Gba1b-/-flies show a progressive deterioration of MT morphology and function.

A) Representative images (10× magnification) and quantification of Coracle (CORA; grayscale) immunostaining at day 1 and 21, illustrating the progressive loss of tubule junctional integrity in aged Gba1b-/- flies compared to controls (ctrl) (two-way ANOVA with Fisher’s LSD multiple comparisons, Day 1 p=0.9547, Day 21 ***p<0.0001), Scale-bar = 100µm. B) Representative images of tubules stained with BODIPYTM (green) and DAPI (blue) and quantification showing increased neutral lipid and RSC proliferation in 3-week-old Gba1b-/- fly tubules compared to ctrl (BODIPYTM quantification, unpaired t-test, *p=0.0374; DAPI RSC proliferation, unpaired t-test, ***p<0.0001)). Scale-bar = 200µm. C) Representative images and quantification of stellate cell (SC) number in 3-week-old ctrl and Gba1b-/- flies, visualised using c724>CD8::RFP, show a reduction in SCs in Gba1b-/- flies compared to ctrl (unpaired t-test, ***p<0.0001). Scale-bar = 250µm. D) Representative images and quantification of lysosomal staining intensity in MTs, visualised with LysoTrackerTM Red, showing increased lysosomal enrichment in 3-week-old Gba1b-/- flies compared to controls (unpaired t-test, *p=0.0144). Scale-bar = 50µm. E) Representative images of the mitochondrial morphology in the tubules of ctrl and Gba1b-/- flies, visualised using tub>mitoGFP and quantified for sphericity with the Mitochondrial Analyser plugin. The analysis reveals that mitochondria in Gba1b-/- fly tubules are less fused and exhibit a more punctate morphology compared to ctrl (ctrl vs Gba1b-/-, **p=0.0032, unpaired t-test). Scale-bar = 25µm. F) Representative images and quantification of MitoTracker™ Red CM-H₂XRos staining in renal tubules, showing reduced mitochondrial membrane potential in 3-week-old Gba1b-/- flies compared to ctrl (unpaired t-test, **p=0.0036). Scale-bar = 200µm. G) Example images of a FRUMS assay, showing the clearance of a systemically injected dye from the hemolymph, the tubule and the whole fly. Gba1b-/- flies display reduced filtration and removal of the dye from the haemolymph and whole body compared to ctrl (ctrl vs Gba1b-/- cleared from the hemolymph, ***p<0.0001; ctrl vs Gba1b-/- cleared from whole body; **p=0.0012, log-rank tests).

Gba1b-/- flies exhibit impaired ionic homeostasis

A) 3-week-old Gba1b-/- flies display a greater body water weight and body weight water percentage compared to ctrl flies (unpaired t-test, ***p<0.0001; ***p<0.0001). B) Hemolymph water weight is also increased in Gba1b-/-flies compared to ctrl by hemolymph removal and by wicking (unpaired t-test, water removed; **p=0.0132; percentage body weight removed *p=0.0112). C) Dehydration assay showing that 3-week-old Gba1b-/-flies are more resistant to desiccated food than age-matched ctrl (log-rank test, ***p=0.0013). D) 3-week-old Gba1b-/- flies are more sensitive to a diet supplemented with 4% NaCl and display reduced survival compared to ctrl (log-rank test, ***p<0.0001). E) The survival of Gba1b-/- flies is reduced compared to ctrl on a diet supplemented with 4% KCl (log-rank test, ***p<0.0001). F) A natriuresis assay showing the survival curves for Gba1b-/- and ctrl flies on diet supplemented with 4% NaCl with or without addition of H2O source (log-Rank test, ctrl vs Gba1b-/- flies, ***p < 0.0001; ctrl vs ctrl + H2O, ***p<0.0001; Gba1b-/- vs Gba1b-/- + H2O, ***p < 0.0001). G) 3-week-old ctrl flies are strongly sensitive to cold shock compared to Gba1b-/- flies (log-rank test, ***p < 0.0001).

