JAK-STAT pathway activation compromises nephrocyte function in a Drosophila high-fat diet model of chronic kidney disease
- Center for Precision Disease Modeling, Department of Medicine, University of Maryland School of Medicine, United States
- Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland School of Medicine, United States
Figures
Figure 1 with 1 supplement
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.
Figure 1—figure supplement 1
High-fat diet leads to lipid droplet accumulation in the nephrocytes.
(A) Nephrocytes from Drosophila w1118 fed a regular diet (normal fat diet, NFD) or high-fat diet (HFD, NFD supplemented with 14% coconut oil). Nile red stains lipid droplets in red. Scale bar: 50 μm. (B) Quantitation of Sns-mRuby3 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=6 flies (7-day-old females).
Figure 2 with 1 supplement
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.
Figure 2—figure supplement 1
High-fat diet changes nephrocyte morphology.
(A) Nephrocytes from Drosophila (sns-mRuby3, 7-day-old females) fed a regular diet (normal fat diet, NFD) or high-fat diet (HFD, NFD supplemented with 14% coconut oil). Sns-mRuby3 is in green. Upper panels show cortical surface; Scale bar: 5 μm. Lower panels show subcortical regions; Scale bar: 5 μm. (B) Quantitation of Sns-mRuby3 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.
Figure 3
High-fat diet activates the Janus kinase/signal transducer and activator of transcription (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.
Figure 4
Janus kinase/signal transducer and activator of transcription (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 occur. A 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 Flippase (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 labeled 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 five neighbor cells.
Figure 5 with 1 supplement
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 Janus kinase/signal transducer and activator of transcription (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-polychaetoid (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 (one nephrocyte/fly) from 7-day-old female flies.
Figure 5—figure supplement 1
Upd2-GFP is secreted from the fat body and transported to the nephrocytes.
(A) Representative confocal images of nephrocytes. Genotype: pp-Gal4>UAS GFP. GFP is shown in green. DAPI stains the nuclei in blue. Scale bar: 20 µm. (B) Representative confocal images of nephrocyte cortical regions. Anti-polychaetoid (Pyd) is shown in red. Scale bar: 4 µm.
Figure 6 with 2 supplements
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, Signal-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).
Figure 6—figure supplement 1
Stat92E depletion rescues HFD-caused nephrocyte functional decline.
(A) Representative confocal images of nephrocytes from female adults that were fed on a regular diet (normal fat diet, NFD) or high-fat diet (NFD supplemented with 14% coconut oil, HFD) for 7 days. Genotype: Control (Dot-Gal4-Gal4/+); Stat92E depletion (Dot-Gal4-Gal4/+; UAS-Stat92E-IR_#2/+). Dextran is shown in red. Scale bar: 40 µm. (B) Box plot shows the quantitation of the relative fluorescence intensity of 10 kD dextran uptake based on images in (A); middle line depicts the median and whiskers show min to max. Statistical analysis was performed with a two-way ANOVA corrected with Tukey; ****p<0.0001; ns, not significant; n=12 flies.
Figure 6—figure supplement 2
Stat92E depletion rescues HFD-caused Sns-mRuby3 distribution defects in the nephrocytes.
(A) Representative confocal images of nephrocytes from female adults that were fed on a regular diet (normal fat diet, NFD) or high-fat diet (NFD supplemented with 14% coconut oil, HFD) for 7 days. Genotype: Control (Dot-Gal4-Gal4, sns-mRuby3/+); Stat92E depletion (Dot-Gal4-Gal4, sns-mRuby3/+; UAS-Stat92E-IR_#2). Sns-mRuby3 is shown in red. Scale bar: 5 µm. (B) Box plot shows the quantitation of Sns-mRuby3 protein distribution (cytoplasmic vs membrane) based on images in (A); middle line depicts the median and whiskers show min to max. Statistical analysis was performed with a two-way ANOVA corrected with Tukey; ****p<0.0001; ns, not significant; n=10 flies.
Figure 7 with 2 supplements
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 Janus kinase/signal transducer and activator of transcription (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.
