse) (G) adult MTs raised at the restrictive temperature (29ºC) demonstrating hyperplasia of associated trachea supplying the MTs.(H1-3) Details of MTs of 15 D SC SskRNAi (Furuse) adults raised at the permissive temperature (18ºC) demonstrating normal distribution of trachea.(I1-3) Details of MTs of 15 D c724GAL4>mGFP adults raised at the restrictive temperature (29ºC) demonstrating normal distribution of trachea.(J1-3) Details of MTs of 15 D SC SskRNAi (Furuse) adults raised at the restrictive temperature (29ºC)