High-fat diet compromises nephrocyte function Nephrocytes from control Drosophila (w1118, females) fed a regular diet (normal fat diet, NFD) or high-fat diet (NFD supplemented with 14% coconut oil, HFD) for 7 days from eclosion.

(A) Representative confocal images of nephrocytes show green fluorescence indicative of FITC-albumin uptake. Scale bar: 50 μm. (B) Box plot shows the quantitation of the relative fluorescence intensity of FITC-albumin shown in (A); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n = 6 flies. (C) Representative confocal images of Drosophila nephrocytes (w1118, 7-day old females) show red fluorescence indicative of 10 kD dextran uptake. Scale bar: 50 μm. (D) Box plot shows the quantitation of the relative fluorescence intensity of 10 kD dextran shown in (C); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two-tailed Student’s t-test; **, P<0.01; n = 6 flies.

High-fat diet changes nephrocyte morphology Nephrocytes from control Drosophila (w1118, 7-day-old females) fed a regular diet (normal fat diet, NFD) or high-fat diet (NFD supplemented with 14% coconut oil, HFD).

(A) Representative confocal images of Drosophila nephrocytes immunostained with anti- polychaetoid (Pyd) in green. Upper panels show cortical surface; Scale bar: 5 μm. Lower panels show subcortical regions; Scale bar: 5 μm. (B) Quantitation of Pyd protein distribution (cytoplasmic vs membrane); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two-tailed Student’s t-test; ***, P<0.001; n = 8 nephrocytes (1 nephrocyte/fly) from 7-day-old female flies. (C) Transmission electron microscopy (TEM) images of Drosophila nephrocyte (w1118, 7-day-old females) cortical regions. Scale bar: 0.5 µm. (D) Quantitation of lacuna channel (LC)-LC distance based on images in (C); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two-tailed Student’s t-test; **, P<0.01; n = 60 LC- LC distance measurements obtained in 10 nephrocytes from six 7-day-old female flies for each group. (E) TEM images of Drosophila nephrocyte (w1118, 7-day-old-females) cytoplasmic regions. Red asterisks indicate large vacuoles. Scale bar: 0.5 µm. (F) Quantitation of the vacuoles that contain electron dense structures based on images in (E). The middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n = 12 nephrocytes for NFD and 29 nephrocytes for HFD from six 7-day-old female flies.

High-fat diet activates the JAK-STAT pathway in nephrocytes.

(A) Table lists human genes encoding JAK-STAT pathway components, along with their Drosophila homologs, the DRSC Integrative Ortholog Prediction Tool (DIOPT) score (maximum score = 15), and their function. (B) Graphical representation of the JAK-STAT signaling pathway and interaction between its components. Domeless, Dome; JAK Hopscotch, Hop; Signal-transducer and activator of transcription 92E, Stat92E; Suppressor of cytokine signaling at 36E, Socs36E; Unpaired, Upd. (C) Representative confocal images of nephrocytes from control Drosophila (10xStat92E-GFP, 7-day-old females) fed a regular diet (normal fat diet, NFD) or high-fat diet (HFD, NFD supplemented with 14% coconut oil). 10xStat92E-GFP is shown in green fluorescence; DAPI (blue) stains DNA to visualize the nucleus. Scale bar: 50 μm. (D) Box plot shows the quantitation of the relative fluorescence intensity of 10xStat92E-GFP based on images in (C); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two-tailed Student’s t-test; ***, P<0.001; n = 6 flies.

JAK-STAT pathway activation compromises nephrocyte function.

(A) Schematic illustration of targeted UAS-hop.Tum expression in the nephrocytes; hopscotch.Tumorous-lethal, dominant gain-of-function, constitutively activates JAK-STAT. Temperature sensitive Gal80ts binds to Gal4 and acts as a negative regulator of the Gal4 transcriptional activator at 18°C. A temperature switch to 29°C releases Gal80ts inhibition as it can no longer bind Gal4, thus allowing UAS-hop.Tum expression driven by Gal4 to occure. Timeline for temperature switches of the fly at different stages of development have been indicated. (B) Representative confocal images of FITC-albumin fluorescence (green) in nephrocytes from control flies (Dot-Gal4/+; tub-Gal80ts/+) and those with activated JAK- STAT (Dot-Gal4/UAS-hop.Tum; tub-Gal80ts/+). Scale bar: 50 μm. (C) Box plot shows the quantitation of the relative fluorescence intensity of FITC-albumin based on images in (B); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n = 6 flies (7-day-old females). (D) Representative confocal images of 10 kD dextran fluorescence (red) in nephrocytes from control flies (Dot-Gal4/+; tub-Gal80ts/+) and those with activated JAK- STAT (Dot-Gal4/UAS-hop.Tum; tub-Gal80ts/+); DAPI (blue) stains DNA to visualize the nucleus. Scale bar: 50 μm. (E) Box plot shows the quantitation of the relative fluorescence intensity of 10 kD dextran uptake based on images in (D); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two- tailed Student’s t-test; **, P<0.01; n = 6 flies (7-day-old females). (F) Schematic illustration of the Flp-out clone strategy to induce UAS-hop.Tum expression. Heat shock induces the expression of Flp recombinase, which excises a stop cassette to initiate Gal4 expression. Gal4 binding to the upstream activation sequences (UAS) drives the expression of GFP (as a marker for positive Flp-out clones) and UAS-hop.Tum. (G) Representative confocal images of 10 kD dextran fluorescence (red) in nephrocytes from flies with a GFP labelled Flp-out UAS-hop.Tum clone (hs-Flp122/+; UAS-FlpJD1/UAS-hop.Tum; Act5C>CD2>Gal4S, UAS- mCD8GFPLL6/+). (H) Box plot shows the quantitation of the relative fluorescence intensity of 10 kD dextran fluorescence uptake based on images in (G); middle line depicts the median and whiskers show minimum to maximum. Control (neighbor of Flp-out clone; UAS-hop.Tum (clone). Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n= 5 clones and 5 neighbor cells.

