High-fat diet compromises nephrocyte function

Nephrocytes from control Drosophila (w1118, 1-day-old 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 Tukey. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n = 30 nephrocytes from six 1-day-old females (5 nephrocytes/fly). (C) Representative confocal images of Drosophila nephrocytes (w1118, 1-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 Tukey. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n = 30 nephrocytes from six 1-day-old females (5 nephrocytes/fly).

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 Tukey. 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 Tukey. 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 Tukey. 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, 1-day-old females) fed a regular diet (normal fat diet, NFD) or high-fat diet (NFD supplemented with 14% coconut oil, HFD). 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 Tukey. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n = 30 nephrocytes from six 1-day-old females (5 nephrocytes/fly).

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 Tukey. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n = 30 from six 1-day-old females (5 nephrocytes/fly). (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 Tukey. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n = 30 from six 1-day-old females (5 nephrocytes/fly). (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 Tukey. 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 from four 3-day-old females.

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 Tukey. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n= 30 from six 1-day-old females (5 nephrocytes/fly). (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 Tukey. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n= 30 from six 1-day-old females (5 nephrocytes/fly). (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 Tukey. Statistical analysis was performed with a two-tailed Student’s t-test; ****, P<0.0001; n= 30 from six 1-day-old females (5 nephrocytes/fly).

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 Tukey. Statistical analysis was performed by two-way ANOVA with Sidak correction; **, P<0.01; ****, P<0.0001; ns, not significant; n = 30 from six 1-day-old females (5 nephrocytes/fly). (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 Tukey. Statistical analysis was performed by two-way ANOVA with Sidak correction; ***, P<0.001; ****, P<0.0001; ns, not significant; n = 30 from six 1-day-old females (5 nephrocytes/fly).

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 Tukey. Statistical analysis was performed by two-way ANOVA with Sidak correction; ****, P<0.0001; ns, not significant; n = 30 from six 1-day-old females (5 nephrocytes/fly). (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 Tukey. Statistical analysis was performed by Mann-Whitney U test; ****, P<0.0001; ns, not significant; n = 30 from six 1-day-old females (5 nephrocytes/fly). (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.

Methotrexate treatment inhibits JAK-STAT pathway activity

(A) Representative confocal images of 1-day-old female adult nephrocytes (10xStat92E-GFP). Control, incubated in Schneider’s Drosophila Medium (ex vivo for 60 min at room temperature); methotraxate, incubated in 10 µM methotrexate in Schneider’s Drosophila Medium (ex vivo for 60 min at room temperature). 10xStat92E-GFP in green fluorescence. DAPI staining in blue to visualize the nucleus. Scale bar: 20 µm. (B) Box plot shows the quantitation of the relative fluorescence intensity of 10xStat92E-GFP based on the images in (A); middle line depicts the median and whiskers show Tukey. Statistical analysis was performed with a two-tailed t-test; ****, P<0.0001; n = 30 nephrocytes from six 1-day old female adults (5 nephrocytes/fly).