Figure 7—figure supplement 1
Methotrexate treatment inhibits Janus kinase/signal transducer and activator of transcription (JAK-STA)T pathway activity.
(A) Representative confocal images of 7-day-old female adult nephrocytes (10xStat92E-GFP). Control, incubated in Schneider’s Drosophila Medium (ex vivo for 60 min at room temperature); methotrexate, 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<0.01; n=6 flies.
Figure 7—figure supplement 2
Methotrexate treatment restores Sns-mRuby3 distribution defects following a high-fat diet.
(A) Representative confocal images of nephrocytes from control Drosophila (sns-mRuby3; 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. Sns-mRuby3 is in red. Scale bar: 5 μm. (B) Box plot shows the quantitation of Sns-mRuby3 protein distribution (cytoplasmic vs membrane) 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<0.0001; ns, not significant; n=12 flies (7-day-old females).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Chicken polyclonal anti-GFP | Abcam | Cat. ab13970; RRID:AB_300798 | IF(1:1000) |
| Antibody | Mouse monoclonal anti-Pyd | Developmental Studies Hybridoma Bank (DSHB) | RRID:AB_2618043 | IF(1:100) |
| Antibody | Goat anti-mouse Alexa Fluor 488 | Invitrogen | Cat. A11029; RRID:AB_2534088 | IF(1:500) |
| Antibody | Goat anti-chicken Alexa Fluor 488 | Invitrogen | Cat. A11039; AB_2534096 | IF(1:500) |
| Chemical compound, drug | Methotrexate | Sigma-Aldrich | Cas. 06563 | Methotrexate treatment |
| Other | DAPI | Thermo Fisher Scientific | Cat. D1306 | Immunochemistry |
| Other | 10 kD Texas Red-dextran | Thermo Fisher Scientific | Cas. D1828 | FITC-albumin and 10 kD dextran uptake assays |
| Other | FITC-albumin solution | Sigma | Cas. A9771 | FITC-albumin and 10 kD dextran uptake assays |
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: w1118 | Bloomington Drosophila Stock Center (BDSC) | RRID:BDSC_3605 | |
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: Dot-Gal4 | BDSC | RRID:BDSC_67608 | |
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: ppl-Gal4 | BDSC | RRID:BDSC_58768 | |
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: tub-Gal80ts | BDSC | RRID:BDSC_7017 | |
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: 10XStat92E-GFP | BDSC | RRID:BDSC_26198 | |
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: Stat92E-IR_#2 | BDSC | RRID:BDSC_33637 | |
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: Socs36E-IR | BDSC | RRID:BDSC_35036 | |
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: Stat92E-IR | Vienna Drosophila Resource Center (VDRC) | VDRC_106980 | |
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: sns-mRuby3 | Delaney et al., 2024 | ||
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: hs-Flp122; UAS-FlpJD1/CyO, Act-GFPJMR1; Act5C>CD2>Gal4S, UAS-mCD8-GFPLL6/TM6b | Zhao et al., 2015 | ||
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: UAS-hop.Tum | Harrison et al., 1995 | ||
| Genetic reagent (D. melanogaster) | Drosophila melanogaster: UAS-upd2:GFP | Hombría et al., 2005 | ||
| Software, algorithm | FIJI (ImageJ) | Schneider et al., 2012; https://imagej.net/Fiji/Downloads | Fiji-macOS | RRID:SCR_003070 |
| Software, algorithm | Adobe Illustrator | https://www.adobe.com/ | Adobe Illustrator 2022 | RRID:SCR_010279 |
| Software, algorithm | GraphPad Prism | https://www.graphpad.com/scientific-software/prism/ | GraphPad Prism 9 | RRID:SCR_002798 |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/96987/elife-96987-mdarchecklist1-v1.docx
-
Source data 1
Individual values for each condition and genotype shown in the plots.
- https://cdn.elifesciences.org/articles/96987/elife-96987-data1-v1.xlsx
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JAK-STAT pathway activation compromises nephrocyte function in a Drosophila high-fat diet model of chronic kidney disease
eLife 13:RP96987.
https://doi.org/10.7554/eLife.96987.3