Silencing Socs36E in the nephrocytes, or upd2 overexpression in the fat body, leads to nephrocyte dysfunction.

(A) Representative confocal images of FITC-albumin (green) in nephrocytes from control flies (Dot-Gal4/+) and flies with nephrocyte-specific silencing of the Socs36E JAK-STAT inhibitor (Dot-Gal4>Socs36E-IR); DAPI (blue) stains DNA to visualize the nucleus. Scale bar: 50 μm. Socs36E, Suppressor of cytokine signaling at 36E. (B) Box plot shows the quantitation of the relative fluorescence intensity of FITC-albumin uptake based on images in (A); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two-tailed Student’s t-test; **, P<0.01; n= 6 flies (7-day-old females). (C) Representative confocal images of 10 kD dextran fluorescence (red) in nephrocytes from control flies (Dot-Gal4/+) and flies with nephrocyte-specific silencing of the Socs36E JAK-STAT inhibitor (Dot-Gal4>Socs36E-IR); DAPI (blue) stains DNA to visualize the nucleus. Scale bar: 50 μm. Socs36E, Suppressor of cytokine signaling at 36E. (D) Box plot shows the quantitation of the relative fluorescence intensity of 10 kD dextran uptake based on images in (C); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two-tailed Student’s t-test; ***, P<0.001; n= 6 flies (7-day-old females). (E) Representative confocal images of 10 kD dextran fluorescence (red) in nephrocytes from control flies (ppl-Gal4/+) and flies with fat body- specific overexpression of JAK-STAT ligand Upd2 (ppl-Gal4>upd2-GFP); DAPI (blue) stains DNA to visualize the nucleus. Scale bar: 50 μm. ppl, pumpless; upd2, unpaired 2. (F) Box plot shows the quantitation of the relative fluorescence intensity of 10 kD dextran uptake based on images in (E); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n= 6 flies (7-day-old females). (G) Representative confocal images of nephrocytes from control flies (ppl-Gal4/+) and flies with fat body-specific overexpression of Upd2 (ppl-Gal4>upd2-GFP). Anti-Pyd is shown in red. Scale bar: 4 μm. (H) Quantitation of Pyd protein distribution (cytoplasmic vs membrane); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n = 12 nephrocytes (1 nephrocyte/fly) from 7-day-old female flies.

Silencing Stat92E attenuates nephrocyte functional defects caused by a high-fat diet Nephrocytes from control flies (Dot-Gal4/+; tub-Gal80ts/+) and those with Stat92E silencing as adults (Dot-Gal4/UAS-Stat92E-IR; tub-Gal80ts/+).

UAS-Stat92E-RNAi expression was induced at the adult stage (see Figure 4A) for seven days before the uptake assay. Stat92E, Singal-transducer and activator of transcription 92E. (A) Representative confocal images of FITC-albumin fluorescence (green); DAPI (blue) stains DNA to visualize the nucleus. Scale bar: 50 μm. (B) Box plot shows the quantitation of the relative fluorescence intensity of FITC- albumin uptake based on images in (A); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed by two-way ANOVA with Sidak correction; **, P<0.01; ****, P<0.0001; ns, not significant; n = 6 flies (7-day-old females). (C) Representative confocal images of 10 kD dextran fluorescence (red); DAPI (blue) stains DNA to visualize the nucleus. Scale bar: 50 μm. (D) Box plot shows the quantitation of the relative fluorescence intensity of 10 kD dextran uptake based on images in (C); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed by two-way ANOVA with Sidak correction; ***, P<0.001; ****, P<0.0001; ns, not significant; n = 6 flies (7-day-old females).

Methotrexate treatment can restore nephrocyte function following a high-fat diet Nephrocytes from control Drosophila (w1118; 7-day-old females) fed a regular diet (normal fat diet, NFD) or high-fat diet (NFD supplemented with 14% coconut oil, HFD), with or without methotrexate (10 μM; ex vivo 60 min) treatment.

(A) Representative confocal images of FITC-albumin fluorescence (green). Scale bar: 50 μm. (B) Box plot shows the quantitation of the relative fluorescence intensity of FITC-albumin uptake based on images in (A); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed by two-way ANOVA with Sidak correction; ***, P<0.001, ****, P<0.0001; ns, not significant; n = 6 flies (7-day-old females). (C) Representative confocal images of 10 kD dextran fluorescence (red). Scale bar: 50 μm. (D) Box plot shows the quantitation of the relative fluorescence intensity of 10 kD dextran uptake based on images in (C); middle line depicts the median and whiskers show minimum to maximum. Statistical analysis was performed by two-way ANOVA with Sidak correction; ****, P<0.0001; ns, not significant; n = 6 flies (7-day-old females). (E) Graphic of proposed model for high-fat diet-induced nephrocyte defects via an adipose-nephrocyte axis. A high-fat diet upregulates the expression and secretion of the adipokine Unpaired 2 (Upd2), leptin-like hormone, from the fat body. Upd2 is a JAK-STAT ligand, and it activates JAK-STAT signaling at the nephrocytes (Signal-transducer and activator of transcription 92E, Stat92E; Suppressor of cytokine signaling at 36E, Socs36E; JAK Hopscotch, Hop; Domeless, Dome). The overactive JAK-STAT pathway disrupts the integrity of the slit diaphragm (SD) filtration structure and thereby leads to nephrocyte dysfunction.