Aged Gba1b-/- flies display diminished hemolymph clearance and nephrocyte function

A). Representative 63x and 10x images and quantification of nephrocytes from ctrl and Gba1b-/-flies at 4 weeks of age following uptake of Dextran 10,000 MWA. Dextran uptake (Red) represents endocytic activity and DAPI (Blue) labels nuclei. In Gba1b-/-fly nephrocytes, a subset of cells lack Dextran (arrow) (unpaired t-test, ***p < 0.0001). Scale bar for 10x and 63x = 100 µm. B) The soluble protein content of the hemolymph of Gba1b-/- flies is higher than in ctrl flies as assessed by a BCA assay (unpaired t-test, *p=0.0180), indicating impaired filtration. C). Lifespan survival curves of klf15-/-; Gba1b-/- double mutants and ctrl. Loss of klf15 significantly reduces lifespan in both ctrl and Gba1b-/- flies (log-rank test, Gba1b-/- vs klf15-/-; Gba1b-/-, ***p < 0.0001). D) Representative 20× images of LysoTrackerTM (Red)-stained brains from klf15-/-; Gba1b-/- flies and ctrl. Quantification of lysosomal area and puncta number shows no significant difference between Gba1b-/- and klf15-/-; Gba1b-/- genotypes (one-way ANOVA with Tukey’s test, p=0.9830). Scale bar = 50 µm. E) Representative 20x images of FK2/polyubiquitin (grayscale) immunostaining in brains from klf15-/-; Gba1b-/- flies and ctrl. Quantification of polyubiquitin puncta and area reveals no significant difference between Gba1b-/- and klf15-/-; Gba1b-/- genotypes (one-way ANOVA with Tukey’s test: FK2 puncta p=0.4015; FK2 area p=0.5228). Scale bar = 50 µm.

Disruption of redox homeostasis in Gba1b⁻/⁻ flies drives disease-associated phenotypes.

A) Whole-fly total glutathione (GSH) abundance is significantly decreased in 3-week-old Gba1b-/- flies compared to ctrl flies (unpaired t-test, *p=0.019). B) Representative 20× images and quantification of the 405 nm (oxidised)/488 nm (reduced) excitation ratio using the mitochondrial-targeted Grx1-roGFP2 redox biosensor in tub>mito-Grx1-roGFP2 control and Gba1b-/-flies. Gba1b⁻/⁻ fly tubules exhibt a significantly lower 405/488 ratio, indicating a shift toward a more reduced glutathione redox state (unpaired t-test, ***p<0.0001). Reduced signal is shown in magenta, oxidised in green. Scale bar = 100 µm. C) Example 20× images and quantification of the 405 nm (oxidised)/488 nm (reduced) ratio in tub>cyto-Grx1-roGFP2 ctrl and Gba1b-/-fly MTs. Quantification shows a significant increase in oxidation in Gba1b-/- tubules (unpaired t-test, ***p < 0.0001). Scale bar = 100 μm. D) Representative 20× images and quantification of dihydroethidium (DHE) fluorescence intensity in control and Gba1b-/- MTs. DHE staining is significantly reduced in Gba1b-/- fly tubules compared to ctrl (unpaired t-test, *p = 0.0151). Scale bar = 200 µm. E) Representative images and quantification of lipid peroxidation in MTs stained with the polyunsaturated lipid peroxidation sensor BODIPYTM 581/591. Oxidised lipids are shown in green (excitation: 488 nm), and reduced lipids in magenta (excitation: 568 nm). Gba1b-/- mutant MTs exhibit significantly elevated oxidised/reduced lipid ratios compared to ctrl (unpaired t-test, *p =0.0242). Scale bar = 100 µm.

Induction of antioxidant effector Nrf2 worsens renal and neurological phenotypes in Gba1b-/-flies

A) Overexpression of Nrf2 in the MTs with the c42-Gal4 driver modestly but significantly reduces lifespan in ctrl flies and leads to a marked reduction in lifespan in Gba1b-/- flies (log-rank test: c42> vs c42>Nrf2, ***p=0.0017; Nrf2 vs c42>Nrf2, ***p=0.0002 ; c42; Gba1b-/- vs c42>Nrf2; Gba1b-/-, ***p<0.0001; Nrf2; Gba1b-/- vs c42>Nrf2; Gba1b-/-, ***p<0.0001). B) Representative 10x images and quantification of proliferating RSCs in c42>Nrf2; Gba1b-/- flies and ctrl. Overexpression of Nrf2 in the tubules leads to increased proliferation of RSCs with a significantly greater effect observed in Gba1b-/- mutants (one-way ANOVA with Tukey’s multiple comparisons test: c42 vs c42; Gba1b-/-, ***p<0.0001; c42>Nrf2 vs c42>Nrf2; Gba1b-/-, ***p<0.0001). The arrow indicates the ureter fork. Scale bar = 100 μm. C) Water weight and percentage body water content of c42>Nrf2; Gba1b-/-flies and ctrl. Nrf2 overexpression significantly increases body water content in ctrl flies, but not in Gba1b-/- flies (one-way ANOVA with Tukey’s multiple comparisons test: c42> vs c42>Nrf2, ***p<0.0001; Nrf2 vs c42>Nrf2, ***p<0.0001; c42; Gba1b-/- vs c42>Nrf2; Gba1b-/-, ***p<0.0001; Nrf2; Gba1b-/- vs c42>Nrf2; Gba1b-/-, ***p<0.0001). D) Representative 20x images and quantification of LysoTrackerTM (Red)-stained brains from c42>Nrf2; Gba1b-/- flies and ctrl. Overexpression of Nrf2 in the renal tubules results in a significant increase in lysosomal puncta number in the brains of Gba1b-/- flies (one-way ANOVA with Tukey’s test: c42; Gba1b-/- vs c42>Nrf2; Gba1b-/-, **p = 0.0076). Scale bar = 50 μm. E) Representative 20x images and quantification of FK2/polyubiquitin and Ref(2)p immunostaining in the brains of c42>Nrf2; Gba1b-/- flies and ctrl. Overexpression of Nrf2 in renal tubules leads to a significant increase in polyubiquitin accumulation and FK2-positive puncta and area in Gba1b-/- fly brains (one-way ANOVA with Tukey’s test: FK2 puncta c42; Gba1b-/- vs c42>Nrf2; Gba1b-/-, ***p< 0.0001; FK2 area c42; Gba1b-/- vs c42>Nrf2; Gba1b-/-, ***p=0.0002; Ref(2)p puncta c42; Gba1b-/- vs c42>Nrf2; Gba1b-/- **p=0.0061, Ref(2)p area c42; gba1b-/- vs c42>Nrf2; Gba1b-/-, ***p=0.0002). Scale bar = 50 μm.

Ubiquitous or tubule-specific induction of redox dyshomeostasis shortens the lifespan of Gba1b-/-flies

A) Survival curves of ctrl and Gba1b−/− flies raised on standard SYA food with or without 20 mM ascorbic acid (AA). AA supplementation significantly reduces lifespan in Gba1b−/− flies (log-rank test: Gba1b-/- vs Gba1b-/- +AA ***p < 0.0001; ctrl vs ctrl +AA p=0.071). (B–D) Survival curves showing the effects of ubiquitous overexpression (tub-GAL4) of antioxidant enzymes CatA (B), Sod1 (C), and Sod2 (D) in Gba1b−/− flies. All conditions resulted in significant lifespan shortening in the context of Gba1b loss (log-rank tests: (B) tub>; Gba1b-/- vs tub>CatA; Gba1b-/-, ***p<0.0001; CatA;Gba1b-/- vs tub>CatA;Gba1b-/-, ***p<0.0001. (C) tub>; Gba1b-/- vs tub>Sod1;Gba1b-/-, ***p<0.0001; Sod1;Gba1b-/- vs tub>Sod1;Gba1b-/-, ***p< 0.0001; (D) tub>; Gba1b-/- vs tub>Sod2; Gba1b-/-, ***p<0.0001; Sod2; Gba1b-/-vs tub>Sod2; Gba1b-/-, ***p<0.0001). (E–F) Lifespan analysis of Gba1b−/− flies with c42-GAL4 overexpression of Sod1 (E) and Sod2 (F), showing significant reduction in lifespan (log-rank test: tub>;Gba1b-/- vs c42>Sod1; Gba1b-/-, ***p<0.0001; Sod1;Gba1b-/- vs c42>Sod1;Gba1b-/-, ***p<0.0001. (F) c42>;Gba1b-/-vs c42>Sod2; Gba1b-/-, ***p<0.0001; Sod2;Gba1b-/-vs c42>Sod2; Gba1b-/-, ***p<0.0001). (G–H) Ubiquitous (G) and tubule (H) overexpression of the Nrf2 repressor Keap1 in Gba1b−/− flies significantly shortens lifespan (log-rank test: tub>; Gba1b-/- vs tub>Keap1;Gba1b-/-, ***p<0.0001; Keap1;Gba1b-/- vs tub>Keap1;Gba1b-/-, ***p<0.0001. (H) c42>;Gba1b-/- vs c42>Keap1; Gba1b-/-, ***p<0.0001; Keap1; Gba1b-/- vs c42>Keap1;Gba1b-/-, ***p < 0.0001).

Parkin PD model flies exhibit severe renal degeneration and mitochondrial redox imbalance

(A) Water content measurements reveal significantly increased water weight and percentage body water in park1/1 flies compared to ctrl (unpaired t-test, ***p<0.0001). (B) park1/1 flies display over proliferation of tubule RSCs in the ureter relative to ctrl (unpaired t-test, ***p<0.0001). (C) Representative images and quantification of mitochondrial ROS (mitoROS) in the MTs show reduced mitochondrial membrane potential in park1/1 flies compared to ctrl (unpaired t-test, **p=0.0014). Scale bar = 100 µm. (D) Quantification of mitochondrial glutathione redox state using the c42>mitoGrx1 sensor demonstrates a significant shift toward a reduced state in park1/1 fly MTs (unpaired t-test, ***p<0.0001). Scale bar = 100 µm. (E) Expression of the mitophagy reporter c42>mitoQC reveals a strong reduction in acidified mitolysosomes in park1/1 fly tubules, indicating impaired mitophagy (unpaired t-test, ***p<0.0001). Scale bar = 100 µm. (F) Dextran uptake assay (10×, 63×) shows a complete loss of functional nephrocytes in park1/1 flies, as quantified by the number of endocytically inactive cells (unpaired t-test, ***p<0.0001). Scale bar = 100 µm. (G) park1/1 flies display increased survival following cold shock compared to ctrl (log-rank test, ***p=0.0007). (H) FRUMS assay reveals significantly reduced dye clearance from the hemolymph in park1/1 flies (***p<0.0001), though total dye clearance from the whole fly was not significantly affected (log-rank test, p =0.4599). (I) Overexpression of park in PCs (c42>park) partially rescues body water homeostasis in park1/1 flies. Water weight: c42; park1/1 vs c42>park; park1/1, *p=0.0471; +>park; park1/1vs c42>park; park1/1, p = 0.4201. Percentage body water: c42; park1/1 > vs c42>park; park1/1, ***p=0.0006; +>park; park1/1vs c42>park; park1/1, **p=0.0059 (one-way ANOVA with Tukey’s multiple comparisons test).

Rapamycin differentially rescues renal defects in park1/1 and Gba1b-/- flies

(A) Percentage body water weight is significantly reduced in Gba1b-/-flies upon rapamycin treatment (**p=0.0017, one-way ANOVA with Tukey’s multiple comparisons test). (B) Quantification of RSC over proliferation using DAPI staining from the ureter shows a significant reduction in Gba1b-/- fly tubules following 3-week rapamycin treatment (one-way ANOVA with Tukey’s multiple comparisons test, ***p<0.0001). (C) Lipid peroxidation, assessed using BODIPYTM 581/591 (oxidised = green, reduced = magenta; excitation at 488 nm and 568 nm), is elevated in Gba1b-/- fly tubules compared to ctrl and rescued by rapamycin (one-way ANOVA with Tukey’s multiple comparisons test, ***p<0.0001; **p=0.0042). Scale bar = 100 µm. (D) Expression of a Mitophagy reporter (tub>mitoQC) shows no significant change in mitophagy in the MTs of Gba1b-/- flies following treatment with rapamycin (one-way ANOVA with Tukey’s multiple comparisons test, p=0.7293). Scale bar = 100 µm. (E) Mitochondrial membrane potential visualised by MitoTracker™ Red CM-H₂XROS is significantly reduced in Gba1b-/- fly tubules compared to ctrl and is not rescued by rapamycin (one-way ANOVA with Tukey’s multiple comparisons test, ***p<0.0001; p=0.9705). Scale bar = 100 µm. (F) Lysosomal content, visualised with LysoTracker™ Red, is significantly decreased in park1/1 fly MTs (unpaired t-test, ***p<0.0001). (G) Water retention is not significantly reduced by rapamycin treatment in park1/1flies (one-way ANOVA, p=0.3152). (H) Mitochondrial GSH redox state in park1/1fly MTs, visualised using c42>mitoROGFP2-Grx1, is not rescued by rapamycin (one-way ANOVA with Tukey’s multiple comparisons test, p=0.8582). Scale bar = 100 µm. (I) Rapamycin treatment reduces RSC overproliferation in park1/1 fly tubules (ctrl vs park1/1, ***p<0.0001, rapa ctrl vs rapa park1/, *p<0.0109, park1/1 vs rapa park1/1, ***p<0.0001, one-way ANOVA with Tukey’s multiple comparisons test). Scale bar = 100 µm. (J) BODIPYTM 581/591 C11 staining shows elevated lipid peroxidation in park1/1 fly tubules, which is significantly reduced by rapamycin treatment (one-way ANOVA with Tukey’s multiple comparisons test, **p<0.0001; p=0.0109). Scale bar = 100 µm.